AKNOWLEDGMENTS

First, I would like to thank the members of my thesis committee for being here during four years, in the good and difficult times. Thank you very much Sylvina Bouleau, Emilie Brasset and Fabienne Malagnac.

I would also like to thank the member of my thesis jury. Thank you for accepting to be part of it, for the advice and the usefull discussions.

When I started to study Biology, I thought "I will never work on developmental genes, they are sooooo boring", and later on, "I will never use 3C technics, I don't like them"… sometimes life is funny.

Sébastien, merci de m'avoir donné ma chance afin de faire mon M2 puis ma thèse. La première fois que nous avons eu cours avec toi à Versailles, je me souviens m'être dit « quel gros nounours, il parle vite et fort mais il à l'air cool ». Je ne m'étais pas trompée ! Tu m'as appâtée avec un projet Droso/souris ribosome, et puis finalement, les gènes Hox chez la Drosophile. J'ai appris à les aimer, et maintenant je les trouve fascinants. Je suis ravie d'avoir été ta thésarde, tu es un bel être humain. Tu n'hésites pas à te remettre en question, tu as été là autant que tu as pu, tu es généreux et attentionné. Merci d'être toi et je suis persudadée que tu resteras un super prof qui passionnera des centaines d'élèves comme tu as pu le faire avec moi.

Daan, thank you for accepting this crazy idea to work on Drosophila in… a mammalian lab! I know it has been tough for you to dive into Drosophila, this weird tiny organism with funny gene names, but I thank you for doing it. I was really glad to be your Ph.D student, I learned a lot during this 4 years. I will always cite you as a model, as a Ph.D supervisor and a successful team leader.

Benoit, je ne te remercierai jamais assez d'exister ! Je pense que ça n'a pas dû être facile de m'encadrer alors que toi aussi tu démarrais dans l'équipe. Tu as dû te mettre à la Drosophile pour moi, j'espère que tu as quand même un peu apprécié. Dès le début, j'ai été impréssioné par ton cerveau : comment une personne peut-elle savoir autant de chose !?! Ça va être dur de ne plus avoir super Benoit à mes côtés… Merci d'avoir été là, tout le temps et sans hésitation.

I would like to thank all the other members of the team CHRODY, past and present, I was so lucky to have you all! I think this is rare to have your colleagues truly becoming friends. You were all there, to laugh, to share ideas, to share difficult moments (and of course to work). Among all my beloved colleagues, special big love to Li-Hsin and Mélanie, thank you girls for all the beers, parties and sssssoooo good moments.

I also would like to thanks all the other members of the Institut I met. Even if it was short, thank you the Crabbe team for the shared labmeetings and good moments! Thanks to all the teams of the building 26, you guys rock! Thank you also to the Drosophila teams of building 21, for your help, for the fun and for all the good moments. Thanks to the sequencing platform and especially Céline Hernandez and Yan Jaszczyszyn.

I was honored to be president of YOUR-I2BC and I am gratefull it exists, thanks to the past and present members for gathering students from different teams and department to share convivial moments.

J'ai également eu l'opportunité d'être monitrice à l'Université d'Orsay et remercie chaleureusement l'équipe enseignante, vous avez été parfaits. Merci de m'avoir acceuillie, formée et fait confiance.

Je tiens aussi à remercier tous les enseignants de Versailles pour m'avoir transmis leurs passions. Choisir Versailles comme Université a été un des meilleurs choix que j'ai pu faire. J'y ai trouvé une petit fac merveilleuse peuplée d'enseignants dédiés, attentifs, pédagogues, passionnés et gentils. Merci ! Line, Manon, Romane, Pierrick, on l'a fait! GG à nous.

Et pour finir ces remerciements mielleux, merci à tous mes amis, d'ici et d'ailleurs. Merci d'avoir été là, de m'avoir supporté (dans tous les sens du terme). Merci à ma famille, Tata, Maman, d'avoir été présentes, et à ma belle famille. Xavier, quel courage de m'avoir supporté.

Merci du fond du coeur.

Papa, je sais que tu aurais été fier de moi. All living organisms are made of cell(s) that contain essential molecules for survival. Among the best studied molecules is the DNA (Deoxyribonucleic Acid), which encodes the genetic information. All the information that is contained in the DNA is called "genome" [START_REF] Alberts | The Structure and Function of DNA[END_REF]. The genomic information is arranged as chromosome(s) within the cell. For example, in most eukaryotes, it is mainly found as multiple linear chromosomes whereas in prokaryotes, chromosomes are mostly circular. In diploid cells, chromosomes are arranged by pairs (autosomes) except for the sexual X and Y chromosomes (allosomes, Z and W in birds). Human cells possess 22 pairs of autosomes and one pair of allosomes making 46 chromosomes per diploid cells in total, apart from haploid cells that contain half of the genetic information.

List of figures

Across eukaryotic species, genome size varies dramatically but surprisingly the number of genes varies much less. For example, the genome size in Saccharomyces cerevisiae is around 12 million bp, in Arabidospis thaliana 125 million bp, in Drosophila melanogaster 170 million bp (referred after as Drosophila) and 2.5 billion bp in mus musculus (referred after as mouse)

whereas their (protein-coding) gene numbers are respectively in the order of 6,000, 25,000, 14,000 and 30,000 [START_REF] Pray | Eukaryotic Genome Complexity | Learn Science at Scitable[END_REF]. Moreover, within the same group, genome size can dramatically vary. It is particularly the case for flowering plants (angiosperm) where genome sizes vary by a factor 2,400 [START_REF] Dodsworth | Genome size diversity in angiosperms and its influence on gene space[END_REF]. Thus, genome size does not directly correlate with gene numbers. In a typical human cell, the DNA length of the combined 46 chromosomes is around 2 meters, which has to fit in a nucleus of around 10µm of diameter.

This implies that DNA (present in almost every cell of an organism) is highly compacted to fit in a small nucleus.

The DNA sequence contains the raw genetic information, but the substrate for essential biological processes is not naked DNA but rather chromatin: the DNA associates with specific proteins forming together the nucleosomes. Nucleosomes are composed of an octamer of histones (two copies each of the H2A, H2B, H3, H4 histones) with 145-147 bp of DNA wrapped around them [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 Å resolution[END_REF]. Each nucleosome is connected to its neighbor by a short fragment of linker DNA (10-80 bp depending on species, cell type, developmental stage and genomic position) thus creating the 10nm chromatin fiber, which resembles a "beads-on-astring". This polynucleosome structure can be folded by the binding of histone linker H1 to

Regulatory elements of gene expression

The size of the genome varies widely among species, thereby affecting the fraction of the genome that encodes proteins. For example, in humans, only 1-2% of the genome codes for proteins (The ENCODE Project Consortium, 2012) whereas this percentage reaches 20% in Drosophila melanogaster and 70% in Saccharomyces cerevisiae. At first, the remaining nonprotein coding regions were seen as junk DNA but many regulatory elements lay in this noncoding part of the genome including promoters, enhancers and insulators.

a. Promoters

The transcription of a genes starts at its TSS (Transcription Start Site) that is embedded within a core promoter, a short DNA sequence surrounding the TSS. The core promoter serves as a docking platform for the Pre-Initiation Complex (a multi-protein complex containing general transcription factors and the RNA polymerase II holoenzyme) that can then prime transcription [START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF].

An important layer of transcriptional regulation is the choice of alternative promoters for a single gene. It has been shown that 30 to 50 % of the human, 50% of the mouse and at least 40% of Drosophila genes possess alternative promoters [START_REF] Batut | High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression[END_REF][START_REF] Davuluri | The functional consequences of alternative promoter use in mammalian genomes[END_REF]. Alternative promoter choice leads to the production of mRNA molecules that differ by their 5'-end. These may give rise to a longer 5' UTR or different proteins that possess different functions, which may be exploited to dynamically change transcriptional programs between cell types (Fig. 1.2).

As said before, packaging of DNA into chromatin forms a condensed structure that reduces the accessibility for the transcription machinery. To allow transcription, this condensed structure needs to be relaxed (promoter clearance). In many species, the promoter and terminator regions are generally depleted in nucleosomes to allow the access of the transcription machinery [START_REF] Mavrich | Nucleosome organization in the Drosophila genome[END_REF][START_REF] Schones | Dynamic Regulation of Nucleosome Positioning in the Human Genome[END_REF]. However, in most cases, promoters alone have a low basal activity and do not drive robust gene expression [START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF].

Additional actors are therefore necessary to further enhance the levels of transcription.

b. Enhancers (and Silencers)

Enhancers are another type of non-coding element that regulate gene expression. Enhancers are short DNA elements that can be located near a promoter, within the gene body, at a large distance away from promoters (rarely over 1MB) or even on a different chromosome [START_REF] Schaffner | Enhancers, enhancers -from their discovery to today's universe of transcription enhancers[END_REF] (Fig. 1.3). They can physically interact with their target promoters by creating chromatin loops to amplify gene expression. They are bound by transcription factors and diverse co-factors [START_REF] Noordermeer | Joining the loops: β-Globin gene regulation[END_REF][START_REF] Vieira | Recruitment of Transcription Complexes to the β-Globin Gene Locus in Vivo and in Vitro[END_REF] and increase gene expression independently of their orientation or position [START_REF] Banerji | Expression of a β-globin gene is enhanced by remote SV40 DNA sequences[END_REF]. Enhancers can even be on different chromosome and still activate gene expression by transvection in Drosophila [START_REF] Geyer | Tissue-specific transcriptional enhancers may act in trans on the gene located in the homologous chromosome: the molecular basis of transvection in Drosophila[END_REF]. This phenomenon has been proposed to occur in mouse as well [START_REF] Lomvardas | Interchromosomal Interactions and Olfactory Receptor Choice[END_REF]. As enhancers are not linearly close to promoters, they can be difficult to identify but their epigenetic signature (histone modifications) can help their identification (see paragraph 3). Moreover, enhancers can be detected because they lack nucleosomes (i.e.

DNAse HS sites) and are bound by transcription factors [START_REF] Shlyueva | Transcriptional enhancers: from properties to genome-wide predictions[END_REF].

Cell-type specific enhancers are needed to control correct spatial and temporal gene expression.

Their tissue-specific epigenetic signature (as seen by ChIP-seq) confirms their tissue-specific activity [START_REF] Visel | ChIP-seq accurately predicts tissue-specific activity of enhancers[END_REF]. In mammals, most enhancers have been reported to engage in contact with only one promoter [START_REF] Zhang | Chromatin connectivity maps reveal dynamic promoterenhancer long-range associations[END_REF]. In contrast, in Drosophila, some enhancers were reported to interact with on average two promoters [START_REF] Ghavi-Helm | Enhancer loops appear stable during development and are associated with paused polymerase[END_REF]). An interesting feature of enhancer-promoter contacts is that it can occur before or during activation of transcription [START_REF] Ghavi-Helm | Enhancer loops appear stable during development and are associated with paused polymerase[END_REF][START_REF] Rubin | Lineage-specific dynamic and pre-established enhancer-promoter contacts cooperate in terminal differentiation[END_REF].

Analogous to enhancers, regulatory elements with silencing functions also exist. The best characterized examples are Polycomb Responsive Elements (PRE) in Drosophila, that have been implicated in loop formation as well [START_REF] Müller | Polycomb response elements and targeting of Polycomb group proteins in Drosophila[END_REF]. Silencers will not be further developed in this manuscript and PREs will be discussed in Chapter III.

Enhancers (and silencers) can be located hundreds of kb's away from their target promoters and are often separated by intermediate genes. Hence, a puzzling question is how they can find each other to ensure gene expression (or repression)? One clue how chromatin loops between enhancers and promoters can occur is with the help of boundary elements, insulator proteins and other proteins such as the Mediator complex [START_REF] Malik | Mediator: A Drawbridge across the Enhancer-Promoter Divide[END_REF].

Boundary elements are also involved in the formation of chromatin loops that have two opposite consequences: they either put enhancers and promoters in close proximity to trigger gene expression or, they prevent the contact between them (Fig. 1.4).

In this second section, we have described a first level of gene regulation, highlighting the importance of promoters, enhancers and boundary elements. These cis-acting elements are encoded by DNA, annotated and consensus sequences can be found across species. These three actors act together and are crucial to regulate gene expression and form together a firstdimension organization (1D genome organization) for gene regulation. Yet, chromatin compaction can greatly affect DNA accessibility and consequently modulate gene transcription, together with DNA replication and DNA repair. In the next section, we will focus on the epigenetic machineries that controls chromatin structure and gene expression.

Epigenetic regulation and its actors

Nearly all cells in an organism possess the same DNA sequence, but during development, cells acquire different identities that are maintained by a memory process through cell division.

Epigenetics is the study of mitotically and/or meiotically heritable (but reversible) changes in gene function that cannot be explained by changes in DNA sequences and that persist in the absence of the factors that induce these changes [START_REF] Riggs | Epigenetic Mechanisms of Gene Regulation[END_REF]. This additional level of gene regulation relies on different supports such as DNA methylation, histone modifications and long non-coding RNAs (lncRNAs).

In mammals, DNA methylation is restricted to CpG dinucleotides, with 70-80% of them being methylated in somatic cells [START_REF] Li | DNA Methylation in Mammals[END_REF]. Highly methylated sequences are found in satellites regions, repetitive sequences (i.e transposable elements), gene bodies and nonrepetitive intergenic DNA. In contrast, 70% of annotated promoters contain CpG island (enrichment of many CpG dinucleotides) that remain mostly unmethylated and are usually permissive to transcription. However, hypomethylated promoters are not always transcribed as RNA Pol II is not recruited [START_REF] Siegfried | DNA methylation and gene expression[END_REF]. Conversely, the methylation of CpG islands found in gene promoters can either directly interfere with the binding of the transcription machinery, or indirectly recruit Methyl-CpG-binding proteins (MBPs) that have a silencing effect through interaction with repressive histone modifying complexes [START_REF] Klose | Genomic DNA methylation: the mark and its mediators[END_REF]).

Yet, some studies have demonstrated that some transcription factors can bind to hypermethylated CpG islands and activate gene transcription [START_REF] Rishi | CpG methylation of half-CRE sequences creates C/EBP binding sites that activate some tissue-specific genes[END_REF][START_REF] Yin | Impact of cytosine methylation on DNA binding specificities of human transcription factors[END_REF]. But generally, hypomethylated promoters are active whereas hypermethylated ones are repressed. DNA methylation is established and maintained by DNA Methyl Transferase proteins (DNMTs). Cytosine methylation is catalyzed by DNMTs (DNA Methyltransferase) using SAM (S-adenosyl methionine) as methyl donor. Cytosine methylation (5-mC) is associated with heterochromatin and repressed genes but this DNA modification is reversible.

Active DNA demethylation is achieved by the TETs family (Ten Eleven Translocation) that oxidizes 5-mC to 5-hydroxymethlcystosine (5-hmC). Oxidation of the 5-hmC can continue leading to 5-formylcytosine (5-fC), then 5-carboxylcytosine (5-caC). These oxidized forms of cytosine are then excised of the genome and repaired by the Base Excision Repair (BER) mechanism [START_REF] He | Tet-Mediated Formation of 5-Carboxylcytosine and Its Excision by TDG in Mammalian DNA[END_REF][START_REF] Li | DNA Methylation in Mammals[END_REF]. Importantly, 5-hydromethylecytosine, produced by the oxidation of 5-methylcytosine, has be found to be associated with elevated transcription levels and associated with active chromatin [START_REF] Ficz | Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation[END_REF].

One striking example of how DNA methylation can induce differential gene expression is genomic imprinting. Genomic imprinting is an epigenetic driven mechanism by which genes are expressed in a parent-of-origin dependent manner (monoallelic parental expression). In mouse, around 200 imprinted genes have been identified (see Catalog of Parent of Origin Effects http://igc.otago.ac.nz/home.html). One example is the Igf2/H19 locus. The Igf2 gene is expressed only from the paternal allele whereas the H19 ncRNA gene is expressed from the maternal allele. This differential expression is due to the methylation of a DMR (Differentially Methylated Region) only in the paternal allele. When methylated, the insulator protein CTCF cannot bind the paternal allele and therefore allows the formation of chromatin loop with a downstream enhancer. Thus, depending on the methylation status of the DMR, either Igf2 or H19 can interact with an enhancer and be expressed in a mono-allelic manner [START_REF] Murrell | Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops[END_REF].

b. Chromatin and histone modifications

The Amino termini (N-ter) of histones (also known as histone tails) protrude from the nucleosome cores, with a considerable number of amino acid residues that can be subjected to post-translational modifications (PTM).

These modifications are dynamic as Writers and Erasers are balancing the level of histone marks: Writer add PTMs to histones whereas Erasers remove specific PTMs. The combinations of several PTM influence gene expression, by either directly affecting chromatin compaction and/or by acting as a landing platform to recruit repressive or active factors. The effect of Writers and Erasers is then achieved by Readers that will identify the PTMs and recruit other proteins in order to increase or decrease gene expression for instance [START_REF] Gillette | Readers, Writers, and Erasers: Chromatin as the Whiteboard of Heart Disease[END_REF][START_REF] Jenuwein | Translating the Histone Code[END_REF].

At least eight different types of histone modifications (or histone marks or epigenetic marks)

have been described, including acetylation, methylation, phosphorylation, sumoylation, ADP ribosylation and deamination [START_REF] Kouzarides | Chromatin Modifications and Their Function[END_REF][START_REF] Lawrence | Lateral Thinking: How Histone Modifications Regulate Gene Expression[END_REF]. Hereafter, I will focus on histone acetylation and methylation, whose role on gene regulation and chromatin structure as deeply studied (Fig. 1.5).

Histone acetylation

The first report of histone acetylation was in 1964 by Allfrey and colleagues [START_REF] Allfrey | Acetylation and methylation of histones and their possible role in the regulation of rna synthesis[END_REF]) and since then it has been shown that the writing and erasing of histone acetylation is a dynamic process. Acetylation of lysine residues is mediated by histone lysine acetyltransferases (HATs), such as CBP/P300 or PCAF/GCN5, whereas the mark is removed by histone deacetylases (HDACs) [START_REF] Kouzarides | Chromatin Modifications and Their Function[END_REF][START_REF] Lawrence | Lateral Thinking: How Histone Modifications Regulate Gene Expression[END_REF].

Acetylation is generally associated with activation of gene expression, as it neutralizes the positive charge of the many lysine residues of the histone N-ter tails. Thus, acetylation of histones H3 and H4 leads to chromatin decompaction [START_REF] Garcia-Ramirez | Modulation of Chromatin Folding by Histone Acetylation[END_REF][START_REF] Tse | Disruption of Higher-Order Folding by Core Histone Acetylation Dramatically Enhances Transcription of Nucleosomal Arrays by RNA Polymerase III[END_REF] and increases DNA accessibility [START_REF] Hebbes | Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken f-globin chromosomal domain[END_REF][START_REF] Krajewski | Reconstitution of hyperacetylated, DNase Isensitive chromatin characterized by high conformational flexibility of nucleosomal DNA[END_REF].

(or a PHD finger) and are then able to transduce the signal to recruit protein complexes and transcription factors [START_REF] Marmorstein | Writers and Readers of Histone Acetylation: Structure, Mechanism, and Inhibition[END_REF].

As a nuance, some counterexamples of the function of histone acetylation can also be found. In Drosophila and yeast (Saccharomyces cerevisiae), acetylation of lysine 12 of histone H4 is associated with repressed chromatin (heterochromatin) [START_REF] Braunstein | Efficient transcriptional silencing in Saccharomyces cerevisiae requires a heterochromatin histone acetylation pattern[END_REF][START_REF] Turner | Histone H4 lsoforms Acetylated at Specific Lysine Residues Define Individual Chromosomes and Chromatin Domains in Drosophila Polytene Nuclei[END_REF].

In summary, despite some exceptions, acetylation is mainly associated with enhanced gene expression, which appears mediated by increased modified DNA accessibility, chromatin decompaction and the recruitment of activating protein complexes.

Histone methylation

Histone methylation is an even more contrasting PTM. Lysine and Arginine residues of histone tails can be mono-, di-, and in the case of lysines, tri-methylated. The transcriptional consequences of histone methylation depend on the amino acid residue modified (K or R), the degree of methylation, and if the methylation is symmetric or asymmetric for arginines.

Importantly, lysine residues can either be acetylated or methylated, as the same amine group is targeted by both modifications. Methylation is added by histone methyltransferases (HMTs)

and was initially considered a stable modification. More recently, the methylation of histones was found to be a dynamic process, involving histone demethylases as well (HDMs).

The first lysine methyltransferase (HKMT) discovered was Suv39H1 in mammals [START_REF] Rea | Regulation of chromatin structure by site-specific histone H3 methyltransferases[END_REF], which contains the catalytic SET (Su(var)3-9, Enhancer of Zeste, and Trithorax) domain. Based on the SET homology search, numerous lysine methyltransferases (HKMTs) have since then be found.

Some HKMTs have been associated with transcriptional silencing [START_REF] Entrevan | Regulation of Genome Architecture and Function by Polycomb Proteins[END_REF]:

• In Drosophila: E(z) (Enhancer of Zeste) which is the catalytic subunit of the Polycomb Repressive complex 2 (PRC2), and Su(var)3-9 in Drosophila.

• In human and mouse: The Enhancer of Zeste Homologs 1 and 2 (EZH1 and 2) which are alternative catalytic subunits of mammalian PRC2 complex, and SUVAR39H1 and SUVAR39H2.

Different lysine residues are targeted to repress gene expression: Lysine 9 on histone H3 (H3K9) for Su(var)3-9 and SUVAR39H1/H2 (H3K9m2/me3), and Lysine 27 on histone H3 (H3K27) for EZH1 and 2 (H3K27me3) [START_REF] Dillon | The SET-domain protein superfamily: protein lysine methyltransferases[END_REF]. These PTMs are associated with more condensed chromatin and decreased DNA accessibility.

On the contrary, other HKMTs are promoting gene expression, such as Trx (Trithorax) and ASH1 (Absent, Small, or Homeotic discs 1) for Drosophila and MLL1-4 in human and mouse.

These HKMTs specifically tri-methylate lysine 4 on histone H3 (H3K4me3), which are associated with less condensed chromatin and increased DNA accessibility [START_REF] Dillon | The SET-domain protein superfamily: protein lysine methyltransferases[END_REF].

Another important histone modification is the tri-methylation of histone H3 on lysine 36 (H3K36me3). It is associated with transcribed region 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] Vakoc | Profile of Histone Lysine Methylation across Transcribed Mammalian Chromatin[END_REF]. H3K36me3 is enriched in actively-transcribed gene bodies, its enrichment being maximal towards the 3' end of the gene, which coincides with the dynamics of Ser2 phosphorylation of the RNAPII C-terminal domain (RNAPII PSer2) [START_REF] Barrera | Genome-wide mapping and analysis of active promoters in mouse embryonic stem cells and adult organs[END_REF].

As histone methylation is a dynamic process, histone demethylases (HDMs) also intervene in the regulation of gene expression. HDMs are specific to a single methylated lysine residue; for example, SU(VAR)3-3 demethylates lysine 4 whereas UTX acts on lysine 27 in Drosophila.

Unlike acetylation, methylation does not alter the charge of the histone tails. Several protein domains, in Readers, can recognize methylated lysines, including Chromodomains, PHD fingers, Tudor domain [START_REF] Gillette | Readers, Writers, and Erasers: Chromatin as the Whiteboard of Heart Disease[END_REF][START_REF] Musselman | Perceiving the epigenetic landscape through histone readers[END_REF]. The Readers can then recruit various factors depending on which lysine is modified and on the degree of methylation.

In summary: the methylation of lysine residues in histone tails can have opposite effects on chromatin, depending on the modified lysine and the degree of methylation: the outcome can either be repression and condensed the chromatin, or enhanced gene transcription and opening up of chromatin.

ChIP-seq to map histone modifications genome-wide Chromatin Immuno-Precipitation followed by high-throughput sequencing (ChIP-seq) allows to map the distribution of histone modifications along the genome. [START_REF] Zhao | NONCODE 2016: an informative and valuable data source of long non-coding RNAs[END_REF].

lncRNAs are involved in numerous biological processes, including gene regulation at the transcriptional, post-transcriptional and translational levels, as well as the sub-cellular localization of proteins. lncRNAs, acting as guide RNAs, allow chromatin modifiers recruitment and chromatin complexes scaffolding and are implicated in many epigenetic processes such as genomic imprinting, dosage compensation, chromatin modifications (histone and DNA methylation) and chromatin remodeling [START_REF] Zhang | Mechanisms and Functions of Long Non-Coding RNAs at Multiple Regulatory Levels[END_REF].

Among the examples where lncRNAs are implicated in epigenetic processes, I will focus on dosage compensation in mouse and Drosophila.

As mentioned previously, male Drosophila upregulate gene expression of their single X chromosome to achieve dosage compensation by covering it with active histone marks:

H4K16ac. This covering is achieved by two lncRNAs (roX1 and roX2) that associate with several proteins (MSL1, MSL2, MSL3, MOF encoded by male-absent on first, and MLE encoded by maleless) to form the Male-Specific Lethal (MSL) complex. Once the complex is formed, it coats the X chromosome and drives its acetylation thanks to the HAT activity of the MOF proteins [START_REF] Ilik | Tandem Stem-Loops in roX RNAs Act Together to Mediate X Chromosome Dosage Compensation in Drosophila[END_REF].

Unlike Drosophila, mouse have a different strategy for sex dosage compensation: in female cells, one X is randomly silenced. Xist lncRNA coats the future inactive X chromosome and serves as a landing platform for the (direct or indirect) recruitment of numerous chromatin modifiers. This includes PRC1 and PRC2, two protein complexes of the Polycomb group proteins that respectively deposit uH2A and H3K27me3 repressive histones marks on the Xi chromosome [START_REF] Żylicz | Molecular Mechanisms of Facultative Heterochromatin Formation: An X-Chromosome Perspective[END_REF].

In these two examples, lncRNAs are crucial to achieve dosage compensation even if in one case it implies transcription activation and in the other one gene repression.

In this first chapter, we have seen that gene expression is not only driven by the primary DNA sequence that encodes regulatory sequences (promoters, enhancers, boundary elements, etc.) but also by various epigenetic mechanisms. Indeed, DNA methylation, histone modifications and lncRNAs can either enhance or repress gene expression and influence chromatin structure.

These epigenetic actors create global chromatin environments where some regions of the genome are maintained in a transcriptionally silent state and others are maintained in an active state. In the next chapter, I will describe how the genome is sub-divided in 3D space of nucleus within the nucleus and how it correlates with epigenetic marks.

CHAPTER 2 -Introduction II: 3D genome organization and its impact on gene expression

To fit within the confined space of the nucleus, chromatin is extensively compacted. Yet, this compaction is not random and one major challenge is to store the genetic information in a way that it remains accessible, functional and allows dynamic changes. For example, we have seen that enhancers and promoters, even if located megabases away from each other, can interact physically together. This implies that chromatin adopts a functional three-dimensional (3D) conformation by the formation of chromatin loops. Similarly, histone modifications create active and repressed domains that need to be physically insulated to prevent deleterious gene activation or repression.

Techniques to study the 3D chromosome conformation

First, I will introduce the techniques used to determine chromosome conformation, as they are essential for the remainder of the introduction and for the results sections. The methods presented thereafter have deepen our knowledge on how chromatin is organized in 3D, and its functional impact on gene regulation.

a. Microscopy

In the early 1900s, cells and nuclei were only observed by conventional microscopy, providing limited insights into the 3D-organization of the chromatin. Then, electron microscopy was developed in the 1950s, allowing higher resolution in the nucleus. But it is only with the development of fluorescence in situ hybridization (FISH) and confocal microscopy that we learned more about the functional organization of chromatin and chromosomes in the cell [START_REF] Rapkin | A view of the chromatin landscape[END_REF].

Recently, the emergence of super resolution microscopy (SRM) has allowed the detection of chromatin conformation, compaction, localization and even its correlation with epigenetic status. SRM can be divided in three main categories: structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED) and single molecule localization microscopy including optical reconstruction of chromatin architecture (ORCA) [START_REF] Boettiger | Advances in Chromatin Imaging at Kilobase-Scale Resolution[END_REF][START_REF] Xu | Probing Chromatin Compaction and Its Epigenetic States in situ With Single-Molecule Localization-Based Super-Resolution Microscopy[END_REF].

Historically, microscopy-based techniques initiated the idea that chromatin organization is not random in the nucleus, albeit with certain cell-to-cell variability. They provided single cell insights, as for example frequencies of colocalization within a cell population, but with limited resolution. With the recent development of SRM, breakthrough discoveries about chromatin organization were made at the single cell level but both at high cost and in time-consuming and hypothesis driven projects. However, these single results are complementary to highest resolution "Chromosome Conformation Capture"-based assays that are performed in cell population (see below).

b. 3C techniques

The biochemical "Chromosome Conformation Capture" (3C) approach, and its more recent adaptations that include genomics-based read-outs have extensively improved our understanding of chromatin architecture. Unlike single-cell microscopic analysis, 3C-based approaches allow the determination of chromatin topology in a cell population with a high resolution and, when coupled to high-throughput sequencing, on a genome-wide scale. 3Cbased methods rely on the fact that DNA regions that physically interact can be chemically cross-linked using formaldehyde. Once the conformation of the chromosome has been fixed, the DNA is fragmented using enzymatic digestion. Digested chromatin fragments are then religated within the constrained space of the intact nucleus (in-nucleus ligation), thereby promoting ligations between fragments that were cross-linked together due to their initial proximity. Cross-links are subsequently reversed resulting in a 3C library that consists of complex chimeric DNA molecules composed of DNA fragments initially found within the same chromatin hub. These chimeric DNA molecules are analyzed to detect ligation events among restriction fragments, serving as a read-out for their physical contacts in the nucleus [START_REF] Dekker | Capturing Chromosome Conformation[END_REF].

In the original 3C approach, pairwise ligation events were detected by PCR [START_REF] Dekker | Capturing Chromosome Conformation[END_REF] or qPCR [START_REF] Hagège | Quantitative analysis of chromosome conformation capture assays (3C-qPCR)[END_REF], where the abundance of the PCR products reflects the frequency of ligation between the two pre-selected loci in the nucleus (One vs One).

More recent genomics adaptations allow for more systematic and unbiased detection of these pairwise interactions. Here, I will discuss two adaptations in detail: 4C-seq (One vs All) and Hi-C (All vs All). An overview of the other assays is shown in Fig 2 .1.

C: In ChIA-PET, immunoprecipitation against a protein of interest is performed after crosslinking.

Biotinylated DNA linkers are then added to immunoprecipitated fragments and ligated. ChIA-PET junctions are then excised with enzymatic digestion and fragments are analyzed by NGS. Recently, HiChIP has been developed, which incorporates both aspects of Hi-C and ChIA-PET (not shown)

Figure adapted from [START_REF] Sati | Chromosome conformation capture technologies and their impact in understanding genome function[END_REF] Particularly relevant for my PhD project is 4C-seq (Circular Chromosome Conformation Capture followed by sequencing). Here, the decrosslinked 3C libraries are submitted to a second round of enzymatic digestion and ligation, which generates a library consisting of small chimeric DNA circles. Among all the small circles, some will contain a pre-selected locus of interest, called thereafter the "viewpoint". To identify the interacting partners of a viewpoint, two inverse primers are designed within this viewpoint. This inverse PCR, which amplifies the interacting partners of the viewpoint, is followed by Illumina sequencing. The reads are then aligned to the genome, permitting the genome-wide identification of all the interacting partners of the viewpoint [START_REF] Matelot | Determination of High-Resolution 3D Chromatin Organization Using Circular Chromosome Conformation Capture (4C-seq)[END_REF][START_REF] Simonis | Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C)[END_REF][START_REF] Van De Werken | Robust 4Cseq data analysis to screen for regulatory DNA interactions[END_REF][START_REF] Zhao | Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intraand interchromosomal interactions[END_REF]. Thus, 4C-seq allows to identify and quantify the interactions of one viewpoint with the entire genome (One vs All). A particularly powerful method to compare 4Cseq datasets is to use the ratio between two conditions (cell types, mutants, etc.), thereby allowing the quantitative detection of differences in 3D interactions between conditions.

Hi-C (All vs All) is another widely used derivative of 3C that allows to detect all interactions in a genome-wide and unbiased fashion. In this assay, 5' ends overhangs of DNA after the first enzymatic digestion are repaired and filled-in with biotinylated nucleotides. Chromatin is then ligated (blunt-end ligation), the crosslink is reversed and DNA purified. DNA is later on sheared and ligation junctions are finally pulled-down using streptavidin beads [START_REF] Lieberman-Aiden | Comprehensive Mapping of Long-Range Interactions Reveals Folding Principles of the Human Genome[END_REF]. DNA sequences on either side of the ligation junction are then sequenced and identified by extensive bioinformatical analyses to result in the detection of all interacting fragments present within the cell population.

c. DamID, MadID, CUT&RUN and CUT&Tag

ChIP-seq has been, over the last 15 years, the most widely used tool for the characterization of genome-wide protein-DNA binding and epigenetic mark distribution [START_REF] Barski | High-Resolution Profiling of Histone Methylations in the Human Genome[END_REF][START_REF] Johnson | Genome-Wide Mapping of in Vivo Protein-DNA Interactions[END_REF]. Other alternatives exist though. DNA adenine methyltransferase identification (DamID) allows to map DNA-protein contacts within living cells. In this system, cells are transfected with a fusion protein containing the E. coli Dam DNA methyl transferase and the protein of interest. As a result, DNA in close proximity to the fusion protein will be methylated on adenines in a GATC context. As methylated adenine does not exist in most eukaryotes [START_REF] Mondo | Widespread adenine N-6methylation of active genes in fungi[END_REF][START_REF] Bochtler | DNA methylation in eukaryotes: Enzymatic mark or a form of DNA damage?[END_REF], after sequencing, it allows to detect chromatin regions that were in close proximity to the fusion protein (For review see [START_REF] Aughey | Dam it's good! DamID profiling of protein-DNA interactions: Dam it's good! Wiley Interdiscip[END_REF]. DamID has been used to characterize chromatin states in Drosophila cell lines [START_REF] Filion | Systematic protein location mapping reveals five principal chromatin types in Drosophila cells[END_REF] and chromatin regions in close proximity to the nuclear lamina in mammalian cells [START_REF] Guelen | Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions[END_REF]. It has the advantages that no specific antibody is needed, as compared to ChIP experiments. Yet, a drawback of DamID is that it only methylates adenine in GATC motifs that are not evenly distributed in the genome. In 2018, Sobecki and colleagues [START_REF] Sobecki | MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells[END_REF] developed an unbiased version of DamID known as MadID. In this technique, they fuse the protein of interest with M.EcoGII that is able to methylate any adenine, in any genomic motif. They were able to develop a technic to robustly identify protein-DNA interaction genome wide [START_REF] Sobecki | MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells[END_REF].

Recently, two other techniques were developed to profile protein-DNA binding: CUT&RUN (Cleavage Under Targets and Release Using Nuclease) and CUT&Tag (Cleavage under Targets and Tagmentation) [START_REF] Kaya-Okur | CUT&Tag for efficient epigenomic profiling of small samples and single cells[END_REF][START_REF] Skene | An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites[END_REF][START_REF] Skene | Targeted in situ genome-wide profiling with high efficiency for low cell numbers[END_REF]. In the CUT&RUN technique, cells are bound to beads and then permeabilized. Cells are then incubated with an antibody against a protein of interest and a Modified pA-MNAse (Protein A/Micrococcal Nuclease) is added. pA-MNAse will then cut genomic DNA around the antibody where it is fixed after addition of Ca 2+ . Fragments are then release in the supernatant and prepared for sequencing [START_REF] Skene | An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites[END_REF][START_REF] Skene | Targeted in situ genome-wide profiling with high efficiency for low cell numbers[END_REF]. CUT&Tag is an adaptation of CUT&RUN that uses a modified pA-Tn5 (Protein A/Transposase 5) directly loaded with sequencing adapters that will be targeted to a secondary antibody that recognize the primary antibody against the protein of interest [START_REF] Kaya-Okur | CUT&Tag for efficient epigenomic profiling of small samples and single cells[END_REF]. Both technics can be used in fewer cells, at a lower cost and faster than ChIP-seq (see for comparison https://www.epicypher.com/resources/blog/cut-and-run-vs-cut-and-tag-which-one-is-rightfor-you/).

Heterochromatin and euchromatin: global regulation of gene expression

In [START_REF] Heitz | Das Heterochromatin der Moose[END_REF], Emil Heitz (Heitz, 1928) observed by conventional microscopy the interphase nuclei of moss and Drosophila and noticed that some regions did not undergo decondensation after mitosis. These condensed regions of chromosomes were called heterochromatin, whereas the decondensed ones that spread out diffusely were called euchromatin. Thus, chromatin inside the nucleus is canonically divided into 2 categories, euchromatin and heterochromatin.

Euchromatin has a relatively relaxed structure, is enriched in active genes and is subjected to changes according to cell cycle. In contrast, heterochromatin is more condensed and is enriched in repetitive sequences such as telomeres and centromeres.

In the nucleus, these two chromatin states globally segregate: heterochromatin tends to be localized at the periphery whereas euchromatin tends to be constrained in the center of the nucleus. This is true for numerous species such as mammals [START_REF] Fedorova | Nuclear architecture and gene regulation[END_REF] and Drosophila [START_REF] Heitz | Der Bau der somatischen Kerne von Drosophila melanogaster[END_REF], but this is not universal as yeast, rod photoreceptor cells of night vision animals and specific human cell types have opposite patterns [START_REF] Brickner | Gene Recruitment of the Activated INO1 Locus to the Nuclear Membrane[END_REF][START_REF] Egecioglu | Gene positioning and expression[END_REF][START_REF] Solovei | Nuclear Architecture of Rod Photoreceptor Cells Adapts to Vision in Mammalian Evolution[END_REF]. Thus, these two compartments are different in their active gene numbers, gene density, chromatin compaction and in their location within the nucleus. Moreover, below I will discuss how these chromatin types differ in their epigenetic signature: both are differently marked by histone modifications and associated proteins that bind (Fig. 2.2).

genome coverage, number of domains, gene average, histone modifications and protein binding profiles. There are two heterochromatin types: Blue (facultative heterochromatin) and Green (constitutive heterochromatin).

1. The "Blue" chromatin corresponds to facultative heterochromatin, characterized by enrichment of Polycomb protein complexes (PcG) and H3K27me3 histone marks. It is defined as euchromatin regions that becomes packaged into a heterochromatin like structure and has the potential to become again transcriptionally active under certain conditions (developmental stage, localization in the nucleus, parental expression) [START_REF] Trojer | Facultative Heterochromatin: Is There a Distinctive Molecular Signature?[END_REF]. PcG chromatin domains are broad and were first identified to regulate developmental genes in Drosophila.

2. The "Green" chromatin corresponds to constitutive heterochromatin and is marked by HP1 ( Heterochromatin Protein 1, a very conserved protein among animals), Su(var)3-9 [START_REF] Schotta | Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing[END_REF], HP-1 interacting proteins as well as H3K9me2 and H3K9me3 marks [START_REF] Saha | Epigenomic and genomic landscape of Drosophila melanogaster heterochromatic genes[END_REF]. This type of heterochromatin marks large domains, often around centromeric regions. Constitutive heterochromatin is stable and can spread to induce gene silencing. One major example is the Position Variegation Effect (PEV) observed in the eye of Drosophila. Translocation of the white gene (that gives the red color to the eye) by UV radiation nearby heterochromatin leads to the stochastic silencing of the white gene, in some but not all cells, due to variable spreading of heterochromatin. As a result, a mosaic variegated eye is observed (red and white patches) [START_REF] Elgin | Position-Effect Variegation, Heterochromatin Formation, and Gene Silencing in Drosophila[END_REF][START_REF] Muller | THE FREQUENCY OF TRANSLOCATIONS PRODUCED BY X-RAYS IN DROSOPHILA[END_REF]. PEV was the first concrete clue that chromatin structure and gene regulation were linked.

The blue and green domains are non-overlapping [START_REF] De Wit | High-Resolution Mapping Reveals Links of HP1 with Active and Inactive Chromatin Components[END_REF] showing they truly represent different chromatin units.

3. The third chromatin type is "Black" chromatin that is also repressive, but appears devoid of specific histone marks. This "Black" chromatin represents the majority of the genome [START_REF] Filion | Systematic protein location mapping reveals five principal chromatin types in Drosophila cells[END_REF] and is gene poor compared to the other chromatin types.

4. The two last chromatin types ("Yellow" and "Red") constitute euchromatin: they are enriched in active genes, active histone marks, RNA polymerase and produce abundant amount of mRNA. Filion and colleagues found that the "Red" and "Yellow" active chromatin types segregate according to protein binding profiles they analyzed and that the "Yellow" chromatin encompasses constitutively expressed housekeeping genes, but to date no other studies were able to recapitulates differences between "Red" and "Yellow" chromatin.

Later one, thanks to chromosome painting (DNA FISH) and Hi-C, chromosome territories were shown in the cells of various species, including human [START_REF] Boyle | The spatial organization of human chromosomes within the nuclei of normal and emerin-mutant cells[END_REF], chicken [START_REF] Cremer | Chromosome territories, nuclear architecture and gene regulation in mammalian cells[END_REF] and Drosophila (Rosin et al., 2018) (Fig. 2.5). Even though chromosomes segregate in different chromosome territories their separation is not strict. Neighboring chromosomes can invade each other's territories and they can intermingle at their boundaries [START_REF] Branco | Intermingling of Chromosome Territories in Interphase Suggests Role in Translocations and Transcription-Dependent Associations[END_REF]. In mammals, chromosomes undergo another level of organization where chromosomes have preferential localization in the nucleus. Indeed, in human cells, genepoor chromosomes tend to be located at the periphery of the nucleus whereas gene-rich chromosomes localize preferentially in the inner space. In human cells, this is particularly clear for Chromosome 18 and 19, which are similar in size but not in gene density, replication timing and DNA composition. Chromosome 19 is gene rich, early replicating and has abundant hyperacetylated histone H4 whereas chromosome 18 has far fewer genes and is late replicating.

In lymphoblastic human cells, chromosome 19 tends to localize in the inner space of the nucleus compared to chromosome 18 that tends to localize at the periphery [START_REF] Croft | Differences in the Localization and Morphology of Chromosomes in the Human Nucleus[END_REF].

However, this correlation seems to be cell-type specific as no significant difference could be seen in flat fibroblasts [START_REF] Croft | Differences in the Localization and Morphology of Chromosomes in the Human Nucleus[END_REF].

At a large scale, it has been demonstrated that heterochromatin and euchromatin are located in different regions of the nucleus and that at the chromosome scale, interphasic chromosomes preferentially reside in different chromosome territories.

Nuclear compartments and Chromatin hubs

Historically, nuclear compartments were discovered by light microscopy and later confirmed by electron microscopy or by staining of specific proteins and/or RNA.

a. Polycomb bodies

Polycomb group (PcG) acts as large protein complexes to silence gene expression as mentioned before. In Drosophila, they bind at Polycomb Responsive Element (PRE), which are cis-DNA regulatory elements that are essential and sufficient to recruit the PcG machinery. PREs can cluster together, forming large repressed Polycomb domains covered by H3K27me3 (Schuettengruber and Cavalli, 2009;[START_REF] Schwartz | Genome-wide analysis of Polycomb targets in Drosophila melanogaster[END_REF] that are particularly involved in the regulation of Hox gene expression.

Using conventional microscopy, the immunodetection of PcG proteins or H3K27me3 mark revealed large and numerous nuclear PcG foci in Drosophila cell lines [START_REF] Messmer | Analysis of the functional role of the Polycomb chromo domain in Drosophila melanogaster[END_REF] and later in Drosophila embryos [START_REF] Buchenau | The Distribution of Polycomb-Group Proteins During Cell Division and Development in Drosophila Embryos: Impact on Models for Silencing[END_REF]. These nuclear clusters were named Polycomb bodies. Recently, super resolution microscopy revealed a high number of small PcG foci in Drosophila S2 cells in addition to the already previously identified large ones. They determined that PcG foci have disparity in size, ranging from 30 to 700 nm with the majority being over 100 nm, and they could estimate around 600-800 PcG foci per S2 cells [START_REF] Wani | Chromatin topology is coupled to Polycomb group protein subnuclear organization[END_REF]. This high number of PcG loci that Wani and colleagues found might be due to the use of high-resolution microscopy as previously, it was postulated that cells contain between 30 to 50 PcG bodies only with size ranging from 200 to 450 nm by microscopy [START_REF] Grimaud | RNAi Components Are Required for Nuclear Clustering of Polycomb Group Response Elements[END_REF].

Genes present within a PcG bodiy can contact each other, as long as they are all repressed, thereby adopting a higher order 3D structure as seen for the Bithorax complex (BX-C) in Drosophila [START_REF] Lanzuolo | Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex[END_REF]. Long range contacts on the same chromosome arm in Drosophila can also be detected between the BX-C and the Antennapedia (ANT-C) homeotic complexes when genes are repressed [START_REF] Bantignies | Polycomb-Dependent Regulatory Contacts between Distant Hox Loci in Drosophila[END_REF][START_REF] Tolhuis | Interactions among Polycomb Domains Are Guided by Chromosome Architecture[END_REF] even though the two clusters are separated by 10Mb (Fig. 2.6). Polycomb bodies could act as repressive factories that allow to mutualize repressive factors. However, when gene became active under the proper stimulus, they can loop out of the Polycomb body and join an active environment [START_REF] Bantignies | Polycomb-Dependent Regulatory Contacts between Distant Hox Loci in Drosophila[END_REF][START_REF] Lanzuolo | Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex[END_REF] The same can be observed in mammals. In tissues in which all genes from the Hoxb complex are repressed, these genes cluster within a single PcG body. In contrast, in other tissues expressing a subset of Hoxb genes, active genes dissociate from the repressed hub [START_REF] Isono | SAM Domain Polymerization Links Subnuclear Clustering of PRC1 to Gene Silencing[END_REF]. Similar tendency have been described for the Hoxd cluster in mammals in which newly activated genes progressively escape the repressive H3K27me3 environment and loop out of the PcG body [START_REF] Noordermeer | The Dynamic Architecture of Hox Gene Clusters[END_REF]. Moreover, PRC1, a component of the PcG machinery, has been shown to be essential to regulate Hox chromatin organization in ES cells. In ES cells, the four Hox clusters are repressed. Promoter-promoter and enhancer-promoter contacts are maintained in a repressed state. PRC1 removal induced a disruption of promoter-promoter interactions but enhancer-promoter interactions remained unchanged. Instead, enhancerpromoters contacts adopted active chromatin marks and become expressed. Thus, PcG can physically constrained Hox genes in ES cells in a poised repressed domain and, under stimulus, genes can escape the repressed environment.

b. Lamina Associated domains

Chromatin organization is also influenced by its interaction with the lamina, the inner layer of the nuclear envelope. Lamina Associated Domains (LADs) were first identified using the DNA adenine methyltransferase identification (DamID) technique, where the E. coli adenine methyltransferase was fused to various lamina components. Chromatin associating with the nuclear envelop could subsequently be revealed by microscopy, micro-arrays or genome-wide sequencing.

LADs have been detected in various species, including C. elegans [START_REF] Ikegami | Caenorhabditis elegans chromosome arms are anchored to the nuclear membrane via discontinuous association with LEM-2[END_REF], Drosophila [START_REF] Pickersgill | Characterization of the Drosophila melanogaster genome at the nuclear lamina[END_REF], mouse [START_REF] Peric-Hupkes | Molecular Maps of the Reorganization of Genome-Nuclear Lamina Interactions during Differentiation[END_REF] and human [START_REF] Guelen | Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions[END_REF][START_REF] Kind | Single-Cell Dynamics of Genome-Nuclear Lamina Interactions[END_REF]. Genes present in LADs are generally repressed or lowly expressed and the integrity of the nuclear lamina is essential for the spatial organization of chromatin and the maintenance of the repressed state in Drosophila [START_REF] Ulianov | Nuclear lamina integrity is required for proper spatial organization of chromatin in Drosophila[END_REF].

LADs are enriched for histone marks characteristics of constitutive or facultative heterochromatin, such as H3K9me2/3 and H3K27me3. LADs and inter-LADs (iLADs) strongly correlate with the transcription state corresponding respectively to repressed and active chromatin [START_REF] Vieux-Rochas | Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain[END_REF]. In general, LADs are then a repressed compartment and when repressed chromatin from LADs is forced to relocalize into an active hub, it loses its repressive marks [START_REF] Robson | Tissue-Specific Gene Repositioning by Muscle Nuclear Membrane Proteins Enhances Repression of Critical Developmental Genes during Myogenesis[END_REF].

c. Transcription factories

Repressive chromatin hubs are well established in the nucleus but evidence for active hubs exists as well. Transcription factories are formed by the focal accumulation of RNA polymerases allowing the concomitant transcription of at least two DNA loci. These subnuclear compartments are enriched in actively transcribed genes and contain high concentration of hyper-phosphorylated RNAPII [START_REF] Eskiw | Ultrastructural study of transcription factories in mouse erythroblasts[END_REF][START_REF] Grande | Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II[END_REF][START_REF] Iborra | Active RNA polymerases are localized within discrete transcription 'factories[END_REF][START_REF] Rosenberg | Cell-cycle specific association of transcription factors and RNA polymerase ii with the human β-globin gene locus: Protein/DNA Interactions During Cell Cycle[END_REF]. As the number of active genes in a cell outnumber the number of transcription factories [START_REF] Pombo | The Localization of Sites Containing Nascent RNA and Splicing Factors[END_REF], it is likely that genes in transcription factories gather to share and increase the concentration of transcription factors necessary for gene expression.

These subnuclear structures are dynamic and have a crucial role in gene regulation and their nuclear domain are well defined. But how and why these dynamic sub-nuclear structures exist?

The location of chromosomes and nuclear sub-compartments remain probabilistic, they do not have absolute coordinates present in every cell. Thus, a system is needed to clearly separate repressed in active regions.

d. Liquid-liquid phase separation

Recently, a new paradigm has emerged to explain how membraneless sub-nuclear compartments are formed: liquid-liquid phase separation. In these models, chromatin modifications, chromatin-associated protein and other molecules organize and are organized within droplets, based on their shared chemical properties. The compartments formed by liquidliquid phase separation are reversible and may, for example, disassemble during mitosis and reassemble after [START_REF] Bhat | Nuclear compartmentalization as a mechanism of quantitative control of gene expression[END_REF][START_REF] Strom | The liquid nucleome -phase transitions in the nucleus at a glance[END_REF]. Within these compartments, the local concentration of specific factors can be remarkably high, potentializing de facto the cellular processes that takes place in the compartment, such as gene repression or activation.

Liquid-Liquid Phase separation has been shown to be governing the clustering of certain types of distal enhancers with targeted promoters during gene activation [START_REF] Cho | Mediator and RNA polymerase II clusters associate in transcription-dependent condensates[END_REF]. The high local concentration of transcription factors in a physically constrained condensate may also help to explain how one enhancer can activate several genes simultaneously and the transvection mechanism in Drosophila where an enhancer from one chromosome can activate a promoter located on another one.

e. A and B compartments

Besides the separation in chromosome territories, a zoom in on the 3D organization of chromosomes reveals finer structures. Repressed regions (gene deserts and heterochromatin)

tend to be condensed and to preferentially interact together. In contrast, active and gene-rich regions are less condensed and interact with other active regions. This separation was initially observed by 4C [START_REF] Simonis | Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C)[END_REF]. These two chromatin compartments were later called A (Active) and B (inactive) as characterized by Hi-C (Lieberman-Aiden et al., 2009) and single cell microscopy [START_REF] Wang | Spatial organization of chromatin domains and compartments in single chromosomes[END_REF] in mammals. A and B compartments are also found in other species such as Drosophila [START_REF] Rowley | Evolutionarily Conserved Principles Predict 3D Chromatin Organization[END_REF]. A and B compartments also correlate with other aspects of nuclear organization described before as, for instance, B compartments correlate nicely with LADs (van Steensel and Belmont, 2017).

TADs and chromatin looping

Enhancers and their target promoters usually reside in the same chromosomal domains called Topologically Associating domains (TAD) [START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF], restricting gene interactions to a defined chromatin domain. TADs were first identified by Hi-C in mammals [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Nora | Spatial partitioning of the regulatory landscape of the X-inactivation centre[END_REF] and Drosophila [START_REF] Sexton | Three-Dimensional Folding and Functional Organization Principles of the Drosophila Genome[END_REF], and are defined as 3D chromatin domains enriched in intra-domain interactions as compared to their surroundings. TAD borders and the genomic regions contained within the TAD are mostly invariant between cell types [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF]. In Drosophila, enhancer-promoter loops precede TADs formations [START_REF] Espinola | Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development[END_REF], so TADs could help to isolate this regulatory domain from spurious contacts and facilitate the initiation of gene transcription. Hence, TADs appear as functional units of the genome.

a. TADs in mammals

TADs in mammals have a reported median size of around 880kb. In mouse embryonic stem cells (ES cells), around 90% of the genome is organized into TADs [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF]. Coregulated genes can be found within the same TAD [START_REF] Flavahan | Insulator dysfunction and oncogene activation in IDH mutant gliomas[END_REF][START_REF] Dily | Distinct structural transitions of chromatin topological domains correlate with coordinated hormone-induced gene regulation[END_REF][START_REF] Nora | Spatial partitioning of the regulatory landscape of the X-inactivation centre[END_REF], but many are composed by a mix of ubiquitously expressed genes and celltype specific genes. Thus, sharing the same TAD is not sufficient to instruct coordinated gene expression. Epigenetic factors such as histone modifications, the formation of sub-TADs and the establishment of specific enhancer-promoter loops may also be necessary. Indeed, Hi-C studies have revealed that TAD borders are mostly invariant between cell types and that some

TADs can be further sub-divided in sub-TADs. These smaller chromatin domains and their borders vary between cell types upon cell differentiation [START_REF] Phillips-Cremins | Architectural Protein Subclasses Shape 3D Organization of Genomes during Lineage Commitment[END_REF]. Sub-TADs median size is around 185kb. Loci within the same sub-TAD tend to show correlated epigenetic modifications as well as correlated transcriptional status [START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF].

A notable feature of mammalian TAD boundaries is that 75% of them are enriched for the binding of the CCCTC-binding factor (CTCF) and the Cohesin complex [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF]. The consensus CTCF binding motif has an orientation and TAD boundaries on either side of TADs are enriched in convergent CTCF motifs [START_REF] De Wit | CTCF Binding Polarity Determines Chromatin Looping[END_REF][START_REF] Dowen | Control of Cell Identity Genes Occurs in Insulated Neighborhoods in Mammalian Chromosomes[END_REF][START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF]. The orientation has been shown to be important as inversion or removal of a single CTCF binding site can be sufficient to weaken TAD boundaries and reduce TAD insulation [START_REF] Guo | CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function[END_REF]. Even though the Cohesin complex and CTCF are present at TAD boundaries, their binding is frequently detected within TADs as well [START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF].

The question of how TADs are created and how CTCF sites are selected to become a TAD boundary then emerged.

TADs are thought to be formed through loop extrusion model. The ring-shaped Cohesin complex is loaded on the DNA, which then slides through its lumen, initiating the formation of a DNA loop. When Cohesin encounters a CTCF bound site, the extrusion is blocked on one side of the DNA but the sliding continues on the other side. The loop extrusion is then stopped by a second CTCF bound site [START_REF] Alipour | Self-organization of domain structures by DNA-loopextruding enzymes[END_REF][START_REF] Fudenberg | Formation of Chromosomal Domains by Loop Extrusion[END_REF][START_REF] Sanborn | Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes[END_REF]. Convergent CTCF motifs have been shown to be crucial since up to 90 % of DNA loops are established between convergent CTCF binding sites [START_REF] De Wit | CTCF Binding Polarity Determines Chromatin Looping[END_REF]Rao et al., 2014) (Fig. 2.7). Studies have revealed that the N-ter of CTCF protein can block loop extrusion in a more stable manner than the C-ter clarifying why orientation is important [START_REF] Li | The structural basis for cohesin-CTCF-anchored loops[END_REF][START_REF] Nora | Molecular basis of CTCF binding polarity in genome folding[END_REF][START_REF] Pugacheva | CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention[END_REF]. Motif orientation thus appears important for the formation of TAD. CTCF is essential in the TAD formation process since acute depletion of CTCF by an inducible Auxin-mediated degron (mAID degron system) shows that chromatin compaction remains intact but insulation between TADs is reduced [START_REF] Nora | Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization[END_REF][START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF]. TAD formation is dynamic and both the Cohesin complex and CTCF, transiently associate with DNA. The WAPL protein is essential for Cohesin unloading from DNA. The depletion of WAPL, resulting in longer Cohesin-DNA association times, induces larger DNA loops, meaning that Cohesin is able to pull more DNA through its ring [START_REF] Haarhuis | The Cohesin Release Factor WAPL Restricts Chromatin Loop Extension[END_REF]. This also implies that, upon WAPL depletion, Cohesin will stay longer on DNA compared to CTCF.

Other questions arise about the role of CTCF in stopping loop extrusion. Does CTCF, in the right orientation, stop Cohesin because of physical blocking or because Cohesin directly binds CTCF?

In conclusion, in (somatic) mammalian cells, the main actors for the formation and maintenance of TAD structure are the Cohesin complex, its unloader WAPL and CTCF, the only insulator protein described in mammals to date. Defects affecting either specific TAD boundaries or one of these factors can be associated with human disease. For example, the deletion, inversion or duplication at TADs spanning the WNT6/IHH/EPHA4/PAX3 locus are responsible for various polydactyly in humans [START_REF] Lupiáñez | Disruptions of Topological Chromatin Domains Cause Pathogenic Rewiring of Gene-Enhancer Interactions[END_REF]. Disruption of CTCF binding at TAD boundaries, either through genetic or epigenetic mechanisms, can cause oncogene activation due to TAD fusions and 'enhancer-hijacking' [START_REF] Flavahan | Insulator dysfunction and oncogene activation in IDH mutant gliomas[END_REF][START_REF] Flavahan | Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs[END_REF][START_REF] Hnisz | Activation of proto-oncogenes by disruption of chromosome neighborhoods[END_REF]. Moreover, CTCF binding sites are among the most frequently mutated sites in cancer cells [START_REF] Hnisz | Activation of proto-oncogenes by disruption of chromosome neighborhoods[END_REF][START_REF] Katainen | CTCF/cohesin-binding sites are frequently mutated in cancer[END_REF]. Thus, TADs in mammals appears as structural and fundamental units of the genome.

b. TADs in Drosophila

In Drosophila, the median size of TADs is around 100 kb and loci within the same TAD harbor correlated epigenetic signature [START_REF] Sexton | Three-Dimensional Folding and Functional Organization Principles of the Drosophila Genome[END_REF]. Hence, they resemble more like mammalian sub-TADs. Drosophila TADs have been classified in four different types [START_REF] Sexton | Three-Dimensional Folding and Functional Organization Principles of the Drosophila Genome[END_REF]:

1. Transcriptionally active and enriched in H3K4me3.

2. Repressed PcG enriched in H3K27me3 (facultative heterochromain).

3. Repressed and devoid in histone marks (black chromatin).

4. Repressed and enriched in H3K9me3 and HP1 protein (constitutive heterochromatin).

These four types are reminiscent of the chromatin types that were proposed by Filion and colleagues [START_REF] Filion | Systematic protein location mapping reveals five principal chromatin types in Drosophila cells[END_REF] in 2010.

More recent studies have revealed that Drosophila TADs may be of even smaller sizes (tens of kb) [START_REF] Wang | Sub-kb Hi-C in D. melanogaster reveals conserved characteristics of TADs between insect and mammalian cells[END_REF]. Heurteau and colleagues [START_REF] Heurteau | Insulator-based loops mediate the spreading of H3K27me3 over distant microdomains repressing euchromatin genes[END_REF] further proposed the existence of micro H3K27me3 domains within euchromatic TADs. Even if these findings could be due to the different set of tools used to detect TADs from Hi-C experiments, these studies agreed that repressive TADs occupy the majority of the Drosophila genome [START_REF] Hou | Gene Density, Transcription, and Insulators Contribute to the Partition of the Drosophila Genome into Physical Domains[END_REF][START_REF] Sexton | Three-Dimensional Folding and Functional Organization Principles of the Drosophila Genome[END_REF][START_REF] Ulianov | Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains[END_REF][START_REF] Wang | Sub-kb Hi-C in D. melanogaster reveals conserved characteristics of TADs between insect and mammalian cells[END_REF]. Super-resolution microscopy confirmed these chromatin structures of Drosophila chromatin, where repressed H3K27me3 compartments form globular domains interspersed by less condensed H3K4me3 chromatin [START_REF] Cattoni | Single-cell absolute contact probability detection reveals chromosomes are organized by multiple lowfrequency yet specific interactions[END_REF][START_REF] Szabo | TADs are 3D structural units of higher-order chromosome organization in Drosophila[END_REF].

Another major difference between Drosophila and mammalian TADs is that boundaries in Drosophila are not enriched for CTCF and the Cohesin complex [START_REF] Cubeñas-Potts | Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture[END_REF][START_REF] Ulianov | Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains[END_REF]. What makes a boundary in Drosophila then became an essential question.

Studies combining Hi-C, ChIP-seq and motif search have helped answering this question.

TADs boundaries in Drosophila emerge from a combination of different factors. First, several studies have shown that TADs boundaries coincide with regions of actively transcribed genes, including housekeeping genes, marked by H3K4me3 [START_REF] Cubeñas-Potts | Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture[END_REF][START_REF] Hou | Gene Density, Transcription, and Insulators Contribute to the Partition of the Drosophila Genome into Physical Domains[END_REF][START_REF] Hug | Chromatin Architecture Emerges during Zygotic Genome Activation Independent of Transcription[END_REF][START_REF] Ulianov | Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains[END_REF]. Most TAD boundaries (77%) occur at gene promoters and in 70% of them, their orientations are divergent [START_REF] Ramírez | High-resolution TADs reveal DNA sequences underlying genome organization in flies[END_REF]. Secondly, Drosophila CTCF (dCTCF) is the only insulator protein conserved in mammals [START_REF] Baniahmad | Modular structure of a chicken lysozyme silencer: Involvement of an unusual thyroid hormone receptor binding site[END_REF][START_REF] Lobanenkov | A novel sequence-specific DNA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5'-flanking sequence of the chicken c-myc gene[END_REF]. Even though dCTCF is not involved in TADs formation or maintenance this insulator protein is important for the regulation of Hox genes in Drosophila (developed in Chapter III). In Drosophila many Insulator proteins have been described. They bind to Boundary elements in combination and appear to segregate chromatin states. They can be sub-divided in three categories (Fig. 2.8):

• I) DNA sequence specific insulator proteins including: dCTCF [START_REF] Moon | CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab-8 insulator[END_REF], GAF [START_REF] Schweinsberg | The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites[END_REF], Ibf1/ Ibf2 [START_REF] Cuartero | Ibf1 and Ibf2 are novel CP190-interacting proteins required for insulator function[END_REF], PITA, ZIPIC [START_REF] Maksimenko | Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin[END_REF], Zw5 [START_REF] Gaszner | The Zw5 protein, a component of the scs chromatin domain boundary, is able to block enhancer-promoter interaction[END_REF], BEAF-32 [START_REF] Zhao | Visualization of chromosomal domains with boundary element-associated factor BEAF-32[END_REF].

• II) Non-sequence specific proteins CP190 [START_REF] Pai | The Centrosomal Protein CP190 Is a Component of the gypsy Chromatin Insulator[END_REF] and multiple protein isoforms encoded by mod(mdg4) [START_REF] Dorn | Transgene analysis proves mRNA transsplicing at the complex mod(mdg4) locus in Drosophila[END_REF].

• III) Modulators of enhancer blocking effect or tissue/stage specific: Elba1, 2, 3, Shep [START_REF] Aoki | Elba, a novel developmentally regulated chromatin boundary factor is a hetero-tripartite DNA binding complex[END_REF][START_REF] Matzat | Tissue-Specific Regulation of Chromatin Insulator Function[END_REF].

The binding patterns of these insulator proteins have been extensively studied over the years to understand if they have specific or redundant functions. It revealed that several insulator proteins can bind to the DNA [START_REF] Nègre | A Comprehensive Map of Insulator Elements for the Drosophila Genome[END_REF][START_REF] Schwartz | Nature and function of insulator protein binding sites in the Drosophila genome[END_REF] and that insulator proteins can form multiprotein complexes [START_REF] Cuartero | Ibf1 and Ibf2 are novel CP190-interacting proteins required for insulator function[END_REF][START_REF] Maksimenko | Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin[END_REF][START_REF] Pai | The Centrosomal Protein CP190 Is a Component of the gypsy Chromatin Insulator[END_REF]. Drosophila TAD boundaries are enriched for the binding of BEAF-32/Chromator, BEAF-32/CP190 [START_REF] Ramírez | High-resolution TADs reveal DNA sequences underlying genome organization in flies[END_REF][START_REF] Wang | Sub-kb Hi-C in D. melanogaster reveals conserved characteristics of TADs between insect and mammalian cells[END_REF] and M1BP [START_REF] Ramírez | High-resolution TADs reveal DNA sequences underlying genome organization in flies[END_REF], and more generally the co-occupancy of multiple architectural protein [START_REF] Van Bortle | Insulator function and topological domain border strength scale with architectural protein occupancy[END_REF].

As mentioned before, actively transcribed genes are an important feature of Drosophila TAD boundaries. To test whether gene transcription is a key step in TADs formation, Drosophila embryonic development is a perfect model. At the beginning of Drosophila development, the fertilized embryo undergoes 13 synchronous nuclear division (nc). During the first hours, the zygotic genome is mostly transcriptionally silenced. It is only from nc 14 (around 2.5 hours after fertilization) that the Zygotic Genome is Activated (ZGA) [START_REF] De Renzis | Unmasking Activation of the Zygotic Genome Using Chromosomal Deletions in the Drosophila Embryo[END_REF][START_REF] Foe | Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in drosophila embryogenesis[END_REF]. Importantly, at nc13, RNA pol II is already massively recruited to the chromatin [START_REF] Blythe | Zygotic Genome Activation Triggers the DNA Replication Checkpoint at the Midblastula Transition[END_REF]. In 2017, Hug and colleagues [START_REF] Hug | Chromatin Architecture Emerges during Zygotic Genome Activation Independent of Transcription[END_REF] compared chromatin architecture of embryos at nc 12, 13 and 14 to examine the effect of ZGA and transcription on TAD formation and maintenance. They found that before ZGA, the majority of the genome is unstructured, except for 180 regions enriched in housekeeping genes.

TAD structure arises with the recruitment of RNA pol II during nc 13. They also found that TAD boundaries are enriched in housekeeping genes, as already shown previously [START_REF] Ramírez | High-Affinity Sites Form an Interaction Network to Facilitate Spreading of the MSL Complex across the X Chromosome in Drosophila[END_REF][START_REF] Ulianov | Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains[END_REF][START_REF] Weber | Support for multiple classes of local expression clusters in Drosophila melanogaster, but no evidence for gene order conservation[END_REF] and later confirmed by Ramirez and colleagues in 2018 [START_REF] Ramírez | High-resolution TADs reveal DNA sequences underlying genome organization in flies[END_REF], who demonstrated that housekeeping genes are a major feature of TAD boundaries in Drosophila (Fig. 2.9). Through development, TADs become more and more structured and TAD boundaries remain invariant, as previously shown in cell lines [START_REF] Ulianov | Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains[END_REF]. Interestingly, injection of RNA polymerase II inhibitors prior to TAD formation does not delay their nucleation but does reduce their insulation [START_REF] Hug | Chromatin Architecture Emerges during Zygotic Genome Activation Independent of Transcription[END_REF]. In conclusion, TADs formation in Drosophila seems to be independent of gene transcription but, transcriptional processes might be essential to maintain chromatin architecture.

Thus, in Drosophila, chromatin state and TAD structures are tightly linked. As epigenetic domains strongly coincide with TAD boundaries, gene transcription and insulator proteins could be sufficient to shape chromatin 3D organization.

present in human and around 54% of boundaries in human being present in mouse. Another study compared cells of four different mammalian species (mouse, rabbit, dog and macaque)

and highlighted again similarities for syntenic regions [START_REF] Vietri Rudan | Comparative Hi-C Reveals that CTCF Underlies Evolution of Chromosomal Domain Architecture[END_REF]. Importantly, not only boundaries and TADs are conserved but also CTCF motifs and orientation.

Within Drosophila species, extensive TAD re-shuffled occurred between Drosophila melanogaster and Drosophila triauraria but, interestingly, TADs containing developmental genes remained well conserved [START_REF] Torosin | 3D genome evolution and reorganization in the Drosophila melanogaster species group[END_REF].

In Caenorhabditis elegans, the Dosage Compensation Complex (DCC) binds to both X chromosomes of hermaphrodite XX worms, reduce its expression level by half to achieve dosage compensation between genders. In hermaphrodites, self-interacting chromatin domains of around 1Mb are detected on the DCC-bound X chromosomes. It resembles the TAD structure found in mammals but no obvious 3D compartmentalization seems to be observed on autosomes [START_REF] Crane | Condensin-driven remodelling of X chromosome topology during dosage compensation[END_REF]. TAD structuration in Arabidopsis thaliana is less evident but chromatin domains insulated from the surrounding are found. Compacted repressed domains enriched in interactions and repressive histone modifications are found as well. In total, more than 1000 boundaries between TAD-like structures are found [START_REF] Wang | Genome-wide analysis of local chromatin packing in Arabidopsis thaliana[END_REF]. In the unicellular eukaryote Schizosaccharomyces pombe, self-interacting domains of around 50-100kb are found and their borders are enriched in Cohesin [START_REF] Mizuguchi | Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe[END_REF].

Rowley and colleagues [START_REF] Rowley | Evolutionarily Conserved Principles Predict 3D Chromatin Organization[END_REF] compared the chromatin organization between Drosophila, Arabidopsis, Neurospora crassa, C. elegans and Plasmodium falciparum and demonstrated that their genome are partitioned by transcriptional activity (Fig. 2.10). This nonexhaustive list indicates that genome partitioning seems to be universally conserved with the formation chromatin structures correlating with gene activity and epigenetic landscape. Even in prokaryotes nucleoids exhibit functional 3D genome organization (not discussed here).

Moreover, a recent study compared the 3D genome organization in 24 diverse species, including animals, plants and fungi, and correlated it with the presence or absence of Condensin II. They found that, across species, chromosome territories in interphase nuclei are maintained through either the clustering of centromeres or telomeres, or because they are polarized in the nucleus. Moreover, the difference between these three organizations seems to be linked to the presence or absence of Condensin II [START_REF] Hoencamp | 3D genomics across the tree of life reveals condensin II as a determinant of architecture type[END_REF].

Hox gene function and organization

Bilaterians are organisms that possess a left/right symmetry from embryos to adulthood (with the exception of the echinoderms). This implies that they exhibit a dorsal-ventral (D/V) and an anterior-posterior (A/P) axis. To establish identities along the A/P axis, the homeoboxcontaining Hox genes are essential [START_REF] Garcia-Fernandez | The genesis and evolution of homeobox gene clusters[END_REF][START_REF] Mcginnis | Homeobox genes and axial patterning[END_REF][START_REF] De Rosa | Hox genes in brachiopods and priapulids and protostome evolution[END_REF]. Hox genes encode transcription factors that provide cellular identity along the A/P axis. Importantly though, they do not provide the instruction to form segments, limbs or appendages per se. The misexpression of Hox genes (expression in the wrong cells/segments or expression of the wrong combinations of Hox genes) can lead to homeotic transformation, whereby specific body segments adopt incorrect positional identities [START_REF] Emerald | Homeotic transformation in Drosophila[END_REF][START_REF] Kessel | Variations of Cervical Vertebrae after Expression of a Hex-7.1 Transgene in Mice[END_REF]).

An intriguing feature of Hox genes is that they are, in most bilaterians, organized in clusters,

where their genomic position corresponds to their expression domain along the A/P axis. This spatial collinearity along the genome was discovered in 1978 by Ed Lewis [START_REF] Lewis | A gene complex controlling segmentation in Drosophila[END_REF] and was found to be conserved in most tested bilaterians [START_REF] Duboule | The structural and functional organization of the murine HOX gene family resembles that of Drosophila homeotic genes[END_REF][START_REF] Gaunt | Spatially restricted domains of homeo-gene transcripts in mouse embryos: relation to a segmented body plan[END_REF][START_REF] Graham | The murine and Drosophila homeobox gene complexes have common features of organization and expression[END_REF][START_REF] Kmita | Organizing Axes in Time and Space; 25 Years of Colinear Tinkering[END_REF][START_REF] Krumlauf | Hox genes in vertebrate development[END_REF]. Besides spatial collinearity, some species also undergo temporal Hox gene activation, as for mouse, where anterior genes are expressed earlier during the embryonic development (Izpisúa-Belmonte and [START_REF] Izpisúa-Belmonte | Homeobox genes and pattern formation in the vertebrate limb[END_REF]. Although Hox genes in most bilaterians are organized in clusters, these can both differ in the number of genes and their overall structuration (see [START_REF] Lappin | HOX GENES: Seductive Science, Mysterious Mechanisms[END_REF] and Figure 3.1). For example:

• Drosophila melanogaster possesses 8 Hox genes that are distributed into two clusters on the same chromosome.

• Caenorhabditis elegans (nematode) has 5 Hox genes in a single cluster.

• The mouse (Mus musculus) has a total of 39 Hox genes divided over four clusters that arose after two events of genome duplications, followed by diverse cluster-specific modifications. The hypothetical ancestor complex is shown in grey.

Hox expression domains in the central nervous system of human are schematized as well as the four paralogous Hox clusters in mammals.

Possible phylogenic relationship are shown by the arrows between the clusters with matching colors.

Figure reproduced from [START_REF] Mark | Homeobox Genes in Embryogenesis and Pathogenesis[END_REF].

Hox genes and their regulation in mammals a. Hox gene expression and histone modification landscape in mouse

The mouse, which is frequently used as a template for Hox gene organization in mammals, Mechanistically, the collinear activation of Hox genes is, at least to some extent, regulated by a switch of the histone modification landscape. In mouse embryonic stem cells, which represent the ground state before activation of homeotic genes, Hox genes are covered by a broad domain of H3K27me3, with some H3K4me3 peaks found at unmethylated CpGs [START_REF] Bernstein | A Bivalent Chromatin Structure Marks Key Developmental Genes in Embryonic Stem Cells[END_REF][START_REF] Noordermeer | Chromatin Architectures and Hox Gene Collinearity[END_REF][START_REF] Noordermeer | Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci[END_REF][START_REF] Schuettengruber | Genome Regulation by Polycomb and Trithorax Proteins[END_REF][START_REF] Soshnikova | Epigenetic Temporal Control of Mouse Hox Genes in Vivo[END_REF]. Hox genes are thus in a bivalent state where they are transcriptionally repressed but ready to be activated, thus harboring both active and repressed histone modifications [START_REF] Azuara | Chromatin signatures of pluripotent cell lines[END_REF][START_REF] Bernstein | A Bivalent Chromatin Structure Marks Key Developmental Genes in Embryonic Stem Cells[END_REF]. During temporal collinear Hox gene activation, as observed in vivo in the mouse, newly activated genes progressively loose the repressed H3K27me3 and gain the active H3K4me3 marks [START_REF] Noordermeer | Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci[END_REF][START_REF] Soshnikova | Epigenetic Temporal Control of Mouse Hox Genes in Vivo[END_REF]. This is further accompanied by an increase in RNA PII recruitment and increased levels of H3 acetylation [START_REF] Soshnikova | Epigenetic Temporal Control of Mouse Hox Genes in Vivo[END_REF].

Hence, temporal collinearity involves a sequential Hox gene activation and progressive changes in histone modifications. Other studies looked at the histone modification landscape associated with the maintenance of Hox genes (i.e. spatial collinearity) at a fixed later-stage developmental timepoint (embryonic day 10.5: E10.5) but at different position along the A/P axis. Hox genes are all repressed in the E10.5 mouse forebrain, Hoxd1 to Hoxd9 are expressed in the anterior trunk and Hoxd1 to Hoxd13 in the posterior trunk (Fig. 3.3). Consequently, in the forebrain all Hox genes are covered by H3K27me3, whereas in the anterior trunk, the active Hox genes are marked by H3K4me3 and the inactive Hox genes are marked by H3K27me3 [START_REF] Noordermeer | The Dynamic Architecture of Hox Gene Clusters[END_REF] (Fig. 3.3).

Besides the presence or absence of histone modifications, it has been shown that Hox gene expression is Polycomb dosage dependent. During motor neuron differentiation in the developing mouse (one of two cell types that express Hox genes along the primary A/P axis), a decrease in H3K27me3 and binding of Bmi1 (a component of PRC1) is observed along the A/P axis. Elevation or attenuation of Bmi1 levels alters the expression domain of Hox genes along the A/P axis, causing changes to motor neuron fate [START_REF] Golden | Polycomb repressive complex 1 activities determine the columnar organization of motor neurons[END_REF].

During limb development, a third type of collinearity, "quantitative collinearity" is observed.

This type of collinearity is characterized by two waves of transcription with two distinct biological roles. Early activated Hox genes are responsible for the development of the arm and forearm whereas during the second wave of transcription, digits are specified [START_REF] Andrey | A Switch Between Topological Domains Underlies HoxD Genes Collinearity in Mouse Limbs[END_REF][START_REF] Montavon | A Regulatory Archipelago Controls Hox Genes Transcription in Digits[END_REF][START_REF] Tarchini | Control of Hoxd Genes' Collinearity during Early Limb Development[END_REF]. In this mechanism, Hoxd9 to Hoxd13 are regulated by a series of enhancers located on the telomeric side of HoxD (in a TAD named the "T-DOM"), whereby the more telomeric a gene is located, the more it is expressed (i.e. Hoxd9 >>> Hoxd13) (Fig. 3.3). In digits, the activity pattern of these genes switches, due to the use of enhancers on the centromeric side (C-DOM). As a result, now the most centromeric genes become most expressed (i.e. Hoxd13 >>> Hoxd9) [START_REF] Andrey | A Switch Between Topological Domains Underlies HoxD Genes Collinearity in Mouse Limbs[END_REF] (Fig. 3.3). Similar to temporal and spatial collinearity, this quantitative collinearity is concomitant with a switch in the histone modification landscape.

3D chromatin organization of Hox genes in mouse

As discussed in Chapter II, the histone modification landscape is tightly linked to 3D chromatin organization. Narendra and colleagues dissected the role of CTCF, a master player of 3Dgenome organization (see Chapter II), in the control of Hox gene expression and 3D chromatin organization [START_REF] Narendra | CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation[END_REF][START_REF] Narendra | CTCF-mediated topological boundaries during development foster appropriate gene regulation[END_REF]. In ES cells, the HoxA cluster is fully repressed and coated by H3K27me3 [START_REF] Narendra | CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation[END_REF][START_REF] Narendra | CTCF-mediated topological boundaries during development foster appropriate gene regulation[END_REF][START_REF] Noordermeer | Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci[END_REF]. When the cells differentiate towards motor-neurons, Hoxa1-a6 become expressed and enriched in H3K4me3

whereas Hoxa7-a13 remain inactive. Deletion of the CTCF binding site located between Hoxa6

and Hoxa7 results in spreading of H3K4me3 into the repressed domain in differentiated motorneurons. In this case, Hoxa7 becomes expressed and interacts with the active part of the HoxA cluster whereas in WT cells it contacts the repressed portion. Thus, this CTCF binding site is essential to physically separate the active and repress HoxA domains in motor-neurons [START_REF] Narendra | CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation[END_REF]. Similarly, the deletion of a CTCF bound sub-TAD within the HoxA cluster in mouse embryos leads to homeotic transformation where no ribs are formed at the T13 position [START_REF] Narendra | CTCF-mediated topological boundaries during development foster appropriate gene regulation[END_REF].

The same 3D distribution of Hox genes was observed for both temporal and spatial collinearity in all Hox clusters in mouse embryos. Fully repressed Hox clusters (either in ES cells, as model for the earliest stages of embryogenesis or in the E10.5 mouse forebrain) are constrained within an inactive H3K27me3 3D compartment [START_REF] Noordermeer | The Dynamic Architecture of Hox Gene Clusters[END_REF][START_REF] Noordermeer | Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci[END_REF]. Upon activation, Hox genes progressively relocate to a transcriptionally active compartment, coinciding with their switch of epigenetic marks as described in the previous paragraph [START_REF] Noordermeer | The Dynamic Architecture of Hox Gene Clusters[END_REF][START_REF] Noordermeer | Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci[END_REF]) (Fig. 3.4). Thus, the 3D organization of Hox clusters directly mirrors histone modification domains and gene expression.

Interestingly, repressed H3K27me3 Hox genes are able to form long-range interactions with other H3K27me3-marked genes, including the other inactive Hox clusters [START_REF] Denholtz | Long-Range Chromatin Contacts in Embryonic Stem Cells Reveal a Role for Pluripotency Factors and Polycomb Proteins in Genome Organization[END_REF][START_REF] Vieux-Rochas | Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain[END_REF]. Importantly, the interactions among specific genes occur at low frequency though and are directly linked to their genomic distance. These long-range contacts may thus have more global regulatory functions, where they may be used to concentrate or spread the repressive machinery, as recently suggested in a preprint [START_REF] Kraft | Polycomb-mediated Genome Architecture Enables Long-range Spreading of H3K27 methylation[END_REF].

Hox genes and their regulation in Drosophila a. Hox gene expression and maintenance

Drosophila development begins in a surprising way compared to mammals. After fertilization, the egg undergoes several rounds of nuclear division (nuclear cycles, nc) but without cell division. The first 9 cycles of division are really rapid and synchronous (every 8 minutes) and create a monolayer cloud of nuclei, without cellularization, called syncytial blastoderm. The next four nuclear division are accompanied by the formation of cellular membranes, giving rise to approximately 6,000 well separated cells and creating the cellular blastoderm [START_REF] Alberts | The Structure and Function of DNA[END_REF]. Up to the nc14 (nuclear cycle 14), the zygotic genome is mostly silenced and rely on maternal mRNAs that were deposited in the egg before fertilization. Several maternal mRNAs form a gradient in the embryo setting up the different polarity axis. Soon after fertilization, maternally-deposited mRNAs start being translated into proteins, which creates a protein gradient across the embryo [START_REF] Gilbert | Developmental Biology[END_REF]. Proteins location, concentration and combination will afterwards determine the pattern of genes expression (Zygotic Genome Activation) [START_REF] Driever | The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner[END_REF]. With the ZGA, the first zygotic segmentation genes become expressed and the Drosophila embryo starts forming repetitive units. In Drosophila, the body can be sub-divided into parasegments or segments (Fig. 3.5).

The molecular signature of the parasegments derives from the patterned expression of segmentation genes along the A/P axis. Segments, which match visible anatomic markers in the adult (such as thoracic or abdominal segments), are directly related to parasegments, as one Segment is composed of two halves (called Compartments) of neighboring Parasegments (PS) (Fig. 3.5). 2.5 hours after fertilization, Gap genes shape the first rough segmentation of the embryo, followed by additional segmentation by the pair-rule genes creating 7 parasegments in the embryo [START_REF] Nüsslein-Volhard | Mutations affecting segment number and polarity in Drosophila[END_REF]. Finally, segment-polarity genes create the last segmentation leading to 14 parasegments (Martinez-Arias and [START_REF] Martinez-Arias | Parasegments and compartments in the Drosophila embryo[END_REF]. Hox genes are the last to be involved and do not specify the number of segments but rather give them their unique identity [START_REF] Carroll | Temporal and spatial relationships between segmentation and homeotic gene expression in Drosophila embryos: distributions of the fushi tarazu, engrailed, Sex combs reduced, Antennapedia, and Ultrabithorax proteins[END_REF][START_REF] Casanova | Double and triple mutant combinations of bithorax complex of Drosophila[END_REF][START_REF] Lewis | A gene complex controlling segmentation in Drosophila[END_REF][START_REF] Mcginnis | Homeobox genes and axial patterning[END_REF][START_REF] Sánchez-Herrero | Genetic organization of Drosophila bithorax complex[END_REF] (Fig. 3.5).

Importantly, several Hox genes possess alternative promoters and/or are subjected to alternative splicing, which increases functional diversity of embryonic domains. Both mechanisms generate different transcripts that can be translated into different proteins with distinct functions. The Antp gene possesses two alternative promoters [START_REF] Jorgensen | Function and misfunction of the two promoters of the Drosophila Antennapedia gene[END_REF][START_REF] Laughon | Structure of Transcripts from the Homeotic Antennapedia Gene of Drosophila melanogaster: Two Promoters Control the Major Protein-Coding Region[END_REF][START_REF] Schneuwly | Structural organiization and sequence of the homeotic gene Antennapedia of Drosophila melanogaster[END_REF] with specific expression patterns, both in the embryo and in imaginal discs in the larvae [START_REF] Bermingham | Different patterns of transcription from the two Antennapedia promoters during Drosophila embryogenesis[END_REF][START_REF] Jorgensen | Function and misfunction of the two promoters of the Drosophila Antennapedia gene[END_REF]. The activity of these two promoters is controlled independently by different gap genes [START_REF] Ingham | The correct activation of Antennapedia and bithorax complex genes requires the lushi tarazu gene[END_REF][START_REF] Irish | Spatial regulation of the Antennapedia and Ultrabithorax homeotic genes during Drosophila early development[END_REF]. Additionally, alternative splicing creates a further diversity of Antp transcripts, leading to 4 highly similar but potentially different proteins [START_REF] Bermingham | Developmentally regulated alternative splicing of transcripts from the Drosophila homeotic gene Antennapedia can produce four different proteins[END_REF]. To date, the functional relevance of this alternative splicing and promoter use of Antp remains to be elucidated. If functional domains are impaired, they could have very different regulatory functions. For example, DNA binding affinity could be modified or protein-protein interactions domains could be changed. Alternatively though, the effects could be more subtle, for instance by affecting the stability of the protein, or by directly competing for interaction partners with isoforms that are functionally different. As promoter 1 and 2 are strictly used in different segments and tissues, it is not unlikely that the protein diversity of Antp has a functional and biological relevance.

The Abd-B gene, which is most relevant for my thesis, possesses 6 alternative promoters (flybase-JBrowse-AbdB) with alternative splicing of the most 5' exons giving rise to 10 different transcripts and at least two different proteins (Fig. 3.6). The two different proteins are produced from transcripts that originate from different sets of promoters, which have known differential functions [START_REF] Celniker | The molecular genetics of the bithorax complex of Drosophila: characterization of the products of the Abdominal-B domain[END_REF][START_REF] Delorenzi | Evidence that the Abdominal-B r element function is conferred by a trans-regulatory homeoprotein[END_REF][START_REF] Kuziora | Different transcripts of the Drosophila Abd-B gene correlate with distinct genetic sub-functions[END_REF][START_REF] Zavortink | The morphogenetic and regulatory functions of the Drosophila Abdominal-B gene are encoded in overlapping RNAs transcribed from separate promoters[END_REF]):

• The A transcript (also called m or a) encodes for the ABD-B-M protein (also named ABD-B I) and is produced by the A promoter

• The B (also called b), C and g transcripts all encode for the ABD-B-R protein (also named ABD-B II) and are produced by promoter B, C and g respectively

The other two promoters have not yet been characterized and will be called promoters 2 and 3.

However, based on the coding sequences (CDS), transcripts resulting from promoters 2 and 3 are likely to also be translated in ABD-B-R protein (flybase-JBrowse-AbdB).

determines PS identity, by activating one specific Initiator element. The activation of this element, in turn, will activate one cis-regulatory region that controls the activity of one Hox gene [START_REF] Casares | Regulation of the infraabdominal regions of the bithorax complex of Drosophila by gap genes[END_REF][START_REF] Drewell | Deciphering the combinatorial architecture of a Drosophila homeotic gene enhancer[END_REF][START_REF] Iampietro | Initiator Elements Function to Determine the Activity State of BX-C Enhancers[END_REF][START_REF] Maeda | The open for business model of the bithorax complex in Drosophila[END_REF][START_REF] Starr | Molecular dissection of cis-regulatory modules at the Drosophila bithorax complex reveals critical transcription factor signature motifs[END_REF]. Hox genes are expressed during the entire life cycle of the fly whereas gap and pair-rule genes are only expressed transiently during early development, up to the gastrulation stage. As a consequence, after gap and pair-rule products have disappeared, the expression patterns of Hox genes need to be maintained through a different mechanism. In drosophila, Polycomb-Group (PcG) proteins are recruited at PREs, where they provide a memory mechanism that maintains the off state of target genes [START_REF] Duncan | Polycomblike: a gene that appears to be required for the normal expression of the bithorax and antennapedia gene complexes of drosophila melanogasteR[END_REF][START_REF] Jürgens | A group of genes controlling the spatial expression of the bithorax complex in Drosophila[END_REF][START_REF] Kennison | Dosage-dependent modifiers of polycomb and antennapedia mutations in Drosophila[END_REF][START_REF] Lewis | A gene complex controlling segmentation in Drosophila[END_REF][START_REF] Simon | Ten different Polycomb group genes are required for spatial control of the abdA and AbdB homeotic products[END_REF]. Conversely, Trithorax-Group proteins (trx-G) are recruited at TREs, where they maintain the on state of active genes [START_REF] Kassis | Polycomb and Trithorax Group Genes in Drosophila[END_REF][START_REF] Kuroda | Dynamic Competition of Polycomb and Trithorax in Transcriptional Programming[END_REF][START_REF] Shearn | The ash-1, ash-2 and trithorax genes of Drosophila melanogaster are functionally related[END_REF] (Fig. 3.9).

Through the involvement of PcG and trx-G proteins in the maintenance of Hox genes expression pattern, histone modifications are deposited over the Hox clusters. As in mammals, several studies have shown the correlation between Hox gene expression and the histone modification landscape in Drosophila. In the Sg4 cell line, Abd-B is expressed and enriched by H3K4me3, as well as H3K27ac and trx-G proteins such as ASH-1. In contrast, the repressed remainder of the BX-C is coated by H3K27me3. Conversely, in BG3 cells, the BX-C is completely silent and covered by H3K27me3 [START_REF] Schwartz | Alternative Epigenetic Chromatin States of Polycomb Target Genes[END_REF]. Chip-seq experiments using whole Drosophila embryos, show that the ANT-C and BX-C appear covered by H3K27me3 (Schuettengruber et al., 2009). However, the full H3K27me3 coverage of the Hox clusters in embryos might be due to the heterogenous nature of expression: as each individual

Hox genes is expressed in only a few segments, hence the majority of cells in the embryo harbors a repressed copy of the gene. In 2014, Bowman and colleagues [START_REF] Bowman | H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex[END_REF] circumvented the problem by setting up a FACS sorting technique to isolate cells from specific PS from Drosophila embryos. By doing that, they found that each PS has a unique signature of histone modifications (Fig. 3.10). In PS4 where no BX-C Hox genes are expressed, the cluster is covered by H3K27me3 and this repressive modification retracts in a collinear manner in PS5, PS6 and PS7 in favor of H3K27ac. These data joins the "open for business" model proposed by Robert Maeda and François Karch [START_REF] Maeda | The open for business model of the bithorax complex in Drosophila[END_REF] where along the A/P axis, the chromatin progressively opens up by gaining activating histone modifications. As a result, the sequential activation of cis-regulatory regions is achieved due to the colinearity.

However, if the chromatin and cis-regulatory regions of the BX-C open up progressively along the A/P axis, how does one cis-regulatory region activate only one gene (in contrast to mammalian Hox gene clusters)? An explanation is found in the posterior prevalence rule of Drosophila Hox genes, which states that the expression of a posterior gene inhibits the transcription of more anterior genes [START_REF] Gummalla | Hox gene regulation in the central nervous system of Drosophila[END_REF]. Thus, technically, only one Hox gene per cell can be expressed. Two other exceptions have been reported though:

1. In the central nervous system (CNS) of Drosophila, Abd-B fails to repress abd-A gene in PS10 to PS12 [START_REF] Gummalla | Hox gene regulation in the central nervous system of Drosophila[END_REF].

2. Similarly, abd-A and Abd-B can be co-expressed in few cells of the female genital discs [START_REF] Foronda | Requirement of abdominal-A and Abdominal-B in the developing genitalia of Drosophila breaks the posterior downregulation rule[END_REF](Foronda et al., ). 2008(Foronda et al., , 2010;;[START_REF] Karch | Mcp and Fab -7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster[END_REF][START_REF] Kyrchanova | Complete reconstitution of bypass and blocking functions in a minimal artificial Fab-7 insulator from Drosophila bithorax complex[END_REF][START_REF] Postika | Boundaries mediate long-distance interactions between enhancers and promoters in the Drosophila Bithorax complex[END_REF][START_REF] Wolle | Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex[END_REF].

The BX-C BEs are flanked with PREs, which are essential for BE function [START_REF] Mihaly | In situ dissection of the Fab-7 region of the bithorax complex into a chromatin domain boundary and a Polycombresponse element[END_REF].

Thus, boundary use coincides with the histone modification landscape, with both histone modification and BEs contributing to the collinear activation and maintenance of Hox gene activity in BX-C (Fig. 3.10).

An interesting remaining question is how the collinear opening of the chromatin influences the choice of alternative promoters of Hox genes. As mentioned, the Abd-B gene uses 6 alternative promoters that gives rise to different transcripts and at least 2 different proteins. How BE use and the histone modification landscape correlate with Abd-B promoter choice remains to be studied to both better understand how collinearity is achieved at the smallest scales and how this helps creating the required transcriptional programs for the specification of segments and resulting organs.

c. Fab-7, a model Boundary Element in the BX-C

Fab-7 is one of the most studied BEs of the BX-C. Its separates iab-6 from iab-7, which both control the expression of Abd-B gene. At the sequence level, the Fab-7 element is composed of a BE and a PRE. Deletion of the PRE alone does not induce an observable phenotype [START_REF] Mihaly | In situ dissection of the Fab-7 region of the bithorax complex into a chromatin domain boundary and a Polycombresponse element[END_REF]. On the contrary, the deletion of the BE alone induces a homeotic phenotype where the A6 segment adopts the identity of the A7 segment due to activation of iab-7 in segment A6.

The combined deletion of the Fab-7 BE and PRE (comprising a DNA segment of around 3.6 kb) enhances the phenotype that is observed when only the BE is absent [START_REF] Singh | Specific combinations of boundary element and Polycomb response element are required for the regulation of the Hox genes in Drosophila melanogaster[END_REF] (Fig. 3.11).

Uniquely, Fab-7 is the only BE in the BX-C that does not contain binding sites for dCTCF and therefore relies on other insulator proteins for its function [START_REF] Wolle | Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex[END_REF]. Moreover, its by-pass and blocking activity are orientation independent [START_REF] Rodin | New Properties of Drosophila Fab-7 Insulator[END_REF]. Two protein complexes are essential for the activity of Fab-7: Elba during early embryogenesis from 0 to 6h post-fertilization [START_REF] Aoki | Elba, a novel developmentally regulated chromatin boundary factor is a hetero-tripartite DNA binding complex[END_REF] and the LBC (Late Boundary Complex) for the remaining fly's life [START_REF] Wolle | Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex[END_REF]. The Elba complex is composed of the DNA-binding proteins Elba1 and 2, and Elba3 which links the other together [START_REF] Aoki | Elba, a novel developmentally regulated chromatin boundary factor is a hetero-tripartite DNA binding complex[END_REF]. The LBC complex is a large (>700 kDa) multiproteic complex composed of Mod(mdg4), GAF and E(y)2. GAF subunit, an insulator protein, appears essential as mutations in the all GAGA binding sites within the Fab-7 element impair both GAF binding and the function of the BE [START_REF] Nègre | A Comprehensive Map of Insulator Elements for the Drosophila Genome[END_REF][START_REF] Schwartz | Alternative Epigenetic Chromatin States of Polycomb Target Genes[END_REF][START_REF] Schweinsberg | The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites[END_REF].

Previously, it was thought that each BE element of the BX-C had a unique ability to drive Hox gene expression. Indeed, replacement of Fab-7 by Fab-6 failed to rescue the Fab-7 deletion [START_REF] Postika | Mapping of functional elements of the Fab-6 boundary involved in the regulation of the Abd-B hox gene in Drosophila melanogaster[END_REF]. Interestingly, a recent study showed that Fab-7 deletion can be fully rescued by replacing it with a 337 bp DNA fragment from the Fab-8 BE. This fragment contains dCTCF binding sites that recapitulate the Fab-7 function when they are placed in the same orientation as the endogenous Fab-8 [START_REF] Kyrchanova | Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex[END_REF][START_REF] Kyrchanova | Complete reconstitution of bypass and blocking functions in a minimal artificial Fab-7 insulator from Drosophila bithorax complex[END_REF]). An essential capacity of the more distant BEs that regulate Abd-B is their capacity to by-pass the intervening BEs. Near the Fab-7 and the Fab-8 BEs lie so-called Promoter Targeting Sequences (PTSs) that permit the by-pass of these BE in order to activate the Abd-B gene [START_REF] Chen | Allocation and specification of the genital disc precursor cells in Drosophila[END_REF][START_REF] Zhou | A Novel cis -Regulatory Element, the PTS, Mediates an Anti-Insulator Activity in the Drosophila Embryo[END_REF]. As such, both Fab-7 and Fab-8 share more proprieties than originally thought. The generic bypass and blocking activity of BEs could thus be due to redundant functionalities [START_REF] Chen | Allocation and specification of the genital disc precursor cells in Drosophila[END_REF][START_REF] Zhou | A Novel cis -Regulatory Element, the PTS, Mediates an Anti-Insulator Activity in the Drosophila Embryo[END_REF]:

1. A PTS for the enhancer blocking activity.

2. An insulator function, encoded in hypersensitive sites 1 and 2 (HSs).

3. A PRE, encoded in hypersensitive site 3 (HS3) for the maintenance of the on/off status.

Off note, even if dCTCF is not involved in TAD boundary maintenance or formation, it has a crucial role for Hox gene regulation. Indeed, maternal and zygotic depletion of dCTCF induce Drosophila lethality at the pupal stage and causes homogenous Abd-B expression in the abdomen [START_REF] Gambetta | The Insulator Protein CTCF Is Required for Correct Hox Gene Expression, but Not for Embryonic Development in Drosophila[END_REF].

d. Role of 3D conformation in Hox gene regulation

Considering that enhancers within the iabs regulate Abd-B promoters, and enhancer regulatory landscape are linked to 3D genome organization (see Chapter II), higher-order chromatin structures were sought in various studies.

At the scale of Abd-B activation, Fab-6, Fab-7 and Fab-8 can interact with a sequence upstream of the Abd-B promoters, the so-called Promoter Tethering Element (PTE) [START_REF] Holohan | CTCF Genomic Binding Sites in Drosophila and the Organisation of the Bithorax Complex[END_REF][START_REF] Kyrchanova | Functional Interaction between the Fab-7 and Fab-8 Boundaries and the Upstream Promoter Region in the Drosophila Abd-B Gene[END_REF]Maeda andKarch, 2007, 2009;[START_REF] Sipos | Transvection in the Drosophila Abd-B Domain: Extensive Upstream Sequences Are Involved in Anchoring Distant cis-Regulatory Regions to the Promoter[END_REF]. Dam-ID experiments confirmed that a BE could loop in a repressed context, by showing that Fab-7 can contact the Abd-B promoter A when the gene is repressed [START_REF] Cléard | Probing long-distance regulatory interactions in the Drosophila melanogaster bithorax complex using Dam identification[END_REF]. To establish proper enhancer/promoter contacts, BEs containing dCTCF can also contact each other, but However, if this Hox gene de-repression is a cause or consequence of chromatin decompaction remained unknown at the time. To answer this question, Cheutin and colleagues performed RNA and DNA-FISH in WT and PcG mutant embryos and analyzed the asynchrony between chromatin decompaction and gene activation [START_REF] Cheutin | Loss of PRC1 induces higher-order opening of Hox loci independently of transcription during Drosophila embryogenesis[END_REF]. In Polyhomeotic (Ph)

or Pc mutant embryos, chromatin decompaction is observed prior to Hox de-regulation.

Moreover, the ectopic expression of the Hox genes follows a collinear pattern: the first genes and correlated their data with segment specific ChIP [START_REF] Bowman | H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex[END_REF][START_REF] Mateo | Visualizing DNA folding and RNA in embryos at single-cell resolution[END_REF].

As expected, they found that in the T2 segment, where Ubx, abd-A and Abd-B are repressed and covered by H3K27me3, the BX-C is folded into a single inactive contact domain. In contrast, in the Ubx expressing T3 segment, Ubx gene is found in its own domain with limited contacts with the rest of the H3K27me3-rich BX-C. Surprisingly, in segments A2 to A4 where Ubx and abd-A are both expressed, the region devoid in H3K27me3 is splitted into two small contact domains. The authors suggested that these two small contact domains could correspond

SCOPE OF THE THESIS

In the first three chapters of my thesis, I discussed how gene regulation in eukaryotic cells is a multi-level process which incorporates, but is not strictly limited to, cis-regulatory elements, the epigenetic landscape and 3D genome organization.

• In Chapter 1, I summarized the organization of eukaryotic chromosomes into chromatin and I introduced the first two aspects of chromatin organization that are involved in gene regulation:

o Cis-regulatory elements like enhancers, silencers and Boundary Elements (BEs).

o Epigenetic regulation like DNA methylation, histone modifications and lncRNAs.

• In Chapter 2, I introduced the different levels of three-dimensional (3D) organization of eukaryotic genomes. Included in this chapter were the relationship between 3D genome organization and cis-regulatory elements, particularly at the level of Topologically

Associating Domains (TADs), and the links between 3D genome organization and epigenetic regulation, which can be found at most levels.

• In Chapter 3, I introduced the Hox genes in both Drosophila and mammalian systems.

Particularly, I summarized how these well-characterized loci have served as model loci to study the mechanisms and functions of gene regulation, cis-regulatory elements, epigenetic regulation and 3D genome organization.

Although large numbers of studies have addressed the links between gene regulation and other levels of chromatin organization, it is rare that they incorporate the full range of regulatory processes that I introduced in Chapter 1 and 2. Moreover, in many cases these links either remain limited to genome-wide correlations, which complicates the (formal) establishment of causality.

The These high-resolution revealed unexpected differences in the distribution of histone modifications between cells, which are directly linked to BE use, 3D genome organization and the choice of alternative Hox gene promoters. I followed up on these observations by comparing 3D genome organization and promoter choice in mutant flies where a specific BE is absent.

These studies confirmed the intimate link between BE function, 3D genome organization, the histone modification landscape for collinear Hox gene activation and (1) they revealed an unexpected dynamic in the organization of histone modifications at the highest resolution and

(2) they identified a new function for BEs in regulating alternative promoter choice.

In Chapter 6, I present the results from my efforts to establish three experimental systems to test the involvement of BEs and insulator proteins in collinear Hox gene regulation in cell lines.

Due to various technical limitations, I did not manage to establish these systems. They were nonetheless important for the overall scientific strategy of the project, and they provide important background for the analyses done in Chapter 5. Moreover, my efforts and conclusion may prove useful for other scientists that plan to take similar approaches. For S2 and S3 cells, classical Gibco FBS (ThermoFisher Scientific) was used whereas Sg4 and Kc167 were grown with Gibco Performance FBS. Cells were grown at 25°C and split every 2-4 days (inoculated at 1.10 6 to 3.10 6 cells/ mL).

Fly stocks

Flies were cultured in standard cornmeal yeast extract medium at 25°C. Fab7 1 flies were a gift from François Karch and White (w 1118 ) flies were a gift from Jacques Montagne.

In Fab7 1 flies, the Fab-7 element has been removed (4.3 kb region between Abd-B and abd-A).

These flies were generated in 1990 by H. Gyurkovics and colleagues [START_REF] Gyurkovics | A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating two domains of regulation[END_REF] and were found among the progeny of X-rayed Oregon-R-369 flies (4000 rads; 1000rads/min, 0.5 mm A1 filter) by observation of ectopic activation of the Abd-B gene in anterior abdominal segments.

RT-qPCR

3.10 6 to 5.10 6 cells were lysed in 1 mL Trizol (ThermoFisher Scientific) and total RNA was purified using the NucleoSpin RNA kit (Macherey-Nagel). Reverse-transcription (RT) was performed using SuperScript IV and Random hexamers following the manufacturer's instructions (ThermoFisher Scientific). A similar protocol was followed for whole larvae ( 20L3 larvae) and imaginal discs (50 wing discs or 150 genital discs). Between 88 ng and 1 µg was used for the RT reaction. A 6 fold dilution of the cDNA preparation was amplified in 10 µL reaction mixture in the presence of 5 µL of Advanced Universal SYBRGreen Supermix (BioRad) and 0.5 µM of corresponding primers. Primers were designed over exon boundaries to prevent amplification from genomic DNA. Primers were designed using the online version of Primer3 software (https://primer3.ut.ee/). Primers were annealed at 55°C or 60°C and Realtime PCR was performed using a LightCycler 480 instrument (Roche). Gene expression was • In the selected restriction fragments, the distance between the DpnII sites (RE1-RE1) is at least 700 bp (in order to allow efficient inter-molecular DNA ligation)

• The distance between the DpnII and NlaIII (RE1-RE2) sites is at least 250 bp (minimum size for DNA circularization)

• The inverse forward primers (primer iF) were designed within 30-35 bp of the DpnII site and the inverse reverse primer (primer iR) within 50 bp of the NlaIII site.

Primers were designed using the online version of Primer3 software (https://primer3.ut.ee/)

with the following parameters:

• "Primer size": min=18, opt=20 max=27 bp

• "Tm": min=54, opt=55, max=57 °C.

Primers did not report multiple completely homologous hits and few related hits when blasted against the dm6 Drosophila genome (FlyBLAST).

In a first step, primer sets without adapter sequences were validated by 4C PCR and validation of electrophoresis migration pattern on an agarose gel. Once validated, sequencing primers (PAGE-purified) containing Illumina Tru-seq adapters and indexes were ordered (Eurogentec)

to produce the 4C-seq sequencing libraries (primers listed in Table 3).

b. 4C-seq in cell lines

4C-seq was performed as published previously [START_REF] Matelot | Determination of High-Resolution 3D Chromatin Organization Using Circular Chromosome Conformation Capture (4C-seq)[END_REF] Restriction buffer, permeabilized by the addition of 7.5 µL 20 % SDS and incubated for 10 min at 60°C, followed by 50 min at 37°C. The SDS was sequestered by adding 50 µL 20 % Triton

(1 hr at 37°C). The cross-linked chromatin was digested with 400U of DpnII (NEB) for 4 hr at 37°C, followed by the addition of another 400U of the DpnII enzyme and an overnight incubation at 37°C. After validation of digestion efficiency by agarose gel electrophoresis, the cross-linked chromatin was ligated in a total volume of 7 mL by addition of 100 U of T4 DNA Ligase (Promega) and incubated 4 hrs at 16°C. The chromatin was de-cross-linked overnight at 65°C in presence of Proteinase K (NEB), and DNA purified using a phenol/chlorophorm/isoamyl alcohol (IAA) extraction followed by ethanol precipitation. DNA concentration was measured using the Qubit dsDNA Broad-Range assay (Thermo-Scientific)

and 500 ng examined on a 1% agarose gel to control for ligation efficiency. DNA was then further digested overnight at 37°C with the secondary restriction enzyme NlaIII (1.5U/µg of DNA), purified using a phenol/chlorophorm/IAA extraction and ethanol precipitation. DNA was finally circularized with 200 U T4 DNA Ligase (Promega) for 4 hr at 16°C, in a total volume of 14 mL, precipitated and column-purified (QIAquick PCR Clean-up, Qiagen). The DNA concentration was measured using the Qubit dsDNA Broad-Range assay (Thermo-Scientific). For each viewpoint, 12 PCR reactions containing 12 ng of 4C material were amplified and pooled. The PCR was done using Expand Long Template PCR System (Roche) (PCR program is: 2 min at 94°C; 30 cycles of (15 sec at 94°C, 1 min at 55° and 3 min at 68°C), then 7 min at 68°C) on a Thermocycler (Bio-Rad C1000) and reactions were pooled and purified using a PCR Clean up kit (Qiagen). Up to 23 viewpoints were finally mixed in equimolar ratio and directly sequenced as multiplexed reactions in one lane by Illumina Nextseq500 75 bp Single End (High Throughput Sequencing Platform -I2BC). Primer sequences for 4C-seq are listed in Table 3.

c. 4C-seq in imaginal discs

Imaginal discs (genital or wing discs) were dissected in cold 1X PBS over multiple sessions that lasted maximum 1hr. Chromatin crosslinking and cell lysis (as previously described) were performed right after dissection and samples were stored at -80°C. A total of 1600 genital discs and 400 wing discs were pooled for the 4C samples. The procedure for 4C on imaginal disks is based on the previous section (4C-seq on cell lines) and on [START_REF] Tolhuis | Chromosome conformation capture on chip in single Drosophila melanogaster tissues[END_REF] but with two modifications: (1) an extra step for the cell lysis, using a glass douncer with pestle A was added and (2) all volumes in the procedure were adjusted by a factor 5 to 20 to account for the low numbers of cells.

d. 4C-seq bio-informatic analysis

Initially, 4C-sequencing reads were sorted, aligned, and translated to restriction fragments using the 4C-seq module of the HTSstation service, hosted by the Swiss Institute of Bioinformatics [START_REF] David | HTSstation: A Web Application and Open-Access Libraries for High-Throughput Sequencing Data Analysis[END_REF] After this service was stopped, a stand-alone version of this module with identical output (C4CTUS, Benoît Moindrot -unpublished) was used. The whole C4CTUS pipeline is described in https://github.com/NoordermeerLab/c4ctus .

Reads coming from different biological samples were demultiplexed using their Illumina Index.

Reads coming from individual viewpoints of the same samples were demultiplexed using the first 18 bases sequenced (viewpoint-specific primer). After removing the sequence of the viewpoint-specific primer, the remainder of the reads were mapped onto the drosophila genome (dm6). Reads in a region directly surrounding the viewpoint were highly enriched and showed considerable experimental variation rather than biological significance, thereby severely influencing overall fragment count. To minimize these effects, the viewpoint itself, the directly neighboring 'undigested' fragment and fragments 2 kb up-and downstream were excluded during the procedure. The fragment counts were then normalized per one million reads (reads between chr3R:4,661,427-8,999,228 for the ANT-C and chr3R:14,656,623-18,972,236 for the BX-C). Merck-Millipore), 4 µL (1/250) GAF (kind gift from Giacomo Cavalli, IGH Montpellier) and 3 µL (1/333) CP190 (kind gift from Elissa Lei, NIH, Bethesda). 60 µL of Salmon Sperm DNA/Protein A Agarose beads were added to precipitate the immune complex. Beads were then sequentially washed for 5 min at 4°C followed by low speed centrifugation in between:

ChIP-seq and

• 1 mL Low Salt Immune Complex Wash Buffer (20 mM Tris-Hcl, 150 mM NaCl, 2 mM EDTA, 0.1 % SDS and 1 % Triton X-100)

• 1 mL High Salt Immune Complex Wash Buffer (20 mM Tris-HCl, 500 mM NaCl, 2mM EDTA, 0.1 % SDS, 1 % Triton X-100)

• For ChIP-seq, enrichment of histone marks was first validated by qPCR. ChIP libraries were subsequently prepared using the NEBNext Ultra FS II kit (NEB) and sequenced using Illumina

Next-seq 500 75 bp Single End reads (High Throughput Sequencing Platform -I2BC). Primer sequences for control ChIP-qPCR are listed in Table 4.

For qPCR, enrichment was determined relative to Input using the D Ct method. Primer sequences for hIP-qPCR are listed in Table 5 and6.

b. ChIP-seq bio-informatics analysis

After Fast-QC filtering, reads were mapped to the Drosophila genome (dm6, UCSC) using bowtie2 2.3.0, with filtering for multiple alignments. Low quality reads (<30) and PCR duplicates were removed using SAMtools. Bedgraphs were generated using BAMCoverage.

To assess the significance of detected peaks detected with bedgraphs, peak calling was performed using MACS2 (with broad option for H3K27me3).

Hi-C and data analysis

For Sg4 cells, Hi-C was performed on 15.10 6 cells using Arima Hi-C kit, following manufacturer's instructions. Published data for S2 R+ Drosophila cells were obtained from GEO dataset GSE99107 [START_REF] Szabo | TADs are 3D structural units of higher-order chromosome organization in Drosophila[END_REF].

For Sg4 and S2 R+ cells, reads were mapped to dm6 genome using HiC-Pro version 2.11.1 and bowtie version 1.1.2 with default settings to remove duplicates, assign reads to DpnII restriction fragments and filter for valid interactions [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF][START_REF] Servant | HiC-Pro: an optimized and flexible pipeline for Hi-C data processing[END_REF].

Hi-C paired matrices of 5kb resolution were generated from the valid interactions and

b. Single cell RNA-seq on imaginal discs

Imaginal discs from WT and Fab-7 1 larva were dissected within 20 min in 400 µL cold PBS (without MgCl2) + 0.04% BSA. To obtain roughly equivalent numbers of cells for each type of imaginal disc, the following discs were dissected and pooled:

• 4 eye/antenna discs

• 8 leg 1 discs • 4 leg 2 discs
• 4 leg 3 discs

• 8 halter discs

• 2 wing discs

• 16 genital discs Pools of discs were then dissociated in 1 mL Trypsin EDTA 0.05% (Thermofisher Scientific)

during 8 minutes at 37°C 900 rpm in a Thermomixer (Eppendorf). After spinning 4 min at 1000 g 4°C, cells were resuspended in 100 µL cold PBS (without MgCl2) + 0.04% BSA and counted on a Biorad TC20 cell counter. Reverse transcription and preparation of the sequencing library were then performed according to the manufacturer's instructions (10X Genomics) using the Chromium Next GEM Single Cell 5' kit and supplied reagents. The resulting libraries were sequenced on the Illumina Next-seq 550 using 75 bp Paired end reads (High Throughput Sequencing Platform -I2BC).

c. Bio-informatic analysis of Single cell RNA-seq data

Cell Ranger version 3.1.0 was used to produce FastQ files for the scRNA-seq data and to align, filter and quantify reads on the dm6 reference genome and to produce feature-barcode matrices.

Downstream analyses were done following Seurat version 4.0.1 (https://satijalab.org/seurat/).

Cells with UMI lower than 350 and mitochondrial genes above 5.5% were excluded from the analysis. Data were then Log normalized and the vst method was used to select the top 2,000 variable features. Datasets for WT and Fab-7 1 were then integrated using the dataset with the highest number of cells as reference and the first 10 dimensions. Clustering was performed with the functions FindNeighbors and FindClusters, that are based on the shared nearest approach, using the first 10 dimensions from PCA. Identification of clusters was done using FindConservedmarkers function in Seurat. All visualizations were performed using Seurat as well.

Sg4 cell line modifications a. Set up of CRISPR/Cas9 Fab-7 deletion

Creation of plasmid containing Cas9 and sgRNA Deletion plasmids containing the Cas9 and sgRNA allowing Fab-7 deletion were made from pAC-sgRNA-Cas9 (Plasmid #49330, Addgene) containing the Cas9 coding-sequence, a puromycin resistance marker and BspQI restriction sites to insert sgRNA. sgRNA design was performed following [START_REF] Bassett | Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9[END_REF] and with the help of FlyCRISPR and CRISPOR websites [START_REF] Concordet | CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens[END_REF]. sgRNA target sequences were designed on both sides of the Fab-7 element and are 20nt sequences preceding an NGG PAM sequence. Primers to insert sgRNA are provided in see Table 9. Forward and Reverse Oligos were annealed to generate compatible overhangs with ligation into the BspQI-digested pAC-sgRNA-Cas9

vector. The ligation reaction was transformed into the SURE (Stop Unwanted Recombination

Events) E. coli strain after consistent unsuccessful outcomes using the classical DH5a strain.

Resulting clones were characterzied by enzymatic digestion and Sanger sequencing.

Cell transfection

Transfection of Sg4 cells was performed as described in the Result section in serum free medium. In brief, Effectene (Qiagen), Fugene HD (Promega) and Amaxa Nucleofector kit V (Lonza) were tested on Sg4 cells. Between 2.10 6 and 5.10 6 cells per well were used for transfection. Ratio of transfection reagent / plasmid was respectively: 12.5:1, 4:1 and 9:1.

Complete medium was then added to allow cell growth. After 3 days, 0.8 µg/mL of puromycin was added during 10 days to select transfected cells. In the first attempt, all cells were recovered to test transfection by PCR and qPCR or freeze for further analysis.

In the Dilution approach, cells were selected with puromycin as described before and after 3 to 10 days of selection, cells were diluted in individual 96 well plates wells to statistically obtain 1 cell / well. To insure cell growth, cell were cultured in "conditioned" medium, meaning complete medium were confluent cells have grown [START_REF] Franz | Generation of genome-modified Drosophila cell lines using SwAP[END_REF].

To test the efficiency of CRISPR/Cas9-mediated Fab-7 deletion, PCR and qPCR were performed. Primers are listed in Table 7 andTable 8.

b. Set up of SUN-TAG system

Plasmid construction for the Sun-Tag

The tripartite Sun-Tag system [START_REF] Chavez | Comparison of Cas9 activators in multiple species[END_REF] was obtained from Addgene: pAct:dCas9-GCN4 (#78899), pAct:dCas9-scFv-VP64 (#78900) and pCFD4-U6:1_U6:3tandemgRNAs (#49411). pCFD4-U6:1_U6:3tandemgRNAs was modified to insert pairs of gRNAs against the abd-A promoter as shown in Fig. 6.4.A.

Plasmid construction for the dual luciferase assay

Plasmids containing Firefly and Renilla under the control of Tubulin promoter were obtained from Hélène Thomassin-Bourrel (Université Paris-Sorbonne). Two modifications were made to the two plasmids. First, Firefly and Renilla sequences were replaced by a Drosophila codonoptimized versions from Addgene (#83343 and #83342). Secondly, theTubulin promoter was replaced by the abd-A promoter by conventional cloning. In the end, four different plasmids were created by Sylvain Bouvard, the M2 student who performed these experiments: abd-A-Firefly, abd-A-Renilla, Tub-Firefly and Tub-Renilla.

Cell transfection

Cell transfection was performed as described in the Result section using Fugene HD on 1.10 6 cells with a ratio of transfection reagent / plasmid of 4:1.

Dual luciferase assay

The assay was performed using DualGlo Luciferase kit (Promega). 3 days after transfection, one fourth of transfected cells were transferred to black 96 well plates (ThermoFisher Scientific). To measure Firefly activity, 1:1 volume of Luciferase reagent was added to wells containing cells of the 96 well plate and incubated for 10 min at RT. Firefly activity was recorded with TECAN Infinite 200 pro luminometer. To quantify Renilla activity, Stop&Glo reagent was added and fluorescence was measured after 5 min of incubation at RT. For each sample, 10 sec acquisition was performed in duplicate for Firefly and Renilla.

c. Set up of RNAi in Sg4 cells

Delivery of RNAi in Sg4 cells 4.10 6 Sg4 cells were seeded at a concentration of 1.10 6 cells/mL in 6 well plates in M3+BPYE serum-free medium. Different quantities of dsRNA were added to the cells (10 µg, 20 µg or 40 µg) and incubated for 45 min at RT. Complete medium (with serum) was then added. Every 2.5 days, cells were counted and a sample was taken for Western Blot or qPCR analysis.

Production of RNAi against GAF protein

RNAi against GAF and LacZ (negative control) were designed as described in [START_REF] Duarte | Transcription factors GAF and HSF act at distinct regulatory steps to modulate stressinduced gene activation[END_REF][START_REF] Fuda | GAGA Factor Maintains Nucleosome-Free Regions and Has a Role in RNA Polymerase II Recruitment to Promoters[END_REF].

RNAi against other GAF exons were designed using VDRC, UP-TORR and SnapDragon.

dsRNAs were produced from a dsDNA amplicon containing T7 promoter at both ends. dsDNA templates were generated by PCR from genomic DNA of L3 Drosophila larvae. The PCR product was produced in two independent PCR reactions with only one primer containing the T7 promoter (either Forward or reverse). T7 transcription was performed using the T7 Ribomax Express RNAi system (Promega) following the manufacturer's instructions, primers are listed in Table 10. Table 1:

Tables

GAF3 Forward AATTGGATTGCCGATACGAG GAF3 + T7

Reverse TAATACGACTCACTATAGGGACAAAGGGAGTTGGAAAAGCA

Introduction

Hox genes encode crucial developmental regulators for the specification of segmental identities along the embryonic Antero-Posterior (A-P) body axis. A unique feature of Hox genes is that they are organized along the chromosome in their order of expression along the A-P axis [START_REF] Mcginnis | Homeobox genes and axial patterning[END_REF]. This feature, named collinearity, was first discovered in Drosophila melanogaster, and later also detected in other bilaterians including mammals [START_REF] Duboule | The structural and functional organization of the murine HOX gene family resembles that of Drosophila homeotic genes[END_REF][START_REF] Lewis | A gene complex controlling segmentation in Drosophila[END_REF]. In mouse, correct spatial expression patterns along the A-P axis require the Hox clusters to be intact [START_REF] Tschopp | Uncoupling Time and Space in the Collinear Regulation of Hox Genes[END_REF]. In contrast, in Drosophila melanogaster, the eight Hox gene paralogs are organized in two different clusters on chromosome 3R: the Antennapedia cluster (ANT-C) and the Bithorax cluster (BX-C). Unlike their compact mammalian counterparts, the Hox clusters in Drosophila spread out over large genomic intervals (300-400kb) and are interspersed by lncRNA which functions are poorly understood. Hox genes contained within these clusters can possess multiple large introns, can be subjected to alternative splicing and can use multiple transcriptional start sites (TSSs) [START_REF] Gummalla | Hox gene regulation in the central nervous system of Drosophila[END_REF][START_REF] Noordermeer | Chromatin Architectures and Hox Gene Collinearity[END_REF][START_REF] Reed | Alternative Splicing Modulates Ubx Protein Function in Drosophila melanogaster[END_REF]. Yet, despite these different constraints on Hox cluster organization, the spatially collinear distribution of Hox genes along the A-P axis is similarly established in both Drosophila and mammalian cells.

Mammalian Hox genes are activated in a temporal collinear fashion, whereby (at least for more posterior genes), a repressive H3K27me3 3D domain in resolved into an active H3K4me3 3D domain [START_REF] Noordermeer | Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci[END_REF][START_REF] Soshnikova | Epigenetic Temporal Control of Mouse Hox Genes in Vivo[END_REF]. This dynamic opening, probably, provides sequential access to cis-regulatory elements located in-between the genes [START_REF] Tschopp | Uncoupling Time and Space in the Collinear Regulation of Hox Genes[END_REF]. Subsequent fixation of organization allows the long-term stabilization of spatial colinear transcriptional programs along A-P axis.

Moreover, knock-down of a Polycomb component (EZH2) impaired the silencing of the HoxA cluster in human cells showing the link between the histone modification landscape and Hox gene expression [START_REF] Ferraiuolo | The three-dimensional architecture of Hox cluster silencing[END_REF]. Mammalian Hox clusters are interspersed by CTCF binding sites and the CTCF insulator protein is essential for the regulation of 3D organization of Hox clusters and their correct expression. Deletions of CTCF binding sites at boundary elements and TAD boundaries in the HoxA and HoxC clusters lead to modification of 3D chromatin architecture and homeotic transformations du to ectopic Hox gene expression [START_REF] Narendra | CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation[END_REF][START_REF] Narendra | CTCF-mediated topological boundaries during development foster appropriate gene regulation[END_REF]. Recently, it has been suggested that CTCF sites with Hox clusters do not all exhibit the same function [START_REF] Amândio | Sequential in-cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster[END_REF]. By performing systematic CTCF binding sites deletion at a strong TAD boundary of the Hoxd cluster, they found that the CTCF sites could have either insulating or enhancer-promoter tethering activity, depending on the mouse tissue [START_REF] Amândio | Sequential in-cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster[END_REF]. Thus, in mammals, correct Hox gene expression is coordinated by Polycomb proteins that decorate Hox cluster and by the 3D chromatin architecture via the fixation of CTCF insulator protein at boundary elements.

To achieve similar spatial collinear Hox gene expression patterns along the A-P axis, the developing Drosophila embryo employs similar regulatory components but with certain noticeable differences. The BX-C, which contains the posterior Ubx, abd-A and Abd-B Hox genes, is divided into 10 cis-regulatory regions that are called abx/bx, bxd/pbx and infraabdominal segments (iabs). Each iab, which measure between 10 and 60 kb in size, contains many ncRNAs, cell type and parasegment specific enhancers that ensure the correct activity of (in most cases) a single Hox gene at the correct region along the A-P axis [START_REF] Busturia | Silencers in abdominal-B, a homeotic Drosophila gene[END_REF][START_REF] Pirrotta | Chromatin complexes regulating gene expression in Drosophila[END_REF][START_REF] Simon | Regulatory elements of the bithorax complex that control expression along the anterior-posterior axis[END_REF]. In-between each pair of iabs, a BE is localized that defines these domains [START_REF] Barges | Fab-8 boundary defines iab-7 domain[END_REF][START_REF] Gyurkovics | A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating two domains of regulation[END_REF][START_REF] Karch | Mcp and Fab -7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster[END_REF][START_REF] Mihaly | In situ dissection of the Fab-7 region of the bithorax complex into a chromatin domain boundary and a Polycombresponse element[END_REF]. In contrast to mammals, multiple iabs and BEs are located between pairs of Hox genes, coinciding with a further fine-tuning of functions. For example, the Mcp BE that separates the iabs for Ubx and abd-A regulation has enhancer blocking activity only. In contrast, the Fab-7 BE that separates iab-6 and iab-7 (both responsible for Abd-B expression) has both enhancer-blocking and boundary by-pass activity [START_REF] Iampietro | Boundary swapping in the Drosophila Bithorax complex[END_REF][START_REF] Karch | Mcp and Fab -7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster[END_REF][START_REF] Kyrchanova | Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex[END_REF][START_REF] Wolle | Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex[END_REF]Deletion of the complete element (Fab-7 1 mutant) leads to the regulatory fusion of iab-6 and iab-7, thereby inducing a homeotic phenotype caused by the ectopic expression of Abd-B in more anterior segments [START_REF] Gyurkovics | A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating two domains of regulation[END_REF][START_REF] Karch | Mcp and Fab -7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster[END_REF][START_REF] Mihaly | In situ dissection of the Fab-7 region of the bithorax complex into a chromatin domain boundary and a Polycombresponse element[END_REF][START_REF] Singh | Specific combinations of boundary element and Polycomb response element are required for the regulation of the Hox genes in Drosophila melanogaster[END_REF].

To achieve their function, BEs are coupled with Polycomb Responsive Elements (PRE) [START_REF] Mihaly | In situ dissection of the Fab-7 region of the bithorax complex into a chromatin domain boundary and a Polycombresponse element[END_REF] and BEs are bound by insulator proteins. Unlike in mammals, where CTCF is the only known somatic insulator protein, Drosophila possesses a collection of insulator proteins that can co-occupy BEs, including the DNA binding proteins dCTCF and GAF, and the non-DNA binding CP190 factor [START_REF] Nègre | A Comprehensive Map of Insulator Elements for the Drosophila Genome[END_REF][START_REF] Wolle | Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex[END_REF]. ChIPseq for dCTCF detected its presence at every BE of the BX-C, except at Fab-7. At Fab-7, the GAF proteins binds instead in a multi-protein complex called Late Boundary Complex (LBC) where its binding via multiple GAGA sites is essential for its activity [START_REF] Kyrchanova | The bithorax complex iab-7 Polycomb response element has a novel role in the functioning of the Fab-7 chromatin boundary[END_REF][START_REF] Kyrchanova | Complete reconstitution of bypass and blocking functions in a minimal artificial Fab-7 insulator from Drosophila bithorax complex[END_REF][START_REF] Schweinsberg | The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites[END_REF][START_REF] Wolle | Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex[END_REF].

A further layer of Hox gene regulation is their histone modification landscape. Histone modifications closely follow Hox gene expression, with repressed Hox genes being covered by the H3K27me3 mark and the active Hox genes carrying both the H3K4me3 and H4ac or H3K27ac marks [START_REF] Beisel | Comparing active and repressed expression states of genes controlled by the Polycomb/Trithorax group proteins[END_REF][START_REF] Bowman | H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex[END_REF]. Interestingly, associated iabs are also enriched in positive histone modifications confirming the regulatory functions of these elements [START_REF] Beisel | Comparing active and repressed expression states of genes controlled by the Polycomb/Trithorax group proteins[END_REF][START_REF] Bowman | H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex[END_REF][START_REF] Schwartz | Genome-wide analysis of Polycomb targets in Drosophila melanogaster[END_REF][START_REF] Schwartz | Alternative Epigenetic Chromatin States of Polycomb Target Genes[END_REF]. Hi-C and 4C experiments in Drosophila and mammals have shown that domains of histone modifications coincide with 3D contact domain organization, including at the Hox clusters [START_REF] Bantignies | Polycomb-Dependent Regulatory Contacts between Distant Hox Loci in Drosophila[END_REF][START_REF] Noordermeer | Chromatin Architectures and Hox Gene Collinearity[END_REF][START_REF] Noordermeer | The Dynamic Architecture of Hox Gene Clusters[END_REF][START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF][START_REF] Sexton | Three-Dimensional Folding and Functional Organization Principles of the Drosophila Genome[END_REF]. Moreover, microscopy, 4C and Hi-C assays have revealed that, when repressed, the ANT-C and BX-C are condensed and can interact together over long-distance when located within the same Polycomb body [START_REF] Bantignies | Polycomb-Dependent Regulatory Contacts between Distant Hox Loci in Drosophila[END_REF][START_REF] Cheutin | Loss of PRC1 induces higher-order opening of Hox loci independently of transcription during Drosophila embryogenesis[END_REF][START_REF] Mateo | Visualizing DNA folding and RNA in embryos at single-cell resolution[END_REF][START_REF] Sexton | Three-Dimensional Folding and Functional Organization Principles of the Drosophila Genome[END_REF]. ). This suggest that they are in a so-called "bivalent state", where either they simultaneously carry the active and repressive mark on the same nucleosome (true bivalent domains) or their distribution is mutually exclusive in the population, with cells (or alleles within cells) either carrying the active or repressive mark.

c. Histone modification landscapes mirror alternative Abd-B promoter use in cell lines

As we had detected similar overall levels of Abd-B expression by RT-qPCR in the Sg4 and S3

cell lines, we wondered if the different patterns of histone modifications could mirror preferred use of alternative promoters. We therefore evaluated alternative Abd-B promoter use by performing calibrated RT-qPCR using a plasmid containing all sequences unique to each isoform and specific primers (Fig. 5.1D). Comparison between Sg4 and S3 cells revealed mostly similar mRNA levels generated from the A, C and g promoters, with the latter two being detected at low levels. mRNA from the 2 promoter was significantly more prevalent in S3 cells, whereas mRNA from the 3 and B promoters was significantly overrepresented in Sg4 cells (Fig.

5.1D

). These data closely mirror the choice of promoters as previously reported in Sg4 cells, with the exception of the lowly expressed C promoter [START_REF] Schwartz | Genome-wide analysis of Polycomb targets in Drosophila melanogaster[END_REF]. The

d. The abd-A and Abd-B gene can be co-expressed in a fraction of Sg4 cells

Two previous studies have shown that the abd-A and Abd-B genes can be co-expressed in (subsets) of cells in the embryonic central nervous system and both embryonic and larval genital discs [START_REF] Foronda | Requirement of abdominal-A and Abdominal-B in the developing genitalia of Drosophila breaks the posterior downregulation rule[END_REF][START_REF] Gummalla | Hox gene regulation in the central nervous system of Drosophila[END_REF]. Our RT-qPCR analysis detected slightly

g. Active Abd-B dissociates from the repressive BX-C contact domain in Sg4 cells

Previously, a 3D study reported that viewpoints within Abd-B and its regulatory domains had reduced interaction with the repressed BX-C in S3 cell line where Abd-B is active compared to in S2 cells where the BX-C is repressed [START_REF] Lanzuolo | Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex[END_REF]. To gain a more comprehensive insight into this 3D organization, we performed Hi-C and 4C-seq in Sg4 cells, where Abd-B is active. To exclude that 3D genome organization is globally different in this cell line, we first supported by microscopy data in both embryos and cell lines, where the active Abd-B gene increases the average distance from the repressed bxd regulatory region that is located between abd-A and Ubx [START_REF] Lanzuolo | Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex[END_REF]. This mechanism of dissociation upon activation resembles the mouse Hox gene clusters, where active and inactive genes are dynamically localized in different contact domains that exactly match the domains of active and repressive histone modifications [START_REF] Noordermeer | The Dynamic Architecture of Hox Gene Clusters[END_REF][START_REF] Noordermeer | Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci[END_REF].

h. Active Abd-B dissociates from the repressive BX-C contact domain, but within different contact domains in cell lines and larval Genital discs

We next wondered if the same contact domain organization is present in cells from the larval Genital discs and the cultured S3 cell line, where the Abd-B gene is active (Fig. 5.1A). 4C-seq using the Abd-B 2 promoter revealed a similar loss of interactions with the upstream part of the BX-C where the repressed genes are located (Fig. S6). Like in Sg4 cells, the Abd-B regulatory domain forms a single active contact domain in the larval Genital discs and in the S3 cell line, which dissociates from the inactive genes and regulatory domains ( previously reported for the mouse Hox clusters [START_REF] Noordermeer | The Dynamic Architecture of Hox Gene Clusters[END_REF][START_REF] Noordermeer | Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci[END_REF].

Next, we focused our attention on 3D interactions in Genital disc cells (Fig. 5.3C). Here the 4C-seq signal indicated a more heterogeneous organization, with sudden drops of interactions detected both at the Fab-7 and Fab-8 BEs. Considering that the Genital disc is comprised of cells from parasegments 12-14, we speculated that this pattern reflected the presence of (at least) 2 cell populations, where either the Fab-7 or Fab-8 BE acted as the upstream boundary of the active contact domain. This raises the intriguing possibility that the Sg4 and S3 cell lines represented different cell populations within the Genital discs, with the former resembling parasegment 12 (with the active contact domain spanning iab-7 to iab-9) and the latter resembling parasegment 13 (with the active contact domain spanning iab-8 and to a lesser extent iab-9).

i. Differential BE function occurs without major changes to insulator protein binding

To gain further insights how BEs isolate the active and repressive contact domains, we characterized the binding of the insulator proteins GAF and CP190. Both these proteins have previously been implicated in the function of BEs in the BX-C [START_REF] Kyrchanova | The bithorax complex iab-7 Polycomb response element has a novel role in the functioning of the Fab-7 chromatin boundary[END_REF][START_REF] Kyrchanova | Complete reconstitution of bypass and blocking functions in a minimal artificial Fab-7 insulator from Drosophila bithorax complex[END_REF][START_REF] Nègre | A Comprehensive Map of Insulator Elements for the Drosophila Genome[END_REF][START_REF] Schweinsberg | The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites[END_REF][START_REF] Wolle | Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex[END_REF]. Using ChIP-qPCR in Sg4, S3 and S2 cells, we found that GAF was enriched at the Fab-7, Fab-8 and B BEs in all three cell types, albeit with some cell-type specificity (Fig. 5.3D, left panel). Particularly, this included a significantly increased GAF binding in Sg4 cells at Fab-7. CP190 was enriched in all three cell types only at the C element, which is the most downstream BE used as BX-C boundary in these cells (Fig. 5.3D, right panel). Some cell-type specificity was detected as well, with the CP190 signal increased at the C element in S3 cells, and a further more minor peak of enrichment detected in the same cells at the Mcp BE. Overall, we found that the function of the Fab-7 or Fab-8 BE as boundary between the active and repressive contact domain, as compared to the situation where they were located within a contact domain, was not accompanied by a differential pattern of GAF or CP190 binding at these BEs.

j. Absence of the Fab-7 BE causes a minor reduction in overall Abd-B activity in

Genital discs

To further characterize the (potential) functions of BEs in structuring contact domains and guiding gene activity, we took profit from the availability of a Drosophila mutant where the Fab-7 element is deleted (Fab-7 1 In this mutant, a 3.6 kb DNA region is deleted, encompassing both the Fab-7 BE and PRE [START_REF] Mihaly | In situ dissection of the Fab-7 region of the bithorax complex into a chromatin domain boundary and a Polycombresponse element[END_REF]. Deletion of this element was previously shown to cause homeotic transformations in the adult fly [START_REF] Gyurkovics | A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating two domains of regulation[END_REF][START_REF] Karch | Mcp and Fab -7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster[END_REF][START_REF] Singh | Specific combinations of boundary element and Polycomb response element are required for the regulation of the Hox genes in Drosophila melanogaster[END_REF], whereas our results suggested it functions as a boundary in subsets of Genital disc cells (Fig. 5.3C). We first analyzed global Hox gene expression in Genital disc cells from WT and Fab-7 1 larvae. Upon deletion of Fab-7 in Wing disc cells, no significant changes in gene expression were detected (Fig. 5.4A). In Genital discs cells, all Hox genes remained similarly expressed (or repressed), with the exception of the Abd-B gene whose activity was mildly reduced (Fig. 5.4A). In the absence of the Fab-7 element, neither the global activity of the abd-A or Abd-B gene was drastically changed in the genital discs.

k. Absence of Fab-7 induces a moderate reorganization of contact domains in Genital disc cells

Next, we determined if chromatin architecture at the BX-C was reorganized in the absence of the Fab-7 BE. Using 4C-seq, we first compared WT and Fab-7 1 wing disc cells, where the abd-

We next compared 4C-seq profiles between WT and Fab-7 1 Genital disc cells, where Abd-B activity was mildly reduced when the Fab-7 BE is absent (Fig. 5.4A). Without Fab-7, the Abd-B viewpoint globally engaged in more interactions over the BX-C, which was particularly prominent towards the upstream part of the BX-C, which contains the repressed Ubx and abd-A genes (Fig. 5.4B, black arrows). This may suggest that within the heterogeneous cell population of the Genital discs, the Abd-B gene associates with the repressive contact domain in a larger fraction of cells when Fab-7 is absent. This increased association with the repressive contact domain may explain the moderate decrease in overall Abd-B expression (without changes in Ubx or abd-A expression).

More detailed comparison between the WT and Fab-7 1 4C-seq data revealed the absence of the drop in signal at the Fab-7 BE in the mutant cells (Fig. 5.4B, purple bar and arrow). We hypothesized that this was caused by a reduced separation between iab-6 and iab-7, with the later now more frequently associating with the repressive contact domain. As such, in Genital disc cells, the deletion of the Fab-7 BE may thus extend, within in a subset of cells, the repressive contact domain towards iab-7.

m. Absence of the Fab-7 BE influences the association of alternative promoter choice in individual Genital disc cells

To overcome the limitations of comparing alternative promoters in the heterogeneous cell populations of the WT and Fab-7 1 Genital disc, we performed a single-cell RNA-seq analysis on pools of micro-dissected larval imaginal discs (Fig. 5.5B). In this analysis, we sequenced from the 5' end of mRNA, which allowed us to distinguish transcripts that originated from the different promoters in single WT and mutant cells. After filtering of low quality cells, we obtained single-cell RNA-seq data for 8242 WT cells and 5582 Fab-7 1 mutant cells. Further quality control revealed that the median number of unique mRNA molecules detected per cell was slightly increased in mutant over WT cells (WT: 6117 Unique Molecular Identifiers (UMIs) / cell; Fab-7 1 : 8035 UMIs / cell). This, in turn, resulted in the median detection of 1899 unique genes in WT cells and 2193 genes in mutant cells (Fig. 5.S9). Whereas we detected fewer mutant cells, we concluded that the data quality of these mutant cells (as measured by the number of unique mRNA molecules / cells) slightly exceeds those of the WT cells.

The analysis of this data is still ongoing, but already has revealed a number of interesting observations regarding the influence of the Fab-7 BE on alternative promoter choice. Clustering of the combined WT and Fab-7 1 single-cell data from pools of imaginal discs, using the Seurat v4.0.4 tool [START_REF] Hao | Integrated analysis of multimodal single-cell data[END_REF], revealed the existence of 13 distinct cell populations (Fig. 5.5C).

Based on selected marker genes, we could trace back clusters that contained individual imaginal discs. Moreover, distinct clusters containing dead cells, muscle cells and neuronal cells were identified. Importantly, all these clusters contained a mix of WT and mutant cells, confirming that the absence of the Fab-7 BE did not have a major influence on the identification of clusters (Fig. 5.5C).

The presence of Abd-B transcripts, for any promoter, was to a very large extent restricted to the Genital disc and muscle clusters, both for the WT and mutant cells (Fig. 5.5D). For the remainder of the analysis, we restricted our analysis on the cells in the Genital disc cluster. We detected 1117 WT cells in the Genital disc cluster, of which 731 cells had accumulated Abd-B (65.4% of cells). Similarly, we detected 783 Fab-7 1 cells in the Genital cluster, of which 389 cells had accumulated Abd-B (49.46% of cells).

Next, we interpreted the 5' aspect of the single cell analysis, to quantitatively determine alternative Abd-B promoter use within single cells. mRNA molecules originating from the A, 2 and B promoters were detected in the largest numbers of both WT and Fab-7 1 mutant cells (Fig.

5.5E

). Detection of mRNA molecules in individual cells therefore showed a high degree of Our single cell RNA-seq analysis thus indicated that the absence of the Fab-7 BE, with function of the Fab-8 BE maintained, promoted the activity of the 2 promoter over the B promoter.

Importantly, the 2 promoter is located more upstream in the Abd-B regulatory domain as compared to the B promoter. As such, this provides further in-vivo evidence that the Fab-7 BE influences alternative Abd-B promoter choice, where it has a positive effect on the production of longer mRNA isoforms. In parallel to our comparative studies, I had planned to get mechanistic insights into the role of BEs and 3D genome organization in the regulation of Hox genes at the BX-C using genetic and epigenetic editing of Drosophila cell lines. For this purpose, had three strategies in mind:

1. Deletion of the Fab-7 element in Sg4 cells.

2. Global knock-down (KD) of insulator proteins in Sg4 cells.

3. Forced activation of abd-A in S2 and Sg4 cells.

In this chapter, I will summarize these strategies and discuss the practical problems that prevented the generation of the corresponding cell lines.

Deletion of the Fab-7 element in Sg4 cells

How deletion of a BE in cell lines affects 3D organization, the epigenetic landscape and Hox gene expression has not been reported. We decided to focus on the Fab-7 element in Sg4 cells, as our ChIP-seq experiments had indicated that this is the boundary between the H3K27me3and H3K4me3-marked domain in these cells. We hypothesized that the deletion of the Fab-7

element could have two opposite outcomes in Sg4 cells.

1. The deletion of the BE could affect the 3D organization of Bithorax cluster by extending

Abd-B contacts up to the next BE: Fab-6. This would allow the H3K4me3 domain to extend up to the Fab-6 BE, thus activating the iab-6 and potentially increasing the activity of Abd-B. We considered the first hypothesis the most likely, as deletion of Fab-7 induces a fusion of the iab-6 and iab-7 cis-regulatory regions in the fly, leading to ectopic activation of Abd-B in the more anterior segments.

The deletion of the

To delete the Fab-7 BE, we designed a CRISPR-Cas9 deletion approach using a published expression vector for Cas9 and gRNA (Bassett et al., 2014) (Adgene #49330). Details of the plasmid are described in the material and method section. As this plasmid relies on a Puromycin marker for selection of transfected clones, I first determined its optimal concentration for Sg4 cells. After testing various concentrations, I found that 0.8 µg/mL of Puromycin resulted in at least 98% of cell death after 10 days.

In parallel, transfection conditions were optimized as the experimental procedures described in [START_REF] Bassett | Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9[END_REF] did not work well for our Sg4 cells. For this purpose, I tested two different transfection reagents to transfect an eGFP plasmid: Fugene HD (Promega; as used in [START_REF] Bassett | Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9[END_REF]) and Effectene (Qiagen). Various combinations of cell number, ratio of plasmid versus the quantity of transfection reagents and incubation time were tested, followed by determination of transfection efficiency by counting green cells in mock versus GFPtransfected cells (fluorescent microscopy, data not shown). The optimal conditions and obtained transfection rates for Fugene and Effectene are recapitulated in Fig. 6.1A. In these tests, Fugene performed better than Effectene, yet the maximum transfection efficiency never exceeded 20%.

Despite low transfection rates, we started transfections to delete the Fab-7 BE in Sg4 cells, under the assumption that transfected cells would be selected by puromycin treatment.

Sg4 cells were transfected with the deletion plasmid using the optimal conditions as determined for the Fugene HD transfection agent. After 10 days of puromycin selection, no viable cells were detected in the non-transfected control. In our three independent experiments, PCR and qPCR on the cell population detected cutting efficiencies of 45%-50%. Indeed, the expected PCR product for the Fab-7 deletion (DFab-7) was detected, confirming successful deletion of the element. However, WT bands on PCR gel could also still be detected by PCR, indicating that either a fraction of cells remained in the WT configuration or that the deletion was heterozygous To overcome this problem of having a considerable fraction of non-deleted cells present, I tried two strategies:

1. "re-CRIPSR" of transfection cells to achieve a further increased rate of deletion.

2. Enrich for individual or small sets of clones by limited dilution.

In the first strategy, the cells which had undergone a first round of CRISPR cutting were subjected to a second round of transfection with either the same gRNA or other gRNA targeting a nearby sequence at the Fab-7 BE (several bp away). The outcome of this strategy did not noticeably improve the cutting efficiency (not shown).

In the second strategy, cells were transfected and selected with puromycin as before. After 3 to 10 days of selection, they were diluted in 96 well plates to obtain on average 1 cell per well Fig. 6.2.A. To maximize cell recovery, isolated single cells were fed with "conditioned medium", meaning medium in which other cells had grown [START_REF] Franz | Generation of genome-modified Drosophila cell lines using SwAP[END_REF]. Cells were checked every day under a microscope and "conditioned medium" was added when needed.

Attempts to isolate clones from the "re-CRISPR" strategy was also done. In both cases, very few cells gave rise to viable cell cultures: 5 cell cultures out of initial 48 wells with observed cell division after single-transfection and 3 cell cultures out of initial 37 wells with observed cell division after re-CRISPR. Pictures of isolated clones are shown in Fig. 6.2.A.

PCR and qPCR were done to assess the deletion efficiency of Fab-7 element in isolated clones.

As shown in Fig. 6.2.B (1 single-transfection and 2 "re-CRISPR" cell culture), recombination events had happened in all cultures, as extra bands had appeared after PCRs and the qPCR experiment did not generate signal for subsets of primers. Moreover, an internal qPCR primer for Fab-7 consistently continued to show enrichment in all three cell cultures, arguing in favor of (complex) re-arrangements rather than full deletion after transfection. As we could not resolve the complex genotype, potentially incorporating incomplete or heterozygous deletion of the Fab-7 element, the isolated cell cultures were not further considered for future experiments.

In a final effort to obtain Sg4 cells with a deleted Fab-7 BE, I tried to transfect the cells using the Amaxa Cell line Nucleofector kit V. This with the hope that electroporation could improve transfection efficiency. I optimized the transfection condition by testing 5 different programs (not shown). Using electroporation, I was able to reach up to 60% of survival after electroporation and 85% of transfection efficiency with control eGFP plasmid (FACS, Fig. Ultimately, we therefore decided to not generate Sg4 cells that carry the Fab-7 deletion, but rather to address a more limited set of questions in Drosophila larvae carrying the Fab-7 deletion (Fab7 1 flies from [START_REF] Gyurkovics | A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating two domains of regulation[END_REF]). The results of the analysis of gene expression and 3D genome organization were presented in the previous chapter, where we globally find both increased interactions with iab-6 and the further downstream H3K27me3marked part of the BX-C (Fig. 5.4.B). Considering that we find a minor, but global, downregulation of Abd-B gene activity (Fig. 5.4.A), these studies suggest that our second hypothesis was in fact more accurate.

Knock-down (KD) of insulator proteins in Sg4 cells

As a complementary strategy to our Fab-7 deletion experiment, we decided to knock-down (KD) the GAF insulator protein in Sg4 cells using RNAi, as this insulator protein is directly involved in the regulation of the Abd-B gene via its interaction with the Fab-7 BE [START_REF] Nègre | A Comprehensive Map of Insulator Elements for the Drosophila Genome[END_REF][START_REF] Schwartz | Alternative Epigenetic Chromatin States of Polycomb Target Genes[END_REF][START_REF] Schweinsberg | The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites[END_REF] . Here, we hypothesized that the absence of binding could weaken the insulating function of the Fab-7 BE. This in turn could have the same outcomes as hypothesized in the previous section (i.e. either upregulation of Abd-B through the activation of iab-6 or repression of Abd-B through increased interactions with the further upstream H3K27me3-marked domain).

I optimized the RNAi delivery for Sg4 cells starting using a published protocols [START_REF] Duarte | Transcription factors GAF and HSF act at distinct regulatory steps to modulate stressinduced gene activation[END_REF][START_REF] Fuda | GAGA Factor Maintains Nucleosome-Free Regions and Has a Role in RNA Polymerase II Recruitment to Promoters[END_REF]. First, I performed dose response experiments by treating the cells for 2.5, 5 and 7 days with different concentration of GAF RNAi: 10 µg, 20 µg and 40 µg days did not improve GAF KD (not shown). To further improve KD efficiency, I designed analyze these cells, but rather to focus on the Drosophila larvae carrying the Fab-7 deletion (Fab7 1 flies, [START_REF] Gyurkovics | A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating two domains of regulation[END_REF]).

Forced activation of abd-A in S2 and Sg4 cells

As a final strategy, we wanted to determine how the forced activation of a Hox gene would affect the 3D genome organization of the BX-C cluster. For this project, I co-supervised an M2 student, Sylvain Bouvard, who performed most of the work. The practical goal of his project was to use the Sun-Tag system in the S2 and Sg4 cell lines to activate the abd-A gene. The Sun-

Tag system was previously used in Drosophila cells to activate gene expression [START_REF] Chavez | Comparison of Cas9 activators in multiple species[END_REF]. and the Genital disc have been observed that co-express these genes (as discussed in the introduction [START_REF] Foronda | Requirement of abdominal-A and Abdominal-B in the developing genitalia of Drosophila breaks the posterior downregulation rule[END_REF][START_REF] Gummalla | Hox gene regulation in the central nervous system of Drosophila[END_REF]. Moreover, we have now confirmed using single-cell RNA-seq, that in Sg4 cells this co-expression also occurs in a subset of cells (although not necessarily from the same allele).

In
The Sun-Tag is a tripartite system that relies on the expression of 3 components, encoded by three different vectors that need to be transfected together Fig. 6.4.A) (#78899, #78900, #49411):

1. The pAct:dCas9-GCN4 vector expresses the inactive dCas9 fused to 10 x GCN4 epitopes.

2. The pAct:scFv-VP64 vector expresses a scFv-VP64 protein where a single-chain variable fragment (scFv) antibody that recognizes the GCN4 epitope is fused to the VP64 transcriptional activator.

3. The pCFD4-U6:1-U6:3tandem_gRNAs vector expresses a couple of guide-RNAs.

fusion, thereby ultimately allowing the recruitment of several VP64 activators to the abd-A promoter.

Three couples of gRNA targeting abd-A TSS region were designed Fig. 6.4.A, top. To validate the system, the Sun-Tag system was first targeted in Sg4 cells to a non-chromatin template consisting of a dual reporter assay with plasmids containing Firefly under the control of the abd-A promoter and Renilla luciferase under the control of a constitutively expressed promoter, or the reverse. This dual Luciferase assay allows the internal normalization of the luminescent signal to the constitutively expressed promoter. The reporter plasmids, together with the Sun-Tag system plasmids were transfected with Fugene HD (using the optimal conditions as determined in the previous section; see Fig. 6.1.A). Each pair of guides (referred to A, B, and C in Fig. 6.4.A) were able to up-regulate abd-A promoter in a non-chromatin template, despite the low transfection rate (data not shown).

Next, we focused our attention on the activation of the endogenous abd-A gene after transfecting the tripartite Sun-Tag system in WT Sg4 cells. Different combination of pairs of gRNAs were tested, alone or in combination Fig. 6.4.B. A minor increase in abd-A activity could be observed, with additive effects when several pairs of gRNAs were transfected together.

We nonetheless considered this activation insufficient to merit further investigation of chromatin reorganization. We hypothesized that this insufficient increase could have three explanations:

1. Low transfection efficiency (as discussed in the previous section).

2. The repressive H3K27me3 histone mark preventing the dCas9 to bind the abd-A promoter.

3. The repressive H3K27m3 histone mark preventing the VP64 to activate the abd-A promoter.

As time was limited in this M2 project (and my PhD project), and we were not easily able to distinguish between these three hypotheses, we decided to stop these experiments.

CHAPTER 7: Discussion, conclusions and perspectives

During my PhD, I determined the relationships between 3D chromatin organization, the histone modification landscape, boundary elements (BEs) and gene expression at the Drosophila Hox gene clusters. As expected, I found that these elements are tightly linked, including a number of novel observations how the Fab-7 boundary guides multi-level process.

3D chromatin organization of the repressed ANT-C cluster

We used three different cell types to study Hox genes in a repressed context:

• In-vivo context: imaginal Wing discs from L3 larvae.

• In-vitro context: S2 and Kc167 cell lines that are derived from Drosophila embryos.

In Wing discs, the three Hox genes in the BX-C are efficiently silenced. In the ANT-C, a low level of Antp gene expression can be detected (Fig. 5.1). Detection of this gene in the wing discs is not surprising as it was recently shown that Antp is essential to achieve wing formation during development [START_REF] Paul | Hox dosage contributes to flight appendage morphology in Drosophila[END_REF]. Nonetheless, considering the low level of Antp activity, we considered wing discs as a tissue where Hox genes globally are repressed. In the S2 and Kc167

cell lines, we did not detect any meaningful levels of Hox gene activity. We thus considered

Wing disc cells and the S2 and Kc167 cell lines as "fully" Hox repressed cell types.

The ANT-C cluster is folded into a repressive contact domain in all three cell types (Fig. 5.S2, Hi-C analysis in the S2 cell line revealed that the ANT-C in S2 cells is sub-divided in three sub-TADs: one containing the Lab, pb, bcd and Dfd genes, one containing the Scr gene and the last one containing the Antp gene. The existence of these sub-domains may aid in the gene-specific regulation of the Hox genes that are generally active in non-overlapping domains [START_REF] Kosman | Multiplex Detection of RNA Expression in Drosophila Embryos[END_REF]. How the Lab, pb and Dfd genes, that are grouped together in the first domain, are individually regulated remains to be determined. Interestingly, this first domain includes the interspersed bcd (Bicoid) gene, a homeobox gene that appears to have lost its collinear pattern of expression [START_REF] Bürglin | Homeodomain proteins: an update[END_REF][START_REF] Pick | Hox genes, evo-devo, and the case of the ftz gene[END_REF]. It remains to be determined if and how this gene benefits from the co-localization with the other Hox genes for its regulation, and how it can overcome the repressive context as part of its non-collinear activity.

We could also use three different cell types to study the Ubx and abd-A genes at the BX-C in a repressed context:

• In-vivo context: imaginal Genital discs from L3 larvae.

• In-vitro context: Sg4 and S3 cell lines that are derived from Drosophila embryos.

Importantly, the ANT-C in all these cells is in a fully repressed context (Fig. depending on cell type. The functional relevance of these differential boundaries remains to be determined. Moreover, at this stage we can't formally exclude that this difference is caused by the (minor) differences in the protocol used to generate the Hi-C data (S2: [START_REF] Szabo | TADs are 3D structural units of higher-order chromosome organization in Drosophila[END_REF]; Sg4: my thesis).

3D chromatin organization of the repressed BX-C cluster

The BX-C is also efficiently repressed in Wing discs and the S2 and Kc167 cell lines.

Intersection between Hi-C and ChIP-seq data in S2 cells confirmed that it is organized into a repressed contact domain containing H3K27me3-marked chromatin (Fig. Alternatively, a strategy could be considered to delete this BE in S2 and Kc167 cells.

To repeat similar studies at the ANT-C, where the functional identification of BEs has received less attention over the past years, it would be interesting to use systems where Hox genes have a differential expression status, such as the Antp-expressing CI8+ cell line [START_REF] Cherbas | The transcriptional diversity of 25 Drosophila cell lines[END_REF] or different imaginal leg discs (which express either Antp or Scr). Such studies, both in repressed and active contexts, and including the removal of sub-TAD boundaries, would greatly help to dissect the regulatory logic of the ANT-C in cell lines and in-vivo.

3D organization of active Abd-B, the exception at the BX-C?

To study multi-level gene regulation of the Abd-B gene at the BX-C, we compared three cell types as well:

• In-vivo context: imaginal Genital discs from L3 larvae.

• In-vitro context: Sg4 and S3 cell lines that are derived from Drosophila embryos.

Of interest, we initially included the Sg4 cell line because it had been reported to express both the Dfd and Abd-B Hox gene [START_REF] Beisel | Comparing active and repressed expression states of genes controlled by the Polycomb/Trithorax group proteins[END_REF][START_REF] Schwartz | Genome-wide analysis of Polycomb targets in Drosophila melanogaster[END_REF] but we only detected expression of Abd-B. Both the Sg4 and S3 cell lines are derived from embryos and Abd-B was detected at the same level by RT-qPCR. Hence, we did not expect noticeable differences between the two cell types. ChIP-seq revealed that in Sg4 the region between the Fab-7 and C

BEs was enriched in H3K4me3 (Fig. 5.1B). This region spans the iab-7 to iab-9, and includes all Abd-B promoters except the g promoter. A ChIP-qPCR comparison between Sg4 and S3 cells showed that in the latter, only iab-8 and the upstream part of iab-9 (comprising the A and 2 promoters of Abd-B) were strongly enriched for H3K4me3. In contrast, enrichment for H3K4me3 on the 3, B and C promoters was strongly reduced. Importantly though, this enrichment was still considerably higher than in the fully repressed S2 cell line (Fig. 5.1C).

Upon observation of these differences, we decided to quantify the activity of alternative Abd-B isoforms using RT-qPCR with absolute quantitation relative to an external standard (Fig. 5.1D).

Comparison of Sg4 and S3 cells revealed that in Sg4 cells, the A, 2, 3, B promoters, and to a lesser extent the C promoter, all generated robust levels of mRNA. In S3 cells, only the A and 2 promoters showed similar robust levels, whereas the 3, B and C promoters all displayed moderate signal. Hence, even if the global Abd-B expression level is similar between the two cell types, they achieve such output using different alternative promoters. In turn, this is directly linked to the activated iabs that contain the enhancers used for Abd-B activated: when iab-7 to iab-9 are fully active (Sg4 cells) the alternative Abd-B promoters are more uniformly used, whereas when iab-8 and the upstream part of iab9 are activated (S3 cells), the balance shifts towards the more upstream promoters. How particularly the strong increase in the activity of the 2 promoter is achieved when the iab7 and iab9 are in a (more) repressed state remains to be determined.

To determine how 3D chromatin organization correlated to the changes in Abd-B expression, we performed Hi-C in Sg4 cells (Fig. 5.3A). Here we found that the 3D organization of the BX-C was different from S2 cells, with the Fub BE maintaining the separation of the Ubx gene from the rest of the cluster, but in this cell type, the abd-A and Abd-B genes are also located in different sub-TADs. Our combined Hi-C, 4C-seq and ChIP-seq experiments further allowed to pinpoint the boundaries of this sub-TAD / active contact domain in Sg4 cells, showing it is bordered by the Fab-7 BE on the 5' end and by the C BE on the 3' end.

Our multi-level analysis confirms the direct link between 3D genome organization and histone modifications, resulting in the formation of "contact domains" [START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF][START_REF] Sexton | Three-Dimensional Folding and Functional Organization Principles of the Drosophila Genome[END_REF]. Moreover, we identify the involvement of BEs in this process, with the Fab-7 and Fab-8 BEs dynamically creating boundaries between the active and repressed contact domains at BX-C. Intriguingly, our ChIP-qPCR analysis for the GAF and CP190 insulator proteins revealed limited differences in binding between these insulator proteins (Fig. when a BE was removed from the mouse HoxC cluster in in-vitro differentiated stem cells, which resulted in the expansion of the active domain [START_REF] Narendra | CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation[END_REF].

This multi-level organization of the Abd-B also raises questions about its organization in other cell types, particularly when its activity relies on the iab-5 and iab-6 infra-abdominal segments:

• How do these iabs direct alternative promoter choice, and how is this linked to domains of histone modifications in iab-8 and iab-9 where these promoters are located?

• Is a single active contact domain formed in these cells as well, or do the intervening inactive iabs remain in a repressed state, which may cause them to loop out from the active domain?

• Do the BEs that border iab-5 and iab-6 (Mcp and Fab-6) have similar insulating functions, and are the intervening iabs important for 3D structure as well?

Overall though, the complex organization of the Abd-B regulatory domain, including 5 iabs, at least 6 BEs and the presence of at least 6 alternative promoters (summarized in Fig. 7.1) suggest that the Abd-B gene has a unique regulation among the Hox genes, which may be reflected in uniquely dynamic 3D organization as well.

Abd-B may not follow the "open for business" model

Maeda and Karch [START_REF] Maeda | The open for business model of the bithorax complex in Drosophila[END_REF] 1. The differences between the in-vivo and in-vitro models could be due to artifacts between the cell types. The S3 and S2 cell lines were isolated from embryos, followed by derivation of the Sg4 line derived from S2 cells. Although these cells have the same embryonic origin, it may be that the prolonged in-vitro culture has resulted in an invasion of the H3K27me3 mark into the more anterior domain.

We consider this option less likely, as our 4C-seq in WT Genital discs suggest a strong physical separation between the inactive Ubx and abd-A genes and the active Abd-B gene, indicating that the inactive genes are organized within their own contact domain.

2. The study by [START_REF] Bowman | H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex[END_REF] was performed in PS where the Abd-B gene is repressed. It may thus be that the Abd-B gene, and its associated regulatory domain, has a unique capacity to impose a repressed state and 3D architecture on the anterior part of the BX-C. Moreover, such an organization may be unique to the larval stage. Further insights into the behavior of the Abd-B gene may be extracted from an ORCA study in A9 segments of the embryo [START_REF] Mateo | Visualizing DNA folding and RNA in embryos at single-cell resolution[END_REF]. In the A9 segment the sub-TADs that contain the repressed Ubx and abd-A genes fuse, whereas the active Abd-B sub-TAD (comprised of the regulatory regions iab-5 to iab-9) adopt a more relaxed and decondensed configuration that is apart from the repressive sub-TAD. Although we do not know the histone modification landscape in these cells, their repressed status and organization in a single contact domain in A9 cells suggests that they are marked by H3K27me3. This result thus also argues against the "open for business" model when Abd-B is active in the embryo.

To formally distinguish between these two hypotheses, it would be necessary to continue the work of Bowman and colleagues [START_REF] Bowman | H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex[END_REF] in more posterior PS. A hint of a potential outcome from these experiments may be obtained from a DNA-FISH study in all PS of the embryo [START_REF] Cheutin | Loss of PRC1 induces higher-order opening of Hox loci independently of transcription during Drosophila embryogenesis[END_REF]. In this study, the distance between Hox genes was determined in all PS of the embryo. Starting from PS10 (where Abd-B is first expressed), the distance between Abd-B and the other homeotic genes gradually increases until the most posterior PS14. As such, this work suggests that it is unlikely that the active Abd-B gene occupies the same contact domain as the inactive Ubx and abd-A genes.

An interesting hypothesis could be that the "open for business" model is in fact a reflection of the need for co-expression of the Ubx and abd-A genes, which occurs from PS7 to PS10. In contrast, except for small subsets of cells in the embryo and larvae, the Abd-B gene is not coexpressed with neither Ubx, nor abd-A. In turn, this may provide an explanation for the boundary function of the Fub BE in inactive cells, as it could provide a means to activate the Ubx gene prior to the activation of abd-A.

Is alternative promoter choice of Abd-B associated with a localized reversal of colinearity?

Among the most distinguishing characteristics of Hox genes is their collinear activation along the A-P axis according to their position within the gene clusters, which was at the root of the Nobel prize awared to Ed Lewis [START_REF] Lewis | A gene complex controlling segmentation in Drosophila[END_REF]. In this model of collinearity, the cis-regulatory regions (from abx/bx to iab-9) should be activated one after the other along the A-P axis in the embryo. This sequential activation is subsequently controlled by histone modifications (through the Polycomb and Trithorax complexes), with further precision added by BEs. Consequently, according to this model, iab-5, 6, 7, and iab-8/9 should become depleted in H3K27me3 and enriched in H3K4me3 in PS10, 11, 12 and 13/14 respectively. Thus, in PS10 only iab-5 should be active, whereas in PS13/14, all iabs should be devoid of repressive marks.

However, we made several observations that break with this model. These observations are primarily based on our work in cell lines, but support is found in our in-vivo analyses as well:

• In Sg4 cells, the active Abd-B contact domain is bordered by the Fab-7 and the C BEs.

Our ChIP-seq analysis showed that this entire domain, consisting of iab-7 to iab-9, is covered by active the H3K4me3 mark and fully devoid of the repressive H3K27me3 mark. In S3 cells, the fully active Abd-B contact domain is bordered by the Fab-8 and B

BEs, thus comprising iab-8, whereas iab-9 adopts a more bivalent or heterogeneous organization with both the presence of the H3K4me3 and H3K27me3 marks. The increased presence of the H3K27me3 mark directly mirrors both the reduced activity of the alternative promoters in iab-9 and 3D contacts with the Abd-B 2 promoter that is located in iab-8. Both this effect on promoter choice and 3D contacts is detected in Genital discs when Fab-7 is absent. Combined, these results show that the upon activation of the more posterior Fab-8 element (PS13/PS14), the levels of H3K27me3 increase within iab-9, which is an inversion of collinearity at a localized scale as compared to the situation when the Fab-7 element is active (PS12).

• Similarly, if collinearity was operating for Abd-B promoters, we would expect 3' promoters to be active in cells where a more 3' BE is used and more promoters towards the 5' to be active when the BE is more on the 5' end of the cluster. Again, our observations in both cell lines and the Genital discs argue that this process is inversed, thus providing evidence for localized inversion of colinearity both in cell lines and invivo.

• Finally, an inversion of collinearity may be observed on the other side of the Abd-B regulatory domain as well. Rather than seeing a sequential addition of iabs to the domain, we in fact detect a renewed coverage by H3K27me3: our ChIP-seq analysis confirmed that iab-5 and iab-6 are covered by H3K27me3 in Sg4 cells (when Fab-7 is active), whereas our ChIP-qPCR show the increased presence of H3K27me3 in iab-7 (when Fab-8 is active). As a result, we find that the 3' boundary of the active Abd-B domain is not fixed along the A-P axis. Although this is not necessarily an inversion of collinearity, it does break with the model that iabs are increasingly recruited to the active regulatory domain of a Hox gene.

Although we do not know the underlying mechanism that explains this reorganized pattern of collinearity, it's interestingly to notice that the relative organization of the Ubx and abd-A genes within their regulatory domains is different from the Abd-B gene:

• The promoters of the Ubx and abd-A genes are located in the most 3' iabs (respectively abx/bx and iab-2), whereas additional infra-abdominal segments are located more towards the 5' end.

• The various alternative promoters of the Abd-B gene are located in iab-8 and iab-9, whereas the uniquely regulatory iab-5 -iab-7 are located on the 3' of these promoters.

As such, the apparent inversion of the Abd-B regulatory landscape (but not the orientation of the gene itself) may explain the requirement for this inversion of collinearity.

Re-arrangements of 3D chromatin organization and Abd-B promoter choice in the absence of Fab-7

Genital discs represent a heterogenous cell population that is formed by cells coming from the A8 and A9 segments [START_REF] Foronda | Requirement of abdominal-A and Abdominal-B in the developing genitalia of Drosophila breaks the posterior downregulation rule[END_REF]. In agreement with this origin, iab-8/9 should control the segment identity, which coincided with our observation that our 4C-seq signal sharply dropped after the Fab-8 BE. We further detected a drop after the Fab-7 BE, which suggests a certain involvement in the 3D architecture of Abd-B in genital discs as well.

After our initial efforts to remove the Fab-7 element from Sg4 cells were unsuccessful (Chapter 6), we decided to use an existing Drosophila mutant where the Fab-7 BE is absent (Fab-7 1 ).

Previous studies have shown that these flies carry homeotic transformations, confirming its active involvement in Abd-B regulation. Indeed, due to ectopic activation of Abd-B in more anterior segments, the 6 th segment adopts the identity of the 7 th segment in adult flies, which is thought to be due to the fusion of iab-6 and iab-7. Consequently, iab-7 becomes prematurely activated in segment 6 while it is supposed to be expressed in segment 7 [START_REF] Singh | Specific combinations of boundary element and Polycomb response element are required for the regulation of the Hox genes in Drosophila melanogaster[END_REF].

Considering the phenotypic consequences of the Fab-7 deletion in the fly, we were expecting that in Fab-7 1 mutants, Abd-B contacts would spread over iab-6 and stop at the Fab-6 BE.

However, what we detected is rather the opposite. In Fab-7 1 Genital discs, Abd-B indeed gained contacts towards the 3' of the cluster, but this is not limited to iab-6. Moreover, the overall expression level of Abd-B was decreased in the mutant Genital disc cells as compared to WT.

We thus interpreted the increased contact frequencies of Abd-B with the upstream part of the BX-C as an increase in repressed contacts. Relevant the homeotic transformation, it may be necessary to focus on cells where iab-5 and/or iab-6 are active. Our single-cell RNA-seq analysis in larvae does not provide evidences for a large population of imaginal discs cells where Abd-B is expressed, thus requiring other means to isolate such cells (identification of cells lines, sorting from embryos or adult flies).

As mention before, genital discs represent an heterogenous cell population. We therefore consider it likely that the reorganization of 3D contacts does not occur, or less prominent, in cells where the Fab-8 BE is used as the main separation between the active and inactive domains. This heterogeneity complicated the analysis of alternative promoter choice by RT-qPCR, as we expect that changes in gene expression will only reflect the subset of cells where Fab-7 was the active BE. To overcome this limitation, we decided to use 5' single cell RNAseq to detect promoter expression at the single cell level. Interestingly, using this analysis we were able to detect RNA from all 6 promoters in the same cell, confirming that all promoters can be used by the same cell (albeit not necessarily from the same allele, and within the life time of the mRNA molecules). In the large majority of cells, both WT and mutant, we detected between 1 to 3 isoforms though. Despite this number of detected promoters not being differing, we did nonetheless confirm that the "shorter" mRNA from the promoters A and 2 are more often detected together in mutant single cells, whereas mRNA from the A and B promoters in the WT cells. As such, this result can indeed be interpreted as if Fab-8 becomes maintains the expression of the more 3' located Abd-B promoters, whereas the absence of Fab-7 has resulted in the repression of more 5' located promoters.

Previous studies on the Fab-7 element have confirmed its involvement in the prevention of premature activation of Abd-B expression [START_REF] Singh | Specific combinations of boundary element and Polycomb response element are required for the regulation of the Hox genes in Drosophila melanogaster[END_REF] and nucleation of H3K27me3 spreading in a repressive context [START_REF] Schuettengruber | Polycomb Domain Formation Depends on Short and Long Distance Regulatory Cues[END_REF]. My work further expands Fab-7 function by showing that in an active context, Fab-7 act as boundary between active and inactive histone modification, as reported for other BEs in the Drosophila genome as well [START_REF] Heurteau | Insulator-based loops mediate the spreading of H3K27me3 over distant microdomains repressing euchromatin genes[END_REF]. Uniquely though, this coincides with a localized inversion of collinearity at a distance (in iab-9) and subsequently influences the relative use of the alternative Abd-B promoters.

Introduction

Conclusion

Pendant ma thèse, j'ai déterminé les liens qui existent entre organisation 3D de la chromatine, les modifications d'histones, les éléments frontières et l'expression des gènes homéotiques de Drosophile. Comme attendu, j'ai montré que ces éléments sont étroitement liés mais j'ai également trouvé de nouvelles fonctions aux éléments frontières.

  and cell lines display comparable Hox gene expression patterns ........... b. The histone modification landscapes in cell lines mirror their global Hox gene expression status .. c. Histone modification landscapes mirror alternative Abd-B promoter use in cell lines ....................d. The abd-A and Abd-B gene can be co-expressed in a fraction of Sg4 cells .................................... e. Repressed Hox clusters are folded into a single repressed contact domain in the S2 cell line ......... f. Repressed Hox clusters are folded into a single repressed contact domain in Kc167 cell lines and in larval Wing discs .................................................................................................................................. g. Active Abd-B dissociates from the repressive BX-C contact domain in Sg4 cells .......................... h. Active Abd-B dissociates from the repressive BX-C contact domain, but within different contact domains in cell lines and larval Genital discs ........................................................................................ i. Differential BE function occurs without major changes to insulator protein binding...................... j. Absence of the Fab-7 BE causes a minor reduction in overall Abd-B activity in Genital discs .......k. Absence of Fab-7 induces a moderate reorganization of contact domains in Genital disc cells....... l. Absence of the Fab-7 BE reduces activity of all Abd-B promoters in Genital disc cells ................. m. Absence of the Fab-7 BE influences the association of alternative promoter choice in individual Genital disc cells .................................................................................................................................. CHAPTER 6 -Thesis project: Results Cell lines modifications to test the involvement of boundary elements and insulator proteins in the regulation of Hox genes and 3D chromatin organization . 1. Deletion of the Fab-7 element in Sg4 cells .............................................................................. 2. Knock-down (KD) of insulator proteins in Sg4 cells ............................................................ Is alternative promoter choice of Abd-B associated with a localized reversal of colinearity?

Figure 1 . 1 :

 11 Figure 1.1: Model of the 10 nm chromatin fiber. ........................................................................

Figure 1 . 2 :

 12 Figure 1.2: Alternative promoter use can differ between cell types ..........................................

Figure 1 . 3 :

 13 Figure 1.3: Regulatory elements of gene expression ..................................................................

Figure 1 . 4 :

 14 Figure 1.4: Mode of action of Boundary Elements .....................................................................

Figure 1 . 5 :

 15 Figure 1.5: Best characterized histone modifications .................................................................

Figure 1 . 6 :

 16 Figure 1.6: Overview of the ChIP procedure (for ChIP-seq and ChIP-qPCR) .........................

Figure 1 . 7 :

 17 Figure 1.7: Distribution of Histone modifications at and around active and repressed genes .

Figure 2 . 1 :

 21 Figure 2.1: Chromosome Conformation Capture and its adaptations ........................................

Figure 2 . 2 :

 22 Figure 2.2: Proprieties of Euchromatin and Heterochromatin ...................................................

Figure 2 . 3 :

 23 Figure 2.3: Chromatin states in Drosophila and the associated proteins/histone modifications ........................................................................................................................................................

Figure 2 . 4 :

 24 Figure 2.4: Different levels of 3D chromatin organization in the nucleus ................................

Figure 2 . 5 :

 25 Figure 2.5: Chromosome territories .............................................................................................

Figure 2 . 6 :

 26 Figure 2.6: Polycomb body dynamics and Hox gene recruitment .............................................

Figure 2 . 7 :

 27 Figure 2.7: TADs in mammals and the principle of loop extrusion...........................................

Figure 2 . 8 :

 28 Figure 2.8: Insulator proteins in mammals and Drosophila .......................................................

Figure 2 . 9 :

 29 Figure 2.9: Characteristics of TAD formation in Drosophila .....................................................

Figure 2 .

 2 Figure 2.10: Comparison of Hi-C maps between different species ...........................................

Figure 3 . 1 :

 31 Figure 3.1: Schematic representation of Hox gene organization and expression in Drosophila and mammals .................................................................................................................................

Figure 3 . 2 :

 32 Figure 3.2: Classification of the different types of Hox cluster organization............................

Figure 3 . 3 :

 33 Figure 3.3: Hoxd cluster colinear regulation in the mouse embryo and during limb formation in mouse .............................................................................................................................................

Figure 3 . 4 :

 34 Figure 3.4: Hoxd colinear activation during the mouse embryonic development ....................

Figure 3 . 5 :

 35 Figure 3.5: Schematic of Drosophila segmentation ....................................................................

Figure 3 . 6 :

 36 Figure 3.6: The Abd-B gene, its coding-sequences, its sequenced cDNAs and TSSs ..............

Figure 3 . 7 :

 37 Figure 3.7: Abd-B transcript localization in the Drosophila embryo .........................................

Figure 3 . 8 :

 38 Figure 3.8: Hox gene expression in Drosophila embryo segments ............................................

Figure 3 . 9 :

 39 Figure 3.9: The BX-C in Drosophila melanogaster ....................................................................

Figure 3 .

 3 Figure 3.10: H3K27me3 distribution over BX-C correlates with segment-specific cis-regulatory domain activity ..............................................................................................................................

Figure 3 .

 3 Figure 3.11: Fab-7 deletion and its phenotypic consequence .....................................................

Figure 3 .Figure 5 . 4 :

 354 Figure 3.12: Model for the 3D chromatin organization of the repressed and active BX-C. .... 82 Figure 3.13: Impact of the Fub and Fab-7 BE deletions on 3D chromatin organization of the BX-C as detected by ORCA ......................................................................................................... 84 Figure 4.1: Plasmid created by Gibson assembly for the quantification of Abd-B isoforms ... 90 Figure 5.1: Gene expression and histone modification landscape of the BX-C ...................... 117 ...................................................................................................................................................... 120 Figure 5.2: 3D chromatin organization of the repressed BX-C................................................ 120 Figure 5.3: 3D chromatin organization of the BX-C when Abd-B is active ............................ 123 Figure 5.4: Hox gene expression and 3D chromatin organization in WT and Fab-7 deleted imaginal discs .............................................................................................................................. 126 Figure 5.5: Analysis of Abd-B promoter activity by calibrated RT-qPCR and 5' single cell RNA-seq ...................................................................................................................................... 129 Figure 5.S1: Epigenetic landscape of the ANT-C in S2 and Sg4 cells .................................... 131 Figure 5.S2: 3D chromatin organization of the ANT-C in S2 cells ......................................... 132 Figure 5.S3: 3D chromatin organization of the repressed BX-C in Wing discs, S2 and Kc167 cells .............................................................................................................................................. 133 Figure 5.S4: 3D chromatin organization of the repressed ANT-C in Wing discs, S2 and Kc167 cells .............................................................................................................................................. 134 Figure 5.S5: 3D chromatin organization of the ANT-C in Sg4 cells ....................................... 135 Figure 5.S6: 3D chromatin organization of the BX-C when Abd-B is active in S3 and Genital discs .............................................................................................................................................. 136 Figure 5.S7: 3D chromatin organization of the BX-C in Genital discs, Sg4 and S3 cells ..... 137 Figure 5.S8: 3D chromatin organization of the BX-C in WT and Fab-7 deleted cells ........... 138 Figure 5.S9: Single cell RNA-seq data quality in WT and Fab-7 deleted imaginal disc cells ...................................................................................................................................................... 139 Figure 5.S10: Abd-B promoter detection in WT and Fab-7 deleted Genital discs ................. 140 Figure 6.1: Optimal transfection conditions and assessment of cut efficiency by CRISPR/Cas9 in Sg4 cells ................................................................................................................................... 143 Figure 6.2: Fab-7 deletion transfection results for the "re-CRISPR" and CRISPR once Sg4 cells ...................................................................................................................................................... 145 Figure 6.3: Fab-7 deletion transfection results for the "re-CRISPR" and CRISPR once Sg4 cells ...................................................................................................................................................... 147 Figure 6.4: Sun-Tag system and result of endogenous abd-A activation ................................ 149

Figure 2 . 7 :

 27 Figure 2.7: TADs in mammals and the principle of loop extrusion

Figure

  Figure reproduced from[START_REF] Chang | TADs and Their Borders: Free Movement or Building a Wall?[END_REF].

Figure 3 . 1 :

 31 Figure 3.1: Schematic representation of Hox gene organization and expression in Drosophila and mammals

  possesses four copies of Hox clusters on four different chromosomes, called the HoxA to HoxD clusters[START_REF] Mcginnis | Homeobox genes and axial patterning[END_REF] Scott, 1992) (Fig.3.1). All four clusters are activated in a spatial and temporal manner along the primary A/P axis during embryogenesis (Izpisúa-Belmonte and[START_REF] Izpisúa-Belmonte | Homeobox genes and pattern formation in the vertebrate limb[END_REF]. The emergence of these four clusters after the genome duplications, resulting in the presence of 2 -4 paralogous copies of each gene, suggest that Hox products may act in tandem during development. Indeed, paralogs from the same Hox group but from different clusters (e.g. Hoxa4-Hoxd4) occupy highly similar domains of expression[START_REF] Duboule | The structural and functional organization of the murine HOX gene family resembles that of Drosophila homeotic genes[END_REF]) (Fig.3.1). Importantly, after being activated as part of the temporal collinear activation program, Hox gene activity state is maintained along the A/P axis. As a result, a pattern of spatial collinear activities is created whereby increasing numbers of Hox genes from different groups overlap along the trunk of the mouse embryo[START_REF] Deschamps | Developmental regulation of the Hox genes during axial morphogenesis in the mouse[END_REF].

  to be deregulated during early embryogenesis are Antp and Ubx, and only later on the other genes also become derepressed. This work suggests that the PcG machinery maintains silencing when activating factors are present by promoting chromatin compaction. The importance of PcG bodies in Hox genes repression has also been highlighted. 4C-seq experiments, together with DNA-FISH showed that Abd-B from the BX-C and Antp from the ANT-C can interact together within the same PcG body in a subset of cells of the embryonic head where both genes are repressed. This long-range contact is not observed in caudal tissues where only Abd-B is expressed. Upon its discovery, this phenomenon was named "Hox kissing" (Fig. 2.6). These very long-distance interactions are not limited to the Hox cluster though, as the Polycomb repressed NK-C (NK homeobox Complex) gene, located in-between the two Hox clusters, also interacts with ANT-C and BX-C in a subset of cells. This tripartite interactions may not occur in the same nucleus though and thus resembles the more probabilistic pattern of long-range interactions described in mammalian cells. Moreover, in mutant embryos for Polycomb or Polycomb-like (Pcl), colocalization of Antp and Abd-B loci is reduced (Bantignies et al., 2011). Together, these studies show the important role of PcG proteins and PREs in the maintenance of the repressed status of Hox genes and in the 3D organization of the BX-C. It is therefore important to study how this 3D organization correlates with the histone modification landscape (H3K27me3 and H3K4me3). Mateo and colleagues used ORCA (Optical Reconstruction of Chromatin Architecture, based on high-resolution microscopy combined with oligopaint and RNA-FISH) to analyze 3D organization of the BX-C in single cells or cryo-dissected embryos

  Hox gene clusters provide an excellent model to dissect such cause-consequence relationships for a number of reasons. As summarized in Chapter 3, a large number of studies have confirmed the essential contribution of cis-regulatory elements, epigenetic regulation and 3D genome organization to the collinear transcriptional regulation of these genes. Moreover, Hox gene clusters are extremely well characterized at the genetic level, with particularly for Drosophila a wealth of biological models available (cell lines, mutants).In the last ten years, the collinear activation of the mouse Hox gene clusters has been dissected in detail, including by high-resolution genomics-based approaches (see Chapter 3). The collinear activation of Drosophila Hox gene clusters share a number of regulatory mechanisms with the mouse clusters, but also has a number of noticeably differences (presented in Chapter 3). A similar in-depth characterization of the Drosophila Hox gene loci will thus improve our understanding how different types of collinear transcriptional programs are organized.In the years prior to the start of my PhD thesis, the dissection of the Drosophila Hox gene clusters had received considerably less attention, particularly incorporating high-resolution genomics-based approaches. We therefore decided to systematically investigate the interplay of histone modifications, insulator proteins and 3D genome organization in transcriptional regulation of Hox genes through the generation and intersection of different types of genomics data. In parallel, we aimed to set up a number of experimental models to test the direct implication of BEs and insulator proteins in these processes. In Chapter 4, I summarize the practical aspects of my work.In Chapter 5, I discuss the main results of my PhD project. I first present an in-depth characterization of gene expression, the histone modification landscape and 3D genome organization of the Antennapedia and Bithorax clusters, both in imaginal discs from Drosophila larvae and in various cell lines that differ in their Hox gene expression status.

Finally, in

 in Chapter 7 I draw more generalized conclusion about the results of my thesis work and I discuss future perspectives how this work can be continued. Particularly, I discuss how my current work has provided a better understanding of the links between BEs, epigenetic regulation and 3D genome organization at the Bithorax cluster. Moreover, I discuss future directions to further characterize the link between BEs and alternative promoter choice, both at the Hox gene clusters and elsewhere in the genome. Finally, I briefly reflect on potential underlying causes why the establishment of all three experimental systems in our chosen cell line remained out of reach. grown according to the manufacturer's instructions in Schneider medium (ThermoFisher Scientific) whereas Sg4, S3 and Kc167 cells were grown in M3+BPYE medium (Sigma-Aldrich). Culture mediums were supplemented with 10 % (S2) or 12,5% (Sg4, S3 and Kc167) Fetal Bovine Serum (FBS) and 1 % penicillin/streptomycin (ThermoFisher Scientific).

  with minor modifications. Briefly, 50.10 6 S2, Sg4, S3 or Kc167 cells were cross-linked for 10 min at Room Temperature in 2 % formaldehyde. After cross-linking, the reaction was quenched by adding glycine to 125 mM. Fixed cells were harvested by centrifugation and lysed in a buffer containing 50 mM Tris-HCl pH7.5, 150 mM NaCl, 5 mM EDTA, 0.5 % NP-40, 1 % Triton and 1X Complete Protease Inhibitors (Roche). Nuclei were then resuspended in 500 µL 1.2X DpnII

  ChIP-qPCR on cell lines a. ChIP protocol 50.10 6 S2, Sg4 cells or S3 were cross-linked for 10 min at Room Temperature in 1 % formaldehyde. After quenching the cross-link reaction by adding glycine to 135 mM, cells were harvested by centrifugation and lysed in LBI buffer (10% Glycerol, 50 mM Tris-Hcl, 140 mM NaCl, 1 mM EDTA, 0.5 % NP-40, 0.25 % Triton X-100 and 0.33X Complete Protease Inhibitors (Roche)) for 10 min on ice. The cell pellet was then resuspended in LB2 buffer (10 mM Tris-HCl, 0.2 M NaCl, 1 mM EDTA and 0.33X Complete Protease Inhibitors) and incubated for 10 min on ice. The cell pellet was then resuspended in LB3 buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 0.25% SDS, 0.1% NaDeoxycholate and 1X Complete Protease Inhibitors) during 10 min on ice. Samples were then sonicated using a Covaris with the following parameters: Peak Power: 110W, Duty Factor: 15%, Cycler/burst: 200, Duration: 1200 sec. The solubilized chromatin was then collected by centrifugation and its concentration measured using Qubit dsDNA HS. Chromatin was then aliquoted and stored at -80°C. An aliquot was used to confirm the sonication efficiency by agarose gel electrophoresis.3 µg of sonicated chromatin was diluted in 10X ChIP dilution buffer (16 mM Tris-HCl, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100 and 1x Comple Protease Inhibitors (Roche)) and precleared for 30 min at 4°C with Salmon Sperm DNA/Protein A Agarose beads (Merck-Millipore) then centrifuged. An aliquot (10%) of the supernatant was taken as Input and the remainder was incubated with the specific antibody overnight at 4°C. The following antibodies were used: 2 µL H3K4me3 (07-473, Merck-Millipore), 5 µg H3K27me3 (17-622

  normalized with Iterative Correction and Eigenvector decomposition methode (ICE) from HiC-Pro Tadtool was used to call TAD boundaries with a window size of 51kb and cutoff of 47 (S2 cells) or 75 (Sg4 cells). 8. Single cell RNA-seq (scRNA-seq) a. Single cell RNA-seq on cell lines 1.10 6 S2 and Sg4 cells were diluted in 1 mL (final concentration 1000 cells/µL) of cold PBS (without MgCl2) + 0.04% BSA. Reverse transcription and preparation of the sequencing library were performed according to the manufacturer's instructions (10X Genomics) using the Chromium Next GEM Single Cell 3' kit and supplied reagents. The resulting libraries were sequenced on the Illumina Next-seq 550 using 75 bp Paired end reads (High Throughput Sequencing Platform -I2BC).

  sequences for 4C-seq experiments Adaptor, index and locus specific sequences are respectively shown in blue, red and black.

  At a more local scale, 3C experiments in the Drosophila S2 and S3 cell lines showed that when the Abd-B gene is active, restriction fragments covering the gene and its regulating iabs had reduced contacts in the H3K27me3-marked domain containing the repressed Ubx and abd-A of the BX-C[START_REF] Lanzuolo | Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex[END_REF].How the functions of BEs, histone modification landscapes and 3D contact domains are integrated to regulate Drosophila Hox gene expression remains to be determined. In this study, we combined ChIP-seq, 4C-seq, Hi-C and single cell RNA-seq to investigate chromatin status of Abd-B within the BX-C, both in Drosophila imaginal discs and cell lines. As expected, we detected a close coincidence between 3D chromatin organization and histone modification landscape. Our detailed genomics-centered studies revealed that different BEs can act as boundaries between the active and inactive histone modification landscape and 3D domains. Moreover, our bulk and single-cell transcription analyses reveal that the use of different BEs is associated with a different preference for distal Abd-B alternative promoter choice. Interestingly, this choice of promoters coincides with highly localized inversion of collinearity. We thus identified a new function for BEs in Drosophila, where they instruct alternative promoter choice through regulation of histone modification landscapes. imaginal discs and cell lines display comparable Hox gene expression patterns To establish a platform to study how BEs, histone modifications and 3D contact domains dynamically interact relative to gene expression at the Drosophila BX-C, we investigated Hox gene expression in two micro-dissected larval imaginal discs (Genital and Wing discs) and four cultured cell lines (Sg4, S3, S2 and Kc167 cell lines). Using RT-qPCR, we determined mRNA levels for six different homeotic genes, including Abd-B (Fig. 5.1A). We detected large amounts of Abd-B and lower levels of abd-A, Ubx, Antp and Dfd in the Genital discs, whereas only trace amounts of Antp and Ubx were found in the Wing discs (Fig. 5.1A). Similar to the Genital discs, Abd-B was by far the most detected in the S3 and Sg4 cell lines. Abd-B accumulation, relative to the Actin and GAPDH house-keeping genes, was more modest though in the cell lines compared to genital discs. The S2 and Kc167 cell lines resembled the Wing discs, showing no substantial activity of any of the homeotic genes (Fig. 5.1A). Our analysis thus confirms that cell lines can have similar gene expression patterns at the BX-C, with Genital discs sharing high mRNA levels for Abd-B alone with the Sg4 and S3 cell lines, and Wing discs sharing no substantial Hox gene expression with the S2 and Kc167 cell lines.b. The histone modification landscapes in cell lines mirror their global Hox gene expression statusTo determine how active and repressive histone modifications at Hox clusters correlate with expression patterns, we performed ChIP-seq experiments for H3K4me3 and H3K27me3 in S2 and Sg4 cell lines. In both cell lines, the repressed ANT-C was similarly covered by H3K27me3 and depleted in H3K4me3 (Fig.5.S1). In contrast, the patterns at the BX-C were different between cell lines. In S2 cells, the repressed BX-C was covered by H3K27me3, with no major peaks of H3K4me3 detected (Fig.5.1B). In Sg4 cells, the inactive Ubx and abd-A genes remained H3K27me3-marked, but here the active Abd-B gene and its surroundings (up to the Fab-7 BE element) were enriched in H3K4me3 (Fig.5.1B). Interestingly, the boundaries between the H3K4me3 and H3K27me3 domains in the Sg4 cells were the BEs Fab-7 at the 5' (purple bar, left) and C at the 3' (grey bar, right) of the Abd-B locus. The Abd-B regulatory domain in Sg4 cells thus comprises the infra-abdominal segments iab-7 to iab-9, which includes all the Abd-B alternative promoters, except the g promoter. In contrast, the upstream iab-5 and iab-6, which regulate Abd-B in the more anterior parasegments 10 and 11 of the embryo and larvae, are repressed by H3K27me3 in Sg4 cells.To determine if these patterns of histone modifications were similar between Sg4 and S3 cell lines expressing Abd-B, we performed ChIP-qPCR at specific sites in the Abd-B regulatory domain, analyzing inactive S2 cells as a control (Fig.5.1C, left and right panels). Interestingly, the pattern of H3K27me3 enrichment was different between these Abd-B expressing cells.Confirming our ChIP-seq data, in the Sg4 cells all interrogated sites between Fab-7 and the C element were devoid of H3K27me3. In contrast, in S3 cells we detected a strong enrichment for this repressive mark in iab-7 (i.e. between the Fab-7 and Fab-8 BEs) and on the 3, B and C Abd-B promoters (albeit lower as compared to the fully repressed S2 cell line) (Fig.5.1C, left panel). Analysis of the active H3K4me3 mark provided a further nuance though. In Sg4 cells, this mark is enriched on all promoters except on the g. In S3 cells, the enrichment on the 3, B and C promoters is considerably lower as compared to Sg4, whereas it is increased at the 2 promoter (Fig.5.1C, right panel). In conclusion, we find that in Sg4 cells, the H3K27me3 and H3K4me3 marks are mutually exclusive (Fig.5.1B, 5.1C). In contrast, in S3 cells we detect a relatively moderate enrichment for both the H3K27me3 and H3K4me3 marks at the 3, B and C promoters (Fig.5.1C

A:

  Hox gene expression in S3, Sg4, S2 and Kc167 cell lines as well as genital and wing discs. Gene expression is normalized over two housekeeping genes (Actin and GAPDH) and to whole larvae. B: ChIP-seq in S2 and Sg4 cells at the BX-C (in between dash lines). Known Boundary elements are shown by vertical bars(Fub, B and C in grey, in purple and Fab-8 in pink). Regulatory Infra-abdominal segments are represented only for Abd-B (iab-5 to iab-9) C: ChIP-qPCR for H3K27me (left) and H3K4me3 (right) in S2, S3 and Sg4 cells. Data are normalized over Input. Error bars indicate Standard Deviation. D: Calibrated RT-qPCR to quantify Abd-B promoter activity in S3 and Sg4 cells. Data are normalized to shared exons between all isoforms and to an external plasmid containing all isoform specific amplification product. Error bars indicate Standard Deviation. E: t-sne maps of 3' single cell RNA-seq data in S2 (pink) and Sg4 cells (blue) (left). t-sne projection to visualize Abd-B and abd-A detection are also shown (middle and right).

  Sg4 and S3 cell lines thus combine different combinations of alternative transcripts to achieve similar overall Abd-B output. Importantly, these patterns of alternative promoter choice mirror the different levels of the H3K4me3 mark at the different Abd-B promoters (Fig. 5.1C, 5.1D). The reduced H3K4me3 levels at the 3 and B promoters in S3 cells, and associated reduced mRNA levels, further coincide with the moderate enrichment for the repressive H3K27me3 mark, confirming what we observed for the iabs epigenetic landscape.

  Fig. 5.S3, Fig. 5.S4). The repressive Hox clusters in larval Wing disc cells and the in-vitro cultured S2 and Kc167 cell lines thus adopt a similar repressive contact domain organization.

  focused on the ANT-C, where all Hox genes remain in a repressed state(Fig 5.S5). 3D organization, as assessed both by Hi-C and 4C-seq, remained highly comparable to the patterns observed in S2 cells. We thus conclude that the inactive ANT-C in Sg4 cells remained organized as a H3K27me3-marked contact domain, with a further sub-division into three sub-domains (Fig.5.S5).In contrast, the BX-C in Sg4 cells was organized markedly different though. Our Hi-C data revealed that each Hox gene in the BX-C is located in its own sub-domain (Fig. 5.3A). Whereas the boundary between Ubx and abd-A remained at the Fub BE, a new boundary had appeared between abd-A and Abd-B as well. 4C-seq experiments in Sg4 cells confirmed this observation, particularly when comparing the interactions of the Abd-B promoter between Sg4 and S2 cells (Fig. 5.3B). Indeed, the ratio of gained/lost 4C-seq interactions exactly overlapped this newly identified boundary, confirming the reorganized 3D interactions of Abd-B in the Sg4 cell line. This new boundary coincided with the Fab-7 BE (purple line), which we previously identified as the boundary between the H3K4me3 and H3K27me3-marked domains (Fig. 5.1B, Fig. 5.3A, 5.3B). The identified Abd-B regulatory domain in Sg4 cells, comprising iab-7 to iab-9, thus formed a single active contact domain. Based on the large reduction of interactions with the remainder of the BX-C (Fig. 5.3A, 5.3B), we further concluded that this active Abd-B contact domain dissociates from the upstream repressed contact domain. Such interpretation is

Fig. 5 .

 5 3B, Fig. 5.S6). Direct comparison of Abd-B interactions between the Genital discs and the Sg4 and S3 cell lines revealed a noticeably difference as well (Fig. 5.3C). The strongest drop in 4C-seq interaction signal in the Sg4 cell line was detected at the Fab-7 BE (purple line and arrow), which confirmed its role of boundary between the active and inactive contact domains. In contrast, in the S3 cell line the drop in signal coincided with the Fab-8 BE (pink line and arrow) (Fig. 5.3C). Comparison of the ratio track further confirmed this observation, with a strong enrichment of interactions in the Sg4 cells observed within the iab-7 (Fig. 5.3C). Conversely, the repressed Ubx and abd-A viewpoints increased interactions with iab-7 in S3 cells (Fig. 5.S7). In parallel, contacts in Sg4 cells were enriched over the B and C promoters, both located in iab-9 B: 4C-seq in S2 and Sg4 cells for Abd-B viewpoint. Differences in the log ratio of interactions are highlighted by the orange rectangles showing increase of interactions in S2 cells and by arrows showing decrease of interactions in Sg4 cells. C: 4C-seq in Genital discs, Sg4 and S3 cells for Abd-B viewpoint. The steep drop of interactions between the active Abd-B gene and the repressed BX-C is highlighted by arrows. Purple arrows show drops of interactions that coincide with Fab-7 whereas pink arrows show with Fab-8. In figures A, B and C, BX-C is delimited by dashed lines, viewpoints are indicated by arrows and boundary elements by vertical bars. Iabs controlling Abd-B expression are depicted in between boundary elements. In between each 4C-seq tracks, log ratio of interactions are shown. Increased frequencies in one cell type over the other is depicted by the corresponding color. D: ChIP-qPCR for GAF and CP190 insulator proteins in S2, S3 and Sg4 cells. Data are normalized over the Input. Error bars indicate Standard Deviation. (Fig. 5.3C). The Abd-B contact domains as detected in these two cell lines thus differed in their extent, with either the Fab-7 or Fab-8 BE acting as the upstream boundary and iab-9 contacts differing as well. Importantly, these differences in contacts matched the different extent of the domain of active histone modifications and alternative promoter use (Fig. 5.1B-D, Fig. 5.3C). These data thus further reinforced the direct link between contact domain formation, the extent of the histone modification domains and alternative promoter use, similar to what we reported here based on our Hi-C and ChIP-seq data (Fig. 5.1A-D, Fig 5.3A, 5.3B) and what was

l.

  Absence of the Fab-7 BE reduces activity of all Abd-B promoters in Genital disc cellsOur 4C-seq and calibrated RT-qPCR in cell lines showed that the use of different BEs as boundary guided the incorporation of iab-7 in the active (Sg4 cells) or repressed (S3 cells) contact domain, which subsequently affected the choice of alternative Abd-B promoters (Fig.5.1C, 5.1 D, Fig.5.3C). We thus wondered if in the absence of Fab-7, we could detect a reorganized choice of Abd-B alternative promoters in the heterogeneous Genital disc cell population as well. To address this question, we assessed Abd-B promoter choice by calibrated RT-qPCR in WT and Fab-7 1 genital disc cells (Fig.5.5A). We noticed a general reduction of mRNA level for transcripts originating from all Abd-B promoters in the mutant cells, confirming our previous observation that global Abd-B levels are reduced (Fig.5.4A). Although some promoter specificity in the reduction of expression was be observed upon the removal of the Fab-7 BE, no major effect could be identified from the comparison of these heterogeneous cell populations (Fig.5.5A).

A

  : Calibrated RT-qPCR to quantify Abd-B promoter activity in WT and Fab-7 deleted genital disc cells. Data are normalized to shared exons between all isoforms and to an external plasmid containing all isoform specific amplification products. Error bars indicate Standard Deviation. B: Strategy to perform single-cell RNA-seq on imaginal discs. Discs were dissected to obtain approximately the same number of cells per discs. C: UMAP plots to visualize clustering of imaginal disc cells in single cell RNA-seq data. D: UMAP plots showing the presence of Abd-B mRNA in single cell RNA-seq data. E: Histogram showing the number of cells for each isoform detected in WT and Fab-7 Genital disc cells. F: Histogram showing the relative ratio (Fab-7 deleted / WT) of mRNA originating from A, 2 and B promoters. G: Histogram showing the fraction of cells in WT and Fab-7 deleted were mRNA from two promoters are detected. concordance with expression levels as determined by promoter-specific RT-qPCR (Fig. 5.5A). Next, we focused on the presence of RNA originating from the most abundant promoters (A, 2, B) in cells where at least one Abd-B isoform was detected (Fig. 5.5F, Fig. 5.S10A). Whereas the relative abundance of mRNA from the A promoter remained largely unchanged, we noticed an opposite dynamic for the 2 and B promoters: mRNA originating from 2 promoter became relatively more abundant in mutant cells, whereas mRNA from the B promoter become more abundant in WT cells. Moreover, when comparing cells where multiple isoforms were detected, we noticed that the combination of mRNA from the A and 2 promoters was relatively enriched in mutant cells and the combination of A and B was enriched in WT cells (Fig 5.5G, S10B).

Figure 5 .

 5 Figure 5.S1: Epigenetic landscape of the ANT-C in S2 and Sg4 cells

  Fab-7 element could result in a less discrete Abd-B contact domain where more interactions are formed with the H3K27me3 domain covering abd-A. As a result, this repressive influence could be reinforced, with the H3K27me3 mark invading the Abd-B regulatory landscape and causing down-regulation of Abd-B.

  Fig 6.1.B and Fig 6.1.C

  6.2.C, left ). Subsequent co-transfection of our Sg4 cells with the deletion plasmids and an eGFP plasmid, followed by sorting based on GFP, resulted in both a very low transfection efficiency and C: qPCR with primers as shown on the cartoon to assess Fab-7 deletion efficiency. qPCR is normalized over the WT and the Mcp Boundary element region that is not affected by Fab-7 deletion survival rate Fig.6.2.C right . Despite efforts to use FACS sorting to recover transfected cells, I never was able to successfully expand cells that carried the deletion.

Fig. 6

 6 Fig. 6.3.A and Fig. 6.3.B Treating the cells for 7 days with 20 µg of GAF RNAi had the highest KD efficiency compared to shorter treatments with less RNAi, although this never exceed 70% of decrease. Increasing the quantity of RNAi (up to 40 µg) and/or extending treatment up to 10

  S2 cells, where no Hox genes are expressed and the BX-C is covered by H3K27me3, we wondered if abd-A expression could be induced by the Sun-Tag despite the fully repressed and compact BX-C environment. If yes, would the abd-A gene loop out of its repressive contact domain and would this domain be restricted by BEs? Would such activation influence the activity of the surrounding genes? In Sg4 cells, where the neighboring Abd-B gene is active, we envisioned two possible outcomes: either abd-A and Abd-B would independently loop out of the repressive contact domain or abd-A would join Abd-B in its active compartment. Whereas the Abd-B and abd-A genes are usually not active at the same time, small subset of cells in the Central nervous system

Fig. 5 .

 5 Fig. 5.S4). Based on the direct overlap with the H3K27me3 mark in S2 cells, and the wellestablished function of this mark in the repression of Hox genes (Gentile and Kmita, 2020). I consider it highly likely that this contact domain contains the H3K27me3-marked domain in all three cell types. 4C-seq on the active Alh gene, located just outside the ANT-C, showed a sharp boundary at the start of ANT-C, with subsequent few interactions between Alh and the ANT-C, and many more with active neighbor genes (Fig. 5.S2), further confirming the discrete nature of the ANT-C contact domain.

  5.1), thus expanding our collection of cell types to investigate its inactive configuration. Hi-C in Sg4 cells for the ANT-C revealed a mostly similar organization, although we also noticed some minor differences (Fig. 5.S2 versus Fig. 5.S5). In Sg4 cells, three sub-TADs at ANT-C are detected as well, but they do not strictly coincide with those detected in S2 cells. The upstream sub-TAD spans a larger distance, here containing the Lab, pb, bcd and Dfd genes and a part of the Scr gene body. The middle sub-TAD contains the Scr promoter and the upstream promoter of Antp and the downstream sub-TAD contains the downstream promoter of Antp and the rest of the ANT-C. This result suggests that the repressed ANT-C uses different internal boundaries,

  5.2). When silenced, the BX-C is divided in two sub-TADs separated by the Fub boundary: one containing the Ubx gene and the other containing the abd-A and Abd-B genes.Although the BX-C and ANT-C are approximately the same size, there are some structural differences between the two clusters. These differences are most likely due to the different gene content of the clusters: they contain a different number of Hox genes, the ANT-C cluster contains additional non-homeotic genes and Hox genes in the ANT-C can be found in both orientations. Hence, I speculate that the Hox genes in the different clusters may be differently subjected to the different gene regulation mechanisms (cis-regulatory elements, BEs, histone modifications), which may require a different structural organization to insure proper gene activation and repression.Our 4C-seq studies in WT and Fab-7 1 Wing discs showed that the absence of the Fab-7 BE did not dramatically affect the 3D genome organization of the BX-C, nor did it interfere with the repression of the Hox genes (Fig.5.4A, Fig.5.S8). I thus argue that within the repressed contact domain, this internal BE is not essential to maintain the repressed state or the overall chromatin folding. However, our Hi-C data identified two sub-TADs boundaries within the repressed ANT-C, and only one boundary within the repressed BX-C (coinciding with the Fub BE). The absence of a visible boundary at the Fab-7 in a repressed context thus appears not a general feature of all BEs at the Drosophila Hox clusters. It would thus be interesting to delete one or several of these visible boundaries, followed by the determination how this influences chromatin organization and gene expression in a repressed context. Here, it would be interesting to use Drosophila mutant missing the Fub boundary element[START_REF] Bender | The Border Between the Ultrabithorax and abdominal-A Regulatory Domains in the Drosophila Bithorax Complex[END_REF], which was shown that the first abdominal segment adopts the identity of the second abdominal segment. This is due to activation of iab-2 in PS6 leading to ectopic expression of abd-A.

  have proposed an "open for business" model for the BX-C, which states that the BX-C chromatin should open from the anterior (Ubx) to the posterior (Abd-B). Along the anterior to the posterior body axis, genes and cis-regulatory regions should lose H3K27me3 progressively and gain positive histone marks. Hence, in PS4all BX-C Hox genes should be covered by repressive histone modifications and transcriptionally inactive, while in PS13/14, the entire BX-C should be in an active state and harbor positive histone modifications[START_REF] Maeda | The open for business model of the bithorax complex in Drosophila[END_REF].According to the "open for business" model, the chromatin in the BX-C should open linearly from its anterior (Ubx) to the posterior (Abd-B) side. Practically, it should thus progressively lose the H3K27me3 mark and gain positive histone marks. Indeed, this was observed when nuclei were sorted from the embryonic parasegments (PS) 4 to 7[START_REF] Bowman | H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex[END_REF]. In PS4 all BX-C Hox genes are repressed, followed by activity of Ubx in PS5 and PS6, up to PS7 where both Ubx and abd-A are expressed. Matching the "open for business" model, a progressive loss of H3K27me3 and an enrichment of H3K27ac was observed from PS4 to PS7. Unfortunately, the study did not include the more posterior parasegments where Abd-B starts to be activated.How to reconcile what happens in more anterior embryonic PSs (where the Ubx and abd-A genes are expressed) and what we detected in cells lines where Abd-B is active? I speculate that two possibilities exist:

  Différents niveaux de régulation de gènesL'activité transcriptionnelle est amplifiée grâce à des éléments appelés enhancer qui entrent en contact avec les promoteurs des gènes. Les enhancers et les promoteurs ciblent des gènes peuvant se situer à de grandes distances. Les éléments frontières (EFs) peuvent aider à les rapprocher spatialement dans le noyau en s'associant entre eux, créant ainsi une boucle chromatinienne.Toutefois, des éléments répresseurs, comme les EFs, peuvent empêcher ce contact activateur et ainsi induire la répression des gènes. Ainsi, selon le contexte cellulaire, les EFs peuvent faciliter l'activation des gènes ou au contraire les inhiber.Les EFs sont liés par des protéines insulatrices qui se retrouvent en nombre chez la Drosophile contrairement aux mammifères. Il existe trois classes de protéines insulatrices : (1) celles qui se lient directement à l'ADN comme GAF, dCTCF ou BEAF-32, (2) celles qui ne se lient pas directement à l'ADN comme CP190 et (3) les modulateurs tissus spécifiques comme Elba1. Le rôle des EFs pourrait donc être en partie conditionner par la combinaison de protéines insulatrices qui s'y lient. Au sein de la même cellule coexistent des gènes exprimés et des gènes réprimés. Afin de maintenir l'état transcriptionnel de ces gènes au cours des divisions cellulaires, les marques d'histones sont impliquées. Parmi toutes les marques d'histones, nous nous sommes intéressés à deux : La tri-méthylation de la lysine 4 sur l'histone H3 (H3K4me3) et la tri-méthylation de la lysine 27 sur l'histone H3 (H3K27me3). H3K4me3 est une marque d'histone positive et se retrouve sous forme de pics aux promoteurs de gènes exprimés. A contrario, H3K27me3 est une marque répressive qui recouvre les promoteurs ainsi que le corps des gènes réprimés. Les marques d'histones forment ainsi différents domaines chromatiniens de gènes actifs ou réprimés. Afin de séparés ces compartiments, les EFs sont impliqués pour éviter l'envahissement d'un état chromatinien d'un domaine à l'autre. L'activité des gènes est régulée par plusieurs facteurs dont les marques d'histones, la liaison aux éléments frontières de protéines insulatrices et l'organisation tridimensionnelle de la chromatine. Les gènes homéotiques de Drosophila melanogaster comme modèle d'étude Afin d'étudier les liens qui existent entre ces différents éléments, nous avons décidé d'utiliser les gènes Hox de Drosophila melanogaster. Tous les organismes bilatériens possèdent une symétrie droite/gauche et donc des axis Dorso/Ventral (D/V) et Antero/Postérieur (A/P). Les gènes homéotiques sont essentiels pour mettre en place l'axe A/P. Ils codent pour des facteurs de transcription qui donnent l'identité cellulaire le long de l'axe A/P. Chez la plupart des organismes bilatériens, les gènes Hox sont organisés en cluster où leurs positions génomiques le long du (des) chromosome(s) reflètent leurs domaines d'expression le long de l'axe A/P. Chez Drosophila melanogaster, les gènes Hox sont organisés en deux clusters situés sur le Chr 3R et espacés d'environ 10Mb : le complexe Antennapedia (ANT-C) et le complexe Bithorax (BX-C). Le complexe ANT-C est composé des gènes labial, proboscipedia, Deformed, Sex comb reduced et Antennapedia ainsi que d'autres gènes non homéotiques et d'ARN non codants alors que le complexe BX-C est formé des gènes Ultrabithorax (Ubx), abdominal A (abd-A) et Abdominal B (Abd-B), d'un autre gène codant pour un transporteur de sucres et de nombreux ARN non codants. Les deux clusters, malgré leurs différences de composition sont de tailles similaires (environ 350kb). Les gènes Hox sont activés durant l'embryogénèse précoce par des gènes de segmentation et déterminent l'identité des différents parasegments de l'embryon de Drosophile. Les gènes Hox sont majoritairement exprimés dans les segments non chevauchants. Cependant, quelques exceptions peuvent être notées : Ubx et abd-A peuvent être co-exprimés dans l'abdomen ; abd-A et Abd-B peuvent être actifs dans les mêmes cellules dans certaines parties du système nerveux central et du disque génital de Drosophile. Le BX-C a été extensivement étudié et des études de génétiques ont permis de mettre en avant son organisation. En plus des trois gènes homéotiques Ubx, abd-A et Abd-B, le BX-C est divisé 9 régions cis-régulatrices segments-spécifiques : abx/bx, bxd/pbx, et iab-2 to iab-8. Ces régions contiennent des enhancers qui permettent d'initier l'expression des gènes homéotiques pendant le développement. Par exemple, les régions cis-régulatrices iab-5 à iab-9 régulent l'expression d'Abd-B dans les parasegments 10 à 14. Une fois les gènes activés, leurs expressions ou répressions sont maintenues par les groupes de protéines Trithorax et Polycomb via la déposition de marques d'histones positives aux TRE (Trithorax Responsive Element) ou négatives aux PRE (Polycomb Responsive Element) respectivement. Les différentes régions cis-régulatrices sont délimitées par des EFs auxquels des protéines insulatrices et les protéines Polycomb peuvent se fixer. Des expériences de tries de noyaux provenant de parasegments spécifiques d'embryons ont montré que les gènes Ubx et abd-A sont activés de manière colinéaire et accompagné de changements du paysage de modification d'histones délimités par des EFs. Dans le PS4 où les gènes du BX-C sont réprimés, tout le BX-C est couvert par H3K27me3. Dans les PS5 et PS6 où seul Ubx est exprimé, ce gène et ses régions régulatrices deviennent déplétés en marques répressives H3K27me3. Dans le PS7 où Ubx et abd-A sont actifs, H3K27me3 est absent et remplacé par H3K4me3. Ainsi, Ubx et abd-A suivent une activation colinéaire délimitée par les EFs dans l'embryon de Drosophile. Ces deux gènes suivent l'hypothèse du 'Open for business' proposé dans les années 2000 qui stipule que les gènes du BX-C sont activés de manière colinéaire le long de l'axe antéro-postérieur. Ce modèle va même plus loin et propose que la chromatine devienne active selon l'axe A/P, en étant contrôlée par les EFs qui agissent comme des barrières dans les différents PS. Qu'en estil du gènes Abd-B, le gène le plus postérieur du BX-C ? Abd-B est un gène complexe qui possède 6 promoteurs alternatifs (A, 2, 3, B, C et g) et produisent au moins 2 protéines connues : -Le transcrit A code pour la protéine ABD-B M et est produit par le promoteur A -Les transcrits B, C et g codent pour la protéine ABD-B R et est produit par les promoteurs B, C et g respectivement. Jusqu'à aujourd'hui, les promoteurs 2 et 3 n'ont pas été caractérisés et l'analyse des CDS sousentend qu'ils pourraient produire la protéine ABD-R. Pour le moment, seulement deux protéines ABD-B ont été détecté, mais il n'est pas à exclure que les protéines issues des transcrits et promoteurs, 3, B, C et g soient légèrement différentes dans leurs domaines fonctionnels et puissent ainsi avoir des rôles distincts. Une question qui reste en suspens est comment l'ouverture colinéaire de la chromatine influence le choix de promoteurs alternatifs des gènes Hox et surtout d'Abd-B. Comment le chromatine. Nous avons donc décidé de conduire cette analyse complète dans des disques imaginaux de Drosophile et dans des lignées cellulaires. Résultats Le statut transcriptionnel des gènes homéotiques corrèle avec les modifications d'histones Nous avons analysé l'expression des gènes homéotiques dans différentes modèles : les disques imaginaux d'ailes et génitaux de Drosophile ainsi que dans les lignées cellulaires S2, S3, Sg4 et Kc167. Nous avons déterminé que les disques imaginaux d'ailes ainsi que les cellules S2 et Kc167 n'exprimaient pas de gènes homéotiques de manière conséquente alors que les disques imaginaux génitaux et les cellules S3 et Sg4 expriment Abd-B. Nous avons ensuite réalisé des expériences de ChIP-seq dans les cellules S2, S3 et Sg4. Dans les trois cas, le complexe réprimé ANT-C est couvert par la marque d'histone répressive H3K27me3. Dans les cellules S2, le complexe BX-C est entièrement enrichi en H3K27me3 alors que dans les cellules S3 et Sg4 le gènes Abd-B et certaines séquences cis-régulatrices sont positives pour H3K4me3. Cependant, les profils de modifications d'histones sont différents entre les cellules S3 et Sg4. Dans les cellules S3, le domaine H3K4me3 est compris entre l'élément frontière Fab-8 et le promoteur 3 d'Abd-B alors que dans les cellules Sg4 il est encadré par Fab-7 et l'élément C. Comme le domaine H3K4me3 englobe différents promoteurs d'Abd-B, nous avons réalisé des expériences de quantification absolue de l'expression des promoteurs d'Abd-B. Nous avons ainsi déterminé que dans les cellules S3, les promoteurs A et 2 sont fortement exprimés alors que les promoteurs 3, B, C et g sont moins ou pas exprimés. En revanche, dans les cellules Sg4, seul le promoteur g est réprimé. Ainsi, bien qu'exprimant Abd-B au même niveau, les cellules S3 et Sg4 diffèrent par leurs paysages épigénétiques et leurs utilisations de promoteurs d'Abd-B. Le complexe BX-C réprimé forme un domaine inactif Nous avons ensuite étudié l'organisation 3D du complexe BX-C dans les disques imaginaux d'ailes et dans les cellules S2 et Kc167. Dans ces trois types cellulaires réprimés, nous avons réalisé des expériences de 4C-seq et de Hi-C dans les cellules S2. Le Hi-C dans les cellules S2 a permis de mettre en évidence que le complexe BX-C forme un TAD réprimé qui est subdivisé en deux sub-TADs qui peuvent interagir ensemble : l'un contenant Ubx et ses séquences cis-régulatrices et l'autre abd-A et Abd-B. Il est intéressant de noter que la frontière entre ces deux TADs coïncide avec l'élément frontière Fub qui régule l'expression d'Ubx. Les expériences de 4C-seq ont permis de montrer que les promoteurs des gènes Ubx, abd-A et Abd-B pouvaient interagir avec l'ensemble du complexe BX-C réprimé et que ces interactions sont principalement localisées au sein du cluster. Le gène Abd-B actif se dissocie du complexe BX-C Des études précédentes ont déjà montré que le gène Abd-B et ses séquences cis-régulatrices perdent leurs interactions avec le reste du complexe quand il est exprimé. Afin d'approfondir nos connaissances sur ce mécanisme, nous avons fait une analyse de l'organisation 3D de la chromatine par Hi-C et 4C-seq dans les cellules S3, Sg4 et les disques génitaux. Les analyses de Hi-C ont révélé que l'organisation du BX-C change considérablement dans les cellules Sg4 où Abd-B est exprimé. Ubx utilise toujours l'élément frontière Fub pour le séparer d'abd-A. En revanche, un nouveau sub-TAD est créé qui contient le gène Abd-B et utilise l'élément Fab-7 comme frontière avec abd-A. Ainsi, dans les cellules Sg4, le complexe BX-C est organisé en trois sub-TAD contenant chacun un gène homéotique et les séquences cisrégulatrices associées. Une fois actif, le gène Abd-B et les séquences cis-régulatrices iab-7 à iab-9 qui sont couvertes par H3K4me3 se dissocient du reste du complexe réprimé dans les cellules Sg4. Nous nous sommes ensuite demandé si ce phénomène de dissociation se reproduit dans les cellules S3 et dans les disques génitaux. Dans tous les cas, le gène Abd-B et des séquences cis-régulatrices perdent leurs interactions avec le reste du cluster mais une analyse comparative entre les cellules S3, Sg4 et les disques génitaux a montré des différences significatives entre les trois systèmes. Comme dit précédemment, la baisse d'interaction coïncide avec l'élément Fab-7 dans les cellules Sg4. Cependant, la baisse d'interaction correspond à l'élément Fab-8 dans les cellules S3. Dans les cellules Sg4, le gène Abd-B et tous ses promoteurs (hormis le g) se dissocient du reste du cluster avec les séquences régulatrices iab-7 à iab-9 alors que dans les cellules S3, seuls les promoteurs A et 2 ainsi que les séquences cis-régulatrices iab-8 et une partie de iab-9 perdent leurs interactions avec le reste du cluster. Ces résultats coïncident parfaitement avec le paysage La délétion de l'élément Fab-7 induit un changement de l'usage de promoteurs d'Abd-B en cellule unique L'impossibilité de détecter des changements d'usage de promoteurs entre WT et mutant par qPCR peut être dû à l'hétérogénéité cellulaire du disque génital qui masque les effets de la délétion. Nous avons donc réalisé des expériences de 5' single cell RNA-seq dans les disques imaginaux de Drosophile afin d'étudier l'hétérogénéité cellulaire. Nous avons analysé les données en suivant la pipeline Seurat. Cela nous a permis de retrouver tous les disques imaginaux de Drosophile grâce à leurs signatures d'expression de gènes. Nous nous sommes ensuite focalisés sur le disque génital et plus particulièrement sur les cellules où Abd-B est détecté. A l'échelle du disque génital, nous avons détecté moins de cellules où Abd-B est exprimé dans le mutant comparé au WT, ce qui est cohérent avec les analyses de RT-qPCR effectuées précédemment. Nous avons ensuite étudié les données 5' nous permettant de détecter les changements d'usage de promoteur d'Abd-B à l'échelle de la cellule unique. Nous n'avons pas détecté de changements massifs dans l'utilisation des promoteurs dans l'absolue. Nous avons plutôt mis en évidence que les associations de promoteurs d'Abd-B changent entre le WT et le mutant. Dans les cellules de disque WT, toutes les isoformes peuvent être détectées, et les promoteurs co-exprimés sont le plus souvent des « longs ». En revanche, dans les cellules mutantes du disque génital, les promoteurs « courts » sont plus souvent exprimés ensemble. Notre analyse de single cell RNA-seq a démontré que l'absence de l'élément Fab-7, alors que Fab-8 est toujours actif, promeut l'expression de promoteurs dit « courts » comme le promoteur 2 au détriment des promoteurs « longs » comme le promoteur B. Ce résultat nous confirme que in vivo comme in vitro, le choix d'élément frontière comme Fab-7 influence l'expression de promoteur d'Abd-B.

  Tout d'abord, nous avons montré que la délétion de l'élément Fab-7 n'influence pas la répression et l'organisation 3D des clusters homéotiques réprimés. Dans tous les types cellulaires, le complexe ANT-C adopte la même configuration. De même, le cluster BX-C dans les cellules S2 et Kc167 ainsi que dans les disques imaginaux d'ailes sont similaires et la délétion de Fab-7 n'influence pas l'organisation et la répression des gènes du BX-C dans les disques d'ailes. Nous avons ensuite comparé l'organisation 3D du BX-C dans les cellules S3 et Sg4 et dans les disques génitaux et avons mis en évidence que bien que Abd-B soit le seul gène homéotique actif dans les trois types cellulaires, différents éléments frontières sont utilisés. Les cellules S3 utilisent Fab-8 et les Sg4 Fab-7, induisant ainsi un changement de l'expression des promoteurs d'Abd-B. Dans les disques génitaux, les éléments frontières Fab-8 et Fab-7 sont utilisés. La délétion de l'élément frontière Fab-7 induit une légère diminution globale de l'expression d'Abd-B et un gain d'interactions du promoteur 2 de ce gène avec le reste du cluster réprimé. De plus, au niveau de la cellule unique, nous avons mis en évidence que la délétion de l'élément frontière influence l'utilisation des promoteurs d'Abd-B induisant un shift d'expression vers les promoteurs les plus courts. Nous avons donc montré que les éléments frontières sont directement corrélés aux modifications d'histones et à l'organisation 3D de la chromatine. De plus, nous avons mis en évidence qu'ils sont impliqués dans le choix de promoteurs alternatifs, au moins pour le gène Abd-B.

  The promoter, the TSS and gene body are all covered by H3K9me2/3 and/or H3K27me3.

	Figure adapted from (Lim et al., 2010)
	c. Long non-coding RNA
	The epigenetic regulation of gene expression also relies on a third actor: long non-coding RNA
	(lncRNAs), which can be linked to histone modifications and DNA methylation. lncRNAs are
	long RNA molecules (>200 bp) that are not translated into proteins and that can bind to RNA,
	DNA or proteins. Similar to mRNA transcripts, most lncRNAs are transcribed by RNA
	polymerase II are capped and polyadenylated. They are abundantly detected in many species,
	with at least 167,150 lncRNAs identified in humans, 130,558 in mouse and 54,818 in
	Drosophila

In this technique, chromatin is crosslinked to fix DNA-protein interactions (optional in native ChIP). Chromatin is then sonicated (or digested with MNase) to shear chromatin into mono-or di-nucleosomes. Specific antibodies against the protein of interest (or specific PMTs) are then used for chromatin Right: repressed gene •

Table 2 :

 2 Primers used for RT-qPCR (Hox gene expression) Primers used for the Absolute quantification of Abd-B isoforms Primers are sorted from the shortest to the longest isoform. The isoform 1 is the same primers set as Abd-B from table 1 and is designed on the common exons that is present in all Abd-B

	Target gene	Primer sequence
	Abd-B Forward	GCACATCGACGTCCAGCTAT
	Abd-B Reverse	GCTCCCACGGATAATCCACC
	abd_A Forward	GCTTACAGACTGGATGGGAA
	abd_A Reverse	GGTTGAAGTGAAACTCCTTCTC
	Ubx Forward	TCGCAGGTAAGAGATACTCAG
	Ubx Reverse	GATTCGTGTGGAACTCCTTC
	Antp Forward	AGATTAGGCTACGCAACTGTAC
	Antp Reverse	GTGATCGTGACTGTGACTCTG
	Scr Forward	CGTGGATGAAGCGAGTAC
	Scr Reverse	GTAGCGGTTGAAGTGGAAC
	Dfd Forward	GCGAAGAAACCCACCAAG
	Dfd Reverse	CCGATACTCTCCAAACTGTC
	Act42A Forward	AAGTGTGTGCAGCGGATAACT
	Act42A Reverse	GTTGTCGACCACTAAAGCTGC
	Gapdh2 Forward	CCCGAGTTTTCGCCCATAGA
	Gapdh2 Reverse	CCGATGCGACCAAATCCATTG

Table 4 :

 4 Primer sequences for Test ChIP-qPCR experiments before sequencing

	Histone mark	Target region		Primer sequence
	Enriched in H3K27me3	Antp promoter Forward CAATTTCTCTGACGGTGTTTT
	(Depleted in H3K4me3)	Antp promoter Reverse GCAGATCGTCATAAACCCA
	Enriched in H3K4me3	CG16952 Forward	promoter	GACTTCTGGTTCTTGATG
	(Depleted in H3K27me3)	CG16952 Reverse	promoter	CTGCACAATCGTTAAATTC
	Table 5: Primers for ChIP-qPCR GAF and CP190
	Target	Sequence
	Mcp_ChIP_Forward	CTGTTGGAAACGCATTA
	Mcp_ChIP_Reverse	TGTGTAAGGAGGAAGAC
	Fab6_ChIP_Forward	TTGCCAGATAAATCCAAGAT
	Fab6_ChIP_Reverse	GCCGATTAAAGCAGTCC
	Fab7_ChIP-Forward	GCTCACTAACACATAGATAA
	Fab7_ChIP-Reverse	CTCTCTTGCTTACCAATAC
	Fab8_ChIP_Forward	GGTTGGTCTGCATATTG
	Fab8_ChIP_Reverse	CGGATTTCTGCTTTCTG
	B_ChIP_Forward	TGTGACTTGGAAGAGAG
	B_ChIP_Reverse	GTGTACGTGTGCATAAC
	C_ChIP-Forward	TGCGCAGAGTCTTATAC
	C_ChIP-Reverse	CTAGTGAACAGACCATCTA

Table 6 :

 6 Primers for ChIP-qPCR H3K27me3 and H3K4me3

	Target	Sequence
	AbdB(A)_ChIP_Forward	GGCTGTTGTTGTAGTTG
	AbdB(A)_ChIP_Reverse	CGTCAGTGTAGATTTGTG
	AbdB(2)_ChIP_Forward	GTGATAAGACCAAAGGAATA
	AbdB(2)_ChIP_ Reverse	CGGGCCGATATTTAATG
	AbdB(3)_ChIP_Forward	GGCTGCTTCATGTTTAC
	AbdB(3)_ChIP_ Reverse	GCGGCCAAGTTATTAAG
	AbdB(B)_ChIP_Forward	GCGGTACTAGTGCTTTA
	AbdB(B)_ChIP_ Reverse	CCGAGTATTTACCACTTTAC
	AbdB(C)_ChIP_Forward	CAATGCCAAACAAAGTATC
	AbdB(C)_ChIP_ Reverse	GAGCTTATGCGAATGTC
	AbdB(g)_ChIP_Forward	AACAACCGCACAAATAC
	AbdB(g)_ChIP_ Reverse	AACCGAGAACCCATTAG
	Table 7: PCR primers to test Fab-7 deletion efficiency
	Target	Sequence
	Fab-7 UP Forward	TGGGCCCGGTTAAGATTGAG
	Fab-7 UP Reverse	GACGAGCAACGGACGTCATA
	Fab-7 Down Forward	TGTGTGCGTTGCGCATTTTA
	Fab-7 Down Reverse	TGATTTAGCAGCGGAACGGT
	Fab-7 Full Forward	GTGGGCCCGGTTAAGATTGA
	Fab-7 Ful Reverse	CAAAAGTCCGCTTGTGACGG

Table 8 :

 8 qPCR primers to test Fab-7 deletion efficiencyMcp is another Boundary Element that should not be affected by the deletion of Fab-7. It is used as an internal control to quantify Fab-7 deletion efficiency.

	Target	Sequence
	qPCR Fab-7 UP Forward	GCAGTGGGAAGTCGTATTT
	qPCR Fab-7 UP Reverse	GTTCACCGGCTTCTGTATT
	qPCR Fab-7 Down Forward	GACCTCCACTACGGGTAAAT
	qPCR Fab-7 Down Reverse	TGCTAGTGCTGCTGAAAC
	qPCR Mcp UP Forward	CAACGTTTTGAATTCCGCTC
	qPCR Mcp UP Reverse	GGACAATCAAAGTTTAATCCGC
	qPCR Mcp Down Forward	ACATGGGCATACGGACTG
	qPCR Mcp Down Reverse	TGTAGTGTCCGACCAATTGAG

Table 9 :

 9 Primers for cloning of sgRNA sequences in pAc-sgRNA-Cas9

	Sequence gDNA	Cloning Primers
	Fab7 Up TCCTTAGTCACCAATGGCTATGG	sense anti-sense	TTCGtccttagtcaccaatggcta AACtagccattggtgactaaggaC
	Fab7 Up TCCTTAAAAAGTCACTTGAACGG	sense anti-sense	TTCGtccttaaaaagtcacttgaa AACttcaagtgactttttaaggaC
	Fab7 D ATCGAGATTGCCTGGATTCCTGG	sense anti-sense	TTCGatcgagattgcctggattcc AACggaatccaggcaatctcgatC
	Fab7 D TATCGCGTTCGATTGTTGACGGG	sense anti-sense	TTCGtatcgcgttcgattgttgac AACgtcaacaatcgaacgcgataC

PAM sequences are indicated in green. For forward primers, TTC was added to the 5' end, AAC was added for the reverse primer (blue). If the sequence did not start with a G, a G was added to allow higher transcription from the U6 promoter (red).

Table 10 :

 10 Primers to produce dsDNA template for RNAi

	Target	Sequence
	GAF + T7 Forward	GAATTAATACGACTCACTATAGGGATGGTTATGTTGGCTGGCGTCAA
	GAF Reverse	TCTTTACGCGTGG TTTGCGT
	GAF Forward	TGGTTATGTTGGCTGGCGTCAA
	GAF +T7 Reverse	GAATTAATACGACTCACTATAGGGATCTTTACGCGTGG TTTGCGT
	LacZ Forward	GAATTAATACGACTCACTATAGGGAGAGATATCCTGCTGATGAAGC
	LacZ Reverse	GAATTAATACGACTCACTATAGGGAGAGCAGGAG CTCGTTATCGC
	GAF2 + T7 Forward	TAATACGACTCACTATAGGGATTGTGCTGGTGTTATCTCGC
	GAF2 Reverse	CTACGGCACCGAAAATAAGC
	GAF2 Forward	TTGTGCTGGTGTTATCTCGC
	GAF2 + T7 Reverse	TAATACGACTCACTATAGGGACTACGGCACCGAAAATAAGC
	GAF3 + T7 Forward	TAATACGACTCACTATAGGGAAATTGGATTGCCGATACGAG
	GAF3 Reverse	CAAAGGGAGTTGGAAAAGCA

seq at ANT-C for S2 and Sg4 cells

  ChIP-seq in S2 and Sg4 cells at the ANT-C (in between dashed lines)

	CHAPTER 6 -
	30 -0 _ 17 -0 _ 30 -0 _ 30 -0 _ A-ChIP-Lab S2 H3K4me3 S2 H3K27me3 Sg4 H3K4me3 Sg4 H3K27me3	pb	bcd	Antp	Scr
										Antp
	chr3R:
	6,600,000	6,650,000	6,700,000	6,750,000	6,800,000	6,850,000	6,900,000	6,950,000	7,000,000	7,050,000	7,100,000	7,150,000

A:

Thesis project: Results Cell lines modifications to test the involvement of boundary elements and insulator proteins in the regulation of Hox genes and 3D chromatin organization

  5.3C). The differential function of BEs, and particularly Fab-8, is therefore likely mediated by the differential binding of other insulator proteins or other types of regulatory proteins. Our in-vitro studies on Fab-7 1 mutant Genital disc cells further suggest that the Fab-7 element in these larval cells primarily functions to prevent the spreading of the H3K27me3 mark into iab-7. At this stage, we can't discriminate between a direct or indirect effect at this stage, e.g. mediated by a structural/functional interaction between Fab-7 and other cis-regulatory elements located in iab-7. It's interesting to notice though that the effect is opposite to what has been observed