Michele Goodhardt

Sophie Polo

Valérie Lallemand-Breitenbach

Christophe Escudé

François Ouimette

David Garrick

Antoine Canat

Adeline Veillet

Robert S Illingworth

Emmanuelle Fabre

Pierre Therizols
email: pierre.therizols@inserm.fr.

DAXX safeguards pericentromeric heterochromatin formation in embryonic stem cells

Omnis cellula e cellula " Cellularphatologie, Rudolf Virchow, 1858 " -La science, mon garçon, est faite d'erreurs, mais d'erreurs qu'il est bon de commettre, car elles mènent peu à peu à la vérité. " Voyage au centre de la terre, Jules Verne, 1864 " […] a genome may react to conditions for which it is unprepared but to which it responds in a totally unexpected manner.

Je remercie également tous les membres de l'équipe (la nouvelle, GeBi bien sûr). Merci à Pascale, pour son attention, sa bonne humeur et sa passion des éléments transposables. Merci à Anastasia, pour avoir été ma voisine durant ces dernières années et avoir partagé des discussions sur tout et rien ainsi que des discussions scientifiques. Merci à Renaud, bien qu'arrivé plus tardivement dans notre équipe, avec qui j'ai partagé un nombre inimaginable de café, de repas, et de discussions sur beaucoup de chose. Merci à Amandine, pour ses remarques toujours avisées ; à Amna, pour ses pauses café partagées ; à Arthur bien sûr pour nos longues heures en salle de culture à discuter tout en écoutant ouiFM ; à Joëlle, pour ses histoires et sa profonde gentillesse ; à Alessia, pour sa bonne humeur ; à Noé, pour ses fameuses pizzas du jeudi. Je remercie également Anna, pour ses (très) longues discussions, mais aussi ses conseils sur l'après thèse ; et Laura, pour nos (trop rares) discussions. Merci aux anciens membres naturellement, à Yasmine, pour être ellemême, et nous faire voyager dans son monde à elle, et aussi pour l'élaboration des scripts que je continue à utiliser ; à Julie, pour son dynamisme ; à Alice, pour sa gentillesse ; à Charlotte, pour son caractère bien trempé ; ainsi que toutes les personnes passées par notre équipe au fil des ans.

Parce que l'équipe n'était pas seule dans ce bâtiment Jean Bernard, je remercie nos voisins de palier, l'équipe Amara : Alexis, pour toutes ses anecdotes ; Sarah, pour nos manifestations partagées ; Laurent, pour tes blagues, mais aussi Ali, Athéna, Estelle, Lamine, Lucie, Claudia, Ophélie, Vasiliya, Stéphane, Julien et tous ceux qui ont travaillé au 2 ème étage. La thèse ce n'est pas seulement un travail en laboratoire, mais une petite partie de vie, partagée également en dehors du labo. Ainsi je remercie tous mes amis hors-paillasse : Claire, sans ses toutes ses sous unité, la polymérase fait trop d'erreurs, Nicolas, pour nos discussions, nos tennis, nos rires et nos lectures partagées ; bien sûr Sam & Paul, on s'imaginait soutenir nos thèses en même temps, tu as changé de direction en cours de route mais tu restes ma meilleure amie ;

Je remercie

Mathilde & Clément, Rosie & Julien, Eléonore & Norman, Anaïs, et Marie, pour votre amitié. Je remercie le Magistère de génétique, pour m'avoir permis ses rencontres formidables, aussi bien professionnelles que personnelles. Je pense également à Théo, à Caro, à Salomé, et à Cordi pour toutes ces bières bues ensembles ! Devenir chercheur n'était certes pas inscrit dans mes gènes, mais je crois qu'ils ont été l'environnement propice pour le faire émerger, merci à toute ma famille pour leur soutien, parfois sans bien savoir ce que je faisais dans mon laboratoire. Je remercie surtout mes parents, pour la liberté qu'ils m'ont accordé de trouver ma voie, pour leur bienveillance à toute épreuve, pour la curiosité qu'ils m'ont transmise, et pour leur amour. Je pense fortement à mes grands-parents, qui n'auront pas connu la fin de cette aventure, mais qui sont avec moi, dans mes pensées.

Enfin, pour ta patience et ton soutien durant toutes ses années, malgré mon caractère et mes retards chroniques, malgré tous mes défauts et mes angoisses, tu m'as soutenu, poussé et aidé, merci Pauline. Merci de partager ma vie… Carrier of the genetic information, deoxyribonucleic acid (DNA) is the basic unit of life.

TABLE OF CONTENTS

Table of contents

Acknowledgments_____________________________________________________

Constituent of DNA, the 4 nucleic acids were identified early on by Albrecht Kossel at the University of Berlin, although there was no evidence that DNA was indeed the molecule responsible for hereditary information. The first observations of DNA in cells were made by Walther Flemming. He coined the term of chromatin as the compact DNA he observed was refractory to light under the microscope. He observed that the chromatin was passed on from the mother to the daughter cell during a process that he named mitosis.

A -Structure and organization of chromatin

The first complete theory that DNA was the molecule required for inheritance of traits was published by Walter Sutton and Theodor Bovery in 1902. The actual set of experiments clearly demonstrating the role of DNA were carried by Oswald Avery in 1944. He qualified DNA as the "transforming principle". This discovery led many groups to try to identify the structure of this molecule. The famous double-helix structure was identified by James Watson, Francis Crick, Maurice Wilkins and Rosalind Franklin in 1953 [START_REF] Watson | Molecular structure of nucleic acids[END_REF].

All cells of the organism carry the same DNA sequence and however, different cells and tissues coexist in one individual. How can it be that the same information gives rise to different outcomes? Although DNA serves as template, not every gene is transcribed within the same cell.

In 1942, in a seminal paper, Conrad Waddington coined the term 'epigenetics' to bring together the different phenotypes exhibited by different cells. This is notably the case during embryonic development. From the zygotic single cell to a whole organism, many cell divisions and cell identities will be required.

In this first part, I will describe the organization of DNA into chromatin and the two main types of chromatin structure.

Nucleosome and chromatin

The DNA molecule is not merely floating naked into the nuclear space but is rather found has a complex structure of nucleic acids and proteins. DNA folds around small basic and positively charged proteins called histones. Four types of canonical histones exist in eukaryotes -H2A, H2B, H3 and H4. They form multimeric complexes -H2A-H2B and H3-H4 that assembles into an octamer to form a nucleosome. DNA wraps itself onto nucleosomes, where a sequence of 148bp of DNA surrounds one nucleosome. This organization of nucleosome-DNA forms the chromatin and implicates repeating units all along the DNA molecule that could give the observed flexibility of this molecule [START_REF] Kornberg | Chromatin Structure : Oligomers of the Histones[END_REF][START_REF] Kornberg | Chromatin structure: a repeating unit of histones and DNA[END_REF]. DNA wraps 1.75 turns around a nucleosome. The crystal structure of nucleosomes interaction with DNA was only obtained in 1997 (Figure 1A) [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 Å resolution[END_REF]).

X-ray crystallography of a nucleosome-DNA complex at high resolution demonstrated the folding of chromatin, but also that some part of the histones where not facing the inside of the structure but were rather turned outside to DNA contacts. These domains are the N-terminal domains of the histones and called histone "tails" that can carry many subsequent post-translational modifications that are essential for chromatin functions. These modifications will be addressed in the second part of the introduction. Although all eukaryotes display a nucleosomal organization of chromatin, the nucleosomes are not equally distributed among the genome. Furthermore, the spacing of nucleosome is of great importance and varies from species to species [START_REF] Baldi | Nucleosome positioning and spacing: From genome-wide maps to single arrays[END_REF]. The spacing between nucleosomes have different impacts on chromatin processes, notably for transcription as the presence of nucleosomes can profoundly impact the binding of transcription factors (Zhu et al. 2018a). In Saccharomyces cerevisiae, the baker's yeast, the average spacing between nucleosome is about 15bp, whereas in mouse it is mostly about 45bp. Furthermore, it was observed across eukaryotes that evolution favored a spacing biased toward 10n+5 (15, 25, 35…) but depleted for 10n (10, 20, 30…) [START_REF] Gibson | Organization of Chromatin by Intrinsic and Regulated Phase Separation[END_REF][START_REF] Voong | Insights into Nucleosome Organization in Mouse Embryonic Stem Cells through Chemical Mapping[END_REF]) (Figure 1B). Very recently, it was demonstrated that distances between nucleosomes could even participate in the phase-separation properties of nucleic acids [START_REF] Gibson | Organization of Chromatin by Intrinsic and Regulated Phase Separation[END_REF]. Gibson et al. showed that histone proteins have the intrinsic ability to form molecular condensates. Phase-separation capacities could be modified according to histone post-translational modifications and transcription factor bindings. These results suggest that chromatin organization and chromatin processes might be regulated by physical principles. The stability of the nucleosome depends on its genomic localization. Interestingly, most species display around the transcription start site (TSS) of genes a nucleosome-depleted regions which is surrounded by strongly phased nucleosomes as observed by MNAse studies [START_REF] Hughes | A Functional Evolutionary Approach to Identify Determinants of Nucleosome Positioning : A Unifying Model for Establishing the Genome-wide Pattern[END_REF].

Using a chemical mapping of nucleosomes, it was recently shown that at the TSS and transcription termination site lied 'fragile' nucleosomes which are unstable and easily displaced during transcription [START_REF] Voong | Insights into Nucleosome Organization in Mouse Embryonic Stem Cells through Chemical Mapping[END_REF]) (Figure 1C).

Between nucleosomes, DNA can be enriched into linker histones (Figure 1D). Linker histones have been conserved along evolution, although more diversity is observed than for nucleosomal-histones. Histone composition can drastically change the biophysical properties of the chromatin fiber and histone concentrations can vary between different regions of the genome.

Mammals have eleven linker histone H1 proteins and it is likely that they have overlapping yet different roles according to cell type and metabolic state. In mouse, H1 protects around 20bp in mouse, and genetic deletion of H1 lead to embryonic lethality, suggesting a crucial role for H1 on the cell physiology [START_REF] Fan | H1 Linker Histones Are Essential for Mouse Development and Affect Nucleosome Spacing In Vivo[END_REF]. Linker histones H1 are tightly linked to chromatin condensation, even though they have been historically less studied. Indeed, H1 presence is usually correlated with a higher chromatin condensation, as observed in for rod photoreceptors of the mouse retina where it corresponds to the increase of the nucleosomal repeat length from 190 to 206bp together with H1 accumulation [START_REF] Popova | Developmentally regulated linker histone H1c promotes heterochromatin condensation and mediates structural integrity of rod photoreceptors in mouse retina[END_REF]. Other studies have shown that H1 mediates local chromatin compaction and subsequent gene silencing [START_REF] Kim | Linker histone H1.2 establishes chromatin compaction and gene silencing through recognition of H3K27me3[END_REF][START_REF] Chen | PTEN Interacts with Histone H1 and controls chromatin condensation[END_REF].

These results illustrate that the very nature of chromatin, the nucleosomal organization, the spacing between nucleosomes and the presence of linker histones can modify DNA-related processes.

Chromatin states: euchromatin and heterochromatin

The mammalian genome measures roughly 2 meters long, yet it needs to fit inside a 10 µmdiameter nucleus. Thus, chromatin must be tightly condensed. The mammalian genome measures roughly 2 meters long, yet it needs to fit inside a 10 µm-diameter nucleus. Thus, chromatin must be tightly condensed. The firsts observations of chromatin using DNA dyes allowed the identification of two main types of chromatin -heterochromatin and euchromatin. Euchromatin corresponds to open, decompacted and transcribed regions of the genome, whereas heterochromatin dovetails with close, compacted and repressed regions (Figure 2A). Subsequent molecular and genomic techniques gave more insights into the meaning of these two-chromatin types categorization.

The classical view of DNA folded around nucleosomes gave rise to the vision of a 10nmfiber that allows two physical properties -the positive charges of the histones are neutralized by the negative charges given by the phosphate DNA backbone and secondly, it provides clues of how the 2m of the genome can be compacted to fit inside a 10µm nucleus. The 10nm fiber was observed by electron microscopy (EM) in 1975 and then referred in textbooks as the bead-on-astring model [START_REF] Olins | Spheroid Chromatin Units ( v Bodies )[END_REF]. As this 6-fold compaction given by the 10nm fiber organization could not account for the different chromatin states observed by microscopy studies, it was proposed that chromatin could be organized as a repeated higher-order structure -the 30nm fiber [START_REF] Finch | Solenoidal model for superstructure in chromatin[END_REF]) (Figure 2B). Furthermore, euchromatin is sought to represent only 10% of the genome, which clearly indicates that most of the genome is organized into the more condensed heterochromatin state [START_REF] Fussner | Living without 30nm chromatin fibers[END_REF]. The 30nm fiber was thus suggested to be a physical organization of chromatin which involves DNA compaction. Several 30nm fiber models were proposed that recapitulated the physical properties of such chromatin as the solenoid, the helix zigzag, the cross-linker or the supranucleosome models [START_REF] Fussner | Living without 30nm chromatin fibers[END_REF]. Each of these models proposed a view for nucleosome arrangements within the three-dimensional space that relied of EM observations of extracted chromatin and theoretical modeling. Yet, the very existence of 30nm fiber remains controversial and some authors argues that the 10nm folding could well-enough explain the different compaction levels and that the 30nm fiber was never actually observed in vivo in another model that the starfish sperm nuclei [START_REF] Horowitz | The Three-dimensional Architecture of Chromatin In Situ: Electron Tomography Reveals Fibers Composed of a Continuously Variable Zig-Zag Nucleosomal Ribbon[END_REF][START_REF] Fussner | Living without 30nm chromatin fibers[END_REF].

Evidence of large-scale chromatin compaction was established a long time ago for the inactive X chromosome. In mammals, females carry two X chromosomes coming from the two parents. Yet, only one remains active in the cell. The other one is silenced and compact. This compaction form the long-known Barr body [START_REF] Barr | A Morphological Distinction between Neurones of the Male and Female , and the Behaviour of the Nucleolar Satellite during Accelerated Nucloprotein Synthesis[END_REF]. Already visible with a DNA dye staining, FISH studies have further confirmed this phenomenon, the inactive X chromosome appearing as a small and compact FISH signal whereas the active X is less compacted and takes more space with a more diffuse fluorescence signal. This was also appreciated on polytene chromosomes of Drosophila melanogaster for which active regions also appear to be more decompacted [START_REF] Berkaeva | Functional analysis of Drosophila polytene chromosomes decompacted unit: The interband[END_REF]. A later study demonstrated that the same-sized Mb domains occupied different proportion of the nucleoplasm depending on the gene-content of the regions. This indicates that different degrees of chromatin compaction are observed, and it seems to correlate with the transcriptional status of the region.

To assess the molecular compaction state of chromatin at specific loci, different strategies have been adopted. The firsts used are molecular techniques such as DNAseI and MNAse. Indeed, these strategies stemmed from early characterization of promoter regions. These techniques have then be used to assess genome-wide the regions that display a more open or closed chromatin state, when the accessibility to digestion was high or low respectively [START_REF] Henikoff | Epigenome characterization at single base-pair resolution[END_REF]. The development of the HiC technique to probe all chromatin interactions genome-wide revealed that the genome globally segregates into two particular compartments -A and B, which correspond respectively to an active and inactive chromatin states. A and B compartments spatially segregate from one another and form the basis of the three-dimensional organization of chromatin that I will deeply describe in a next chapter. The HiC technique that probe all chromatin interactions genome-wide could also be used to assess chromatin compaction state of one locus as it is the intensity of close-by interactions for each locus. It has been used to study the impact of polycomb on gene repression during differentiation [START_REF] Kundu | Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation[END_REF].

Correlation with ChIP-seq data have revealed that compacted and decompacted regions were each enriched for particular set of proteins and histone modifications, and also correlated with the A and B chromatin compartments. Indeed, the strongest correlation include the enrichment of histone acetylation for open regions, whereas closed regions are enriched for histone methylation, that I will describe in a later chapter. Interestingly, Filion et al. proposed that in addition to the observed open or closed chromatin, more subtle chromatin compaction states coexist within the cell and might arise from the action of different set of factors [START_REF] Filion | Systematic Protein Location Mapping Reveals Five Principal Chromatin Types in Drosophila Cells[END_REF].

These results suggest that while chromatin can either belong to the A or B compartments, compaction state is finely regulated to allow the different DNA-related processes to happen.

More recent studies have used a novel assay for transposase-accessible chromatin (ATAC) followed by high-throughput genome sequencing [START_REF] Buenrostro | Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position[END_REF]. This technique allows for a genome-wide cartography of chromatin accessibility. It has been improved to look at allelespecific gene transcription, which gives a deeper understanding of genomic regulation, at the level of specific gene-units [START_REF] Xu | Landscape of monoallelic DNA accessibility in mouse embryonic stem cells and neural progenitor cells[END_REF]. The authors observed an unexpected allele-specific regulation of chromatin compaction, yet the allelic choice could be randomly established and did not rely on the parental origin. This study points out that not only chromatin state regulation happens at specific regions of the genome, but that it can also be regulated at particular gene-unit and at the level of the chromatid.

It was though for a long time that chromatin compaction was a direct visualization of the transcriptional state of the locus. It is now demonstrated that it is not always the case. Chromatin decondensation can indeed precede gene activation, and it is now believed that decompaction of a locus might prime a gene to allow a faster activation when needed. This was observed for the immunological memory of T cells and macrophages [START_REF] Bevington | Chromatin priming elements establish immunological memory in T cells without activating transcription[END_REF]. This was also hypothesized and then demonstrated for the upregulation upon differentiation of specific genes in pluripotent cells [START_REF] Peric-Hupkes | Molecular Maps of the Reorganization of Genome-Nuclear Lamina Interactions during Differentiation[END_REF]Therizols et al. 2014).

Since the 1960's, studies have tried to understand lymphocyte activation. It was observed early on that upon activation, the cells would drastically change their nuclear size, RNA production levels and would increase global histone acetylation. It was only recently elucidated how activation of lymphocytes could trigger overall chromatin decondensation. Chromatin firstly goes away from the repressive nuclear periphery, and then a large MYC-dependent genome reorganization takes place, where long-range contact decrease and short-range contacts increase. This MYC-dependent reorganization is visible as histone nanodomain clusters are dissolve into mononucleosome domains [START_REF] Kieffer-Kwon | Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation[END_REF]. Interestingly, chromatin condensation state can vary when cells are kept in culture, suggesting that in vitro culture conditions might hamper the possible conclusions [START_REF] Oh | Nuclear DNA Methylation and Chromatin Condensation Phenotypes Are Distinct Between Normally Proliferating / Aging , Rapidly Growing / Immortal , and Senescent Cells ABSTRACT[END_REF].

Compaction state of peculiar loci have been confirmed using fluorescent in situ labeling (FISH). It has notably been used during the natural decompaction of specific loci upon differentiation of mouse pluripotent cells [START_REF] Williams | Neural induction promotes large-scale chromatin reorganisation of the Mash1 locus[END_REF]. Further characterization of compaction state have been assessed by live-cell imaging of particular loci, displaying striking differences between a compacted and a decompacted locus (Figure 2C) [START_REF] Giri | Orc5 induces large-scale chromatin decondensation in a GCN5-dependent manner[END_REF]). However, it remains to understand how biophysical properties of the chromatin fiber is controlled.

B -Epigenetic regulations of chromatin

The term 'epigenetics' was developed in 1942 by the developmental biologist Conrad Waddington. He introduced the concept of epigenetics to link genotypes and phenotypes in a will to gain insights into the role of the genetic regulation of development. Nowadays, epigenetics usually refers to the heritable genetic information that does not involve changes in DNA sequences.

Epigenetic information thus relies onto the chemical modification of either DNA or histones. DNA folds around nucleosomes, which are composed of the four canonical histones in most of the chromatin. However, other histones have emerged during evolution for specific functions. Histones are considered as very stable proteins with half-lives of several months to year in non-dividing cells, opening the question of how chromatin could be maintained in case of unwanted disruption such as transcription or damages.

Canonical histones are transcribed during S-phase and are crucial for proper DNA replication. Histone genes are short genes, with no introns, organized in multiple tandem repeats of 10 to 17 gene units to ensure large production of proteins. Histone genes have no polyadenylation sequence on 3' end but end with a stem loop followed by a purine-rich region that is complementary to the U7 snRNA. Histone mRNAs are regulated by the stem loop binding protein and the U7 snRNA [START_REF] Marzluff | The Human and Mouse Replication-Dependent Histone Genes[END_REF]. Histone variants have the capacity to be transcribed outside of mitosis, suggesting an important role, particularly in non-dividing cells.

Histone variants genes often contain introns and are only present in one or two copies in the genome. Different variants have been identified for the histones H2A and H3, that I will describe in the following subsection. Interestingly, H4 does not have any variant, and it is one of the most slowly evolving protein, with divergence only described for unicellular eukaryotes [START_REF] Malik | Phylogenomics of the nucleosome[END_REF]. Histone variants usually differ from canonical ones by only a few amino acid substitutions, but these changes are often found within the globular core of the proteins, thereby modifying their interactions between histone-histone and histone-DNA. I will hereby present some H2A and H3 variants.

All the nucleosomal histones can be post-translationally modified. These modifications have been linked to chromatin regulation and compaction state. Some proteins can modify histones, and act as 'modifiers', some can read the modifications, they act as 'readers' and some can remove it, acting as 'erasers'. It was proposed that these proteins act in a coordinated manner, thus forming a dynamic system that Jenuwein, Strahl and Allis called the 'histone-code' hypothesis [START_REF] Strahl | The language of covalent histone modifications[END_REF][START_REF] Jenuwein | Translating the Histone Code[END_REF]). An important number of studies now supports this theory and research is now focusing on disease-related histone modification, implicating either histone or histone-modifying proteins mutations. Changes in the histone composition of specific modifications of histone are crucial to regulate chromatin properties such as the compaction state.

Histone post-translational modifications

For a long time, histones were considered as passive organizational proteins Since then, many histone modifications were observed, yet their role revealed a great complexity system. Many types of post-translational modifications were identified -acetylation and methylation are the major ones, but there is also phosphorylation, ubiquitylation, sumoylation, citrullination, glycosylation, carbonylation, crotonylation, propionylation, ADP-ribosylation, biotinylation, N-formylation, butyrylation, proline and aspartic acid isomerization and the recently discovered dopaminylation, that have been observed so far (Figure 3). The histone code is getting more and more complex as some residues like lysine 9 and 27 of histone H3 can be modified by different post-translational modifications, suggesting that the dynamic and combination of post-translational modifications is crucial for proper chromatin regulation. Globular core of histones can be modified, but many of the associated modifications studied in relation to genome regulation lie at the N-terminal tails of histones. I will only focus on the two most common -acetylation and methylation.

Acetylation

Acetylation is carried by histone acetyltransferase (HAT) enzymes. The major HAT in mammals are the TIP60, the P300/CBP and GCN5 complexes, but more proteins have the capacity to acetylate histone tails. All canonical histones can be acetylated, but the amino acids vary from one to another. Some lysines of newly synthesized H3 and H4 are modified before incorporation and then deacetylated, before properly modified to establish genomic patterns [START_REF] Sobel | Conservation of deposition-related acetylation sites in newly synthesized histones H3 and H4[END_REF]. Lysines are the most often acetylated residues and only one acetyl group can be added. It was the first histone modification associated with gene regulation [START_REF] Brownell | Tetrahymena Histone Acetyltransferase A : A Homolog to Yeast Gcn5p Linking Histone Acetylation to Gene Activation[END_REF][START_REF] Kuo | Transcription-linked acetylation by GcnSp of histones H3 and H4 at specific lysines[END_REF][START_REF] Taunton | A Mammalian Histone Deacetylase Related to the Yeast Transcriptional Regulator Rpd3p[END_REF]. Acetylated histones are associated with open chromatin as the negative charge can neutralize the positive one from the lysine and weakens the interactions between the DNA and the nucleosomes [START_REF] Shahbazian | Functions of Site-Specific Histone Acetylation and Deacetylation[END_REF]. Histone acetylation increases before the mRNA production, suggesting that this modification is indeed preceding and regulating transcription [START_REF] Pogo | RNA SYNTHESIS AND HISTONE ACETYLATION DURING THE COURSE OF GENE ACTIVATION IN LYMPHOCYTES *[END_REF]. Deacetylation is carried by histone deacetylases (HDAC), and HAT and HDAC act dynamically to ensure transcriptional regulation of the genome (Figure 4A). The most common modifications observed are H3K9ac, H3K14ac, H3K27ac and H4K16ac. At the chromatin levels, they mostly appear as peaks present at regulatory regions.

Although acetylation is mainly linked to activation, some reports observed that in specific conditions, HDAC recruitment was correlated with activation, suggesting a more complex view with cycles of acetylation-deacetylation taking place at active genes, yet it remains clear that silenced heterochromatin is devoid of acetylation [START_REF] Wang | Requirement of Hos2 Histone Deacetylase for Gene Activity in Yeast[END_REF][START_REF] Wang | Genomewide Mapping of HATs and HDACs Reveals Distinct Functions in Active and Inactive Genes[END_REF]. Histone acetylation is associated with high accessibility of DNAseI digestion and decompacted chromatin [START_REF] Ridsdale | Histone Acetylation Alters the Capacity of the Hl Histones to Condense Transcriptionally Active / Competent Chromatin *[END_REF][START_REF] Hebbes | DNase I sensitivity in the chicken f-globin chromosomal domain[END_REF]. Genome-wide, histone acetylation is enriched at active promoters but not within gene bodies. H3K9ac and H3K14ac co-occupy many regulatory elements in murine embryonic stem cells (mESCs) and their enrichment correlates with transcription intensity [START_REF] Karmodiya | H3K9 and H3K14 acetylation co-occur at many gene regulatory elements , while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells[END_REF]. The authors observed that H3K9ac and H3K14ac-rich promoters display low levels of DNA methylation, suggesting interplay between different types of epigenetic modifications. HATs are crucial for cell regulation, and their deletion results in either embryonic lethality, as observed for Gcn5 and P300, or defects at later stages of differentiation as reported for CBP [START_REF] Phan | GCN5 and p300 Share Essential Functions During Early Embryogenesis[END_REF][START_REF] Kasper | CBP / p300 double null cells reveal effect of coactivator level and diversity on CREB transactivation[END_REF].

Interestingly, acetylation of histones has also been implicated in transcriptional repression.

Indeed, H4K16 must be deacteylated to enable chromatin compaction [START_REF] Shogren-Knaak | Histone H4-K16 Acetylation Controls Chromatin Structure and Protein Interactions[END_REF].

It renders HDAC proteins as crucial in proper organization of chromatin, as indicated by the disruption of heterochromatin clusters when cells are treated with the HDAC inhibitor trichostatine A (TSA) [START_REF] Taddei | Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases[END_REF]. HDAC functions have been conserved along evolution as both yeast and mESCs were shown to regulate telomeric heterochromatin via histone deacetylation [START_REF] Dan | Roles for Histone Acetylation in Regulation of Telomere Elongation and Two-cell State in Mouse ES Cells[END_REF].

Acetylation of histones can be recognized by different proteins, often containing a bromodomain such as BRD4, which has been implicated in many cancers and genetic syndromes [START_REF] Olley | BRD4 interacts with NIPBL and BRD4 is mutated in a Cornelia de Lange -like syndrome[END_REF]. Other domains have the capacity to recognize and bind to acetylated histones (Figure 4B). Genome regulation appears to be a combination of histone modifications and binding of different factors, indicating multiple layers of control and complexity.

Methylation

Long before to be understood as histone methylase, factors crucial for proper development were discovered in genetic screens in Drosophila and called trithorax and polycomb proteins [START_REF] Schuettengruber | Genome Regulation by Polycomb and Trithorax : 70 Years and Counting[END_REF]. It was later demonstrated that these proteins were not only implicated in the regulation of Hox gene expression but had a broad variety of functions, explained by the fact that they regulate histone methylation profiles. Histone methylation can be associated with different genomic regions depending on the residue. Methylation is performed by histone methyltransferase enzymes, both arginine and lysine residues can be methylated, but as the main methylated sites identified are lysine residues, these enzymes are called lysine methyltransferases (KMTs). Many proteins belong to this family of enzymes, and each one is specialized for one residue, yet some can achieve the same enzymatic process to ensure the robustness of the system.

For each residue, a histone lysine demethylase (KDMs) exist that perform the opposite action of removing the methyl site (Figure 5A). Histone lysine methylation can exist in three states, according to the number of methylation group added -mono-(me1), di-(me2) or tri-(me3) methylation. Combination of the methylation state and the underlying residue specify different chromatin states. The catalytic activity of KMTs often reside in their SET domain [START_REF] Rea | Regulation of chromatin structure by site-specific histone H3 methyltransferases[END_REF].

H3K4me1 is an activating mark only found at enhancers, whereas tri-methylation of the same residue is specific to promoters [START_REF] Heintzman | Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[END_REF]). H3K36 and H3K79 methylation are found over gene bodies of active genes [START_REF] Bannister | Spatial Distribution of Di-and Tri-methyl Lysine 36 of Histone H3 at Active Genes *[END_REF][START_REF] Wang | Combinatorial patterns of histone acetylations and methylations in the human genome[END_REF]. Methyltransferase catalyzing H3K4 methylation mainly belong to the myeloid/lymphoid or mixed-lineage leukemiaassociated (MLL) family and disruptions of MLL is often observed in leukemia by fusions with additional proteins and different genetic syndromes via point mutations [START_REF] Winters | MLL-Rearranged Leukemias -An Update on Science and Clinical Approaches[END_REF][START_REF] Meyer | The MLL recombinome of acute leukemias in 2013[END_REF][START_REF] Ng | Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome[END_REF]).

The early identified polycomb group proteins has revealed to be H3K27me3 methylase.

Two main complexes coexist in cells -polycomb repressive complex 1 (PRC1) and 2 (PRC2). PRC2 is composed of one of the two enhancer of zeste (EZH), the embryonic ectoderm development (EED), the suppressor of zeste (SUZ12) and the CAF1 histone-binding proteins RBBP4 and RBBP7 [START_REF] Schuettengruber | Genome Regulation by Polycomb and Trithorax : 70 Years and Counting[END_REF]. The enzymatic activity is mediated by EZH1 or EZH2 [START_REF] Cao | Role of Histone H3 Lysine 27 Methylation in Polycomb-Group Silencing[END_REF]. Identified for its role on body patterning by regulating Hox genes, polycomb proteins have now been implicated in many processes including X inactivation, genomic imprinting or cell cycle control. Furthermore, mis-regulation of polycomb was observed in many cancers, suggesting a broad range of gene regulation in many different cell types [START_REF] Plath | Role of Histone H3 Lysine 27 Methylation in X Inactivation[END_REF][START_REF] Terranova | Polycomb Group Proteins Ezh2 and Rnf2 Direct Genomic Contraction and Imprinted Repression in Early Mouse Embryos[END_REF][START_REF] Hidalgo | Ezh1 Is Required for Hematopoietic Stem Cell Maintenance and Prevents Senescence-like Cell Cycle Arrest[END_REF][START_REF] Comet | Maintaining cell identity : PRC2mediated regulation of transcription and cancer[END_REF]. Polycomb proteins have been linked to the maintenance of ES cell identity and depletion of PRC1 components leads to derepression of lineage-commitment genes [START_REF] Endoh | Polycomb group proteins Ring1A / B are functionally linked to the core transcriptional regulatory circuitry to maintain ES cell identity[END_REF]. Interestingly, in both flies and mammals, polycomb proteins display a spatial organization into small nuclear foci that stabilizes transcriptional silencing [START_REF] Saurin | The human polycomb group complex associates with pericentromeric heterochromatin to form a novel nuclear domain[END_REF][START_REF] Grimaud | RNAi Components Are Required for Nuclear Clustering of Polycomb Group Response Elements[END_REF][START_REF] Terranova | Polycomb Group Proteins Ezh2 and Rnf2 Direct Genomic Contraction and Imprinted Repression in Early Mouse Embryos[END_REF]. At the level of the chromatin, polycomb proteins have been implicated in the regulation of local chromatin loops but also higher-order organization and chromatin compaction states [START_REF] Vieux-Rochas | Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain[END_REF][START_REF] Entrevan | Regulation of Genome Architecture and Function by Polycomb Proteins[END_REF][START_REF] Boyle | A central role for canonical PRC1 in shaping the 3D nuclear landscape[END_REF]. Specific targeting of EZH2 to a locus induced increased contacts with other H3K27me3-rich domains, showing that the presence of polycomb can reorganize higher-order chromatin folding [START_REF] Wijchers | Cause and Consequence of Tethering a SubTAD to Different Nuclear Compartments[END_REF] Histone 3 methylation of lysine 9 are associated with constitutive heterochromatin, whereas methylation of lysine 27 is associated with facultative heterochromatin and developmental processes. The two enzymes Prdm3 and prdm16 have been described as H3K9me1 methylases [START_REF] Pinheiro | Prdm3 and Prdm16 are H3K9me1 methyltransferases required for mammalian heterochromatin integrity[END_REF]. H3K9me2 is catalyzed by the G9a and G9a-like protein (GLP), whereas K9me3 is performed by the Suv39H1 and 2 and SETDB1 (also named ESET) (Figure 5A).

H3K9me2 is a hallmark of chromatin lying at the nuclear periphery, whereas H3K9me3 is a hallmark of pericentromeric and telomeric heterochromatin, regions that will be covered in chapter 2. Although Suv39H1/2 and SETDB1 have different preferences in genomic localization, they can act within the same complex, further illustrating the redundancy that can take place [START_REF] Fritsch | A Subset of the Histone H3 Lysine 9 Methyltransferases Suv39h1, G9a, GLP, and SETDB1 Participate in a Multimeric Complex[END_REF] Many demethylases, named lysine demethylases (KDMs) have been identified, such as the jumonji-domain-containing (JMJD) family that notably removes H3K9me3, or ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) that mainly removes H3K27me3 (Figure 5A) [START_REF] Agger | UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development[END_REF][START_REF] Lee | Demethylation of H3K27 Regulates Polycomb Recruitment and H2A Ubiquitination[END_REF]. Demethylases, like their methylase counterparts often carry specific affinity for a modified residue, although there is some level of redundancy between KDMs, such as lysine specific demethylase 1 (LSD1) which demethylates H3K4me1 and me2, but also H3K9me1 and me2 as well, suggesting more variability than previously imagined [START_REF] Shi | Histone Demethylation Mediated by the Nuclear Amine Oxidase Homolog LSD1[END_REF][START_REF] Metzger | LSD1 demethylates repressive histone marks to promote androgen-receptordependent transcription[END_REF].

Both methylases and demethylases carry essential roles, yet to date it seems that although they act redundantly, many of them show embryonic lethality at different times of development when depleted (Figure 5B). For example, deletion of SetdB1 results in very early embryonic lethality before the implantation stage, whereas deletion of MLL1 display organogenesis defects for myeloid and lymphoid lineages and lethality around 7 to 10 days after implantation (Figure 5C) [START_REF] Jambhekar | Roles and regulation of histone methylation in animal development[END_REF]. Furthermore, overexpression of LSD1 is frequently observed in cancers, indicating that proper regulation of both methylation and demethylation processes are crucial for genome regulation [START_REF] Kahl | Androgen Receptor Coactivators Lysine-Specific Histone Demethylase 1 and Four and a Half LIM Domain Protein 2 Predict Risk of Prostate Cancer Recurrence[END_REF]. Unlike acetylation, histone methylation does not per se impact chromatin compaction and its impact on chromatin state likely resides in the activity of specific 'readers'.

The use of genome-wide analysis of ChIP experiments has allowed a deeper understanding of histone modification repartition throughout the genome. Histone methylation profiles show striking features with H3K4 or H3K27me being locally restricted to regulatory regions, whereas H3K36me goes over the Kbs length of gene bodies, and H3K9me spreads over Mbs domains.

These assays performed in a large variety of conditions and cell-types has given a comprehensive resource to better understand the gene regulation mechanisms but also has helped apprehending the chromatin folding into chromatin states.

The many proteins identified in the methylation processes suggests both specific roles for each enzyme, but also redundant pathways that act to ensure robustness of the gene regulation programs. Histone methylation can be recognized by different proteins, notably via chromodomain-containing proteins, giving rise to an additional layer of regulation. Finally, KMT activities are not limited to histones and many non-histone proteins can be post-translationally modified and then regulate gene regulation.

DNA methylation

Not only histones can have additional modification, but the DNA itself can be epigenetically marked, notably via the methylation. Methylation of 5 th carbon of cytosine (5mC) is a well-known and characterized epigenetic modification of DNA. It has been deeply investigated in many cellular contexts. It is well conserved as it is found in the animal kingdom, in plants and some fungi, although it is not present in the baking yeast Saccharomyces cerevisiae and barely detectable in Drosophila melanogaster [START_REF] Takayama | Genome methylation in D . melanogaster is found at specific short motifs and is independent of DNMT2 activity[END_REF].

Cytosine methylation is mainly restricted to CpG contexts. Interestingly, the genome is globally depleted for CpG sequences with about 28 million sites. Originally, CpG were described at promoters where it was observed as 1 or 2 Kbs of methylation-rich sequences called CpG islands.

CpG islands are often found unmethylated across all tissues. In fact, only about 10% of genomic CpG lie in promoters. For the majority of CpG sites scattered in the genome, 60 to 80% is methylated [START_REF] Smith | DNA methylation : roles in mammalian development[END_REF].

DNA methylation is established by different enzymes called DNA-methyl transferases (DNMTs) (Figure 6A). Highly conserved enzymes, three DNMTs exist in mammals, with an additional fourth enzyme specific to rodents. DNMT3A and DNMT3B are de novo enzymes that will catalyze the reaction onto an unmethylated DNA sequence. Unique to rodents, DNMT3C emerged via duplication of the DNMT3B gene and methylates promoters of young retrotransposons in male germ cells [START_REF] Barau | The DNA methyltransferase DNMT3C protects male germ cells from transposon activity[END_REF]. DNMT2 enzyme carries no effect on DNA and its activity is instead limited to transfer RNA (tRNA) cytosines [START_REF] Goll | Methylation of tRNA Asp by the DNA Methyltransferase Homolog Dnmt2[END_REF]. DNMTs activities rely on the DNMT3L co-factor that stabilizes and facilitates DNMT3A activity [START_REF] Chedin | The DNA methyltransferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a[END_REF][START_REF] Veland | DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells[END_REF]. DNA methylation is a very dynamic process. Indeed, once added during development, 5mC must be re-established at each mitosis, or it will be lost in a passive way.

If there is no maintenance of the DNA methylation mark, it is diluted as an unmodified cytosine is incorporated in the newly synthesized strand. DNMT1 is a maintenance enzyme that, upon recognition of a hemi-methylated DNA fiber, will add a 5mC onto the complementary strand.

DNMT1 is constitutively expressed, yet it undergoes an increased abundance at the entry of the Sphase to add the 5mC at each hemi-methylated CpG replicated [START_REF] Kishikawa | Control elements of Dnmt1 gene are regulated in cellcycle dependent manner[END_REF]. DNMT1 is recruited to replication forks via interactions with proliferating cell nuclear antigen (PCNA) and ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). UHRF1 binds to the parental methylated sites via its SET and RING associated (SRA) domain and recruits DNMT1 to methylated the newly synthetized DNA strand [START_REF] Bostick | UHRF1 Plays a Role in Maintaining DNA Methylation in Mammalian Cells[END_REF][START_REF] Arita | Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism[END_REF][START_REF] Avvakumov | Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1[END_REF].

Furthermore, in order to provide gene regulation, DNA methylation must be erased at need. It is the role of the ten-eleven translocation (TET) family of enzymes, which have the capacity to convert 5-methylcytosine into 5-hydroxymethylcytosine (5hmC) (Figure 6B) [START_REF] Tahiliani | Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1[END_REF]. It was observed that TET1 bind to CpG-rich promoters of active genes, yet downregulation of Tet1 alone does not lead to a downregulation of all its targets, indicating redundant functions via TET2 and TET3 enzymes [START_REF] Wu | Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells[END_REF]. Interestingly, TET1 is also important for gene silencing as it recruits PRC2 at developmental gene promoters [START_REF] Wu | Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells[END_REF]. TET proteins display differential tissue expression with TET1 being strongly expressed in embryonic stem cells.

TET1 is essential for ESC maintenance in vitro, as TET1 depletion downregulates expression of the pluripotency factor Nanog [START_REF] Ito | Role of Tet proteins in 5mC to 5hmC conversion , ES-cell self-renewal and inner cell mass specification[END_REF]. Mouse deficient for either TET1 or TET2 or both are viable, yet the TET1/TET2 double KO displayed reduced 5hmC and increased 5mC patterns and presented postnatal abnormalities with notably some female infertility [START_REF] Dawlaty | Combined Deficiency of Tet1 and Tet2 Causes Epigenetic Abnormalities but Is Compatible with Postnatal Development[END_REF].

Deletion of Tet3 results in neonatal lethality [START_REF] Gu | The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes[END_REF]. Recruitment of TET1 to promoters does not only rely onto 5mC as depletion of DNA methylation does not prevent its binding. With the expected possible dilution of DNA methylation in case there would be no maintenance, TET enzymes provide an additional and active mechanism of DNA demethylation. It is likely that these two mechanisms act redundantly to ensure efficient DNA demethylation and epigenetic erasure (Hackett et al. 2013b(Hackett et al. , 2013a)). Recent studies suggested that TET enzymes have the ability to convert 5mC to 5hmC and then 5mC to 5-formylcytosyne (5fC) and then to 5-carboxylcytosine (5caC), other cytosine derivatives (Figure 6C) [START_REF] Ito | Tet Proteins Can Convert 5-Methylcytosine to 5-Formylcytosine and 5-Carboxylcytosine[END_REF].

The enzymatic activity of TET enzymes creates oxidation and can induce cytidine deaminase (AICDA)-mediated deamination [START_REF] Cortellino | Thymine DNA Glycosylase Is Essential for Active DNA Demethylation by Linked Deamination-Base Excision Repair[END_REF]. The uracil-guanidine and hydroxyuracil-guanidine produces a mismatch recognized by the thymine DNA glycosylase (TDG), a protein from the base-excision repair (BER) pathway. In vivo, TDG depletion leads to embryonic lethality, as it is required for active demethylation of lineage-specific promoters [START_REF] Cortellino | Thymine DNA Glycosylase Is Essential for Active DNA Demethylation by Linked Deamination-Base Excision Repair[END_REF]. TDG only removes 5fC and 5caC but not 5hmC and requires the DNA replication machinery [START_REF] Maiti | Thymine DNA Glycosylase Can Rapidly Excise 5-Formylcytosine and 5-Carboxylcytosine[END_REF]. The action of TDG creates abasic sites in the genome that are resolved through BER and restores an unmodified cytosine (Figure 6C). TET proteins can bind to any modified cytosine, yet it has higher affinity for 5mC and 5hmC [START_REF] Hu | Structural insight into substrate preference for TET-mediated oxidation[END_REF]. Hu et al. suggested that 5hmC is a relatively stable mark that could act as a regulatory modification. The action of TDG and subsequent BER can generate a single-strand break (SSB) via the action of the AP endonuclease 1 (APE-1). The SSB is restored by XRCC1 and the DNA ligase 3 [START_REF] Weber | Biochemical reconstitution of TET1-TDG-BER-dependent active DNA demethylation reveals a highly coordinated mechanism[END_REF]). Yet, because CpG are present as a dyad in the genome, both cytosyine can be differentially oxidized. The action of TET and TDG onto both strands can then lead to two SSB occurring at the same time, thus generating a double-strand break (DSB), although it seems to be a rare event, suggesting that active demethylation of the two strands occurs iteratively to avoid destabilization of the locus [START_REF] Weber | Biochemical reconstitution of TET1-TDG-BER-dependent active DNA demethylation reveals a highly coordinated mechanism[END_REF].

The presence or absence of a modification will induce the recruitment and action of different co-factors. CpG sequences not only recruit DNMTs when methylated to maintain its status but also numerous additional partners. Notably, the methyl-binding domain-containing family of enzymes (MBD) is heavily recruited to 5mC. A prominent member of the MDB family is the ubiquitously expressed MeCP2 protein, yet other proteins contain a MDB and can be differentially expressed [START_REF] Wood | Tagging methyl-CpG-binding domain proteins reveals different spatiotemporal expression and supports distinct functions[END_REF]. MecP2 functions as a transcriptional repressors, notably via the recruitment of the NCor/SMRT co-repressor complex [START_REF] Lyst | Rett syndrome mutations abolish the interaction of MeCP2 with the NCoR/SMRT co-repressor[END_REF]. ChIP experiments revealed that MeCP2 binds to CpG across all the genome, suggesting that it acts mainly as a global repressor rather than in a promoter-specific manner [START_REF] Skene | Neuronal MeCP2 Is Expressed at Near Histone-Octamer Levels and Globally Alters the Chromatin State[END_REF]. Indeed, MeCP2 deficiency results in global changes in genes and repeated elements transcriptional upregulation. Unmethylated CpG at promoters can be bound by CXXC finger protein 1 (CFP1) that recruits the H3K4 methyltransferase SETD1, which allows to maintain an open, unmethylated region [START_REF] Thomson | CpG islands influence chromatin structure via the CpG-binding protein Cfp1[END_REF]. Interestingly, depletion of Cfp1 reduced the levels of H3K4me3 at CFP1targeted promoters without affecting the transcriptional level, suggesting that it does not per se regulate transcription [START_REF] Clouaire | Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells[END_REF].

DNA methylation acts in close relationship with histone modifications. There is a strong genome-wide correlation between DNA methylation and H3K9me3 [START_REF] Meissner | Genome-scale DNA methylation maps of pluripotent and differentiated cells[END_REF].

DNMT3A can bind to unmethylated H3K4 via its ADD domain and this binding abolishes HP1 tethering to methylated H3K9 (Figure 6A). But in other contexts, DNMTs can bind to HP1 or H3K9 methylases and be recruited to H3K9me3-rich regions, suggesting synergistic action of H3K9me3 and 5mC. Another example is the UHRF1 protein that can physically interact with methylated H3K9 and this interaction increases the stability of the UHRF1 complex, suggesting additional mechanisms of recruitment and stabilization of DNMT complexes depending of the chromatin state of the targeted locus [START_REF] Rothbart | Association of UHRF1 with methylated H3K9 directs the maintenance of DNA methylation[END_REF]. The H3K27me3-modification can be observed at CpG-rich promoters that are devoid of DNA methylation. Furthermore, PRC2 can interact with TET1, further demonstrating the antagonist relationship between H3K27me3 and 5mC [START_REF] Gu | DNMT3A and TET1 cooperate to regulate promoter epigenetic landscapes in mouse embryonic stem cells[END_REF]. Thus, DNA methylation, together with H3K9me3 is a hallmark of constitutive heterochromatin.

The DNA methylation machinery is crucial for cellular processes. Depletion of UHRF1 is sufficient to induce a global loss of DNA methylation and associated phenotypes, including embryonic lethality [START_REF] Sharif | The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA[END_REF]. Highly similar, DNMT3A and DNMT3B act in a redundant manner, yet they both display embryonic lethality and some tissue-specificities, suggesting specific functions [START_REF] Li | Targeted Mutation of the DNA Methyltransferase Gene Results in Embryonic Lethality[END_REF][START_REF] Okano | DNA Methyltransferases Dnmt3a and Dnmt3b Are Essential for De Novo Methylation and Mammalian Development[END_REF]. Cells in culture are also dependent on their DNA methylation machineries as their depletion generally results in cell death, except for embryonic stem cells which have the capacity to overcome the loss of DNA methylation [START_REF] Tsumura | Maintenance of selfrenewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b[END_REF][START_REF] Dodge | Inactivation of Dnmt3b in Mouse Embryonic Fibroblasts Results in DNA Hypomethylation , Chromosomal Instability , and Spontaneous Immortalization *[END_REF][START_REF] Jackson-Grusby | Loss of genomic methylation causes p53-dependent apoptosis and epigenetic[END_REF]. Murine embryonic fibroblasts (MEFs) deficient for DNMTs display high levels of chromosome instabilities and apoptosis, and require P53 mutation to partially overcome the loss of DNA methylation [START_REF] Jackson-Grusby | Loss of genomic methylation causes p53-dependent apoptosis and epigenetic[END_REF][START_REF] Dodge | Inactivation of Dnmt3b in Mouse Embryonic Fibroblasts Results in DNA Hypomethylation , Chromosomal Instability , and Spontaneous Immortalization *[END_REF].

ESCs are thus a useful model to study epigenetic modifications.

One well-characterize disease associated with impaired DNA methylation machinery is the ICF syndrome (immunodeficiency, centromeric instability and facial anomalies), a rare autosomal disorder with about 50 cases described worldwide [START_REF] Jefferson | Altered intra-nuclear organisation of heterochromatin and genes in ICF syndrome[END_REF]. These patients present a mutation into Dnmt3b gene that gives a hypofunctional enzyme, due to a point mutation in 20q11.2 genomic Dnmt3b region. These patients notably display immunological disorders, such as reduced number of T-cells, and lymphocytes also present a characteristic centromeric instability of three chromosomes -1, 9 and 16 that show a typical loss of DNA methylation of their pericentromeric satellite repeats of class II. These phenotypes also happen in an ICF mouse model with the same point mutation of Dnmt3b [START_REF] Ueda | Roles for Dnmt3b in mammalian development: a mouse model for the ICF syndrome[END_REF]). Interestingly, other mutated genes have since been discovered such as ZBTB24, CDCA7 and HELLS [START_REF] Velasco | Comparative methylome analysis of ICF patients identifies heterochromatin loci that require ZBTB24, CDCA7 and HELLS for their methylated state[END_REF]. Although these genes are not directly involved in the DNA methylation machinery, their mutation all lead to hypomethylation of pericentromeric satellites [START_REF] Thijssen | Mutations in CDCA7 and HELLS cause immunodeficiency-centromeric instability-facial anomalies syndrome[END_REF]. Generation of a zebrafish model bearing a mutation in the zbtb24 gene resulted in the phenotypes of ICF syndrome [START_REF] Rajshekar | Pericentromeric hypomethylation elicits an interferon response in an animal model of ICF syndrome[END_REF]. Rajshekar et al. further observe that the loss of DNA methylation at pericentromeric induces the increase of satellite repeats which ultimately triggers an interferon response.

Pathologies can arise with defects not directly linked to the DNA methylation proteins but to 5mC 'readers'. Hemizygous mutations in the X-linked MeCP2 gene inducing a loss of function is lethal for males and results in the Rett syndrome for females [START_REF] Amir | Rett syndrome is caused by mutations in Xlinked MECP2 , encoding methyl-CpGbinding protein 2[END_REF]. Rett syndrome is a severe neurological disorder, suggesting a crucial role for DNA methylation and MeCP2 in the brain, the tissue where it is the highly expressed. The majority of MeCP2-reported mutation affect its methyl-binding domain, suggesting that it is its role as 5mC binding protein that is essential [START_REF] Tillotson | The Molecular Basis of MeCP2 Function in the Brain[END_REF].

DNA methylation has been the focus of many studies as it is frequently altered in many cancers. Interestingly, cancer cells usually display altered DNA methylation profiles, with lower levels at some locations and higher elsewhere. It can result from mutation arising within genes of the DNA methylation pathway or within genes controlling the precise localization of 5mC deposition. Most of the studies have focused on the CpG islands of promoters. Indeed, the TET enzymes are often mutated, as in myeloid cancers in which TET2 is mutated [START_REF] Delhommeau | Mutation in TET2 in Myeloid Cancers[END_REF]. DNA methylation defects not only arise from mutations in the DNA methylation machinery but can be induced by hypoxia in solid cancers, indicating that multiple pathways can lead to altered epigenetic modifications [START_REF] Thienpont | Tumour hypoxia causes DNA hypermethylation by reducing TET activity[END_REF]. However, it is now proposed that DNA methylation profiles undergo many changes along the aging of any individuals. Altered DNA methylation might arise in healthy tissue and only favor tumorigenesis upon mutation of another gene [START_REF] Klutstein | DNA methylation in cancer and aging[END_REF]. Furthermore, tumors often emerge as clonal cell population, and DNA methylation patterns can vary from one cell to the other within the same cancer, highlighting the essential role of this epigenetic modification [START_REF] Brocks | Intratumor DNA methylation heterogeneity reflects clonal evolution in aggressive prostate cancer[END_REF].

Variants of H2A

Many variants of H2A have been identified, and a lot of them are restricted to specific species or tissues. Five full-length variants have been identified in mammals -H2A.X, H2A.Z, macroH2A, H2A.Bdb and H2A.J (not represented in the figure) (Figure 7). I will only describe two of them -macroH2A and H2A.X, which are involved in heterochromatin and genome stability respectively.

macroH2A

MacroH2A was first described in 1992 [START_REF] Pehrson | MacroH2A , a Core Histone Containing a Large Nonhistone Region[END_REF]. It is an unusual histone as it is composed of a tripartite structure where the N-terminal domain is linked to a non-histone macro domain through a lysine rich H1-like region. Thus, macroH2A is larger than its canonical counterpart as the macro domain itself is about 28kDa and originated from the highly conserved macro proteins. Two genes exist in mammals giving two macroH2A proteins 1 and 2 that can carry Maze et al., Nat. Rev. Gen. 2014 different roles. MacroH2A is usually associated with repressed chromatin, notably onto the inactive X chromosome and during senescence when it localizes into senescence-associated heterochromatin foci [START_REF] Zhang | Formation of MacroH2A-Containing Senescence-Associated Heterochromatin Foci and Senescence Driven by ASF1a and HIRA[END_REF][START_REF] Costanzi | Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals[END_REF]. MacroH2A1 can associate with ATRX, which regulates its stability [START_REF] Ratnakumar | ATRX-mediated chromatin association of histone variant macroH2A1 regulates a -globin expression[END_REF]. Loss of Atrx increases macroH2A1 deposition at the α-globin gene and induces its silencing. Furthermore, recent reports suggest that macroH2A might important functions in chromatin organization. Indeed, macroH2A1 can associate with the nuclear lamina in interphase of mouse liver cells [START_REF] Fu | MacroH2A1 associates with nuclear lamina and maintains chromatin architecture in mouse liver cells[END_REF]. Fu et al. further characterized the genome-wide localization of macroH2A and observed that it is enriched at the boundaries of lamina-associated domains, which I will define in a next section. While depletion of macroH2A does not impair LAMIN B distribution, it disrupted H3K9me3 localization. Another study indeed demonstrated that the loss of macroH2A disrupted H3K9me3 localization from heterochromatin and the compaction state of pericentromeric heterochromatin in embryonic stem cells [START_REF] Douet | MacroH2A histone variants maintain nuclear organization and heterochromatin architecture[END_REF].

However, recent studies have challenged this view as they point out that macroH2A might also be a positive regulator of transcription [START_REF] Buschbeck | The histone variant macroH2A is an epigenetic regulator of key developmental genes[END_REF][START_REF] Gamble | The histone variant macroH2A1 marks repressed autosomal chromatin , but protects a subset of its target genes from silencing[END_REF]. Genome regulation via macroH2A can have important impact on cell identity as the depletion of macroH2A leads to the upregulation of the CDK8 colorectal oncogene and increased malignant phenotypes [START_REF] Kapoor | The histone variant macroH2A suppresses melanoma progression through regulation of CDK8[END_REF]).

H2A.X

H2A.X is mostly known for its ability to be phosphorylated at the Serine in position 139 in human in response to DNA damages. In addition, H2A.X differs from H2A.1 by only four amino acids, that might give it specific nucleosomal properties, but this is currently not known. Interestingly, H2A.X is transcribed during S-phase the same way as canonical histones -its mRNA thus folds in a stem loop structure without polyA tails [START_REF] Mannironi | H2A.X. a histone isoprotein with a conserved C-terminal sequence, is encoded by a novel mRNA with both DNA replication type and polya 3′ processing signals[END_REF][START_REF] Nagata | Polyadenylated and 3′ processed mRNAs are transcribed from the mouse histone H2A.X gene[END_REF].

However, as a histone variant, it can be transcribed outside of S-phase that produces a longer mRNA that carries a polyA tail. Although some organisms, such as Caenorhabditis elegans and Saccharomyces cerevisiae does not possess a H2A.X variant, their respective H2A can be phosphorylated in response to DNA damage, indicating that the variant itself might not be necessary, yet the phosphorylation signaling is conserved throughout evolution. H2A.X represent from 2 to 25% of H2A in mammalian cells, depending on cellular types. Interestingly, H2A.X is not essential for mouse development, although mice displayed severe phenotypes such as growth retardation, immunodeficiency and male-specific infertility [START_REF] Celeste | Genomic instability in mice lacking histone H2AX[END_REF]. Celeste et al. described H2A.X deficiency results in increased levels of chromosomal instabilities due to impaired recruitment of subsequent proteins of the DNA damage response pathway, implicating H2A.X as a crucial factor for DNA repair.

There are as many as 10 4 to 10 5 varied DNA lesions per day per mammalian cell due to both endogenous and exogenous sources [START_REF] Hoeijmakers | DNA Damage Response and Repair Mechanisms[END_REF]. Organisms have evolved and created several pathways known as the DNA-damage response (DDR) that allows them to repair their DNA. Among these are the homologous recombination (HR) which is mostly error-free and non-homologous end joining (NHEJ) which is more error-prone than HR [START_REF] Heyer | Regulation of homologous recombination in eukaryotes[END_REF]. After DNA lesions, specific kinases are activated and phosphorylates H2A.X on its Serine 139. γ-H2A.X signal fuses into nuclear foci in the minutes following DSBs [START_REF] Rogakou | Megabase chromatin domains involved in DNA doublestrand breaks in vivo[END_REF]. At the chromatin levels, γ-H2A.X signals spreads over distances that vary from species to species but is Mbs-long in mammals. It is estimated that ~0.03% of all H2A.X molecules are phosphorylated per doublestrand break (DSB) [START_REF] Kinner | Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin[END_REF]. Following DSBs, the ataxia telangiectasia mutated (ATM) kinase phosphorylates H2A.X in a process that requires nibrin 1 (NBS1) for optimum efficacy [START_REF] Fernandez-Capetillo | DNA damageinduced G 2 -M checkpoint activation by histone H2AX and 53BP1[END_REF][START_REF] Falck | Conserved modes of recruitment of ATM , ATR and DNA-PKcs to sites of DNA damage[END_REF]. γ-H2A.X accumulates around the site of the lesion in an asymmetrical manner and γ-H2A.X domains can extend up to 1.7Mb in human [START_REF] Iacovoni | High-resolution profiling of c H2AX around DNA double strand breaks in the mammalian genome[END_REF]. Deletion of both γ-H2A.X and ATM results in embryonic lethality [START_REF] Zha | Complementary functions of ATM and H2AX in development and suppression of genomic instability[END_REF]. In response to other types of DNA damages, such as single-strand breaks resulting from replication stresses or UV damages, the other kinase ATR is active and can also phosphorylate H2A.X [START_REF] Hanasoge | H2AX phosphorylation after UV irradiation is triggered by DNA repair intermediates and is mediated by the ATR kinase[END_REF][START_REF] Ward | Histone H2AX Is Phosphorylated in an ATR-dependent Manner in Response to Replicational Stress *[END_REF]. γ-H2A.X stands as a general signal for DNA lesions and occurs in both HR and NHEJ, rendering the H2A.X histone variant a crucial factor for genome stability.

Variants of H3

Different variants of histone H3 have emerged along evolution, although they do not present much genetic differences between them. About 8 variants have been described, yet only two are ubiquitously expressed, the other being transcribed in a tissue-specific manner. These two widely expressed H3 variants are H3.3 and CENP-A. I will briefly describe CENP-A and will then focus onto the H3.3 variant.

CENP-A, the centromeric H3

One peculiar chromosomal locus is the centromere. Indeed, it must be specifically recognized during mitosis to ensure faithful segregation of chromosomes.

Centromeric nucleosomes are enriched with the centromeric histone variant CENP-A (Centromeric protein A), which is only found there. Centromeric histone defines the centromeric identity more than the underlying sequence [START_REF] Allshire | Epigenetic regulation of centromeric chromatin: Old dogs, new tricks?[END_REF]. CENP-A is crucial for centromere identity and direct kinetochore assembly during mitosis (Müller and Almouzni 2014).

The loss of Cenp-A results in the disorganization of the kinetochore [START_REF] Goutte-Gattat | Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function[END_REF].

CENP-A is deposited by a specific chaperone, the Holliday junction recognition protein (HJURP) in a cell-cycle dependent manner [START_REF] Dunleavy | HJURP Is a Cell-Cycle-Dependent Maintenance and Deposition Factor of CENP-A at Centromeres[END_REF][START_REF] Foltz | Centromere-Specific Assembly of CENP-A Nucleosomes Is Mediated by HJURP[END_REF]. In telophase/early G1, HJURP binds to centromeric DNA and incorporates CENP-A. The timely control of HJURP depends on its phosphorylation (Müller et al. 2014). Crystal structure of CENP-A-containing chromatin was determined and described slight structural differences with H3-containing nucleosomes with notable differences in entrance and exit of DNA that display more flexibility [START_REF] Tachiwana | Crystal structure of the human centromeric nucleosome containing CENP-A[END_REF]. The flexible ends of 13bp observed for CENP-A-containing nucleosome are essential for proper mitotic fidelity as it prevents H1 binding and compaction [START_REF] Roulland | The Flexible Ends of CENP-A Nucleosome Are Required for Mitotic Fidelity Article The Flexible Ends of CENP-A Nucleosome Are Required for Mitotic Fidelity[END_REF]. CENP-A nucleosomes are transmitted across S-phase in a HJURP-dependent manner, which in addition with newly synthesized CENP-A during G1-phase enables a stable inheritance of centromere identity [START_REF] Zasadzińska | Inheritance of CENP-A Nucleosomes during DNA Replication Requires HJURP[END_REF].

CENP-A can be overexpressed in different cancers. Its expression correlates with malignant progression and could be used a predictive biomarker for breast cancers [START_REF] Sun | Elevated expression of the centromere protein-A(CENP-A)-encoding gene as a prognostic and predictive biomarker in human cancers[END_REF].

In colon cancer, CENP-A is upregulated and assembled ectopically via the DAXX and HIRA chaperones [START_REF] Nye | HJURP antagonizes CENP-A mislocalization driven by the H3.3 chaperones HIRA and DAXX[END_REF]. This ectopic formation of CENP-A nucleosomes can recruit kinetochore components at multiples localization and enhance the genomic instability and levels of DNA damages.

The H3.3 histone variant

H3.3 variant is encoded by two genes which are H3f3a and H3f3b in mouse and humans.

The two proteins are identical, yet the two genes possess different untranslated regions [START_REF] Krimer | Induction of H3 . 3 replacement histone mRNAs during the precommitment period of murine erythroleukemia cell differentiation[END_REF]. H3.3 has only 4 to 5 amino acids different from canonicals H3.1 and H3.2, with only one amino acid change in the N-terminal tail and the other belonging to the globular histone core (Figure 8A). H3.3 is one of the most conserved protein among eukaryote, suggesting crucial roles for the cell. The two genes are expressed differentially depending on the cell type [START_REF] Jang | Histone H3 . 3 maintains genome integrity during mammalian development[END_REF].

H3.3 incorporation modify the structural properties of the chromatin. In the early embryonic development, H3.3 maintains a decondensed chromatin state and prevents genome instabilities [START_REF] Jang | Histone H3 . 3 maintains genome integrity during mammalian development[END_REF][START_REF] Lin | Histone variant H3.3 maintains a decondensed chromatin state essential for mouse preimplantation development[END_REF]. In vitro, H3.3 was observed to change the folding of chromatin arrays, inducing a more open conformation with increased spacing between nucleosomes (Figure 8B) (Chen et al. 2013). Chromatin condensation observed in H3.3 knock down correlates with higher levels of linker histone H1, but does not induce large transcriptional changes [START_REF] Lin | Histone variant H3.3 maintains a decondensed chromatin state essential for mouse preimplantation development[END_REF]. Indeed, at the chromatin levels H3.3 is generally anti-correlated with the presence of the linker histone H1 [START_REF] Braunschweig | Histone H1 binding is inhibited by histone variant H3.3[END_REF]). H3.3 was firstly observed at euchromatin enriched for active marks such as H3K4me2 and high PolII occupancy in Drosophila and latter in mammals [START_REF] Ahmad | The Histone Variant H3 . 3 Marks Active Chromatin by Replication-Independent Nucleosome Assembly[END_REF][START_REF] Mito | Genome-scale profiling of histone H3.3 replacement patterns[END_REF][START_REF] Jin | H3 . 3 / H2A . Z double variant -containing nucleosomes mark ' nucleosome-free regions ' of active promoters and other regulatory regions[END_REF][START_REF] Goldberg | Distinct Factors Control Histone Variant H3.3 Localization at Specific Genomic Regions[END_REF]. H3.3 is mainly observed enriched all along gene bodies, with peaks of occupancy at the promoter, yet preceded by a dip at the transcription start site, and the replacement rate of H3.3 is proportional to PolII densities, suggesting that H3.3 is incorporated during transcription to compensate the loss of H3.1 during the progression of the PolII [START_REF] Mito | Genome-scale profiling of histone H3.3 replacement patterns[END_REF]. When looking at specific inducible genes such as Ifit1, Tamura et al. had showed that upon interferon treatment, Ifit1 is transcribed and H3.3 occupancy increases, yet H3.3 stays enriched long after the transcription ceased demonstrating that H3.3 enrichment at gene bodies is not merely the result of the elongation process [START_REF] Tamura | Inducible Deposition of the Histone Variant H3 . 3 in Interferon-stimulated Genes[END_REF]. Interestingly, finer characterization of H3.3 occupancy revealed that it is not only enriched at genes and promoters, but also to regulatory elements, and unexpectedly to heterochromatin regions such as telomeres, indicating previously unappreciated roles for this H3 variant [START_REF] Goldberg | Distinct Factors Control Histone Variant H3.3 Localization at Specific Genomic Regions[END_REF]. Assessment of the dynamic of H3.3 incorporation revealed different turnover rates along the genome (Figure 8C). Promoters and enhancers presented a fast H3.3 turnover rate, whereas gene bodies and transcription end sites displayed a lower turnover rate [START_REF] Ha | Genome-wide analysis of H3.3 dissociation reveals high nucleosome turnover at distal regulatory regions of embryonic stem cells[END_REF][START_REF] Kraushaar | Genome-wide incorporation dynamics reveal distinct categories of turnover for the histone variant H3.3[END_REF][START_REF] Deaton | Enhancer regions show high histone H3.3 turnover that changes during differentiation[END_REF].

Interestingly, in euchromatin regions, H3.3 is enriched at nucleosome-free regions, notably the +1 nucleosome at the promoter, possibly creating a high nucleosome turnover at this position, thus enhancing the accessibility for additional transcriptional factors [START_REF] Jin | H3 . 3 / H2A . Z double variant -containing nucleosomes mark ' nucleosome-free regions ' of active promoters and other regulatory regions[END_REF]. This nucleosomal instability comes from the presence of both H3.3 and H2A.Z in the same nucleosome [START_REF] Yukawa | Genome-wide analysis of the chromatin composition of histone H2A and H3 variants in mouse embryonic stem cells[END_REF]. Furthermore, the gap created by nucleosome eviction at sites of transcription are filled by H3.3, as observed for the inducible Hsp70 gene in Drosophila [START_REF] Schneiderman | Nucleosome-depleted chromatin gaps recruit assembly factors for the H3.3 histone variant[END_REF]. Many genes retain a memory of their transcriptional state. It was demonstrated using nuclear-transplant embryos that the MyoD muscle-lineage gene could keep a transcriptional state for 24 cell divisions without the proper network of transcription factors. Defined as a memory, the transcriptional state of MyoD was shown to be dependent on the lysine 4 of H3.3, pointing to a possible role for H3.3 in epigenetic marking of specific promoters in the genome [START_REF] Ng | Epigenetic memory of an active gene state depends on histone H3.3 incorporation into chromatin in the absence of transcription[END_REF]. It was observed that non-nucleosomal histones are post-translationally modified before incorporation into chromatin, and that could impact on the later modifications of histones, indicating another layer of regulation by histone variants [START_REF] Loyola | PTMs on H3 Variants before Chromatin Assembly Potentiate Their Final Epigenetic State Short Article[END_REF].

In embryonic stem cells, H3.3 carry additional roles that are lost during cellular differentiation. Firsts observations that H3.3 was present at heterochromatin regions came during cell-cycle observations of the H3.3-specific phosphorylation of serine 31 that displayed a pericentromeric localization during metaphase [START_REF] Hake | Serine 31 phosphorylation of histone variant H3.3 is specific to regions bordering centromeres in metaphase chromosomes[END_REF]. Although observed at heterochromatin loci in somatic cells, H3.3 is particularly important for heterochromatin in ESCs.

It is observed enriched at different DNA tandem repeats such as telomeres and transposable elements [START_REF] Goldberg | Distinct Factors Control Histone Variant H3.3 Localization at Specific Genomic Regions[END_REF]. The presence of H3.3 is crucial for the proper silencing of these regions. Indeed, loss of H3.3 enrichment at telomeres is associated with a decrease in H3K9me3 signal and subsequent telomere integrity [START_REF] Ivanauskiene | The PML-associated protein DEK regulates the balance of H3.3 loading on chromatin and is important for telomere integrity[END_REF][START_REF] Udugama | Histone variant H3.3 provides the heterochromatic H3 lysine 9 trimethylation mark at telomeres[END_REF]. H3.3 occupancy at endogenous retroviruses (ERVs) was also demonstrated to be crucial for their transcriptional repression and associated H3K9me3 enrichment [START_REF] Elsässer | Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells[END_REF]. Interestingly, heterochromatic H3.3 possess a low turnover rate, except at satellite sequences where it has a high dissociation rate, yet dependent on the replication machinery [START_REF] Ha | Genome-wide analysis of H3.3 dissociation reveals high nucleosome turnover at distal regulatory regions of embryonic stem cells[END_REF]. H3.3 is essential for mouse development. Mice lacking only one H3f3a or b gene is viable but generates fertile individuals. Depletion of both genes results in early embryonic lethality at the implantation stage [START_REF] Jang | Histone H3 . 3 maintains genome integrity during mammalian development[END_REF]. In agreement with the genetic experiments performed by Jang et al., the knock-down of one or both H3.3 genes by zygotic microinjection led to the same conclusions [START_REF] Lin | Histone variant H3.3 maintains a decondensed chromatin state essential for mouse preimplantation development[END_REF]. Impossibility to reach the blastocyst stage has been proposed to rely on the lysine 27 residue of H3.3 [START_REF] Santenard | Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3[END_REF]. Moreover, H3.3 is essential for proper gametogenesis [START_REF] Tang | Contribution of the Two Genes Encoding Histone Variant H3.3 to Viability and Fertility in Mice[END_REF]. Even embryonic stem cells, which have a particular chromatin state, that I will develop in the last part of the introduction, and cannot survive more than a few divisions without H3.3 because of increased genome instabilities, thus resembling to heterochromatin-related defects (Figure 8D) [START_REF] Jang | Histone H3 . 3 maintains genome integrity during mammalian development[END_REF].

H3.3-associated chaperones

This 'double face' of the histone variant H3.3 arise from two main deposition pathways [START_REF] Szenker | The double face of the histone variant H3.3[END_REF]. In euchromatin, H3.3 is deposited via the histone regulator A (HIRA) complex [START_REF] Quivy | HIRA Is Critical for a Nucleosome Assembly Pathway Independent of DNA Synthesis[END_REF]. HIRA is in a complex with two other proteins -calcineurin binding protein 1 (CABIN1) and ubinuclein (UBN). H3.3/H4 dimers bind to the HRD domain of UBN1 [START_REF] Ricketts | Ubinuclein-1 confers histone H3.3-specific-binding by the HIRA histone chaperone complex[END_REF]. H3.3 is deposited at heterochromatin sites by the death-domain associated protein (DAXX) and altpha-thalassemia/mental retardation syndrome X-linked protein (ATRX) complex, that I will describe in the next sub-section [START_REF] Drané | The death-associated protein DAXX is a novel histone chaperone involved in the replicationindependent deposition of H3.3[END_REF]. Apart from these main identified deposition pathways, other chaperone can bind and incorporate H3.3 within the chromatin (Figure 8C). In euchromatin regions, H3.3 often co-localizes with the histone variant H2A.Z at 'nucleosome-free' regions of regulatory elements [START_REF] Jin | H3 . 3 / H2A . Z double variant -containing nucleosomes mark ' nucleosome-free regions ' of active promoters and other regulatory regions[END_REF]. In human cells, it was observed that the E1A binding protein p400 (EP400) that is part of the pre-initiation complex and a known chaperone of H2A.Z could, together with H2A.Z, incorporate H3.3 at promoter and enhancer regions [START_REF] Pradhan | EP400 Deposits H3.3 into Promoters and Enhancers during Gene Activation[END_REF]. Pradhan et al. demonstrated that the presence of both H2A.Z and H3.3 enhanced transcription in vitro. H3.3 was implicated in the myogenic differentiation in mouse.

Skeletal muscle differentiation relies on the action of the master regulator MyoD, a transcription factor that activate myogenic-lineage genes to induce differentiation. Harada et al. reported that the chromodomain helicase DNA-binding domain 2 (CHD2) binds to both MyoD and H3.3 during myogenic differentiation. CHD2 incorporates H3.3 at MyoD targeted promoters prior to differentiation, suggesting that H3.3 serves as a poised mechanism for subsequent activation [START_REF] Harada | Chd2 interacts with H3.3 to determine myogenic cell fate[END_REF]. Deletion of Chd2 in this context does not impair H3.3 deposition at housekeeping transcribed genes. The related chromatin remodeling factor CHD1 was also interacting with HIRA and regulating H3.3 deposition in Drosophila embryos [START_REF] Konev | CHD1 Motor Protein Is Required for Deposition of Histone Variant H3.3 into Chromatin in Vivo[END_REF].

In humans, both CHD1 and CHD2 can modulate transcription-coupled deposition of H3.3 [START_REF] Siggens | Transcription-coupled recruitment of human CHD1 and CHD2 influences chromatin accessibility and histone H3 and H3.3 occupancy at active chromatin regions[END_REF]. These results indicate that although two main H3.3 deposition pathways exist in all cell types; alternative chaperones might have emerged for specialized functions in different tissues or time of life.

In line with the idea of specialized roles of H3.3 according to specific cell types, additional roles for H3.3 were described in murine ESCs. Some developmental genes are silenced by the polycomb repressive compex 2 (PRC2) and the recruitment of PRC2 is facilitated by H3.3 deposition at these promoters [START_REF] Banaszynski | Hira-Dependent Histone H3.3 Deposition Facilitates PRC2 Recruitment at Developmental Loci in ES Cells[END_REF]. Indeed, deletion of H3.3 impaired PRC2 presence and ESCs presented difficulties to differentiate, highlighting the crucial role played by H3.3 in pluripotent cells. Interestingly, H3.3-rich PRC2 loci display a low histone turnover rate [START_REF] Deaton | Enhancer regions show high histone H3.3 turnover that changes during differentiation[END_REF]. During the early embryonic development, in the zygote, methylation of the lysine 27 of H3.3, achieved by polycomb, is essential for developmental progression, strengthening the connection between polycomb regulation and H3.3 [START_REF] Santenard | Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3 . 3[END_REF]).

Many factors have been identified interacting with H3.3-containing complexes. In Drosophila and human cell lines, the oncogene DEK was proposed to act as a H3.3-chaperone [START_REF] Sawatsubashi | A histone chaperone, DEK, transcriptionally coactivates a nuclear receptor[END_REF]. Interestingly, the RPA complex was found in a shRNA screen as a partner of HIRA-H3.3 complexes [START_REF] Zhang | RPA Interacts with HIRA and Regulates H3 . 3 Deposition at Gene Regulatory Elements in Mammalian Cells[END_REF]. Replication protein A (RPA) is a singlestrand DNA binding protein implicated in DNA replication and DNA repair. In human cells, it was demonstrated that RPA regulated transcriptional directionality at gene promoters. RPA enabled HIRA-mediated H3.3 deposition at regulatory elements, and its deletion impaired transcription [START_REF] Zhang | RPA Interacts with HIRA and Regulates H3 . 3 Deposition at Gene Regulatory Elements in Mammalian Cells[END_REF]. Although implicated in DNA replication, a well-studied role of RPA lies during the repair of DNA damages. Because of its capacity to be transcribed during the whole cell cycle, H3.3 was thought to be important for DNA repair mechanisms to reconstitute the chromatin. In human cells, UV exposure generates DNA damages that induce the recruitment of HIRA. HIRA deposits newly synthesized H3.3 histones at the lesion site and that primes the locus for later transcriptional reactivation [START_REF] Adam | Transcription recovery after DNA damage requires chromatin priming by the H3.3 histone chaperone HIRA[END_REF]. In chicken DT40 cells, H3.3 was also identified to be crucial for UV-damage recovery, notably to complete DNA replication (Frey et al. 2014). Depletion of H3.3 renders the cell more sensitive to UV damages, suggesting a broad effect of this variant on both replication fork progression and DNA repair mechanisms. Luijsterburg et al. proposed that one role of H3.3 at sites of DNA damage could be chromatin expansion. Indeed, the authors observed that CHD2 is recruited at DNA breaks and incudes chromatin expansion and H3.3 deposition [START_REF] Luijsterburg | PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining[END_REF]. The presence of H3.3 is not merely for chromatin reconstitution as it can impact later steps of NHEJ (non-homologous end joining) repair.

C -The DAXX-complex chaperone

DAXX is a very interesting protein. Historically described as a pro-apoptotic protein, Daxx is a 120KDa protein encoded on the chromosome 17 in mouse. It has diverse been reported to act on diverse cellular pathways. Indeed, DAXX is a multi-task protein that has been identified as an interactor of many proteins in proteomic screens [START_REF] Yang | Daxx , a Novel Fas-Binding Protein That Activates JNK and Apoptosis[END_REF]. Yang et al. showed that DAXX induced apoptosis response when overexpression with the intracellular domain of FAS.

Furthermore, DAXX could activate the JNK kinase ASK1 (also called Map3K5) via direct interaction, then leading to an apoptosis response (Figure 9A) [START_REF] Chang | Activation of Apoptosis Signal -Regulating Kinase 1 ( ASK1 ) by the Adapter Protein Daxx[END_REF]. However, opposite results suggested that DAXX might also repress apoptosis [START_REF] Meinecke | Modification of nuclear PML protein by SUMO-1 regulates Fasinduced apoptosis in rheumatoid arthritis synovial fibroblasts[END_REF]).

DAXX interacts with the tumor-suppressor protein P53 upon overexpression [START_REF] Gostissa | The Transcriptional Repressor hDaxx Potentiates p53-dependent Apoptosis *[END_REF]). P53 is a key signaling protein implicated in the cellular response upon DNA damages or oncogene-activation and leads to cell-cycle arrest, apoptosis or senescence. P53 is a short-lived protein and its stability is controlled by other proteins such as the mouse double minute 2 (MDM2) protein. DAXX notably controls the stability of MDM2 via direct interaction. Upon DNA damages, DAXX dissociates from MDM2, which induces its auto-degradation and allows P53 signaling [START_REF] Tang | Critical role for Daxx in regulating Mdm2[END_REF][START_REF] Zhao | Negative Regulation of p53 Functions by Daxx and the Involvement of MDM2 *[END_REF].

DAXX-regulation of apoptosis signaling may happen via other protein interactions. Indeed, DAXX have been implicated in the regulation of the phosphate and tensin homolog (PTEN) tumor-suppressor protein via PTEN deubiquitinylation [START_REF] Song | The deubiquitinylation and localization of PTEN are regulated by a HAUSP -PML network[END_REF]. In glioblastoma, a study reported that PTEN bind to DAXX and could regulate H3.3 deposition [START_REF] Benitez | PTEN regulates glioblastoma oncogenesis through chromatin-associated complexes of DAXX and histone H3.3[END_REF]. In the common PTEN deficient glioblastoma, depletion of DAXX presented a synthetic growth defect phenotype.

The first hint that Daxx was indeed interacting with histone proteins was that it could interact with the centromeric CENP-C during interphase [START_REF] Pluta | Interphase-specific association of intrinsic centromere protein CENP-C with HDaxx , a death domain-binding protein implicated in Fas-mediated cell death[END_REF]. In 2010, Drané et al. purified the human H3.3 histone variant from human HeLa cells and performed mass spectrometry experiments to isolate interactant of H3.3. They observed that DAXX and ATRX interacted with H3.3 and regulated its heterochromatin deposition [START_REF] Drané | The death-associated protein DAXX is a novel histone chaperone involved in the replicationindependent deposition of H3.3[END_REF]. H3.3 binding to DAXX is due to the recognition of H3.3 glycine 90 residue and DAXX glutamine 225 residue in human (Figure 9B). DAXX then wraps 40% of the accessible surface of the dimer H3.3/H4, highlighting how a single amino acid substitution in histone can have a crucial impact [START_REF] Elsässer | DAXX envelops a histone H3.3-H4 dimer for H3.3-specific recognition[END_REF][START_REF] Lewis | DAXX is an H3.3specific histone chaperone and cooperates with ATRX in replicationindependent chromatin assembly at telomeres[END_REF].

DAXX localizes at PML nuclear bodies (NBs) within the nucleus (Figure 9C). Its localization and activities are regulated by post-translational modifications such as SUMOylation.

Small-ubiquitin like modifier (SUMO) are small peptides that can bind to proteins to modify its function and stability. DAXX contains two sumo-interacting motifs (SIM), at the C and N-termini, but the most important one is the N-terminal SIM which controls its interaction with PML. I will deeply describe the DAXX-PML interaction in the PML part of this introduction. However, C- terminal SIM seems implicated in other functions such as the regulation of transcription factors' interactions [START_REF] Escobar-Cabrera | Characterizing the N-and Cterminal small ubiquitin-like modifier (SUMO)-interacting motifs of the scaffold protein DAXX[END_REF].

Intriguingly, Daxx -/-mice die at an early embryo stage, E9.5, and present high levels of apoptosis markers [START_REF] Michaelson | Loss of Daxx , a promiscuously interacting protein , results in extensive apoptosis in early mouse development[END_REF]. A recent paper confirmed the essential role of Daxx and presented a more detailed picture where Daxx-deficient embryo die even before gastrulation at the morula or blastocyst stage, suggesting an essential role in first stages of development (Figure 9D) [START_REF] Liu | SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin[END_REF]. At the zygote and 2-cell stage, DAXX and H3.3 are recruited to pericentromeric heterochromatin in a DPPA3-dependent manner [START_REF] Arakawa | Stella controls chromocenter formation through regulation of Daxx expression in 2-cell embryos[END_REF]. Moreover, deletion of Dppa3 impaired Daxx and pericentromeric satellites expressions, and ectopic expression of DAXX restored H3.3 deposition and pericentromeric expression.

Because H3.3 can be incorporated during all the cell cycle and not only during S-phase, post-mitotic tissues like accumulate H3.3, and this effect increases along the life [START_REF] Piazzesi | Controls Animal Lifespan through the Regulation of Pro-longevity Transcriptional Programs[END_REF][START_REF] Maze | Critical Role of Histone Turnover in Neuronal Transcription and Plasticity[END_REF][START_REF] Tvardovskiy | Accumulation of histone variant H3.3 with age is associated with profound changes in the histone methylation landscape[END_REF]. In post-mitotic neurons, DAXX is phosphorylated in response a calcium-dependent signaling, and in turn incorporates H3.3 at regulatory regions [START_REF] Michod | Calcium-Dependent Dephosphorylation of the Histone Chaperone DAXX Regulates H3.3 Loading and Transcription upon Neuronal Activation[END_REF]. Furthermore, H3.3-specific and DAXX or ATRX driver mutations were observed in pediatric glioblastoma, indicating heterochromatin-associated H3.3 is crucial even in somatic tissues [START_REF] Schwartzentruber | Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma[END_REF][START_REF] Wu | Somatic histone H3 alterations in pediatric diffuse intrinsic pontine[END_REF]. The observed mutations for H3. 3 were the K27M and G34R/V. Furthermore, H3.3 might physiologically be impacted in finer phenotypes, particularly in the brain where its transcription and incorporation is increases in human and mouse suffering from depression [START_REF] Lepack | Aberrant H3.3 dynamics in NAc promote vulnerability to depressive-like behavior[END_REF]. H3.3 plays a critical role in the nucleus accumbens that regulates the reward circuitry and mood. Blocking H3.3 activity was performed by antidepressants therapeutics and released the depressive disorder. DAXX-mediated H3.3 deposition is implicated in the neuronal activation of a particular set of genes and loss of Daxx impairs their transcriptional induction [START_REF] Michod | Calcium-Dependent Dephosphorylation of the Histone Chaperone DAXX Regulates H3.3 Loading and Transcription upon Neuronal Activation[END_REF]. The histone variant H3.3 and its chaperone DAXX are of particular importance as they carry multiple roles, during embryonic development, but also in somatic cells.

Together, these results indicate that H3.3 is implicated in a wide diversity of processes. At heterochromatin regions, H3.3 is deposited via the DAXX-ATRX complex. However, DAXX itself may carry additional ATRX and H3.3-independent roles as observed for ERV regulation, showing another layer of complexity [START_REF] Hoelper | Structural and mechanistic insights into ATRXdependent and -independent functions of the histone chaperone DAXX[END_REF].

II -Spatial compartmentalization of the nuclear space.

Genome activity is regulated at different levels. At the chromatin state, epigenetic modifications such as histone post-translational modifications or DNA methylation, can modify the compaction state of the chromatin and thus regulates binding of transcription factors.

Furthermore, it has recently emerged that the chromatin is not randomly positioned within the nucleus and its three-dimensional organization acts as an important regulator for all chromatin processes, from transcription to DNA repair and replication (Bickmore and Van Steensel 2013).

In mammalian genomes, only about 2% of the genome encodes form proteins. Thus, the vast majority of the genome was qualified as "junk DNA" [START_REF] Ohno | So much "junk" DNA in our genome[END_REF]. It was observed that an important part of this non-coding genome overlapped with previous microscopic descriptions of heterochromatin. Recently, NGS techniques have enabled a more comprehensive view of these genomic regions. An important amount of evidence has emerged to suggest that heterochromatin subcompartments serve as nuclear landmarks for spatial chromatin organization and genome regulation.

I will firstly describe the 3D organization of chromatin within the nucleus. I will then define the two major spatial landmarks of heterochromatin -the lamina-associated domains located at the nuclear periphery, and the chromocenters within the center. Another smaller heterochromatin feature consists of the telomeric regions. Lastly, spreading between spatial heterochromatin landmarks and euchromatin, the transposable elements are part of the heterochromatin components that compose half of the genome but are scattered and do not assemble into a unique compartment (Figure 10). I will present each of these four heterochromatin features with the associated network of proteins that regulate them.

A -Three-dimensional folding and radial positioning of chromatin

This higher-order organization of chromatin was observed at first using microscopy-based techniques. In 1928, using DNA-staining dyes, Emil Heitz coined the term "heterochromatin" as he noticed that some regions of the chromosomes were densely-stained. He went even further in this characterization as he could specify that some regions are always densely-stained, whereas other are only heterochromatic in some cells [START_REF] Passarge | Emil Heitz and the Concept of Heterochromatin : Longitudinal Chromosome Differentiation was Recognized Fifty Years Ago[END_REF][START_REF] Allshire | Ten principles of heterochromatin formation and function[END_REF].

The idea that chromosomes must have delimited spaces in the nucleus was proposed early on by Carl Rabl and later developed by Theodor Boveri, whom worked on horse worms and coined the term of chromosome territory (CT) [START_REF] Boveri | Die Blastomerenkerne von Ascaris megaloce-phala und die Theorie der Chromosomenindividualität[END_REF]). Yet, EM studies have diminished this theory as it only reported an intermingling of chromatin in interphase nuclei. In the 1990's and 2000's, researchers have studied the organization of the genome into CTs using hybridization techniques.

These advances were mainly due to the development of chromosome-specific probes that could be used in FISH experiments. Once probes were designed for specific chromosomes, it enabled the visualization of the different CTs coexisting in cells [START_REF] Bolzer | A complete set of repeat-depleted, PCR-amplifiable, human chromosomespecific painting probes[END_REF]. CTs were observed to exist for each human chromosome in lymphoid cells [START_REF] Boyle | The spatial organization of human chromosomes within the nuclei of normal and emerin-mutant cells[END_REF][START_REF] Bolzer | Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes[END_REF].

Interestingly, this organization is conserved in primates and in mice, suggesting a putative functional role) [START_REF] Tanabe | Evolutionary conservation of chromosome territory arrangements in cell nuclei from higher primates[END_REF].

The CT organization reflects that each chromosome segregates in the nuclear volume.

Further studies have examined the radial organization of the genome -how the chromosomes positioned themselves in accordance to nuclear lamina. At chromosomal scale, it was observed that radial positioning was regulated by the gene-content, the higher the gene-density the more central the chromosome appears. Yet, even for one chromosome, finer-scale observations revealed a general rule for which gene-dense regions tend to more centrally located (Figure 11). This Different landmarks define nuclear heterochromatin sub-compartments. The nucleus is defined by a nuclear envelope segregating the nucleus from the cytoplasm. The nuclear lamina is only interrupted by the nuclear pore complexes. Heterochromatin regions lie at the nuclear periphery. PCH clusters into large foci serving as anchors for heterochromatin regions. Half of telomeres are located close to the PCH foci because of the acrocentric structure of murine chromosomes, the other half is randomly distributed within the nuclear space. The nucleolus serves as anchoring landmark for heterochromatin regions. All heterochromatin-associated landmarks are enriched for specific histone modifications such as H3K9me2 and H3K9me3. PCH = pericentromeric heterochromatin, HP1 = heterochromatin protein 1, NPC = nuclear pore complexes, LBR = lamin B receptor. Adapted from Canat et al., Brief. Funct. Genomics 2020. localization is sought to be dependent on the transcription activity (Bickmore and van Steensel 2013).

Figure 11|

Localizations of chromosomes within the nuclear space. On the left is a modeling of the chromosome territories observed in a mammalian nucleus. Each color represents a particular chromosome. In the middle is a modeling of the A and B chromatin compartment with the A compartment in blue encompassing open and transcribed regions and lies mostly within the nuclear interior. In red is the B compartment that corresponds to closed and inactive regions of the genome that lies notably at the nuclear periphery but also in the nuclear interior where it can associate to nuclear landmarks such as PCH. On the right is a modelling of the genome based on LAD status. LADs are displayed in yellow and are, by definition, mostly enriched at the nuclear periphery, although a substantial fraction of LADs are found within the nuclear interior, probably associated with pericentromeric clusters, illustrating the high overlap between heterochromatin compartments. Non-LAD regions are mostly gene-rich domains that are highly transcribed and represented in blue and mostly observed around the center of the nuclear space. LAD = lamina-associated domains. From Buchwalter, Kaneshiro and Hetzer, Nat. Rev. Genetics 2019.

The study led by Lieberman-Aiden et al. showed that the genome could be organized as fractal globules, yet the HiC realized was only 1Mb-resolution scale. In 2012, three groups codiscovered, using high-resolution HiC that each chromosome was organized into topologicallyassociated domains (TADs). Organization into TADs was observed in mammalian cells [START_REF] Nora | Spatial partitioning of the regulatory landscape of the X-inactivation centre[END_REF][START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF], as well as in drosophila at the same time [START_REF] Sexton | Three-Dimensional Folding and Functional Organization Principles of the Drosophila Genome[END_REF]. TADs are Mb-domains partitioning of the mammalian genomes, and of about 100kb-size in Drosophila. They are defined as the intra-TAD contacts are much higher than the inter-TAD contacts. The overall TAD-organization of chromosomes is mostly invariant between cell types, although a number of TAD boundaries appear to be cell-type specific [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF]. TADs correspond to the basal topological organization of the genome and it correlates with other genomic features, such as the DNA replication domain [START_REF] Pope | Topologicallyassociating domains are stable units of replication-timing regulation[END_REF]. TADs were firstly described in cell population. The use of single-cell HiC techniques provided a more comprehensive picture [START_REF] Nagano | Single-cell Hi-C reveals cell-to-cell variability in chromosome structure[END_REF]. Nagano et al. observed that whereas intra-chromosomal domains were conserved, interchromosomal domains were not and displayed a high cell-to-cell variability. This study reinforced the chromosomal territory organization and noted that only active regions of the chromosome tended to localize at the territory's boundaries.

Altogether, these studies reveal a complex spatial organization at multiple scales.

Chromosomes tend to segregate into specific territories and chromatin regions displaying the same state have increased contacts. In addition to chromosome-specific organization, inter-chromosome interactions arise from spatial compartmentalization of the genome.

B -Lamina-Associated Domains

The major heterochromatin subnuclear compartment correspond to the periphery of the nuclear space. Genomic regions that localize close to the nuclear envelope are called laminaassociated domains (Figure 11).

Nuclear envelope and definition of the lamina-associated domains

The periphery of the nucleus is defined by the nuclear envelope. The nuclear envelope is a double membrane barrier with one side, the inner nuclear envelope, facing the nuclear space and the other, the outer nuclear envelope, facing the cytoplasm. The outer nuclear membrane is contiguous with the endoplasmic reticulum, indicating a strong interconnection between nuclear and cytoplasmic events. Yet, the inner and outer nuclear envelopes have different protein networks and regulation. The nuclear envelope is underlined by the nuclear lamina, a filamentous meshwork composed of lamin proteins. The nuclear lamina and the inner nuclear envelope are highly interacting networks. Four lamin proteins have been described -A, B1, B2 and C. Lamins A and C are derived from an alternative splicing event of the same gene unit, whereas lamins B1 and B2 comes from two separate genes [START_REF] Fisher | cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins[END_REF][START_REF] Mckeon | Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins[END_REF][START_REF] Weber | Protein chemical analysis of purified murine lamin B identifies two distinct polypeptides B1 and B2[END_REF]). Lamins B1 and B2 are ubiquitous, whereas expression of LAMINA/C is tissue-specific [START_REF] Eckersley-Maslin | Lamin A / C is expressed in pluripotent mouse embryonic stem cells © 2013 Landes Bioscience . Do not distribute[END_REF][START_REF] Röber | Differential timing of nuclear lamin A / C expression in the various organs of the mouse embryo and the young animal : a developmental study[END_REF][START_REF] Constantinescu | Lamin A/C Expression Is a Marker of Mouse and Human Embryonic Stem Cell Differentiation[END_REF][START_REF] Solovei | LBR and Lamin A / C Sequentially Tether Peripheral Heterochromatin and Inversely Regulate Differentiation[END_REF]. Other transmembraneproteins have been identified that are located within the nuclear envelope. Entitled NET (nuclear envelope transmembrane) proteins have been mostly identified in the last two decades. Tens of NET proteins have been described, many of them only expressed in a cell-type specific manner [START_REF] Wong | NET gains and losses: The role of changing nuclear envelope proteomes in genome regulation[END_REF][START_REF] Schirmer | Nuclear Membrane Proteins with Subtractive Proteomics[END_REF].

The lamin meshwork and the nuclear envelope are only interrupted by the nuclear pore complexes that allow the complex communication between the nucleus and the rest of the cell. It was suggested early on during the 1960's by electron microscopy that the nuclear lamina forms a repressive environment with a visible layer of dense and compact chromatin surrounding the nuclear space (Figure 12A, Figure 12B, Figure 12C) [START_REF] Comings | The rationale for an ordered arrangement of chromatin in the interphase nucleus[END_REF][START_REF] Van Steensel | Lamina-Associated Domains: Links with Chromosome Architecture, Heterochromatin, and Gene Repression[END_REF]. The observations that the inactive X chromosome in female cells was often seen at the periphery suggested that the peripheral localization might concern only some genomic regions [START_REF] Dyer | Molecular cytological differentiation of active from inactive X domains in interphase: implications for X chromosome inactivation[END_REF]. Indeed, further FISH (fluorescent in situ hybridization) analysis using defined genomic sequences reported that the peripheral localization correlated with gene-poor domains as the gene-rich chromosome 19 was reported to be more centrally located than the gene-poor chromosome 18 which is preferentially found at the periphery [START_REF] Croft | Differences in the localization and morphology of chromosomes in the human nucleus[END_REF]. Interestingly, translocation of a 20Mb region of chromosome 18 was demonstrated to be sufficient for a peripheral localization [START_REF] Croft | Differences in the localization and morphology of chromosomes in the human nucleus[END_REF]). This correlation is not restricted to chromosomes 18 and 19 but is a general rule for all human chromosomes [START_REF] Boyle | The spatial organization of human chromosomes within the nuclei of normal and emerin-mutant cells[END_REF].

Genome-wide mapping of peripherally positioned genes were produced using a technique called DNA adenine methyltransferase identification (DamID). The DamID relies on the bacterial DNA methyltransferase that adds a methyl group onto an adenine instead of a cytosine, thus the only adenine methylated observed during the genome-wide DNA sequencing results from the DamID [START_REF] Van Steensel | Identification of in vivo DNA targets of chromatin proteins using tethered Dam methyltransferase[END_REF]. To identify regions interacting with the nuclear lamina, the methyltransferase is fused to LAMINB (Figure 12D). Widely used in Drosophila melanogaster and human cells, the DamID has produced a comprehensive view of the genomics regions preferentially in close proximity to the nuclear lamina that were named lamina-associated domains (LADs) [START_REF] Pickersgill | Characterization of the Drosophila melanogaster genome at the nuclear lamina[END_REF][START_REF] Guelen | Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions[END_REF]. LADs have been confirmed by other approaches such as ChIP-seq, and in other organisms like mice or Caenorhabditis elegans [START_REF] Poleshko | Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction Article Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction[END_REF][START_REF] González-Aguilera | Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans[END_REF]. The general rules assessed by FISH experiments were confirmed and refined. In mammalian cells, over 1000 LADs are observed. LADs are large domains of 0.1 to 10Mb long, that in total represent about 40% of the genome. They carry heterochromatin hallmarks as LADs are A/T-rich regions and gene-poor regions. Interestingly, LADs are overall conserved, devoid of synteny breakpoints, and although the sequence is not, the A/T-rich feature is constant between mouse and human, suggesting that the radial positioning is evolutionary important [START_REF] Meuleman | Constitutive nuclear lamina -genome interactions are highly conserved and associated with A / T-rich sequence[END_REF]. Furthermore, the genes present in LADs are lowly expressed. As heterochromatin regions, LADs replicate during the late S phase [START_REF] Hiratani | Genomewide dynamics of replication timing revealed by in vitro models of mouse embryogenesis[END_REF].

Assayed in population of millions of cells, no information could be obtained about the number of LADs effectively contacting the nuclear lamina in each nucleus. In fact, only about 30% of LADs are positioned at the periphery in each cell [START_REF] Kind | Single-Cell Dynamics of Genome-Nuclear Lamina Interactions[END_REF]. Using a memory-approach of the DamID via a fluorescent m6A-tracer, the authors could visualize the LADs contacting the nuclear lamina but also follow their fate during the next cell cycle. They observed that during interphase LADs displayed a confined dynamics at the nuclear periphery but they are dislodge during mitosis and do not seem to act as anchoring points for the novel domains that will associate with the nuclear lamina during this second interphase [START_REF] Kind | Single-Cell Dynamics of Genome-Nuclear Lamina Interactions[END_REF]. Single cell genome-wide characterization of DamID sequencing signal then provided evidence that association with the nuclear lamina involve multiple lamina interactions rather than long stretches of association.

Regions contacting the lamina are extremely gene-poor segments, suggestion a potential structural role for these contact points (Kind et al. 2015a).

What have these DNA sequences special to be located at the nuclear periphery, is it encoded in the DNA or is it an epigenetic feature? Apart from the A/T enrichment, a GAGA motif was frequently observed in the Igh and Cyp3a LADs. Furthermore, it was demonstrated the GAGA motif-containing sequence of a few Kbs was sufficient to induce lamina association and transcriptional repression at another genomic location [START_REF] Zullo | DNA sequence-dependent compartmentalization and silencing of chromatin at the nuclear lamina[END_REF]. In another study by Bian et al., a dissection of the beta-globin Hbb locus identified three regions sufficient for peripheral targeting that were also a few Kbs in length [START_REF] Bian | β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications[END_REF]. Sequence-specific targeting mechanisms seems to be conserved throughout evolution as it was also proposed in yeast that some particular sequences called DNA zip code, here located in some gene promoters, were sufficient to target other genomic regions to the nuclear periphery [START_REF] Ahmed | DNA zip codes control an ancient mechanism for gene targeting to the nuclear periphery[END_REF]. However, GAGA repeats do not seem to be particularly in LADs genome-wide, suggesting additional mechanisms [START_REF] Guelen | Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions[END_REF].

It was described that the Igh gene and the neuronal regulator Mash1 were localized at the periphery of progenitor cells and were relocalized to the nuclear interior in more differentiated cells and correlated with their transcriptional activation [START_REF] Kosak | Subnuclear Compartmentalization of Immunoglobulin Loci During Lymphocyte Development[END_REF][START_REF] Williams | Neural induction promotes large-scale chromatin reorganisation of the Mash1 locus[END_REF].

Observed for particular genes, it was then demonstrated that association with the nuclear lamina reflected the transcriptional status for a large majority of LAD-genes [START_REF] Peric-Hupkes | Molecular Maps of the Reorganization of Genome-Nuclear Lamina Interactions during Differentiation[END_REF][START_REF] Kind | Single-Cell Dynamics of Genome-Nuclear Lamina Interactions[END_REF]. Some regions always associate with the nuclear lamina and are called constitutive LADs (cLADs), whereas some only associate in a cell-type specific manner and are named facultative LADs (fLADs), that only partially correlate with the transcriptional status of the region [START_REF] Peric-Hupkes | Molecular Maps of the Reorganization of Genome-Nuclear Lamina Interactions during Differentiation[END_REF].

Studies have observed in many tissues the sequences and underlying genes interacting with the nuclear lamina, yet it remains to understand the protein composition of the nuclear periphery that allows such a silencing and positioning effects.

Protein composition of the nuclear periphery

The first genome-wide characterization of the sequences associated with the nuclear lamina revealed that the genes present in genes were repressed [START_REF] Pickersgill | Characterization of the Drosophila melanogaster genome at the nuclear lamina[END_REF]. LADs strongly correlate with positive enrichment in heterochromatin marks such as -high H3K9me2 and H3K9me3, low H3K4me2 and H4 acetylation and high H3K27me3, CTCF and YY1 binding at the LAD borders [START_REF] Guelen | Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions[END_REF][START_REF] Wen | Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells[END_REF][START_REF] Harr | Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins[END_REF]. LADs are not enriched with high DNA methylation levels [START_REF] Berman | Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains[END_REF]. In Drosophila, depletion of the B-type lamin induced a loss of nuclear lamina interaction for many genes [START_REF] Kohwi | Developmentally Regulated Subnuclear Genome Reorganization Restricts Neural Progenitor Competence in Drosophila[END_REF][START_REF] Shevelyov | The B-type lamin is required for somatic repression of testis-specific gene clusters[END_REF]. Interestingly, in mESCs, lamins are not essential for LADs, showing that additional proteins are required to maintain this localization [START_REF] Amendola | Nuclear lamins are not required for laminaassociated domain organization in mouse embryonic stem cells[END_REF]. It was observed that A-type lamins could interact with chromatin regions, intriguingly both hetero-and euchromatin [START_REF] Gesson | A-type Lamins bind both heteroand euchromatin, the latter being regulated by lamina-associated polypeptide 2 alpha[END_REF][START_REF] Harr | Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins[END_REF][START_REF] Kubben | Mapping of lamin A-and progerininteracting genome regions[END_REF]. Although, deletion of LaminA induced a relocalization away from the nuclear lamina, it was not associated with a loss of repression, suggesting that the nuclear lamina might be important for peripheral anchoring in some tissues but that the repressive compartment is largely due to other proteins [START_REF] Harr | Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins[END_REF][START_REF] Kubben | Mapping of lamin A-and progerininteracting genome regions[END_REF].

Deeper investigations revealed that the enzymes responsible for the compacted heterochromatin state of LADs were important for their peripheral localization. Zullo et al. identified that a GAGA-containing sequence could drive a nuclear lamina association, and further demonstrated that it relies on the binding of the cKrox factor in complex with HDAC3 and the lamina protein Lap2β [START_REF] Zullo | DNA sequence-dependent compartmentalization and silencing of chromatin at the nuclear lamina[END_REF]. Treatment with the HDAC inhibitor trichostatin A (TSA) led to an increased expression of heterochromatin-genes [START_REF] Finlan | Recruitment to the Nuclear Periphery Can Alter Expression of Genes in Human Cells[END_REF]. It was reported that the function of HDAC3 in LADs was not restricted to its enzymatic activity. Indeed, deletion of HDAC3 impaired the cardiac-lineage gene Actc1 peripheral positioning. While complementation with a catalytically dead HDAC3 mutant was sufficient to restore its nuclear association, an HDAC3 mutant unable to bind Lap2β could not [START_REF] Poleshko | Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction Article Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction[END_REF]. It is interesting to note that HDAC3 -/-mice die during embryonic development, enzymatic-dead HDAC3 mice are viable, possibly highlighting a structural role for HDAC3 [START_REF] You | Nuclear receptor co-repressors are required for the histone-deacetylase activity of HDAC3 in vivo[END_REF]. Tissue-specific NETs could also have a role in proper establishment of LADs and gene activation, as exemplified during myogenesis, where myoblast specific NET tether myotubes genes to the periphery to ensure robust repression [START_REF] Robson | Tissue-Specific Gene Repositioning by Muscle Nuclear Membrane Proteins Enhances Repression of Critical Developmental Genes during Myogenesis[END_REF]. Lamin B-receptor (LBR) is expressed in many cell types.

Interestingly, in the cell-types where LBR is not expressed, such as retinal rod cells of nocturnal mammals or olfactory sensory neurons in mouse, no heterochromatin compartment is visible at the periphery [START_REF] Solovei | Nuclear Architecture of Rod Photoreceptor Cells Adapts to Vision in Mammalian Evolution[END_REF][START_REF] Clowney | Nuclear Aggregation of Olfactory Receptor Genes Governs Their Monogenic Expression[END_REF][START_REF] Solovei | LBR and Lamin A / C Sequentially Tether Peripheral Heterochromatin and Inversely Regulate Differentiation[END_REF]. Furthermore, LBR might be implicated in the recruitment of some loci such as the inactive X chromosome [START_REF] Chen | Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing[END_REF].

Nuclear pores may contribute to gene tethering at the nuclear periphery as reported for NUB93 and NUP153, which binds to enhancers sequences [START_REF] Ibarra | Nucleoporin-mediated regulation of cell identity genes[END_REF]. Deletion of NUP153 in murine ESCs impaired the pluripotency state by the loss of silencing at differentiated genes that correlated with a spatial relocalization away from the periphery [START_REF] Benner | The nucleoporin Nup153 regulates embryonic stem cell pluripotency through gene silencing[END_REF]. In a genomewide screen, many proteins were found to disrupt heterochromatin localization at the nuclear periphery, which included DNA methylation proteins, histone chaperones, but also proteins implicated in the maintenance of repressive histone modifications [START_REF] Shachar | Identification of Gene Positioning Factors Using High-Throughput Imaging Mapping[END_REF]. Many proteins are acting together to ensure the proper localization and silencing of heterochromatin at the nuclear periphery, these proteins can be linked to the nuclear envelope or associated with repressive chromatin state, suggesting a redundancy of pathways to ensure higher robustness of nuclear envelope tethering. High-throughput integration of reporter genes at 27,000 different genomic positions revealed that although LADs globally act as repressive environment, the silencing effect vary from positions to positions, suggesting local effects of particular proteins [START_REF] Akhtar | Chromatin position effects assayed by thousands of reporters integrated in parallel[END_REF].

The prominent histone modifications associated with LADs are H3K9me2 and H3K9me3 and have thus been the focus of many studies (Figure 10, Figure 13). Bian et al. have identified three peripheral targeting sequences within a transgenic HBB locus. They observed that the peripheral anchoring of the transgene was abolished in a Suv39h1/2 double KO [START_REF] Bian | β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications[END_REF].

Intriguingly, H3K9me3 is the prominent feature of pericentromeric heterochromatin (PCH), and PCH do not localize at the nuclear lamina. Deletion of the H3K9 di-methylase induced the lamina detachment of the endogenous beta-globin genes, suggesting that H3K9me2 and H3K9me3 may have partially overlapping function in nuclear tethering, and the two marks ensure a robust peripheral localization [START_REF] Bian | β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications[END_REF]. It was observed that integration of large transgene into the C. elegans genome led to accumulation of H3K9me3 and that these transgene localized at the nuclear periphery [START_REF] Towbin | Repetitive transgenes in C. elegans accumulate heterochromatic marks and are sequestered at the nuclear envelope in a copy-numberand lamin-dependent manner[END_REF]. Interestingly, using a genetic screen for peripheral localization in C. elegans, Towbin et al. discovered that the histone lysine methylase pathways was essential. Indeed, they observed that depletion of either the S-adenosil-methionine proteins or the methyltransferase enzymes led to a central relocalization of their reporter [START_REF] Towbin | Step-wise methylation of histone H3K9 positions heterochromatin at the nuclear periphery[END_REF]. A latter study reported that the CEC-4 protein directly binds to H3K9 mono, di and tri-methylated. CEC-4 localizes at the nuclear envelope independently of H3K9 methylation [START_REF] Gonzalez-Sandoval | Perinuclear Anchoring of H3K9-Methylated Chromatin Stabilizes Induced Cell Fate in C. elegans Embryos[END_REF].

Interestingly, CEC-4 is required for the anchoring of heterochromatin domains at the nuclear lamina, but not for its repression. Furthermore, the action of genome-anchoring of CEC-4 is only visible in worm embryo, but not in adult larvae. Deletion of cec-4 impairs the differentiation of embryo toward a muscle cell-type [START_REF] Gonzalez-Sandoval | Perinuclear Anchoring of H3K9-Methylated Chromatin Stabilizes Induced Cell Fate in C. elegans Embryos[END_REF]. These results suggest that redundant pathways for genome anchoring coexist within the nucleus, and that some of these pathways are tissue-specific. No orthologs of CEC-4 have been identified in mammals, yet, other proteins may carry the same function. The PRR14 protein in human was described as localizing in the inner nuclear envelope in interphase and it has the capacity to bind to HP1 [START_REF] Poleshko | The Human Protein PRR14 Tethers Heterochromatin to the Nuclear Lamina during Interphase and Mitotic Exit[END_REF]. Poleshko et al. proposed that PRR14 functions in recognizing heterochromatin regions via HP1 binding during mitosis and then guide the heterochromatin-bound regions toward the periphery when the nuclear envelope is reformed. It was recently demonstrated that an additional factor acting in differentiated tissues in C. elegans was the MRG1 protein. Intriguingly, MRG-1 binds to euchromatin regions and is involved in histone acetylation, indicating that euchromatin domains also play a role in heterochromatin compartmentalization [START_REF] Cabianca | Active chromatin marks drive spatial sequestration of heterochromatin in C. elegans nuclei[END_REF].

In conclusion, different pathways are at play, from epigenetic histone-modifications and particular 'readers' to nuclear lamina proteins, to ensure proper localization and repression of LADs.

Functions and dysfunctions of LADs

From all the studies characterizing the nuclear periphery, it emerges that it acts as a repressive compartment. Yet, different phenotypes accompany the loss of peripheral anchoring, sometimes leading only to a relocalization, sometimes to a loss of the transcriptional repression. Does this compartmentalization have functional roles in genome regulation? Genetic deletion of B-types lamins in mouse impair organogenesis, although without affecting transcription levels, suggesting a crucial role of the nuclear envelope on the cellular architecture and on the organism [START_REF] Kim | Mouse B-Type Lamins Are Required for Proper Organogenesis But Not by Embryonic Stem Cells[END_REF].

There is a good genome-wide correlation of radial positioning and transcriptional state of a locus in a given cell-type. Yet this correlation is not complete. Indeed, using murine embryonic stem cells (ESCs) and two subsequent differentiated states -neuronal progenitor cells (NPCs) and astrocysts (ACs), it was demonstrated that repressed genes associated with the nuclear lamina in ESCs lose their peripheral localization in NPC if they are activated. Vice-versa, genes that are repressed in NPC gain nuclear lamina association [START_REF] Peric-Hupkes | Molecular Maps of the Reorganization of Genome-Nuclear Lamina Interactions during Differentiation[END_REF]. Although it works as a general rule, some genes detach from the lamina in NPCs, yet are only activated at later developmental stages, suggesting they might enter an "unlocked" state as the authors coined it, so they can be expressed at a later stage more efficiently (Figure 14). Association and disassociation concern single gene units as well as multigene regions. Detachment of nuclear lamina is conserved for other cell differentiation programs such as cardiac or muscle commitment [START_REF] Poleshko | Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction Article Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction[END_REF][START_REF] Robson | Tissue-Specific Gene Repositioning by Muscle Nuclear Membrane Proteins Enhances Repression of Critical Developmental Genes during Myogenesis[END_REF].

It remained to understand the cause-consequence relationship. Artificial locus-tethering to the nuclear periphery resulted in gene silencing, although with different efficiencies depending on the reporter [START_REF] Kumaran | A genetic locus targeted to the nuclear periphery in living cells maintains its transcriptional competence[END_REF][START_REF] Finlan | Recruitment to the Nuclear Periphery Can Alter Expression of Genes in Human Cells[END_REF][START_REF] Harr | Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins[END_REF]). Yet, forced transcriptional activation of a LAD-gene induce relocalization toward the nuclear interior (Therizols et al. 2014). It was further shown that the transcriptional activation of a LAD-gene was not the cause of its detachment but rather a consequence of the change in chromatin state. Indeed, targeting of the particular DEL peptide that only decompacted the locus without transcriptional activation led to the relocalization toward the nuclear interior (Therizols et al. 2014). Furthermore, the central localization is maintained for several mitosis even when the peptide is lost, suggesting that epigenetic mechanisms maintain the decompacted state and radial positioning of the locus (Therizols et al. 2014).

Nuclear periphery as a compartment can encompass up to ~40% of the genome. Although not all LADs are at the periphery in each cell, it represents a large amount of DNA that is subjected to all DNA-related process and might encounter DNA breaks. In flies, reports suggest that DNA breaks occurring in heterochromatin are delocalized toward the nuclear periphery via the structural maintenance of chromatin proteins 5 and 6 (SMC5 and SMC6) [START_REF] Ryu | Heterochromatic breaks move to the nuclear periphery to continue recombinational repair[END_REF]. These results suggest that the nuclear periphery might act as a compartment for heterochromatin repair. A recent study further demonstrate that histone-modification are important in the DNA damage response as demethylation of H3K9 via KDM4A is essential to complete DNA repair only at heterochromatin loci [START_REF] Janssen | Timely double-strand break repair and pathway choice in pericentromeric heterochromatin depend on the histone demethylase dKDM4A[END_REF]. Interestingly, DNA lesions occurring in LADs in mammals display a delay in the DNA damage response activation and are less resolved using homologous recombination (HR) but rather via non-homologous end joining (NHEJ) [START_REF] Lemaître | Nuclear position dictates DNA repair pathway choice[END_REF]. Despite the discrepancies between organisms, it seems that the radial positioning of chromatin can influence repair pathway choice and kinetics.

Descriptions of the nuclear envelope and lamina-associated domains in various tissues revealed that many pathologies presented an unusual nuclear chromatin compartment, either from the chromatin-associated modifications or directly from the nuclear envelope. A whole family of pathologies is called 'laminopathies' as the common ground of these disorders is the impaired nuclear lamina, and notably mutations in the Lamin A gene [START_REF] Mattout | Nuclear lamins, diseases and aging[END_REF]. Laminopathies are degenerative disorders with a broad range spectrum, such as the Hutchinson-Gilford progeria syndrome (HGPS) which is a premature aging pathology or the Emery-Dreifuss muscular dystrophy that display perturbed heterochromatin [START_REF] Perovanovic | Laminopathies disrupt epigenomic developmental programs and cell fate[END_REF]. Laminopathies result from an accumulation of mutant LAMIN A that induces a disturbed nuclear lamina [START_REF] Goldman | Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome[END_REF]). Since LAMIN A has been implicated in the spatial distribution of heterochromatin, heterochromatin localization in laminopathies has been scrutinized [START_REF] Solovei | LBR and Lamin A / C Sequentially Tether Peripheral Heterochromatin and Inversely Regulate Differentiation[END_REF]. Using a C. elegans model, Harr et al. demonstrated that a point mutation in the unique Lamin A gene mimicked many of the Emery-Dreifuss syndrome phenotypes, including hyper-sequestration of heterochromatin at the nuclear periphery. Interestingly, deletion of the CEC-4 protein, which is involved in H3K9me3-binding, rescued the effect of the Lamin A mutation, suggesting a strong interconnection between nuclear lamina and histone-modifications in the peripheral tethering and phenotypic impact of laminopathies [START_REF] Harr | Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model[END_REF].

Radial positioning can be affected in numerous diseases such as cancers. Indeed, changes in Lamin A gene is often observed in human cancers [START_REF] Willis | Lamin A/C Is a Risk Biomarker in Colorectal Cancer[END_REF][START_REF] Johnson | A-type lamins regulate retinoblastoma protein function by promoting subnuclear localization and preventing proteasomal degradation[END_REF]). Furthermore, by looking at the spatial localization of cancer-associated genes, Meaburn et al.

described a global reorganization of the 3D-genome organization during normal and tumorigenic cell differentiation. A number of genes changed their radial localization in cancerous cells, independently of their transcriptional status [START_REF] Meaburn | Locus-specific and activity-independent gene repositioning during early tumorigenesis[END_REF]. Specific gene repositioning was reproducible between individuals in the case of breast cancer, suggesting a potential function of this repositioning [START_REF] Meaburn | Disease-specific gene repositioning in breast cancer[END_REF]. For particular breast cancer subtypes, it was demonstrated that large multiple gene-unit regions were undergoing transcriptional changes [START_REF] Rafique | Estrogen-induced chromatin decondensation and nuclear reorganization linked to regional epigenetic regulation in breast cancer[END_REF]. Rafique et al. observed that the regions displaying changes in expression were concomitantly decompacted and relocalized away from the nuclear periphery in an estrogendependent manner. Although, the presence at the nuclear periphery was not assessed, large regions encompassing differences in chromatin compaction were described for bladder cancers as well [START_REF] Vallot | PRC2-independent chromatin compaction and transcriptional repression in cancer[END_REF]. These reports suggest that environmental clues such as hormones might regulate the chromatin folding changes observed in cancers and that the nuclear periphery might be a key contributor to the gene-expression alterations.

Lamina-associated domains encompass ~40% of the mammalian genomes. Chromatin association with the nuclear lamina is stochastic but controlled by diverse epigenetic pathways thus ensuring proper formation this nuclear sub-compartment. As an important repressive heterochromatin domain, the LAD-genes are lowly if expressed. The integrity of the nuclear lamina and LADs is crucial for the cell biology and defects in LADs positioning and silencing if often observed in diseases.

C -Pericentromeric heterochromatin

A large portion of the genome consists of repeated DNA sequences. One well-known example is the pericentromeric heterochromatin (PCH) regions that surround centromeres.

Pericentromeric heterochromatin and chromocenters organization

PCH regions are mainly composed of satellite sequences tandemly repeated that can vary from one specie to another. In human, pericentromeric regions consist of three different satellite sequences that are tandemly repeated over large genomic distances (>4Mb). These three different repeat sequences are called satellites. In human, the I, II and II satellites are dispersed in a chromosome-specific pattern. In mouse, tandem repeats of pericentromeric regions are the major satellites, whereas centromeric repeats are the minor satellites. Major satellite sequences are A/Trich 234bp tandemly repeated in a head to tail fashion for 1,000 to 10,000 times per chromosomes, forming Mbs-long domains (Figure 15A) [START_REF] Martens | The profile of repeatassociated histone lysine methylation states in the mouse epigenome[END_REF]. Although major satellites are virtually identical between all chromosomes, recent reports suggest that some chromosomespecific tandem repeats might exist [START_REF] Komissarov | Tandemly repeated DNA families in the mouse genome[END_REF]. In human, satellite sequences represent ~3% of the genome and in mouse, satellites represent from 3 to 8% of the genome depending on the report [START_REF] Martens | The profile of repeatassociated histone lysine methylation states in the mouse epigenome[END_REF][START_REF] Komissarov | Tandemly repeated DNA families in the mouse genome[END_REF]. Major satellites display high sequence similarities [START_REF] Vissel | Mouse major (γ) satellite DNA is highly conserved and organized into extremely long tandem arrays: Implications for recombination between nonhomologous chromosomes[END_REF]. The importance of satellites in the genome can vary as it is ~20% in Drosophila melanogaster and even ~50% in some plant species (Garrido-Ramos 2017).

Murine chromosomes are all acrocentric with one arm being only composed of satellite and telomeric sequences. However, human chromosomes can be either metacentric, sub-metacentric and acrocentric, with different lengths of the two arms.

Although satellite repeats are dispersed within the nucleoplasm, some species such as D. melanogaster, mice and some plants show a striking clustering of PCH into chromocenters (Figure 10, Figure 15A) [START_REF] Guenatri | Mouse centric and pericentric satellite repeats form distinct functional heterochromatin[END_REF]. This could also be the case for human in particular condition like blood lymphocytes [START_REF] Alcobia | Spatial associations of centromeres in the nuclei of hematopoietic cells: Evidence for cell-type-specific organizational patterns[END_REF]. This clustering was first identified by DNA-staining dies. Specific purification of chromocenters in murine cells followed by DNA sequencing suggest that while the majority of DNA sequences in chromocenters are major satellites, ~30% might be other repeated elements such as LINE1 and ERVs [START_REF] Ostromyshenskii | Mouse chromocenters DNA content: Sequencing and in silico analysis[END_REF]. It is not perfectly understood how this clustering is mediated but a AT-hook protein named D1 in Drosophila, conserved from mouse ( HMGA1) to flies, was recently identified [START_REF] Jagannathan | A conserved function for pericentromeric satellite DNA[END_REF]).

Furthermore, it was showed that this clustering is mediated by a dynamic interplay between different players. Indeed, the Prod protein, which binds to the particular {AATAACATAG}n satellite sequence was found to act with D1 in chromocenter clustering in flies [START_REF] Jagannathan | The modular mechanism of chromocenter formation in Drosophila[END_REF]). H4K20me3 also seems required for proper PCH clustering, as deletion of the Suv4-20H1/2 enzymes result clustering defects [START_REF] Hahn | Suv4-20h2 mediates chromatin compaction and is important for cohesin recruitment to heterochromatin[END_REF]. Interestingly, deletion of the Suv39H1/2 enzymes does not impact chromocenter formation. However, deletion of the H3K9me1 methyltransferases Prdm3 and Prdm16 completely disintegrate DAPI-rich foci and impairs major satellite clustering [START_REF] Pinheiro | Prdm3 and Prdm16 are H3K9me1 methyltransferases required for mammalian heterochromatin integrity[END_REF].

All murine cells display chromocenters, as observed by DAPI-dense foci. Yet, not all cells display the same number of chromocenters. Most somatic cells present small and round DAPI foci, while embryonic stem cells have more diffuse chromocenters, which might be a consequence of the more decondensed chromatin state contained in pluripotent cells. The radial positionning of the chromocenters changes along development [START_REF] Solovei | Positional changes of pericentromeric heterochromatin and nucleoli in postmitotic Purkinje cells during murine cerebellum development[END_REF]. This spatial organization is of major importance for the maintenance of the heterochromatin properties of these compartments as forced-tethering to the nuclear enveloppe prevents proper establishment of heterochromatin marks during early embryo [START_REF] Jachowicz | Heterochromatin establishment at pericentromeres depends on nuclear position[END_REF]. But in mouse embryonic stem cells, it was reported that PCH regions were bound by Lamin proteins, suggesting an interconnection between heterochromatin compartments [START_REF] Saksouk | Redundant Mechanisms to Form Silent Chromatin at Pericentromeric Regions Rely on BEND3 and DNA Methylation[END_REF]. Some cells present a chromocenter clustering pattern strikingly different, such as retinal cells of nocturnal mammals which only have a unique, large and central DAPI-dense focus [START_REF] Solovei | Nuclear Architecture of Rod Photoreceptor Cells Adapts to Vision in Mammalian Evolution[END_REF]. Intriguingly, this conformation seems to be evolutionary favored as a physical way to improve light collection, suggesting crucial role of PCH organization for cellular and tissue physiology.

These chromocenters composed the majority of the so-called constitutive heterochromatin.

Yet, it also serves as a anchoring landmark for some facultative heterochromatin regions. FISH observation of unique loci revealed that the α and β-globin gene relocalized away from chromocenters upon activation [START_REF] Francastel | A functional enhancer suppresses silencing of a transgene and prevents its localization close to centromeric heterochromatin[END_REF][START_REF] Brown | Expression of α-and βglobin genes occurs within different nuclear domains in haemopoietic cells[END_REF]. Using the 4C technique to genome-wide map the regions interacting with the major satellite repeats, de Laat's lab has identified around 1 000 regions they called pericentromere-associated domains (PADs) [START_REF] Wijchers | Characterization and dynamics of pericentromere-associated domains in mice[END_REF]. These regions show a great overlap with LADs, together with more distinctive feature like H4K20me3 enrichment, which is enriched at chromocenters. This redudancy between compartment can be explained by a competition between the two compartments -the nuclear periphery and the chromocenters. This competition phenomenon was nicely illustrated using a transgene intergration in mouse cells [START_REF] Bian | β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications[END_REF]. It is proposed that the choice between the two compartments might be based on H3K9me3 content [START_REF] Bian | β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications[END_REF]Kind et al. 2015b). Interestingly, they observed that higher levels of H3K9me3 was associated with a peripheral localization, although LADs are enriched in H3K9me2 whereas PCH are enriched in H3K9me3.

In ESCs, LADs form large domains spanning an average of 1Mb. PADs form smaller domains.

Upon differentiation, LADs sizes remain constant whereas PADs increases, indicating a reinforcement of PADs contact with PCH. Furthermore, Wijchers et al. observed that differentiation-induced PADs are transcriptionnaly silenced before their association with PCH, suggesting that this association is not the primary cause of their silencing but rather a compartmentalization of repressed regions. These results suggest that different mechanisms regulate peripheral and pericentromeric heterochromatin, and these regulation are likely influenced by cell-type particularities.

Protein composition of PCH and maintenance mechanisms

PCH are maintained via epigenetic-based protein modifications, that are dynamically regulated. It serves as a good model to study heterochromatin. These regions were identified almost twenty years ago as enriched in specific epigenetic modifications such as depletion in histone acetylation, H3K9me3 and subsequent HP1 binding, and DNA methylation (Figure 15B).

The Suv39h enzyme trimethylates H3K9 to allow HP1a binding [START_REF] Martens | The profile of repeatassociated histone lysine methylation states in the mouse epigenome[END_REF]. There is an interplay between Suv39h 'writer' enzyme and the HP1a 'reader' protein that helps heterochromatin spreading along these regions by avoiding competition between these two proteins [START_REF] Al-Sady | Division of labor between the chromodomains of HP1 and Suv39 methylase enables coordination of heterochromatin spread[END_REF]. Other heterochromatin marks are also found at PCH (Figure 16A). Some other histone modifications are dependent on H3K9me3 for establishment, such as H4K20me3 [START_REF] Schotta | A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin[END_REF]). H4K20me3 is established via the Suv420h enzyme. This enzyme not only methylates histone H4 but is also important for PCH compaction as its loss results in a decompaction and a scattering of chromocenters. Suv420h can also interact with cohesins [START_REF] Hahn | Suv4-20h2 mediates chromatin compaction and is important for cohesin recruitment to heterochromatin[END_REF]. The histone modification H3K64me3 however appears to be dependent of the presence of H3K9me3, but not on DNA methylation or HP1, suggesting that multiple pathways act independently to ensure proper heterochromatin conformation of the PCH [START_REF] Lange | Dissecting the role of H3K64me3 in mouse pericentromeric heterochromatin[END_REF].

Multiple isoforms of HP1 exist in mammalian cells -α, β and γ. Of interest, each isoform can carry different roles, and they are found at different positions within the nucleus. Although the most known heterochromatic protein, it was recently observed that depletion of HP1α, but not the other two, led to an even more compact heterochromatin state [START_REF] Bosch-Presegué | Mammalian HP1 Isoforms Have Specific Roles in Heterochromatin Structure and Organization[END_REF]. HP1 was also found to interact with structural proteins such as CTCF. These data suggest that PCH are finetune heterochromatic compartment with thigh links to genome organization.

DNA methylation is another important feature of pericentric regions and is maintained there during DNA replication via UHFR1 and DNMT3A and DNMT3B (Figure 16A) [START_REF] Papait | Np95 Is Implicated in Pericentromeric Heterochromatin Replication and in Major Satellite Silencing ed[END_REF][START_REF] Bachman | Dnmt3a and Dnmt3b Are Transcriptional Repressors That Exhibit Unique Localization Properties to Heterochromatin[END_REF]. However, loss of DNA methylation does not impact the formation of chromocenters [START_REF] Saksouk | Redundant Mechanisms to Form Silent Chromatin at Pericentromeric Regions Rely on BEND3 and DNA Methylation[END_REF]. Pluripotent cells with low DNA methylation levels display increased levels of H3K27me3 at PCH [START_REF] Tosolini | Contrasting epigenetic states of heterochromatin in the different types of mouse pluripotent stem cells[END_REF]. Because of its importance as huge DNA repeats, PCH has been used as a model to study the dynamic protein network implicated in the heterochromatin maintenance [START_REF] Müller-Ott | Specificity, propagation, and memory of pericentric heterochromatin[END_REF]. A more detailed view was obtained using pitch technique and revealed that loss of DNA methylation or H3K9me3 was compensated by the Bend3 protein that then recruited PRC2 and the NuRD complex [START_REF] Saksouk | Redundant Mechanisms to Form Silent Chromatin at Pericentromeric Regions Rely on BEND3 and DNA Methylation[END_REF]. In addition to the numerous proteins that acts at PCH, there is a dynamic interplay between all these players.

Long-though to be transcriptional activators, some transcription factors may help for the heterochromatinization process of PCH. PAX3 and PAX9 transcription factors bind within PCH regions and repress transcription of these repeats and their depletion leads to a dramatic derepression of the mouse major satellites [START_REF] Bulut-Karslioglu | A transcription factor-based mechanism for mouse heterochromatin formation[END_REF]. Additional transcription factors are implicated in major satellite regulation. Indeed, the SNAIL1 protein via interactions with the H3K4 deaminase LOXL2 controls pericentromeric transcription [START_REF] Millanes-Romero | Regulation of Heterochromatin Transcription during Epithelial-to-Mesenchymal Transition[END_REF]. This SNAIL1-LOXL2 pathway acting at pericentromeres is crucial for the epithelial-tomesenchymal transition of mesenchymal cells, suggesting a broad impact of heterochromatin regulation. Furthermore, it was observed that the major pluripotency factor NANOG could also regulate heterochromatin organization as its deletion leads to a decompaction of the chromocenter in embryonic stem cells, and the forced expression in epiblast stem-cells when it is not supposed to be active could also decompact PCH, showing an intricate relationship between heterochromatin organization and pluripotency [START_REF] Novo | The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells[END_REF]. NANOG directly binds to pericentromeric repeats and additional factors such as SALL1, which controls Nanog-mediated regulation of PCH [START_REF] Novo | The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells[END_REF]. The regulation of PCH is not only dynamic according to cell state, but also for dividing cells, each mitosis represents a challenge. Indeed, at each mitosis, heterochromatin must be reinstalled after DNA replication. KAP1 protein that is usually involved in retrotransposon repression is involved in this process in MEF cells [START_REF] Jang | KAP1 facilitates reinstatement of heterochromatin after DNA replication[END_REF].

The heterochromatin state is a barrier to transcription. However, transcription of major satellites and the production of RNAs seems important for proper establishment of the repressive environment (Figure 16B). Indeed, deletion of Dicer in murine ESCs led to elevated transcription of PCH satellites together with a loss of H3K9me3 [START_REF] Kanellopoulou | Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing[END_REF][START_REF] Murchison | Characterization of Dicer-deficient murine embryonic stem cells[END_REF]. Deletion of Dicer results in early embryonic lethality [START_REF] Bernstein | Dicer is essential for mouse development[END_REF]. Major satellites are constitutively transcribed, however in a cell-cycle dependent manner with the more abundant transcripts observed in late G1 phase [START_REF] Lu | Proliferationdependent and cell cycle-regulated transcription of mouse pericentric heterochromatin[END_REF]. Quiescent cells do not have detectable major satellite transcripts. PCH RNAs are produced by RNA polymerase II and can be sense or anti-sense, although both are not expressed at the same levels. Upon stress exposure, human cells display an elevated levels of satellite III transcripts (Figure 16B) [START_REF] Jolly | Stress-induced transcription of satellite III repeats[END_REF][START_REF] Valgardsdottir | Transcription of Satellite III non-coding RNAs is a general stress response in human cells[END_REF]. Satellite III RNAs accumulate at nuclear stress granules and disturb segregation of chromosomes during the following 24h, suggesting a role in satellite RNAs in chromosomal instability [START_REF] Giordano | Heat Shock Affects Mitotic Segregation of Human Chromosomes Bound to Stress-Induced Satellite III RNAs[END_REF]. It was later observed, in mouse embryonic stem cells, that the RNA produced from major satellite repeats could stabilize SUV39h binding, allowing a better enzymatic activity [START_REF] Velazquez Camacho | Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation[END_REF]. Interestingly, SUV39h protein has a higher affinity for RNA than DNA and this binding is independent of H3K9me3, indicating its ability to induce heterochromatin formation [START_REF] Shirai | Impact of nucleic acid and methylated H3K9 binding activities of Suv39h1 on its heterochromatin assembly[END_REF]). During early development, a strand-specific burst in major satellite transcription is necessary to properly form PCH clustering [START_REF] Probst | A Strand-specific burst in transcription of pericentric satellites is required for chromocenter formation and early mouse development[END_REF]. Intriguingly, ESCs with high levels of DNA methylation show correspondingly higher transcription of major satellites than lowly methylated ESCs [START_REF] Tosolini | Contrasting epigenetic states of heterochromatin in the different types of mouse pluripotent stem cells[END_REF]. Furthermore, not only major satellite RNAs but potentially additional RNAs might be important to establish the heterochromatin environment at chromocenters. In muscle cells, it was observed that a lncRNA, Chro1, helps targeting DAXX/ATRX to pericentromeric regions and deposit H3.3 [START_REF] Park | Long non-coding RNA ChRO1 facilitates ATRX/DAXX-dependent H3.3 deposition for transcription-associated heterochromatin reorganization[END_REF]. Loss of either DAXX, ATRX, H3.3, Chro1 or RNA transcription affects the 3Dorganization of PCH and the muscle differentiation. Interestingly, DAXX can relocate away from PML NBs toward centromeric and pericentromeric repeats in human cells subjected to heat shocks [START_REF] Morozov | Dualistic function of Daxx at centromeric and pericentromeric heterochromatin in normal and stress conditions[END_REF]. Morozov et al. demonstrate that the lack of Daxx represses satellite transcription and destabilizes histone-modifications associated at pericentromeric regions. These results suggest that DAXX might carry an important role in the reformation of heterochromatin following stress-induced transcription of satellites. ATRX usually localizes at pericentromeric regions [START_REF] Mcdowell | Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes[END_REF].

Together, these studies show an additional and essential layer of regulation whereby heterochromatin must be transcribed to establish a proper repressive state.

Once H3K9me3 is established, HP1a can bind to this modification. In addition, it was observed that induced SUMOylation of HP1a could induce its targeting to pericentric heterochromatin even in the absence of Suv39h and H3K9me3 [START_REF] Maison | SUMOylation promotes de novo targeting of HP1 ± to pericentric heterochromatin[END_REF]. Additional studies advanced our comprehension of this mechanism showing that SENP7, a SUMO protease, was necessary for HP1a retention at PCH [START_REF] Maison | The SUMO protease SENP7 is a critical component to ensure HP1 enrichment at pericentric heterochromatin[END_REF]. This retention is modulated by the two PxVxL domains of SENP7 that binds HP1a and the presence of SENP7 directly regulates HP1a mobility [START_REF] Romeo | The SENP7 SUMO-Protease Presents a Module of Two HP1 Interaction Motifs that Locks HP1 Protein at Pericentric Heterochromatin[END_REF]. Other proteins than HP1 can also be SUMOylated. This is the case of IKAROS, a zinc-finger protein that binds to PCH repeats [START_REF] Cobb | Targeting of Ikaros to pericentromeric heterochromatin by direct DNA binding[END_REF]. IKAROS localization if independent of SUMOylation but its enzymatic activity is, indicating that not only the right proteins must be targeted to the right compartment but they then need additional modification to play their role [START_REF] Gómez-Del Arco | Ikaros SUMOylation: Switching Out of Repression[END_REF]. These data demonstrate that pericentric heterochromatin foci are the result of a dynamic interplay between histone and nonhistone proteins, that can all be post-translationally modified, thus adding complexity but also robustness in heterochromatin formation and maintenance.

Figure 16| Heterochromatin composition and transcriptional state of pericentromeres. (A)

Heterochromatin state of pericentromeres is maintained via the action of multiple proteins. DNMT proteins maintain high levels of DNA methylation, which is then bound by MBD-containing proteins. Histone H3 are notable modified on lysine 9 by SUV39H1/2 enzymes that tri-methylates this residue, which can then recruit HP1. Histone H4K20me3 is established by SUV4-20H1/2 enzymes. Histone deacetylases remove histone acetylation, further contributing to the compact state of chromatin. (B) Satellite repeats can be physiologically transcribed under some circumstances. Transcription of pericentromeres is important for heterochromatin formation as observed in G1-phase or during the early embryonic development. However, transcription of satellite sequences is observed under pathological conditions as in case of cellular stresses such as heat shock, or in some cancers. From Saksouk, Simboeck and Déjardin, Epigenetics & Chromatin 2015.

A new perspective regarding chromocenter has recently emerged. Indeed, HP1, a main component of chromocenter was shown to undergo liquid-liquid phase separation (LLPS) in vitro and in vivo [START_REF] Strom | Phase separation drives heterochromatin domain formation[END_REF][START_REF] Larson | Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin[END_REF]. Binding of HP1 results in the reshaping of nucleosome and increases nucleosome-nucleosome interaction, thus favoring a compact heterochromatin state [START_REF] Sanulli | HP1 reshapes nucleosome core to promote phase separation of heterochromatin[END_REF]. HP1-marked chromocenters exhibit dynamic patterns of foci fusions during in the embryo [START_REF] Strom | Phase separation drives heterochromatin domain formation[END_REF]. Formation of biomolecular condensates seems to be a key feature in the dynamic organization of PCH. However, the exact capacity of HP1 to mediate undergo phase-separation is still under debate [START_REF] Erdel | Mouse Heterochromatin Adopts Digital Compaction States without Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separation Article Mouse Heterochromatin Adopts Digital Compaction States without Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separa[END_REF]. Nucleosomes carry some LLPS properties when incorporated in chromatin, adding a force to the formation of particular subcompartments [START_REF] Gibson | Organization of Chromatin by Intrinsic and Regulated Phase Separation[END_REF]. Moreover, major satellite transcripts can interact with the scaffold attachment factor B (SAFB) that promotes PCH organization via LLPS [START_REF] Huo | The Nuclear Matrix Protein SAFB Cooperates with Major Satellite RNAs to Stabilize Heterochromatin Architecture Partially through Phase Separation[END_REF]. It is thus likely that PCH organization results from LLPS and non-LLPS mechanisms.

Biological functions of pericentromeric heterochromatin

Chromocenters serve as anchoring landmarks for PADs [START_REF] Wijchers | Characterization and dynamics of pericentromere-associated domains in mice[END_REF]. Using a Lac0 integrated in a non-PAD genomic locus, Wijchers et al. observed that targeting of a LacR in fusion with the chromodomain of Suv39H1 was sufficient to induce repositioning of the LacO array near chromocenter and induced its transcriptional repression. Interestingly, the same group latter demonstrated that recruitment of Suv39H1 indeed reproduced the chromodomain recruitment, however, the recruitment of a Suv39H1 lacking its chromodomain resulted in an efficient silencing of the LacO locus, but not in its repositioning toward chromocenters [START_REF] Wijchers | Cause and Consequence of Tethering a SubTAD to Different Nuclear Compartments[END_REF]. These results suggest that, although chromocenters act as a tether for repressed PAD regions, repression of a locus is not sufficient to induce its spatial relocalization.

Pericentromeric regions were first though to protect centromeres, as the deletion of heterochromatin proteins can lead to genomic instabilities. It is the case for the SUV39H1/2 and the SUV4-20H1 and 2 enzymes that both display higher levels of chromosomal abnormalities [START_REF] Peters | Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability[END_REF]. Depletion of SUV4-20H1/2 enzymes resulted in impaired cohesin recruitment at major satellites and increased chromosomal segregation defects in MEF cells (Figure 17B) [START_REF] Hahn | Suv4-20h2 mediates chromatin compaction and is important for cohesin recruitment to heterochromatin[END_REF]. Furthermore, depletion of HMGA1, which is involved in chromocenter clustering results in elevated levels of DNA damages and formation of micronuclei (Figure 17A) [START_REF] Jagannathan | A conserved function for pericentromeric satellite DNA[END_REF]. It is now clear that heterochromatin regions must be tightly repressed and spatially organized to ensure proper cell physiology.

Defects in the DNA methylation machinery also lead to chromosomal instability, as it is the case in the ICF syndrome where Dnmt3b is mutated [START_REF] Xu | Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene[END_REF]. In this syndrome, a specific instability of the chromosomes 1, 9 and 16 is observed, which are more GC-rich than other satellites and thus more prone to DNA demethylation. This is consistent with the fact that they carry more HSat II than other chromosomes, and this phenotype also happens when using the 5azacytidine drug to impair DNA methylation [START_REF] Prada | Satellite 2 demethylation induced by 5-azacytidine is associated with missegregation of chromosomes 1 and 16 in human somatic cells[END_REF]. Intriguingly, Alzheimer patient cells are more prone to 5-azacytidine-induced DNA demethylation at pericentromeres of chromosomes 1, 9 and 16 (Marques Paya et al. 1998). Alzheimer disease results from the appearance of intracellular aggregates of the microtubule-associated protein TAU in neuronal cells. In neuronal cultures, TAU localizes at chromocenters. Depletion of Tau or aggregation of Tau in Alzheimer disease disturb PCH organization and localization of H3K9me3 and HP1 at PCH [START_REF] Mansuroglu | Loss of Tau protein affects the structure, transcription and repair of neuronal pericentromeric heterochromatin[END_REF]. Misregulation of TAU generates widespread heterochromatin decondensation, aberrant gene expression and higher levels of DNA damages [START_REF] Frost | Tau promotes neurodegeneration through global chromatin relaxation[END_REF]. These studies established a novel role of TAU in PCH organization that impact neurodegeneration.

Frontotemporal dementia and amyotrophic lateral sclerosis are neurological disorders resulting from hexanucleotide G4C2 (Proline-Arginine -PR) repeat expansions in the C9orf72 locus. Expression of the poly(PR) peptide revealed its association with PCH (Zhang et al. 2019b).

Zhang et al. observed that poly(PR) localization at chromocenters disrupted HP1α transcription

and localization and induced the upregulation of many families of transposable elements. The presence of poly(PR) diminished HP1a LLPS capacities in vitro. Upregulation of repeated elements is observed in patients suffering from frontotemporal dementia and amyotrophic lateral sclerosis [START_REF] Prudencio | Repetitive element transcripts are elevated in the brain of C9orf72 ALS/FTLD patients[END_REF].

Furthermore, upregulation of satellite sequences is often seen in different cancers, such as pancreatic and breast cancers [START_REF] Ting | Aberrant Overexpression of Satellite Repeats in Pancreatic and Other Epithelial Cancers[END_REF][START_REF] Zhu | BRCA1 tumour suppression occurs via heterochromatin-mediated silencing[END_REF]. In breast cancer, it is proposed that the tumor-suppressive role of BRCA1 could results from its role in maintaining heterochromatin organization, and Brca1 depletion leads to a drastic change in chromocenter organization and strong transcriptional de-repression of the DNA satellite sequences in both mice and human [START_REF] Zhu | BRCA1 tumour suppression occurs via heterochromatin-mediated silencing[END_REF]. Satellite RNAs alone can generate DNA damages and recruit BRCA1, which ultimately induces tumor formation, as observed from RNA microinjection experiments (Zhu et al. 2018b).

Increased levels of satellite transcription in breast cancer cells destabilizes DNA replication forks, that in turn increases the levels of R-loops and can ultimately generates genomic instabilities, further highlighting the importance of transcriptional regulation of satellite repeats (Figure 17C).

It is proposed that heterochromatin is more refractory to γ-H2A.X accumulation in human cells, although in murine, specifically-induced DSBs within constitutive heterochromatin regions induces γ-H2A.X foci formation [START_REF] Cowell | yH2AX Foci Form Preferentially in Euchromatin after[END_REF][START_REF] Tsouroula | Temporal and Spatial Uncoupling of DNA Double Strand Break Repair Pathways within Mammalian Heterochromatin[END_REF]. Interestingly, DNA lesions at PCH in G1-phase are stably positioned within chromocenters and mostly repaired via NHEJ whereas DSBs occurring in S or G2-phase are relocalized toward the periphery of the chromocenters and mostly repaired via HR, suggesting different heterochromatin environment at PCH regions along the cell cycle (Figure 17D) [START_REF] Tsouroula | Temporal and Spatial Uncoupling of DNA Double Strand Break Repair Pathways within Mammalian Heterochromatin[END_REF].

Pericentromeric heterochromatin represent an important fraction of the nuclear constitutive heterochromatin. PCH assemble into DAPI-dense chromocenters in multiple species and its repressive environment is redundantly and dynamically established and maintained.

Pericentromere clustering into chromocenters has been implicated in genome integrity and the high level of divergence among satellite sequences has been proposed to underlie hybrid incompatibility [START_REF] Jagannathan | Function of Junk : Pericentromeric Satellite DNA in Chromosome Maintenance[END_REF]. PCH dysregulation can lead to genome instability and diseases.

D -Telomeres

Telomeres constitute the other main constituent of the constitutive heterochromatin and its regulation is particularly important for cell viability.

Localization and definition of telomeric heterochromatin.

Telomeres are the regions located at each end of the chromosomes, that comprise hundreds of repeats of the {TTAGGG} DNA sequence, highly conserved in eukaryotes [START_REF] Meyne | Conservation of the human telomere sequence ( TTAGGG ). among vertebrates[END_REF][START_REF] Moyzis | A highly conserved repetitive DNA sequence, (TTAGGG)n present at the telomeres of human chromosomes[END_REF]. Interestingly, not all organisms possess particular telomeric sequences as the Drosophila melanogaster for which chromosome ends consist of an array of non-LTR retrotransposons [START_REF] Pardue | Retrotransposons Provide and Evolutionarily Robust Non-Telomerase Mechanism to Maintain Telomeres[END_REF]. Telomeric tandem repeats assemble into a lariatlike structure (T-Loop) which shields the DNA end (Figure 18A) [START_REF] Shay | Telomeres and telomerase: three decades of progress[END_REF][START_REF] Griffith | Mammalian Telomeres End in a Large Duplex Loop[END_REF]. The length of the telomeres varies greatly from species to species, from 300bp in yeast to 10-15Kbs in human and 20-60Kbs in mice [START_REF] Jain | Telomeric Strategies : Means to an End[END_REF]. From 300 to 8,000 telomeric repeats represents about 0.1% of the genome.

In mammals, the double-stranded part of telomeres is composed of nucleosome like other regions of the genome, whereas yeast telomeres are devoid of nucleosomes [START_REF] Makarov | Nucleosomal Organization of Telomere-Specific Chromatin in Rat[END_REF][START_REF] Jain | Telomeric Strategies : Means to an End[END_REF]. Furthermore, it appears that the nucleosome density at telomeres can vary in a cell-cycle dependent manner [START_REF] Galati | TRF2 Controls Telomeric Nucleosome Organization in a Cell Cycle Phase-Dependent Manner[END_REF]. Telomeric repeats are transcribed and produce the well-conserved G-rich RNA TERRA (telomere repeat-containing RNA), which lengths from 0.1 to 10Kbs [START_REF] Azuara | Telomeric Repeat-Containing RNA and RNA Surveillance Factors at Mammalian Chromosome Ends[END_REF].

In yeast, it has been known for year that telomeres cluster in 3 to 6 foci, 8 times less than the number of telomeres, and are located close to the nuclear envelope [START_REF] Kupiec | Biology of telomeres: Lessons from budding yeast[END_REF].. These foci are dynamics and are generated by random associations-dissociations of individual telomeres [START_REF] Hozé | Spatial telomere organization and clustering in yeast Saccharomyces cerevisiae nucleus is generated by a random dynamics of aggregation-dissociation[END_REF]. Telomeres spatial positioning within the nuclear space is less clear in mammals (Figure 10). A first report in human lymphocyte indicated that telomeres are not close to the nuclear lamina and rather tended to be within the nuclear interior [START_REF] Amrichová | Nuclear and territorial topography of chromosome telomeres in human lymphocytes[END_REF]. More recent studies showed that human telomeres organization depends on the cell-cycle and that in G1 telomeres are associated with the nuclear lamina [START_REF] Crabbe | Human Telomeres Are Tethered to the Nuclear Envelope during Postmitotic Nuclear Assembly[END_REF]. This was confirmed by genome-wide studies using the new MadID technique [START_REF] Sobecki | MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells[END_REF]. Furthermore, reports have shown light on the interaction between telomeric proteins and the nuclear lamina network, suggesting interconnections between heterochromatin sub-compartments [START_REF] Wood | TRF2 and lamin A/C interact to facilitate the functional organization of chromosome ends[END_REF].

Interestingly, mice have acrocentric chromosomes. This creates a specific spatial organization of telomeres as half of the telomeres are located near to chromocenters, and the other half are dispersed throughout the nucleoplasm (Figure 10). In human, chromosomes can be metacentric, sub-metacentric or acrocentric. Such chromosome organization might be important for the generation of nuclear subcompartments.

Protein composition of telomeres

In somatic cells, there is a telomere attrition at each mitosis, which is responsible of the 'Hayflick limit' that postulates that each cell possess only a limited number of cell division before activating senescence signaling [START_REF] Hayflick | The serial cultivation of human diploid cell strains[END_REF][START_REF] Shay | Senescence and immortalization: Role of telomeres and telomerase[END_REF][START_REF] Daniali | Telomeres shorten at equivalent rates in somatic tissues of adults[END_REF]. Interstingly, telomere shortening along the life resulting from DNA replication and the many damages it can generate acts as a barrier to tumor formation, as a minimal length of only one chromosome ends can trigger a cellular growth arrest signaling [START_REF] Hemann | The Shortest Telomere , Not Average Telomere Length , Is Critical for Cell Viability and Chromosome Stability[END_REF]. . Because of the replication process that uses a DNA template, chromosome ends face the 'end replication problem' [START_REF] Watson | Origin of concatemeric T7 DNA[END_REF]. Evolution has selected the use of the telomerase protein that can extend the 3' ends of chromosomes [START_REF] Shay | Telomeres and telomerase: three decades of progress[END_REF]. Telomere ends are maintained via the telomerase (TERT) protein, which bears a reverse transcriptase activity (Figure 18B) [START_REF] Greider | The Telomere Terminal Transferase of Tetrahymena Is a Ribonucleoprotein Enzyme with Two Kinds of Primer Specificity[END_REF]. Indeed, ectopic expression of telomerase rescues the shortening of telomeres and extend the life-span of cells [START_REF] Bodnar | Extension of Life-Span by Introduction of Telomerase into Normal Human Cells[END_REF]. TERT functions with an RNA component (TERC) that provides a template for its activity, the dyskerin protein (DKC1) and other associated proteins such as TCAB1, NHP2 and NOP10 [START_REF] Shay | Telomeres and telomerase: three decades of progress[END_REF]. The structure of the telomerase bound to DNA was only recently obtained [START_REF] Nguyen | Cryo-EM structure of substrate-bound human telomerase holoenzyme[END_REF].

Telomerase expression is limited to embryonic and adult stem cells, whereas most somatic tissues have no detectable telomerase expression [START_REF] Cohen | Protein Composition of Catalytically Active Human Telomerase from Immortal Cells[END_REF]. When expressed, telomerase elongates only seven per cent of the shortest telomeres at each S-phase, suggesting an efficient control of telomere elongation (Teresa [START_REF] Teixeira | Telomere Length Homeostasis Is Achieved via a Switch between Telomerase-Extendible and -Nonextendible States[END_REF]).

Many proteins act throughout the cell-cycle to ensure telomere-protection from DNA damages. Among these proteins are the TRF proteins that can control telomere length (van Steensel and de Lange 1997). TRF1, TRF2, RAP1 and TIN2 together form the shelterin complex that act in different ways to protect telomeres and avoid DNA damages (Figure 18B)(de Lange 2018). The sheltering complex does not act alone but rather recruits, when needed other proteins like TPP1, POT1, RPA or 53BP1. 53BP1 was indeed demonstrated as promoting non-homologous end joining by increasing chromatin mobility, suggesting that telomere localization and movement within the nucleoplasm is of particular importance [START_REF] Dimitrova | 53BP1 promotes nonhomologous end joining of telomeres by increasing chromatin mobility[END_REF]. Telomere protein composition is dynamic and can be cell specific. Studies have focused on identifying all the proteins present at particular loci, notably telomeres by developing the PICh technology [START_REF] Déjardin | Purification of Proteins Associated with Specific Genomic Loci[END_REF]. TRF2, via its basic N-terminal domain, can bind DNA in a sequence-independent manner to branched DNA, which notably includes the T-Loop structure [START_REF] Schmutz | TRF2 binds branched DNA to safeguard telomere integrity[END_REF]. Many cancer cells for example have evolved a telomerase-independent pathway called ALT (Alternative-Lengthening of Telomeres) that stabilizes telomere length using recombination when heterochromatin might be compromised [START_REF] Voon | Compromised telomeric heterochromatin promotes ALTernative lengthening of telomeres[END_REF]. This process was recently showed to be highly dynamic and rely on phase-separation mechanism [START_REF] Min | Clustered telomeres in phase-separated nuclear condensates engage mitotic DNA synthesis through BLM and RAD52[END_REF]. Indeed, in ALTpositive cancer cells, telomeres tend to cluster in PML NBs. Min et al. have constructed an artificial system to generate phase-separation clustering of telomeres using an array of polySUMO chains.

They observe that, when co-overexpressed with Bloom (BLM) helicase in human cells, this induced ALT-phenotypes that relied on both TRF2 and RAD52. Interestingly, TRF2 must be SUMOylated to induce the ALT-pathway, a SUMOylation process favored by PML-concentration of SUMO peptides. Interestingly, although embryonic stem cells express TERT, they display a related telomeric organization with high levels of PML NBs colocalization [START_REF] Chang | PML bodies provide an important platform for the maintenance of telomeric chromatin integrity in embryonic stem cells[END_REF]. PML NBs contain DAXX, which increases its telomeric localization upon DNA demethylation [START_REF] He | The Daxx/Atrx Complex Protects Tandem Repetitive Elements during DNA Hypomethylation by Promoting H3K9 Trimethylation[END_REF]. DAXX and PML maintain the heterochromatin state of telomeres via H3.3 deposition and subsequent lysine 9 trimethylation [START_REF] Udugama | Histone variant H3.3 provides the heterochromatic H3 lysine 9 trimethylation mark at telomeres[END_REF]. Indeed, telomeric heterochromatin is enriched in H3K9me3-histone modification. Although Suv39H1/2 dKO cells display telomere defects, more recent observations suggest that the main enzyme responsible for H3K9me3 at telomeres might be SETDB1 [START_REF] Karimi | Article DNA Methylation and SETDB1 / H3K9me3 Regulate Predominantly Distinct Sets of Genes , Retroelements , and Chimeric Transcripts in mESCs[END_REF][START_REF] Elsässer | Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells[END_REF][START_REF] Gauchier | SETDB1dependent heterochromatin stimulates alternative lengthening of telomeres[END_REF][START_REF] García-Cao | Epigenetic regulation of telomere length in mammalian cells by the Suv39h1 and Suv39h2 histone methyltransferases[END_REF].

Telomere length vary greatly between species due to the differences in telomere constitution and maintenance mechanisms. Protection of DNA ends is essential to prevent undesired recombination events and DNA damages which can arise at DNA ends.

Influence of telomeres

The main function of telomeres is to ensure proper DNA replication in S-phase and to protect chromosome ends from DNA damages during interphase. However, the heterochromatin state of telomeres regulates gene expression over long distances because of the 3D organization (TTAGGG)n are tandemly repeated over large distances. Telomeric DNA is bound and protected by the shelterin complex composed of TRF1, TRF2, POT1, TIN2 and RAP1 proteins. The shelterin complex enables higher-order organization of telomeres that forms T-loops. (B) In pluripotent cells and in 85 to 90% of cancer cells, telomeres are maintained via the telomerase complex, composed of the the reverse-transcriptase TERT, the associated RNA TERC and additional proteins DKC1, TCAB1, NOP10 and NHP2. However, about 10 to 15% of cancer cells use a telomerase-independ ALT pathway to maintain telomeric lengths. ALT = Alternative-lengthening of telomeres. A is adapted from de Lange, Ann. Rev. Gen. 2018. B is from Shay and Wright, Nat. Rev. Gen. 2019. [START_REF] Robin | Telomere position effect: Regulation of gene expression with progressive telomere shortening over long distances[END_REF]. The length of the telomere can influence expression of the Isg15 gene which is not in contact with telomeric heterochromatin nor activated via DNA damages signaling at the DNA ends [START_REF] Lou | Telomere length regulates ISG15 expression in human cells[END_REF]. Using an adapted HiC technique, Robin et al. have mapped genomic association with telomeres and described that at least three genes located in subtelomeric regions.

Telomeric loops can extend to up to 10 Mb away from DNA ends and may thus regulate chromatin state of many loci in a mechanism the authors called telomere position effect over long distances [START_REF] Robin | Telomere position effect: Regulation of gene expression with progressive telomere shortening over long distances[END_REF]. Telomere position effect has been conserved throughout evolution as it is also important in yeast where telomere loops can regulate expression of genes located up to 20 Kbs away [START_REF] Gottschling | Position Effect at S . cerevisiae Telomeres : Reversible Repression of Pol II Transcription[END_REF][START_REF] Bruin D De | Telomere looping permits gene activation by a downstream UAS in yeast[END_REF]. These results highlight that heterochromatin telomeric chromatin might have additional roles in global chromatin organization and gene silencing that extend beyond its locus.

A rare event can lead to upregulation of the telomerase gene, which is propagated within the subsequent cancer. The TERT promoter is embedded within a CpG island. Changes in TERT promoter is often associated with TERT transcription in cancers [START_REF] Lee | DNA hypermethylation within TERT promoter upregulates TERT expression in cancer[END_REF][START_REF] Liu | TERT Promoter Hypermethylation in Gastrointestinal Cancer : A Potential Stool Biomarker[END_REF].

TERT transcription is regulated by transcription factors bindings its promoter and aberrant transcription factor such as MYC can enhance transcriptional activity of the telomerase gene (Wang et al. 1998a). Furthermore, genomic mutation can arise in TERT promoter and promotes its expression such as observed for melanoma [START_REF] Huang | Highly Recurrent TERT Promoter Mutations in Human Melanoma[END_REF]. The vast majority of cancer cells display TERT expression, yet ~10 -15% use the ALT-mechanism (Figure 18B) [START_REF] Bryan | Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell line[END_REF]. Marzec et al. decided to assess all genome locations where TRF2 was binding in ALTpositive cells. They observed that TRF2 binds to many locations throughout the genome, and interestingly at NR2C/F binding sites (a hormone regulated nuclear receptor). NR2C/F promotes spatial proximity of telomeres and favors telomere-telomere recombination necessary for ALT. It can trigger insertion of telomeric sequences into the genome, creates fragile sites and promotes genomic instability. Thus, ALT-cancer cells-genomic instability might be partially explained by telomere defects [START_REF] Marzec | Nuclear-Receptor-Mediated Telomere Insertion Leads to Genome Instability in ALT Cancers[END_REF]. ALT-positive cancer cells often display deactivating mutations in Daxx, H3.3 or Atrx genes [START_REF] Jiao | DAXX/ATRX, MEN1, and mTOR Pathway Genes Are Frequently Altered in Pancreatic Neuroendocrine Tumors[END_REF][START_REF] Heaphy | Altered Telomeres in Tumors with ATRX and DAXX mutations[END_REF][START_REF] Ahvenainen | Loss of ATRX / DAXX Expression and Alternative Lengthening of Telomeres in Uterine Leiomyomas[END_REF][START_REF] Schwartzentruber | Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma[END_REF][START_REF] Clynes | Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX[END_REF][START_REF] Napier | ATRX represses alternative lengthening of telomeres[END_REF][START_REF] Barthel | Systematic analysis of telomere length and somatic alterations in 31 cancer types[END_REF][START_REF] Voon | Compromised telomeric heterochromatin promotes ALTernative lengthening of telomeres[END_REF]. ATRX regulates cohesion of sister chromatid and when lost, cancer cells display defective cohesion and increased non-homologous recombination leading to genomic instability [START_REF] Ramamoorthy | Loss of ATRX Suppresses Resolution of Telomere Cohesion to Control Recombination in ALT Cancer Article Loss of ATRX Suppresses Resolution of Telomere Cohesion to Control Recombination in ALT Cancer Cells[END_REF]. Interestingly, it was recently observed that ALT-mechanism is not a consequence of a lack of heterochromatin at telomeres, but rather results from heterochromatin formation via the SETDB1 methyl-transferase [START_REF] Gauchier | SETDB1dependent heterochromatin stimulates alternative lengthening of telomeres[END_REF]. Gauchier et al. report that ALT charasteristics appear as an excess of heterochromatin features like H3K9me3 provided by SETDB1 and that depletion of Setdb1 can suppress ALT phenotypes. These results suggest that telomeric heterochromatin must be tighly regulated to avoid genomic instabilities.

Mutations of the main composent of telomere maintenance are also implicated in the dyskeratosis congenita disorder, such for telomerase and dyskerin [START_REF] Mitchell | A telomerase component is defective in the human disease dyskeratosis congenita[END_REF][START_REF] Armanios | Haploinsufficiency of telomerase reverse transcriptase leads to anticipation in autosomal dominant dyskeratosis congenita[END_REF]. Dyskeratosis congenita is a rare disease affecting different tissues. Patients display skin hyperpigmentation, nail dystrophy and aplastic anemia, which can be accompanied from pulmonary and liver fibrosis. It mainly affects regenerative tissues via the increase of chromosome instability due to lower levels of telomerase and associated shorter telomeres [START_REF] Mitchell | A telomerase component is defective in the human disease dyskeratosis congenita[END_REF].

Nowadays, many genetic diseases have been linked to telomere dysfunction and are refered to as telomeropathies [START_REF] Holohan | Telomeropathies : An emerging spectrum disorder[END_REF].

Telomeres are important constituent of the constituve heterochromatin. Their localization and maintenance mechanisms can differ from cell to cell. Their stability is particularly important for the cell and defects in telomere protein can lead to enhanced genomic instabilities and diseases.

E -Transposable Elements

About 70 years ago, Barbara McClintock proposed that a phenotypic characteristic of maize could be explained by transposition of mobile genetic elements [START_REF] Mcclintock | Controlling Elements and the Gene[END_REF]. Although this claim was welcomed with skepticism at that time, she was later rewarded for her discovery. In the early 2000's, the publication of the human genome shed the light on the fact that protein-coding genes account for merely 2% of the entire genome. In total, about half of mammalian genomes are composed of transposable elements (TE) and virtually all organisms contain TEs, which are scattered all along the genome.

Localization and definition of TEs

Two classes of TEs exist. Class I corresponds to retrotransposons that can have LTR sequences or non-LTR. Class I retrotransposon uses a reverse-transcription phase that allows these elements to undergo duplicative transposition -at each transposition, their number increases. Class II are DNA transposons that can be either autonomous or non-autonomous. Class II DNA transposons do not use a reverse-transcription step and integrate with a cut and paste mechanism.

They are first excised from the genome and then integrated elsewhere, thus they do not increase their abundance. Class I TEs use an RNA intermediate and they are transcribed by the RNA Polymerase II. Thus, they must be tightly regulated. TEs have coevolved with their host genomes and transcription and integration events have been used by the host as an adaptative force yet important regulation pathways have emerged to restrain this capacity to avoid disastrous integrations. Many families coexist within mammalian genomes and the sum of TEs present within an organism is called its endovirome [START_REF] Friedli | The Developmental Control of Transposable Elements and the Evolution of Higher Species[END_REF].

Class II TEs are DNA transposons that move by cycles of excision-insertion and as such their number stays constant and are thus the less important group of TEs (Figure 19). They possess short terminal inverted repeats (TIR) and members of this family include the Tigger and Charlie elements [START_REF] Smit | Tiggers and other DNA transposon fossils in the human genome[END_REF]. DNA transposons have given rise to essential human genes such as the RAG recombinase that generates the wide variety of antibodies [START_REF] Huang | Discovery of an Active RAG Transposon Illuminates the Origins of V ( D ) J Recombination Article Discovery of an Active RAG Transposon Illuminates the Origins of V ( D ) J Recombination[END_REF]Zhang et al. 2019a). Yet, as no such transposons are still active in humans, I will mainly focus on the TEs class I.

Class I TEs can be referred to as endogenous retroelements (EREs) and is divided into two categories -long terminal repeats (LTR) and non-LTR elements. EREs account for 90% of all TEs in mouse and human genomes [START_REF] Beck | LINE-1 Elements in Structural Variation and Disease[END_REF]. While LTR elements encode the gag and pol proteins that are related from retroviral proteins, yet they do not transcribe the envelope protein, which precludes their existence within the host cell. These elements are named endogenous retroviruses (ERVs) (Figure 19). ERVs are 7 to 9 Kbs length but LTR sequences might recombine and leave solitary 'solo' LTR throughout the genome, that represent the majority of the observed LTR sequences [START_REF] Chuong | Regulatory activies of transposable elements : from conflicts to benefits[END_REF]. In human, at least 31 families of ERVs have been described based on sequences homology and they display almost no transposition activity, except for one HERVK [START_REF] Mayer | A revised nomenclature for transcribed human endogenous retroviral loci[END_REF][START_REF] Marchi | Unfixed Endogenous Retroviral Insertions in the Human Population[END_REF][START_REF] Feschotte | Endogenous viruses : insights into viral evolution and impact on host biology[END_REF]. In mouse however, more recent ERVs are described -the intracisternal A particles (IAP) and ETn/MusD which are still active and are responsible for about 10% of the spontaneous mutations [START_REF] Maksakova | Retroviral Elements and Their Hosts : Insertional Mutagenesis in the Mouse Germ Line[END_REF].

LTR-containing endogenous retroviruses arise from an ancient infection of germ cells by an exogenous retrovirus as it was exemplified for IAP which has lost its envelope gene but gained a reticulum endoplasmic targeting signal allowing a complete intracellular replicative cycle [START_REF] Ribet | An infectious progenitor for the murine IAP retrotransposon : Emergence of an intracellular genetic parasite from an ancient retrovirus[END_REF]. Non-LTR class I TEs comprise the short and the long interspaces nuclear elements (SINEs and LINEs). SINEs are 300 bp length but present in about 1.8 million copies, the larger member of this family being the Alu element in human and the B1 and B2 families in mouse, from which only ~1,000 are still active (Figure 19). SINEs are non-autonomous elements as they do not encode for any proteins and thus rely on the transcription of the LINEs, which impacted their rate of insertions that diverge greatly among species. Alu elements encode for small components of RNA polymerase III promoter and a polyA tail. LINEs are about 6 Kbs length and encode for two proteins -ORF1 and ORF2. ORF1 encodes for an unknown protein and ORF2 encodes for a protein that carries endonuclease and reverse-transcriptase activities. LINE1 possess a CpG-rich internal promoter recognized by the RNA Pol II. LINEs represent ~20% of the human and mouse genomes with ~950,000 copies found (Figure 19). However, many of these copies are degenerated sequences and only 10,000 are full-length elements, from which ~100 are active [START_REF] Friedli | The Developmental Control of Transposable Elements and the Evolution of Higher Species[END_REF]. Each family of young TEs display very high similarities among members, making it difficult to assess by sequencing technologies, but along evolution, each element will degenerate.

The different TEs display diverse integration preferences. SINEs are mainly observed in gene-rich euchromatin regions, however the exact insertion site varies between intergenic, intronic and even exonic regions [START_REF] Versteeg | The Human Transcriptome Map Reveals Extremes in Gene Density , Intron Length , GC Content , and Repeat Pattern for Domains of Highly and Weakly Expressed Genes[END_REF]). Interestingly, younger Alu seems to exhibit a LINElike integration profiles which is enriched in heterochromatin regions. Prescott Deininger proposes that young Alu elements are removed from heterochromatin during evolution [START_REF] Deininger | Alu elements : know the SINEs[END_REF]. Furthermore, euchromatin highly-transcribed regions might be more accessible for Alu elements.

On the contrary, older LINE-1 and ERVs are preferentially observed in heterochromatin regions, suggesting that they are evolutionary removed from euchromatin [START_REF] Lander | Initial sequencing and analysis of the human genome[END_REF]). These discrepancies between integrations preferences rely on the chromatin state and associated-proteins that are recruited at each localization.

Regulation of TEs

To avoid any detrimental retrotransposition of LINE and ERVs, cells have evolved diverse repression mechanisms. Repressive mechanisms rely either on chromatin modifications to prevent transcription, or on the use of viral protections machineries.

Cellular factors have emerged to prevent integration of exogenous and endogenous retroviruses which are part of an innate immunity system. Such pathways use proteins like the APOBEC family, which have a cytidine deaminase activity and carry a post-transcriptional control over TEs [START_REF] Harris | DNA Deamination Mediates Innate Immunity to Retroviral Infection[END_REF][START_REF] Mangeat | Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts[END_REF][START_REF] Esnault | APOBEC3G cytidine deaminase inhibits retrotransposition of endogenous retroviruses[END_REF]. APOBEC enzymes convert C-to-U during the reverse transcription of the nascent retroviral DNA, acting as a general mechanism to limit the effect of retroviruses by a G-to-A hypermutation. Other proteins such as the three-prime-repair exonuclease 1 (TREX1) mediates reverse transcripts-degradation of LINE1 and IAP [START_REF] Stetson | Trex1 Prevents Cell-Intrinsic Initiation of Autoimmunity[END_REF]. TREX1 is part of the IFN-stimulatory DNA (ISD) response that senses viral infection within the cell, suggesting common mechanisms at play. Other factors of the ISD response that actively suppresses retroviral infection, notably the TRIM5α which recognizes retroviruses capsid could have evolved to prevent endogenous retroviral integration along the evolution [START_REF] Kaiser | Restriction of an Extinct Retrovirus by the Human TRIM5 a Antiviral Protein[END_REF][START_REF] Pertel | TRIM5 is an innate immune sensor for the retrovirus capsid lattice[END_REF]. Moreover, the density and positioning of nucleosomes at a genomic locus can also modulate the integration frequency for murine leukemia virus (MLV) [START_REF] Benleulmi | Intasome architecture and chromatin density modulate retroviral integration into nucleosome[END_REF]. Other cellular strategies can exert a post-translational control over retroviral proteins. The cellular factor TEX19.1 is expressed upon hypomethylation of pluripotent cells and can bind to and stimulates the polyubiquitination of the ORF1 protein produced by LINE1 elements [START_REF] Maclennan | Mobilization of LINE-1 retrotransposons is restricted by Tex19.1 in mouse embryonic stem cells[END_REF]. Polyubiquitination of ORF1 protein leads to its degradation and prevents new genomic insertions. These results suggest that selected networks of cellular immunity to prevent viral infection might apply or be rewired to counteract endogenous retroviruses integrations.

Contrary to exogenous viral infection, the cellular factors preventing ERV insertions are only necessary if they are expressed in the first place. Chromatin modifications associated with heterochromatin are established to maintain proper silencing of these sequences. DNA methylation at CpG is a common mechanism in mammals to ensure transcriptional repression of TEs (Figure 20). Indeed, loss of DNA methylation via the depletion of Uhrf1 for example, results in the upregulation of LINE1 and IAPs [START_REF] Bostick | UHRF1 Plays a Role in Maintaining DNA Methylation in Mammalian Cells[END_REF][START_REF] Sharif | The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA[END_REF]. DNA methylation likely exerts its function via methyl-binding proteins such as MeCP2 as patients suffering from Rett syndrome display higher levels of LINE1 expression in the brain, indicating that TEs are regulated in a tissue-specific way [START_REF] Muotri | L1 retrotransposition in neurons is modulated by MeCP2[END_REF]. Other proteins act to ensure the proper establishment of DNA methylation profiles. The pluripotency factor DPPA3 protects the specific genomic localizations against DNA demethylation and its loss results in the diminution of IAP but not LINE1 DNA demethylation [START_REF] Nakamura | PGC7 / Stella protects against DNA demethylation in early embryogenesis[END_REF]. Upon depletion of the lymphoid-specific helicase 1 (LSH1), cells display large DNA demethylation, notably at IAP, LINE1 and SINE B1, which results in the loss of repressive histone modifications and upregulation of their transcription [START_REF] Huang | Lsh , an epigenetic guardian of repetitive elements[END_REF][START_REF] Dennis | Lsh , a member of the SNF2 family , is required for genome-wide methylation[END_REF][START_REF] Yan | Association of Lsh, a Regulator of DNA Methylation, with Pericentromeric Heterochromatin Is Dependent on Intact Heterochromatin[END_REF][START_REF] Yu | Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways[END_REF].

Histone methylation, notably the H3K9me3 is observed at a wide variety of TEs -LTR and non-LTR ones [START_REF] Bulut-Karslioglu | Suv39h-Dependent H3K9me3 Marks Intact Retrotransposons and Silences LINE Elements in Mouse Embryonic Stem Cells[END_REF][START_REF] Kondo | Enrichment for Histone H3 Lysine 9 Methylation at Alu Repeats in Human Cells[END_REF][START_REF] Martens | The profile of repeatassociated histone lysine methylation states in the mouse epigenome[END_REF][START_REF] Mikkelsen | Genome-wide maps of chromatin state in pluripotent and lineage-committed cells[END_REF]). However, deletion of each protein from the H3K9me3 maintenance pathway display a somehow specific deregulation pattern. Deletion of Suv39H1 and 2 seems to impair repression of full-length LINEs in pluripotent but not differentiated cells [START_REF] Bulut-Karslioglu | Suv39h-Dependent H3K9me3 Marks Intact Retrotransposons and Silences LINE Elements in Mouse Embryonic Stem Cells[END_REF].

Interestingly, pluripotent embryonic stem cells present an ERV-dependent repressive H3K9me3 mediated by the other H3K9me3 methylase SETDB1 [START_REF] Matsui | Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET[END_REF]. Matsui et al. demonstrated that LTR-containing TEs such as IAP and MusD were regulated by H3K9me3mediated deposition of SETDB1, but not non-LTR elements like LINE1. Although H3K9me3 is enriched at these elements, it is not the only histone modification as H4K20me3 is observed too.

Yet, deletion of H4K20me3 methylases Suv420H1 and 2 did not impair neither H3K9me3 enrichment not ERV expression, suggesting that it is not required for proper repression but might only strengthen the repressive environment. H3K9me3 enrichment correlates with the presence of KAP1 (also named TRIM28) (Figure 20) [START_REF] Matsui | Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET[END_REF]. In fact, KAP1 recognizes endogenous retroviruses such as IAPs, but also exogenous retroviruses such as MLV and then recruits SETDB1 to establish an H3K9me3 chromatin state, as depletion of KAP1 induces the loss of H3K9me3 at IAPs [START_REF] Rowe | KAP1 controls endogenous retroviruses in embryonic stem cells[END_REF][START_REF] Wolf | TRIM28 Mediates Primer Binding Site-Targeted Silencing of Murine Leukemia Virus in Embryonic Cells[END_REF]. Furthermore, KAP1 act as a scaffold for a large complex comprising SETDB1 but also the histone deacetylase containing NuRD complex and the H3K9me3-binding protein HP1, thus creating a local heterochromatin state [START_REF] Wolf | Primer Binding Site-Dependent Restriction of Murine Leukemia Virus Requires HP1 Binding by TRIM28[END_REF][START_REF] Macfarlan | Endogenous retroviruses and neighboring genes are coordinately repressed by LSD1 / KDM1A[END_REF][START_REF] Schultz | Targeting histone deacetylase complexes via KRAB-zinc finger proteins : the PHD and bromodomains of KAP-1 form a cooperative unit that recruits a novel isoform of the Mi-2 ␣ subunit of NuRD[END_REF][START_REF] Sripathy | The KAP1 Corepressor Functions To Coordinate the Assembly of De Novo HP1-Demarcated Microenvironments of Heterochromatin Required for KRAB Zinc Finger Protein-Mediated Transcriptional Repression[END_REF]. The KAP1-mediated silencing of ERVs is limited to the embryonic development, when the level of DNA methylation is low [START_REF] Rowe | KAP1 controls endogenous retroviruses in embryonic stem cells[END_REF]. MLV is targeted by KAP1 because of its primer binding site (PBS), a sequence complementary to proline tRNA [START_REF] Wolf | TRIM28 Mediates Primer Binding Site-Targeted Silencing of Murine Leukemia Virus in Embryonic Cells[END_REF]. Transcriptional repression of the two alleles of exogenous Moloney murine leukemia virus (MMLV) is asynchronously mediated and it is likely that it reflects an iterative silencing process that may also control endogenous TEs [START_REF] Schlesinger | Asynchronous transcriptional silencing of individual retroviral genomes in embryonic cells[END_REF]. Histone-modification based silencing mechanisms are particularly important in murine embryonic stem cells. Indeed, mESCs are refractory to exogenous viral infections [START_REF] Teich | Virus Infection of Murine Teratocarcinoma Stem Cell Lines[END_REF]. Furthermore, mESCs can overcome the absence of DNA methylation for proper survival and ERV repression. It is proposed that additional chromatin pathways specific to pluripotent cells have emerged to ensure the transcriptional silencing of TEs and avoid any mutational potential during the embryonic development [START_REF] Leung | Silencing of endogenous retroviruses : when and why do histone marks predominate ?[END_REF]. Yet, further studies will likely present a finer comprehension of TE repressive processes and a better understanding of the reciprocal impact of both histone modification-based and DNA methylation mechanisms and how they both influence each other and ensure a complex and robust silencing of TEs [START_REF] Turelli | Interplay of TRIM28 and DNA methylation in controlling human endogenous retroelements[END_REF]).

KAP1 association with chromatin is mediated by its DNA-binding co-factors -the krüppelassociated box domain-zinc finger (KRAB) proteins, one of the largest family of transcription factors in mammals [START_REF] Huntley | A comprehensive catalog of human KRAB-associated zinc finger genes : Insights into the evolutionary history of a large family of transcriptional repressors[END_REF]. KRAB zinc-finger proteins (ZFPs) are composed of one KRAB and two C2H2 zinc-finger domains. The KRAB domain allows interaction with KAP1, whereas the zinc-finger domains mediate the binding to specific DNA sequences [START_REF] Yang | The Role of KRAB-ZFPs in Transposable Element Repression and Mammalian Evolution[END_REF].

During the evolution path, TEs have degenerated from their original sequence, yet they still need to be suppressed by cellular factors. KRAB-ZFPs are rapidly evolving proteins, and notably the zinc-finger domains [START_REF] Liu | Deep Vertebrate Roots for Mammalian Zinc Finger Transcription Factor Subfamilies[END_REF]. This particular feature of KRAB-ZFPs have likely arise as a co-evolution arms race between TEs, that try to escape transcriptional silencing, and repressive cellular factors, that are selected on their ability to bind a diverse range of targets [START_REF] Thomas | Coevolution of retroelements and tandem zinc finger genes[END_REF][START_REF] Jacobs | An evolutionary arms race between KRAB zinc-finger genes ZNF91/93 and SVA/L1 retrotransposons[END_REF]. Although essential for the embryonic development, the role of KRAB-ZFPs seems restricted to the pluripotent cells, suggesting that these proteins initiate the silencing of ERVs during mammalian development and become dispensable upon cell differentiation [START_REF] Wolf | The KRAB zinc finger protein ZFP809 is required to initiate epigenetic silencing of endogenous retroviruses[END_REF]. Furthermore, from genome-wide sequencing data, it seems that KRAB-ZFP-repressive role happens at ERVs, but also at non-LTR retrotransposons including LINEs and SINEs [START_REF] Jacobs | An evolutionary arms race between KRAB zinc-finger genes ZNF91/93 and SVA/L1 retrotransposons[END_REF].

At the chromatin level, it was recently observed that the histone chaperone complex DAXX/ATRX could regulate the transcriptional activities of a subset of ERVs via H3.3 deposition in embryonic stem cells (Figure 20) [START_REF] Elsässer | Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells[END_REF]. Using a ChIP-reChIP strategy, Elsässer et al. observed that H3.3K9me3 is particularly enriched at ERV sequences, as well as DAXX and ATRX. Depletion of H3.3 leads to a loss of the transcriptional repression of IAPs and MusD repeats, suggesting a particular function for H3.3 in the maintenance of K9me3-heterochromatin compartment in pluripotent cells, which is coherent with its role in K9me3-enrichment at telomeres in these cells [START_REF] Udugama | Histone variant H3.3 provides the heterochromatic H3 lysine 9 trimethylation mark at telomeres[END_REF]. The amount of H3.3K9me3 is drastically reduced in Setdb1 KO cells, suggesting that DAXX/ATRX-deposition of H3.3 functions in the same repressive pathway than the SETDB1-KAP1 complex, and reciprocally, the levels of KAP1 are reduced when cells lack the H3.3 variant [START_REF] Elsässer | Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells[END_REF]. The authors observed a physical interaction between DAXX and KAP1, that is independent of the presence of H3.3, and thus proposed that DAXX functions in the KAP1-SETDB1 complex at a subset of ERVs to establish the local heterochromatin environment, which could then be maintained by SUV39H1/2 enzymes (Bulut- [START_REF] Bulut-Karslioglu | Suv39h-Dependent H3K9me3 Marks Intact Retrotransposons and Silences LINE Elements in Mouse Embryonic Stem Cells[END_REF]. Intriguingly, DAXX/ATRX complex-binding sites increases upon DNA demethylation, and notably at LTR-containing ERVs, concomitantly with an increase of H3K9me3 signal [START_REF] He | The Daxx/Atrx Complex Protects Tandem Repetitive Elements during DNA Hypomethylation by Promoting H3K9 Trimethylation[END_REF]. Furthermore, depletion of Daxx or Atrx results in the upregulation of IAPs, that the author attributes to the loss of H3K9me3. However, DAXX-mediated silencing of ERVs can happen via an ATRX-dependent and an ATRX-independent pathway [START_REF] Hoelper | Structural and mechanistic insights into ATRXdependent and -independent functions of the histone chaperone DAXX[END_REF]. Another report assessed the role of ATRX in creating a local heterochromatin environment at IAP sequences [START_REF] Sadic | Atrx promotes heterochromatin formation at retrotransposons[END_REF]. Sadic et al. further reported that ATRX binds to a short region of 160bp form the gag gene present in over 600 IAP elements. This short sequence was sufficient to induce the silencing of an exogenous DNA fragment and was thus called SHIN standing for short heterochromatin inducing sequence. These results suggest that multiple factors are acting differentially to ensure the proper silencing of TEs.

Because of the high diversity of TE sequences, many cellular proteins target only a subset of elements to create a common local heterochromatin environment consisting in DNA methylation and H3K9me3. This local heterochromatin compartment can vary according to the cell type, with the DNA methylation being the prominent repressive factor in somatic cells, whereas H3K9me3 pathway seems to be key during the embryonic development.

Diversity of functions of the transposable elements

TEs have had a great impact on the evolution of their host genomes. When they are embedded into a local repressive environment, TEs are part of the broader heterochromatin and B compartment in the nucleus. Because of their important number of individuals, TEs likely play a role in the maintenance of the three-dimensional folding of chromatin [START_REF] Cournac | The 3D folding of metazoan genomes correlates with the association of similar repetitive elements[END_REF]. In Drosophila, the gypsy DNA retrotransposon contains many binding sites for transcription factors and acts as an insulator domain. The insulation role of gypsy is mediated by the formation of a local loop for regions surrounding the insulator, suggesting that gypsy retrotransposon can regulate higherorder chromatin folding [START_REF] Byrd | Visualization of chromatin domains created by the gypsy insulator of Drosophila[END_REF]. Furthermore, mammalian genomes are notably organized via the action of the CTCF transcription factors. Interestingly, Schmidt et al. analyzed CTCF-binding sites across diverse mammalian species and discovered highly conserved motifs corresponding to a two-part sequence separated by 20 or 21bp [START_REF] Schmidt | Waves of Retrotransposon Expansion Remodel Genome Organization and CTCF Binding in Multiple Mammalian Lineages[END_REF]. Interestingly, the CTCF-binding sequences in mouse, rats, dogs and opossum are present within SINEs. The authors proposed that repeat-drive CTCF-binding sites expansion result from waves of SINE retrotransposons activities over the evolution of mammalian genomes [START_REF] Schmidt | Waves of Retrotransposon Expansion Remodel Genome Organization and CTCF Binding in Multiple Mammalian Lineages[END_REF]. TEs may thus represent an important source of evolution of the spatial organization of the genome.

LINE1 expression can be regulated by the condensing II protein, which suggest that although LINEs might maintain the 3D-folding of the genome, their expression is also controlled by the spatial organization [START_REF] Ward | Condensin II and GAIT complexes cooperate to restrict LINE-1 retrotransposition in epithelial cells[END_REF].

Furthermore, upon the loss of repressive marks and the subsequent transcriptional activation, TEs become a strong force to modulate the global compaction state of the genome. This particular feature is exploited by mammalian genomes during the early embryonic development. Indeed, following fertilization, the embryo possess a densely compact genome and upon the loss of DNA methylation, the 2-cell state correlates with peaks of expression of LTR and non-LTR endogenous retrotransposons [START_REF] Fadloun | Chromatin signatures and retrotransposon profiling in mouse embryos reveal regulation of LINE-1 by RNA[END_REF]. It was proposed that the global transcription of repeated elements was a mean to achieve a fast and robust general decompaction of the genome coinciding with the first transcription activity of the zygotic genome [START_REF] Jachowicz | LINE-1 activation after fertilization regulates global chromatin accessibility in the early mouse embryo[END_REF]). I will discuss this point more thoroughly in the last part of the introduction.

While cells adopt a wide variety of factors to repress the expression of TEs, some have been 'domesticated' and their expression is tolerated or even essential for cell survival [START_REF] Lowe | 29 Mammalian Genomes Reveal Novel Exaptations of Mobile Elements for Likely Regulatory Functions in the Human Genome[END_REF]. Lowe and Haussler searched for putative regulatory elements that originated from mobile elements such as SINE, LINE and ERVs, and they detected over 280,000 possible sequences that presented cross-species constraint and a purifying selection, without any detectable RNA transcript. The authors propose that about 20% of regulatory sequences in human might have emerged from the domestication of TEs, suggesting that TEs have indeed served as a source of non-coding regulatory sequences. Demonstration of such 'exaptation' was performed by Bejerano et al. which described an enhancer perfectly conserved in human, mouse and rat that originated from a SINE retrotransposon active in an ancient organism and still active in the Indonesian coelacanth [START_REF] Bejerano | A distal enhancer and an ultraconserved exon are derived from a novel retroposon[END_REF]. The authors thus called this element a 'living fossil' as it is still active in one specie and shown that this element indeed could serve as a regulatory element for a reporter gene. Differentiation of regulatory T cells depends on FOXP3 factor and it was observed that a conserved enhancer of FOXP3 that lie in one of its introns is derived from a retrotransposon and is specific to placental mammals [START_REF] Samstein | Extrathymic Generation of Regulatory T Cells in Placental Mammals Mitigates Maternal-Fetal Conflict[END_REF]). This regulation is crucial for the proper development of regulatory T cells to allow maternal-fetal tolerance during pregnancy. This hypothesis was already though to be important for promoter regulation of a gene, such as for the salivary amylase gene which in human requires the presence of an ERV to maintain its tissue-specific expression [START_REF] Ting | Endogenous retroviral sequences are required for tissue-specific expression of a human salivary amylase gene[END_REF]). These results indicate that co-option of TEs to become regulatory elements have happened and these events likely arise as an additional mean to confer specific spatio-temporal control of gene expression [START_REF] Thompson | Long Terminal Repeats : From Parasitic Elements to Building Blocks of the Transcriptional Regulatory Repertoire[END_REF]. TEs are crucial modulator for the evolution of their host genome, however, trans-species transmission of TEs must be happening via exogenous source of retroviruses and propagation as endogenous retroviruses could then shape the new host genome. Endogenization of a retrovirus is ongoing in koalas where the koala retrovirus (KoRV) has invaded only a subpopulation of animals, but is present in germ cells and is transmitted to the progeny [START_REF] Tarlinton | Retroviral invasion of the koala genome[END_REF][START_REF] Fiebig | Transspecies Transmission of the Endogenous Koala Retrovirus[END_REF].

These results indicate that not only the present ERVs, but also exogenous retroviruses could play a role in genome regulation.

Although important player for genomic evolution, TE transcription and integration can be detrimental for the cell. Indeed, about 10 % of spontaneous mutations in mouse and 50% in Drosophila arise from de novo insertions of TEs and in human, more than 120 pathologies are linked to de novo insertions [START_REF] Cosby | Host-transposon interactions: Conflict, cooperation, and cooption[END_REF]. TEs overexpression is sometimes a consequence of particular disease, such as the Rett syndrome for which patients display elevated LINE1 retrotranspositions [START_REF] Muotri | L1 retrotransposition in neurons is modulated by MeCP2[END_REF]. LINE1 upregulation of transcription or retrotransposition is a hallmark of many cancers [START_REF] Burns | Transposable elements in cancer[END_REF]. Loss of DNA methylation is often observed in certain cancers, which thus express higher levels of LINE1 RNA due to a loss of repressive marks.

Hypomethylation of LINE1 was correlated to enhanced genomic instability and poor prognosis for non-small-lung, colon and breast cancers [START_REF] Daskalos | Hypomethylation of retrotransposable elements correlates with genomic instability in non-small cell lung cancer[END_REF][START_REF] Ogino | A Cohort Study of Tumoral LINE-1 Hypomethylation and Prognosis in Colon Cancer[END_REF][START_REF] Van Hoesel | Hypomethylation of LINE-1 in primary tumor has poor prognosis in young breast cancer patients : a retrospective cohort study[END_REF][START_REF] Saito | Long Interspersed Nuclear Element 1 Hypomethylation Is a Marker of Poor Prognosis in Stage IA Non -Small Cell Lung Cancer[END_REF]. Upregulation of TEs can lead to the increase of the interferon response in acute myeloid leukemia [START_REF] Cuellar | Silencing of retrotransposons by SET DB1 inhibits the interferon response in acute myeloid leukemia[END_REF]. In lung cancers, it is observed that LINE1 upregulation correlates with enhanced new insertions [START_REF] Iskow | Natural Mutagenesis of Human Genomes by Endogenous Retrotransposons[END_REF]. Furthermore, the capacity to perform genome-wide DNA and RNA sequencing as led to propose a role for TEs in tumorigenesis, as LINE1 insertions in somatic cancer cells are preferentially observed at genes that are frequently mutated in these cancers [START_REF] Lee | Landscape of Somatic Retrotransposition in Human Cancers[END_REF]. Colorectal cancer often originates from a mutation in the adenomatous polyposis coli (APC) tumor suppressor gene. It was reported early on that a LINE1 insertion could occur within the APC gene as it was observed for patient cells [START_REF] Miki | Disruption of the APC Gene by a Retrotransposal Insertion of LI Sequence in a[END_REF]. It recently demonstrated that LINE1 integration events could indeed lead to the disruption of one APC allele as a consequence of the derepression of an active LINE1 in nontumor cells [START_REF] Scott | A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer[END_REF]. When combined with a spontaneous mutation on the other APC allele, it could lead to cancer. Thus, active TEs represent an important source of variation that can potentially drive tumorigenesis, as well as creating genomic instability when it happens as passenger mutations.

In conclusion, transposable elements are important modulator of genome function and evolution. TE sequences can be coopted by the host genome to generate new regulatory system that take advantages of their activities and transcription factor binding sites. Yet the lack of repression exerted by the host cells can lead to undesired mutations and pathologies.

F -Nuclear Bodies

Different nuclear bodies have been observed in cells. Contrary to the heterochromatin compartments presented above, the main common characteristic of nuclear bodies is the enrichment of certain proteins and not specific chromatin domains. Indeed, high concentration of some proteins can generate membrane-less organelles, which can carry specific functions.

The Nucleolus

The nucleolus was the first sub-nuclear compartment identified. However, comprehensive understanding of its assembly and functions required a long time.

Definition of the nucleolus

Firstly observed during the 19 th century, the nucleolus was not the center of attention of many biological studies until the realization that nucleolus arises at specific loci that was called the nucleolus organizer regions (NORs) by Barbara McClintock in 1934 [START_REF] Pederson | The Nucleolus[END_REF]. Within the nucleolus are the rDNA genes encoding for ribosomal RNAs which are actively transcribed. The nucleolus organizes as a tripartite organelle. The fibrillar center (FC) correspond to the multiple rDNA genes, the dense fibrillar compartment (DFC) is made from the synthesis of rRNAs and the granular component (GC) is the place of ribosome assembly [START_REF] Tiku | Nucleolar Function in Lifespan Regulation[END_REF]. Because of its function of ribosome biogenesis, the nucleolus regulates the metabolic state of the cell. Thus, active cells have large and numerous nucleoli whereas cell cycle arrest reduces the size of the nucleolus. In mammalian cells, nucleoli must reassemble after each mitosis around NORs. RDNA transcription is carried by the efficient RNA Polymerase I. Many additional small nucleolar RNAs and proteins reside within the nucleolus and participate into the transcription and maturation processes. Not all rDNA genes are transcribed within the same cell at the same time. Furthermore, associated proteins such as UBF are essential in this process as depletion of UBF changes rDNA genes from an active to an inactive state [START_REF] Sanij | UBF levels determine the number of active ribosomal RNA genes in mammals[END_REF]. The nucleolus is a specialized sub-nuclear compartment that comprises DNA, RNA and proteins and carries the essential ribosomal biogenesis function.

The nucleolus act as a nuclear landmark and can associate with chromatin regions [START_REF] Canat | Genome anchoring to nuclear landmarks drives functional compartmentalization of the nuclear space[END_REF]. Human centromeres were observed to specifically localize closely to the nucleolus [START_REF] Carvalho | Chromosomal Gdark bands determine the spatial organization of centromeric heterochromatin in the nucleus[END_REF]. Furthermore, entire large regions could interact with the nucleolus as reported for the X inactive chromosome, in which case, the nucleolar association seems necessary for proper silencing [START_REF] Zhang | Perinucleolar Targeting of the Inactive X during S Phase: Evidence for a Role in the Maintenance of Silencing[END_REF]. Initial genome-wide identification of genomic regions that could associate with the nucleolus described the nucleolus-associated domains (NADs) which encompassed ~4% of the human genome and confirmed that is mostly corresponds to heterochromatin regions such as satellite repeats and transposable elements [START_REF] Németh | Initial genomics of the human nucleolus[END_REF].

Further characterization of NADs revealed that the ~1600 heterochromatin regions in fact cover ~40% of the human genome [START_REF] Dillinger | Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation[END_REF]. Human NADs are ~800Kb long on average and correspond to classical heterochromatin regions with a low gene content and enrichment of repressive histone modifications and present a high overlap with LADs. Interestingly, although nucleoli are strongly reorganized upon senescence, NADs largely retain their nucleolar association and display only moderate changes in expression. The regions displaying the major reorganization upon senescence being centromeric and pericentromeric satellites, which disassociate from the nucleolus [START_REF] Dillinger | Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation[END_REF]. Intriguingly, peripheral tethering of LADs is compromised in senescent cells, suggesting different anchoring pathways to nuclear landmarks for LADs and NADs [START_REF] Lenain | Massive reshaping of genome-nuclear lamina interactions during oncogeneinduced senescence[END_REF]. Characterization of NADs in mouse cells revealed a more complex picture.

The nucleolus as dynamic compartment

The nucleolus is a particularly dynamic membrane-less organelle. Indeed, it selfassemble via liquid-liquid phase separation (LLPS), which depends on its transcriptional activity [START_REF] Berry | RNA transcription modulates phase transition-driven nuclear body assembly[END_REF]. Each sub-compartment (FC, DFC and GC) forming the nucleolar undergo LLPS and assemble into distinct liquid phases which do not mix together [START_REF] Feric | Coexisting Liquid Phases Underlie Nucleolar Subcompartments[END_REF]. Feric et al. propose that each liquid phase's properties facilitated sequestration, transcription and maturation of the ribonucleoproteins. Furthermore, not only the transcription process but also the nascent transcripts drives the formation of the nucleolar sub-compartments [START_REF] Yao | Nascent Pre-rRNA Sorting via Phase Separation Drives the Assembly of Dense Fibrillar Components in the Human Nucleolus Article Nascent Pre-rRNA Sorting via Phase Separation Drives the Assembly of Dense Fibrillar Components in the Human Nucleolus[END_REF]. Treatment with 1,6-hexanediol, which impairs LLPS seems to increase the number of nucleoli and prevented association of NAD, with a stronger effect for class II NAD, suggesting differences in anchoring mechanisms [START_REF] Vertii | Two contrasting classes of nucleolusassociated domains in mouse fibroblast heterochromatin[END_REF].

The number of nucleoli is dependent on the metabolic state of the cell. For example, fusion events happen upon senescence, with only one giant nucleolus visible [START_REF] Dillinger | Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation[END_REF]). In ESCs, rDNA genes lack repressive histone modifications due to lower levels of DNA methylation, which results in extensive transcription [START_REF] Savić | lncRNA Maturation to Initiate Heterochromatin Formation in the Nucleolus Is Required for Exit from Pluripotency in ESCs[END_REF]. RDNA genes obtain heterochromatin feature upon differentiation and inhibition of heterochromatin formation impairs ESC differentiation, underlying the importance of this mechanism. Around half of the ~400 rDNA genes are expressed in somatic cells. During the early development, one copy of each NOR is marked for later inactivation in somatic cells in a process that is clonally retain and resemble the X inactivation memory [START_REF] Schlesinger | Allelic inactivation of rDNA loci[END_REF]. Nucleoli are strongly reorganized during embryogenesis. Transcription of rDNA genes only starts at the end of the 2-cell state but it is essential for further division (Coura [START_REF] Koné | Three-Dimensional Distribution of UBF and Nopp140 in Relationship to Ribosomal DNA Transcription During Mouse Preimplantation Development1[END_REF]. From the zygote to the 4-cell state, the nucleolus appears as the nucleolus precursor body (NPB) which are mostly devoid of activity as transcription of the rDNA genes happens at the periphery of the NPB (Kresoja-rakic and Santoro 2019). In the zygote and 2-cell state, pericentromeres appear as a ring-shape around NPBs and only organize as chromocenters from the 4-cell state (Aguirre-Lavin et al. 2012).These results highlight that the nucleolus is a dynamic sub-nuclear compartment, both in terms of assembly but also as an organelle that undergo changes in shape and composition.

Diseases associated with nucleolar dysfunction

Center of ribosome biogenesis, the nucleolus bears a critical role in the cell.

However its functions extends beyond this role as it has been implicated in many other cellular processes such as tRNA processing, pre-mRNA splicing and telomerase sequestration [START_REF] Wong | Subnuclear shuttling of human telomerase induced by transformation and DNA damage[END_REF][START_REF] Bertrand | Nucleolar localization of early tRNA processing[END_REF][START_REF] Falaleeva | Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing[END_REF]. Furthermore, the nucleolus act as sensor for signaling pathways. Indeed, upon insulin treatment, the insulin receptor substrate 1 translocates within the nucleus, localizes specifically at the nucleolus and an increase in rRNA synthesis [START_REF] Tu | Nuclear translocation of insulin receptor substrate-1 by oncogenes and Igf-I: Effect on ribosomal RNA synthesis[END_REF].

Heterochromatin surrounding the nucleolus provides stability for the silenced rDNA genes. Alteration of heterochromatin using the 5-azacytidine drug or by deleting the Su(var)3-9 gene in Drosophila results in rDNA instability and generates extrachromosomal rDNA circles that ultimately leads to senescence [START_REF] Gagnon-Kugler | Loss of Human Ribosomal Gene CpG Methylation Enhances Cryptic RNA Polymerase II Transcription and Disrupts Ribosomal RNA Processing[END_REF][START_REF] Peng | H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability[END_REF]. Furthermore, because of the high levels of transcription, the nucleolus undergoes many RNA -DNA hybrids, which are called R-loops and must be resolved via topoisomerases. Indeed, preventing topoisomerases activity impairs nucleolus organization [START_REF] Govoni | Inhibition of Topoisoerase II Activity and its Effect on Nucleolar Structure and Function[END_REF][START_REF] Christensen | Distinct effects of topoisomerase I and RNA polymerase I inhibitors suggest a dual mechanism of nucleolar/nucleoplasmic partitioning of topoisomerase I[END_REF].

RDNA genes also appear to be prone to recombination, especially in some cancers [START_REF] Stults | Human rRNA gene clusters are recombinational hotspots in cancer[END_REF]. Intriguingly, ALT-positive cancer cells and ESCs depleted for Atrx display high levels of rDNA copy loss [START_REF] Udugama | Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers[END_REF]. Udugama et al. describe that ribosomal DNA instability in Atrx-mutated results from impaired H3.3 deposition and subsequent heterochromatin formation at rDNA genes, suggesting that rDNA repeats are partly silenced via the same pathway than the more typical heterochromatin.

PML NBs

Promyelocytic (PML) proteins are ubiquitously expressed and assemble into 5 to 30 nuclear bodies (NBs) of 0.1 to 1 µm in diameter dispersed within the nuclear space. PML NBs have been implicated in many biological processes.

Definition and roles of PML NBs

PML is essential for the formation of the PML NBs and recruits many proteins such as SP100, DAXX or SUMO peptides. It acts as a hub for protein post-translational modifications like SUMOylation and ubiquitination and have been associated to a wide variety of DNA-related processes (Lallemand-Breitenbach and de Thé 2010). PML was identified as an oncoprotein in fusion with the retinoic acid receptor α (RARα) in acute promyelocytic leukemia (APL) [START_REF] Dyck | A Novel Macromolecular Structure Is a Target of the Promyelocyte-Retinoic Acid Receptor Oncoprotein[END_REF][START_REF] Daniel | PML Protein Expression in Hematopoietic and Acute Promyelocytic Leukemia Cells[END_REF][START_REF] Weis | Retinoic Acid Regulates Aberrant Nuclear Localization of PML-RARa in Acute Promyelocytic Leukemia Cells[END_REF]. PML and RARα fusion arise from the t(15 -17) translocation and leads to the mis-localization of RARα in the nucleus and expression of PML-RARα is sufficient to lead to APL phenotypes in mouse. Furthermore, APL therapy -a combination of retinoic acid and arsenic induces the degradation of PML-RARα and the reorganization of PML NBs [START_REF] Nasr | Eradication of acute promyelocytic leukemia-initiating cells through PML-RARA degradation[END_REF][START_REF] Ablain | Activation of a promyelocytic leukemia-tumor protein 53 axis underlies acute promyelocytic leukemia cure[END_REF]. PML belongs to the tripartitemotif (TRIM) family and carries a RING domain that can bind to the SUMO E2 ligase UBC9, two B-boxes and a coiled-coiled domain required for homodimerization (Figure 21A) [START_REF] Duprez | SUMO-1 modification of the acute promyelocytic leukaemia protein PML : implications for nuclear localisation[END_REF][START_REF] Tao | Structure of the MID1 Tandem B-Boxes Reveals an Interaction Reminiscent of[END_REF][START_REF] Kastner | Structure , localization and transcriptional properties of two classes of retinoic acid receptor fusion proteins in acute promyelocytic leukemia ( APL ): structural similarities with a new family of oncoproteins[END_REF]). Seven isoforms have been described in human, but only two in mouse, that in each species are generated by alternative splicing of the C-terminal region [START_REF] Condemine | Characterization of Endogenous Human Promyelocytic Leukemia Isoforms[END_REF]. The different isoforms might carry distinct function, either for PML or for NB formation, which would likely explain the differences of NBs observed across cell types and conditions. PML is mainly a nuclear protein as it carries a nuclear localization sequence (NLS) on its exon 6, yet, about 10% of PML proteins might reside or shuttle into the cytoplasm where it can have other functions such as regulation of signaling pathways [START_REF] Giorgi | PML Regulates Apoptosis at Endoplasmic Reticulum by Modulating Calcium Release[END_REF][START_REF] Lin | Cytoplasmic PML function in TGF-b signalling[END_REF][START_REF] Carracedo | The nuclear bodies inside out : PML conquers the cytoplasm[END_REF].

Acting as a hub for post-translational modifications, PML NBs concentrate the SUMO E2 ligase UBC9 and PML is heavily SUMOylated, along with the additional proteins sequestered into PML NBs. SUMOylation might be a recruitment pathway via the SUMO-SIM (SUMO-interacting motif) interactions of the different proteins. Indeed, PML mutants that cannot be SUMOylated can still generate NBs but subsequent partners recruitment is impaired [START_REF] Shen | The Mechanisms of PML-Nuclear Body Formation[END_REF][START_REF] Sahin | Oxidative stress-induced assembly of PML nuclear bodies controls sumoylation of partner proteins[END_REF]. Although PML NBs are easily visible under the microscope, 90% of PML proteins are diffuse in the nuclear space (Lallemand-Breitenbach and de Thé 2010). The consensus mechanism for NB formation is that PML shell forms via PML-PML interactions, and that subsequent SUMOylation favors recruitment of SIM-containing proteins (Figure 21B). Recently, a biophysical model was proposed to explain PML NBs formation that could undergo a liquid-liquid phase separation process, which could be a physical principle to enhance PML concentration and render NB formation easier [START_REF] Shin | Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome[END_REF][START_REF] Banani | Biomolecular condensates: Organizers of cellular biochemistry[END_REF][START_REF] Banani | Compositional Control of Phase-Separated Cellular Bodies[END_REF]. It was also reported that polySUMO-polySIM interactions could induce liquid-liquid phase separation, thus suggesting that SUMOylation of PML and other partners might reinforce the stability of PML NBs (Min et al.

2019).

PML is an important sensor of cellular stresses, such as oxidative stress, which induces assembly of NBs. In fact, PML does not form many NBs in vivo in physiological cells but only display high number of NBs in inflammatory tissues, suggesting that in vitro cultured cells exhibit constitutively high levels of oxidative stress [START_REF] Terris | PML Nuclear Bodies Are General Targets for Inflammation and Cell Proliferation1[END_REF]. PML NBs often contain P53 protein and it was shown that PML is required for ROS sensing and subsequent P53 activation, via the regulation of P53 post-translational modifications in mouse [START_REF] Niwa-Kawakita | PML is a ROS sensor activating p53 upon oxidative stress[END_REF]. Thus, PML regulates ROS levels in the nucleus. PML-dependent regulation of P53 is also true in human where reformation of PML NB upon arsenic treatment of APL cells induces a P53-senescence pathway [START_REF] Ablain | Activation of a promyelocytic leukemia-tumor protein 53 axis underlies acute promyelocytic leukemia cure[END_REF]. However, deletion of Pml gene does not impair growth and survival of mice, yet defects in hematopoietic and apoptotic functions are observed (Gang Wang et al. 1998;Wang et al. 1998b). Yet, Pml -/-mice exhibit neocortex hematopoietic defects, suggesting important roles in further differentiation and maintenance of tissues [START_REF] Regad | The tumor suppressor Pml regulates cell fate in the developing neocortex[END_REF][START_REF] Wang | Role of PML in Cell Growth and the Retinoic Acid Pathway[END_REF]. PML is also implicated in the control of protein quality as it can promote degradation of misfolded proteins via SUMOylation of the misfolded protein and recruitment of the SUMOdependent ubiquitin ligase RNF4. In a spinocerebellar ataxia disease, a polyglutamine (polyQ) syndrome, PML NBs colocalize with the misfolded proteins and it was further demonstrated that depletion of Pml could increase polyQ phenotypes in mouse [START_REF] Janer | PML clastosomes prevent nuclear accumulation of mutant ataxin-7 and other polyglutamine proteins[END_REF][START_REF] Guo | A cellular system that degrades misfolded proteins and protects against neurodegeneration[END_REF].

These results indicate that PML is an important player in the regulation of cellular homeostasis.

The shape of PML NBs can vary according to the cell state. In human embryonic stem cells, PML NBs were reported to adopt different conformations upon differentiation and can appear as linear rods or rosettes [START_REF] Butler | Changing nuclear landscape and unique PML structures during early epigenetic transitions of human embryonic stem cells[END_REF]). These unusual PML NBs appear to spatially interact with LaminA and centromeres, suggesting a role for PML in the organization of the heterochromatin in the nucleus. Upon LaminA depletion, PML NBs are perturbed, further indicating that PML interacts with the nuclear lamina, as it was proposed in a descriptive study where the isoform II of PML tend to associate with the inner membrane of the nuclear envelope [START_REF] Stixova | Trajectories and nuclear arrangement of PML bodies are influenced by A-type lamin deficiency[END_REF][START_REF] Condemine | Characterization of Endogenous Human Promyelocytic Leukemia Isoforms[END_REF]. Intriguingly, the unprocessed LaminA progerin accumulates at PML NBs in the aging disease HGPS (Hutchinson-Gilford Progeria Syndrome) [START_REF] Harhouri | MG 132 -induced progerin clearance is mediated by autophagy activation and splicing regulation[END_REF]. In HGPS, PML NBs display increased abnormal shapes such as long threads inside the nucleoplasm resulting from PML isoform 2 along aging, and these peculiar PML NBs perturb DNA repair processes and transcriptional regulation of PML NB-associated genes [START_REF] Wang | PML2-mediated thread-like nuclear bodies mark late senescence in Hutchinson -Gilford progeria syndrome[END_REF]. Shape and localization of PML NBs have been described in stressed and senescent human cells, which display large PML NBs at the nucleolus [START_REF] Condemine | A nucleolar targeting signal in PML-I addresses PML to nucleolar caps in stressed or senescent cells[END_REF]). The unusual rods and rosettes PML NBs do not contain the classical other partners SP100, DAXX nor SUMO, suggesting that protein composition of the nuclear bodies might influence its functions [START_REF] Butler | Changing nuclear landscape and unique PML structures during early epigenetic transitions of human embryonic stem cells[END_REF]).

Interplay of PML with DAXX, H3.3 and chromatin

Classical PML NBs do not contain chromatin, yet it contains the histone chaperone DAXX, its co-factor ATRX and sometimes HIRA (Figure 22A). DAXX and HIRA are the two histone chaperones specific for the histone variant H3.3, which is observed at PML NBs in some cells and notably upon overexpression of H3.3 (Figure 22B) [START_REF] Delbarre | DAXXdependent supply of soluble (H3.3-H4) dimers to PML bodies pending deposition into chromatin[END_REF]. The presence of HIRA within PML NBs seems to be quite rare in normal conditions but increases in senescence cells, where HIRA transiently localizes at PML NBs [START_REF] Zhang | Formation of MacroH2A-Containing Senescence-Associated Heterochromatin Foci and Senescence Driven by ASF1a and HIRA[END_REF]. Senescence cells are characterized by a strong higher-order chromatin reorganization with the appearance of senescence-associated heterochromatin foci (SAHF) [START_REF] Narita | Rb-Mediated Heterochromatin Formation and Silencing of E2F Target Genes during Cellular Senescence[END_REF]. In senescence cells, DAXX also associates with PML NBs and recruits there the newly synthesized H3.3 [START_REF] Corpet | Dynamics of histone H3.3 deposition in proliferating and senescent cells reveals a DAXXdependent targeting to PML-NBs important for pericentromeric heterochromatin organization[END_REF]. Newly synthesized H3.3 is mostly observed at sites of active transcription, but although it is not found in SAHF, enrichment of H3.3 increases at constitutive heterochromatin regions such as pericentromeres. Interestingly, PML NBs act as a storage center for H3.3 and depletion of PML impairs the deposition at both euchromatin and heterochromatin, highlighting a strong interconnection between PML NBs and H3.3 [START_REF] Corpet | Dynamics of histone H3.3 deposition in proliferating and senescent cells reveals a DAXXdependent targeting to PML-NBs important for pericentromeric heterochromatin organization[END_REF]. The link between H3.3 and PML is mediated by DAXX as only DAXX, and not ATRX, is crucial for H3.3-H4 dimers targeting to PML NBs [START_REF] Delbarre | DAXXdependent supply of soluble (H3.3-H4) dimers to PML bodies pending deposition into chromatin[END_REF].

In interphase nuclei, it was reported that PML NBs can associate preferentially with different loci such as the MHC class I gene cluster and the TP53 locus and regulates its transcriptional activity [START_REF] Shiels | PML bodies associate specifically with the MHC gene cluster in interphase nuclei[END_REF][START_REF] Kumar | Functional interaction between PML and SATB1 regulates chromatin-loop architecture and transcription of the MHC class I locus[END_REF][START_REF] Sun | Specific interaction of PML bodies with the TP53 locus in Jurkat interphase nuclei[END_REF]. The fact that PML might associate to chromatin loci shed new light on the role of the NBs as regulators of the compartmentalization of the nucleus. Attempts to characterize the chromatin interactions with PML NBs observed that they tend to preferentially associate with active regions, suggesting a role for PML NBs in the transcriptional activity [START_REF] Wang | Promyelocytic leukemia nuclear bodies associate with transcriptionally active genomic regions[END_REF][START_REF] Ching | Identifying gene locus associations with promyelocytic leukemia nuclear bodies using immuno-TRAP[END_REF]. PML can regulate gene expression, it is notably the case for the Id2 gene. At the Id2 promoter, PML mediates SETDB1 binding and repression of the locus. Interestingly, SETDB1 is a constitutive member of PML NBs since the early embryogenesis (Figure 22C). Furthermore, it is an essential member of PML NBs as Setdb1 depletion results in the loss of NBs without affecting the total level of PML proteins [START_REF] Cho | Dual functions of histone-lysine Nmethyltransferase Setdb1 protein at promyelocytic leukemia-nuclear body (PML-NB): Maintaining PML-NB structure and regulating the expression of its associated genes[END_REF]. PML NBs have also been linked to the KAP1 repressor as KAP1 forms foci adjacent to PML NBs upon differentiation [START_REF] Briers | KRAB zincfinger proteins localise to novel KAP1containing foci that are adjacent to PML nuclear bodies[END_REF]. PML was reported to control the expression of many genes, especially in ESCs where PML is proposed to be an important regulator of pluripotency, although the action of PML in genome regulation could be indirect [START_REF] Hadjimichael | Promyelocytic Leukemia Protein Is an Essential Regulator of Stem Cell Pluripotency and Somatic Cell Reprogramming[END_REF].

Interestingly, a recent study described a role for PML in regulating the higher-order organization of chromatin and loss of Pml resulted in the alteration of heterochromatin compartments [START_REF] Delbarre | PML protein organizes heterochromatin domains where it regulates histone H3 . 3 loading by ATRX / DAXX[END_REF]. The regions regulated by PML are named PML-associated domains and are enriched in H3K9me3, a mark that they tend to lose upon Pml depletion because of an impaired DAXX-mediated H3.3 deposition. Yet other regions can be affected by the loss of Pml. Pml-null mouse fibroblasts displayed higher levels of H3.3 into constitutive heterochromatin regions such as subtelomeres, centromeres and pericentromeres [START_REF] Spirkoski | PML modulates H3.3 targeting to telomeric and centromeric repeats in mouse fibroblasts[END_REF].

The role of the histone variant H3.3 on heterochromatin maintenance is clearly visible in embryonic stem cells as H3.3 provides the telomeric H3K9me3 [START_REF] Udugama | Histone variant H3.3 provides the heterochromatic H3 lysine 9 trimethylation mark at telomeres[END_REF]. Moreover, PML NBs colocalize with some telomeres in ESCs and cancer cells with the alternative-lengthening of telomeres (Figure 22D) [START_REF] Draskovic | Probing PML body function in ALT cells reveals spatiotemporal requirements for telomere recombination[END_REF][START_REF] Chang | PML bodies provide an important platform for the maintenance of telomeric chromatin integrity in embryonic stem cells[END_REF]. Depletion of Pml decreases H3.3 levels and affects telomere integrity in embryonic stem and ALT-positive cancer cells [START_REF] Ivanauskiene | The PML-associated protein DEK regulates the balance of H3.3 loading on chromatin and is important for telomere integrity[END_REF][START_REF] Osterwald | PML induces compaction, TRF2 depletion and DNA damage signaling at telomeres and promotes their alternative lengthening[END_REF]. In murine embryonic stem cells, the DAXXmediated deposition of H3.3 at telomeres is further regulated by the oncoprotein DEK. Indeed, loss of DEK induces the relocalization of PML and DAXX away from telomeres and increased the incorporation of H3.3 into chromosome arms [START_REF] Ivanauskiene | The PML-associated protein DEK regulates the balance of H3.3 loading on chromatin and is important for telomere integrity[END_REF]. Ivanauskiene et al. propose that non-nucleosomal H3.3 is stored into PML NBs and from then can be recruited by its chaperones. After depletion of DEK, the pool of non-nucleosomal H3.3 is reduced and H3.3 is no longer observed at PML NBs. On the opposite, in the case of Daxx depletion, the soluble pool of H3.3 increases as it is less incorporated in the chromatin. Thus, it emerges a model whereby newly synthesized H3.3 would be directed to PML NBs in a DAXX-dependent manner and be stored until further necessity.

PML NBs can associate with chromatin regions. One of the functions of PML is the activation of an antiviral response. It was further observed that in response to herpes simplex virus (HSV) infection, the HSV genome is embedded within PML NBs and is chromatinized via H3.3 incorporation and H3K9-methylation [START_REF] Cohen | Promyelocytic leukemia ( PML ) nuclear bodies ( NBs ) induce latent / quiescent HSV-1 genomes chromatinization through a PML NB / Histone H3 . 3 / H3 . 3 Chaperone Axis[END_REF]. Interestingly, H3.3 incorporation is mediated by both chaperones DAXX and HIRA in this context, but this mechanism is dependent on PML. PML NB formation also occurs during HIV and other retroviruses infections, where it mediates a DAXX-dependent control of HIV reverse-transcription [START_REF] Dutrieux | PML/TRIM19-Dependent Inhibition of Retroviral Reverse-Transcription by Daxx[END_REF]. From all these studies, it appears that PML might be an important regulator of heterochromatin, notably via its interactions with DAXX and H3.3.

In the ICF syndrome, which is a consequence of a mutation in the DNMT3B gene, a reported phenotype is the instability of the centromeres of three chromosomes -1, 9 and 16.

These three centromeres are the most GC-rich and they undergo the strongest DNA demethylation. It was observed that lymphocytes of ICF patients display atypical PML NBs that are bigger than classical ones (Figure 22E). Called 'giant NBs', they form a scaffold for the classical SUMO, SP100 and DAXX proteins, and interestingly, they contain chromatin regions marked with HP1 [START_REF] Luciani | Subcellular distribution of HP1 proteins is altered in ICF syndrome[END_REF]. It was observed that giant PML NBs form onto the pericentromeric regions of the chromosomes 1, 9 and 16 and are enriched in DNA repair proteins such as BLM, BRCA1 and TOPOIIIα [START_REF] Luciani | PML nuclear bodies are highly organised DNAprotein structures with a function in heterochromatin remodelling at the G2 phase[END_REF]). These structures are exclusively observed in G2 phase and the authors proposed that it could be a mechanism to reform heterochromatin after S-phase. Not only in the ICF syndrome could PML NBs sequester DNA repair protein. Proteins such as BRCA1, RAD51 or MRE11 can transit through PML NBs [START_REF] Boichuk | Functional Connection between Rad51 and PML in Homology-Directed Repair[END_REF][START_REF] Carbone | PML NBs associate with the hMre11 complex and p53 at sites of irradiation induced DNA damage[END_REF]. Furthermore, PML NBs could act as sensors of DNA damages for upon DNA damages, the number of PML NBs increases due to fission events of the existing NBs and this mechanism is dependent on DNA damage response (DDR) checkpoints ATM, ATM, NBS1 and CHK2 [START_REF] Dellaire | Promyelocytic leukemia nuclear bodies behave as DNA damage sensors whose response to DNA double-strand breaks is regulated by NBS1 and the kinases ATM, Chk2, and ATR[END_REF]. The function of PML in DDR seems to be at a late stage, as the loss of Pml does not impair DNA damages signaling such as yH2AX and RAD51 foci formation, but diminished by up to 20 fold the efficiency of homologous recombination [START_REF] Yeung | Promyelocytic Leukemia Nuclear Bodies Support a Late Step in DNA Double-Strand Break Repair by Homologous Recombination[END_REF]). Yet a more recent study suggest that the initial recognition of DNA damage might be delayed when Pml is absent (Di [START_REF] Masi | PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL[END_REF]. Because of the possible localization of BRCA1 and RAD51 inside PML NBs, it was proposed that defects in DDR might arise from the mislocalization of these proteins if there is no PML NBs and that could be an important feature of APL pathogenesis [START_REF] Voisset | PML nuclear body disruption cooperates in APL pathogenesis and impairs DNA damage repair pathways in mice[END_REF]. Together, these studies indicate that PML is a multivalent interactor that plays different roles in the regulation of DNA-related processes. One of the functions of PML might reside in its ability to form NBs that could help concentrating proteins at specific nuclear localizations.

III -Embryonic stem cell as a model of embryonic development and chromatin specifies.

All cells of the organism originate from a unique one, the zygote, created by the fusion of the two parental gametes. Cell-types is not a DNA property, rather the expression of a defined set of genes. A lot of phenotypic changes happen during the embryonic development, from zygote to birth. Indeed, many differentiation programs start after the implantation stage, during gastrulation, with the appearance of the three layers that will compose all the organism -mesoderm, endoderm and ectoderm. Cells then commit lineage differentiation, a definitive process, that erase their pluripotent capacities.

Pluripotency has intrigued biologists for a long time and is still not completely understood.

I will here present the early mouse embryonic development, then the use of pluripotent cells in cultures and their use to gain developmental insights. Finally, I will present pluripotency from a chromatin viewpoint.

Embryonic development and embryonic stem cells

Understanding the developmental properties of human and mouse has been the focus of many research groups for above a century. Yet, this has proven to be a complex question to tackle. Embryonic development is an extremely complex suite of events that includes cell differentiation, tissue morphogenesis and growth of the organism. The use of pluripotent cells has been a key technology to better understand the early embryogenesis. In vivo experiments have yielded important discoveries and remain crucial to assess the relevance of molecular events identified in vitro. However, because of the inherent difficulties to get in vivo materials, the use of pluripotent stem cells in culture is particularly adapted to dissect molecular events.

Definition of the embryonic development and pluripotent cells

Embryonic development consists of the route from the zygote to birth, that includes many stages [START_REF] Saiz | Early cell fate decisions in the mouse embryo[END_REF].

Pre-implantation development starts in the oviducts until the blastocyst stage when the uterine implantation takes place at around E4.5 in mouse and E7 in human (Figure 23).

Totipotency is defined as the capacity to generate all cellular tissues, including the embryo and extra-embryonic tissues. Embryonic cells are totipotent up to the 4-cell state. Between the 4-cell and blastocyst state, some of the embryonic cells differentiate to give rise to the extraembryonic tissues -the trophectoderm and the primitive endoderm which are observed at the periphery of the embryo. The trophectoderm will give the placenta, whereas the primitive endoderm will form the yolk sac. The rest of the cells at the blastocyst stage form the inner cell mass (ICM) which consist of pluripotent cells able to differentiate into all tissues of the organism [START_REF] Shahbazi | Deconstructing and reconstructing the mouse and human early embryo[END_REF].

During these cell divisions, many transcriptional changes arise, giving specific transcriptomic signatures. Within the blastocyst, the three tissues present distinct signatures -ICM cells composed of the naïve pluripotent epiblast present high OCT4, NANOG and REX1, KLF2, KLF17 and ESSRβ whereas the trophectoderm cells display high Cdx2, EOMES and KRT8 transcription, and the primitive endoderm high GATA6, SOX17 and GATA4 signaling [START_REF] Shahbazi | Deconstructing and reconstructing the mouse and human early embryo[END_REF]. At the implantation stage, specific signaling pathways act to ensure proper polarized specialization of the embryo. One key signal is the activation of NODAL by the epiblast, that activates BMP4 in the adjacent extraembryonic tissue, which in turn activates WNT3 in the epiblast [START_REF] Ben-Haim | The Nodal Precursor Acting via Activin Receptors Induces Mesoderm by Maintaining a Source of Its Convertases and BMP[END_REF]. These transcriptomic signatures have getting more and more exhaustive and precise with the arrival of new NGS (next-generation sequencing) technologies such as single cell RNA sequencing. Single-cell RNA-seq (scRNA-seq) studies have generated a comprehensive timeline of gene activation from mouse to human [START_REF] Xue | Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing[END_REF]. ScRNA-seq has determined that for about 12 to 24% of autosomal genes there is a random monoallelic expression that occurs independently of the other allele [START_REF] Deng | Single-Cell RNA-Seq Reveals Dynamic, Random Monoallelic Gene Expression in Mammalian Cells[END_REF]. It has given unprecedent deepness for cross-species comparisons and helped identify transcriptional variation and conservation such as GATA6, SOX17 and GATA4 sequential activation in primitive endoderm from primates to humans [START_REF] Boroviak | Single cell transcriptome analysis of human , marmoset and mouse embryos reveals common and divergent features of preimplantation development[END_REF]. Many of the transcriptional changes seen before gastrulation are linked to chromatin regulation, which I will discuss in the last part of this introduction. This deeper understanding of the pathways implicated during acquisition and loss of pluripotency have been under the focus on researches for decades. It was necessary to understand the in vivo transient pluripotency to get to in vitro maintained pluripotency cultures.

The 1950's have witnessed the emergence of the in vitro culture conditions for embryonic cells that have paved the way for today's culture conditions [START_REF] Hammond | Recovery and Culture of Tubal Mouse Ova[END_REF][START_REF] Whitten | Culture of Tubal Mouse Ova[END_REF].

Researchers have firstly tried to cultivate the whole pre-implantation embryo until 1981 when two teams isolated embryonic stem cells (ESCs) from mouse ICM blastocysts [START_REF] Evans | Establishment in culture of pluripotential cells from mouse embryos[END_REF][START_REF] Martin | Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells Developmental Biology[END_REF]). In the post-implantation embryo, ICM cells have converted into the epiblast before giving rise to the other tissues. Epiblast stem cells have been derived and cultured in vitro to help elucidate this first primed state of pluripotency [START_REF] Brons | Derivation of pluripotent epiblast stem cells from mammalian embryos[END_REF][START_REF] Tesar | New cell lines from mouse epiblast share defining features with human embryonic stem cells[END_REF]. The defined set of additional factors necessary for pluripotent epiblast stem cell-derived cultures FGF2 and ACTIVIN-A can be used to generate epiblast-like stem cells from cultured ESCs, however epiblast stem cells do not rely onto the LIF factor essential for ESCs [START_REF] Hayashi | Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells[END_REF].

Extraembryonic ectoderm cells can also be cultured in-vitro [START_REF] Tanaka | Promotion to trophoblast stem cell proliferation by FGF4[END_REF]. Furthermore, mixing ESC and TSC can generate blastocyst-like structures that highly resemble to in vivo blastocysts, including the formation of a pro-amniotic cavity, indicating that ESCs possess essential intrinsic properties [START_REF] Rivron | Blastocyst-like structures generated solely from stem cells[END_REF]).

These studies have shed light on the early embryonic development and have further shown the differences between human and mouse development. Moreover, not all in-vitro mouse stem cell cultures can be applied to humans. Murine and human ESCs share some resemble but also some differences indicating that the in vitro culture of mESC or hESC does not correspond to the exact same pluripotency, however it can be smoothen by particular culture conditions [START_REF] Davidson | The pluripotent state in mouse and human[END_REF][START_REF] Gu | Rapid conversion of human ESCs into mouse ESC-like pluripotent state by optimizing culture conditions[END_REF]. Indeed, whereas murine ESCs grow as domed colonies, human ESCs grow as more flat colonies like murine epiblast stem cells. Moreover, they do not rely onto the Two cell-type will form the extra-embryonic tissues: the trophectoderm in green that will give the placenta and the primitive endoderm in purple which will give the yolk sac. The inner cell mass in yellow will differentiate into the whole organism. At E4.5, the blastocyst can implant within the uterus and then form the epiblast. ICM = inner cell mass. Adapted from Shahbazi and Zernicka-Goaetz, Nature Cell Biol. 2018 and Saiz and Plusa, Reproduction 2013 exact same signaling pathways. Human embryonic stem cells in culture resemble to both murine epiblast stem cells and embryonic stem cells, suggesting that pluripotency might to some extent be specie-specific [START_REF] Schnerch | Distinguishing Between Mouse and Human Pluripotent Stem Cell Regulation : The Best Laid Plans of Mice and Men[END_REF].

Human ESCs presented a high therapeutic interest until 2007. Indeed, the possibility to differentiate ESC to any cell-type held great promises and many in vitro differentiation pathways have been described. From 2007 and during the following years, induced pluripotent stem cells (iPSCs) were generated from murine and human somatic cells, by overexpressing the pluripotent factors -OCT4, KLF4, SOX2 AND C-MYC [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors[END_REF][START_REF] Takahashi | Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors[END_REF]. Induced pluripotent stem cells display an ESC-like transcriptomic regulation and 3Dchromatin organization, suggesting common pluripotency networks for embryonic and somatic cells (Krijger [START_REF] Lodewijk | Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming Article Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming[END_REF][START_REF] Guenther | Chromatin Structure and Gene Expression Programs of Human Embryonic and Induced Pluripotent Stem Cells[END_REF]).

Different levels of pluripotency in in vitro cultures

ESCs pluripotency is partly defined as the capacity to generate chimera embryos when injected in a blastocyst. Yet, in in-vitro studies, researchers often only look at transcriptional markers associated with pluripotency that I will briefly describe here. Classical pluripotency markers usually consist of the so-called 'Yamanaka factors' that are sufficient to reprogram somatic cells -OCT4, KLF4, SOX2, C-MYC; and some non-Yamanaka as NANOG, ESSRΒ and REX1. These factors are transcription-factors that act within complex signaling pathways. OCT4 is transcribed from the Pou5f1 gene that stands for octamer-binding transcription factor 4 (Kellner and Kikyo 2010).

Although OCT4 is not involved in the initiation of totipotency, it appears to be crucial for pluripotency in both mouse and humans, as deletion of the Pou5f1 impairs embryogenesis after the blastocyst stage [START_REF] Nichols | Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4[END_REF][START_REF] Fogarty | Genome editing reveals a role for OCT4 in human embryogenesis[END_REF]. Inactivation of Oct4 in human and murine ESCs induce a loss of pluripotency and differentiation into neural ectoderm cell-type [START_REF] Bertero | Optimized inducible shRNA and CRISPR / Cas9 platforms for in vitro studies of human development using hPSCs[END_REF][START_REF] Thomson | Pluripotency Factors in Embryonic Stem Cells Regulate Differentiation into Germ Layers[END_REF]. The Pou5f1 gene is under the control of OCT4 and SOX2 binding, which creates a regulatory feedback loop [START_REF] Okumura-Nakanishi | Oct-3 / 4 and Sox2 Regulate Oct-3 / 4 Gene in Embryonic Stem Cells[END_REF]. KLF4 stands for Krüppel-Like Factor 4, it is also a transcription factor. Its proper expression is crucial to normal development, as mice lacking this gene die rapidly after birth [START_REF] Villarreal | Defining the regulation of KLF4 expression and its downstream transcriptional targets in vascular endothelial cells[END_REF]. SOX2 belongs to the SRY-related HMG box family of transcription factors and is expressed in the preimplantation embryo, but also later in the epiblast [START_REF] Huang | Molecular basis of embryonic stem cell self-renewal : from signaling pathways to pluripotency network[END_REF]. Deletion of Sox2 impairs pluripotency both in vivo and in vitro, likely via its crucial role in OCT4 regulation [START_REF] Masui | Pluripotency governed by Sox2 via regulation of Oct3 / 4 expression in mouse embryonic stem cells[END_REF][START_REF] Avilion | Multipotent cell lineages in early mouse development depend on SOX2 function[END_REF]. NANOG is a Q50 homeodomain-containing transcription factor. Interestingly, NANOG is not essential for embryonic development, yet it is required for germline formation [START_REF] Chambers | Nanog safeguards pluripotency and mediates germline development[END_REF].

Culturing embryonic stem cells requires special medium conditions. First culture media in the 1980's relied on mouse embryonic fibroblasts (MEFs) as feeders for ESCs in addition to fetal bovine serum. Yet, it was identified that MEFs produced the leukemia inhibitory factor (LIF), which activates the JAK-STAT3 signaling pathway. So, ESCs could be kept in culture without MEF feeder when LIF was added to the serum-based medium [START_REF] Williams | Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells[END_REF]). These cells exhibit the classical pluripotency-associated factors -OCT4, KLF4, NANOG and ESSRβ expression. They retain their pluripotency capacities as injection into blastocysts could generate chimeras, they are thus naïve stem cells. I will refer to this culture condition as the 'standard' ESC serum/LIF condition.

Although well-defined, the 'standard' serum culture condition uses a fetal bovine serum, which we do not know its complete chemical composition. Furthermore, it has been realized that 'standard' medium culture of ESCs was sufficient for chimera's formation [START_REF] Nagy | Derivation of completely cell culture-derived mice from early-passage embryonic stem cells[END_REF][START_REF] Bradley | Formation of germline chimaeras from embryo-derived teratocarcinoma cell lines[END_REF]. However, in vitro pluripotency differs from its in vivo counterpart observed in the ICM as ESCs display some transcriptional changes [START_REF] Plusa | Embryonic stem cell identity grounded in the embryo[END_REF][START_REF] Boroviak | The ability of inner-cellmass cells to self-renew as embryonic stem cells is acquired following epiblast specification[END_REF]. Research groups have tried to use serum-free medium for ESC culture conditions to get pluripotent cells that would match in vivo ICM cells. In 2008, a study led by Austin Smith described the 3i (3 inhibitors) medium able to maintain naive pluripotency of mESCs that they called the ground state of pluripotency without LIF [START_REF] Ying | The ground state of embryonic stem cell self-renewal[END_REF]. Ying et al. were not only able to cultivate ESC in a serum-free medium, but they also demonstrated that the activation of the STAT3 signaling is dispensable for maintaining of pluripotency, and ESCs instead required the complete inhibition of differentiation-signaling, indicating intrinsic pluripotent capacities of these cells. An alternative 2i/LIF medium was adopted to overcome the reduced proliferation observed in 3i medium. 2i/LIF-medium is obtained using small-molecule inhibitors for the GSK3 and MEK3 pathways (Figure 24). Intriguingly, the MEK-ERK signaling pathway is inhibited in this medium, yet genetic ablation of ERK impedes ESC culture, suggesting a MEK-independent role for ERK signaling [START_REF] Chen | Erk signaling is indispensable for genomic stability and self-renewal of mouse embryonic stem cells[END_REF]. Furthermore, differences in signaling pathways at play in 2i/LIF or serum cultures are exemplified by the observation that the Klf2 knock-out, a factor regulated by ERK, is only lethal in the 2i/LIF medium [START_REF] Yeo | Klf2 is an essential factor that sustains ground state pluripotency[END_REF]. A proteomic study confirmed those transcriptomic and genetic studies, the authors also observed differences in metabolism between the two medium conditions and reported a lower reactive oxygen species levels in 2i/LIF [START_REF] Taleahmad | Proteome Analysis of Ground State Pluripotency[END_REF]. Cells cultured in 2i/LIF condition retain proper differentiation capacities, indicating that a metastable state, such as serum-based condition, is not necessary for lineage-commitment [START_REF] Marks | The transcriptional and epigenomic foundations of ground state pluripotency[END_REF]. Both serum and 2i/LIF culture conditions present the classical dome-shaped colonies formed by ESCs multiplication. Nonetheless, not all ESCs in in-vitro culture dish display the same morphology and transcriptional profile, suggesting a high heterogeneity. It was demonstrated using GFP-tagged REX1 marker, which should only be expressed in the ICMderived cells (i.e. ESCs) and not in Epiblast stem cells. Yet, in serum-based culture, REX1-positive coexist with a REX1-negative population [START_REF] Toyooka | Identification and characterization of subpopulations in undifferentiated ES cell culture[END_REF]. Furthermore, even if REX1negative cells are sorted, after five days of culture, half of the population present a Rex1-positive profile, indicating that a population of ESCs is a mix of transient and reversible states toward naïve and primed pluripotent cells [START_REF] Toyooka | Identification and characterization of subpopulations in undifferentiated ES cell culture[END_REF]. Deeper population-level genome-wide studies of the 'standard' and 2i/LIF conditions have been conducted. It was observed that 2i/LIF ESCs presented lower expression of differentiation-related genes that correlated with an increase of RNA Pol II-pausing at promoters, and importantly an increased expression of Nanog, while conserving the same differentiation capacities [START_REF] Marks | The transcriptional and epigenomic foundations of ground state pluripotency[END_REF]. These results were thought to arise from a more homogenous cell-population in 2i/LIF, as assessed by scRNA-seq [START_REF] Guo | Serum-Based Culture Conditions Provoke Gene Expression Variability in Mouse Embryonic Stem Cells as Revealed by Single-Cell Analysis[END_REF]). This view is challenged by scRNA-seq suggesting that there is the same level of heterogeneity in the two culture conditions, although it does not concern the same set of genes, with lineage-commitment genes being completely silenced in 2i/LIF conditions [START_REF] Kolodziejczyk | Single Cell RNA-Sequencing of Pluripotent States Unlocks Modular Transcriptional Variation Resource Single Cell RNA-Sequencing of Pluripotent States Unlocks Modular Transcriptional Variation[END_REF]. Differences between the two cultures arises in part from changes in the epigenome states, notably characterized by a lower DNA methylation level in 2i/LIF, which I will discuss in the next sub-part. Finally, cells in culture display features of pluripotency, one should keep in mind that during the embryonic development, pluripotency is a transient state before lineage commitment. However, in vitro ESCs can be kept in cultures virtually indefinitely and it is likely that subtle changes may accumulate during prolonged culture.

Pluripotency in a chromatin context

Pluripotency results from signaling pathways that ultimately induce transcription factors.

Yet, as defined in the second part of the introduction, chromatin is not merely binding sites for proteins, but also bears a complex three-dimensional organization. During development, many chromatin changes happen, some are large-scale like the building of the different heterochromatin compartments, and some are visible at finer-scale such as promoter reorganization.

Chromatin changes along development affect genomic loci with differences in histone modifications enrichment. This is notably the case for promoters which can only bear in pluripotent cells both H3K4me3 and H3K27me3-enrichments [START_REF] Azuara | Telomeric Repeat-Containing RNA and RNA Surveillance Factors at Mammalian Chromosome Ends[END_REF]). These promoters were thus called bivalent, because these two histone modifications do not usually colocalize. The genes are inactive but poised for later activation, although it was reported that they might be expressed at low levels even in ESCs [START_REF] Herz | The Curious Case of Bivalent Marks[END_REF]). Bivalent promoters have been identified from the 8-cell stage to the blastocyst in the embryo, but also in other adult stem cell as hematopoietic or mesenchymal stem cells. Interestingly, it seems that the bivalency might have a function during development as it is conserved in zebrafish [START_REF] Vastenhouw | Chromatin signature of embryonic pluripotency is established during genome activation[END_REF]. Furthermore, the number of bivalent promoters is reduced in ground-state ESCs compared to 'standard' serum ESCs, suggesting that the DNA methylation levels might play a role in promoter identities [START_REF] Marks | The transcriptional and epigenomic foundations of ground state pluripotency[END_REF]. In pluripotent cells, many genes display monoallelic expression, suggesting an even finer level of regulation [START_REF] Xu | Landscape of monoallelic DNA accessibility in mouse embryonic stem cells and neural progenitor cells[END_REF]. It was demonstrated that the monoallelic expression of some factors such as Nanog was essential at the ground state of pluripotency as the removal of one allele of Nanog impaired proper development of the ICM (Miyanari and Torres-Padilla 2012).

Thus, transcriptional regulation is important in the maintenance of pluripotency. At the zygote stage, there is no transcription from both parental genomes, all the proteins at play arise from the oocyte transcripts. An important event at the 2-cell stage in mouse, 4-cell stage in human, is the zygotic genome activation (ZGA), when the embryo starts its own transcription for the first time. At the ZGA, the chromatin becomes globally decondensed. How could the genome decompact all at once? It was noted that retrotransposon transcription is correlated with the pluripotent state, with a transcriptional burst at the 2-cell stage and a slow decrease along the development, that is following a loss of active marks onto the chromatin (Figure 25B) [START_REF] Fadloun | Chromatin signatures and retrotransposon profiling in mouse embryos reveal regulation of LINE-1 by RNA[END_REF]. It was further demonstrated that the activation of LINE1 elements was modulating global chromatin accessibility and that this activation and the subsequent silencing at later stages was required for proper maturation toward blastocyst [START_REF] Jachowicz | LINE-1 activation after fertilization regulates global chromatin accessibility in the early mouse embryo[END_REF].

The first 4 to 5 days of the embryonic development are moments of intense changes. The major change, observed early on, is the wave of DNA demethylation from the zygote to the blastocyst, then the methylation is progressively restored [START_REF] Howlett | Methylation levels of maternal and paternal genomes during preimplantation development[END_REF]. Genome-wide assessment of 5mC observed that the loss is global and at almost all genomic locations, excepts for imprinted-loci and some ERVs such as the young IAPs (Figure 25B) [START_REF] Lane | Resistance of IAPs to Methylation Reprogramming May Provide a Mechanism for Epigenetic Inheritance in the Mouse[END_REF]. Right after the zygote stage, the paternal genome becomes fully unmethylated, the maternal genome on the other hand will lose its DNA methylation over the next cell divisions [START_REF] Ladstätter | Genomic insights into chromatin reprogramming to totipotency in embryos[END_REF]. Around 20 imprinting control regions (ICRs) have been identified in both human and mouse [START_REF] Barlow | The mouse insulinlike growth factor type-2 receptor is imprinted and closely linked to the Tme locus[END_REF][START_REF] Zwart | Bidirectional action of the Igf2r imprint control element on upstream and downstream imprinted genes[END_REF][START_REF] Ac | Embryological and molecular investigations of parental imprinting on mouse chromosome 7[END_REF]. ICRs are often CpG-rich regions that display differential methylation pattern in a parent-of-origin manner (Greenberg and Bourc'his 2019). Imprinted genes are thus methylated on only one of the two alleles such as IGF2 for which only the paternal allele is expressed, or the closely located H19 for which only the maternal allele is expressed, the reciprocal allele being methylated [START_REF] Dechiara | Parental Imprinting of the Mouse Insulin-like Growth Factor II Gene[END_REF][START_REF] Bartolomei | Parental imprinting of the mouse H19 gene[END_REF].

ESCs in culture display ~75% of DNA methylation for all possible CpGs, indicating a strong level of DNA methylation which likely results from the in vitro serum culture conditions [START_REF] Stadler | DNA-binding factors shape the mouse methylome at distal regulatory regions[END_REF]. Recapitulating the changes in DNA methylation levels during early embryogenesis was only achieved with the use of the chemically defined 2i/LIF medium for ESCs [START_REF] Ying | The ground state of embryonic stem cell self-renewal[END_REF]. The scale and pattern of the observed DNA demethylation observed in culture resemble the one seen in the ICM at the blastocyst stage, as assessed by genome-wide bisulphite sequencing [START_REF] Ficz | FGF signaling inhibition in ESCs drives rapid genomewide demethylation to the epigenetic ground state of pluripotency[END_REF]). The addition of vitamin C to the 2i/LIF medium could further decrease the global level of 5mC and increase the level of 5hmC at the same time, via enhanced activity of the TET enzymes (Figure 24) [START_REF] Blaschke | Vitamin C induces Tet-dependent DNA demethylation and a blastocystlike state in ES cells[END_REF]. The decrease of 5mC was shown to be dependent on synergistic mechanisms, both passive and active. Indeed, the increase of the PRDM14 protein represses the action of the de novo methylases, whereas TET enzymes enhanced the conversion from 5mC to 5hmC, inducing a rapid and genome-wide decrease of DNA methylation (Hackett et al. 2013a). A latter study complemented these results showing that the TET activation relied on the stabilization of JMJD2C, directly induced by MEK inhibition, and the PRDM14 activity was mediated by the G9A methyltransferase activity toward DNMT3A and DNMT3B [START_REF] Sim | 2i Maintains a Naive Ground State in ESCs through Two Distinct Epigenetic Mechanisms[END_REF]. Interestingly, depletion of the three DNMT enzymes only partially upregulates the transcription of transposable elements, yet, converting the cells to the 2i/LIF medium induces a strong upregulation of TEs, suggesting synergistic mechanisms at play [START_REF] Matsui | Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET[END_REF]. Walter et al. proposed that conversion of ESCs from a serum to a 2i/LIF medium induces a rapid genome-wide DNA demethylation that is comparable to what happens in the early embryo. Furthermore, they observed that cells respond to the loss of DNA methylation by an upregulation of TEs transcription during the first cell division only [START_REF] Walter | An epigenetic switch ensures transponson repressioin upon dynamic loss of DNA methylation in embryonic stem cells[END_REF]. Indeed, once in 2i/LIF for ~6 days, ESCs undergo a switch from a DNA methylation-based repression of TEs toward a histone modification-based control, which correlates with the downregulation of TEs. These results emphasize the capacity of ESCs to overcome the loss of DNA methylation, which mimics the physiological path of the early embryo.

The spatial architecture of chromatin is largely reorganized from the 2-cell to the epiblast.

At the zygote and 2-cell stage, heterochromatin is mainly observed surrounding the pre-nucleolar bodies. Firstly, the pre-nucleolar bodies merge to give rise to the nucleolus where transcription of the rDNA takes place. Constitution of pericentromeric heterochromatin blocks into chromocenters is visible at the 4-cell stage (Figure 25A). Chromocenter formation arise during the 2-cell state of the embryo and coincides with a burst of transcription for the major satellites (Figure 25B) [START_REF] Probst | A Strand-specific burst in transcription of pericentric satellites is required for chromocenter formation and early mouse development[END_REF]. It was further demonstrated that chromocenter formation required only reverse pericentric transcripts, but not the replication of the pericentromeric regions [START_REF] Casanova | Heterochromatin Reorganization during Early Mouse Development Requires a Single-Stranded Noncoding Transcript[END_REF]. Interestingly, the two parental pericentromeric regions display different timing of replication, and knock-down of major satellite transcription impacted differentially maternal and paternal heterochromatin organization but was in any case sufficient to impair progression toward 4-cell embryo [START_REF] Casanova | Heterochromatin Reorganization during Early Mouse Development Requires a Single-Stranded Noncoding Transcript[END_REF]. Furthermore, the spatial organization in itself is important for proper acquisition of histone modifications and heterochromatin regulation as forced-targeting of pericentromeric regions at the 2-cell stage to the nuclear lamina impaired the development toward blastocyst stage [START_REF] Jachowicz | Heterochromatin establishment at pericentromeres depends on nuclear position[END_REF]. Interestingly, DAXX accumulates to pericentromeric regions at the zygotic and 2-cell stage, specifically in the male pronucleus in a, respectively, ATRX, DPPA3 and PRC1-dependent manners (Figure 26A) [START_REF] La Fuente | ATRX contributes to epigenetic asymmetry and silencing of major satellite transcripts in the maternal genome of the mouse embryo[END_REF][START_REF] Arakawa | Stella controls chromocenter formation through regulation of Daxx expression in 2-cell embryos[END_REF][START_REF] Liu | SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin[END_REF]. Loss of Atrx results in the overexpression of major satellite transcripts in the zygote, whereas the loss of Dppa3 induces a lower levels of major satellite RNAs at the 2-cell state. DPPA3 protects the genome against DNA demethylation at the zygotic state, notably at IAP elements [START_REF] Nakamura | PGC7 / Stella protects against DNA demethylation in early embryogenesis[END_REF]. Depletion of PRC1 components or Daxx itself resulted in chromosomal instabilities and ultimately reduced the number of embryos reaching the blastocyst stage [START_REF] Liu | SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin[END_REF]. Totipotent and pluripotent cells display constitutively high levels of DNA damage signaling, such as high expression of γH2A.X. It is proposed that the high levels of γH2A.X in the early embryo might not be linked to actual DNA damages as γH2A.X foci are not colocalized with 53BP1 foci [START_REF] Ziegler-Birling | phosphorylated H2A . X during mouse preimplantation development in the absence of DNA damage[END_REF]. Intriguingly, the loss of DPPA3 induces 5hmC and γH2A.X accumulation in pre-implantation embryos, suggesting that loss of silencing of TEs in the embryo and zygotic genome activation must be tightly regulated to avoid detrimental accumulation of DNA damages (Figure 26B) [START_REF] Nakatani | Stella preserves maternal chromosome integrity by inhibiting 5 hmC-induced yH2AX accumulation[END_REF]. The DNA demethylation in 2i/LIF is notably visible at pericentromeric satellites [START_REF] Tosolini | Contrasting epigenetic states of heterochromatin in the different types of mouse pluripotent stem cells[END_REF]. Although DNA methylation is an important epigenetic mark associated with constitutive heterochromatin maintenance, ESCs can overcome the loss of this modification. As mentioned in the second part of the introduction, pericentromeric organization also rely on the histone modification H3K9me3. Intriguingly, these two marks are dependent on one another as deletion of DNMTs results in the diminution of H3K9me3 and deletion of Suv39H1/2 induces the loss of DNA methylation [START_REF] Saksouk | Redundant Mechanisms to Form Silent Chromatin at Pericentromeric Regions Rely on BEND3 and DNA Methylation[END_REF]. Indeed, H3K9me3 and H3K27me3 are nonoverlapping modifications because of steric hindrance (Figure 26C). However, loss of DNA methylation or H3K9me3 enhances the recruitment of polycomb and NuRD proteins at nearly all major satellites to maintain the heterochromatin state, resembling the polycomb recruitment observed in the early embryo [START_REF] Saksouk | Redundant Mechanisms to Form Silent Chromatin at Pericentromeric Regions Rely on BEND3 and DNA Methylation[END_REF][START_REF] Cooper | Targeting Polycomb to Pericentric Heterochromatin in Embryonic Stem Cells Reveals a Role for H2AK119u1 in PRC2 Recruitment[END_REF]. Saksouk et al. further shown that polycomb recruitment depended on the BEND3 protein (Figure 26D). Thus, heterochromatin state of pericentromeric regions can be maintained via multiple pathways according to the cellular state.

As developed in the second part of the introduction, heterochromatin is organized in multiple sub-nuclear compartments, one is the peripheral compartment with the LADs that are near the nuclear lamina. The dynamics of LADs during differentiation has been of great interest, although technical limitations made it difficult to explore within the pre-implantation embryo until very recently. The authors developed a single-cell DamID-LaminB1 approach with a low number of cells and performed it at four different time points from the zygote to the blastocyst [START_REF] Borsos | Genome-lamina interactions are established de novo in the early mouse embryo[END_REF]. They observed that zygotes displayed a LAD profile, whom 86% overlap with blastocystidentified LADs, yet at the 2-cell stage 42% of the zygotic LADs reposition toward the nuclear interior, from which the majority were previously defined as cLADs, suggesting a broad and unusual LAD reorganization at the 2-cell stage. From the 2-cell stage to the blastocyst, some LADs lose their association, whereas other gain DamID signal in a transcription-dependent manner, as it was observed for the differentiation of mESCs (Peric-Hupkes et al. 2010). Interestingly, there are less transcriptional changes between LAD and non-LAD at the 2-cell stage than in other cell types, possibly a consequence of the more open chromatin state in these totipotent cells. Furthermore, TADs formation in the early embryo was not dependent on the DNA replication, but LAD acquisition was dependent on the parent-of-origin genome, with paternal LADs being broader, suggesting a stronger association. The authors observed that proper TADs establishment in the early embryo was dependent on the histone demethylase KDM5B, suggesting a strong importance of chromatin modification in this mechanism. To date, no study has reported differences in chromatin architecture between 2i/LIF and serum-based mESCs culture conditions. Formation of TADs in the early embryo correlate with the ZGA but does not rely on transcription [START_REF] Hug | Chromatin Architecture Emerges during Zygotic Genome Activation Independent of Transcription[END_REF][START_REF] Du | Allelic reprogramming of 3D chromatin architecture during early mammalian development[END_REF]. Furthermore, zygote cells have a low level of chromatin organization, and organization into TAD and A and B compartments is progressively acquired during the early development with strengthening of these features [START_REF] Du | Allelic reprogramming of 3D chromatin architecture during early mammalian development[END_REF]. One could propose that at each division in the embryo, chromatin-related factors reinforce genome organization, thus preventing undesired conversion toward a more naïve state.

Pluripotent cells have an overall less condensed chromatin state, as it was described by assessing histone mobilities in ES and differentiated cells [START_REF] Meshorer | Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells[END_REF]). In concordance with the less compacted chromatin, mESCs display high global transcriptional levels and differences in nucleosome distribution with a more homogeneous repartition in pluripotent cells, suggesting less defined chromatin subcompartments [START_REF] Efroni | Global Transcription in Pluripotent Embryonic Stem Cells[END_REF][START_REF] Ricci | Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo[END_REF].

Interestingly, it is proposed that this peculiar chromatin state could be a crucial regulator of pluripotency. Indeed, it was observed in high-throughput screenings that conversion from pluripotent ESCs to totipotent 2cell-like cells in vitro was enhanced when knocking-down chromatin-related factors [START_REF] Rodriguez-Terrones | A molecular roadmap for the emergence of early-embryoniclike cells in culture[END_REF][START_REF] Ishiuchi | Early embryonic-like cells are induced by downregulating replicationdependent chromatin assembly[END_REF][START_REF] Cossec | SUMO Safeguards Somatic and Pluripotent Cell Identities by Enforcing Distinct Chromatin States[END_REF][START_REF] Wu | SETDB1-Mediated Cell Fate Transition between 2C-Like and Pluripotent States[END_REF]. The histone variant H3.3 is essential for embryonic development in mice, its lysine 27 residue being crucial for 2cell-state division [START_REF] Santenard | Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3[END_REF][START_REF] Lin | Histone variant H3.3 maintains a decondensed chromatin state essential for mouse preimplantation development[END_REF]. H3.3 was also implicated in the maintenance of proper chromatin state. Indeed, depletion of H3.3 renders reprogramming more efficient [START_REF] Fang | Global H3.3 dynamic deposition defines its bimodal role in cell fate transition[END_REF]. The SUMO-ligase UBC9 was also reported as a guardian of epigenetic state. Its removal allows ESCs to convert in a 2cell-like state, but also improves reprogramming efficiency of somatic cells into iPSCs [START_REF] Cossec | SUMO Safeguards Somatic and Pluripotent Cell Identities by Enforcing Distinct Chromatin States[END_REF]. These results suggest that totipotent, pluripotent and somatic cells possess differences in their chromatin organization, yet some factors used to maintain chromatin identity can play a role in different cellular states. Each state is not definitive, as reprogramming can be achieved. An emerging picture is that chromatin-related factors are crucial actors in maintaining cellular identities.

Many of the epigenetic-related factors mentioned in the second part of the introduction have been studied in different model organisms when possible. Intriguingly, most of these proteins were reported as embryonic lethal, although at different stages, such as the double knock-out of Dnmt3a and b, the H3K9-trimethylase Setdb1, the H3K9-dimethylases G9a and GLP [START_REF] Li | Targeted Mutation of the DNA Methyltransferase Gene Results in Embryonic Lethality[END_REF][START_REF] Okano | DNA Methyltransferases Dnmt3a and Dnmt3b Are Essential for De Novo Methylation and Mammalian Development[END_REF][START_REF] Dodge | Histone H3-K9 methyltransferase ESET is essential for early development[END_REF][START_REF] Tachibana | G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis[END_REF][START_REF] Tachibana | Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9[END_REF]. For the others H3K9trimethylases Suv39h1 and 2, the double knock-out resulted in an increase in embryonic lethality and the remaining mice developed but were smaller and infertile [START_REF] Peters | Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability[END_REF]. A similar observation was made for the knock-out of the Suv4-20h1 and 2 for which single KO did not present developmental defects but the double KO displayed perinatal lethality [START_REF] Schotta | A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin[END_REF].

OBJECTIVE OF THE THESIS

Objective of the thesis Evaluation of the role of Daxx in heterochromatin maintenance in pluripotent cells

About half of mammalian genomes are composed of repeated DNA sequences that must remain silenced heterochromatin at all time. It is now well-established that heterochromatin maintenance is essential for proper cell physiology. Defects in heterochromatin are commonly observed in different diseases and can even drive tumorigenesis.

In somatic cells, heterochromatin regions, both dispersed and pericentromeric are constitutively silenced via high levels of DNA methylation, H3K9me3-modifications and subsequent binding of HP1α. This peculiar heterochromatin organization arises during the early embryonic development. At the 2-cell stage, a major DNA repeats transcriptional burst, notably of the LINE1 family is proposed to facilitate genome-wide chromatin decompaction necessary for the zygotic genome activation. Interestingly, transcription of the major satellite repeats, composing the pericentromeric heterochromatin, is essential for the establishment of pericentromeric regionsclustering into chromocenters and mis-regulation of major satellite transcription at the 2-cell stage impairs further developmental capacity. Transcription of DNA repeats then decreases and is repressed from the blastocyst stage and later via high DNA methylation levels. Transcription of DNA repeats from the 2-cell to the blastocyst stage must then be tightly regulated independently of 5mC-based mechanisms.

Embryonic stem cells display lower levels of DNA methylation than differentiated cells. Indeed, DNA methylation marks are erased at the beginning of the embryonic development and are only re-established from the blastocyst stage from which are derived the embryonic stem cells.

A useful model for the study of the embryonic development is the embryonic stem cells which can be cultivated in vitro. ESCs display all the characteristics of pluripotent cells -self-renewal and generation of all the tissues of the organism. However, when in culture, ESCs tend to accumulate DNA methylation increasing the differences from their blastocyst counterparts along time. A new culture medium was described in 2008 by Ying et al. which is composed of 2 inhibitors (2i) for the GSK3β-and MEK3-pathways and convert the cells to a more physiological state with lower DNA methylation. DNA methylation can be further decreased upon addition of vitamin C into the 2i medium. Thus, pluripotent cells easily overcome the absence of DNA methylation, suggesting the existence of additional heterochromatin maintenance pathways in pluripotent cells. Some factors have already been described such as BEND3 and polycomb proteins at pericentromeres, and DAXX and SETDB1 for dispersed endogenous retroviruses.

Interestingly, DAXX localizes at PCH in the lowly methylated paternal genome in the first stages of development. However, genetic deletion of Daxx only impairs embryonic development at later stages, and Daxx -/-embryos could not develop to the blastocyst state, suggesting a strong importance of DAXX in transitioning the embryonic cells toward a pluripotent state, when chromocenter formation arise.

DAXX is the heterochromatin-specific chaperone of the histone variant H3.3. H3.3 is a peculiar histone variant as it is enriched in both euchromatin and heterochromatin regions. Its deposition at euchromatin is achieved via the HIRA complex. H3.3 is essential for ESC viability and regulates chromosomal stability of both in vitro ESCs and in the embryo, suggesting crucial heterochromatin-related functions. Furthermore, it provides the H3K9me3-modification at telomeric regions and help heterochromatin silencing at some endogenous retrovirus elements.

However, its role at major satellite repeats has mainly been envisioned as a transcriptional facilitator.

In the present work, I used ESCs as a model to study the role of DAXX in the maintenance of heterochromatin and gain deeper understanding of pericentromere regulation in pluripotent cells.

Introduction

Most of the mammalian genome is composed of DNA repeats. In mouse, the major satellites form Mbs-arrays of constitutive heterochromatin that surround centromeres 1 . Loss of silencing of satellite DNA is a hallmark of various cancer types and contributes to genomic instability. At the molecular level, constitutive heterochromatin exhibits epigenetic marks such as DNA methylation, H3K9me3 and HP1. Maintenance of H3K9me3 at major satellites rely on the SUV39H1/2 methyltransferases, whereas it strongly depends on the other K9me3 methylase SETDB1 at dispersed DNA repeats and telomeres 2, 3, 4 .

The pericentromeric heterochromatin (PCH) of different chromosomes tend to aggregate to form large DAPI-dense heterochromatin clusters, called chromocenters. Defect in satellite clustering has been described different pathologies such as Alzheimer's disease or breast cancers and is often associated with higher DNA damages or chromosome segregation defects 5, 6, 7, 8 . Chromocenters formation occurs at the 4-cell stage and expression of major satellite transcripts is necessary for the proper reorganization of pericentromeric heterochromatin proper developmental progression 9, 10 . Factors responsible for the PCH clustering remain largely unknown. Interestingly, DAXX, a chaperone of the histone variant H3.3 specifically binds to PCH during the first stages of development, yet loss of Daxx only impairs development at later stages 11, 12, 13 . However, the function of Daxx at PCH remains unknown in pluripotent cells.

In this paper, we decipher the role that DAXX plays at PCH in pluripotent embryonic stem cells. We found that DAXX is essential for ground-state ESCs survival. Furthermore, we observe that DAXX localizes to PCH to recruit SETDB1, facilitating heterochromatin reformation through H3K9me3 modification after DNA damages induced by active DNA demethylation.

Results

DAXX is essential for ES cell survival upon ground-state conversion.

To assess the role of Daxx in pluripotent cells, we generated a Daxx knock-out (KO) ES cell line. We targeted the exon 3 of the Daxx locus using the CRISPR/cas9 technology and confirmed the absence of DAXX mRNA and protein (Fig. 1A). The generated Daxx KO ES cell line did not present obvious changes in H3.3 protein levels. Consistent with previous reports, the lack of Daxx did not impair cell growth in serum-based ESCs culture condition 14 . In contrary of most epigenetic factor knockouts, the loss of Daxx did not impair the differentiation process.

No growth nor morphological defects could be detected in Daxx KO ESCs after 5 days of culture in presence of retinoic acid (Fig. 1B). Since Daxx contributes to alternative pathways of heterochromatin formation independently of DNA methylation, Daxx KO and WT ESCs were grown into 2i medium supplemented with Vitamin C 15,16 . In this medium, the level of 5mC is greatly reduced (Fig. S1A), allowing ESCs to reach the ground-state of pluripotency.

Consistent with previous reports, no growth defect was detected for Daxx KO lines after 4 days of conversion (Fig. 1B) 17 . However, prolonged culture into 2i+VitC medium induced a severe decreased in cell viability. Compared to the parental WT line, only 12.6% of Daxx KO ESCs remained after 8 days of culture in 2i+VitC (Fig. 1B). No surviving cells could be detected after 9 to 10 days of conversion. These results indicate an essential role for Daxx in the maintenance of pluripotent cell survival upon ground-state conversion.

To evaluate the effect of the loss of Daxx onto the transcriptional landscapes of Daxx KO ESCs, we performed an RNA sequencing experiment for each of the culture conditions.

Previous studies have demonstrated that Daxx binds to specific ERVs such as the IAP and ERVK elements, facilitating their silencing in ESCs 17 . As expected, we found that many ERV families were upregulated in Daxx KO ESCs in serum and ground-state conditions (Fig. 1C).

Several LINE1 elements, notably the L1Md T and L1Md A families, which have not been reported to be regulated by DAXX before were also upregulated in pluripotent Daxx KO ESCs (Fig. 1C,S1B). The role of Daxx on transcriptional silencing is not limited to interspersed repeats, we detected a strong upregulation major satellites RNA in both serum and groundstate condition (Fig. 1C,S1B). The effect of Daxx onto DNA repeats was restricted to repeats transcribed by RNA-PolII as the SINEs, transcribed by RNA-PolIII, were not affected. The misregulation of repeated element expression were significantly reduced upon retinoic acid differentiation supporting a specific role for DAXX in transcriptional silencing of DNA repeats when the level of DNA methylation is reduced. At the level of gene transcripts, the greatest differences between Daxx KO and WT cells were observed in pluripotent cells. With 1250 genes down-regulated and 1345 up-regulated ones (-1>Log(FC)>1; Pvalue<0.05), groundstate was the most affected condition (Fig. 1D). Interestingly, nearly all the transcriptional changes were abolished upon differentiation with only 125 down-regulated and 8 up-regulated genes (Log(FC)>1; Pvalue<0.05). Compared to WT, DaxxKO cells showed similar response to medium changes for both 2i+VitC and retinoic acid addition, suggesting the loss of viability observed in 2i+VitC is not caused by a conversion defect (Fig. S1C).

To understand the potential cause for the reduced cell survival of ground-state Daxx KO ESCs, we searched for transcriptional pathways specifically deregulated in ground-state.

Using a gene ontology algorithm for the most significantly mis-regulated genes (Pvalue<0.001), we revealed that Daxx KO cells presented an overexpression of the type II interferon response and apoptosis, which is coherent with the reduced cell viability, and also transcriptional pathways linked to DNA repair such as P53, Toll-like receptor and EGFR1 signaling (Fig. S1D) 18,19,20 . We noticed that among the most down-regulated pathways were the different types of DNA damage response (DDR) (Fig. 1E). We hypothesize that the reduced cell survival might arise from defects in DNA damage repair in the absence of Daxx.

DAXX accumulates at PCH upon ground-state conversion and DNA damages but is not part of DDR.

To better characterize the role of DAXX in ground-state ESCs, we decided to assess its localization by immunofluorescence. DAXX is a known partner of the PML nuclear bodies (NBs), membrane-less structures devoid of chromatin, forming hubs for post-translational modification 21 . As expected, in serum ESCs, DAXX is present as round foci of 0.3 µm in diameter on average that localizes at PML NBs (Fig. 2A, Fig. S2A). Intriguingly, upon ground-state conversion, DAXX also accumulates at heterochromatin H3K9me3-rich foci surrounded by PML, forming large structures of 1.1 µm in diameter (Fig. 2A, Fig. S2A). H3K9me3/DAPIrich foci in mouse correspond to chromocenters, formed by the clustering of pericentromeric regions of multiple chromosomes. We confirmed that the H3K9me3 regions were the pericentromeric heterochromatin by DNA FISH of the major satellites (Fig. 2B). DAXX relocalization at PCH is visible in half of ground state ESCs (Fig. 2C). Amongst those cells, 62% displayed only one PCH marked by DAXX, the remaining presenting 2 or ≥3 PCH-marked per nucleus. We conclude that upon ground-state conversion, DAXX relocalizes to major satellite repeats.

However, the presence of DAXX signal at a small fraction of chromocenters is different from the synchronized major satellite replication profile 1 . Thus, Daxx recruitment does not result simply from replicative heterochromatin maintenance but requires a specific stimulus. The loss of Daxx induced the up-regulation of multiple DNA repeats (Fig. 1C). Previous studies have reported that loss of heterochromatin silencing could lead to increased DNA damages 22 . Moreover, we detected that DNA repair proteins were transcriptionally down-regulated in the absence of Daxx (Fig. 1E). We thus decided to assess DNA damage signaling in Daxx KO cells. A key signaling factor of DNA damages is the phosphorylation of the H2AX histone variant, which happens at the early stages of DNA lesion recognition 23 . Because we detected a strong transcriptional upregulation of the major satellites and the spatial relocalization of DAXX, we decided to look for specific accumulation of γH2AX at pericentromeric heterochromatin. We counted the total number of γH2AX foci and evaluated if it was overlapping with a DAPI-rich focus. Interestingly, we observed that γH2AX foci appeared more at chromocenters in both WT and Daxx KO cells upon ground-state conversion (Fig. 2D). Hence, ground-state conversion induces DNA damage signaling at pericentromeric heterochromatin.

As increased levels of DNA damages were visible at chromocenters, we tested whether exogenously induced DNA damages could relocalize DAXX toward chromocenters. Hydroxyurea (HU) creates randomly positioned replication forks collapses that can ultimately generate double-strand breaks (DSBs). Random damages inflected with HU treatment resulted in DAXX and PML accumulation at DAPI-rich foci in 30% of the cells (Fig. 2E). We decided to artificially generate DSBs at PCH to see if that would lead to the recruitment of Daxx. We designed a sgRNA targeting major satellites (Fig. S2B). When we expressed the catalytically dead Cas9 (dCas9), there was no relocalization of DAXX nor PML, while expression of the active Cas9 induced high γH2AX signal at PCH and DAXX and PML accumulation (Fig. 2E,S2B). Yet, each nucleus displayed a limited number of structures like in ground-state. Recruitment of DAXX at pericentromeric regions can thus be triggered by DNA damages occurring at these locations.

Interestingly, γH2AX is only visible within the DAXX-PML PCH-foci in ~50% of the cases during ground-state conversion, suggesting a somehow distinct recruitment mechanism than direct DNA damage response (Fig. S2C). It was reported that specific recruitment of NBS1, which is part of the MRN complex, could induce DNA damage response in the absence of DNA lesions24. We thus artificially recruited NBS1 to major satellites using a Tale protein and while we observed the presence of γH2AX, there was no PML relocalization (Fig. 2F,S2D). As NBS1 failed to recruit Daxx, we propose that DAXX is not part of DDR but rather comes at a later post-repair step of heterochromatin reformation.

DAXX relocalizes to PCH upon DNA damages induced by active DNA demethylation.

A major epigenetic change arising upon ground-state conversion is the strong reduction in DNA methylation levels, particularly at the major satellite repeats 15, 25, 26 . The demethylation was shown to take place both passively and actively 27 . Prolonged culture in ground-state condition drastically impaired Daxx KO cell survival (Fig. 1B). To test whether the loss of DNA methylation was the cause of cell death, we treated the cells with the 5-azacytidine drug that blocks the DNMTs activity. We observed that the loss of Daxx impaired proper cell survival upon 4 days of 5-azacytidine treatment (Fig. 3A). Because DAXX relocalized to pericentromeric regions upon ground-state conversion, we tested Daxx localization in 5azacytidine-treated cells. Indeed, DAXX accumulated at H3K9me3-and DAPI-rich foci upon DNA demethylation and PML formed a giant shell around DAXX foci (Fig. 3B). DAXX-PML accumulation at PCH was visible in 29% of the cells (Fig. 3C).

We noted that upon ground-state conversion ESCs accumulated DNA damages at PCH and that artificially-induced DSBs led to DAXX recruitment (Fig. 2A,2E). Active DNA demethylation via the TET enzymes can ultimately result in DSBs 28 . Because loss of DNA methylation specifically induced the recruitment of DAXX toward pericentromeric regions, we wondered if DNA demethylation could be a cause of the observed DNA damages (Fig. 2D,3B). We decided to recruit the catalytic domain of the TET1 enzyme that converts 5-methylcytosine (5mC) into 5-hydroxymethyl-cytosine (5hmC), using a Tale strategy. Targeted recruitment of TET1-catalytic domain (TET1CD) toward major satellite repeats led to the specific upregulation of major satellite transcripts of 2.5-fold, but not other repeats such as the IAPEz, confirming the specific action of the Tale (Fig. 3C). Targeting of the TET1CD induced a reduction in 5mC signal at DAPI-dense foci (Fig. S3A,S3B). We then performed immunofluorescence to assess if our Tale-construct generated DNA damages at pericentromeric heterochromatin. We noted that the Tale accumulated at DAPI-rich regions as expected and it induced multiple γH2AX foci colocalizing with the Tale-flag signal, indicating enhanced DNA damages at PCH (Fig. 3D). To confirm that DNA damages generated by active DNA demethylation were the main cause of Daxx recruitment, we looked at DAXX and PML localization. We observed that DAXX-PML foci at PCH were visible in the Tale-TET1CD condition and not in the Tale-Δ control (Fig. 3F). Interestingly, specific activation of major satellite repeats in serum condition using a Tale-MajSat-VP64 resulted in strong increase in major satellites RNAs as well as γH2AX foci and DAXX-PML accumulation (Fig. S3D,S3E,S3F). However, as ground-state conversion leads to the silencing of pericentromeric regions, it suggests that the DNA damages arising in our cells do not result from increased activation (Fig. S1C). Hence, we conclude that accumulated DNA damages visible at major satellites can result from the DNA demethylation of pericentromeric regions.

DAXX maintains heterochromatin organization in pluripotent cells.

Changes in major satellites transcription often correlates with impaired spatial organization of chromocenters 5 . Since we observed an upregulation of major satellite repeats in Daxx KO cells, we decided to evaluate the organization of the pericentromeric heterochromatin clusters (Fig. 1C). We performed DNA FISH experiments against the major satellites and counted the number of foci. We observed that the number of chromocenters was increased in the absence of Daxx in both serum and ground-state ESCs (Fig. 4A). In agreement with the absence of phenotype in the Daxx KO cells upon differentiation, we detected the same number of major satellite foci in the differentiated condition. The increased of foci number was correlated with the diminution of the sizes of major satellites foci (Fig. S4A).

Because the spatial organization of pericentromeric heterochromatin was affected, we then assessed the molecular state of these regions. We realized an MNAse digestion of serum ESCs and observed that the lack of Daxx induced an enhanced chromatin accessibility (Fig. S4C). To understand the increased in chromatin accessibility, we expressed a GFP-HP1α in both cell lines and performed FRAP experiments to measure the speed of fluorescence recovery (Fig. 4B). We observed that the half-recovery time was significantly lower in both serum and ground-state conditions in the absence of Daxx, suggesting a faster protein dynamic which agrees with the MNAse accessibility results (Fig. 4B,S4C). However, the mobile fraction of GFP-HP1α remained constant, suggesting that the same amount of proteins was bound to chromatin (Fig. S4B). These results indicate that Daxx is necessary for the proper organization of pericentromeric heterochromatin in pluripotent cells.

Interestingly, HP1 was reported to be able to undergo liquid-liquid phase-separation (LLPS) 29, 30 . LLPS corresponds to particular biophysical properties including fusion-fission events as well as a dynamical behavior. Strom et al. observed that chromocenters boundaries displayed enhanced HP1α variance, indicating a heterochromatin barrier impeding the diffusion of proteins, which is coherent with the tension surface exhibited by LLPS-driven biocondensates. We reasoned that higher HP1α recovery rate might arise from altered heterochromatin barrier. We thus re-examined the FRAP data, looking at the intensity variation along the duration of the movie (Fig. 4C). In WT cells, we noted that the variance levels were low in the nucleoplasm and only increased at chromocenters, reaching the highest peaks at the borders of chromocenters as previously reported. However, the variance from HP1α in Daxx KO cells displayed a strikingly different pattern, with an increased variance levels in the nucleoplasm (Fig. 4C). We measured the variance at non-bleached chromocenters and observed a strong reduction of the peaks at the borders, suggesting a lower heterochromatin barrier. This effect was visible in both serum and ground-state conditions, however we noted that variance peaks in ground-state were higher for both WT and Daxx KO ESCs (Fig. 4C).

Coherently with the increased dynamic of HP1α, we note that the HP1α variance at chromocenters borders is lower in Daxx KO ESCS, which could indicate that the loss of Daxx directly or indirectly affects heterochromatin barriers formed by LLPS capacities of HP1α.

To evaluate the role of Daxx at major satellites, we specifically recruited DAXX using a Tale in Daxx KO ESCs in serum. The recruitment of DAXX induced a strong clustering of the major satellite signal, visible as a decrease in the number of foci by DNA FISH and a correlated increase in foci sizes (Fig. 4D). Furthermore, targeted recruitment of DAXX can overcome the normal spatial clustering as its recruitment in WT cells resulted in the same clustering phenotype (Fig. S4D). Overall, our data indicate that Daxx is sufficient to induce a spatial reorganization of pericentromeric heterochromatin.

DAXX recruits SETDB1 and mediates chromocenter clustering via H3.3K9me3 modification.

We next wondered what the function of DAXX at PCH could be. PCH are regions particularly enriched in H3K9me3 (Fig. 2A). SETDB1 is an H3K9me3 methyl-transferase that localizes within PML NBs 31 . We thus decided to assess whether SETDB1 also accumulated at pericentromeric heterochromatin upon DNA demethylation. In WT cells, we observed a strong signal of SETDB1 at DAPI-rich regions marked with DAXX and PML presence (Fig. 5A).

However, in the absence of Daxx, we did not observe SETDB1 recruitment nor PML to DAPIrich chromocenters but noticed that SETDB1 still localized at PML NBs (Fig. 5A,S5A). As DAXX is the heterochromatin-specific chaperone of H3.3, we knocked-down H3.3 expression and noted a strong reduction in PML-PCH relocalization upon DNA demethylation (Fig. S5B).

We conclude that DAXX accumulates at PCH regions and then recruits PML and SETDB1 in an H3.3-dependent manner.

The main histone H3K9me3 methyl-transferases acting at pericentromeric heterochromatin are the SUV39H1/2 enzymes 22, 32 . However, because SETDB1 is another H3K9me3 methyl-transferase, we wondered if it was recruited for its methylase activity. We took advantage of Suv39H1/2 double knock-out (Suv39dKO) ESCs in which H3K9me3 could only result from SETDB1 activity. Strikingly, Suv39dKO cells presented no growth defects phenotype upon ground-state conversion (data not shown). We confirmed that matched WT ESCs presented DAXX-PML accumulation at H3K9me3-rich PCH (data not shown). We observed that while no H3K9me3 was observed in serum, specific foci were present in groundstate which colocalize with PML NBs at major satellites (Fig. 5B). The presence of H3K9me3 signal at PCH of Suv39dKO ESCs was reported in ground-state condition, yet it remained a mystery 26 . Hence, SETDB1 can be recruited to PCH in a DAXX-dependent manner and exerts its tri-methylase activity.

As the targeted recruitment of DAXX induced a strong major satellites clustering, we asked if SETDB1 was recruited by the Tale-DAXX construct. While no SETDB1 signal was observed upon recruitment of the Tale-Δ, the Tale-DAXX displayed a strong SETDB1 signal (Fig. S5C). We noted that H3.3 was important for the pericentromeric localization of DAXX and wondered if H3.3 was important for SETDB1 recruitment as well (Fig. S5B). The DAXX-H3.3 interaction is mediated by the histone-binding domain of DAXX. It was reported that the tyrosine 222 residue in murine DAXX was responsible for the H3.3-binding 14 . We recruited a DAXX mutant in which we replaced the tyrosine 222 residue by an alanine (Y222A) and observed that the recruitment of SETDB1 was abolished (Fig. S5C). Next, to assess whether DAXX-mediated SETDB1 recruitment generated an enzymatic activity, we recruited our Tale constructs into the Suv39dKO ESCs. The recruitment of a Tale protein to the major satellite sequences did not increase the levels of H3K9me3 signal. However, upon DAXX targeting, we observed a strong accumulation of H3K9me3 signal by immunofluorescence (Fig. 5C). As expected from the absence of SETDB1 recruitment, targeting of the DAXXY222A mutant did not lead to any tri-methylase activity (Fig. 5C). The DAXX-mediated recruitment of SETDB1 induced H3K9 tri-methylation, that we propose to be specific of H3.3. However, to rule out a possible effect of any H3K9 tri-methylation that could arise at canonical H3, we recruited SUV39H1 and SETDB1 to major satellites. Although both constructs resulted in high H3K9me3, H3K9me3-rich chromocenters appeared as small foci different from the action of DAXX (Fig. 5C). Because the recruitment of SUV39H1 and SETDB1 results in a different phenotype from the DAXX recruitment, we reasoned that the function of DAXX-SETDB1 might rely on the modification of H3.3.

To evaluate the function of H3.3 in DAXX-mediated clustering, we first measured the clustering phenotype induced by DAXXY222A mutant recruitment. H3.3 binding-deficient DAXX did not induce any clustering of major satellites foci, like the recruitment of SUV39H1 or SETDB1 (Fig. 5D). We then depleted the endogenous pool of H3.3 using two specific siRNAs and overexpressed individual H3.3 constructs. H3.3-binding to DAXX is mediated by its glycine 90 residue and when it is replaced by the methionine residue, which is the H3.1 residue (G90M), it completely abolishes the H3.3-DAXX interaction 33 . Depletion of endogenous H3.3 impaired the Tale-DAXX-mediated major satellite clustering (Fig. 5E). We confirmed that our H3.3 constructs were expressed at the same levels (Fig. S5E). Complementation of the loss of endogenous H3.3 by a WT H3.3 rescued the tale-DAXX clustering, why complementation with the H3.3G90M mutant led to the symmetrical effect of the Daxx mutant without any clustering. Interestingly, when we expressed the H3.3K9A mutant, which cannot be posttranslationally modified on its lysine 9, we observed an absence of pericentromeric clustering (Fig. 5E). From these results, we propose that the effect of Daxx on the pericentromeric heterochromatin is mediated through the specific tri-methylation of H3.3K9 via the methyltransferase SETDB1.

Discussion

In the present work, we describe a novel role of the H3.3-chaperone DAXX in pluripotent cell maintenance of pericentromeric heterochromatin.

Upon conversion toward a DNA hypomethylated condition, we notice that DAXX massively accumulates to chromocenters and drives PML recruitment (Fig. 2A,3A). Intriguingly, previous reports described DAXX recruitment to pericentromeric regions in the early stage of embryonic development 12, 13 . In the mouse embryo, DAXX-recruitment is controlled by diverse proteins such as DPPA3 and PRC1. Furthermore, upon loss of DNA methylation in pluripotent cells, PRC1 components and BEND3 are targeted to chromocenter to induce H3K27me3 34, 35 . It remains to understand whether similar protein network regulates DAXX relocalization upon ground-state conversion. However, as PRC1 proteins and BEND3 appear at most chromocenters, notably in DnmtKO cells, whereas DAXX only localize at 1 to 3 PCH per nucleus, we think that it may be recruited by alternative mechanisms. Indeed, DAXX is increasingly recruited to ERV repeats in DnmtKO ESCs 17 . Interestingly, it was reported that ERV elements are present interspersed between major satellite repeats 36 . Hence, we propose that DAXX might be recruited to these particular ERV sequences imbedded at chromocenters (PCH-ERVs), possibly after DNA damages.

Targeted recruitment of TET1 to major satellites could generate γH2AX foci accumulation, demonstrating that active DNA demethylation can lead to DNA damages (Fig. 3D). We similarly observe that ground-state conversion drives the increase of the number of damaged chromocenters (Fig. 2D). Since exogenously induced DNA damages and hypomethylation of chromocenters lead to DAXX relocalization, we conclude that DAXX is recruited to major satellites following DNA damage (Fig. 2A,2E,3A). Interestingly, targeting recruitment of NBS1, although sufficient to induce γH2AX accumulation, could not drive DAXX relocalization, suggesting that DAXX may not act within the DNA damage response (DDR), but rather at later steps to reform the heterochromatin state of the locus (Fig. 2F). Active DNA demethylation-induced DNA damages is often resolved via coordinated action of base-excision repair (BER), TDG and TET to avoid DSBs 37, 38, 39 . However, CRISPR/Cas9 targeting leads to DSBs and can induce DAXX accumulation, although in serum, with higher levels of DNA methylation. We observe discrete foci of γH2AX at chromocenters, which could indicate the presence of rare DSBs. Future studies are required to assess the role of DAXX in DDR.

Chromocenters are highly enriched in H3K9me3-modification induced by SUV39H1/2 enzymes 2, 22, 40 . Remarkably, we demonstrate that DAXX recruits SETDB1 to perform H3K9 tri-methylation activity at PCH, an action different from mere binding of SETDB1 or SUV39H1 (Fig. 5A, 5B, 5C). Since KDM enzymes have been implicated in DNA repair pathway choice and repair completion, we propose that the function of SETDB1 is to generate H3.3K9me3 41, 42, 43 . More experiments are necessary to deeply understand the molecular events occurring at DSBs related to loss and gain of H3.3K9me3. Furthermore, the absence of Daxx impairs chromocenter organization (Fig. 4A). It is possible that the same mechanism we describe upon loss of DNA methylation takes place in serum. However, it remains to better characterize whether the binding of DAXX and SETDB1 in the presence of DNA methylation might be transient or localized at specific loci such as PCH-ERVs. Moreover, a recent understanding of chromocenter organization came from the observations that HP1α could undergo LLPS 29, 30 .

Intriguingly, we notice that the absence of Daxx impairs the dynamics and alters heterochromatin barrier formation, suggesting a direct or indirect change in LLPS capacities of HP1α (Fig. 4B,4C). Since we show that the major satellite clustering induced by DAXX relies on the binding of H3.3 and recruitment of SETDB1 (Fig. 5C,5D), there are perhaps undescribed differences of HP1α binding to H3K9me3 and H3.3K9me3.

Finally, we describe an essential role of DAXX for cell survival in prolonged DNA hypomethylated conditions. We further observe that the function of DAXX at chromocenters is to induce H3.3 deposition and SETDB1 recruitment. Intriguingly, the loss of SETDB1 prevents cell survival in both pluripotent and leukemic somatic cells 44, 45 . In that direction, future work should inquire whether this essential DAXX-mediated SETDB1 recruitment is limited to pluripotent cells and assess the vital role of DAXX upon chronic loss of DNA methylation.

Methods

Cell lines

Feeder-free E14 mESCs were used for most experiment excepts otherwise notified. E14 mESCs were kindly provided by Pablo Navaro (Pasteur Institute). DaxxKO cell line was constructed using CRISPR/cas9 editing in E14 mESCs. Guide-RNA was designed using the online CRISPOR tool. Oligos were designed with a BbsI site on 5' to clone them into the pSpCas9(BB)-2A-Puro(pX459) v2.0 vector 46 . Guide was designed carrying two guanines on 5' of the sequence to avoid off-target effects, as previously described 47 . Feeder-free Suv39HdKO and corresponding WT R1 ESCs were obtained from Antoine Peters 48 .

Culture conditions

Pluripotent cells were cultured either in a serum condition, defined as follow -DMEM (Gibco), supplemented with 10% of ESC-certified FBS (Gibco), 2-mercaptoethanol (0.05mM, Gibco), Glutamax (Gibco), MEM non-essential amino acids (0.1mM, Gibco), Penn-Strep (100units/mL, Gibco) and LIF (1000units/mL,) for serum condition. Serum-cultivated cells were grown on 0.1% gelatin-coated plates or stem cell plates (Stem Cell technology) at 37°C with 5% CO2.

Medium was changed every day and cells were passaged every 2 to 3 days. The other culture condition is the chemically defined serum-free 2i + VitC condition defined as follow -Neurobasal -DMEM/F-12 (50 -50, Gibco) medium, supplemented with N2 and B-27 supplements (Gibco), BSA fraction V (0.05%Gibco), 1-thioglycerol (1.5x10 -4 M, Sigma) and ESGRO 2 inhibitors (GSK3i and MEKi) and LIF (Merck). Vitamin C (L-ascorbic acid 2phosphate, Sigma) was added at a concentration of 100µg/mL [START_REF] Blaschke | Vitamin C induces Tet-dependent DNA demethylation and a blastocystlike state in ES cells[END_REF]). Cells were grown on 0.1% gelatin-coated plates. Medium was changed daily. Cells were passed every 2 days at 1 -4 ratio for the first passage then at a 1 -6 ratio. Additional media used were constituted as follow -serum medium supplemented with either 5-azacytidine (2µM, Sigma) or Hydroxy-Urea (2mM, Sigma). Differentiation of mESCs was done by LIF removal for the first 24h. Then, non-LIF medium was supplemented with retinoic acid (10 -6 M,) for 4 days. For cell growth quantification, cells were counted at each passage for at least 3 different experiments.

Vectors and transfections

Cells were harvested using trypsin and one million was plated in a 0.1% gelatin-coated plate and transected with 2.5µg of DNA or 90ng of RNA using the Lipofectamine 2000 reagent (Thermo), following manufacturer's protocol. SiRNAs used are SMARTpools of 4 different siRNAs targeting mRNA of interest (Horizon Discovery). TALE vectors were constructed using previously described methods 49 . A TALE specific DNA binding domain targeting Major Satellite repeats was created by the modular assembly of individual TALE repeats inserted into a backbone vector containing TALE-Nrp1-VP64 previously described 50 . The BamHI-NheI fragment containing VP64 was replaced by PCR products encoding the DAXX protein or corresponding DAXX mutants.

Catalytically dead Cas9 was inserted into the pSpCas9(BB)-2A-Puro(pX459) v2.0 vector to generate a SpdCas9-2A-Puro 46 .

Target sequences for the TALE repeat domains and Cas9 associated guides RNA are listed in Supplementary table 1.

RNA extraction for RNAseq or RT-qPCR

Total RNA was extracted using RNeasy extraction kit (Qiagen) according with manufacturer's protocol including DNAseI treatment for 15min at room temperature (Qiagen). Complementary DNA were generated from 1µg of RNA using the Maxima first strand cDNA synthesis kit (Thermo Fisher), with a second round of DNAseI from the Maxima kit for 15min. Real-time qPCR was carried out using a LightCycler 480 instrument (Roche) and the LightCycler 480 SYBR green master mix (Roche). The qRT-PCR primers used in this study are listed in supplementary table. Three independent biological repeats were obtained for each sample.

For RNAseq experiment, RNA quality was assessed using the Agilent 2100 bioanalyzer.

Libraries were prepared using oligo(dT) beads for mRNA enrichment, then fragmented and reverse transcribed using random hexamers primer. After adaptor ligation, the double-stranded cDNA is completed through size selection of 250-300bp and PCR amplification, then quality of the library is assessed by the Agilent 2100 bioanalyzer. Sequencing was performed in 150bp paired-end reads using an Illumina sequencer platform.

RNA-seq Mapping and Processing

FASTQ files generated by paired end sequencing were aligned to the mouse genome using bowtie2 v2.2.6 (parameters ---local --threads 3; mm9 genome build). Mapped RNA-seq data was processed using tools from the HOMER suite (v4.8). SAM files were converted into tag directories using 'makeTagDirectory' (parameters --format sam -sspe; Referencewill be uploaded to GEO upon publication). Genomic intervals which extended beyond the end of the chromosomes was removed using 'removeOutOfBoundsReads.pl'. bigWig browser track files were generated using 'makeUCSCfile' (parameters --fsize 1e20 -strand + -norm 1e8). For gene expression analysis, read depths were quantified for all annotated refseq genes using analyzeRepeats.pl (parameters -rna mm9 -strand both -count exons -rpkm -normMatrix 1e7 -condenseGenes). For repeat analysis, read coverage was quantified for each repeat and then condensed to a single value for each named entry (parameters -repeats mm9 -strand bothrpkm -normMatrix 1e7 -condenseL1). Read depths were then corrected for the number of instances of each repeat prior to expression analysis.

Expression Analysis

Quantified RNA-seq data was processed using the limma package (R/Bioconductor; Referencewill be uploaded to GEO upon publication) 51 . Following the addition of an offset value (1 RPKM) to each gene or repeat, data was normalised across all samples using 'normalizeBetweenArrays' with method='quantile'. Fold-changes and p-values for differential expression of genes and repeats were determined using empirical Bayes statistics. Briefly, data was fit to a linear model using 'lmFit' and specified contrasts were applied using 'makeContrasts' and 'contrasts.fit'. Data was processed using the 'topTable' function with adjust.method="BH" (Benjamini-Hochberg multiple-testing correction). Differential expression was defined as log2 fold change ≤-1 or ≥ 1 and an adjusted p-value of ≤ 0.01. Three biological replicates for each condition represent independently cultured pools of cells.

Data visualization

Heatmaps and boxplots were generated using Prism GraphPad. Histograms were drawn using either Prism GraphPad or Excel. Volcano plots were generated using the plot function of R.

Pathway analysis was performed using the EnrichR tool 52, 53 .

Immunofluorescence

Murine ESCs were harvested with trypsin (Gibco) and plated for 4-6h onto 0.1% gelatin-coated glass cover slips. Cells were fixed with 4% paraformaldehyde for 10min at room temperature, then rinsed three times with PBS. Cells were permeabilized with 0.1X triton for 12min at room temperature, then rinsed three times with PBS. Blocking was done in 3% BSA solution for 30min at room temperature. All incubations with primary antibodies were performed for either 1h at room temperature or overnight at 4°C with the following antibodies for H3K9me3 (Active Motif, 1:1000), DAXX (Santa Cruz, 1:500), yH2AX (Abcam, 1:1000) and SETDB1 (Proteintech, 1:100). Incubation with secondary antibodies (fluorescently labeled anti-mouse or anti-rabbit, 1:1000) were performed for 1h at room temperature. Mounting was performed using ProLong Diamond with DAPI mounting media (Thermo). Antibodies are listed in supplementary table.

For 5mC staining, cells were fixed with 4% PFA for 10 min at room temperature, then permeabilized using 1% BSA, 0.5% triton X-100 for 30 min at room temperature. Cells were washed 3 times in PBS before incubation with RNAse A (20mg/mL) for 1h at 37°C. Cells washed 3 times in PBS, then denatured in 4M HCl for 10 min at 37°C. Slides are neutralized by extensive washes in PBS, then blocked in 1% BSA, 0.1% Triton X-100 for 30 min at room temperature before incubation with 5mC antibody (Diagenode, 1:1000) overnight at 4°C. After 3 washes in PBS, cells were blocked in 1% BSA, 0.1% Triton X-100 for 30 min at room temperature before incubation with secondary antibody for 1h at room temperature. Mounting was performed using ProLong Diamond with DAPI mounting media (Thermo).

Fluorescent in situ Hybridization

Murine ESCs were harvested with trypsin (Gibco) and plated for 4-6h onto 0.1% gelatin-coated glass cover slips. Cells were fixed with 4% paraformaldehyde (PFA) for 10min at room temperature, then rinsed three times with PBS. Cells were permeabilized with 0.5X triton for 12min at room temperature, then rinsed three times with PBS. Cells were briefly washed in 2X SSC, then treated with RNAseA (100µg/mL, Sigma) for 1h at 37°C. Cells were briefly washed in 2X SSC, then denatured by serial 2min incubation into 70,90 and 100% ethanol. Cover slips were air dried for 15min. Cover slips are incubated with 200nM of PNA probe, placed for 10min at 95°C for denaturation, then placed for 1h at room temperature in the dark for hybridization.

Cover slips were washed twice in 2X SSC 0.1%Tween-20 for 10min at 60°C. Cover slips were immerged at room temperature in 2X SSC 0.1%Tween-20 for 2 min, then in 2X SSC for 2min and 1X SSC for 2min. Mounting is performed using ProLong Diamond with DAPI mounting media (Thermo).

Dot blot experiments

DNA extraction was performed using the Wizard genomic DNA extraction kit (Promega).

Genomic DNA (1µg) was then denatured in 0.1M NaOH for 10min at 95°C before neutralization with 1M NH4OAc on ice for 5min. DNA samples were spotted on a nitrocellulose membrane.

Blotted membrane was washed in 2X SSC and dried at 80°C for 5min before UV cross-linking at 120,000µJ/cm2. Membrane was then blocked using PBS, 5% milk 0.1% tween for 30min at room temperature. Membrane was incubated with 5mC antibody (Diagenode, 1:1000) for 3h at room temperature. After 3 washes of 10min each in PBS, membrane was blocked again for 30min and then incubated with secondary anti-HRP antibody for 1h at room temperature.

Membrane was washed 3 times for 10min in PBS and visualized by chemiluminescence with ECL Plus.

Chromatin accessibility

Chromatin was extracted from desired ES cell lines and digested with MNAse (NEB) for different times and stopped before migration onto 1% agarose gel and subsequent Southern blotting with major satellites probe. Southern blot hybridization was performed with DIGlabeled PCR products and DIG wash-and-block kit (Roche) before visualization with CSPD (Roche).

Fluorescent Recovery After Photobleaching

Murine ESCs transfected with GFP-HP1α were harvested with trypsin (Gibco) and plated for 4-6h onto 0.1% gelatin-coated glass live-cell Nunc slides (Thermo). FRAP experiment was carried with an LSM 800 confocal microscope (Zeiss). The 488nm laser was used to bleach and acquire GFP signal. 1 image was taken before a bleach pulse of 5ms. Bleaching area was set to target a single pericentric domain. Images were acquired every second during 35s postbleach.

Image acquisition and analysis

Images were obtained with an inverted Nikon Ti Eclipse widefield microscope using a 60X immersion objective and LED sources. Z-stacks images were taken and then deconvoluted using a custom ImageJ deconvolution script. Quantifications of images were performed using custom Icy scripts. Number of major satellite foci were counted using a custom Icy script and performed onto the medium focal plane. FRAP analysis was performed using a FRAP analysis ImageJ Jython script, that generated FRAP curves and the associated half-recovery time and mobile fraction parameters. GFP-HP1α variance along time was obtained from ImageJ analysis using z-projection along time. Quantification of heterochromatin barriers were performed using a 2µm line across individual non-bleached chromocenter borders, for which the variance intensity along time was

Western blotting

Total protein extracts were prepared in RIPA buffer with protease inhibitor cocktail (Roche).

Proteins were separated by electrophoresis in 8-15% poly-acrylamide gels then transferred onto nitrocellulose membranes. Membranes were incubated in ponceau then washed in PBS, 0.1% tween. Membranes were blocked in PBS, 0.1% tween, 5% milk for 30min at room temperature before incubation with primary antibody overnight at 4°C. After 3 washed in PBS, 0.1% tween, membranes were incubated with secondary HRP-conjugated antibody for 1h at room temperature. Membranes were washed 3 times in PBS, 0.1% tween and visualized by chemoluminescence using ECL Plus. Other results concerning Daxx at PCH

Supplementay table 1

List of primers used for RT-qPCR

Name Sequence exp-GAPDH-F ATCACCATCTTCCAGGAGCGAG exp-GAPDH-R GACCCTTTTGGCTCCACCCTTC exp-mDAXX-F GAGGGCGAGAGACAGAAGAG exp-mDAXX-R GTCGTCGTCATCATCATCGT exp-L1Tf-F CAGCGGTCGCCATCTTG exp-L1Tf-R CACCCTCTCACCTGTTCAGACTAA exp-IAPLTR1_Mm-F GCGCTGACAGCTGTGTTCTA exp-IAPLTR1_Mm-R GGACGTGTCACTCCCTGATT exp-IAPEz_int-F CAGTGCGCAGACTCATTCAT exp-IAPEz_int-R TATTCTACCGGAGCGTGGAC exp-Mervl-F CTCTACCACTTGGACCATATGAC exp-Mervl-R GAGGCTCCAAACAGCATCTCTA exp-MajSat-F TGGAATATGGCGAGAAAACTG exp-MajSat-F AGGTCCTTCAGTGGGCATTT Oligo used

Methods

Cell lines

Feeder-free E14 mESCs were used for most experiment excepts otherwise notified. E14 mESCs were kindly provided by Pablo Navaro (Pasteur Institute). DaxxKO cell line was constructed using CRISPR/cas9 editing in E14 mESCs. Guide-RNA was designed using the online CRISPOR tool (site). Oligos were designed with a BbsI site on 5' to clone them into the pSpCas9(BB)-2A-Puro(pX459) v2.0 vector (Ran et al. 2013). Guide was designed carrying two guanines on 5' of the sequence to avoid off-target effects, as previously described (Cho et al. 2014).

Culture conditions

Pluripotent cells were cultured either in a serum condition, defined as follow -DMEM (Gibco), supplemented with 10% of ESC-certified FBS (Gibco), 2-mercaptoethanol (0.05mM, Gibco), Glutamax (Gibco), MEM non-essential amino acids (0.1mM, Gibco), Penn-Strep (100units/mL, Gibco) and LIF (1000units/mL,) for serum condition. Serum-cultivated cells were grown on 0.1% gelatin-coated plates or stem cell plates (Stem Cell technology) at 37°C with 5% CO2. Medium was changed every day and cells were passaged every 2 to 3 days. The other culture condition is the chemically defined serum-free 2i + VitC condition defined as follow -Neurobasal -DMEM/F-12 (50 -50, Gibco) medium, supplemented with N2 and B-27 supplements (Gibco), BSA fraction V (0.05%Gibco), 1-thioglycerol (1.5x10 -4 M, Sigma) and ESGRO 2 inhibitors (GSK3i and MEKi) and LIF (Merck). Vitamin C (L-ascorbic acid 2-phosphate, Sigma) was added at a concentration of 100µg/mL [START_REF] Blaschke | Vitamin C induces Tet-dependent DNA demethylation and a blastocystlike state in ES cells[END_REF]. Cells were grown on 0.1% gelatin-coated plates. Medium was changed daily. Cells were passed every 2 days at 1 -4 ratio for the first passage then at a 1 -6 ratio.

Additional media used were constituted as follow -serum medium supplemented with either 5azacytidine (2µM, Sigma) or Hydroxy-Urea (2mM, Sigma). Differentiation of mESCs was done by LIF removal for the first 24h. Then, non-LIF medium was supplemented with retinoic acid (10 - 6 M,) for 4 days. For cell growth quantification, cells were counted at each passage for at least 2 different experiments.

Vectors and transfections

Cells were harvested using trypsin and one million was plated in a 0.1% gelatin-coated plate and transected with 2.5µg of DNA or 90ng of RNA using the Lipofectamine 2000 reagent (Thermo), following manufacturer's protocol. SiRNAs used are SMARTpools of 4 different siRNAs targeting mRNA of interest (Horizon Discovery). TALE vectors were constructed using previously described methods (Zhang et al. 2011). A TALE specific DNA binding domain targeting Major Satellite repeats was created by the modular assembly of individual TALE repeats inserted into a backbone vector containing TALE-Nrp1-VP64 previously described (Therizols et al. 2014). The BamHI-NheI fragment containing VP64 was replaced by PCR products encoding the DAXX protein or corresponding DAXX mutants. CDS of the different proteins were amplified by PCR from cDNAs obtained after RNA extraction of serum E14 mESCs.

Catalytically dead Cas9 was inserted into the pSpCas9(BB)-2A-Puro(pX459) v2.0 vector to generate a SpdCas9-2A-Puro (Ran et al. 2013).

RNA extraction for RNAseq or RT-qPCR

Total RNA was extracted using RNeasy extraction kit (Qiagen) according with manufacturer's protocol including DNAseI treatment for 15min at room temperature (Qiagen). Complementary DNA were generated from 1µg of RNA using the Maxima first strand cDNA synthesis kit (Thermo Fisher), with a second round of DNAseI from the Maxima kit for 15min. Real-time qPCR was carried out using a LightCycler 480 instrument (Roche) and the LightCycler 480 SYBR green master mix (Roche). The qRT-PCR primers used in this study are listed in supplementary table. Three independent biological repeats were obtained for each sample. For RNAseq experiment, RNA quality was assessed using the Agilent 2100 bioanalyzer. Libraries were prepared using oligo(dT) beads for mRNA enrichment, then fragmented and reverse transcribed using random hexamers primer. After adaptor ligation, the double-stranded cDNA is completed through size selection of 250-300bp and PCR amplification, then quality of the library is assessed by the Agilent 2100 bioanalyzer. Sequencing was performed in 150bp paired-end reads using an Illumina sequencer platform.

RNA-seq Mapping and Processing

FASTQ files generated by paired end sequencing were aligned to the mouse genome using bowtie2 v2.2.6 (parameters ---local --threads 3; mm9 genome build). Mapped RNA-seq data was processed using tools from the HOMER suite (v4.8). SAM files were converted into tag directories using 'makeTagDirectory' (parameters --format sam -sspe; Reference -will be uploaded to GEO upon publication). Genomic intervals which extended beyond the end of the chromosomes was removed using 'removeOutOfBoundsReads.pl'. bigWig browser track files were generated using 'makeUCSCfile' (parameters --fsize 1e20 -strand + -norm 1e8). For gene expression analysis, read depths were quantified for all annotated refseq genes using analyzeRepeats.pl (parameters -rna mm9 -strand both -count exons -rpkm -normMatrix 1e7 -condenseGenes). For repeat analysis, read coverage was quantified for each repeat and then condensed to a single value for each named entry (parameters -repeats mm9 -strand both -rpkm -normMatrix 1e7 -condenseL1). Read depths were then corrected for the number of instances of each repeat prior to expression analysis.

Expression Analysis

Quantified RNA-seq data was processed using the limma package (R/Bioconductor; Referencewill be uploaded to GEO upon publication) (Team 2017). Following the addition of an offset value (1 RPKM) to each gene or repeat, data was normalised across all samples using 'normalizeBetweenArrays' with method='quantile'. Fold-changes and p-values for differential expression of genes and repeats were determined using empirical Bayes statistics. Briefly, data was fit to a linear model using 'lmFit' and specified contrasts were applied using 'makeContrasts' and 'contrasts.fit'. Data was processed using the 'topTable' function with adjust.method="BH"

(Benjamini-Hochberg multiple-testing correction). Differential expression was defined as log2 fold change ≤-1 or ≥ 1 and an adjusted p-value of ≤ 0.01. Three biological replicates for each condition represent independently cultured pools of cells.

Lamina-associated domains-genes analysis

List of LADs from embryonic stem cells were obtained from previously published datasets [START_REF] Peric-Hupkes | Molecular Maps of the Reorganization of Genome-Nuclear Lamina Interactions during Differentiation[END_REF]. Constitutive LADs were defined as ubiquitous lamina-association, and facultative LADs as regions interacting with the nuclear lamina only in embryonic stem cells.

Comparison of LAD-genes and interLAD-genes expression was done using the Galaxy platform.

Immunofluorescence

Murine ESCs were harvested with trypsin (Gibco) and plated for 4-6h onto 0.1% gelatin-coated glass cover slips. Cells were fixed with 4% paraformaldehyde for 10min at room temperature, then rinsed three times with PBS. Cells were permeabilized with 0.1X triton for 12min at room temperature, then rinsed three times with PBS. Blocking was done in 3% BSA solution for 30min at room temperature. All incubations with primary antibodies were performed for either 1h at room temperature or overnight at 4°C with the following antibodies for H3K9me3 (Active Motif, 1:1000), DAXX (Santa Cruz, 1 -500), yH2AX (Abcam, 1:1000), H3K9me2 (Active Motif, 1:1000), LaminB1 (Abcam, 1:1000) and ATRX (Santa Cruz, 1:1000). Incubation with secondary antibodies (fluorescently labeled anti-mouse or anti-rabbit, 1:1000) were performed for 1h at room temperature. Mounting was performed using ProLong Diamond with DAPI mounting media (Thermo). Antibodies are listed in supplementary table.

Fluorescent in situ Hybridization

Murine ESCs were harvested with trypsin (Gibco) and plated for 4-6h onto 0.1% gelatin-coated glass cover slips. Cells were fixed with 4% paraformaldehyde for 10min at room temperature, then rinsed three times with PBS. Cells were permeabilized with 0.5X triton for 12min at room temperature, then rinsed three times with PBS. Cells were briefly washed in 2X SSC, then treated with RNAseA (100µg/mL, Sigma) for 1h at 37°C. Cells were briefly washed in 2X SSC, then denatured by serial 2min incubation into 70,90 and 100% ethanol. Cover slips were air dried for 15min. Cover slips are incubated with 200nM of PNA probe, placed for 10min at 95°C for denaturation, then placed for 1h at room temperature in the dark for hybridization. Cover slips were washed twice in 2X SSC 0.1%Tween-20 for 10min at 60°C. Cover slips were immerged at room temperature in 2X SSC 0.1%Tween-20 for 2 min, then in 2X SSC for 2min and 1X SSC for 2min. Mounting is performed using ProLong Diamond with DAPI mounting media (Thermo).

Image acquisition and analysis

Images were obtained with an inverted Nikon Ti Eclipse widefield microscope using a 60X immersion objective and LED sources. Z-stacks images were taken and then deconvoluted using a custom ImageJ deconvolution script. Quantifications of images were performed using custom

Western Blotting

Total protein extracts were prepared in RIPA buffer with protease inhibitor cocktail (Roche).

Proteins were separated by electrophoresis in 8-15% poly-acrylamide gels then transferred onto nitrocellulose membranes. Membranes were incubated in ponceau then washed in PBS, 0.1% tween. Membranes were blocked in PBS, 0.1% tween, 5% milk for 30min at room temperature before incubation with primary antibody overnight at 4°C. After 3 washed in PBS, 0.1% tween, membranes were incubated with secondary HRP-conjugated antibody for 1h at room temperature.

Membranes were washed 3 times in PBS, 0.1% tween and visualized by chemoluminescence using ECL Plus. We thus decided to look for 2C-like genes that were either up or down-regulated specifically in 2Clike as previously described, such as Dppa3, the Zscan4 cluster, Tdpoz4 or the EIF4a1-like genes (Gm5662 and Gm2016) (Figure 27A). Remarkably, upon ground-state conversion, Daxx KO ESCs presented a transcriptional profile that resembled to 2C-like cells (Figure 27B). Interestingly, this effect is lost in serum-based culture or upon differentiation (Figure 27B). Many of the specifically up-regulated genes in 2C-like cells show a strong down-regulation upon differentiation as they correspond to pluripotency-related factors [START_REF] Rodriguez-Terrones | A molecular roadmap for the emergence of early-embryoniclike cells in culture[END_REF]. We confirmed the effect on transcription for 3 genes (Gm5662, Spz1 and Zscan4) and the MERVL ERV family (Figure 27C). These results suggest that DAXX contributes to the repression of the 2C-like transcriptional program. Interestingly, in accordance with our data, DAXX has been found in a large siRNA screen for factors that could enhance the conversion toward a 2C-like state

RESULTS

Deeper characterization of DAXX function in

DAXX contributes to the radial positioning of chromocenters

In embryonic stem cells, chromocenters are randomly distributed throughout the nucleoplasm. However, it has been reported that among the proteins observed at pericentromeric heterochromatin were components of the nuclear lamina [START_REF] Saksouk | Redundant Mechanisms to Form Silent Chromatin at Pericentromeric Regions Rely on BEND3 and DNA Methylation[END_REF]. Interestingly, cultivating the cells in the 2i/LIF medium can result in a more central positioning of chromocenters than that observed in serum-based medium [START_REF] Saksouk | Redundant Mechanisms to Form Silent Chromatin at Pericentromeric Regions Rely on BEND3 and DNA Methylation[END_REF]. Saksouk et al. proposed that the changes in radial positioning of chromocenters could result from differences in LAMINB binding.

We developed an automated approach to quantify the intensity of the major satellite DNA FISH signal at the periphery of the nucleus, such as described previously (Figure 28A) [START_REF] Saksouk | Redundant Mechanisms to Form Silent Chromatin at Pericentromeric Regions Rely on BEND3 and DNA Methylation[END_REF]. We confirmed this central repositioning in our 2i/LIF with vitamin C medium (Figure 28B, Figure 28C). Interestingly, the longer the conversion toward the ground-state of pluripotency, the more central is the repositioning as we noted from 4 to 10 days in culture. All further experiments in ground-state were conducted at 4 days of conversion. Thus, ground-state conversion triggers a repositioning of chromocenters away from the nuclear lamina.

Because the loss of Daxx impacted the spatial organization of major satellites (Figure 4A article), we wondered whether there were changes in their radial positioning as well. Using our automated approach, we observed that Daxx KO ESCs displayed an altered radial localization of major satellites. Indeed, in both serum and ground-state conditions, the lack of Daxx resulted in a more peripheral positioning of pericentromeric clusters (Figure 28D, Figure 28E). Conversion toward the ground-state of pluripotency induced a slight central relocalization of major satellites foci, although it remained more peripheral compared to WT ESCs. Our results indicate that in addition to the altered organization of major satellites, the loss of Daxx induces a peripheral localization of chromocenters. Interestingly, it has been reported that high levels of H3K9me3 on a transgene locus was associated with its peripheral localization [START_REF] Bian | β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications[END_REF]. Since Daxx KO ESCs presented H3K9me3 signal at PCH as WT cells (Figure 28D), our results suggest that subtle differenced, perhaps through the loss of tri-methylation of lysine 9 of H3.3, could impact the spatial localization of chromocenters.

DAXX contributes to peripheral heterochromatin organization and silencing of LADs

As already mentioned, major satellites are enriched in H3K9me3 histone modification, mainly deposited by the SUV39H1/2 enzymes [START_REF] Peters | Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability[END_REF]. However, the nuclear periphery and the lamina-associated domains are mainly enriched in H3K9me2 [START_REF] Kind | Single-Cell Dynamics of Genome-Nuclear Lamina Interactions[END_REF]. As major satellites signal was increased at the nuclear periphery, we next asked how the presence of H3K9me3-rich chromocenters could impact the peripheral heterochromatin compartment (Figure 28C, Figure 28E). We detected no differences in H3K9me2 distribution in ground-state compared to serum-based medium in WT ESCs (Figure 29A). The loss of Daxx did not impact H3K9me2 peripheral localization in serum-based medium, suggesting that peripheral localization of chromocenters is not challenging for proper establishment of H3K9me2. Intriguingly, conversion of Daxx KO cells toward the ground-state of pluripotency impaired H3K9me2 distribution (Figure 29A). Instead of being uniformly distributed along the periphery, H3K9me2 appeared as individual patches. We specifically measured the intensity levels of this histone modification at the nuclear periphery and calculated the standard deviation for the signal intensity of each pixel and quantified the signal heterogeneity in the different conditions. We confirmed the alteration of H3K9me2 signal distribution in ground-state in the absence of Daxx, suggesting a particular feature of ground-state ESCs (Figure 29B). It remains to understand if the peripheral localization of chromocenters in ground-state is implicated in the H3K9me2-ring disruptions.

Next, we wondered if the altered H3K9me2 distribution could result from defects in the nuclear lamina. We performed immunofluorescences assays of the LAMINB1 constituent of the lamina.

We did not observe any changes of LAMINB1 signal in the absence of Daxx in serum-based or ground-state medium (Figure 29C). Our results suggest that the absence of Daxx results in impaired distribution of H3K9me2 at the nuclear periphery independently of lamina integrity defects upon ground-state conversion. It would be informative to assess whether additional nuclear lamina proteins such as NET are still present upon ground-state conditions [START_REF] Czapiewski | Anchoring a Leviathan: How the nuclear membrane tethers the genome[END_REF].

Furthermore, it remains to assess if there are local or global losses of H3K9me2 onto corresponding chromatin.

The histone modification H3K9me2 is highly enriched in LADs [START_REF] Kind | Single-Cell Dynamics of Genome-Nuclear Lamina Interactions[END_REF]). Because we observed that H3K9me2 signal appeared as patches at the periphery, we decided to assess the transcription of the genes specifically located within LADs. We analyzed the RNA-seq experiment performed in WT and DaxxKO ESCs and classified the genes into LAD or inter-LAD (iLAD) categories based on DamID definitions for mESCs [START_REF] Peric-Hupkes | Molecular Maps of the Reorganization of Genome-Nuclear Lamina Interactions during Differentiation[END_REF]. Association with the nuclear lamina is cell-type dependent for some genes, and these regions are thus defined as facultative LADs (fLADs), whereas the regions always lying at the nuclear periphery are named constitutive LADs (cLADs) [START_REF] Peric-Hupkes | Molecular Maps of the Reorganization of Genome-Nuclear Lamina Interactions during Differentiation[END_REF]. We noted that the distribution of the Log2(FoldChange (DaxxKO/WT)) for iLAD-genes presented a high dispersion in ground-state condition compared to serum-based and differentiated cells, which is due to the higher number of differentially transcribed genes in the hypomethylated condition (Figure 30A). The distribution of Log2(FoldChange (DaxxKO/WT)) for both cLAD and fLAD genes were centered to 0 in both differentiated cells and serum-based ESCs, indicating no preferential bias of transcriptional changes. Statistical difference between iLAD and fLAD in differentiated cells is mainly due to the transcriptional changes of fLAD genes upon differentiation.

However, in the hypomethylated DNA condition, we observed a strong bias toward the up-regulation of LAD-genes, both constitutive and facultative (Figure 30A). We deeply investigated some of the LAD-genes displaying differential expression and choose 6 genes for which we designed specific qPCR primers (Figure 30B). We selected five genes belonging to the cLAD category (Adh1, Naip6, Casp1, Casp4 and Casp12) and one from the fLAD category (Samd5), which is activated upon differentiation. We then confirmed the loss of silencing effect for these 6 LAD-genes by RT-qPCR in ground-state compared to serum conditions (Figure 30C).

Intriguingly, we noted that 4 of the 5 constitutive LAD-genes were up-regulated in WT groundstate cells, compared to serum condition, suggesting some changes in peripheral heterochromatin silencing upon ground-state conversion. We observed a strong upregulation of all the 6 genes from 12 to more than 500-fold. Our results indicate that the absence of Daxx results in the upregulation of LAD-genes upon hypomethylation of pluripotent cells. Interestingly, we noticed an alteration of the H3K9me2 profile (Figure 29A). However, these are only correlative data and the causal link between these two observations remains to be confirmed. Furthermore, additional experiments are required to see whether it correlates with delocalization of LAD-genes away from the nuclear lamina. Altogether, our data suggest that the absence of Daxx impairs chromocenter organization in pluripotent cells, whereas peripheral heterochromatin is only altered upon ground-state conversion, suggesting an additional mechanism at play when DNA methylation levels are low.

4. LINE1 elements upregulation contributes to the accumulation of DNA damages in the absence of Daxx

Upon ground-state condition Daxx-deficient ESCs displayed higher rate of γH2AX foci at chromocenters, we thus decided to assess the total level of DNA damage signaling (Figure 2D article). We performed Western blot to quantify the amount of γH2AX in WT and Daxx KO ESCs in serum or upon ground-state conversion. Remarkably, we detected a strong increase in γH2AX levels upon ground-state conversion (Figure 31A). However, the total amount of γH2AX in WT cells was similar between serum and ground-state conditions (Figure 31A). As the difference between WT and Daxx KO ESCs seemed greater in total γH2AX signaling compared to the number of chromocenters displaying γH2AX foci, we reasoned that DNA damage signaling might arise from additional sources.

We demonstrated that activation of major satellites could enhance DNA damage signaling at chromocenters (Figure S3D article). As the absence of Daxx resulted in the up-regulation of multiple families of DNA repeats, we asked whether it could be implicated in the increased DNA damage signaling we observed (Figure 31A). Among the up-regulated DNA repeats was the LINE1 Tf family, which was increased both in serum and ground-state conditions (Figure 1C article). We observed that the absence of Daxx impaired the silencing of LAD genes (Figure 30A).

Because LINE1 are enriched in LADs, we quantified the number of elements present in constitutive or facultative LADs [START_REF] Meuleman | Constitutive nuclear lamina -genome interactions are highly conserved and associated with A / T-rich sequence[END_REF]. The LINE1 Tf family (L1Md_T) was enriched in LADs with almost 48% of the elements present in total LADs (Figure 31B). As a euchromatic control, SINEB1 appeared largely absent from LADs with only 17% of elements.

Loss of silencing of LINE1 elements have often been observed together with increased DNA damages, yet it remains mainly correlative [START_REF] Belgnaoui | Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells[END_REF]. To assess whether the elevated DNA damage signaling was indeed a consequence of heterochromatin derepression, we designed specific sgRNAs targeting the LINE1 Tf family, as this family is already strongly up-regulated in the serum condition. We transfected the cells with a dCas9 fused to the VPR trans-activator to induce the specific upregulation of LINE1 Tf. We detected an up-regulation of the same order of magnitude in the WT and the Daxx KO cells from 2-to 4-fold increase assessed via RT-qPCR (Figure 31C). We then assessed the levels of yH2AX in these conditions by Western-blot and observed that while the dCas9 without a sgRNA did not present any signal, it was strongly increased in LINE1 upregulated conditions (Figure 31D). These results show that the up-regulation of DNA repeats, both pericentromeric or dispersed such as LINE1 contributes to γH2AX signal accumulation. We observed that in ground-state condition active DNA demethylation at PCH could generate DNA damages and DAXX recruitment. Our data suggest that the observed accumulation of γH2AX might results from the loss of silencing of heterochromatin. It yet remains to understand how the activation of LINE1 elements leads to DNA damages and it will be interesting to assess whether it is the transcription per se or the action of LINE1 ORF1 endonuclease. Daxx signal was embedded within a 'giant' Pml NB. Because we observed that the specific targeting of Daxx to major satellite repeats was sufficient to induced Pml relocalization, we wanted to assess the role of Pml. We thus designed a guide RNA targeting the exon 1 of Pml and generated a Pml KO ES cell line (Figure 32A). We confirmed the absence of Pml protein via immunofluorescence The absence of Pml did not impact the cell survival in serum-based or differentiated cells (Figure 32B). However, the loss of Pml prevented cell survival in hypomethylated conditions.

Characterization of the role of PML in

Indeed, upon 4 days of 5-azacytidine treatment or 8 days of ground-state conversion resulted in a strong reduction of the number of cells (Figure 32B). Thus, the absence of PML resulted in the same reduced cell survival upon DNA hypomethylation than the absence of Daxx (Figure 1B article). We confirmed that the impaired cell survival did not result from an alteration of the ground-state conversion and measured the transcription of specific ground-state markers (such as Prdm14, Dnmt3L and Nanog) and detected no difference in Pml KO cells (Figure 32C). We detected the same transcriptional up-regulation for DNA repeats such as LINE1 Tf (Figure 32C). We also confirmed that the absence of Pml did not alter transcriptional response from retinoic acid differentiation and observed the expected activation of Nrp1, Ptn and Nestin and the silencing of Oct4 and Dppa2 (Figure 32D). Our result points toward an essential role of PML in pluripotent cells upon the loss of DNA methylation.

DAXX accumulation at PCH is independent of PML

We observed the relocalization of both DAXX and PML to pericentromeric regions upon DNA hypomethylation and deletion of Pml or Daxx resulted in impaired cell survival in these conditions (Figure 1B article, 2A article, 3B article, Figure 32B). To assess the causal link between DAXX and PML, we converted the Pml KO ESCs into the ground-state condition. We demonstrated that the loss of Daxx prevented Pml accumulation at chromocenters (Figure S5B article). Moreover, while the loss of Daxx did not prevent the formation of PML NBs, the deletion of Pml dispersed DAXX signaling throughout the nucleoplasm, and only few small foci were detectable (Figure 33A). We noted that some of the DAXX signal localized at chromocenters in serum-based medium, although it remained quite rare. Interestingly, upon conversion into groundstate, we noted that DAXX still strongly accumulated at PCH regions despite the absence of Pml, indicating that Pml does not recruit nor confine Daxx (Figure 33A). We quantified the number of DAXX-accumulation at PCH and observed from 1 to 3 DAXX structures were visible per nucleus, similar to the WT cells (Figure 33B). Our data indicate that DAXX is firstly recruited to PCH regions and then recruits PML and that loss of Pml does not impair DAXX accumulation. (Figure 35A). However, upon 2i/VitC conversion, we observed that H3K9me2 signal appeared as patches at the periphery that resembled the signal from Daxx-deficient ESCs (Figure 35A). As previously reported, we assessed the fluorescence homogeneity signal. We noted that in Pmldeficient cells, the signal was strongly heterogeneously distributed compared to WT cells (Figure 35B). We confirmed the integrity of the nuclear lamina via immunofluorescence of LaminB (Figure 35C). Hence, the loss of Pml results in an impaired H3K9me2-signal distribution, which highly resemble to the phenotype observed for Daxx KO cells. The histone modification H3K9me2 is highly enriched in LADs [START_REF] Kind | Single-Cell Dynamics of Genome-Nuclear Lamina Interactions[END_REF]). Because we observed that H3K9me2 signal appeared as patches at the periphery, a phenotype shared with Daxx KO ESCs, and that the absence of Daxx resulted in the upregulation of LAD-genes, we decided to assess the transcription of some genes specifically located within LADs. We performed qRT-PCR experiments onto ground-state and serum ESCs for six genes belonging to constitutive (Naip6,Adh1,Casp1,Casp4,Casp12) or facultative (Samd5) LADs categories We observed a slight transcriptional increase for five of the six investigated genes (Figure 36 A major partner of DAXX is ATRX. ATRX belongs to the SWI-SNF chromatin remodeler family and is thus able to bind to DNA. ATRX has been shown to be important for H3.3 deposition at telomeres in mESCs [START_REF] Goldberg | Distinct Factors Control Histone Variant H3.3 Localization at Specific Genomic Regions[END_REF]. Interestingly, ATRX is commonly observed at chromocenters in different cell types, including mESCs [START_REF] Wong | ATRX interacts with H3.3 in maintaining telomere structural integrity in pluripotent embryonic stem cells[END_REF][START_REF] Mcdowell | Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes[END_REF]. DAXX-binding to ATRX can be abrogated by a mutation in its Atrx-binding domain at the N-terminal [START_REF] Hoelper | Structural and mechanistic insights into ATRXdependent and -independent functions of the histone chaperone DAXX[END_REF]. We constructed a Daxx mutated onto the tyrosine 130 (Y130A). We targeted DAXX to major satellites using a Tale-fusion protein as previously described and we performed immunoFISH to quantify the number of major satellite foci (Figure 37A).

Intriguingly, the recruitment of DaxxY130A was sufficient to induce a major satellite clustering in Daxx KO ESCs in serum (Figure 37B). Targeted recruitment of DAXXY130A was also sufficient to mediate PCH clustering in WT ESCs (Figure 37C). These results are in good agreement with the report that ATRX was implicated chromocenter clustering, yet only in differentiated cells and not in ESCs [START_REF] Marano | ATRX contributes to MECP2mediated pericentric heterochromatin organization during neural differentiation[END_REF]. Our results indicate that the direct interaction between DAXX and ATRX is not required for DAXX-mediated major satellite clustering.

However, as ATRX was reported to bind to PCH regions, we decided to assess whether the loss of either DAXX or PML would prevent this localization. We thus performed immunofluorescence experiments of ATRX in our cells (Figure 37D). In WT cells, ATRX localizes at some or all DAPI-dense chromocenters in 70% of the cells (Figure 37E). In WT cells, ATRX often localizes also at PML NBs. Interestingly, the deletion of either Daxx or Pml results in the same phenotype with more cells displaying ATRX binding at PCH (Figure 37E). The absence of Daxx did not prevent ATRX localization within PML NBs in our cells, contrarily to previous observations [START_REF] Delbarre | DAXXdependent supply of soluble (H3.3-H4) dimers to PML bodies pending deposition into chromatin[END_REF]. These experiments indicate that the ATRXrecruitment toward PCH is independent of DAXX and PML. We observed that ATRX bindingdeficient DAXX was able to induce PCH clustering, however, it remains to assess whether the total absence of Atrx is dispensable for DAXX-mediated clustering (Figure 37B, Figure 37C).

Post-translational regulation of DAXX

We observed that deletion of either Daxx or Pml resulted in similar phenotypes with alterations of pericentromeric heterochromatin organization (Figure 4A article, Figure 34B). [START_REF] Sahin | Oxidative stress-induced assembly of PML nuclear bodies controls sumoylation of partner proteins[END_REF][START_REF] Lin | Role of SUMO-Interacting Motif in Daxx SUMO Modification, Subnuclear Localization, and Repression of Sumoylated Transcription Factors[END_REF]. We constructed a DAXX mutant deficient for the C-terminal SIM.

We recruited the Tale-MajorSatellite-DAXXΔSIM and evaluated its clustering capacity by DNA FISH (Figure 38A). We noted that the loss of its C-terminal SIM motif did not impair major satellite clustering in both WT and Daxx KO ESCs (Figure 38B, Figure 38C). However, we noticed that the clustering phenotype was more pronounced in WT cells compared to Daxxdeficient cells for which the DAXXΔSIM-mediated clustering was statistically different from DAXX clustering, suggesting that the effect in WT cells might be enhanced by the recruitment of endogenous DAXX. Our results suggest that the SIM motif, hence interaction with SUMOylated proteins is not essential for Daxx-mediated clustering, though it may favor this process.

Furthermore, not only DAXX can bind to SUMOylated partners, but it can also be SUMOylated itself onto its lysines. We then constructed a Tale-DAXX mutant for which we replaced the 9 most SUMOylated lysines with arginine [START_REF] Lin | Role of SUMO-Interacting Motif in Daxx SUMO Modification, Subnuclear Localization, and Repression of Sumoylated Transcription Factors[END_REF]. Recruitment of DAXX9KR

did not induce the clustering of major satellites in neither the Daxx KO nor the WT cells (Figure 38D, Figure 38E). These data indicate that post-translational modifications of DAXX seem important for its ability to mediate PCH-clustering. We next wondered whether our Tale-DAXX constructs were active and could recruit PML or H3.3. We thus performed immunofluorescence assays for PML and H3.3 in the Tale-DAXX, DAXXY222A and DAXXΔSIM conditions (Figure 39A). In the Tale-Δ condition, we noted that the H3.3 signal was dispersed throughout the nucleoplasm, with only some small foci sometime visible. Overall, the H3.3 signal was mostly absent from DAPI-rich chromocenters (Figure 39A).

In the Tale-DAXX condition, H3.3 was strongly recruited to virtually all DAPI-rich PCH, whereas the Tale-DAXXY222A, which cannot bind H3.3, did not recruit H3.3 to PCH as expected (Figure 39B). The Tale-Δ did not alter PML localization which only appeared as small and round nuclear bodies (Figure 39A). We observed that Daxx recruited PML which formed like 'giant' shells around major satellite foci, similar to what is visible upon ground-state conversion (Figure 39A, Figure 2A article). However, the number of 'giant' PML NBs at PCH was seen in most of the cells (~80%), but not at all PCH with 3 or more PCH foci in ~40% of the cells (Figure 38B).

Intriguingly, the recruitment of DAXXY222A did not recruit PML, suggesting that the DAXX-H3.3 interaction might be important for the DAXX-PML interaction (Figure 39A). The Tale-DAXXΔSIM that should not bind PML did not recruit PML, however, it still recruited H3.3, which correlates with the clustering phenotype observed with this construct (Figure 38E, Figure 39B, Figure 39C). We thus conclude DAXX tethering to major satellite is sufficient to recruit PML and to induce a visible accumulation of H3. WT vs DaxxKO We observe that in ground-state condition, the loss of Daxx induces transcriptional changes that resemble to the 2C-like ESCs, suggesting that DAXX represses the 2C-like program (Figure 27B, Figure 27C). To confirm the role of DAXX in maintaining pluripotent heterochromatin state, it would be relevant to assess the exact conversion rate toward 2C-like state using an MERVL-GFP reporter, as previously published, and at different time of the conversion. Many factors were reported to enhance the conversion of pluripotent ESCs toward totipotent 2C-like ESCs. Such factors include the PRC1 complex, the EP400-TIP60 complex which is implicated in histone modifications and EP400 has been proposed to be an alternative H3.3 chaperone for regulatory region depositions, the methyl-transferase SETDB1 and the E3 SUMO ligase UBC9 [START_REF] Rodriguez-Terrones | A molecular roadmap for the emergence of early-embryoniclike cells in culture[END_REF][START_REF] Wu | SETDB1-Mediated Cell Fate Transition between 2C-Like and Pluripotent States[END_REF][START_REF] Cossec | SUMO Safeguards Somatic and Pluripotent Cell Identities by Enforcing Distinct Chromatin States[END_REF][START_REF] Pradhan | EP400 Deposits H3.3 into Promoters and Enhancers during Gene Activation[END_REF]. These factors are implicated in chromatin regulation, highlighting that chromatin is a crucial feature of cell type identity. Interestingly, Daxx was reported in a 2C-like repressor siRNA screen, which is in good agreement with our RNA-seq results, and points toward a function for DAXX as a regulator of chromatin state identity [START_REF] Rodriguez-Terrones | A molecular roadmap for the emergence of early-embryoniclike cells in culture[END_REF]. Interestingly, DAXX and H3.3 interact with PRC1 and SETDB1 in different contexts (Figure 5C article, S5D article) [START_REF] Liu | SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin[END_REF].

List of primers used for RT-qPCR

Name Sequence exp-Gm5662-F GCACAGCCTCCTTACACCAT exp-Gm5662-R AAAAACAGGCGCAGAGGTAA exp-Spz1-F CTGCAGGAAGCAGATCACTG exp-Spz1-R GCTTCTGCTTGGTGGATAGC exp-Zscan4-F GAGATTCATGGAGAGTCTGACTGATGAGTG exp-Zscan4-R GCTGTTGTTTCAAAAGCTTGATGACTTC exp-MERVL-F CTCTACCACTTGGACCATATGAC exp-MERVL-R GAGGCTCCAAACAGCATCTCTA exp-Samd5-F CTGAGCAAACCCCCATACTC exp-Samd5-R ATACGCCCCTTTCCAGTCTT exp-Naip6-F CGACATCTTGGACGATGAAA exp-Naip6-R GTTGTCCAGGGCTTGAAAGA exp-Adh1-F TGTGGCTGACTTCATGGCTA exp-Adh1-R AAGTCAGGACGGTACGGATG exp-Casp1-F CGTCTTGCCCTCATTATCTGC exp-Casp1-r CACCATCTCCAGAGCTGTGA exp-Casp4-F TCTCACTGAGGTATGGGGCT exp-Casp4-R GTCTGGTGTTCTGAGAGTGCA exp-Cas12-F CGAACACGTCTGGCTCTCAT exp-Cas12-R TGTCTCCATTTCCTGAGCTGT exp-L1Orf1-F AGAGAACACAAACTGCAGCC exp-L1Orf1-R GCGTTGTTCCCGTTCTTGTA exp-Prdm14-F GTTTCCGTGTTCTCTCTGCC exp-Prdm14-R GCTTGTTCAGGCTGGAAGAC exp-Dnmt1-F CGGGTCTACTGCAGTTCCAT exp-Dnmt1-R GGTACAGGGTCTCGTTCACA exp-Dnmt3L-F GCGGCCCTTCTTCTGGATAT exp-Dnmt3L-R TAGTCTCTGCCACGGACATC exp-Oct4-F CGAGAACAATGAGAACCTTC exp-Oct4-R CCTTCTCTAGCCCAAGCTGAT exp-Nanog-F AAAGGATGAAGTGCAAGCGG exp-Nanog-R CTTCCAGATGCGTTCACCAG exp-L1Tf-F CAGCGGTCGCCATCTTG exp-L1Tf-R CACCCTCTCACCTGTTCAGACTAA exp-Nrp1-F GGCCGATTCAGGACCATAC exp-Nrp1-R ATAGACCACAGGGCTCACCA exp-Ptn-F CGAGTGCAAACAGACCATGA exp-Ptn-R GGCGGTATTGAGGTCACATT exp-Nestin-F GAACTCTCGCTTGCAGACAC exp-Nestin-R AGAAGGATGTTGGGCTGAGG exp-Dppa2-F CTGCAATTTCTTGCTGGACA exp-Dppa2-R TTTTTCTTTGCACGGCTTCT exp-GAPDH-F GACCCTTTTGGCTCCACCCTTC exp-GAPDH-
These results suggest that there might be unidentified functions of H3.3 in regulating the cellular identity of ESCs and that histone variants might contribute to preventing conversion of pluripotent cells toward a more naïve totipotent state during development.

Intriguingly, the E3 SUMO-ligase UBC9 was implicated in the maintenance of pluripotent chromatin state, but also in the maintenance of somatic chromatin states [START_REF] Cossec | SUMO Safeguards Somatic and Pluripotent Cell Identities by Enforcing Distinct Chromatin States[END_REF]. It is remarkable that similar mechanisms can be employed by both pluripotent and somatic stem cells.

In that direction, it would be interesting to assess the role of DAXX in the reprogramming of induced pluripotent stem cells.

Mechanism of DAXX-PML recruitment at PCH upon loss of DNA methylation.

In murine embryonic stem cells, like in most cell types, DAXX localizes within PML NBs which are normally devoid of chromatin. We here show that upon DNA hypomethylated conditions, DAXX is recruited to major satellite repeats and appears as large foci at some chromocenters (Figure 2A article, 3B article). Interestingly, it has previously been reported that DAXX could bind to pericentromeric regions during DNA hypomethylated conditions [START_REF] Baumann | Loss of maternal ATRX results in centromere instability and aneuploidy in the mammalian oocyte and Preimplantation embryo[END_REF][START_REF] Liu | SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin[END_REF][START_REF] Arakawa | Stella controls chromocenter formation through regulation of Daxx expression in 2-cell embryos[END_REF]. During the early development, the paternal nucleus displays lower levels of 5mC and correspondingly higher levels of 5hmC. At this stage of development, DAXX is recruited to pericentromeric regions of the paternal nucleus via different proteins. Indeed, the depletion of either Dppa3, Atrx or PRC1 prevented proper localization of DAXX [START_REF] Baumann | Loss of maternal ATRX results in centromere instability and aneuploidy in the mammalian oocyte and Preimplantation embryo[END_REF][START_REF] Liu | SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin[END_REF][START_REF] Arakawa | Stella controls chromocenter formation through regulation of Daxx expression in 2-cell embryos[END_REF]. It would be interesting to assess whether the same proteins are at play in pluripotent cells. and DAXX [START_REF] Rowe | KAP1 controls endogenous retroviruses in embryonic stem cells[END_REF][START_REF] Wolf | The KRAB zinc finger protein ZFP809 is required to initiate epigenetic silencing of endogenous retroviruses[END_REF][START_REF] Matsui | Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET[END_REF][START_REF] He | The Daxx/Atrx Complex Protects Tandem Repetitive Elements during DNA Hypomethylation by Promoting H3K9 Trimethylation[END_REF]. The DNA binding activity of these proteins is mediated by the zinc finger domains of the KRAB-ZF proteins.

Remarkably, murine chromocenters are not only composed of major satellites. In fact, although the majority of the genomic DNA recovered from chromocenters are major satellites, ~30% belongs to LINE1 and ERV families [START_REF] Ostromyshenskii | Mouse chromocenters DNA content: Sequencing and in silico analysis[END_REF] completely reset all epigenetic information (Hackett et al. 2013b). Furthermore, deamination of 5mC is implicated in the DNA demethylation process in PGCs [START_REF] Popp | Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency[END_REF]. It would thus be interesting to study the role of DAXX in PGCs formation and search for the appearance of DAXX structures at PCH. Overall, it remains to bear in mind that the early embryonic development takes only about 5 days before the implantation and a strong gain in DNA methylation levels. However, ESCs in culture correspond to a somehow artificial environment where the active DNA demethylation takes place for days.

DAXX and SETDB1 participate in re-establishment of heterochromatin state following active DNA demethylationinduced DNA damages.

Upon TET1 targeting to chromocenters, using a Tale strategy, we notice that the decrease of 5mC levels at these locations correlates with an increase in γH2AX foci, demonstrating that active DNA demethylation can induce DNA damages (Figure S3A article, 3D article).

Furthermore, recruitment of TET1 at PCH induces DAXX accumulation (Figure 3E article). It is in agreement with previously published data that observed a correlation between DNA demethylation and DNA damages [START_REF] Nakatani | Stella preserves maternal chromosome integrity by inhibiting 5 hmC-induced yH2AX accumulation[END_REF]. Nevertheless, γH2AX is a broad DNA damage signaling factor and it remains to understand what types of DNA lesions arise from TET1mediated active DNA demethylation. It has been demonstrated that TET activity hydroxylates 5mC into 5hmC, which can then be removed via the action of TDG and the BER pathway [START_REF] Cortellino | Thymine DNA Glycosylase Is Essential for Active DNA Demethylation by Linked Deamination-Base Excision Repair[END_REF][START_REF] Santos | Active demethylation in mouse zygotes involves cytosine deamination and base excision repair[END_REF]. We notice that the number of γH2AX foci occurring at PCH was increased upon ground-state conversion (Figure 2D Intriguingly, the targeted recruitment of NBS1 induce an increase in the number of γH2AX foci at chromocenters, but does not lead to DAXX accumulation, suggesting that DAXX is not acting within the DDR but rather at later steps during heterochromatin reformation (Figure 2G article). Indeed, following DNA lesions, the epigenetic mark of the locus is lost and must be reestablished at the end of the DNA repair process [START_REF] Soria | Prime , Repair , Restore : The Active Role of Chromatin in the DNA Damage Response[END_REF]. We notice that γH2AX signaling was only visible in 50% of the DAXX structures at PCH, which agrees with a late DNA repair step when γH2AX is removed from the chromatin (Figure S2D SUMOylation. Indeed, SUMOylation of XRCC1 facilitates its interaction with TDG and promotes the assembly of BER complexes at DNA lesions [START_REF] Steinacher | SUMOylation coordinates BERosome assembly in active DNA demethylation during cell differentiation[END_REF]). In the same line, it would be interesting to evaluate the impact of DNA damage response-proteins on the recruitment of DAXX in WT cells grown in hypomethylated conditions. Indeed, if DAXX is recruited at late DNA repair stages, it could be interesting to prevent proper DNA repair by knocking-down specific steps such as resection (i.e. SAE2 or EXO1) and see if it abrogates the formation of DAXX structures at PCH and impact cell survival upon ground-state conditions. Finally, to assess the importance of post-repair heterochromatin reformation, it would be informative to measure the ability of Daxx-deficient cells to perform the different types of DNA repair, which was shown to be dependent on the nuclear positioning [START_REF] Lemaître | Nuclear position dictates DNA repair pathway choice[END_REF].

Active DNA demethylation generates AP-sites and can ultimately results in DSBs.

However, the observed DNA damages at PCH could arise from indirect sources. Indeed, we observe that HU treatment resulted in DAXX accumulation at PCH (Figure 2F). We observe a strong recruitment of DAXX at PCH upon DNA hypomethylation (Figure 2A article, 3B article). DAXX recruitment at PCH was observed during the first stages of early embryonic development [START_REF] Arakawa | Stella controls chromocenter formation through regulation of Daxx expression in 2-cell embryos[END_REF][START_REF] Liu | SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin[END_REF]. However, the function of DAXX at this stage is primarily though to deposit H3.3 to participate into the decompaction of heterochromatin regions thus allowing the transcriptional burst of major satellites [START_REF] Liu | SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin[END_REF].

Production of major satellite RNAs is necessary for further development of the embryo [START_REF] Casanova | Heterochromatin Reorganization during Early Mouse Development Requires a Single-Stranded Noncoding Transcript[END_REF]. Furthermore, this is in agreement with the reported role for H3. We observe that DAXX recruits SETDB1 at PCH to perform H3K9 tri-methylation (Figure 5D article). Furthermore, we note that the absence of Daxx impairs heterochromatin organization and silencing (Figure 1C article, 4A article). We thus propose that DAXX, via SETDB1 and H3.3, participate in the reformation of heterochromatin after DNA damages. As SETDB1-mediated tri-methylation seems important, it suggests that the broken chromatin has lost its H3K9me3-previous modification. Intriguingly, it was reported that the action of KDM enzymes are essential for the DNA repair pathway choice and repair completion in both Drosophila and mammalian cells [START_REF] Young | Kdm4b Histone Demethylase Is a DNA Damage Response[END_REF][START_REF] Janssen | Timely double-strand break repair and pathway choice in pericentromeric heterochromatin depend on the histone demethylase dKDM4A[END_REF][START_REF] Colmenares | Drosophila Histone Demethylase KDM4A Has Enzymatic and Nonenzymatic Roles in Controlling Heterochromatin Integrity Drosophila Histone Demethylase KDM4A Has Enzymatic and Non-enzymatic Roles in Controlling Heterochromatin Integrity[END_REF]. It would be interesting to assess in mESCs whether the demethylation of H3K9 also takes place prior or during to the DNA repair, and further dissect if preventing the H3K9 demethylation could impact the formation of DAXX structures. Preliminary experiments reveal that KDM4A targeting to chromocenters does not lead to DAXX recruitments, suggesting that loss of H3K9me3 is not a sufficient information per se (data not shown). However, it remains to assess KDM functions in the DNA damage response. In the same direction, the Suv39H1/2 dKO mice are viable, but not Setdb1 nor Daxx-deficient mice, suggesting that indeed the role of DAXX is more linked to SETDB1 than to SUV39H1/2 enzymes [START_REF] Peters | Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability[END_REF][START_REF] Dodge | Histone H3-K9 methyltransferase ESET is essential for early development[END_REF][START_REF] Michaelson | Loss of Daxx , a promiscuously interacting protein , results in extensive apoptosis in early mouse development[END_REF][START_REF] Liu | SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin[END_REF]. Interestingly, H3.3K9R-expressing embryos, that still express endogenous H3.3, can develop to the blastocyst stage, however, it is still unknown whether arginine methylation can arise [START_REF] Santenard | Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3[END_REF].

DAXX maintains pericentromeric heterochromatin organization.

In but also the Serine 31. Intriguingly, H3.3S31 can be phosphorylated and is observed at pericentromeric regions in somatic cells [START_REF] Hake | Serine 31 phosphorylation of histone variant H3.3 is specific to regions bordering centromeres in metaphase chromosomes[END_REF]. We obtained preliminary results suggesting that H3.3S31 might be involved in PCH clustering (data not shown). It would be interesting to further investigate the interplay between SETDB1, H3.3S31 and PCH clustering and even additional histone residues undergoing post-translational modifications.

Our model is not exhaustive, and additional players might be involved in this heterochromatin maintenance mechanism. One of the major proteins that binds H3K9me3 is HP1α. We do not observe striking differences in H3K9me3 and HP1α presence at chromocenters in Daxx KO ESCs (Figure 4B article, Figure 28A). However, when measuring the dynamic of HP1α using FRAP, we notice that the half-recovery rate was faster in the absence of Daxx (Figure 4B article). Furthermore, when we look at the variance of HP1α signal over time, we detect lower levels of heterochromatin barriers, suggesting that HP1α is either diffusing more rapidly throughout the nucleoplasm to chromocenters, or that its affinity is reduced in the absence of Daxx We propose a model of heterochromatin maintenance at pericentromeric heterochromatin.

However, this mechanism might extend beyond PCH. Heterochromatin compartments serve as landmarks for heterochromatin regions dispersed throughout the genome, such as the pericentromere-associated domains (PADs) or the lamina-associated domains (LADs) [START_REF] Canat | Genome anchoring to nuclear landmarks drives functional compartmentalization of the nuclear space[END_REF][START_REF] Wijchers | Characterization and dynamics of pericentromere-associated domains in mice[END_REF][START_REF] Peric-Hupkes | Molecular Maps of the Reorganization of Genome-Nuclear Lamina Interactions during Differentiation[END_REF]. We notice that in the absence of Daxx, chromocenters were scattered within the nucleoplasm (Figure 4A

article). Because PADs and

LADs present a strong overlap of DNA sequences and that we detected a strong upregulation of genes located within LADs in ground-state Daxx KO ESCs, it is likely that PAD-genes could also be mis-regulated (Figure 30A). Thus, it would be of interest to choose candidate genes belonging to PADs only or both PADs and LADs and perform DNA FISH. It will be interesting to see whether mis-regulated genes have shifted from heterochromatin to euchromatin regions or if the differences in transcription could happen without any changes in radial positioning. The strong overlap between PADs and LADs results from the high redundancy existing between heterochromatin compartments. It would thus be interesting to describe which genes do move from PADs to LADs and which genes are not relocalized, enabling un uncoupling of transcription and radial positioning.

Lastly, we propose that our heterochromatin maintenance model may not be restricted to mESCs. H3K9me3-establishment at chromocenters was mainly though to result from the SUV39H1 and 2 enzymes while SETDB1 is mostly associated with telomeric heterochromatin [START_REF] Martens | The profile of repeatassociated histone lysine methylation states in the mouse epigenome[END_REF][START_REF] Peters | Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability[END_REF][START_REF] Gauchier | SETDB1dependent heterochromatin stimulates alternative lengthening of telomeres[END_REF][START_REF] Burton | Heterochromatin establishment during early mammalian development is regulated by pericentromeric RNA and characterized by non-repressive H3K9me3[END_REF]. We demonstrate that upon hypomethylation, the other H3K9me3-methylase SETDB1 is recruited by DAXX to PCH and actively generates H3K9me3, even in the absence of Suv39H1/2 (Figure 5B article). It agrees with previous observations that Suv39H1/2 dKO ESCs in ground-state presented some H3K9me3 foci onto DAPI-rich chromocenters [START_REF] Tosolini | Contrasting epigenetic states of heterochromatin in the different types of mouse pluripotent stem cells[END_REF]. Our results underlie a previously unexpected redundant action of the different H3K9 tri-methylases at pericentromeric regions. In order to uncouple the action of DAXX from SETDB1, it would be appealing to look at chromocenter organization in Setdb1-deficient cells. It was reported that the loss of Daxx could impact the organization of chromocenters in murine embryonic fibroblasts [START_REF] Rapkin | The histone chaperone DAXX maintains the structural organization of heterochromatin domains[END_REF].

However, it remains to assess the exact function of DAXX and SETDB1 in pericentromeric heterochromatin regulation in somatic cells. Intriguingly, DAXX can be recruited to PCH in highly methylated cells such as myotubes [START_REF] Salsman | Myogenic differentiation triggers PML nuclear body loss and DAXX relocalization to chromocentres[END_REF]. Indeed, in less committed myoblasts, DAXX localizes at PML NBs and upon differentiation into myotubes, there is the disappearance of PML NBs, which correlates with the recruitment of DAXX to chromocenters. Surprisingly, the expression of TET1 and TET2 enzymes in increased upon myotube differentiation, concomitantly with enhanced the level of 5hmC [START_REF] Zhong | Ten-eleven translocation-2 (Tet2) is involved in myogenic differentiation of skeletal myoblast cells in vitro[END_REF]. It would be interesting to dissect whether the observed DAXX recruitment to PCH also relies onto DNA damages-induced active DNA demethylation.

Roles of DAXX-SETDB1 at other genomic regions

As mentioned above, we detect a strong upregulation of LAD-genes in ground-state cells lacking Daxx (Figure 30A). Surprisingly, we note that the peripheral ring of H3K9me2 is altered and appears irregular in these cells, a phenotype never described before (Figure 29A). We hypothesize that it is a loss of H3K9me2 in LADs that leads to the transcriptional upregulation of LAD-genes. To confirm it, one could perform a ChIP-seq experiments onto H3K9me2 mark and look if there is a global decrease or local decreases at specific loci. Furthermore, because we demonstrated that the function of DAXX at pericentromeres relies on SETDB1, it would also be informative to perform an H3K9me3 ChIP-seq. Indeed, reanalyzing the RNA-seq data from a recent study that studied the conversion toward the ground-state of pluripotency of Setdb1deficient cells, we detected that an increase of LAD-genes transcriptions, although in both serumbased and ground-state conditions (data not shown) [START_REF] Wu | SETDB1-Mediated Cell Fate Transition between 2C-Like and Pluripotent States[END_REF]. These data suggest that the function of DAXX and SETDB1 extend beyond pericentromeric satellites.

DAXX and SETDB1 have been implicated in the transcriptional regulation of ERVs, notably in hypomethylated ESCs [START_REF] He | The Daxx/Atrx Complex Protects Tandem Repetitive Elements during DNA Hypomethylation by Promoting H3K9 Trimethylation[END_REF][START_REF] Matsui | Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET[END_REF][START_REF] Fukuda | A CRISPR knockout screen identifies SETDB1-target retroelement silencing factors in embryonic stem cells[END_REF]. We confirm the role of DAXX in regulating diverse families of ERVs upon ground-state conversion (Figure 1C article). We report that some LINE1 elements are also controlled by DAXX, even in serumbased medium, which has not been previously reported (Figure 1C article). The effect of the transcriptional up-regulation of transposable elements onto nearby genes remains controversial [START_REF] Elsässer | Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells[END_REF][START_REF] Jachowicz | LINE-1 activation after fertilization regulates global chromatin accessibility in the early mouse embryo[END_REF]). Yet, because of the important number of genes that are up-regulated within LADs, it would be interesting to assess the importance of transposable elements loss of silencing onto LAD-genes. LINE1 Tf elements are mostly enriched in LADs and can be up-regulated using a dCas9-VPR construct (Figure 31B, Figure 31C). We notice that Taleactivation was often more efficient than dCas9-activation (data not shown). It would thus be informative to up-regulate LINE1 Tf, and perhaps multiple families at once, using a Tale-VP64 approach and perform RNA-seq experiments to assess the transcriptional states of LAD-genes, and genes located near targeted LINE1. Furthermore, because we describe a function of DAXX in heterochromatin reformation after active DNA demethylation-induced DNA damage, it would be interesting to evaluate whether this phenomenon also takes place at retrotransposons using genome-wide mapping of 5hmC, AP-sites and yH2AX signaling. Such ChIP-seq assays could be performed in ESCs transfected with Tale-TET1CD constructs targeting different TE families such as LINE1 Tf and IAPEz.

Genome-wide mapping of DAXX binding revealed that about 20% of DAXX peaks appear at gene promoters [START_REF] He | The Daxx/Atrx Complex Protects Tandem Repetitive Elements during DNA Hypomethylation by Promoting H3K9 Trimethylation[END_REF]. Furthermore, it was recently reported that DAXX physically interact with PRC1 and might thus regulate polycomb-targeted genes. Indeed, in the zygote, PRC1 can recruit DAXX to major satellites and oocytes and 2-cell embryo lacking Daxx presented an upregulation of genes that were highly marked by H3K27me3 [START_REF] Liu | SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin[END_REF]. These results are in good accordance with our transcriptomic as upon preliminary analysis, we noted a good correlation between H3K27me3 presence and up-regulation in the absence of Daxx upon ground-state conversion (data not shown). We notice that the absence of Daxx resulted in a 2C-like transcriptional signature upon ground-state conversion, suggesting that DAXX contributes to the repression of 2C-like program (Figure 27B). Intriguingly, PRC1 is among the complex implicated et al. 2015;[START_REF] Ivanauskiene | The PML-associated protein DEK regulates the balance of H3.3 loading on chromatin and is important for telomere integrity[END_REF]. It would be interesting to assess if the H3.3K9me3modification is carried specifically by SETDB1. In cancer cells displaying ALT-maintenance of telomeres, deletion of Daxx or Setdb1 results in loss of telomeric heterochromatin, suggesting that the crucial role played by DAXX and SETDB1 is also true in somatic cells [START_REF] Gauchier | SETDB1dependent heterochromatin stimulates alternative lengthening of telomeres[END_REF][START_REF] Ahvenainen | Loss of ATRX / DAXX Expression and Alternative Lengthening of Telomeres in Uterine Leiomyomas[END_REF]. Furthermore, H3.3 have been linked to ALT-mechanism as phosphorylation of H3.3 serine 31 is essential for survival of human ALT-cancer cells [START_REF] Chang | CHK1-driven histone H3.3 serine 31 phosphorylation is important for chromatin maintenance and cell survival in human ALT cancer cells[END_REF]. It would be of great interest to deeper investigate the exact role of H3.3 in ALT-cancer, and the possible function H3.3K9. Intriguingly, SETDB1 is the main histone methyl-transferase acting at telomeres, in contrast of the action of SUV39H1/2 at chromocenters. In murine cells, chromosomes are all acrocentric, implying that one telomere is always located close to a pericentromeric region, hence a chromocenter. As observed in the case of TRF2 protein which has been described as a telomeric protein, which has recently been implicated in the proper replication of pericentromeric satellites, it is conceivable that other telomeric proteins may be involved in chromocenter maintenance primarily via spatial localizations, further strengthening the links between heterochromatin compartments [START_REF] Mendez-Bermudez | Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2[END_REF].

Activation of heterochromatin regions can generate DNA damages and IFN signaling

In the second part of the discussion, I proposed a model based on our results that active DNA demethylation at chromocenters could generate DNA damage signaling and recruit DAXX.

Interestingly, we notice that Daxx KO ESCs display a strong increase in total yH2AX levels by

Western blot upon ground-state conversion, that is unlikely to be solely explained by major satellite up-regulation (Figure 31A). Correlations have often been observed between up-regulation of DNA repeat elements and increased DNA damages, albeit no link has clearly been demonstrated for endogenous elements [START_REF] Tharp | Maximizing the ovarian reserve in mice by evading LINE-1 genotoxicity[END_REF][START_REF] Belgnaoui | Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells[END_REF][START_REF] Gasior | The Human LINE-1 Retrotransposon Creates DNA Doublestrand Breaks[END_REF][START_REF] Xiang | Histone Demethylase KDM4B Promotes DNA Damage by Activating Long Interspersed Nuclear Element-1[END_REF]. To assess the direct consequences of LINE1 derepression, we targeted a dCas9-VPR protein to specifically activate LINE1 Tf family. We detect a transcriptional up-regulation of the targeted LINE1 family and observe a concomitant increase of yH2AX signal (Figure 31B, Figure 31C).

Furthermore, the specific activation of major satellite repeats also induce a yH2AX increase at chromocenters (Figure S3E article). These data suggest that the loss of silencing of DNA repeated elements directly contribute to the increase of DNA damages, as it has been described in breast cancer cells where satellite RNAs are sufficient to induce DNA damages (Zhu et al. 2018b). To further confirm these results, it would be interesting to assess yH2AX levels upon specific well. SUMO-SIM interactions was proposed to mediate LLPS, notably for PML NBs formation [START_REF] Min | Clustered telomeres in phase-separated nuclear condensates engage mitotic DNA synthesis through BLM and RAD52[END_REF][START_REF] Banani | Compositional Control of Phase-Separated Cellular Bodies[END_REF]. HP1α binding at chromocenters is affected by SUMOylation in MEFs [START_REF] Maison | SUMOylation promotes de novo targeting of HP1 ± to pericentric heterochromatin[END_REF]. Indeed, the targeting of the SENP1/2 SUMO proteases at PCH can block HP1α at chromocenters and thus diminish its dynamical properties [START_REF] Maison | The SUMO protease SENP7 is a critical component to ensure HP1 enrichment at pericentric heterochromatin[END_REF][START_REF] Romeo | The SENP7 SUMO-Protease Presents a Module of Two HP1 Interaction Motifs that Locks HP1 Protein at Pericentric Heterochromatin[END_REF]. Because DAXX possesses a SIM motif and binds to SUMOylated proteins such as PML, it would be informative to measure the SUMOylation-deSUMOylation of HP1α at In that direction, the loss of Setdb1 prevents cell survival upon ground-state conversion, strengthening its importance [START_REF] Wu | Cell Reports[END_REF]. It yet remains to assess the importance of SETDB1 in H3.3 recruitment. SETDB1

shuttling from the cytoplasm to the nucleoplasm is regulated by ATF7IP, which also localizes at PML NBs [START_REF] Sasai | The Transcriptional Cofactor MCAF1/ATF7IP Is Involved in Histone Gene Expression and Cellular Senescence[END_REF][START_REF] Tsusaka | ATF7IP regulates SETDB1 nuclear localization and increases its ubiquitination[END_REF]. ATF7IP localization and function can be regulated by post-translational modifications such as SUMOylation and ubiquitynation [START_REF] Sasai | The Transcriptional Cofactor MCAF1/ATF7IP Is Involved in Histone Gene Expression and Cellular Senescence[END_REF].

Furthermore, ATF7IP is an important co-factor of SETDB1 and facilitates its enzymatic activity [START_REF] Wang | mAM Facilitates Conversion by ESET of Dimethyl to Trimethyl Lysine 9 of Histone H3 to Cause Transcriptional Repression[END_REF]. Hence, it would be interesting to assess if modifications of ATF7IP, induced by PML or DAXX could differentially regulate the function of SETDB1 in pluripotent cells.

Although previous studies reported the control exerted by PML onto DAXX repressive activity, it is the first report that H3.3 binding is implicated in DAXX-PML interaction [START_REF] Li | Sequestration and Inhibition of Daxx-Mediated Transcriptional Repression by PML[END_REF]. Furthermore, it was observed that H3.3 could accumulated at PML NBs, in a DAXXdependent manner [START_REF] Delbarre | DAXXdependent supply of soluble (H3.3-H4) dimers to PML bodies pending deposition into chromatin[END_REF]. In that direction, it would be interesting to evaluate the role of DAXX-H3.3 interaction on PML NB formation. Furthermore, DAXXΔSIM was able to induce PCH clustering without recruiting PML, strengthening that it is indeed H3.3 that is essential for that phenotype (Figure 38B). However, the clustering effect of DAXXΔSIM was stronger in WT cells than in Daxx KO ESCs, perhaps indicating a recruitment of endogenous DAXX. It would be interesting to assess whether DAXXΔSIM can interact with WT DAXX and other partners using a mass spectrometry strategy. Remarkably, we could not exclude that it is SETDB1 that recruits PML as SETDB1 localizes at PML NBs and can physically interact with PML [START_REF] Cho | Dual functions of histone-lysine Nmethyltransferase Setdb1 protein at promyelocytic leukemia-nuclear body (PML-NB): Maintaining PML-NB structure and regulating the expression of its associated genes[END_REF]. In that direction, it would be useful to specifically recruit SETDB1 at chromocenters using a Tale protein and observe whether it leads to DAXX or PML recruitment. When we recruit the DAXX9KR, we do not observe a strong clustering phenotype, suggesting that SUMOylation of DAXX might be important in its clustering function, and perhaps for H3.3 deposition (Figure 38D). It would be informative to assess H3.3 binding for the DAXX9KR mutant to dissect whether SUMOylation of DAXX can control its chaperone activity.

Roles of DAXX and pericentromeric regions in pathologies

We describe the presence of DAXX-PML structures at PCH upon hypomethylation (Figure 2A article). These DAXX-PML NB are much bigger than the classical PML NBs and could be qualified as 'giant' ones (Figure S2A article). PML NBs do not usually contain chromatin, yet in a genetic disease, the immunodeficiency, centromeric instability and cranial abnormality (ICF) syndrome, PML NBs appear as 'giant' bodies containing the pericentromeric repeats of some chromosomes in lymphocytes of ICF patients [START_REF] Luciani | PML nuclear bodies are highly organised DNAprotein structures with a function in heterochromatin remodelling at the G2 phase[END_REF]. Interestingly, the ICF syndrome results from defects in the DNA methylation machinery, mostly from a mutated DNMT3B [START_REF] Xu | Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene[END_REF]). In the ICF syndrome, hypomethylation occurs notably at pericentromeric repeats of three specific chromosomes 1, 9 and 16 which harbor the particular satellite I and are thus more prone to breakage. Giant PML NBs form around these three peculiar chromosomes and contain DAXX and DNA damage response proteins like BRCA1, TOPIIα, BLM and NBS1 [START_REF] Luciani | PML nuclear bodies are highly organised DNAprotein structures with a function in heterochromatin remodelling at the G2 phase[END_REF].

We demonstrated that the function of DAXX at PCH in pluripotent cells relies onto the action of the SETDB1 enzyme, it would thus be interesting to assess if SETDB1 is also recruited in ICF patients. As we observed that the recruitment of DAXX-SETDB1 might be promoted by DNA damage-induced by TET-mediated active DNA demethylation, it would be interesting to measure the level of DNA damages specifically occurring at PCH of ICF patients. It would further be intriguing to knock-down TET activities in ICF-lymphocytes and ask whether DNA damages and centromeric instabilities might be reduced.

Loss of Daxx impairs pericentromeric heterochromatin organization in pluripotent cells (Figure 4A article). Intriguingly, disruption of chromocenters was observed in a murine mouse model for Brca1-deficient breast cancer [START_REF] Zhu | BRCA1 tumour suppression occurs via heterochromatin-mediated silencing[END_REF]. Furthermore, it was demonstrated that the action of BRCA1 at pericentromeric regions was to repress satellite transcription and pericentromeric RNAs could induce the tumorigenesis [START_REF] Zhu | BRCA1 tumour suppression occurs via heterochromatin-mediated silencing[END_REF](Zhu et al. , 2018b)). Since in ICF syndrome, BRCA1 localizes at DAXX-containing PML NBs, it would be relevant to assess the function of DAXX in BRCA1-mediated heterochromatin maintenance in breast cancers.

Furthermore, breast cancers often display hypomethylation of pericentromeric regions, it would thus be interesting to assess the cell survival of these cells upon Daxx depletion [START_REF] Narayan | Hypomethylation of pericentromeric DNA in breast adenocarcinomas[END_REF].

Furthermore, we observe that the absence of Daxx results in the loss of silencing of heterochromatin regions, including different classes of transposable elements and pericentromeric satellites (Figure 1C article). Over-expression of pericentromeric satellites is observed in different cancer types such as pancreatic cancers [START_REF] Ting | Aberrant Overexpression of Satellite Repeats in Pancreatic and Other Epithelial Cancers[END_REF]. Intriguingly, in lung, kidney, ovary, colon or prostate cancers, up-regulation of satellites correlated with the loss of silencing of LINE1 elements, suggesting broad defects in heterochromatin silencing. Pancreatic cancers notably display higher transcription of LINE1 elements, together with global DNA hypomethylation [START_REF] Marinoni | Hypo-methylation mediates chromosomal instability in pancreatic NET[END_REF]. Pancreatic tumors often bear point mutations in DAXX, which corresponds to high levels of chromosomal instability, suggesting a putative role for DAXX in genome stability in pancreatic cells upon hypomethylation [START_REF] Marinoni | Loss of DAXX and ATRX Are Associated With Chromosome Instability and Reduced Survival of Patients with Pancreatic Neuroendocrine Tumors[END_REF][START_REF] Jiao | DAXX/ATRX, MEN1, and mTOR Pathway Genes Are Frequently Altered in Pancreatic Neuroendocrine Tumors[END_REF]. It would be informative to assess the DNA damages and repair mechanisms in pancreatic tumor cells to dissect the exact function exerted by DAXX. Mutations of H3.3 and DAXX have been reported in pediatric neuroblastoma, and although it mainly seems to affect polycomb-related mechanisms, it remains to understand the exact function of DAXX in these cancers [START_REF] Schwartzentruber | Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma[END_REF][START_REF] Wu | Somatic histone H3 alterations in pediatric diffuse intrinsic pontine[END_REF][START_REF] Dyer | ATRX and DAXX: Mechanisms and mutations[END_REF]. Finally, as mentioned for breast cancers, other cancer types display DNA hypomethylation, it would be interesting to study the function of DAXX in these diseases [START_REF] Ehrlich | DNA hypomethylation in cancer cells[END_REF]. It has been reported that SETDB1 was an essential factor for several acute myeloid leukemia cell lines acting on ERV repression and the IFN response [START_REF] Cuellar | Silencing of retrotransposons by SET DB1 inhibits the interferon response in acute myeloid leukemia[END_REF]).

Since we observed a role for DAXX in recruiting SETDB1 upon DNA hypomethylation, future studies should explore the potential of DAXX as a clinical target.

RESUME SUBSTANTIEL DE LA THESE EN FRANCAIS Introduction

  Hugues et Valérie pour leur intérêt tout au long de l'avancement de mon projet ; ainsi que toute leur équipe. Je pense bien sûr à Cécile et à Caroline, pour tous nos repas et pausescafé partagées ; à Marie-Claude, pour ses conseils et discussions ; mais encore à Michiko, à Florence, à Omar et toutes les personnes croisées au 3 ème étage. Je remercie les membres de la plateforme technologique du bâtiment Hayem et notamment Niclas pour son aide précieuse pour la microscopie. Merci également à Stéphane, pour son enthousiasme et son temps passé avec moi à son microscope, ainsi que Judith, pour ses conseils sur la dynamique des protéines. Je remercie chaudement Rodney, pour m'avoir accueilli dans son équipe pour mon stage de M1, et être depuis resté un véritable mentor. Je remercie la Ligue Nationale Contre le Cancer d'avoir financé ma quatrième, et si importante, année de thèse.

Figure 1|

 1| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 2|

 2| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 4|

 4| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 5|

 5| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 6|

 6| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 7|

 7| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure

  Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 11|

 11| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 16|

 16| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 17|

 17| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 19|

 19| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 20|

 20| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 27|

 27| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 28|

 28| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 29|

 29| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 31|

 31| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 32|

 32| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 33|

 33| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 34|

 34| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 35|

 35| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 38|

 38| Figure 1| Nucleosomal organization of chromatin and nucleosome repartition.Figure 2| Different chromatin compaction states. Figure 3| Landscape of histone post-translational modifications.Figure 4| Schematic of histone acetylation and associated 'readers'.Figure 5| Histone methyltransferases and their associated phenotypes. Figure 6| DNA methylation machinery and mechanism. Figure 7| Histone variants. Figure 8| Histone variant H3.3 structure and function.Figure 9| The H3.3-chaperone DAXX, structure, localization and function. Figure 10| Spatial compartmentalization of the nuclear space.Figure 11| Localizations of chromosomes within the nuclear space. Figure 12| Lamina associated domains. Figure 13| Proteins associated with lamina associated domains. Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. Figure 15| Organization of pericentromeres.Figure 16| Heterochromatin composition and transcriptional state of pericentromeres.Figure 17| Importance of pericentromeric satellite regulation. Figure 18| Organization and composition of telomeric DNA. Figure 19| Overview of the classes of transposable elements in mouse. Figure 20| Multiple proteins ensure transcriptional repression of ERVs. Figure 21| PML proteins assemble into PML nuclear bodies. Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. Figure 23| Murine early embryonic development. Figure 24| Signaling pathways in pluripotent cells in vitro. Figure 25| Chromatin changes along early embryonic development. Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells.Figure 27| Absence of Daxx results in 2C-like transcriptional profile.Figure 28| DAXX contributes to the radial positioning of chromocenters.Figure 29| DAXX contributes to peripheral heterochromatin organization. Figure 30| Loss of Daxx impairs the silencing of genes within LADs.Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx.Figure 32| Generation of Pml KO ESCs.Figure 33| DAXX accumulation at PCH is independent of PML.Figure 34| PML contributes to pericentromeric heterochromatin organization.Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon ground-state conversion. Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction.Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering.

Figure 39|

 39| Figure 39| Recruitment of H3.3 and PML upon DAXX targeting to PCH. Figure 40| Model displaying the function of DAXX at pericentromeric heterochromatin and the consequences of its loss.

Figure

  Figure 1| Nucleosomal organization of chromatin and nucleosome repartition. (A) Crystal structure of a nucleosome consisting of a histone octamer and DNA. (B) Nucleosome spacing in S. cerevisiae and M. musculus that favor two different types of inter-nucleosome distances. (C) MNAse-seq profile displaying nucleosome occupancy near a transcription start site (TSS). In orange is highlighted a fragile nucleosome region which is strongly unstable. (D) DNA wrapped around a nucleosome. At the top of the dyad lies the linker inter-nucleosome sequence. A and D are from Zhou, Gaullier and Luger, Nat. Struc. Mol. Biol. 2019. B is from Gibson et al., Cell 2019. C is adapted from Voong et al., Cell 2016.

Figure

  Figure 2| Different chromatin compaction states. (A) Electron microscopy picture of a mammalian nucleus. In light grey is the decompacted, open and active euchromatin whereas in dark grey lies the compacted, closed and inactive heterochromatin. (B) Theoretical states of chromatin compaction and the corresponding packing ratio obtained from these configurations. (C) Live-cell imaging of a LacO array visualized by a LacI-YFP reporter. On the left is a compact locus, whereas on the right is shown different decompaction state from medium to a very large decondensation. A is adapted from Akthar and Gasser, Nat. Rev. Gen. 2006. B is from Fussner, Ching and Bazett-Jones, Trends in Biochem. Sciences 2011. C is from Giri et al., J. Cell Science 2016.

Figure

  Figure 3| Landscape of histone post-translational modifications. For each canonical histone residues are indicated the reported post-translational modifications. Many of the modifications are observed on the Nterminal tails but the globular cores are also subjected to chemical modifications.

Figure 4|

 4| Figure 4| Schematic of histone acetylation and associated 'readers'. (A) Scheme of a nucleosome displaying one N-terminal histone tail with a lysine residue either acetylated or nor. The acetyl-group is added by a histone acetyl-transferase (HAT) and removed by a histone deacetylase (HDAC). (B) The different domains acting as 'readers' of an acetylated residue. The bromodomain (BRD) being the most common one. From Gong, Chiu and Miller, PLoS Gen. 2016.

Figure

  Figure 5| Histone methyltransferases and their associated phenotypes. (A) The different histone lysine methyl-transferases (KMT) and their corresponding substrate preferences. Must be read as EZH2 catalyzes the methylation of the lysine 27 residue. (B) Scheme of the different stages of development and the corresponding stage of lethality for each KMT in mouse. Must be read as EZH2 is lethal at the developmental stage E6. (C) Representation of the tissues specificities for the viable KMTs. Must be read as MLL4 deficiency impairs muscle development. From Jambhekar, Dhall and Shi, Nat. Rev. Mol. Cell Biol. 2019.

Figure

  Figure 6| DNA methylation machinery and mechanism. (A) Scheme of the four main DNMT proteins and their different peptidic domains. DNMT3 possesses two members A and B that are highly similar. (B) The three main TET enzymes with their corresponding protein motifs. (C) Life cycle of a methylated cytosine. A DNMT first adds a methyl-group onto the cystosine (5mC). A TET enzyme can then oxydize the 5mC into a hydroxymethyl (5hmC). 5hmC can be lost passively through replication or further oxidized into a formyl-cytosine (5fC) or a carboxyl-cytosine (5caC). 5fC or 5caC modifications can ultimately generates double-strand breaks if not resolved via TDG. AM-AR = active modification-active removal, AM-PD = active modification-passive dilution, TDG = Thymine DNA glycosilase, BER = Base excision repair. C is from Wu and Zhang, Nat. Rev. Gen. 2018.

Figure

  Figure 7| Histone variants. Scheme of the various histone variants identified. They often differ by only a few mutations. Only histone H4 does not possess any identified variant. FromMaze et al., Nat. Rev. Gen. 2014

Figure

  Figure 8| Histone variant H3.3 structure and function. (A) Scheme of the H3.3 protein compared to the two canonical H3.1 and H3.2 histones. Highlighted are the 4 to 5 amino acid changes of H3.3 that are mainly observed within the globular core. (B) Nucleosomal chromatin fiber reconstituted in vitro carrying either H3.1 or H3.3-nucleosomes. Presence of H3.3 is associated with a decondensed structure of the chromatin. (C) Scheme of the turnover rate of H3.3 at its enriched positions along the chromosome. Above are indicated the preferential and additional chaperones described for H3.3 that are locus-specific. (D) Scheme of the role of H3.3 in embryonic stem cells. Lack of H3.3 induces defects in chromosome segregation during mitosis. B is from Chen et al., N.A.R. 2013. C is adapted from Huang and Zhu, Bioessays 2014. D is from Jang et al., Genes & Dev. 2015.

Figure

  Figure 9| The H3.3-chaperone DAXX, structure, localization and function. (A) Scheme of DAXX protein and its different domains. (B) Structures of DAXX and H3.3 showing their interaction domain, mediated by the specific amino acid H3.3G90 and DAXXY222 and G225. (C) DAXX localizes within PML nuclear bodies in murine embryonic stem cells. (D) Daxx-deficient embryo die before the implantation stage and the majority does not reach the blastocyst stage. B is from Huang and Zhu, Bioessays 2014. C is from personal unpublished data. D is from Liu et al., EMBO J. 2020.

Figure

  Figure 10| Spatial compartmantalization of the nuclear space. Scheme of a mammalian nucleus.

Figure

  Figure 12| Lamina associated domains. (A) Electron microscope image showing a mammalian nucleus. An electron-dense layer of chromatin is observed at the nuclear periphery. Other electron-dense heterochromatin regions are observed within the nuclear interior, notably around the nucleolus. (B) Imaging of LADs. In green is the m6A-tracer binding to genomic regions methylated by the LaminB-DamID. In red is a staining of Lamin B. (C) Cartoon illustrating the folding of one chromosome. The entire chromosome drawn in blue faces the nuclear interior, whereas LADs of this chromosome, drawn in green, specifically lie at the nuclear periphery. (D) Scheme of a DamID genome-wide profile showing nuclear lamina (NL) contact intensities. LADs are depicted in green. Adapted from van Steensel and Belmont, Cell 2017.

Figure

  Figure 13| Proteins associated with lamina associated domains. Proteins from the nuclear lamina such as LBR, Emerin and LAP2α can bind to chromatin-associated factors. Heterochromatin LADs are enriched in specific histone modifications such as H3K9me2 and me3, subsequently bound by 'readers' like HP1. LaminB is exclusively observed within the nuclear lamina whereas some LaminA can bind to non-LADs of the genome. From Buchwalter, Kaneshiro and Hetzer, Nat. Rev. Genetics 2019.

Figure 14|

 14| Figure 14| Changes in nuclear lamina interactions upon differentiation and 'unlocking' theory. The nuclear periphery acts as a repressive compartment for LADs. Some LADs are cell-type specific and called facultative (fLAD). Inactive neuronal gene on the left localizes at the periphery in ESCs and changes positioning and transcriptional state in NPC.s Active stem cell gene on the right faces the nuclear interior in ESCs and is repressed and gain lamina-interaction upon differentiation into NPCs. The gene in the middle is inactive and localizes at the nuclear periphery in ESCs, upon NPC differentiation, it relocalizes away from the periphery upon NPC differentiation but in a poised state and is only activated upon AC differentiation. ESC = embryonic stem cell, NPC = neural precursor cell, AC = astrocyte. From Peric-Hupker et al., Mol. Cell 2010.

Figure

  Figure 15| Organization of pericentromeres. (A)Left, scheme of mouse chromosome. White zones represent euchromatin regions, whereas dense zones represent heterochromatin regions. Highlighted are the telomere, centromere and pericentromere regions with a zoom onto their repeated sequences. Right, table showing sizes, copy number and percentage of telomere, centromere and pericentromere regions in mouse. Bottom, scheme of the spatial organization of constitutive heterochromatin with the pericentromeres that merge to form big chromocenters. At the periphery of chromocenters are observed the centromere and closely located telomere, as well as pericentromereassociated domains. (B) Representative immunoFISH and immunofluorescence pictures of H3K9me3 histone modification, major satellite repeats and 5mC DNA methylation in murine ESCs. B is from personal unpublished data.

Figure

  Figure 17| Importance of pericentromeric satellite regulation. (A) PCH of different chromosomes cluster together. Loss of clustering impairs cohesion of chromosomes and generates micronuclei formation during interphase and induces accumulation of DNA damages. (B) SUV4-20H1/2 enzymes provide H4K20me3modification. Deletion of Suv4-20H1/2 results in loss of cohesion recruitment at PCH and mis-segregation of chromosomes. (C) High levels of pericentromeric satellites are observed in Brca1-null breast cancers. RNA produced can destabilize DNA replication forks and generates R-loops, ultimately leading to genomic instabilities. (D) Chromocenters represent highly compact constitutive heterochromatin and particular DNA repair strategies have thus evolved to ensure proper stability of PCH regions. In G1-phase of NIH 3T3 cells, DSBs arising in chromocenters are mainly repaired by NHEJ within the sub-nuclear compartment. However, in G2-phase, DSBs relocate toward the periphery of chromocenters and are resolved via HR. A is adapted from Jagannathan and Yamashita, Cold Spring Harbor Symp. Quant. Biol., 2017. B is from Hahn et al. Genes & Dev. 2013. C is from Zhu et al., Mol. Cell 2018. D is from Tsouroula et al. Mol. Cell 2016.

Figure

  Figure 18| Organization and composition of telomeric DNA. (A) Telomeric sequences

Figure

  Figure 19|Overview of the classes of transposable elements in mouse. Class II TEs represent the DNA transposons. Of about 1.4Kbs in length, they only represent up to 0.9% of the murine genome. They transpose via a cut and paste mechanism. Class I TEs are mobile via a copy-paste mechanism, which increases their number at each transposition. LTR retrotransposons, such as IAP elements, possess LTR repeats at ends. About 3.13% of the murine genome is composed of these elements, and many 'solo-LTR' are observed, remnants of past transpositions. Non-LTR elements comprise the LINE and the SINE elements. LINE elements are present within heterochromatin regions and are the most abundant families of TEs. ORF2 carries endonuclease and reverse-transcriptase activities. SINE elements are found within euchromatin regions. They do not encode for proteins and thus rely of the ORF2 of LINE to retrotranspose. Data from Mouse Genome Sequencing Consortium, Nature 2002.

Figure

  Figure 20| Multiple proteins ensure transcriptional repression of ERVs. KRAB-ZFPs bind toDNA and recruit KAP1. KAP1 binds to diverse heterochromatin-related proteins and enables heterochromatin formation at ERV sequences. Among the recruited proteins are DNMTs that methylate DNA, SETDB1 which trimethylates H3K9, subsequently bound by HP1 and NuRD/HDAC complexes that remove histone acetylation. DAXX can also be part of SETDB1/KAP1 complex and deposits H3.3-H4 dimers into these heterochromatin regions. Adapted from Ecco, Imbeault and Trono, Development 2017.

  Hoelper et al. propose that H3.3 stabilizes DAXX, and that H3.3 thus carries a role in the DAXX-H3.3-ATRX silencing pathway; but they suggest that an additional DAXX-SETDB1-KAP1-HDAC1 pathway might favor silencing of specific ERVs without ATRX nor H3.3 incorporation.

Vertii

  et al. observe two classes of NADs coexisting in murine embryonic fibroblasts. Class I NADs correspond to typical heterochromatin domains and present a high overlap with LADs, repressive histone marks and late DNA replication, whereas class II NADs represent a facultative heterochromatin signature with H3K27me3 signal and a more early replication timing[START_REF] Vertii | Two contrasting classes of nucleolusassociated domains in mouse fibroblast heterochromatin[END_REF]. Interestingly, the authors observe using DNA FISH that while class I appears either next to nucleolus or close to the nuclear periphery, the class II exclusively associate with the nucleolus. As the class II NAD is absent in ESCs, the author conclude that class II NAD corresponds to developmental genes which are only silenced upon differentiation[START_REF] Vertii | Two contrasting classes of nucleolusassociated domains in mouse fibroblast heterochromatin[END_REF]

Figure

  Figure 21| PML proteins assemble into PML nuclear bodies. (A) Scheme of the PML protein and its domains. PML, also named TRIM19 (TRI-partite Motif 19) possesses a RING (Really Interesting New Gene) domain, two B1 and B2 zinc-finger boxes and a multimerization coiled-coil domain. Each of these domains is important for nuclear body assembly. (B) PML proteins can dimerize and then multimerize to form nuclear bodies that serve as a storage center for various other proteins that mostly contain a SIM domain. SUMO-SIM interaction allows recruitment of PML NB partners. PML NBs serve as hubs of post-translational modifications and notably SUMOylation. B is from Lallemand-Breitenbach and de Thé, Cold Spring Harbor Persp. Biol. 2010.

Figure 22| PML

 22| Figure 22| PML NBs as a storage center for H3.3-chaperones and interaction with chromatin. (A) H3.3-chaperones HIRA, DAXX and ATRX can localize within PML NBs. (B) Over-expression of H3.3 leads to its accumulation at PML NBs, which serve as storage center before chromatin deposition. (C) The H3K9 tri-methylase enzyme SETDB1 localizes at PML NBs, this is notably visible in the early embryo. Deletion of Setdb1 impairs the formation of PML NBs. (D) In murine embryonic stem cells, PML can be found at some telomeres, visible here with the presence of the telomeric repeats in red and PML in green. (E) Pictures of lymphocytes of control (top) or ICF patients (bottom) (Immunodeficiency, Centromeric instability and Facial abnormalities). The ICF syndrome is caused by mutation in DNMT3B and results in hypomethylation, notably at pericentromeres of the chromosomes 1, 9 and 16. Interestingly, pericentromeric satellites of these three chromosomes are found within PML NBs. A and B are adapted from Delbarre et al., Genome Res. 2013. B is from Cho et al., J. Biol. Chem. 2011. D is from personal unpublished data. E is from Luciani et al., J. Cell Science 2006.

Figure

  Figure 23| Murine early embryonic development. At the fertilization, the two gametes fuse to form the zygote. The first 4 days of development consist in numerous cell division and the embryo reaches a 2-cell stage, 4-cell stage, 8-cell stage, then the morula. Around E4, the embryo forms the blastocyst in which three cell types co-exist.Two cell-type will form the extra-embryonic tissues: the trophectoderm in green that will give the placenta and the primitive endoderm in purple which will give the yolk sac. The inner cell mass in yellow will differentiate into the whole organism. At E4.5, the blastocyst can implant within the uterus and then form the epiblast. ICM = inner cell mass. Adapted from[START_REF] Shahbazi | Deconstructing and reconstructing the mouse and human early embryo[END_REF] Zernicka-Goaetz, Nature Cell Biol. 2018 and[START_REF] Saiz | Early cell fate decisions in the mouse embryo[END_REF] Plusa, Reproduction 2013

Figure

  Figure 24| Signaling pathways in pluripotent cells in vitro. ESCs grow as dome-shaped colonies in both serum-based 'standard' medium and ground-state 2i/LIF + VitC medium. Serum-cultivated ESCs display 75 to 80% of DNA methylation levels, whereas conversion toward 2i/LIF + VitC reduces DNA methylation to 5 to 15%. However, pluripotency goes through different pathways. Serum-based ESCs activate the BMP4 receptor that ultimately represses lineage-specific genes. Addition of LIF to serum medium activates the STAT3 signaling pathways that activates pluripotency factors such as KLF4. Ground-state ESCs have 2 inhibitors in the medium that block the GSK3 and the MEK pathways that sustain activation of pluripotency factors. Ground-state ESCs are more homogeneous than 'standard' serum-grown cells. VitC = Vitamin C. Adapted from Hackett and Surani, Cell Stem Cell 2014. ESCcolonies pictures are from personal unpublished data.

  Consistent with some heterogeneity even for ground-state ESCs, 2cell-like stage observed by Macfarlan et al. are a rare ESC population in culture with only less than 1% of the cells (Macfarlan et al. 2012). 2cell-like ESCs activate a specific gene-set which closely resemble the in vivo 2-cell state embryo and they are totipotent cells as they can even generate extra-embryonic tissues such as the placenta and the yolk sac. Interestingly, activation of 2cell-related transcripts such as the Zscan4 cluster and MERVL transposable elements further increase the DNA demethylation (Eckersley-Maslin et al. 2016). It was observed that conversion toward this peculiar 2cell-like state could be further enhanced via depletion of some factors, notably chromatin-associated proteins that I will describe in the next section (Rodriguez-Terrones et al. 2018).

Figure

  Figure 25| Chromatin changes along early embryonic development. (A) Schematic of the early embryonic development, from the zygotic to the blastocyst stage. Below are depicted the global levels of histone modifications, chromatin features and heterochromatin transcription across pre-implantation embryo. (B) DAPI (4ʹ,6-diamidino-2-phenylindole)-staining of nuclei from the oocyte, the zygote, the 2-cell and 4-cell embryo. DAPI binds to A/T-rich regions, highlighting notably the pericentromeric satellites. PCH localize around nucleolar-like bodies until the 4-cell stage of development, when pericentromeric satellites assemble into well-defined chromocenters. Adapted from Burton and Torres-Padilla, Nat. Rev. Mol. Cell Biol. 2014.

Figure

  Figure 26| Pericentromeric maintenance in totipotent and pluripotent cells. (A) In the paternal nucleus of murine zygote, DAXX accumulates at PCH in a DPPA3 (also named STELLA)-dependent manner. (B) Depletion of DPPA3 induces stronger active DNA demethylation at PCH, which in turns increases the levels of DNA damage. (C) Super-resolution picture of murine ESCs showing H3K9me3 enrichment at DAPI-dense PCH. However, in case of loss of DNA methylation such as in DNMT triple KO cells, the intensity of H3K9me3 signal reduces and chromocenters present overlapping signal with H3K27me3 mark deposited by polycomb proteins. Steric hindrance is visible as there is no colocalization of H3K9me3 and H3K27me3 signals. (D) Diminution of DNA methylation levels in ESCs, either by DNMT triple KO cells or 5-azacytidine treatment results in BEND3 accumulation at PCH. A is from Cooper et al., Cell Rep. 2014. B is from Saksouk et al., Mol. Cell 2014. C is adapted from Arakawa et al., Biochem. Biophys. Res. Comm. 2015 D is adapted from Nagani et al., EMBO Rep. 2015.

Figure 1 Figure

 1 Figure 1

Figure 2 Figure 2 |

 22 Figure 2

Figure 3 Figure 3 |

 33 Figure 3

Figure 4 Figure 4 |

 44 Figure 4

Figure 5 Figure 5 |

 55 Figure 5

  chromocenters from the Tale-TET1CD experiments were measured using a 2µm line across individual DAPI-dense chromocenters. Intensity profiles were obtained and the mean with SEM were calculated for DAPI and 5mC signals for each condition. DNA damage signaling, SETDB1 and H3K9me3-recruitments at chromocenters were counted manually. Statistical analysis performed are indicated in the text, figures or figure legends and all statistical results are available in Supplementary table 1. Pvalues are represented as follow: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

  Icy scripts. Statistical analysis performed are indicated in the text, figures or figure legends and in Supplementary data. Pvalues are represented as follow: * <0.05; **<0.01; ***<0.001; ****<0.0001.

pluripotent cells 1 .

 1 Loss of Daxx results in transcriptional 2C-like signature Transcriptomic analysis of the Daxx KO ESCs in serum-based medium or upon differentiation or ground-state conversions revealed an extensive number of differentially expressed genes especially upon ground-state conversion (Figure 1D article). Furthermore, we noticed that the Daxx KO cells displayed an overexpression of the MERVL elements (Figure 1C article). MERVL upregulation has notably been reported in a rare subpopulation of ESCs in culture called 2-cell like (2C-like), which transcriptionally resemble the 2-cell state of the embryo.

(

  [START_REF] Rodriguez-Terrones | A molecular roadmap for the emergence of early-embryoniclike cells in culture[END_REF]. Most of the identified factors repressing 2C-like transcriptional program are chromatin-related factors such as PRC1, EP400-TIP60 histone acetyl-transferase complex or the histone methyl-transferase SETDB1, suggesting a strong importance for heterochromatin regulation in the totipotency to pluripotency transition[START_REF] Rodriguez-Terrones | A molecular roadmap for the emergence of early-embryoniclike cells in culture[END_REF][START_REF] Wu | SETDB1-Mediated Cell Fate Transition between 2C-Like and Pluripotent States[END_REF].

Figure

  Figure 27| Absence of Daxx results in 2C-like transcriptional profile. (A) Heatmap displaying Log2(FoldChange(WT/KO)) in GS, serum or upon differentiation. The list of up-or down-regulated genes comes from Rodriguez-Terrones et al., Nature Genetics, 2018 and Wu et al., Cell Reports, 2020. 45 up-regulated genes and 21 down-regulated genes are observed. (B) Volcano plot displaying Log2(FoldChange(KO/WT)) for all genes compared to Log10(pvalue). In light blue or light red are highlighted the differentially expressed genes with Log2(FoldChange(KO/WT)) <-1 or >1 and pvalue<0.05. In dark blue or dark red are the same genes as in A. (C) RT-qPCR assay for 3 representative genes (Gm5662, Spz1 and Zscan4) and 1 repeated element (MERVL) up-regulated in 2C-like ESCs. Histogram displays mean and SEM from 3 biological replicates.

Figure

  Figure 28| DAXX contributes to the radial positioning of chromocenters. (A) Representative images of immunoFISH experiments with H3K9me3 staining and DNA FISH of major satellites in the serum-based medium or after 5 or 10 days of ground-state conversion. (B) Scheme of the automatic threshold of DAPI signal and generation of a mask comprising the 10% most peripheral signal. (C) Quantification of the major satellite DNA FISH signal intensity observed within the 10% most peripheral zone compared to the intensity observed within the whole nucleus for the same conditions as in (A). Data from 3 biological replicates except for 10 days in ground-state that comprises 2 biological replicates. n = the total number of cells analyzed (D) Represntative images from immunoFISH experiments as in (A) for both WT and Daxx KO ESCs in serum-based medium or 4 days after ground-state conversion. (E) Quantification of the peripheral signal of major satellite DNA FISH signal as in (C). Data from 3 biological replicates except for 10 days in ground-state that comprises 2 biological replicates. n = the total number of cells analyzed. Two-sided Mann-Whitney was used for statistical analysis. GS = ground-state

Figure

  Figure 29| DAXX contributes to peripheral heterochromatin organization. (A) Representative images of immunofluorescence of H3K9me2 histone modification. On the left are the WT cells in either serum-based or ground-state conditions. On the right are the Daxx KO cells in the two conditions. White arrows indicate the patches of H3K9me2 that are uniquely visible in Daxx KO ESCs converted to ground-state condition. (B) Scheme of the homogeneity score representation where 0 is a perfectly homogenous signal. A line is traced at the nuclear periphery and we calculated the variation of the signal along the line. Boxplot displays the mean of variation for each nucleus. Data from at least 3 biological replicates. n= total number of nuclei analyzed. (C) Representative images of immunofluorescence of LaminB protein, a constituent of the nuclear lamina. On the left are the WT cells in either serum-based or ground-state conditions. On the right are the Daxx KO cells in the two conditions. Two-sided Mann-Whitney was used for statistical analysis. GS = ground-state

Figure

  Figure 30| Loss of Daxx impairs the silencing of genes within LADs. (A) Boxplot of mRNA levels of 904 genes belonging to cLAD, ESC fLAD or ESC iLAD in the different culture conditions. (B) IGV-window example of three genes displaying a strong up-regulation in the absence of Daxx specifically in ground-state. (C) RT-qPCR for 6 genes belonging to either fLAD or cLAD-categories, as indicated. Highlighted is the fold-increased of Daxx KO compared to WT in ground-state. Histogram represents the mean with SEM of three biological replicates. Two-sided unpaired t-tests were used for statistical analysis. GS = ground-state; Diff = Differentiated.

DAXX-mediated pericentromeric heterochromatin organization 1 .

 1 PML is essential for pluripotent cell survival upon DNA hypomethylation DAXX is commonly found at Pml NBs (Lallemand-Breitenbach and de Thé 2010). We confirmed that Daxx localized within Pml NB in serum-based ESCs (Figure 2A article). Upon hypomethylation we noted that Daxx accumulated at pericentromeric regions (Figure 2A article).

Figure

  Figure 31| LINE1 activation contributes to the accumulation of DNA damages in the absence of Daxx. (A) Western blot for γH2AX signaling in serum and ground-state conditions for WT and Daxx KO ESCs. H3 and LaminB1 serve as loading controls. One experiment is shown, from two independent replicates. (B) Diagram showing the percentage of LINE1 Tf or SINEB1 elements found within cLAD, fLAD or iLAD. Computed from UCSC Repeat Masker data and LAD-status obtained from Peric-Hupkes et al., Mol. Cell 2010. (C) Transcriptional activation of LINE1 Tf using a dCas9-VPR in WT or Daxx KO serum ESCs. Cells were either transfected without a LINE1 Tf-targeteing sgRNA, with 1 sgRNA or 3 sgRNAs. Mean with SEM are represented. WT data from 2 biological replicates, Daxx KO data from 2 technical replicates (D) Western blot for yH2AX or two loading controles Lamin B1 or H3 in the different transfections. One experiment is shown from two independent experiments.

Figure

  Figure 32|Generation of Pml KO ESCs. (A) Scheme of the Pml locus located on chromosome . sgRNA was designed to target the exon 1 of the Pml gene. Immunofluorescence of DAXX and PML and Western blot of PML proteins in WT or the selected Pml KO clone. H3 serves as loading control. (B) Quantification of the number of cells compared to WT for each condition. Histogram represents the mean with SEM of 3 biological experiments, except Diff which corresponds to 2 biological replicates. (C) qRT-PCR performed for markers of ground-state conversion. Histogram represents mean with SEM of two biological replicates. (D) qRT-PCR performed for markers of differentiation. Histogram represents mean with SEM of two biological replicates. GS = ground-state; Diff = differentiatied

Figure

  Figure 33| DAXX accumulation at PCH is independent of PML. (A) Representative immunofluorescence pictures of DAXX and PML in WT and Pml KO ESCs in both serum or ground-state conditions. White arrow highlights DAXX-PML accumulation at PCH. (B) Histogram representing the number of DAXX-PML structures at PCH observed in the different conditions. Mean with SEM is represented. At least 2 biological replicates for each condition. n=total number of nuclei analyzed. Chi-square test were used for statistical analysis.

3 .

 3 PML contributes to organization and radial localization of PCHAlthough we have shown that PML was recruited by DAXX, we detected the same loss of survival phenotype. DAXX localized within PML NBs and accumulated at chromocenters around DAXX (Figure2Aarticle). Because the absence of Daxx resulted in the alteration of heterochromatin organization in pluripotent cells, we decided to assess the heterochromatin organization in ESCs lacking Pml. We quantified the number of major satellite foci and observed, as for Daxx KO cells, that the number of foci was significantly increased in pluripotent cells (Figure34A, Figure34B). This effect was abolished upon differentiation of the cells. The increase in the number of major satellite clusters was correlated with a diminution in the sizes of each of these foci (Figure34B). Thus, the loss of Pml in pluripotent cells impairs the clustering of pericentromeric regions, a phenotype that resemble to Daxx-deficient ESCs.The absence of Pml disturbed the organization of pericentromeric heterochromatin.Because the Daxx KO ESCs displayed the same phenotype together with an altered radial positioning of the chromocenters, we decided to measure the peripheral signal of PCH in the absence of Pml. We observed that Pml KO ESCs displayed an altered radial localization of major satellites. Indeed, in both serum and ground-state conditions, the lack of Pml resulted in a more peripheral positioning of pericentromeric clusters (Figure34C). Conversion toward ground-state induced a central relocalization of major satellites foci, although it remained more peripheral compared to WT ESCs (Figure34C). Our results indicate that in addition to the altered organization of major satellites, the absence of Pml induces a peripheral localization of chromocenters.

Figure

  Figure 34| PML contributes to pericentromeric heterochromatin organization. (A) Representative immunoFISH experiments of H3K9me3 histone modification and major satellite repeats of WT and Pml KO cells in serum-based or ground-state condition. (B) Quantification of PCH clustering. Top, number of major satellite foci detected in medium focal plane. Bottom, area of major satellite foci of focal medium plane. Boxplots represents three independent experiments. (C) Quantification of radial positioning of major satellite signal in serum or ground-state condition, using automatic threshold as previously described. Boxplot represents 10%-most peripheral compared whole-nucleus major satellite fluorescence intensities of three independent replicates. Two-sided Mann-Whitney tests were used for statistical analysis. GS = ground-state; Diff = Differentiation.

Figure 35|

 35| Figure 35| Loss of Pml induces alteration of H3K9me2 peripheral signal upon groundstate conversion. (A) Representative immunofluorescence pictures of WT or Pml KO cells in serum or groundstate media. White arrows highlight patches of H3K9me2 only visible in Pml KO cells upon ground-state conversion. (B) Quantification of H3K9me2 heterogeneity. A line is traced at the periphery of the nucleus. A homogeneity score is calculated from signal of each pixel from the line representing the variation from the mean. Boxplot displays homogeneity scores measured from at least three biological replicates. n = total number of nuclei analyzed. (C) Representative pictures of LaminB immunofluorescence in WT or Pml KO cells in serum-based or ground-state conditions. GS = ground-state. Two-sided Mann-Whitney tests were used for statistical analysis.

  ). While the loss of Daxx led to strong upregulation of 12 to 500-fold, the absence of Pml resulted in a moderate increase of 2 to 21-fold. Our results indicate that the absence of Pml results in the same tendency of transcriptional up-regulation of LAD-genes upon hypomethylation of pluripotent cells than Daxxdeficient ESCs.

Figure

  Figure 36| Absence of Pml induces the loss of LAD-genes upon ground-state conversion.Messenger RNA levels of one fLAD and 5 cLAD-genes in WT or Pml KO in serum or upon ground-state conversion. Histogram represents mean expression with SEM from two biological replicates. Highlighted are the up-regulation measured from Pml KO compared to WT in ground-state. GS = ground-state.

Figure

  Figure 37| DAXX-mediated PCH clustering is independent of ATRX interaction. (A) Representative immunoFISH pictures for Tale-Flag and major satellite repeats in Daxx KO-transfected cells. (B) Quantification of the number of major satellite foci measured in Daxx KO-transfected cells. Boxplot represents data from 3 biological replicates, except for DAXX9KR which is 2 biological replicates. n=total number of nuclei analyzed. (C) Quantification of the number of major satellite foci measured in WT transfected cells. Boxplot represents data from 3 biological replicates, except for DAXXΔSIM which is 2 biological replicates. n=total number of nuclei analyzed. (D) Representative immunofluorescence pictures of ATRX and PML in WT, Daxx or Pml KO ESCs in serum-based condition. (E) Quantification of the number of DAPI-dense chromocenters displaying ATRX signal. Histogram represents one of two independent experiments Two-sided Mann-Whitney tests were used for statistical analysis in B and C. Chi-square tests were used for statistical analysis in E.

  Moreover, we demonstrated that specific recruitment of DAXX toward major satellites could drive a strong clustering of chromocenters (Figure4Darticle). DAXX-mediated PCH clustering is independent of ATRX binding. Interestingly, DAXX localizes within PML NBs and possesses sites of post-translational modifications, we thus decided to further investigate the domains of DAXX necessary for proper pericentromeric clustering in relation with PML. Daxx notably possesses two SUMO-interacting motif (SIM) at the N-and C-termini. SIMs are domains that can bind to SUMOylated proteins and can thus participate in protein-protein interactions. It was observed that only the C-terminal SIM was required for recruitment to PML NBs (Escobar-Cabrera et al. 2011;

Figure

  Figure 38| SUMOylation but not SIM domain of DAXX seem implicated in PCH clustering. (A) Representative immunoFISH pictures of major satellites and Flag signal in serum Daxx KO ESCs transfected with the indicated Tale constructs. (B, D) Quantification of the number of major satellite foci detected on medium focal plane in Daxx KO ESCs transfected with the indicated Tale constructs. (C, E) Quantification of the number of major satellite foci detected on medium focal plane in Daxx KO ESCs transfected with the indicated Tale constructs. n=total number of nuclei analyzed from 3 biological replicates, except for DAXX9KR which corresponds to 2 biological replicates. Two-sided Mann-Whitney tests were used for statistical analysis.

  3 and that only H3.3 recruitment correlates with DAXXmediated clustering, strengthening the role of H3.3 in heterochromatin organization.

Figure 39|

 39| Figure 39| Recruitment of H3.3 and PML upon DAXX targeting to PCH. (A) Representative immunofluorescence pictures of PML and H3.3 in WT-transfected cells. (B) Quantification of H3.3-recruitment from the different Tale-DAXX constructs. Histogram displays mean with SEM from 2 biological replicates. n=total number of nuclei analyzed. (C) Quantification of PML-recruitment from the different Tale-DAXX constructs. Histogram displays mean with SEM from 2 biological replicates. n=total number of nuclei analyzed. Chi-square tests were used for statistical analysis.

CONCLUSIONS

  In this work, I have studied the role of the H3.3-chaperone DAXX on heterochromatin maintenance in murine embryonic stem cells. The main conclusions from the article are the following (Figure 40): o DAXX is essential for cell survival upon DNA hypomethylated conditions; o DAXX contributes to the silencing of different heterochromatin regions such as dispersed DNA repeats and pericentromeric satellites; o Active DNA demethylation of major satellites can generate DNA damage signaling and lead to DAXX accumulation at chromocenters, yet DNA damage signaling alone does not trigger DAXX recruitment; o At PCH, DAXX recruits SETDB1 in a H3.3-dependent manner and induces H3.3K9 trimethylation; o DAXX regulates spatial and molecular organization of chromocenters in pluripotent cells. I deeply investigated the functions of DAXX in the second part of the results and observed that (Figure 40): o DAXX contributes to the maintenance of chromatin state identity and its loss induces a 2C-like transcriptional signature upon ground-state conversion; o DAXX controls chromocenters radial positioning; o DAXX contributes to the proper distribution of H3K9me2-peripheral mark and the silencing of genes present within lamina associated domains; o Loss of Pml results in phenotypes highly similar to the loss of Daxx, suggesting that PML might act as a regulator of DAXX.

FigureDISCUSSION 1 .

 1 Figure 40| Model displaying the function of DAXX at pericentromeric heterochromatin and the consequences of its loss. Upon ground-state conversion, active DNA demethylation induced by the TET enzymes can lead to DNA damages at chromocenters. DNA damage response proteins act at the site of the lesion. DAXX accumulates at a limited number of chromocenters and is surrounded by a PML shell. DAXX can then recruit the lysine methyl-transferase SETDB1 to induce H3.3K9 tri-methylation and reform the heterochromatin state.In the absence of Daxx, pericentromeric heterochromatin organization is altered and more chromocenters are visible and major satellites are transcriptionally up-regulated. Upon ground-state conversion, DaxxKO ESCs present a strong increase in DNA repeats expression. Furthermore, genes localized within lamina associated domains are de-repressed and H3K9me2 ring pattern at the nuclear periphery is altered. Many genes are mis-regulated in ground-state condition and a 2C-like transcriptional signature is observed. Finally, prolonged culture in DNA hypomethylated conditions lead to cell death in the absence of Daxx. Depletion of Pml results in phenotypes highly similar to the loss of Daxx such as impaired organization of chromocenters. Ground-state conversion of PmlKO resemble DaxxKO ESCs as an alteration of the H3K9me2 peripheral ring is visible and the concomitant loss of silencing of genes present in LADs. Prolonged culture in DNA hypomethylated conditions results in cell death as well.

  damages, only 1 to 3 chromocenters displayed DAXX accumulation (Figure 2C article, 2E article). Accumulation of DAXX at PCH highly resemble to what is observed in the human ICF syndrome, where only 3 chromosomes present DAXX recruitment toward pericentromeric satellites (Luciani et al. 2006). It raises the possibility that DAXX binding might happen at specific chromosomes upon DNA demethylation. Yet, mouse chromosomes, unlike human counterparts, possess highly similar pericentromeric satellites. It would be informative to perform immunoFISH experiments using probes targeting specifically each chromosomes and count whether DAXX accumulation occurs at different rates for every chromosome. The other possibility is that because we are looking in fixed cells, we might only observe a transient binding of DAXX. Investigating the recruitment along time would require the use of live-cell imaging using both a fluorescent marker for chromocenters and for DAXX and see if the assembly-disassembly of DAXX structures can happen at multiple occasions during a single interphase.We note that SETDB1 is recruited by DAXX at PCH (Figure5A article). Silencing of ERVs in mESCs mostly rely onto the action of KRAB-Zinc finger (ZF) proteins, KAP1, SETDB1

  article). However, to better characterize the active DNA demethylation takes places at PCH, it would be informative to quantify the levels of 5mC and 5hmC in serum-based ESCs and upon ground-state conversion.TDG and BER induce the removal of the 5hmC base, which generates abasic sites (AP-sites). As BER is important for the DNA demethylation process, it would be interesting to evaluate the role of BER proteins such as TDG, XRCC1 or LIG3 onto the accumulation of DAXX at PCH, which would indicate whether AP-sites are driving DAXX recruitment. Upon TET1CD targeting to major satellites, we detect a homogenous reduction of 5mC signal at chromocenters, while γH2AX foci appear as discrete dots, suggesting that the visible γH2AX foci might mark DSB sites (FigureS3Aarticle, 3D article). The observation that broad DNA demethylation results in local DNA damage signaling argues against DDR being a mere consequence of BER. Active DNA demethylation can ultimately results in DSBs, although the coordinated action of TET, TDG and BER proteins usually avoid the formation of DSBs[START_REF] Weber | Biochemical reconstitution of TET1-TDG-BER-dependent active DNA demethylation reveals a highly coordinated mechanism[END_REF]. However, since we target genomic regions that are tandemly repeated over Mbs-long distances, it could result in an important number of AP-sites, which can lead to DSBs if unresolved[START_REF] Kidane | Accumulation of abasic sites induces genomic instability in normal human gastric epithelial cells during Helicobacter pylori infection[END_REF]. Studying the mechanisms of DSB repair at chromocenters, Tsoroula et al.observed that somatic NIH3T3 murine cells relocate DSBs to the periphery of chromocenters to perform HR only in G2-phase, whereas in G1-phase, DSBs remain within heterochromatin foci and undergo non-homologous end joining (NHEJ)[START_REF] Tsouroula | Temporal and Spatial Uncoupling of DNA Double Strand Break Repair Pathways within Mammalian Heterochromatin[END_REF]. Upon ground-state conversion, we observe the presence of discrete γH2AX foci at chromocenters, which agrees with possible rare DSBs occurring at DNA hypomethylated major satellites (Figure2D). Furthermore, exogenous induction of DSBs via HU-treatment or CRISPR/Cas9 targeting major satellites results in the accumulation of DAXX, indicating that DSBs alone can lead to DAXX recruitment (Figure2F). The accumulation of DAXX, after DSBs or ground-state conversion, often appear at the periphery of chromocenters, which is also compatible with the model proposed by Tsoroula et al.(Figure2A). Confirming that active DNA demethylation at PCH could be responsible for DAXX relocalization would require to use the Tale-TET1CD in Dnmt tKO ESCs, which should not lead to DNA damage signaling. Furthermore, it would be interesting to knock-down TET enzymes in ground-state ESCs and look for the disappearance of DAXX foci. Remarkably, the knock-down of TET enzymes could also rescue the survival of Daxx-deficient cells upon ground-state conversion. Symmetrically, the knock-down of DNMT enzymes or the over-expression of TET enzymes in serum-based medium could increase the number of DAXX foci at PCH.

  article). We observe the same pattern using CRISPR/Cas9-or TET1-demethylation-induced PCH DNA damages, with γH2AX foci visible within chromocenters but DAXX mostly localized at the periphery of the DAPI-rich signal (Figure2Farticle, S2C article, 3D article, 3E article). To confirm that DAXX structures contain chromatin undergoing late DNA repair steps, it would be interesting to perform immunofluorescence of proteins involved at different times of the repair, using inducible DNA damages systems. H3.3-deposition has been implicated in replication fork progression after DNA damages, possibly indicating that DAXX-mediated H3.3 deposition might be important upon ground-state condition to overcome DNA lesions[START_REF] Frey | Histone H3.3 is required to maintain replication fork progression after UV damage[END_REF]. It has also been reported that Daxx down-regulation prevented H3.3 relocalization at sites of UV lesions and impaired homologous recombination, in good agreement with our observations that loss of Daxx increases DNA damages[START_REF] Juhasz | ATRX Promotes DNA Repair Synthesis and Sister Chromatid Exchange during Homologous Article ATRX Promotes DNA Repair Synthesis and Sister Chromatid Exchange during Homologous Recombination[END_REF]. Of particular interest, DAXX and PML are important regulators of SUMOylation, and notably in ESCs (S. Tessier, V. Lallemand-Breitenbach, submitted article). Intriguingly, BER proteins necessary upon active DNA demethylation are regulated by

  Interestingly, HU generates replication fork collapses and it thus remains to assess if DNA replication stalks are more frequent upon DNA hypomethylation. It is possible that DNA replication fork collapses are more frequent upon active DNA demethylation as it will generates AP-sites which could slow the replication fork, notably at pericentromeric heterochromatin regions, which are highly methylated. Moreover, in the absence of Daxx, pericentromeric satellites are up-regulated, which might increase the level of R-loops (Figure 1C article). R-loops formation is increased upon transcriptionreplication conflicts and can generate DNA breaks, demonstrating that defects in heterochromatin silencing possibly contributes to genome stability (Hamperl et al. 2017). Interestingly, in the C. elegans worm, it has been observed that the SETDB1 homolog and the BRCA1 homolog are implicated in the silencing of satellite repeats (Padeken et al. 2019). Deletion of the SETDB1 homolog induces a loss of satellite repression that ultimately results in DNA damage-mediated recruitment of BRCA1 homolog. Deletion of both SETDB1 and BRCA1 homologs result in enhanced levels of R-loops. It would thus be interesting to gauge the level of R-loops in Daxxdeficient cells and assess whether RNAseH treatment, that specifically degrades DNA-bound RNAs, could both prevent DAXX recruitment at pericentromeric satellites and cell survival of Daxx KO ESCs upon ground-state conversion. Importantly, the specific transcriptional activation of major satellites using a Tale strategy induced γH2AX foci at PCH, suggesting that strong upregulation of satellites is sufficient to generate DNA damages (Figure S3C article). However, because major satellite transcription decreases upon ground-state conversion, while DNA damages at chromocenters are increased, it seems that DNA damages at PCH can arise from nontranscriptional defects (Figure 2D article, S1B article).

  3-specific lysine 27 residue[START_REF] Santenard | Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3[END_REF]. Mutation of the lysine 27 of H3.3 impaired localization of H3.3 and prevented development toward the blastocyst stage. Developmental defects of H3.3K27R could only be overcome with the addition of major satellite dsRNAs. Deletion of zygotic Daxx results in defects to reach the blastocyst stage, with decreased viability from the 4-cell stage, while maternal Daxx is dispensable for proper development, suggesting an additional role for DAXX apart from the transcriptional activation of pericentromeric repeats at the pluripotent stage[START_REF] Liu | SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin[END_REF].Because the loss of Setdb1 also results in early embryonic lethality, before the blastocyst stage, we propose that novel role of DAXX might act through SETDB1. It would be of great interest to assess the in vivo relevance of the DAXX-SETDB1-H3.3-H3K9me3 pathway we uncovered in pluripotent cells.

(Figure

  4C article). Intriguingly, we demonstrate that DAXX could recruit SETDB1 at pericentromeric regions to induce H3K9me3, that we propose to take place specifically onto the H3.3 variant (Figure5D article). It raises the possibility that HP1α, and perhaps other proteins, have different affinities for H3.3K9me3 than for canonical H3K9me3. To gain deeper understanding of pericentromeric regulation, it would be important to perform biochemistry experiments such as chromatin IP or in vitro affinity assays to assess the importance of the histone bearing the tri-methylated lysine 9 for HP1α binding. As no link has been established so far between DAXX and HP1α, it is intriguing to observe such differences in HP1α dynamics (Figure4Barticle). Furthermore, the absence of Daxx diminishes heterochromatin barrier, possibly via alteration of HP1α LLPS (Figure4C article). It suggests that additional proteins might be able to modulate the LLPS properties of HP1α[START_REF] Strom | Phase separation drives heterochromatin domain formation[END_REF]. It would thus be informative to study the LLPS properties of HP1α in the absence of Daxx using live-cell imaging and treating the cells with the 1,6-hexanediol molecule that abolishes LLPS. Moreover, defects in chromocenter formation might come from additional protein binding to specific DNA sequence or histone modifications. An example is the HMGA1 protein in somatic murine cells that accumulates at chromocenters and is important for major satellite clustering and genome integrity[START_REF] Jagannathan | A conserved function for pericentromeric satellite DNA[END_REF]. It would be informative to look at HMGA1 or other HMG proteins in ESCs in serumbased and ground-state conditions in Daxx KO ESCs. Furthermore, to gain a more comprehensive view of pericentromeric heterochromatin protein network and how it is affected upon the loss of Daxx, it could be informative to perform PICh experiments as previously described[START_REF] Saksouk | Redundant Mechanisms to Form Silent Chromatin at Pericentromeric Regions Rely on BEND3 and DNA Methylation[END_REF]).An additional layer of control may come from major satellite RNAs. Interestingly, SUV39H1/2 enzymes can bind to RNA and transcription of major satellites has been proposed to help SUV39H1/2 action by retaining the enzymes at the right localization[START_REF] Johnson | RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin[END_REF][START_REF] Velazquez Camacho | Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation[END_REF]. We observe that depletion of Daxx in mESCs induces an upregulation of major satellite transcripts (Figure1Carticle). Because Daxx KO ESCs display misregulation of major satellite transcription, it could impair proper binding of SUV39H1/2 or other RNA-binding proteins. We notice that the specific activation of major satellite transcription using a Tale-VP64 could induce DAXX-recruitment at chromocenters (FigureS3D article). Yet, specific activation of PCH generated DNA damages as well, making it difficult to uncouple the functions of the transcription and of the produced RNA from the DNA damages (FigureS3Earticle). It would be interesting to test whether RNA production is important for DAXX recruitment and function at PCH by specifically knocking-down major satellite RNAs or transfecting major satellite dsRNA.

  in 2C-like repression as its depletion enhanced conversion from pluripotent ESCs toward 2C-like totipotent cells[START_REF] Rodriguez-Terrones | A molecular roadmap for the emergence of early-embryoniclike cells in culture[END_REF]. In that direction, it would be interesting to deeper investigate the interplay between DAXX and PRC1 in the control of the 2C-like transcriptional program. PRC1 was demonstrated to participate into the nuclear organization of chromatin in an enzymatic and CTCF-independent manner, suggesting a structural role for PRC1[START_REF] Boyle | A central role for canonical PRC1 in shaping the 3D nuclear landscape[END_REF][START_REF] Vieux-Rochas | Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain[END_REF]. One could hypothesize that the structural function of DAXX and possibly H3.3 extend beyond constitutive heterochromatin regions in pluripotent cells via the regulation of polycomb-target genes and it would be interesting to assess the global genome organization and chromatin compaction at polycomb-marked genes in Daxx-deficient cells.He et al. reported that DAXX can recruit SUV39H1 to maintain telomeric heterochromatin notably upon DNA hypomethylation[START_REF] He | The Daxx/Atrx Complex Protects Tandem Repetitive Elements during DNA Hypomethylation by Promoting H3K9 Trimethylation[END_REF]. Although we notice an increased recruitment of DAXX at telomeres, our data do not support this model (data not shown). Indeed, we observe that Suv39H1/2 dKO could grow well even upon ground-state conversion, indicating nonessential functions upon DNA hypomethylation (Figure5C article). We observe that DAXX rather recruit SETDB1 (Figure5Aarticle). DAXX, PML and SETDB1 are observed at telomeres in mESCs and DAXX-deposited H3.3 can be modified into H3.3K9me3 at this location (Udugama

  Figure 34B, Figure35B). It could be informative to better characterize Pml KO phenotypes as for Daxx KO such as DNA repeats expression and activation of IFN response upon ground-state conversion. Since PML NBs act as a hub for SUMOylation and that DAXX can be regulated via SUMOylation, it remains to assess whether PML contributes to DAXX regulation. Interestingly, we detected that in the absence of Daxx, ground-state ESCs displayed a 2C-like transcriptional signature (Figure27B). If DAXX is regulated by PML, Pml-deficient cells should display the same transcriptional signature. Indeed, the absence of Pml induces the activation of a 2C-like transcriptional profile, providing evidence that PML might regulate DAXX (S. Tessier, V.Lallemand-Breitenbach, submitted article). Interestingly, the only E3 SUMO ligase, UBC9 has been implicated in repression of 2C-like program, indicating a broad SUMOylation network safeguarding chromatin identities[START_REF] Cossec | SUMO Safeguards Somatic and Pluripotent Cell Identities by Enforcing Distinct Chromatin States[END_REF]. Furthermore, as mentioned in the second part of the discussion, DAXX is recruited at PCH regions in the early embryo via PRC1[START_REF] Liu | SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin[END_REF]. Interestingly, Liu et al. reported that PRC1-mediated recruitment of DAXX is regulated via SUMOylation. Additionally, it was reported that PML could regulate PRC2 activity and that deletion of Pml induced a global redistribution of H3K27me3 histone modification[START_REF] Amodeo | A PML/Slit Axis Controls Physiological Cell Migration and Cancer Invasion in the CNS[END_REF][START_REF] Delbarre | PML protein organizes heterochromatin domains where it regulates histone H3 . 3 loading by ATRX / DAXX[END_REF]. Therefore, it would be interesting to assess the role of PML onto polycomb complexes regulation and DAXX-polycomb interaction.Furthermore, in addition to the impaired spatial organization of chromocenters, we observe differences in HP1α dynamics in the absence of Daxx, and we propose that DAXX directly or indirectly controls LLPS properties of HP1α (Figure4C article). It would be interesting to evaluate the impact of Pml loss onto heterochromatin barriers and LLPS properties of HP1α as

  PCH in the absence of Daxx or Pml. Together, these data suggest the existence of a SUMOylation network that might regulate LLPS properties of different proteins such as PML or HP1α at PCH. We demonstrate that targeted recruitment of DAXX to major satellites induces a strong clustering of PCH (Figure 4D article). Upon further characterization of the domains of DAXX necessary for PCH clustering, we notice that interaction with H3.3 is essential as neither DAXXY222A nor expression of H3.3G90M can form big PCH clusters like WT DAXX (Figure 5C article, 5D article). Intriguingly, we observe that DAXX could recruit SETDB1 and PML but DAXXY222A could not (Figure S5C article). Since the role of DAXX at PCH is to recruit SETDB1, it is possible that SETDB1 regulation might play a role upon ground-state conversion. By immunofluorescence, we detect SETDB1 as homogeneously distributed within the nucleoplasm and the cytoplasm in serum-based ESCs, and rarely as nuclear dots (Figure S5A article). However, upon-ground-state conversion, SETDB1 appears as foci colocalizing with PML NBs and 'giant' DAXX-PML structures at PCH (Figure 5A article).

  Près de la moitié du génome est composé de séquences répétées correspondant à l'hétérochromatine, et qui doivent être maintenus éteintes transcriptionnellement. Des défauts de maintenance de l'hétérochromatine sont souvent observés dans différentes pathologies et peuvent mener à la tumorigénèse. Les régions hétérochromatiques peuvent être dispersées ou localisées, notamment autour des centromères. Les régions péricentromériques restent inactives dans les cellules somatiques grâce à l'action combinée de la méthylation de l'ADN et de la modification d'histone H3K9me3, reconnue par différentes protéines dont HP1α. Cette organisation particulière émerge au cours du développement embryonnaire précoce. En effet, au stade 2-cellules à lieu une forte activation transcriptionnelle des régions répétées comme les éléments LINE1 qui permettent une décompaction globale du génome. Au même moment se produit une forte transcription des satellites majeurs composant les régions péricentromériques. Cette activation permet la mise en place dès le stade 4-cellules des chromocentres, régions visibles en DAPI où se regroupent les régions d'hétérochromatine péricentromériques (PCH) de multiples chromosomes. De manière intéressante, l'activation des régions hétérochromatines correspond à une baisse drastique du niveau de méthylation de l'ADN au stade 2-cellules. Ces régions sont ensuite progressivement réduites et à nouveau fortement réprimées à partir du stade blastocyste quand le niveau de méthylation de l'ADN est rétabli. Ainsi, la transcription de l'hétérochromatine depuis le stade 2-cellules jusqu'au stade blastocyste est contrôlée de manière indépendante de la méthylation de l'ADN (5mC). Les cellules souches embryonnaires (ESC) présentent des niveaux faibles de méthylation de l'ADN comparés aux cellules différentiées. Les ESC sont dérivées des cellules de la masse interne du blastocyste et représentent un modèle de choix pour étudier le développement embryonnaire. Les ESC présentent les caractéristiques des cellules pluripotentes et peuvent se diviser en tous types cellulaire formant l'organisme. En revanche, lors de la culture des CSE in vitro, elles accumulent de plus en plus de méthylation de l'ADN. Un nouveau milieu de culture appelé 2i pour 2 inhibiteurs a été développé par Ying et al. En 2008. L'ajout de vitamine C en milieu 2i permet de réduire très substantiellement le niveau de 5mC et les CSE, à l'inverse des cellules différenciées, supportent l'absence de méthylation de l'ADN. Certains facteurs permettant le contrôle de l'hétérochromatine en condition hypométhylée ont été décrit comme BEND3 et les protéines des complexes polycomb aux péricentromères et DAXX et SETDB1 aux rétrovirus endogènes dispersés. De manière intéressante, DAXX localise aux PCH du noyau paternel, fortement déméthylé, dans les tous premiers jours du développement. Cependant, la délétion génétique de Daxx ne perturbe le développement de l'embryon qu'à des stades plus tardifs et empêche la formation du blastocyste, suggérant une forte importance de DAXX pour permettre la transition des cellules embryonnaires vers un état pluripotent, quand les chromocentres se forment. DAXX est la chaperonne du variant d'histone H3.3 spécifique pour les régions d'hétérochromatine. Un enrichissement de H3.3 est observé à la fois à certaines régions de l'hétérochromatine comme de l'euchromatine, mais sa déposition est alors médiée par le complexe HIRA. De manière intéressante, H3.3 est essentiel pour le développement embryonnaire comme pour la maintenance des ESC en culture. Un défaut de H3.3 entraine une instabilité chromosomique, suggérant un rôle crucial au niveau de l'hétérochromatine. De plus, H3.3 fournit la modification H3K9me3 aux régions télomériques et aide au maintien de l'état hétérochromatique à certains rétrovirus endogènes (ERV). Cependant, son rôle aux satellites péricentromériques a surtout été envisagé comme facilitant la transcription. Dans ce travail, j'ai utilisé les cellules souches embryonnaires murines comme un modèle pour étudier le rôle de DAXX dans la maintenance de l'hétérochromatine et obtenir une meilleure compréhension de la régulation des régions péricentromériques dans les cellules pluripotentes. Résultats Afin d'étudier le rôle de DAXX, j'ai généré une lignée inactivée pour le gène Daxx grâce à la technologie CRISPR/Cas9. De manière intéressante, j'ai observé que les cellules DaxxKO étaient capable de se différencier. En revanche, en cultivant les cellules dans un milieu 2i et vitamine C afin qu'elles atteignent l'état naïf de pluripotence, j'ai observé qu'après 8 à 10 jours de culture les cellules DaxxKO périssaient. L'analyse transcriptomique dans ces différents milieux a révélé que l'absence de Daxx entrainait une forte activation de nombreuses familles d'ADN répété, notamment certains ERV, certains LINE1 et les satellites majeurs composant les PCH. De plus, un très grand nombre de gènes sont différentiellement exprimés en l'absence de Daxx en condition naïve de pluripotence. Parmi les gènes moins exprimés se trouvent ceux intervenant dans les différentes voies de réparation de l'ADN. Dans un milieu « classique » contenant du sérum, DAXX localise aux corps nucléaires PML (PML NBs). Après conversion vers l'état naïf de pluripotence, DAXX accumulait à certaines régions péricentromériques dans la majorité des cellules. PML accumulait également autour de ces structures formées par DAXX. De manière intéressante, la conversion vers l'état naïf de pluripotence hausse le nombre de chromocentre présentant des foyers γH2AX. De plus, l'induction des dommages à l'ADN de manière aléatoire ou ciblée spécifiquement aux régions péricentromériques entraine la relocalisation de DAXX aux PCH. Néanmoins, le recrutement de NBS1 aux PCH, induisant l'accumulation de γH2AX ne suffit pas à recruter DAXX. Traiter les cellules avec du 5-azacytidine, réduisant le niveau de méthylation de l'ADN réduisit la survie des cellules DaxxKO de manière similaire à celle observée en milieu 2i et vitamine C. De plus, le traitement avec le 5-azacytidine entrainait également l'accumulation de DAXX aux PCH. Afin de confirmer que la déméthylation de l'ADN était responsable des dommages à l'ADN observés, j'ai recruté une protéine Tale ciblant spécifiquement les satellites majeurs fusionné au domaine catalytique de l'enzyme TET1 (TET1CD). Cette construction Tale-TET1CD permet la déméthylation active spécifique des chromocentres menant à une légère élévation de la transcription des satellites majeurs. De manière remarquable, la déméthylation active entraine l'apparition de foyers γH2AX aux chromocentres ainsi que le recrutement de DAXX, démontrant ainsi que la déméthylation active peut mener à des dommages à l'ADN et suffit à entrainer la relocalisation de DAXX. Bien que DAXX ne soit pas visible aux chromocentres en condition sérum, son absence entraine une forte désorganisation de l'organisation des séquences satellites majeurs. En effet, j'ai observé que la perte de DAXX augmentait le nombre de foyer de satellites majeurs et ceux-ci sont également plus petits en conditions pluripotentes mais cet effet disparait après différentiation des cellules. De plus, l'accessibilité de la chromatine, mesurée par digestion à la MNase est plus forte dans les cellules DaxxKO, suggérant une altération de la compaction de l'hétérochromatine. En mesurant par FRAP la récupération de fluorescence de GFP-HP1α, une protéine reconnaissant la modification H3K9me3 et fortement enrichie aux chromocentres, j'ai montré que l'absence de Daxx augmente la dynamique de HP1α. Il semble également que cette dynamique accrue corrèle avec une altération de la variance du signal GFP-HP1α au cours du temps, ce qui suggère que la perte de Daxx puisse influer sur la capacité de séparation de phase de HP1α. Enfin, le recrutement de DAXX aux satellites majeurs à l'aide d'une protéine Tale entraine une forte diminution du nombre de chromocentres visibles, montrant ainsi que DAXX est capable de générer un rassemblement spatial des régions péricentromériques. La modification H3K9me3 aux chromocentres est mise en place par les enzymes SUV39H1/2. De manière intrigante, la troisième H3K9 tri-méthylase SETDB1 est recrutée aux chromocentres par DAXX. De plus, les enzymes SUV39H1/2 ne sont pas nécessaires à la survie des ESC en condition naïve de pluripotence, et ces cellules présentes également du signal H3K9me3 aux chromocentres uniquement dans cette condition et pas en sérum. A l'aide de Tales, j'ai observé que l'interaction de DAXX avec H3.3 était nécessaire pour recruter SETDB1 et H3K9ME3, et pour former un rassemblement spatial des séquences péricentromériques. De plus, le recrutement de l'enzyme SUV39H1, bien que capable d'induire une forte concentration de H3K9me3, ne forme pas de rassemblement spatial des satellites majeurs. Enfin, grâce à des versions mutantes de H3.3, j'ai démontré que la lysine 9 de H3.3 était nécessaire à cette réorganisation spatiale des satellites majeurs. En outre, l'analyse transcriptionnelle a révélé que l'absence de Daxx entrainait une activation des gènes actifs dans un état ressemblant au stade 2-cellules, suggérant que DAXX participe à la répression de cet état et au maintien de l'identité chromatinienne des cellules pluripotentes. En plus de la perte d'organisation spatiale des chromocentres, j'ai montré qu'en l'absence de Daxx, les chromocentres étaient plus souvent visibles à la périphérie du noyau. Dans le même temps, la modification d'histone associée à la périphérie nucléaire H3K9me2 est altérée dans le DaxxKO, uniquement en condition naïve de pluripotence et apparait de manière discontinue. Cette perturbation de la distribution de H3K9me2 en condition naïve de pluripotence corrèle avec l'activation des gènes présents dans les domaines associés à la lamina, même si la relation de cause à effet reste à démontrer. Lors de la relocalisation de DAXX en conditions hypométhylées, un corps nucléaire géant PML se forme également autour de DAXX. J'ai donc généré une lignée inactivée pour le gène Pml et observé que plusieurs phénotypes étaient communs aux Daxx et Pml KO. En effet, l'absence de Pml empêche la survie des cellules en conditions hypométhylées, entraine une altération de l'organisation des chromocentres, ainsi que l'activation des gènes présent dans les domaines associés à la lamina spécifiquement en condition naïve de pluripotence. Cependant, l'absence de Pml n'impacte pas l'accumulation de DAXX aux chromocentres. De plus, en caractérisant les domaines de DAXX nécessaires au regroupement spatial des satellites majeurs, j'ai observé que seules les lysines de DAXX sujettes à la SUMOylation étaient requises pour cet effet de DAXX. Ces résultats suggèrent que PML pourrait avoir un rôle dans la régulation du mécanisme d'action de DAXX, possiblement par des modifications post-traductionnelles comme la SUMOylation. Discussion Ce travail a mis en évidence un nouveau rôle essentiel de DAXX, la chaperonne du variant d'histone H3.3, dans le maintien de l'hétérochromatine péricentromérique des cellules pluripotentes. J'ai observé que lors de la conversion des ESC vers un milieu diminuant drastiquement le niveau de méthylation de l'ADN, DAXX accumulait fortement aux chromocentres et entrainait la relocalisation de PML autour de ces structures géantes de DAXX. De manière intéressante, une localisation similaire des DAXX aux régions péricentromériques a été décrite au cours des stades précoces du développement embryonnaire. Dans l'embryon, le recrutement de DAXX dépend de plusieurs protéines comme DPPA3 et le complexe PRC1. De plus, la perte de méthylation dans les cellules pluripotentes induit le recrutement de BEND3 et des protéines appartenant au complexe PRC1 aux chromocentres pour induire la modification H3K27me3. Il serait intéressant d'évaluer ces mêmes protéines participent au recrutement de DAXX. Cependant, BEND3 et les protéines du complexe PRC1 sont présents à la plupart des chromocentres, là où le recrutement n'est visible qu'à un nombre très limité de chromocentres lors de la conversion vers la condition naïve de pluripotence. Ces données suggèrent que le recrutement de DAXX pourrait être régulé par des mécanismes différents. Dans les cellules DnmtKO, il a été montré que DAXX se fixait à un certain nombre de d'éléments ERV. Il est intéressant de noter qu'une publication récente par Ostromyshenskii et al. rapportait que certains éléments ERV étaient dispersés entre les répétitions de satellites majeurs et étaient donc localisés dans les chromocentres. Il est possible que DAXX puisse être recruté à ces éléments ERV particuliers localisant aux chromocentres (PCH-ERV). Plus d'expériences seront nécessaires pour confirmer cette hypothèse qui ouvrirait de nouvelles perspectives sur un potentiel rôle des éléments ERV dans l'organisation 3D du génome. Le ciblage de TET1 aux satellites majeurs peut générer l'accumulation de foyers γH2AX aux chromocentres, démontrant que la déméthylation active de l'ADN peut mener à des dommages à l'ADN. De manière similaire, j'ai observé que la conversion vers l'état naïf de pluripotence entrainait une accumulation de foyers γH2AX aux chromocentres. De plus, la génération des cassures double brin par CRISPR/Cas9 est suffisante pour induire le recrutement de DAXX aux satellites majeurs, démontrant que DAXX est relocalisé suite à des dommages à l'ADN. Cependant, le ciblage de NBS1, permettant d'induire une forte présence de γH2AX aux chromocentres ne conduit pas à la relocalisation de DAXX, suggérant ainsi que DAXX ne fasse pas intrinsèquement parti de la voie de réponse aux dommages à l'ADN, mais peut être à un stade plus tardif pour la reformation de l'hétérochromatine. Les chromocentres montrent une forte présence de la modification H3K9me3, déposée à cet endroit par les enzymes SUV39H1/2. Dans ce travail, j'ai montré que DAXX recrutait l'autre enzyme H3K9 tri-méthylase SETDB1 afin d'exercer son activité aux régions péricentromériques. Des enzymes KDM permettant l'enlèvement de la modification H3K9me3 ont été impliqué dans le choix et le déroulement de voies de réparations de l'ADN. Cela suggère que la fonction de SETDB1 soit de rétablir la modification d'histone H3K9me3, éventuellement sur le variant H3.3. De futures études sont nécessaire pour mieux appréhender le mécanisme moléculaire permettant le recrutement des KDM puis de DAXX aux sites de cassures. Il est possible qu'un mécanisme similaire dépendant de l'activité de DAXX et de SETDB1 soit effectif dans les ESC en sérum contenant un fort niveau de méthylation. Toutefois, la présence de méthylation de l'ADN pourrait amener à un niveau de cassure plus bas qui génèrerait un recrutement de DAXX plus transitoire ou plus localisé, comme aux PCH-ERV, empêchant ainsi son observation par nos techniques d'imageries. De plus, une nouvelle compréhension de l'organisation des chromocentres est venue de la réalisation que HP1α peut s'agréger par séparation de phase. Etonnamment, j'ai observé que l'absence de Daxx altérait la dynamique et, de manière directe ou non, la capacité de séparation de phase de HP1α. Comme le rassemblement des régions péricentromériques induit par DAXX repose sur son lien avec H3.3, cela suggère qu'il puisse y avoir des différences encore inconnues de la fixation de HP1α sur H3K9me3 et H3.3K9me3. Enfin, j'ai observé que DAXX était essentiel pour la conversion des ESC vers un milieu diminuant drastiquement le niveau de méthylation de l'ADN. La fonction de DAXX aux chromocentres est de déposé H3.3 et de recruté SETDB1. Il est intéressant de noter que la perte de Setdb1 empêche la survie des cellules pluripotentes et également des cellules somatiques issues de leucémie. Dans cette direction, de futurs travaux devront déterminer si le recrutement essentiel de SETDB1 par DAXX est limité aux cellules pluripotentes, et ainsi étudier un possible rôle vital de DAXX lors de pertes chroniques du niveau de méthylation de l'ADN.

Figure Supp. 4 A) Size of MajSat foci

	DaxxKO-ServsGS tMSDAXX vs DAXXY222A		0,000154933 3,022E-99	WT GS n=209 tMSVP64 n=65 tMSDAXX n=391 tMSDAXX n=74
	ser-ser E) DAXX at PCH D) Sizes foci tMSDAXX in WT tMSΔ vs DAXXY222A		0,522768974 0,00017048	KO FCS n=128 tMSDAXXY222A n=90
	GS-GS Chi-square Mann-Whitney		0,012146258 pval. pval.	KO GS n=200
	E) DAXX at PCH tMSΔ vs VP64 tMSΔ vs DAXX		2,80269E-05 0.0016	tMSΔ n=206 tMSΔ n=1438
	Chi-square		pval.	tMSVP64 n=77 tMSDAXX n=997
	dcas vs cas9		0,003680042	dcas n=182
	dcas vs HU Figure 4 Figure 5		2,94735E-07	cas9 n=210
	cas vs HU A) # of MajSat foci C) H3K9me3-recruitment		0,077123279	HU n=65
	Mann-Whitney Chi-square		pval. pval.
	Figure Supp. 2 WTvsKO-GS tMSΔ vs DAXX		<0.0001 1,8847E-267	WT FCS n=220 tMSΔ n=107
	A) Size of PML NB WTvsKO-FCS tMSΔ vs DAXXY222A		<0.0001 0.24	KO FCS n=179 tMSDAXX n=73
	Mann-Whitney WTvsKO-Diff tMSΔ vs SUV39H1		pval. 0.4685 <0.00001	WT GS n=253 tMSSUV39H1 n=80
	classical' vs 'giant' tMSDAXX vs DAXXY222A		<0.0001 2,3102E-102	classical' n=116 KO GS n=141 tMSDAXXY222A n=63
	tMSDAXX vs SUV39H1		2,66E-220	giant' n=77 WT Diff n=320 tMSSETDB1 n=71
	B) yH2AX + foci tMSDAXXY222A vs SUV39H1		1.268E-18	KO Diff n=399
	Chi-square B) Half-recovery FRAP: tMSΔ vs SETDB1		pval. <0.00001
	dCas9 vs Cas9 Mann-Whitney tMSDAXXvs SETDB1		1,91581E-85 pval. 9,8003E-59	dcas n=240
	WT GS vs KO GS tMSSUV39H1 vs SETDB1		0.0005 0.30	cas9 n=79 WT FCS n=66
	for CRISPR/Cas9 experiments WT FCS vs KO FCS D) yH2AX + foci <0.0001 D) Flag clustering	KO FCS n=59
	Name Chi-square Mann-Whitney		Sequence pval. pval.	WT GS n=41
	Sg-mDaxx tMSΔ vs nbs1 tMSΔ vs DAXX	gGACCTCATCCAGCCGGTTCA 1,1537E-136 <0.0001	tMSΔ n=115 KO GS n=39 tMSΔ n=107
	sg-MajSat D) # foci tMSDAXX in DaxxKO tMSΔ vs SUV39H1	CCATATTCCACGTCCTACAG 0.7736	tMSNBS1 n=123 tMSDAXX n=73
	Targeting sequence D) DAXX at PCH Mann-Whitney tMSΔ vs DAXXY222A	pval. 0.9781	tMSSUV39H1 n=80
	Tale Chi-square tMSΔ vs DAXX tMSDAXX vs SUV39H1		Sequence pval. <0.0001 <0.0001	tMSΔ n=435 tMSDAXXY222A n=63
	Tale-MajSat tMSΔ vs nbs1 tMSDAXX vs DAXXY222A		TGCCATATTCCACGT 0,360385276 <0.0001	tMSΔ n=206 tMSDAXX n=338 tMSSETDB1 n=71
	D) Sizes foci tMSDAXX in DaxxKO tMSDAXXY222A vs SUV39H1		0.59	tMSNBS1 n=82
	Figure 1 Mann-Whitney tMSΔ vs SETDB1		pval. 0.91
	C) Number of genes differentially Figure 3 tMSΔ vs DAXX tMSDAXX vs SETDB1		0.0038 <0.0001	tMSΔ n=2064
	expressed B) DAXX at PCH tMSSUV39H1 vs SETDB1		0.73	tMSDAXX n=1108
	RNAseq -1<Log(FC KO/WT)>1; pvalue<0.05 GS Chi-square E) # foci H3.3 complementation Serum vs 5aza Mann-Whitney Serum noH3.3 vs H3.3WT D) yH2AX + foci Mann-Whitney H3.3WT vs G90M Diff Chi-square WTvsKO-GS H3.3WT vs H3.3K9A tMSΔ vs Tet1CD WTvsKO-FCS H3.3G90M vs H3.3K9A Figure 2 C) DAXX at PCH Chi-square Serum vs ground-state WTvsKO-Diff B) Mobile fraction FRAP: Figure Supp.5 E) DAXX at PCH Chi-square Mann-Whitney B) DAXX at PCH tMSΔ vs Tet1CD WTGSvsKOGS Chi-square WTFCSvsKOFCS WT vs siH3.3 WT vs DaxxKO		Up pval. down 8,16745E-24 pval. Up <0.0001 down <0.0001 pval. Up down <0.0001 <0.0001 pval. 3,64197E-37 0.0027 0.0829 pval. 4,04122E-08 0.0195 pval. pval. 7,37729E-17 0.53 pval. 0.55 3,59432E-12 4,64151E-20	1345 Serum n=225 1250 noH3.3 n=109 5aza n=190 497 H3.3WT n=135 528 8 n=1213vs1044 H3.3G90M n=134 n=1121vs1143 H3.3K9A n=171 tMSΔ n=115 125 Serum n=225 n=4351vs7008 tMSTet1CD n=136 WT-GS n=41 tMSΔ n=206 KO-GS n=39 WT FCS n=438 tMSTet1CD n=108 WT-FCS n=66 siH3.3 n=118
	siH3.3 vs DaxxKO Figure Supp. 3 D) yH2AX + foci Chi-square WT FCS vs GS D) # foci tMSDAXX in WT C) SETDB1 recruitment D) yH2AX + foci Chi-square Mann-Whitney Chi-square tMSΔ vs VP64 tMSΔ vs DAXX tMSΔ vs DAXX		0.027971171 pval. 0,000663871 pval. pval. pval. 8,67827E-22 <0.0001 1,12458E-87	GS n=438 KO-FCS n=59 DaxxKO n=83 WT FCS n=155 tMSΔ n=115 tMSΔ n=398 tMSΔ n=178

  Upon the loss of DNA methylation, in both ESCs and the first stages of development, polycomb proteins are recruited to pericentromeric heterochromatin, presumably to protect the heterochromatin state. Polycomb and subsequent H3K27me3 signals are not observed at PCH in basal conditions because of steric hindrance withEzh2 in ground-state ESCs to assess if it impedes DAXX-PML relocalizations. We noted that the DAXX-ATRX interaction did not seem essential for the DAXX-mediated clustering of major satellites (Figure37B). However, because ATRX is still present at PCH, even in the absence of Daxx, it remains possible that other proteins implicated in PCH clustering might be recruited via However, unmethylated DNA does not necessarily induce DAXX recruitment at PCH. Indeed, in Dnmt tKO ESCs, ChIP-seq experiments reported the increased binding of DAXX at dispersed repeated DNA elements such as ERVs, but not at PCH, suggesting an additional layer of control[START_REF] He | The Daxx/Atrx Complex Protects Tandem Repetitive Elements during DNA Hypomethylation by Promoting H3K9 Trimethylation[END_REF]. We notice that DAXX recruitment at major satellites could arise from DNA damages, as observed from HU and

	ATRX (Figure 37E). As ATRX is essential in mESCs, it would require a knock-down, or a
	conditional knock-out strategy to interrogate its role in DAXX-mediated clustering and DAXX
	recruitment upon hypomethylation.
	We detect a strong relocalization of DAXX toward PCH regions upon DNA
	hypomethylation (Figure 2A article, 3B article).
	H3K9me3-modificiation. PRC1 is recruited to PCH in Dnmt tKO in a BEND3-dependent
	manner, and then recruits PRC2 to induce H3K27me3-modification in mESCs (Saksouk et al. 2014;
	Cooper et al. 2014). Yet, these relocalizations of BEND3, PRCs and H3K27me3 appear at all
	chromocenters, suggesting a general heterochromatin pathway. Intriguingly, in our conditions, we

only detected DAXX at 1 to 3 chromocenters, suggesting that its recruitment might rely on additional signaling than the H3K27me3-based mechanisms. To formally rule this out, it would be interesting to knock-down Bend3, PRC1 factors such as Ring1B and PRC2 factors such as Eed or CRISPR/Cas9 inducible DSBs (Figure

2E article

). Intriguingly, we notice that upon ground-state conversion or exogenously-induced DNA

  . It would thus be interesting to test if the reported LTRs from Ostromyshenskii et al. also localizes at chromocenters in mESCs and if they carry a role in DAXX-recruitment. In that direction, one could generate Tale-DAXX constructs targeting PCH-LTR sequences and assess if there is a major satellite clustering as well.Moreover, the targeting of Tale-TET1CD toward pericentromeric ERVs could lead to increased DNA damages at chromocenters and DAXX recruitment. Among the proteins acting at ERVs is the repressor KAP1 and physical interaction between DAXX and KAP1 was observed in mESCs[START_REF] Elsässer | Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells[END_REF]. However, KAP1 recruitment at PCH is frequent at multiple PCH, even in the absence of DAXX (data not shown). It would still be interesting to use some KRAB-ZF-deficient ESCs and to knock-down KAP1, because it is essential in ESCs, to see if it abolishes the accumulation of DAXX at PCH[START_REF] Walter | An epigenetic switch ensures transponson repressioin upon dynamic loss of DNA methylation in embryonic stem cells[END_REF]. If this hypothesis is correct, it would then represent a potential new function for ERVs in global genome organization and will require assessing the role of PCH-ERVs and KRAB-ZF in the formation of chromocenters in the early embryo.

We hypothesize that in vitro conversion of ESCs and the embryonic development are quite different in term of chromatin modifications. Indeed, while serum-based ESCs display fairly high levels of DNA methylation, the conversion toward ground-state conditions generates a strong active DNA demethylation. Active DNA demethylation occurs at the paternal nucleus during the very early steps of development, while the maternal genome is mostly passively demethylated

[START_REF] Lee | Reprogramming the methylome: Erasing memory and creating diversity[END_REF][START_REF] Messerschmidt | DNA methylation dynamics during epigenetic reprogramming in the germline and preimplantation embryos[END_REF]

. Nevertheless, a second wave of active DNA demethylation takes place during the development of primordial germ cells (PGCs) at ~E9.5 in order to

  the third part of this discussion, we proposed a model whereby DAXX can accumulate These data indicate that we might be observing two distinct mechanisms -the maintenance of chromocenter organization on one hand and the restoration of heterochromatin state following DNA damages on the other hand. Nevertheless, the role of DAXX in serum or ground-state conditions might be similar at PCH, but only visible by immunofluorescence upon ground-state conversion. Indeed, some γH2AX foci are already visible at chromocenters in serum condition. Thus, it is conceivable that some DNA damages could happen at chromocenters, probably as rare events. In that direction, DAXX can be visible at PCH in serum only after high levels of DNA damages, suggesting that in serum takes place a DNA methylation-related mechanism to avoid such levels of DNA damage or chromatin reformation (Fig.2Earticle). It remains possible that DAXX and SETDB1 could bind to chromocenters in serum-based medium but in a transient, such as DNA damages, or local, such as pericentromeric ERVs, manners preventing their observation by immunofluorescence. Deeper exploration is required to decipher whether the same triggering mechanisms exist in serum condition such as DAXX and SETDB1 ChIP-seq and live-cell imagingSilencing of ERV requires DAXX-mediated H3.3[START_REF] Elsässer | Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells[END_REF]. In serum condition, there might be DAXX-mediated H3.3 deposition at PCH-specific ERVs. DAXX could then recruit SETDB1 to induce H3.3K9me3 like in ground-state, but as it would happen at a lesser rate to reform heterochromatin and enable transcriptional silencing of PCH-specific ERVs, and as rare or very local binding, it is not visible by immunofluorescence. H3.3 differs from canonical H3 from only 4 to 5 amino acids, among which are residues giving specificities for particular chaperones,

	recruit SETDB1 to induce H3K9 tri-methylation (Figure 5C article). Furthermore, the
	recruitment of DAXX in ESCs expressing H3.3K9A was not able to induce PCH clustering
	(Figure 5D article). Intriguingly, the recruitment of SUV39H1 does not lead to PCH clustering
	(Figure 5C article) Preliminary data suggests that the targeted recruitment of SETDB1 does not
	drive PCH clustering neither yet performing H3K9me3 activity (data not shown). Since DAXX
	can recruit H3.3, whereas SUV39H1 and SETDB1 were not reported to be able to bind H3.3, we
	propose that SETDB1 might tri-methylates DAXX-deposited H3.3K9 and that this specific
	modification controls PCH clustering. Altogether, we propose a general model where this function
	of DAXX is strongly visible upon ground-state conversion with important accumulation of DAXX
	at chromocenters following DNA damages induced by active DNA demethylation. However, the
	same heterochromatin maintenance might happen in serum condition, but as a more local event.
	Pinheiro et al. 2012). Moreover, deletion of the H3K9me1 methyl-transferases Prdm3 and Prdm16
	in MEFs resulted in altered major satellite clustering, likely via impairment toward further
	methylation to di or tri-methylated states (Pinheiro et al. 2012). Further investigating the role of
	SETDB1 in PCH organization will give a better understanding of the mechanism at play. Moreover,
	deletion of the Suv4-20H1/2 enzymes that catalyzes H4K20me3 histone modification results in
	defects in chromocenter clustering in ESCs and impaired recruitment of cohesins at
	pericentromeric regions, which enhanced chromosome segregation defects (Hahn et al. 2013). It
	would thus be interesting to assess the levels of other heterochromatin-associated histone
	modifications in serum-based and ground-state Daxx-deficient ESCs to dissect whether it could
	explain the observed loss of chromocenter clustering.
	We demonstrate that the ability of DAXX to mediate chromocenter clustering depends
	onto the histone variant H3.3 (Figure 5D article). Indeed, recruitment of DAXXY222A that
	cannot bind H3.3 or the knock-down of H3.3 genes prevents DAXX-mediated PCH clustering
	(Figure 5C article, 5E article). To further characterize the role of DAXX and H3.3 in
	chromocenter organization, it would be informative to complement Daxx-deficient ESCs with WT
	DAXX or DAXXY222A and assess major satellites clustering. We demonstrate that DAXX could

at chromocenters upon DNA damages and then recruit SETDB1 to reform heterochromatin state after DNA repair took place (Figure 2A article, 2F article, 5A article). However, we observe that DAXX maintains pericentromeric heterochromatin spatial and molecular organization in both serum and ground-state conditions (Figure 4A article, 4B article, 4C article).

experiments. Depletion of Daxx does not prevent H3K9me3 at chromocenters, likely deposited by SUV39H1/2 (Figure

28A

). Intriguingly, deletion of only the Suv39H1/2 enzymes does not prevent PCH clustering, but the triple Suv39H1/2 and Setdb1 does, suggesting that SETDB1 and perhaps DAXX carry a function at PCH even in DNA methylated conditions

[START_REF] Peters | Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability[END_REF]

Acknowledgments Acknowledgments Français

Les génomes des mammifères sont largement composés de séquences ADN répétées. Les régions d'hétérochromatine péricentromérique (PCH) sont formées de nombreuses répétitions de satellites majeurs. Les régions d'ADN répétés sont souvent réprimées par des mécanismes similaires comme la méthylation de l'ADN, la modification H3K9me3 et HP1. Des défauts de l'hétérochromatine sont observés dans différents cancers et contribuent à l'instabilité génomique. Les PCH des différents chromosomes se rassemblent et forment de larges chromocentres. La formation des chromocentres a lieu au cours de l'embryogénèse précoce, un moment où le niveau de méthylation de l'ADN est faible. Cette réorganisation est essentielle pour la poursuite du développement, et pourtant les mécanismes moléculaires la régulant dans les cellules pluripotentes précoces restent inconnus. Ce travail se concentre sur le rôle du variant d'histone H3.3, et de sa chaperonne DAXX, sur l'organisation de l'hétérochromatine dans les cellules souches embryonnaires (ESC). Premièrement, nous avons observé que la perte de Daxx altérait l'organisation des chromocentres dans les cellules pluripotentes. Les ESC peuvent être converti ver un état naïf de pluripotence, qui présentent une très forte diminution de leur niveau de méthylation de l'ADN. Nous avons découvert que DAXX était essentiel pour la survie des ESC lors de la conversion vers l'état naïf de pluripotence. De plus, nous avons observé que la perte active de la méthylation de l'ADN pouvait générer des dommages à l'ADN aux PCH, ce qui induit une forte accumulation de DAXX. En disséquant le rôle de DAXX, nous avons découvert que l'interaction DAXX-H3.3 était impliquée dans le recrutement de SETDB1 aux chromocentres pour reformer l'hétérochromatine. Enfin, nous avons montré que le rôle de DAXX s'étendait à d'autres régions de l'hétérochromatine tels que les domaines associés à la lamina. Dans l'ensemble, ce travail met en évidence un nouveau rôle DAXX dans le maintien de l'hétérochromatine des cellules pluripotentes. Mots-clefs : hétérochromatine ; variants d'histone ; H3.3 ; DAXX ; cellules souches embryonnaires ; méthylation de l'ADN.

References 1.Guenatri et al. J. Cell Biol. 2004 2.Martens et al. EMBO J. 2005 3.Matsui et al. Nature 2010 4.Fukuda et al. Genome Res. 2018 5.Zhu et al. Nature 2011 6.Mansuroglu et at. Sci. Rep. 2016 7.Jagannathan et al. eLife 2018 8.Hahn, et al. Genes & Dev. 2013 9.Probst et al. Developmental Cell 2010 10.Casanova et al. Cell Rep. 2013 11.Lewis et al. P.N.A.S. 2010 12.Arakawa et al. Biochem. Biophys. Res. Comm. 2015 13.Liu et al. EMBO J. 2020 14.Elsässer et al. Nature 2015 15.Ying et al. Nature 2008 16.Blaschke et al. Nature 2013 17.He et al. Cell Stem Cell 2015 18.Chou et al., Dev. Cell 2014 19.Williams and Schumacher, Cold Spring Harbor Persp. Med. 2016 20.Russo et al., Science 2019 21.Lallemand-Breitenbach and de Thé Curr. Op. Cell Biol. 2018 22.Peters et al. Cell 2001 23.Stucki and Jackson DNA Repair 2006

LIST OF ABBREVIATIONS

LIST OF FIGURES

These results indicate the crucial role played by epigenetic modifiers during development, but they also demonstrate the redundancy existing between these different enzymes as observed for the Suv39h and Suv4-20h enzymes. Specific deletion of HP1 proteins α, β or γ present different defects but no developmental arrest, although the triple KO is not reported [START_REF] Brown | HP1γ function is required for male germ cell survival and spermatogenesis[END_REF]. Only HP1β KO present defects in cerebral cortex development [START_REF] Aucott | HP1-beta is required for development of the cerebral neocortex and neuromuscular junctions[END_REF]). Altogether, it seems that the heterochromatin state seems crucial in pluripotency control.

The early embryonic development follows different chromatin state, from totipotency to pluripotency to differentiated cells. Specific signaling pathways, that converge to chromatin regulation, correlate with each state. Indeed, it arises from recent studies that the regulation of cell physiology might happen from specific chromatin states. Notably, heterochromatin regulation seems essential in safeguarding cellular identity.

Appendix to the introduction Review 1

Antoine CANAT, Adeline VEILLET, Amandine BONNET and Pierre THERIZOLS. Genome anchoring to nuclear landmarks drives functional compartmentalization of the nuclear space.

Briefings in functional genomics, 2020.

RESULTS

Supplementary figure 1

Supplementary data

CONCLUSIONS DISCUSSION

DAXX interactions and post-translational modifications

We observe that in serum ESCs, DAXX localizes at PML NBs (Figure 2A article).

However, upon ground-state conversion, DAXX is massively recruited toward chromocenters to form 'giant' structures encompassing major satellite repeats (Figure 2A article, Figure 2B article).

Yet, 'classical' small PML NBs are still visible in ground-state condition that contain DAXX as in serum, suggesting that the formation of 'giant' structures do not arise merely as a loss of 'classical' NBs. We demonstrate that in the absence of Daxx, PML is not recruited to PCH, whereas the lack of Pml do not impair DAXX recruitment to PCH (Figure S5B article, Figure 33A). Furthermore, upon Daxx depletion, PML NBs are still visible, but the loss of Pml alters the organization of DAXX into NBs, with only few foci observed that are likely to be telomeres (Figure S5A article, Figure 33A). These results indicate that while DAXX localization is impacted by PML, its recruitment toward PCH seems independent of PML. It resembles the observations made during myogenic differentiation where in myoblasts, DAXX localized at PML NBs but upon myotubes differentiation, PML expression was abolished and DAXX relocalized toward chromocenters [START_REF] Salsman | Myogenic differentiation triggers PML nuclear body loss and DAXX relocalization to chromocentres[END_REF]. However, in myotubes, DAXX was present at all chromocenters, which differs from our observations that DAXX accumulated at 1 to 3 chromocenters (Figure 2C article). Intriguingly, SIM domain of DAXX is necessary for its pericentromeric relocalization in myotubes but exogenous expression of PML could not rescue this phenotype and DAXX remained at chromocenters, suggesting that post-translational modifications, and not PML itself, are crucial to control DAXX localization [START_REF] Salsman | Myogenic differentiation triggers PML nuclear body loss and DAXX relocalization to chromocentres[END_REF]. We thus propose that PML might contribute to post-translational modifications of DAXX, such as SUMOylation, to regulate its local recruitment at heterochromatin regions. In that direction, it would be informative to better dissect the post-translational modifications of DAXX upon ground-state conversion. Furthermore, confirming the role of the modified residues will require to rescue Daxx KO ESCs with mutant versions of DAXX and assess their capacity to localize at chromocenters. Surprisingly, when prompting DAXX localization in Pml-deficient cells, we noticed that it still accumulates at chromocenters (Figure 33A). These data suggest that the loss of Pml does not impact the recruitment of DAXX, but rather its function. It would thus be informative to study the posttranslational modifications of DAXX in Pml KO ESCs. We hypothesize that post-translational modifications of DAXX could contribute to increase local concentration of proteins, thus facilitating heterochromatin reformation. In cells lacking Pml, the concentrating role of DAXX might be impaired. In that direction, it will be of great interest to see if DAXX can recruit SETDB1 and H3.3 in Pml-deficient cells upon ground-state conversion.

RESUME SUBSTANTIEL DE LA THESE EN FRANCAIS

RESUME RESUME English

Most of mammalian genomes are composed of DNA repeated elements. Pericentromeric heterochromatin (PCH) regions are formed of large arrays of major satellite repeats. DNA repeats are often silenced via similar mechanisms such as DNA methylation, H3K9me3 modification and HP1. Loss of heterochromatin silencing is commonly observed in different cancers and can lead to genomic instability. PCH of different chromosome can assemble into large DAPI-dense foci called chromocenters. Formation of chromocenter arise during the early embryogenesis when the level of DNA methylation is low. Such reorganization is essential for further development of the embryo, yet, the molecular mechanisms regulating its maintenance in the early pluripotent cells remain unknown. This work focuses on the role of the histone variant H3.3 and its chaperone DAXX in heterochromatin organization in embryonic stem cells (ESCs). Firstly, I observed that the loss of Daxx alters the organization of chromocenters in pluripotent cells. ESCs can be converted toward the ground-state of pluripotency which displays a dramatic loss of DNA methylation. I uncovered that DAXX is essential for proper growth of ESC upon ground-state conversion. Furthermore, I observed that active loss of DNA demethylation can generate DNA damages at PCH, which induces a strong accumulation of DAXX. Dissecting the function of DAXX, I discovered that the DAXX-H3.3 interaction was implicated in the recruitment of SETDB1 at chromocenters to reform the heterochromatin state. Finally, I noticed that the function of DAXX extend beyond pericentromeric heterochromatin. Indeed, the absence of Daxx impairs the proper silencing of different heterochromatin regions including the lamina-associated domains. Altogether, this work provides evidence that DAXX is a regulator of heterochromatin maintenance in pluripotent cells. Keywords: heterochromatin; histone variants; H3.3; DAXX; embryonic stem cells; DNA methylation.