François Tronche

Jean- Baptiste Boulé

François Je Remercie Le Professeur Jean

Riou

Cas9 CRISPR associated protein 9 CATD Cenp-A Targeting Domain CCAN Constitutively Centromere Associated Network CDE Centromere DNA Element CENP Centromeric Protein CENP-A Centromeric Protein A CENP-B Centromeric Protein B ChIP Chromatin Immunoprecipitation CIP Chemically Induced Proximity Clr Cryptic Loci Regulator CPC Chromosomal Passenger Complex CRISPR Clustered Regularly Interspaced Short Palindromic Repeats

je remercie les membres du jury d'avoir accepté de juger ce travail.

Figures

Figure 1. Historical drawings by Walter Flemming (1843-1905) showing cell division. ..................................... Figure 2. Organisation of centromeric andpericentromeric region on a chromosome.. ...................................... Figure 3. Classification of repetitive DNA in the human genome. ........................................................................ Figure 4. Centromeric region in different organisms. ........................................................................................... Figure 5. Dicentric chromosome formation. .......................................................................................................... Figure 6. Histone modifications on different histone tails. .................................................................................... Figure 7. Euchromatin andheterochromatin histone tail modifications. .............................................................. Figure 8. The trilaminar kinetochore structure. .................................................................................................... Figure 9. CENP-A is interspersed with H3 on extended chromatin fibres.. .......................................................... Figure 10. Organisation of centromeric heterochromatin. .................................................................................... Figure 11. HP1 domains and interacting partners. ............................................................................................... Figure 12. Formation of pericentromeric heterochromatin.. ................................................................................ Figure 13. Heterochromatin formation in S. pombe. ............................................................................................. Figure 14. Organisation of mouse centromeric region. ......................................................................................... Figure 15. Association of pericentromeric region in mouse cells. ........................................................................ Figure 16. The percent of non-coding to total genomic DNA per haploid genome across . Chromatin organisation at major and minor satellites requires centromeric RNA. ............................ Figure 24. Transcription factor based mechanism for heterochromatin formation. ............................................. Figure 25. Structure of the locked nucleic acid (LNA) monomer (Kauppinen, Vester, & Wengel, 2005). ............ Figure 26. Schematic representation of the TALE protein. ................................................................................... Figure 27. Designer TALE proteins. ...................................................................................................................... Figure 28. Chromatin in vivo assay system employs chemically induced proximity (CIP) to induce chromatin modifications in living cells. ................................................................................................................................... Figure 29. Polydactyl zing finger protein fusion. .................................................................................................. Figure 30. Schematic representation of the LITE system. ..................................................................................... Figure 31 Transcriptional profile of major satellite s revealed by LNA modified oligonucleotides. .................. Figure 32. Complex transcriptional profile revealed by LNA probes.. ............................................................... Figure 33. Comparison of the transcriptional profile of MEF cells revealed with isosequential LNA and 2'-O-Me probe targeting major satellites. .......................................................................................................................... Figure 34. Transcriptional profile revealed by northern blot after different stringency washes. ....................... Figure 35. Transcriptional profile obtained with LNA,DNA and RNA probe targeting major satellite. ........... Figure 36. Strand specific RT PCR analysis of total RNA isolated from MEF cells. .......................................... Figure 37. Northern analysis of RNA from cells treated with TSA and chaetocin. ............................................. Figure 38. Northern analysis of RNA from heat shocked MEF cells. .................................................................. Figure 39. Analysis of RNA from cells treated with different RNA polymerase inhibitors. ................................. Figure 40. Comparison between total RNA and recovered short RNA. ............................................................... Figure 41. Northern blot after the ligation of adapters on the short RNA.. ......................................................... Figure 42. 3' adapter ligation on the U5 RNA. .................................................................................................... Figure 43. Schematic representation of the plasmid pJL156 and different TALE fusion proteins. ..................... Figure 44. Immunofluorescence detection of the TALE N212-GFP binding to major satellites in mouse . Immunofluorescence detection of NIH-3T3 cells transfected with different TALE fusion proteins. .. TANGO. ............................................................................... Figure 48. Effects of the demethylation with TALE N212-mJMJD2D on H3K9me3 signal in Table 1. Functional classes of short non-coding RNA. ................................................................................................................. 54 Table 2. Cells lines and culture conditions used in this study. ................................................................................................. 95 Table 3. Complete list of oligonucleotides used in this study. ............................................................................................... 103 Table 4. Primers used in the study. ................................................................................................................................................... 104 Table 5. Adapter sequences used for RACE experiments ........................................................................................................ 104 Table 6. List of oligonucleotides used in the study. ................................................................................................................... 108 Table 7. Schematic representation of the results of northern hybridization revealed with the major satellite specific probes. .......................................................................................................................................................................................... 115 Table 8. Lengths of the expected PCR products after RT--PCR amplification of major satellite transcripts using the indicated primer pairs. . ................................................................................................................................................................. 120 Table 9. Conditions of treatment with trichostatin A and chaetocin. ............................................................................... 122 Table 10. Conditions of treatment with different RNA polymerase inhibitors. ............................................................. 126 Table 11. Three TALE constructs used in the study. ................................................................................................................... 139 Table 12. Different measured values for non--transcfected cell and cells transfected with different TALE fusion proteins obtained after TANGO analysis. ....................................................................................................................................... 163 Table 13. Different measured values for non--transfected cell and three population of cells transfected with TALE N212--mJMJD2D obtained after TANGO analysis ........................................................................................................

Tables

... 164

Introduction

There's real poetry in the real world. Science is the poetry of reality.

CHAPTER I

Structure and function of centromeric regions Usage of the terms

The terms centromere or centromeric region are often used to refer to both the centromere as the place of kinetochore assembly and the surrounding pericentromeric region.

In this manuscript, I will use the term centromere to refer to the centromere core where the kinetochore is assembled, while the term pericentromeric will be used for the region surrounding the central core, comprised of constitutive heterochromatin.

I. 1. Determining the centromeric region 1.1. First description of the centromere

The process of mitosis was first described in the late 19 th century by Flemming, who used the term Chromosomen to name the fibrous, stainable material in the resting nucleus that eventually become more compact and change into as he calls them, threads, later named chromosomes. Flemming described the threads slowly rearranging to the centre of the nucleus where they are separated into two groups to finally reappear in the two daughter nuclei. He described the filaments from the spindle apparatus, for which he was convinced to be responsible for the transport of the threads. However, he did not describe the centromere, and his images show the threads attached to the spindle fibres throughout the chromosome length (Figure 1). The term centromere was coined by Darlington in 1936 to define the primary constriction on the mitotic chromosome [START_REF] Paweletz | Walther Flemming: pioneer of mitosis research[END_REF][START_REF] Dos | Centromeres in cell division, evolution, nuclear organization and disease[END_REF]. Indeed, the word centromere comes from Greek words "kentron" meaning central and "meros" meaning part, referring to its position in the middle of the metaphase chromosome. Today we know that the centromere, or the primary constriction is not always found in the centre of the chromosome. With a few exceptions, there exists however, one defined locus on the chromosome crucial for accurate segregation of the genetic material. During cell division, sister chromatids of each of the chromosomes are segregated into two daughter cells, exactly how Flemming first described it. Centromeres play a crucial role in this event by serving as a platform for the assembly of a kinetochore. The kinetochore, a multiprotein complex, interacts with the spindle microtubules, ensuring the bi-orientation of chromosomes on the metaphase plate and the accurate segregation of sister chromatids [START_REF] Cheeseman | A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension[END_REF]. With rare exceptions known to date, such as holocentric chromosomes of C.elegans, where a centromere is dispersed along their entire length, providing multiple attachment sites for the microtubules [START_REF] Dernburg | Here, there, and everywhere: kinetochore function on holocentric chromosomes[END_REF][START_REF] Melters | Holocentric chromosomes: convergent evolution, meiotic adaptations, and genomic analysis. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology[END_REF], or dicentric chromosomes that arise due to genome rearrangements [START_REF] Stimpson | Dicentric chromosomes: unique models to study centromere function and inactivation. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology[END_REF] and are stabilised by the inactivation of one of the two centromeres, most eukaryotes have a single functional centromere. Any defects such as centromere loss or formation of multiple centromeres cause inaccurate chromosome segregation that leads to aneuploidy and chromosome breakage, respectively, thus attesting the necessity of having only one functional attachment site per chromosome.

Over the years, it had become clear that the centromere is not only a simple scaffold for the assembly of kinetochore proteins and that it is more complicated in its structure and organisation than previously thought. While the structure of the kinetochore and its role in cell division has been extensively studied, the centromere itself remains somewhat elusive.

The centromere role is conserved across the species and most organisms have a single functional centromere to achieve faithful chromosomal segregation. This chromosomal locus has a distinct chromatin structure and is surrounded by heterochromatin regions (which will be described later in this manuscript). Experimental evidences point to the idea that both the DNA sequence and proteins that modify centromeric chromatin act together to contribute to the establishment and maintenance of a functional centromere. However, we still poorly understand the mechanisms by which they do so. An emerging view is that non-coding RNA synthesised from the surrounding regions play an important role in this processes. They might act as structural components of the centromeric chromatin or may mediate specific modifications. It has become clear that the underlying chromatin is not only important for the function of the kinetochore but also seems to be a self-sustaining region that impacts the functional organisation of the nucleus, ultimately leading to the control of gene expression.

2. Organisation of the centromeric region

There are two distinct chromatin domains, both required for chromosome segregation: the centromere and the pericentromere (Figure 2). These two domains are defined by different sets of proteins and chemical modifications that determine their organisation and function.

The centromere is formed on a locus that is cytogenetically seen as a primary constriction on a metaphase chromosome. Centromere specific proteins associate to this regions, forming a complex network that is a structural core for the assembly of outer kinetochore proteins that will interfere with the microtubules [START_REF] Hori | The CCAN recruits CENP-A to the centromere and forms the structural core for kinetochore assembly[END_REF]. Regions surrounding the centromere are called pericentromeric regions and are made of large blocks of constitutive heterochromatin (see paragraph 2.1). This region assures proper cohesion of sister chromatids during mitosis and preserves chromosome integrity resisting against the strong forces generated by the pulling of microtubules [START_REF] Sullivan | A solid foundation: functional specialization of centromeric chromatin[END_REF]. The centromere function is evolutionary conserved and the centromere locus is faithfully transmitted from one generation to the next. One could therefore imagine that the simplest way for determining the position of the centromere on a chromosome would be a DNA sequence. Surprisingly, most organisms lack a precise consensus sequence that would determine the centromere identity. A unique feature that distinguishes centromeres from other chromosomal regions is the presence of an evolutionary conserved specific histone H3 variant, CenH3, also called CENP-A, which replaces the canonical histone H3 in centromeric nucleosomes, and is the only known signature of a functional centromere [START_REF] Palmer | A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones[END_REF][START_REF] Sullivan | Human CENP-A contains a histone H3 related histone fold domain that is required for targeting to the centromere[END_REF]. Despite the absence of sequence signature, centromeric DNA of virtually all eukaryotic organisms has a characteristic of being AT rich and repetitive.

1.3. DNA at the centromeric and pericentromeric region 1.3.1 Repetitive DNA Only around 2% of the human genome codes for proteins. The remaining 98% is made of non-coding sequences, mostly introns, promoters, and repetitive DNA. Repetitive DNA occupies around 55% of the human genome and this large amount is a characteristic of all multicellular organisms [START_REF] Shapiro | Why repetitive DNA is essential to genome function[END_REF] (Figure 3). It includes both transposable genetic elements and tandem repeats. Transposable genetic elements are interspersed throughout the genome and are able to propagate by transposition, moving from one place in the genome to the other. Four groups of such repeats are known to mammals.

Together, they count for 45% of the human genome. These are the DNA transposons and RNA transposons, including long interspersed elements (LINE), shorts interspersed elements (SINE) and long terminal repeats (LTR). Tandemly repeated sequences, also called satellite sequences occupy around 10% of the human genome. This group of repetitive DNA is characterized by a monomer unit present in multiple copies that extend in millions of bases in a head to tail fashion. Depending on the length of the monomer and the size of the block of the repeated unit, tandem repeats can be classified into satellites, minisatellites and microsatellites (Figure 3).

Diverse functions of repetitive DNA

For a long time repetitive DNA was considered to be a parasite to the genome. This meant that repetitive sequences can spread by duplicating themselves in the genome, while leaving no effect on the phenotype except by introducing occasional mutations due to their insertion within the functional genes [START_REF] Orgel | Selfish DNA: the ultimate parasite[END_REF]. The idea of junk DNA became popular already in the 1960s and this term was quickly adopted to describe all repetitive DNA [START_REF] Palazzo | The case for junk DNA[END_REF]. The repetitive DNA was thought to have no protein coding function nor could it be associated with any other purpose in the genome at the time. It was therefore regarded as functionally unimportant, even parasitic. However, even in these early years it was already speculated that these sequences could hide a potential function. Some sixty years ago Barbara McClintock suggested that certain DNA sequences might move around the genome and thereby influence the expression of genes. We know these sequences today as DNA transposons. Somewhere at the same time, Britten and Davidson proposed the first ideas explaining the mechanisms of gene regulation. They suggested a model where repetitive DNA is a key for gene regulation in complex organisms. The idea of junk DNA persisted and even today, repetitive sequences are still regarded as mostly non-functional.

There is, however, an accumulating evidence that somewhat changed the view of non-coding DNA as functional debris of the genome. Shapiro et al. argue that the repetitive DNA serves to organise the coding information in the genome [START_REF] Shapiro | Why repetitive DNA is essential to genome function[END_REF].

Restraining the functionality of the sequence only to the coding portion and ignoring the rest as an useless parasitic element is not to think about the other aspects of what a functional sequence really is. Genome requires a precise regulation of expression, replication and repair and is organised in functional domains. The repetitive DNA could be thus regarded as an important regulator and organiser of the genome. The simplicity of the information content of the repetitive sequences and its ability to interact with DNA, RNA and proteins is in accordance with the proposed function as being organisers of the genetic information and participating to the control of gene expression [START_REF] Shapiro | Why repetitive DNA is essential to genome function[END_REF].

Repetitive DNA has been implicated in different aspects of gene expression and genome organisation. Different classes of repetitive DNA are associated with various functions in the genome. Transposable elements are found in numerous promoter sequences and many contain SINE and LINE elements. Human LINE-1 act as positive transcriptional regulatory element. Human Alu elements as well as mouse B1 and B2 are implicated in mRNA regulation [START_REF] Allen | The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock[END_REF]. Repetitive DNA has a role in nuclear organisation and architecture. For example, some human LTR retrotransposons and LINE elements act as scaffold/matrix associated regions [START_REF] Rollini | Identification and characterization of nuclear matrix-attachment regions in the human serpin gene cluster at 14q32.1[END_REF], while in Drosophila, gypsy elements determine intranuclear gene localisation and nuclear pore association [START_REF] Labrador | Setting the boundaries of chromatin domains and nuclear organization[END_REF]. Gypsy also acts as an insulator element that inhibits the propagation of silencing [START_REF] Gerasimova | A Chromatin Insulator Determines the Nuclear Localization of DNA[END_REF]. Finally, tandemly repeated DNA act in the structural assembly of distinct chromatin domains, such as centromeric and pericentromeric regions.

Repetitive DNA at the centromeric region

Satellite DNA is localised at the functional centromere and the surrounding pericentromeric heterochromatin. The name satellite DNA is historical, as these sequences were originally identified from the cesium chloride density gradient where they appeared as single, isolated "satellites" due to their higher AT content, in comparison with the rest of the genome [START_REF] Corneo | A satellite DNA isolated from human tissues[END_REF]. Satellite sequences are organized into several megabases long units that are formed by the arrangement of monomers into long arrays of tandem repeats.

Satellite sequences are variable between species and can rapidly change in repeat number and primary sequence. Even today, with the development of the genomic sequencing projects, satellite sequences stay mostly uncharacterized. The homogeneity of the monomers that are present in multiple copies makes them difficult to be sequenced and most importantly to be assembled into large contigs, which is why they are mostly excluded from the genome assemblies [START_REF] Rudd | Analysis of the centromeric regions of the human genome assembly[END_REF][START_REF] Plohl | Satellite DNAs between selfishness and functionality: structure, genomics and evolution of tandem repeats in centromeric (hetero)chromatin[END_REF]).

Variation of centromeric sequences between species

Centromere DNA differs greatly from one species to the next, showing variations in primary sequence, length and quantity of the repeats. Variations exist even in different chromosomes of the same organism. The simplest centromere is found in budding yeast S. cerevisiae, the only organism where a specific DNA sequence determines the centromere locus. In all chromosomes of S. cerevisiae, three AT-rich DNA sequence elements CDEI, CDEII and CDEIII (Centromeric DNA element I, II, III) that span a total length of 125 bp, define a simple point centromere (Figure 4a). CDEIII recruits sequence-specific protein complex necessary for loading of the yeast CENP-A homolog and the formation of a single centromeric nucleosome [START_REF] Hyman | Microtubule-motor activity of a yeast centromere-binding protein complex[END_REF][START_REF] Meraldi | Phylogenetic and structural analysis of centromeric DNA and kinetochore proteins[END_REF]. Other eukaryotic organisms have more complex centromeres, termed regional centromeres, where the kinetochore assembles at a defined chromosomal domain surrounded by long stretches of constitutive heterochromatin. In fission yeast S. pombe, the kinetochore assembles at the nonrepetitive AT-rich central domain surrounded by the flanking innermost repeats, while pericentromeric heterochromatin is formed at the outermost repeats [START_REF] Takahashi | A low copy number central sequence with strict symmetry and unusual chromatin structure in fission yeast centromere[END_REF] (Figure 4b). In Drosophila melanogaster the centromere is assembled on the blocks of simple repeat sequences AATAT and AAGAG, interspersed by transposons [START_REF] Sun | Sequence analysis of a functional Drosophila centromere[END_REF]) (Figure 4c).

In mouse, centromeres are defined by minor satellites, while the surrounding pericentromeric heterochromatin is established on the major satellites [START_REF] Guenatri | Mouse centric and pericentric satellite repeats form distinct functional heterochromatin[END_REF] (Figure 4d). Primate centromeres mostly contain alpha satellite family of repeats that was initially isolated as component α of the highly repetitive DNA fraction of the African green monkey [START_REF] Lee | Human centromeric DNAs[END_REF]. All human centromeric regions contain two types of sequence organisation, monomeric and higher order repeats (HOR). The centromere core is found at the alpha satellite DNA, which consists of 171 bp monomer forming chromosome specific higher-order repeats [START_REF] Masumoto | The role of CENP-B and alpha-satellite DNA: de novo assembly and epigenetic maintenance of human centromeres[END_REF]) (Figure 4e). Pericentromeres contain alpha satellite repeat monomers that are interrupted by several families of heterogeneous sequences called satellite I, II and III as well as beta and gamma-satellites [START_REF] Lee | Human centromeric DNAs[END_REF]). The monomeric repeats can contain interspersed elements such as LINE and SINE or LTR retrotransposons [START_REF] Schueler | Structural and functional dynamics of human centromeric chromatin[END_REF]. The monomer units of different degrees of polymorphism are tandemly repeated along the centromeric region. Arrays of higher order repeats contain stretches of highly identical monomer units that can be distinguished by their internal organisation of monomers in each repeat unit. Contrary to homogeneous sequences that span the centromere of mouse chromosomes, human centromeric sequences are highly polymorphic, showing divergence even between different chromosomes [START_REF] Hayden | Human centromere genomics: now it's personal. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology[END_REF].

Neocentromeres and dicentrics

The variations in centromere sequence between species or even between chromosomes of a single organism point to the idea that there are mechanisms that control the processes of centromere formation and inheritance that go beyond a mere determination by the underlying sequence. Evidence for this come from studies of stable dicentric chromosomes and chromosomes that have established functionally new centromere or neocentromere. Dicentric chromosomes can arise by duplication or other chromosomal rearrangements and can be stable after inactivation of one of the centromeres (Figure 5). The stabilisation is probably induced by partial deletion of a part of alpha satellite sequence containing CENP-A. This abolishes the downstream accumulation of other centromeric proteins and prevents the formation of a functional kinetochore [START_REF] Earnshaw | Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads[END_REF][START_REF] Stimpson | Dicentric chromosomes: unique models to study centromere function and inactivation. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology[END_REF].

Neocentromeres mostly form on rearranged or fragmented chromosomes, but can sometimes appear on non-repetitive, mostly euchromatic loci after the inactivation of the existing centromere, taking over the role of a functional centromere [START_REF] Marshall | Neocentromeres: new insights into centromere structure, disease development, and karyotype evolution[END_REF].

After the discovery of neocentromere formation on a human chromosome on a sequence different from alpha satellite repeat, it became clear the DNA sequence is not sufficient for the centromere formation [START_REF] Voullaire | A functional marker centromere with no detectable alpha-satellite, satellite III, or CENP-B protein: activation of a latent centromere?[END_REF]. These new functional centromeres are able to establish a fully functional kinetochore by binding almost all centromeric proteins except centromeric protein B (CENP-B), which has a binding site in the alpha satellite region.

Formation of neocentromeres on a locus outside of the established repetitive sequence strongly suggested that divers chemical modifications of chromatin could have a role in determination of centromere. These chromatin modifications present epigenetic, heritable changes that do not modify the underlying DNA sequence, but can still influence gene expression and help to the determination of centromere identity. However, common features of most centromere sequences such as high AT content and repetitiveness of the primary sequence testify to the importance of these elements. The centromere might therefore be determined by the synergy of the DNA sequence and epigenetic factors, where a special chromatin structure formed by their interactions determines the centromere identity and activity. The inconsistency between the conservation of the centromere function and the high divergence of the centromeric sequence is know as the centromere paradox [START_REF] Eichler | Repetitive conundrums of centromere structure and function[END_REF]. In order to maintain the centromere function, the associated proteins are evolving in concert with the rapidly changing repetitive sequences.

These changes in the sequence and adjacent proteins could eventually lead to reproductive isolation and eventually speciation [START_REF] Talbert | Spreading of silent chromatin: inaction at a distance[END_REF].

I. 2. Chromatin organisation at the centromere

Histone modifications and the underlying chromatin state

In eukaryotes, DNA is assembled together with histone proteins into chromatin. The principal unit of chromatin is the nucleosome, an octamer formed by two molecules of four histones (H2A, H2B, H3 and H4) wrapped with 147 bases of DNA in 1.7 turns [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF]. Histones undergo various chemical modifications that are used to separate and define different chromatin states connected to their gene expression ability. These post-translational chemical modifications occur on the N-terminal histone tails that extend out of the nucleosome core. There are eight modifications associated with histones: methylation, acetylation, phosphorylation, ubiquitinylation, sumoylation, ADP ribosylation, deimination and proline isomerisation (Figure 6). Due to the large number of histone residues, the number of possible chemical modifications is vast and grows even more complex when we take into account that several modifications can appear in different forms, such as for example mono-, di-or trimethylation [START_REF] Kouzarides | Chromatin modifications and their function[END_REF]). The combination of histone modifications is known under the name "histone code" [START_REF] Jenuwein | Translating the histone code[END_REF]. These modifications, together with DNA methylation and chromatin remodelling are part of epigenetic mechanisms that act together to organise the genome into distinct functional domains, and contribute to the regulation of the output of genetic information.

Already at the beginning of the last century, Heitz observed that certain regions of moss chromosomes show a dense pattern of staining during all phases of the cell cycle when coloured with DNA dyes. It was later discovered that differences in the staining pattern of some regions, when compared to the rest of the genome, tend to reflect differences in their structure and accessibility. Indeed, chromatin exists in two different forms, euchromatin and heterochromatin. In these distinct chromatin environments, different epigenetic modifications determine chromatin accessibility and its transcriptional potential (Figure 7). Euchromatin is associated with gene rich areas of chromatin, it is less condensed and therefore more accessible. Heterochromatin is formed at regions of highly repetitive DNA, flanking the centromeres and at the telomeres, where it stays condensed throughout the cell cycle and is referred to as constitutive heterochromatin. In contrast, facultative heterochromatin can form at genomic loci which chromatin state is easily converted to open, euchromatic state, during different stages of development [START_REF] Grewal | Heterochromatin revisited[END_REF]. Heterochromatin and euchromatin show different patterns of epigenetic modifications. Histone modifications act on chromatin by either changing the chromatin structure altering the histone charge or recruiting different proteins that further act on chromatin. For example, acetylation neutralises basic charge of the lysine, unfolding the chromatin and making it more accessible for transcription [START_REF] Kouzarides | Chromatin modifications and their function[END_REF]. Therefore, distinct chromatin marks are associated with precise chromatin states.

Acetylation of histones H3 and H4 is an epigenetic mark that defines euchromatin and is connected with gene activity. Methylation of histone H3 on lysine 4 marks transcriptionally active sites [START_REF] Santos-Rosa | Active genes are tri-methylated at K4 of histone H3[END_REF][START_REF] Bernstein | Methylation of histone H3 Lys 4 in coding regions of active genes[END_REF][START_REF] Schneider | Histone H3 lysine 4 methylation patterns in higher eukaryotic genes[END_REF]), while H3 lysine 9 methylation is a hallmark of pericentromeric heterochromatin and marks transcriptionally silent regions [START_REF] Rice | Histone Methyltransferases Direct Different Degrees of Methylation to Define Distinct Chromatin Domains[END_REF]. cerevisiae. Downregulation of CENP-A is lethal for all organisms, causing defects in chromosome segregation. The loss of CENP-A results in mislocalization of inner kinetochore proteins and failure of kinetochore assembly leading to aneuploidy and genomic instability [START_REF] Howman | Early disruption of centromeric chromatin organization in centromere protein A (Cenpa) null mice[END_REF][START_REF] Régnier | CENP-A is required for accurate chromosome segregation and sustained kinetochore association of BubR1[END_REF]. Aneuploidy is a mark of almost all tumours and it is most likely caused by CENP-A overexpression and the formation of ectopic neocentromeres as observed in human colorectal cancer. CENP-A overexpression in colorectal cancer cells leads to CENP-A mislocalization to noncentromeric regions of chromosome or even to a complete loss of CENP-A, suggesting disruption of the kinetochore [START_REF] Tomonaga | Overexpression and mistargeting of centromere protein-A in human primary colorectal cancer[END_REF]. Indeed, abolishment of CENP-A overexpression decreases aneuploidy [START_REF] Amato | CENPA overexpression promotes genome instability in pRb-depleted human cells[END_REF]). If CENP-A overexpression is indeed the main reason for chromosomal instability in cancer cells, understanding the mechanisms leading to this event and its consequences might help in developing new approaches in anticancer therapy. How CENP-A incorporation beyond the centromere boundaries is limited is still not known but it might be that the formation of pericentromeric heterochromatin prevents further CENP-A incorporation (Pidoux & Allshire 2005).

A R T K Q T A R K S T G G K A P R K Q L A T K A A R K S A 9! 27 Me H3 S G R G K G G K G L G K G G A K R H R K V L R H4 20 Me A R T K Q T A R K S T G G K A P R K Q L A T K A A R K S A 9! 4! 14 27 H3 Heterochromatin Euchromatin Ac Ac Me S G R G K G G K G L G K G G A K R H R K V L R H4
CENP-A containing nucleosomes carry structural features that distinguish them from the canonical H3-containing nucleosomes. Amino acid sequence of the N-terminal tail of CENP-A differs from histone H3 and is also quite variable between species. However, only the region localized at the C-terminal histone fold domain called the CENP-A targeting domain (CATD) is required to target CENP-A to chromosomes [START_REF] Black | Structural determinants for generating centromeric chromatin[END_REF]). Histone H3 containing the 22 amino acids that make the CATD domain is able to replace CENP-A at the functional centromere [START_REF] Black | Centromere identity maintained by nucleosomes assembled with histone H3 containing the CENP-A targeting domain[END_REF]). Due to the CATD domain, the CENP-A nucleosomes show a more rigid structure to the interface formed with the histone H4 (tenfold more slower hydrogen exchange along the peptide backbone) than the histone H3 [START_REF] Black | An epigenetic mark generated by the incorporation of CENP-A into centromeric nucleosomes[END_REF]).

The canonical histone H3 contains numerous post-translational modifications on its Nterminal tail, while only some are known for CENP-A [START_REF] Bailey | Posttranslational modification of CENP-A influences the conformation of centromeric chromatin[END_REF]. CENP-A is phosphorylated by Aurora B at serine 7 (Ser7), which is similar to Aurora B phosphorylation of histone H3 at Ser 10 [START_REF] Zeitlin | CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis[END_REF]. Other modifications characteristic only to CENP-A are trimethylation of glycine 1 (Gly1) and phosphorylation of Ser16 and Ser18. The double serine phosphorylation motif was shown to form a specific structure that cause intramolecular associations between the N-terminal tails of CENP-A, changing the conformation of CENP-A nucleosomes and resulting in a different chromatin structure at the centromere [START_REF] Bailey | Posttranslational modification of CENP-A influences the conformation of centromeric chromatin[END_REF].

After each round of replication, centromere site must be re-established at the newly synthesised sister chromatid. The CENP-A nucleosomes are loaded at the place of the preexisting CENP-A by the help of a histone chaperone HJURP (Holliday junction recognition protein) during the early G1 phase [START_REF] Dunleavy | Centromeric chromatin makes its mark[END_REF][START_REF] Foltz | Centromere-Specific Assembly of CENP-A Nucleosomes Is Mediated by HJURP[END_REF]).

Centromere associated proteins

The kinetochore is seen by electron microscopy as a three-laminar structure at the primary constriction of chromosomes. It is composed of distinct protein complexes attached on one side to the microtubules and on the other side to the centromeric chromatin, allowing the segregation of sister chromatids during cell division [START_REF] Cheeseman | Molecular architecture of the kinetochore-microtubule interface[END_REF] (Figure 8). The outer kinetochore layer consists out of several protein complexes. These complexes together form the KMN network of proteins, named according to the acronym for the protein components KNL1, Mis12 and Ndc80, that bind to the microtubules [START_REF] Cheeseman | A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension[END_REF][START_REF] Cheeseman | The conserved KMN network constitutes the core microtubulebinding site of the kinetochore[END_REF]. KMN network interacts with proteins of the inner kinetochore together called the CCAN (constitutively centromere-associated network). The CCAN is formed by a group of centromere specific proteins termed CENPs (for centromeric proteins).

There are 16 CENPs that permanently associate with the centromere [START_REF] Foltz | The human CENP-A centromeric nucleosome-associated complex[END_REF][START_REF] Okada | The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres[END_REF]. The localisation of CCAN occurs downstream to CENP-A deposition suggesting that CENP-A marks the position for the CCAN assembly. After the initial recruitment to the centromere, the CCAN acts as a foundation for the assembly of the outer kinetochore proteins [START_REF] Hori | CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochore[END_REF][START_REF] Hori | The CCAN recruits CENP-A to the centromere and forms the structural core for kinetochore assembly[END_REF]. CENP-C localisation to the centromere requires six Cterminal residues of the CENP-A [START_REF] Guse | In vitro centromere and kinetochore assembly on defined chromatin templates[END_REF]. The only centromeric protein that specifically binds to a centromeric sequence is CENP-B. CENP-B binds to a specific 17 bp sequence present at the centromeric region of human and mouse, and it seems to be required for de novo assembly of CENP-A [START_REF] Masumoto | A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite[END_REF]. Chromosomal passenger complex containing Aurora B kinase, INCENP, Survivin and Borealin transiently mark the centromere during mitosis [START_REF] Ruchaud | Chromosomal passengers: conducting cell division[END_REF]. Chromatin between two sister-chromatids is referred to as the inner centromere and is associated with the Chromosome passenger complex proteins such as Aurora B and INCENP (blue). When the microtubules are absent, the fibrous corona stretches out of the outer layer (adapted from Perpelescu & Fukagawa 2011).

2.2.3. Centrochromatin -the chromatin forming at the centromeres Defined by the presence of CENP-A, centromeric chromatin reveals a specific level of organisation and structure. On the extended chromatin fibre, chromatin at the centromere core in both flies and humans is arranged into regions containing CENP-A/CID nucleosomes interspersed with regions containing the canonical histone H3 nucleosomes (Figure 9). 3D organisation of the metaphase chromosomes shows the formation of a unique cylindrical structure where blocks of CENP-A/CID nucleosomes are orientated toward the outer kinetochore plate while H3 containing nucleosomes are placed toward the interior, between the sister chromatids [START_REF] Blower | Conserved organization of centromeric chromatin in flies and humans[END_REF]) (Figure 10). The length of the DNA forming this structure is approximately 500-1500 kb in humans and 200-500 kb in flies [START_REF] Allshire | Epigenetic regulation of centromeric chromatin: old dogs, new tricks? Nature reviews[END_REF]. This distinct chromosomal domain sometimes referred to as centrochromatin is surrounded by long stretches of heterochromatin. These two regions are marked by specific epigenetic marks that distinguishes them one from the other and from the rest of the genome. The core histone H3 at the centromere is uniquely modified carrying posttranslational modifications different from both silent and active chromatin. Centromeric H3 is dimethylated at lysine 4 (H3K4me2) which is an epigenetic mark associated with euchromatin and potentially transcriptionally active regions. However, both histones H3 and H4 of centromeric nucleosomes are hypoacetylated, lacking acetylation marks usually found in euchromatin, and are at the same time deprived of di-or tri-methylation of H3K9, a hallmark of heterochromatin. Heterochromatic regions that flank the centromeric chromatin are enriched for H3K9 di-and trimethylation and show hypoacetylation of both H3 and H4 histones [START_REF] Sullivan | Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin[END_REF].

The reason for the formation of this unique type of chromatin on the centromere is still questioned. The existence of canonical histone H3 nucleosomes at the centromere, carrying distinct epigenetic marks surely has a functional significance. It participates to the formation of the three-dimensional structure on the metaphase chromosome, assuring kinetochore assembly and contacts with the microtubules. Canonical histone carrying the lysine 4 dimethylation mark seems to promote the incorporation of CENP-A in human cells. Indeed, depletion of H3K4me2 at centromeres fails to recruit CENP-A chaperon HJURP causing defectiveness of CENP-A incorporation [START_REF] Blower | Conserved organization of centromeric chromatin in flies and humans[END_REF][START_REF] Bergmann | Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore[END_REF]). The combination of histones together with specific epigenetic marks could promote the incorporation of CENP-A to the locus, marking it for kinetochore assembly in the next generations [START_REF] Allshire | Epigenetic regulation of centromeric chromatin: old dogs, new tricks? Nature reviews[END_REF]. These findings confirm the importance of CENP-A as the foundation for the assembly of kinetochore and centromere function. These structural properties make CENP-A nucleosomes different from the canonical H3 nucleosomes and surely facilitate the assembly of a specific chromatin on the centromere. and histone 4 lysine 20 trimethylation (H4K20me3) marks [START_REF] Peters | Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability[END_REF][START_REF] Kourmouli | Heterochromatin and tri-methylated lysine 20 of histone H4 in animals[END_REF][START_REF] Martens | The profile of repeat-associated histone lysine methylation states in the mouse epigenome[END_REF]. Non-histone proteins such as HP1 associate with pericentromeric heterochromatin.

HP1 was first discovered as a suppressor of position effect variegation (PEV, see paragraph 2.3.4 of this Chapter), when a mutation in Su(var)2-5, a gene coding for HP1, resulted in inhibition of PEV [START_REF] Eissenberg | Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster[END_REF]). Three isoforms of HP1 are known in mammals: HP1α, HP1β and HP1γ. These proteins are evolutionary conserved, and can be found throughout eukaryotic organisms. HP1 homologs in S. pombe are known under the names Swi6 and Chp2 [START_REF] Zeng | HP1: Heterochromatin binding proteins working the genome[END_REF]. Mammalian HP1α and HP1β localize to heterochromatic regions, while HP1γ can be found in euchromatin, where it is implicated in transcriptional activation [START_REF] Minc | HP1γ associates with euchromatin and heterochromatin in mammalian nuclei and chromosomes[END_REF].

Another non-histone component of pericentromeric heterochromatin is SU(VAR)3-9, a histone methyltransferase (HMT) conserved from mammals (Suv39h1 and 2) to fission yeast (Clr4). Suv39h is primarily responsible for trimethylation of H3K9 [START_REF] Rea | Regulation of chromatin structure by site-specific histone H3 methyltransferases[END_REF][START_REF] Rice | Histone Methyltransferases Direct Different Degrees of Methylation to Define Distinct Chromatin Domains[END_REF]. Other methyltransferases such as SETDB1 and G9a also modify H3K9 in mammals, catalysing different levels of methylation (mono-and dimethylation). Suv39h

indirectly controls DNA methylation and methylation of H4K20. Loss of Suv39h activity causes absence of H3K9 methylation mark and loss of HP1 from heterochromatin [START_REF] Bannister | Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain[END_REF][START_REF] Peters | Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability[END_REF]).

HP1 contains three distinct domains that have different binding preferences (Figure 11). HP1 chromodomain present in its N-terminal end tethers HP1 to heterochromatin through the interaction with Suv39h-trimethylated lysine 9 of histone H3 (Lachner et al. 2001a;[START_REF] Bannister | Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain[END_REF][START_REF] Nakayama | Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly[END_REF]). This highly specific interaction cannot be seen in other chromodomain proteins such as Polycomb. Indeed, H3K9me3 binds a conserved motif present in HP1 chromodomain. However, since H3K9me3 is not specific only to

heterochromatin, but appears throughout the nucleus, this interaction seems not to be sufficient for HP1 targeting to heterochromatin. Moreover, the central, variable hinge domain of HP1 is found to strongly bind RNA [START_REF] Muchardt | Coordinated methyl and RNA binding is required for heterochromatin localization of mammalian HP1alpha[END_REF]. HP1 hinge domain is a major target to sumoylation. It was shown that this post-translational modification promotes association of HP1 with RNA and its accumulation to pericentromeric heterochromatin [START_REF] Maison | SUMOylation promotes de novo targeting of HP1α to pericentric heterochromatin[END_REF]). H3K9me3 provides binding sites for HP1 that in turn binds Suv39h enzyme with its chromoshadow domain (Lachner et al. 2001a;[START_REF] Bannister | Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain[END_REF][START_REF] Nakayama | Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly[END_REF]). HP1 chromoshadow domain can also bind other proteins such as Suv4-20h2, methyl-CpG-binding proteins, Dnmt3a and Dnmt3b and the large subunit of chromatin assembly factor CAF, p150, implicated in DNA replication and repair. It also interacts with HP1α and HP1β, a feature that permits homodimerisation of HP1 proteins. 2.3.2. The interaction network which allows the establishment and maintenance of pericentromeric heterochromatin

Diverse epigenetic modifications act on heterochromatin, maintaining this structure conformationally more compact and, as long considered, transcriptionally inactive. However, heterochromatin has been shown to be less transcriptionally silent than previously thought. It has been found that transcription itself and the produced RNA molecules act as important factors for its efficient organisation and maintenance.

Post-transcriptional histone modifications cooperate to establish silent heterochromatin state. These modifications are interdependent, often acting as scaffolds for the recruitment of chromatin modifiers and other chromatin-associated proteins. Maintenance of underacetylated histone tails is crucial for histone methylation and subsequent HP1 accumulation at pericentromeric heterochromatin [START_REF] Rea | Regulation of chromatin structure by site-specific histone H3 methyltransferases[END_REF]. Long term exposure to histone deacetylase inhibitor TSA leads to a loss of HP1 proteins, defects during chromosome segregation and relocalisation of pericentromeric heterochromatin to nuclear periphery in mouse cells [START_REF] Taddei | Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases[END_REF]. Once deacetylated, histone tails accumulate different methylation patterns. The methylation of lysine 9 of histone H3 catalysed by Suv39 is a hallmark of heterochromatin, and both Suv39h and the modification it catalyses are indispensable for the propagation of heterochromatin domain and proper function of the centromere (Figure 12). Modification of histone H4 is mediated by histone methyltransferase Suv4-20h2 in a Suv39h dependent manner. Suv4-20h2 associates with pericentromeric heterochromatin via interaction with HP1, where it contributes to cohesin recruitment to pericentromeres and methylates H4K20 that participates to the establishment of transcriptionally silent heterochromatic state [START_REF] Kourmouli | Heterochromatin and tri-methylated lysine 20 of histone H4 in animals[END_REF][START_REF] Schotta | A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin[END_REF]. It is probable that Suv4-20 interacts with multiple HP1 molecules stabilising their association with heterochromatin and making heterochromatin less accessible by mediating interactions between chromatin fibres [START_REF] Hahn | Suv4-20h2 mediates chromatin compaction and is important for cohesin recruitment to heterochromatin[END_REF]. DNA methylation is also found to be dependent on the Suv39h tri-methylation of lysine 9. DNA methyltransferase Dnmt3b, required for de novo DNA methylation accumulates at pericentromeric heterochromatin via HP1 where it binds to the Suv39h methylated H3K9, forming a repressive complex that help to maintain silent heterochromatin [START_REF] Lehnertz | Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin[END_REF]. The interaction between Swi6 and Clr4 histone methyltransferase in fission yeast is important for the spreading of H3K9 methylation mark (see paragraph 2.3.3 of this Chapter).

Together with the RNA interference pathway, this interaction is important as it provide a selfassembly mechanism that is the basis for the formation of heterochromatin. Clr4 has also been shown to bind to H3K9 through its chromodomain in fission yeast [START_REF] Zhang | Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin[END_REF]). In mammals it has been proposed that Suv39h interacts with multiple factors that are responsible for its tethering to pericentric heterochromatin and spreading of the repressive domain.

It was recently shown that in mammals, the HP1 interaction with Suv39h is not as important as previously thought for spreading of H3K9me3. In Suv39h double null cells, the H3K9me3 mark was completely recovered over heterochromatin when mouse ES cells were rescued with the Suv39h mutant unable to bind HP1. Moreover, Suv39h accumulated to pericentromeric regions and DNA methylation marks were re-established [START_REF] Muramatsu | Pericentric heterochromatin generated by HP1 protein interactiondefective histone methyltransferase Suv39h1[END_REF]. It is therefore possible that the Suv39h/Clr4 interaction with methylated H3K9 creates new binding sites for Suv39h/Clr4 by modification of adjacent H3K9, as well as other chromatin bound proteins and modification marks, promoting in this way the spreading of heterochromatin [START_REF] Müller-Ott | Specificity, propagation, and memory of pericentric heterochromatin[END_REF]. Furthermore, HP1 accumulation also depends on H3K27me3. Indeed, the accumulation of H3K27me3 at pericentric heterochromatin is stabilised by its interaction with Polycomb repressive complex 2 that catalyses H3K27me2/3 [START_REF] Boros | Polycomb repressive complex 2 and H3K27me3 cooperate with H3K9 methylation to maintain heterochromatin protein 1α at chromatin[END_REF].

Heterochromatin assembly in S pombe requires RNA interference

RNA interference is a process by which the expression of a gene is specifically inhibited by double-stranded RNA (dsRNA). This post-transcriptional silencing process (PTGS), initially discovered in plants, was described for the first time in C. elegans [START_REF] Fire | Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[END_REF]. The initial long double-stranded RNA are processed by an endonuclease Dicer to generate short, 21 bp siRNA. These siRNAs are loaded to a ribonucleoprotein complex RISC (RNA induced silencing complex), which is guided by the siRNA to the complementary target RNA. RISC component, protein Argonaut cleaves the target RNA [START_REF] Kurreck | RNA interference: from basic research to therapeutic applications[END_REF].

Post-transcriptional gene silencing can also be induced by endogenous microRNA, where they can inhibit translation of the target RNA by the mechanism of steric blocking. RNA silencing mostly functions at the post-transcriptional level, interfering with the target RNA, but it can also act directly at the level of chromatin. In this case, the siRNA targets a specific genomic locus to direct the formation of repressive chromatin domain. The process has first been described in S. pombe as a transcriptional gene silencing mechanism [START_REF] Almeida | RNA silencing and genome regulation[END_REF]).

In S. pombe, repetitive DNA sequences at pericentromeric region require an RNA interference mechanism for the establishment of repressive epigenetic modifications and heterochromatin assembly (Figure 13). Small RNA complementary to the yeast pericentromeric dh and dg repeats are transcribed and further processed by the RNAi machinery. Transcribed dsRNA is cleaved by Dicer1 to generate double stranded siRNA, which are then bound by Ago1. Ago1, Tas3 and Chp1 assemble to form a RNA interference transcriptional silencing complex (RITS). RITS localizes to heterochromatin, where it associates with RNA-dependent RNA polymerase complex (RDRC). Association of these two complexes, as well as their interaction with siRNA seem to depend on Dicer and Clr4. Loss of Dicer causes delocalization of RITS from heterochromatin, suggesting that the siRNA guides RITS to the complementary sequence on heterochromatin. The histone methyltransferase Clr4

forms Clr-C (cryptic loci regulator complex), a protein complex that is recruited to heterochromatin and catalyses methylation of H3K9, creating new binding sites for chromodomain proteins Swi6 and Chp1. Chp1, a component of RITS, stabilises RITS by binding to methylated H3K9 nucleosomes. RDRC then generates new dsRNA using nascent RNA as a template, contributing to the self reinforcing loop of heterochromatin assembly [START_REF] Verdel | RNAi-mediated targeting of heterochromatin by the RITS complex[END_REF][START_REF] Motamedi | Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs[END_REF][START_REF] Bühler | Tethering RITS to a nascent transcript initiates RNAi-and heterochromatin-dependent gene silencing[END_REF]. Transcription of pericentromeric repeats occurs during the S phase and is mediated by RNA polymerase II [START_REF] Djupedal | RNA Pol II subunit Rpb7 promotes centromeric transcription and RNAi-directed chromatin silencing[END_REF]. At this time, phosphorylation of serine 10 of histone H3 (H3S10) by Aurora kinase causes delocalisation of chromodomain proteins Swi6, Chp1 and Chp2 from heterochromatin. The resulting relaxation of heterochromatin causes a change in transcriptional activity and permits transcription of the repeats. H3K9 methylation mark, disrupted during transcription, is restored by RNAi-mediated transcripts that guide Clr-C to heterochromatin and permits restoration of Swi6 before the end of the S-phase [START_REF] Kloc | RNA interference guides histone modification during the S phase of chromosomal replication[END_REF]).

The proposed model where the targeting of RISC to heterochromatin is caused by its interaction with nascent transcripts has been termed co-transcriptional gene silencing. Bound to heterochromatin, RITS complex releases RNA pol II from heterochromatin and promotes formation of a silent heterochromatin state [START_REF] Bühler | Tethering RITS to a nascent transcript initiates RNAi-and heterochromatin-dependent gene silencing[END_REF][START_REF] Zaratiegui | RNAi promotes heterochromatic silencing through replicationcoupled release of RNA Pol II[END_REF]. In mammals, the regulation of transcription also seems to be cell cycle regulated. Although there is evidence that centromeric RNA in mammals are processed and repressed by RNA interference, there is no clear evidence that support the direct role for the RNA interference in heterochromatin assembly.

Role of pericentromeric heterochromatin

Enriched in cohesin, pericentromeric regions are the last part of sister chromatids to be separated during cell division. When the chromosomes attach to the spindle, the pulling forces of the microtubules act on chromosomes with a force that would immediately detach sister chromatids before their alignment at the metaphase plate.

The attachment of sister chromatids is therefore mediated by the multiprotein complex called cohesin. In budding yeast, the separation of sister chromatids in anaphase depends on the cohesin removal from chromatin by the cleavage of cohesin subunit sister chromatin cohesion 1 (SSC1) [START_REF] Uhlmann | Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1[END_REF]. Only then can the anaphase be activated and the separation of sister chromatids can begin. In fly and vertebrates the cohesin is eliminated from the chromosome arms already in the prophase, long before the sister chromatid separation [START_REF] Darwiche | Characterization of the components of the putative mammalian sister chromatid cohesion complex[END_REF]. At the pericentromeric region, the cohesin remains intact and is retained there until the onset of anaphase [START_REF] Waizenegger | Two distinct pathways remove mammalian cohesin from chromosome arms in prophase and from centromeres in anaphase[END_REF][START_REF] Warren | The Drosophila RAD21 cohesin persists at the centromere region in mitosis[END_REF]. The heterochromatin formed at pericentromeric repeats recruits cohesin complex at this region thereby insuring proper chromosome segregation. Indeed, the loss of HP1 at pericentric heterochromatin leads to defects in chromatid cohesion and aberrant chromosome segregation in mitosis [START_REF] Bernard | Requirement of heterochromatin for cohesion at centromeres[END_REF][START_REF] Nonaka | Recruitment of cohesin to heterochromatic regions by Swi6/HP1 in fission yeast[END_REF]. In mammalian cells, however, cohesin complex does not interact directly with HP1 and is probably recruited to pericentromeric heterochromatin by interaction with histone methyltransferase Suv4-20h2 that requires HP1

proteins for its stable loading to pericentromeric heterochromatin [START_REF] Hahn | Suv4-20h2 mediates chromatin compaction and is important for cohesin recruitment to heterochromatin[END_REF].

Studies in fission yeast suggest that pericentromeric heterochromatin is also required for establishment of CENP-A. It was shown that after introduction of naked centromeric and pericentromeric sequences to fission yeast cells lacking the heterochromatin components, CENP-A could not be established de novo [START_REF] Folco | Heterochromatin and RNAi are required to establish CENP-A chromatin at centromeres[END_REF]. Moreover, regions surrounding neocentromeres in humans contain essential heterochromatin features such as HP1, suggesting that heterochromatin environment is important for the centromere function [START_REF] Amor | Neocentromeres: role in human disease, evolution, and centromere study[END_REF]. Indeed, heterochromatin might be necessary for the integrity of centromeric regions acting by preventing the spreading of CENP-A containing nucleosomes, stabilizing in this way the position of the centromere [START_REF] Maggert | The activation of a neocentromere in Drosophila requires proximity to an endogenous centromere[END_REF]. Heterochromatin itself however, has the possibility to spread, influencing the expression of euchromatic domains by promoting epigenetic silencing. For example, the inactivation of one of the X chromosomes in females occurs by expansion of heterochromatin from one single locus, causing inactivation of the entire chromosome [START_REF] Heard | Delving into the diversity of facultative heterochromatin: the epigenetics of the inactive X chromosome[END_REF]. Euchromatic loci can also be silenced when found in the proximity of heterochromatin. If a gene that is usually expressed repositions by mechanisms of inversion or translocation close to heterochromatin, it will become silenced and the resulting phenotype will be variegated. This effect was first described in Drosophila under the name position effect variegation (PEV) [START_REF] Elgin | Position-effect variegation, heterochromatin formation, and gene silencing in Drosophila[END_REF] repetitive DNA sequences mark mouse centromeric region: minor and major satellites (Figure 14). The mouse centromere core is characterized by the presence of minor satellite sequence.

Its basic repeat unit is 120 bp long and it is repeated more than 2500 times on each chromosome. Pericentromeric regions comprise the more abundant major satellite sequence.

The 234 base pair major satellite is repeated up to 6 megabases in length, making up around 10% of the mouse genome [START_REF] Wong | Sequence organization and cytological localization of the minor satellite of mouse[END_REF][START_REF] Guenatri | Mouse centric and pericentric satellite repeats form distinct functional heterochromatin[END_REF]. The sequence has an unequal distribution of adenine and tymidine bases: one strand is purine and the other pyrimidine rich. Epigenetic modifications that mark mouse centromeric regions clearly differentiate the minor from the major satellites. Minor satellites are the place for kinetochore assembly, where histone H3 combines with its centromeric variant CENP-A. Major satellites carry typical heterochromatin characteristics. HP1 and an RNA molecule transcribed from the major satellite region itself are found to be required for organisation of pericentromeric heterochromatin in mouse [START_REF] Guenatri | Mouse centric and pericentric satellite repeats form distinct functional heterochromatin[END_REF][START_REF] Maison | SUMOylation promotes de novo targeting of HP1α to pericentric heterochromatin[END_REF], see Chapter 2). As in D.

melanogaster and S. pombe, centromeric and pericentromeric domains in mouse replicate during different time frame in the S-phase. Whereas minor satellites replicate late during Sphase, major satellites replicate during mid S-phase. This difference in the replication time is suggested to be a contributing factor for the establishment of different modification patterns and organisation of these two domains [START_REF] Guenatri | Mouse centric and pericentric satellite repeats form distinct functional heterochromatin[END_REF]).

The major satellite repeats are extremely abundant in mouse cells, carrying specific epigenetic marks and forming specific nuclear structures called chromocenters. One of the advantages in dealing with these highly repetitive sequences is that there is no interchromosomal variability since all mouse chromosomes carry the same repeat unit of a known sequence. Minor satellite sequences are located on the centromere (green) while the major satellite sequences (pink) are located around the centromere.

Association of pericentromeric regions

A current assumption is that the organisation of chromatin in the nucleus into the socalled chromatin domains and their localisation in the nucleus are important for functional organisation of the nucleus. It has been observed that heterochromatin has a tendency to be predominantly associated with the nuclear periphery and can be found around the nucleoli borders, while euchromatin mostly occupies the nuclear interior [START_REF] Gibcus | The hierarchy of the 3D genome[END_REF].

The only exception to this nuclear organisation are the rod receptor cells of nocturnal mammals, where the nuclear localization of heterochromatin and euchromatin in these cells are inverted. The inversion takes place during rod cell maturation and affects the optical properties of the retina [START_REF] Solovei | Nuclear architecture of rod photoreceptor cells adapts to vision in mammalian evolution[END_REF]. Heterochromatin is bound to the nuclear periphery by two different nuclear tethers, the inner nuclear membrane protein lamin B receptor LBR, and the lamin A/C (Solovei et al. 2013). This conventional nuclear organisation with the heterochromatin on the inner nuclear membrane seems to facilitate gene interaction and transcriptional regulation by promoting interaction between chromosomes.

Association of heterochromatin with nuclear envelope seems to affect the correct positioning of genes in the nucleus and affect transcription [START_REF] Solovei | Nuclear architecture of rod photoreceptor cells adapts to vision in mammalian evolution[END_REF].

Visible organisation of chromatin into distinct nuclear structures is particularly remarkable in the nucleus of mouse cells and can be observed as well in several other species such are some plants and Drosophila [START_REF] Zhang | The Drosophila salivary gland chromocenter contains highly polytenized subdomains of mitotic heterochromatin[END_REF][START_REF] Fransz | Interphase chromosomes in Arabidopsis are organized as well defined chromocenters from which euchromatin loops emanate[END_REF].

Pericentromeric heterochromatin from different chromosomes associates into distinct domains forming spherical nuclear compartments, so-called chromocenters, in the interphase nuclei (Figure 15). These clusters can be easily seen under the microscope as large bright spots in the nucleus when cells are counterstained with fluorochromes such as Hoechst or DAPI that preferentially intercalate with A/T-rich sequences. Minor satellites appear around the chromocenters as discrete spots. Differences in the organisation of chromocenters, notably their size, number and localization change between different cell types. For example, differentiation of myoblast to myotubes in mouse triggers nuclear reorganisation and clustering of pericentromeric heterochromatin. This activity seems to be dependent on histone deacetylation, which leads to further establishment of heterochromatin marks [START_REF] Terranova | The reorganisation of constitutive heterochromatin in differentiating muscle requires HDAC activity[END_REF]. Study on a wide range of different mouse cells showed that chromocenter size depends largely on the shape of the nucleus [START_REF] Mayer | Common themes and cell type specific variations of higher order chromatin arrangements in the mouse[END_REF]. Cells that possess large, spherical nuclei show the highest degree of clustering, with a small number of large chromocenters (lymphocyes, macrophages and myotubes). On the contrary, cells with an elipsoidal nuclei are associated with lower degree of clustering and contain higher number of small chromocenters (fibroblasts and myoblasts) [START_REF] Mayer | Common themes and cell type specific variations of higher order chromatin arrangements in the mouse[END_REF]. The common feature of the majority of chromocenters is their contact with the nuclear border [START_REF] Mayer | Common themes and cell type specific variations of higher order chromatin arrangements in the mouse[END_REF]). This association is possibly non-random as there could also be preferences for the aggregation of pericentromeric regions between certain chromosomes [START_REF] Vadakkan | Cell-type specific proximity of centromeric domains of one homologue each of chromosomes 2 and 11 in nuclei of cerebellar Purkinje neurons[END_REF]. surrounding the chromocenters as discrete spots [START_REF] Guenatri | Mouse centric and pericentric satellite repeats form distinct functional heterochromatin[END_REF]).

The functional significance of this clustering is still unclear, but it was suggested that it may serve to compartmentalize heterochromatin from the rest of the genome, thus allowing accumulation of epigenetic factors and the maintenance of heterochromatin status [START_REF] Almouzni | Heterochromatin maintenance and establishment: lessons from the mouse pericentromere[END_REF]). It has also been observed that association of euchromatic loci with chromocenters correlates with their silencing, suggesting that chromocenters might participate to the mechanisms of gene regulation [START_REF] Brown | Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin[END_REF]Roldán et al. 2005).

CHAPTER II.

Non-coding RNA from centromeric and pericentromeric regions Discovery of mRNA, tRNA and rRNA or even new classes of small nuclear and nucleolar RNA, all of them dedicated to the production of functional proteins, reinforced the view of the RNA as a template and a platform for protein synthesis [START_REF] Morris | The rise of regulatory RNA[END_REF].

Discovery of transcription factors established the idea of proteins as not only enzymes and structural components but also as molecules capable to directly regulate gene expression [START_REF] Morris | The rise of regulatory RNA[END_REF]). In the late 1960s, after the discovery of retrotransposon sequences, it was suggested that the RNA might be involved in the regulatory network of gene expression in animals [START_REF] Britten | Gene regulation for higher cells: a theory[END_REF][START_REF] Davidson | Sequence organization in animal DNA and a speculation on hnRNA as a coordinate regulatory transcript[END_REF]. Finding that the coding sequences of higher organisms are interspersed with non-coding regions, both of which are transcribed together, was however taken with surprise [START_REF] Berget | Spliced segments at the 5' terminus of adenovirus 2 late mRNA[END_REF][START_REF] Chow | An amazing sequence arrangement at the 5' ends of adenovirus 2 messenger RNA[END_REF]. These non-coding sequences termed introns, were found to be excised from the primary RNA transcript and were interpreted as remnants of no longer functional genes that have taken a new role and are now only significant for the mechanism of alternative splicing.

In the early 1980's two research groups independently discovered that RNA molecules are capable to catalyse chemical reactions [START_REF] Kruger | Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena[END_REF][START_REF] Guerrier-Takada | The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme[END_REF]).

Since then, many of these types of RNAs possessing enzymatic properties, termed ribozymes, have been described. These discoveries supported the now widespread hypothesis of the RNA World [START_REF] Gilbert | Origin of life: The RNA world[END_REF], which postulates that early in the evolution of life, the flow of genetic information was assured by replicating RNA molecules with catalytic activity [START_REF] Orgel | Prebiotic chemistry and the origin of the RNA world[END_REF]).

Finally, discoveries of RNA with no protein coding function such as micro RNA and small interfering RNA put RNA in a new perspective. These were the first evidences for the existence of new classes of RNA, non-coding RNA that turned out to have important and widespread biological functions. The significance of non-coding RNA was further supported by the finding that majority of DNA across species is non-protein coding. While the prokaryotic genomes are composed mostly out of protein-coding sequences and contain a limited number of regulatory RNAs [START_REF] Gottesman | Micros for microbes: non-coding regulatory RNAs in bacteria[END_REF], the genomes of more complex organisms are dominated by non-protein coding sequences.

1.1.2. Non-coding RNA as a key to complexity

According to Taft et al., complexity of an organism is regarded as a combination of metabolic and developmental complexity that reflects the number and type of cells and the degree of cellular organisation [START_REF] Taft | The relationship between non-protein-coding DNA and eukaryotic complexity[END_REF]. It was therefore expected that the complexity would correlate with a high number of genes, defined as protein coding sequences and their associated regulatory elements, allowing regulated expression of proteins necessary to preform diverse functions in such an organism. However, the proportion of protein-coding sequences actually decreases with organismal complexity and this has been called the G-value paradox [START_REF] Hahn | The g-value paradox[END_REF]. The percentage of protein coding genome in prokaryotes is ∼ 90% and this number linearly decreases being ∼68% in yeast, ∼25% in nematodes, ∼17% in insects, ∼9% in pufferfish, reaching ∼2% in chicken and ∼1% in mammals [START_REF] Costa | Non-coding RNAs, epigenetics and complexity[END_REF].

The estimated number of protein coding genes in humans fell from previous estimates that reached 140 000 to around 20 000. Large portions of eukaryotic genomes are, however, expressed but do not code for proteins. There are an ever-increasing number of non-coding transcripts identified in eukaryotes. Two possible explanations have been proposed for the existence of such species. Either these transcripts represent a transcriptional "noise" or they represent a newly discovered network of regulation of genetic information present in the form of the non-coding RNA [START_REF] Mattick | A new paradigm for developmental biology[END_REF]. Recent evidences support the idea of the functionality of these non-coding transcripts. Numerous loci have the ability to express non-coding transcripts that are cell type specific and differentially expressed during development. There is also an increasing number of well characterize functional non-coding RNA [START_REF] Mattick | Non-coding RNA[END_REF]. The recent finding of the ENCODE project [START_REF] Bernstein | An integrated encyclopedia of DNA elements in the human genome[END_REF]) further highlighted the significance of the non-coding portion of the genomes, allowing a more thorough annotations of the regulatory regions and identifying new non-coding transcripts and pseudogenes present in genomes of eukaryotic organisms. Surely, this needed a redefining of the current definition of a gene. In 2007, a new definition of a gene took into account all the recent findings of non protein-coding but actively transcribed and potentially functional transcripts: a gene is a "union of genomic sequences encoding a coherent set of potentially overlapping functional products" [START_REF] Gerstein | What is a gene, post-ENCODE? History and updated definition[END_REF]).

According to Mattick, the ncRNA could be the key to complexity of higher organisms (Figure 16). In other words, the idea is that complex organisms possess an ability to use a "digital programming system" that is based on non-coding RNAs. These RNAs would have for main function to be signalling molecules that coordinate a complex molecular network.

The signalling would be performed at two levels, either by sequence specific interactions or by secondary and tertiary structure through which they are able to target proteins to convey different actions, one of them being a change in chromatin conformation [START_REF] Mattick | RNA regulation: a new genetics? Nature reviews[END_REF][START_REF] Mattick | A new paradigm for developmental biology[END_REF]).

Emerging role for ncRNAs

In 1993, a small, 22 nt RNA was discovered that could inhibit the translation of its complementary target mRNA [START_REF] Lee | The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[END_REF]. This was the first evidence for the existence of non-coding regulatory RNA, a discovery that opened a new field of studies. Today, it is established that a variety of both short and long non-coding RNA are involved in numerous biological processes such as transcriptional and post-transcriptional regulation of gene expression, chromatin structure and nuclear architecture [START_REF] Mattick | RNA regulation of epigenetic processes[END_REF]) (Table 1). There is no general unified classification of non-coding transcripts. However, many authors suggest that the non-coding RNA can be divided into short (>200 nt) and long (>200 nt) transcripts [START_REF] Mercer | Long non-coding RNAs: insights into functions[END_REF]).

Short ncRNA

One of the most studied short RNA are micro RNA (miRNA). These RNA are derived from hairpin precursor sequences and processed by the Dicer nuclease, which cleaves the hairpin structure, releasing the mature transcript [START_REF] Bernstein | Role for a bidentate ribonuclease in the initiation step of RNA interference[END_REF]. Dicer is involved in the formation of another class of endogenous non-coding RNA, called small interfering RNA (siRNA). Both siRNA and miRNA recruit a multiprotein complex, called RISC (RNA induced silencing complex) and are part of the RNA interference process, which presents an elegant mechanism of post-transcriptional gene regulation (Fire et al. 1998, Chapter I, Introduction). Unlike siRNA, which require complete sequence complementarity with its target, miRNA recognize their target RNA by complementarity to a 2-7 nt long seed region in the 3'UTR [START_REF] Birmingham | 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets[END_REF]). miRNA are one of the most abundant regulators of gene expression. They have been shown to regulate a myriad of processes including cell proliferation, cell death or fat metabolism. They constitute around 1% of predicted genes in humans, worms and flies. As they are differentially expressed during development and differentiation, it has been suggested that the miRNA profile could be unique to each cell type [START_REF] Bartel | Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs[END_REF].

Another endogenous class of short ncRNA are germline specific RNA that associate with Argonaut family of proteins called PIWI (Figure 17). These PIWI-interacting RNAs

Long ncRNA

Besides a wide range of small regulatory non-coding RNA, eukaryotic genomes possess a variety of long non-coding transcripts that can perform diverse functions (Figure 18). Long non-coding RNA were first described during large-scale sequencing of mouse cDNA libraries, known as the FANTOM project [START_REF] Okazaki | Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs[END_REF] 2) that trimethylates H3K27, or Trithorax group proteins, which methylates H3K4 [START_REF] Dinger | Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation[END_REF][START_REF] Khalil | Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression[END_REF]) (Figure 19).

Several of these non-coding RNA have been well characterized. One of these ncRNA is HOTAIR (Hox antisense intergenic RNA), one of many ncRNA that are expressed from human homeobox (Hox) loci. HOX loci (HOXA-D) code for transcription factors that control the early development of the embryo by directing the formation of body structure. As such, they are considered as the master regulators of embryonic development [START_REF] Rinn | Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs[END_REF]).

These regions are under epigenetic control, and non-coding RNAs play an important role in the regulation of their expression. HOTAIR, a non-coding RNA expressed from the HOXC cluster, recruits repressive complexes to silence the HOXD locus in trans. HOTAIR binds both the Polycomb remodelling complex PRC2, which trimethylates H3K27 and the lysine specific demethylase LSD1, which demethylates H3K4 [START_REF] Rinn | Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs[END_REF]).

Other long non-coding RNAs have been found to associate with chromatin repressive complexes to silence imprinted gene clusters. Imprinting is a mechanism where one allele of an autosomal gene is epigenetically silenced. The long ncRNAs Kcnqlot1 and Air use a similar mechanism to target repressive epigenetic marks on the silenced allele. Air associates with the H3K9 methyltransferase G9a on a Slc22a promoter, while Kcnq1ot1 interacts with both G9a and the PRC2 complex to direct H3K9 and H3K27 methylation to the Kcnq1 locus from which it is transcribed [START_REF] Nagano | The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin[END_REF][START_REF] Pandey | Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation[END_REF].

Inactivation of X chromosome is a mechanism of dosage compensation in mammalian female genomes, which ensures an equal expression of X-linked genes in both males and females in all cells (Figure 20). It has been studied most extensively in mouse, where the inactivation of the X chromosome is a result of an interaction of several non-coding transcripts. A regulatory region on the X chromosome called X inactivation centre controls the expression of the long ncRNA Xist. Xist coats the X chromosome from which it is expressed in cis to induce epigenetic silencing of the entire chromosome. How exactly Xist induces the inactivation of the entire chromosome is not fully understood. The non-coding RNA RepA, encoded within Xist and that is required for Xist induction was found to bind the PCR2 complex and to target it to the X chromosome to initiate silencing [START_REF] Zhao | Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome[END_REF].

Xist is also able to bind PCR2, and once it covers the inactive X, it can spread the repressive chromatin marks throughout the chromosome. X inactivation is an example of the interplay of several non-coding RNAs. In mouse, the levels of Xist are controlled by another non-coding RNA antisense to Xist, Tsix, that inhibits the expression of Xist at the future active X chromosome and seems to be implicated in the choice of the allele to be expressed during the X inactivation [START_REF] Lee | Targeted mutagenesis of Tsix leads to nonrandom X inactivation[END_REF]Navarro et al. 2006). Another ncRNA, Linx, has been proposed to control the expression of Tsix [START_REF] Nora | Spatial partitioning of the regulatory landscape of the X-inactivation centre[END_REF]. A recent discovery of a novel ncRNA, XACT, that covers the active X chromosome in humans, suggest the role of different non-coding transcripts in the control of a possible species-specific mechanisms of X inactivation [START_REF] Vallot | Long non-coding RNAs and human X-chromosome regulation: a coat for the active X chromosome[END_REF]. Transcription from mouse pericentromeric regions was observed some 20 years ago using isolated mouse genomic clone containing major satellite repeat. Satellite transcripts were detected by northern analysis and in situ hybridization in foetus and different adult tissues [START_REF] Rudert | Transcripts from opposite strands of gamma satellite DNA are differentially expressed during mouse development[END_REF]. Both sense and antisense transcripts were found to be spatially and temporally regulated. Ubiquitous distribution of transcripts was found in foetal tissues. In the cells of the central nervous system, one strand was found highly expressed during 12,5 to 15,5 days post coitum embryos, while the transcripts from the other were detected only during 11,5 days post coitum. These findings indicated to a precise temporal regulation of transcription in the brain. In the adult tissues, satellite transcripts were found only in liver and testis [START_REF] Rudert | Transcripts from opposite strands of gamma satellite DNA are differentially expressed during mouse development[END_REF]. More recent transcriptomic analysis of human centromeric and pericentromeric sequences confirmed that these sequences are generally not expressed in normal somatic tissues, with the exception of embryonic ovary, placenta and liver, where only transcription from the centromere was detected. Pericentromeric transcripts were, as previously, been found in the testis, suggesting that they might be involved in the process of spermatogenesis [START_REF] Eymery | A transcriptomic analysis of human centromeric and pericentric sequences in normal and tumor cells[END_REF]).

Centromeric transcripts as integral components of centromeric chromatin

Several studies revealed that the transcription of the centromere core domain in yeast and vertebrate cells can affect centromere integrity and kinetochore function (see further text).

Transcripts emanating from the centromere core domain might be involved in CENP-A deposition to the centromeres as well as in regulation of the binding of the kinetochore proteins during mitosis.

Centromeric RNAs regulate the kinetochore activity

There are certain indications that non-coding RNAs are important for the targeting of Upon exposure to thermal stress, vertebrate cells respond by activation of heat shock transcription factor 1 (HSF1). HSF1 binds to promoters of heat inducible genes to activate their transcription and the synthesis of specific proteins, called heat shock proteins. These proteins act as protein chaperones that prevent protein denaturation and aggregation that might occur in cells upon heat induced stress. In human cells, HSF1 has also been found to localise to pericentromeric regions of 14 human chromosomes rich in satellite II and III sequences, with a preferential localisation to the 9q12 region of chromosome 12 [START_REF] Jolly | In vivo binding of active heat shock transcription factor 1 to human chromosome 9 heterochromatin during stress[END_REF][START_REF] Denegri | Human chromosomes 9, 12, and 15 contain the nucleation sites of stress-induced nuclear bodies[END_REF][START_REF] Eymery | Heat shock factor 1 binds to and transcribes satellite II and III sequences at several pericentromeric regions in heat-shocked cells[END_REF]. HSF1 is found to activate the transcription of satellite III sequences. Satellite III transcripts are detected as long RNAs that vary in size from 2 to several kilobases, synthesised exclusively from the sense, G-rich strand of the repeat [START_REF] Jolly | Stress-induced transcription of satellite III repeats[END_REF][START_REF] Rizzi | Transcriptional activation of a constitutive heterochromatic domain of the human genome in response to heat shock[END_REF]). After transcription, they remain associated to their transcription sites and together with HSF1 form the so-called nuclear stress bodies [START_REF] Jolly | HSF1 transcription factor concentrates in nuclear foci during heat shock: relationship with transcription sites[END_REF]. Nuclear stress bodies appear transiently and exclusively in human cells and contain, besides HSF1 and satellite III transcripts, several splicing factors and RNA binding proteins.

Satellite III transcription can be induced to different extents by other stress inducing agents such as exposure to UV light and other DNA damaging agents, as well by oxidative stress [START_REF] Valgardsdottir | Transcription of Satellite III non-coding RNAs is a general stress response in human cells[END_REF]. Similar accumulation of satellite transcripts upon stress has been reported in plants and insects but their function is still not clear [START_REF] Pecinka | Epigenetic regulation of repetitive elements is attenuated by prolonged heat stress in Arabidopsis[END_REF][START_REF] Pezer | Satellite DNA-associated siRNAs as mediators of heat shock response in insects[END_REF]. Activation of satellite III transcription in these conditions suggest a conserved role for these transcripts in defence against stress.

It has been argued that these transcripts might serve as an assembly platform for RNAbinding proteins that can be found in nuclear stress bodies. Transcriptional activation of satellite III sequences might recruit factors involved in the regulation of gene expression such as transcription factors and RNA processing factors. Satellite III sequences could thus serve as regulatory domains that control expression of genes in their proximity in response to stress [START_REF] Valgardsdottir | Transcription of Satellite III non-coding RNAs is a general stress response in human cells[END_REF]). These transcripts might also participate to the reestablishment of heterochromatin after its disruption by heat shock. Indeed, after induction of stress, heterochromatin marks such as H3K9 methylation and HP1 become absent from this region, and there is an increase in the acetylation of histone H3, a characteristic of euchromatin [START_REF] Rizzi | Transcriptional activation of a constitutive heterochromatic domain of the human genome in response to heat shock[END_REF].

Another example of the transcriptional activation of repetitive sequences upon heat shock can be found in mouse cells. Even thought there is no evidence for the transcriptional activation of pericentromeric sequences upon thermal stress, the heat shock induces upregulation of two transcripts synthesized from short interspersed elements (SINE) called B1 and B2 RNA. B2 RNA has been found to associate with RNA polymerase II and transiently repress mRNA transcription as a response to stress [START_REF] Allen | The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock[END_REF]) (Figure 21). Microinjection of LNA/DNA gapmers directed against major satellite transcripts results in developmental arrest of the affected embryos. Moreover, the embryos failed to organise their pericentromeric heterochromatin into chromocenters [START_REF] Casanova | Heterochromatin Reorganization during Early Mouse Development Requires a Single-Stranded Noncoding Transcript[END_REF]). To discriminate between the contribution of sense and antisense transcripts to the reorganisation of heterochromatin, the embryos were injected with LNA/DNA gapmers targeting either forward of reverse transcripts. Even though elevated levels of mostly sense transcripts were observed during the 2-cell stage, interference with these transcripts did not affect development in comparison to control embryos. On the other hand, interference with antisense transcripts resulted in developmental arrest and failure of pericentromeric regions to organise into chromocenters. This demonstrates the importance of the antisense transcripts for nuclear reorganisation and embryo development [START_REF] Casanova | Heterochromatin Reorganization during Early Mouse Development Requires a Single-Stranded Noncoding Transcript[END_REF].

Another study showed that the developmental arrest of the embryos could be rescued by the injection of double stranded RNA [START_REF] Santenard | Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3[END_REF]. The existence of such species has not been excluded. It seems so far, however, that the single stranded, antisense RNA has a dominant role in pericentromeric heterochromatin organisation. A link between pericentromeric heterochromatin reorganisation and major satellite transcription has been established in Purkinje cells that are associated to rod receptors in mouse [START_REF] Solovei | Positional changes of pericentromeric heterochromatin and nucleoli in postmitotic Purkinje cells during murine cerebellum development[END_REF][START_REF] Solovei | Nuclear architecture of rod photoreceptor cells adapts to vision in mammalian evolution[END_REF]. During development, the changes in the appearance of chromocenters (their number, localisation and size) are accompanied with unusual modifications at the chromatin level in these cells, where a euchromatic histone mark, H3K4 trimethylation, becomes enriched at major satellites together with the repressive H3K9 trimethylation. The acquisition of activating chromatin marks result in an elevated level of satellite transcription [START_REF] Kishi | Transcriptional activation of mouse major satellite regions during neuronal differentiation[END_REF]).

Normal muscle cell differentiation is accompanied by a global reorganisation of heterochromatin and transcription from pericentromeric region. This process is characterized by centromere clustering and followed by an increase in H3K9 and H4K20 methylation at major satellites, and an accumulation of both major and minor satellite-derived transcripts.

Interestingly, the reorganisation of heterochromatin seems to be dependent on the activity of histone deacetylases, since the treatment of cells with a HDAC inhibitor prevents this reorganisation.

Altogether, these studies provide evidence that transcription from major satellites is involved in the reorganisation of heterochromatin at an early stage in development, when nuclear reorganisation takes place. This points to the existence of important regulatory mechanisms that act upon transcription of pericentromeric heterochromatin in a specific time window in early development [START_REF] Probst | A strand-specific burst in transcription of pericentric satellites is required for chromocenter formation and early mouse development[END_REF]. Only reverse RNA species have been found to be important for heterochromatin reorganisation, as the interference with the other strand seem not to affect this process. However, it can not be excluded that the transcripts from the opposite strand might have other still undefined roles during development [START_REF] Probst | A strand-specific burst in transcription of pericentric satellites is required for chromocenter formation and early mouse development[END_REF][START_REF] Casanova | Heterochromatin Reorganization during Early Mouse Development Requires a Single-Stranded Noncoding Transcript[END_REF]. The fact that the differentiation was not affected in cells which fail to reorganise their heterochromatin into chromocenters might suggest that, in the absence of the spatial organisation of heterochromatin, the heterochromatin structure is nevetherless maintained by other factors, allowing differentiation [START_REF] Terranova | The reorganisation of constitutive heterochromatin in differentiating muscle requires HDAC activity[END_REF].

Non-coding RNA in heterochromatin formation: lessons from fission yeast

The link between the RNAi machinery and pericentromeric transcription is well established in fission yeast [START_REF] Volpe | Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi[END_REF] Numerous examples show evidence for transcription from centromeric and pericentromeric repeats but there are still unanswered questions concerning the exact function of these RNA.

In vertebrate cells, small RNA species are found to be dependent on Dicer, one of the key proteins in RNA interference, which cleaves the dsRNA into small interfering RNAs. The correlation between the accumulation of major satellite transcripts and Dicer downregulation was described in mouse myoblasts, where cells change the expression of Dicer as they go through differentiation [START_REF] Terranova | The reorganisation of constitutive heterochromatin in differentiating muscle requires HDAC activity[END_REF]. Moreover, Dicer ablation causes derepression of both centromeric and pericentromeric transcripts in ES mouse cells [START_REF] Murchison | Characterization of Dicer-deficient murine embryonic stem cells[END_REF][START_REF] Kanellopoulou | Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing[END_REF]. Dicer deficient ES cells showed a reduced level of small RNA species (25-30 nt) while the longer RNA molecules became specifically enriched in comparison with cells expressing the protein [START_REF] Kanellopoulou | Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing[END_REF]). The same effect was observed in Dicer deficient DT40 hybrid chicken-human cell line [START_REF] Fukagawa | Dicer is essential for formation of the heterochromatin structure in vertebrate cells[END_REF]).

Dicer inactivation causes delocalisation of HP1 and loss of H3K9 methylation which is accompanied by mitotic defects such as premature sister chromatid separation. Small transcripts that correspond to centromeric alpha satellite repeats were strongly reduced in these cells [START_REF] Fukagawa | Dicer is essential for formation of the heterochromatin structure in vertebrate cells[END_REF]. It is however unclear whether loss of Dicer has a similar effect on pericentromeric heterochromatin in mouse [START_REF] Kanellopoulou | Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing[END_REF][START_REF] Murchison | Characterization of Dicer-deficient murine embryonic stem cells[END_REF]. Other evidence apart of these for the possible role of the RNAi machinery are scarce.

Long pericentromeric transcripts could be post-transcriptionally processed without the implication of the RNAi machinery. A DNA binding protein WDHD1 has been found to associate with centromeric chromatin during mid to late S phase in mouse cells [START_REF] Hsieh | WDHD1 modulates the post-transcriptional step of the centromeric silencing pathway[END_REF]. Its downregulation causes loss of HP1 and other heterochromatin associated marks, and the increase in histone H4 acetylation, leading to mitotic defects. Cells depleted of WDHD1 show altered sizes of both major and minor satellite-derived transcripts in a fashion similar to Dicer depletion. The small RNA molecules were significantly reduced in the favour of enrichment in large RNA species, suggesting that this protein is involved in posttranscriptional processing of centromeric and pericentromeric RNA [START_REF] Hsieh | WDHD1 modulates the post-transcriptional step of the centromeric silencing pathway[END_REF]).

Non-coding RNA as a component of pericentromeric heterochromatin

Transcription across satellite repeats have been shown to generate both ss-and dsRNA [START_REF] Martens | The profile of repeat-associated histone lysine methylation states in the mouse epigenome[END_REF][START_REF] Wong | Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere[END_REF]). Elevated levels of dsRNA were reported in Suv39dn, Dicer or transcription factor Pax3-deficient cells, confirming that it is important to preserve the correct levels of these transcripts in order to maintain heterochromatin integrity and proper centromere function [START_REF] Martens | The profile of repeat-associated histone lysine methylation states in the mouse epigenome[END_REF][START_REF] Fukagawa | Dicer is essential for formation of the heterochromatin structure in vertebrate cells[END_REF]Bulut-Karslioglu et al. 2012a). Changes in expression of molecules that reside at or remodel this region, such as HP1 and Suv39h1, have been mostly reported to induce transcription, having as a consequence the loss of heterochromatin integrity and detrimental effects on normal centromere function.

Indeed, it seems that pericentromeric RNA acts as a structural component of heterochromatin in mouse [START_REF] Maison | SUMOylation promotes de novo targeting of HP1α to pericentric heterochromatin[END_REF]. Reverse, purine rich major satellite transcripts have been found to associate with SUMO-ylated (Small Ubiquitin-like MOdifier) HP1. The major target of sumoylation is the hinge domain of HP1, which has an RNA binding activity. While the localisation of HP1 to the pre-existing HP1 domains at pericentromeric heterochromatin is possible even without the hinge domain, HP1 lacking the hinge domain is unable to localise to pericentromeric heterochromatin in Suv39h double null cells. This indicates that the transcripts from mouse major satellites are required for de novo HP1 alpha localisation at pericentromeric heterochromatin, probably providing an initial step for its targeting to pericentromeric heterochromatin [START_REF] Maison | SUMOylation promotes de novo targeting of HP1α to pericentric heterochromatin[END_REF]) (Figure 22).

As previously described, a similar interaction between proteins and pericentromericderived RNA could be happening in mouse embryos during first stages of mouse development [START_REF] Probst | A strand-specific burst in transcription of pericentric satellites is required for chromocenter formation and early mouse development[END_REF]. It is not known if the association of RNA with HP1 is cell cycle regulated. Two RNA species detected during G1/S and M phase in mouse cells [START_REF] Lu | Proliferation-dependent and cell cycle regulated transcription of mouse pericentric heterochromatin[END_REF]) could be connected to reestablishment of heterochromatin during replication or could be linked to the loading of HP1 after chromocenter replication or at the end of mitosis.

II.3. Regulation of pericentromeric transcription

Chromatin modifications and transcription

Transcription of centromeric regions is indeed possible despite the numerous histone modifications and DNA methylation marks that are the hallmarks of the so-called silent chromatin [START_REF] Kishi | Transcriptional activation of mouse major satellite regions during neuronal differentiation[END_REF]. Transcription is observed in a variety of cell types in normal growth condition and during differentiation, despite the persistence of H3K9me3 and H4K20me3 marks [START_REF] Martens | The profile of repeat-associated histone lysine methylation states in the mouse epigenome[END_REF][START_REF] Terranova | The reorganisation of constitutive heterochromatin in differentiating muscle requires HDAC activity[END_REF][START_REF] Maison | SUMOylation promotes de novo targeting of HP1α to pericentric heterochromatin[END_REF]) (Figure 23). However, transcriptional activation of centromeric region is usually followed by abrupt changes in chromatin marks.

3.1.1.Histone modifications

Histone methylation

In cells depleted of the histone demethylase Suv39, there is an increase in transcription from both major and minor satellites [START_REF] Lehnertz | Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin[END_REF][START_REF] Martens | The profile of repeat-associated histone lysine methylation states in the mouse epigenome[END_REF]. These transcripts might accumulate as their processing is impaired due to the lack of recruitment of processing factors or RNAi machinery after the loss of H3K9me3 in Suv39-depleted cells [START_REF] Martens | The profile of repeat-associated histone lysine methylation states in the mouse epigenome[END_REF]. The knock out of histone demethylase KDM2A has been shown to increase the levels of H3K36me2, delocalises HP1 from pericentromeres and increase transcription from both human and mouse pericentromeres but not centromeres [START_REF] Frescas | KDM2A represses transcription of centromeric satellite repeats and maintains the heterochromatic state[END_REF]. Moreover, the transcriptional activation of major satellites has also been observed during neuronal development, during which the H3K4me3, an "active" chromatin mark is established despite the existence of H3K9me3 [START_REF] Kishi | Transcriptional activation of mouse major satellite regions during neuronal differentiation[END_REF].

Experiments on human artificial chromosome show that depleting H3K4me2, a mark constitutively found on the centromeres, by tethering the H3K4me2-specific demethylase LSD1 to the HAC kinetochore causes a rapid decrease in centromeric transcription and interferes with the recruitment of HJURP to the centromeres and subsequent CENP-A deposition. This suggests a tight relationship between centromeric transcription and maintenance of chromatin at the centromere [START_REF] Bergmann | Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore[END_REF].

Histone acetylation

Maintaining the hypoacetylated state is required for efficient heterochromatin organisation and genome integrity as it has been shown that changes in histone acetylation lead to defects in centromere function [START_REF] Taddei | Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases[END_REF]. Indeed, deacetylation of histone H3 is required for its subsequent methylation by Suv39h [START_REF] Rea | Regulation of chromatin structure by site-specific histone H3 methyltransferases[END_REF]. Interestingly, treatment of cells with the histone deacetylase inhibitor TSA does not seem to influence transcription from either pericentromeric or centromeric regions in mouse cells. However, it disrupts the organisation of pericentric heterochromatin. HP1 and H3K9me3 are lost from mouse chromocenters in TSA treated cells, indicating that deacetylase activity is necessary for the organisation of heterochromatin [START_REF] Maison | Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component[END_REF].

Maintenance of heterochromatic state is required during S-phase when the replication disrupts heterochromatin domains. During chromocenter replication in mouse cells, newly deposited histones are epigenetically modified in order to allow for the re-formation of heterochromatin. For example, the histone binding protein Np95 recruits histone deacetylase, which subsequently deacetylates histone H4 following their deposition to newly synthesized chromatin, allowing the establishment of heterochromatin state after replication. Absence of Np95 results in the increased transcription of major satellite sequences, as the deacetylation of H4 at lysines 8 (H4K8) and 16 (H4K16) is necessary for the correct establishment of heterochromatin following replication [START_REF] Papait | Np95 is implicated in pericentromeric heterochromatin replication and in major satellite silencing[END_REF]. It is however still not clear whether the increased transcription is due to the increased transcriptional rate or is the observed RNA accumulation due to the inhibition of the processing enzymes, such as Dicer.

Upregulation of human centromeric transcripts in response to stress is accompanied by an increase in acetylated histone H4 acetylation and an accumulation of histone acetyltransferase CPB at nuclear stress bodies, that are forming at the 9q12 locus. Enrichment of acetylated H4K8 and H4K16 at nuclear stress bodies and the loss of conventional chromatin marks such as HP1 and H3K9me3 suggest a different heterochromatin organisation in nuclear stress bodies that could facilitate the stress induced transcription at this locus [START_REF] Jolly | Stress-induced transcription of satellite III repeats[END_REF][START_REF] Rizzi | Transcriptional activation of a constitutive heterochromatic domain of the human genome in response to heat shock[END_REF]).

DNA methylation

DNA methylation is highly enriched at centromeric regions in a variety of organisms where it contributes to epigenetic silencing. Loss of DNA methylation might therefore facilitate transcription of these regions, but the actual link between this epigenetic mark and the levels of centromeric and pericentromeric transcripts is not completely clear.

DNA methylation is correlated with methylation of the lysine 9 of histone H3 in several organisms, including mouse ES cells. The Dnmt3b DNA methyltransferase associate with HP1 and is targeted to mouse heterochromatin by Suv39h-dependent H3K9 trimethylation. Although these heterochromatin marks seem to act together to regulate chromatin silencing, major satellite transcripts were found to be upregulated only in Suv39dn

but not in Dnmt3b or Dnmt1 (the DNA methyltransferase responsible for the establishment of methylation patterns following replication) deficient cells [START_REF] Lehnertz | Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin[END_REF][START_REF] Martens | The profile of repeat-associated histone lysine methylation states in the mouse epigenome[END_REF]. Similarly, a double knock out for these two DNA methyltransferases in human HeLa cells showed no changes in the pericentromeric or centromeric sequence expression.

On the other hand, an accumulation of pericentromeric, but not centromeric, sequences was observed in HeLa cells treated with DNA methylation inhibitor, 5-azacytidine [START_REF] Eymery | A transcriptomic analysis of human centromeric and pericentric sequences in normal and tumor cells[END_REF]. Inversely, treatment of mouse MEL cells with 5-azacytidine resulted in elevated level of only centromeric, minor satellite transcripts [START_REF] Bouzinba-Segard | Accumulation of small murine minor satellite transcripts leads to impaired centromeric architecture and function[END_REF]. These examples suggest that in the case of enzyme knock out, there are other possible mechanisms that act to repress centromeric and pericentromeric transcription. Moreover, the existence of independent pathways that repress transcription of either centromeric or pericentromeric sequences could be envisaged. In human cells, two transcription factors, HSF1 and Tonicity enhancer binding protein (tonEP), have been identified to control expression of pericentromeres during heat induced and osmotic stress, respectively [START_REF] Jolly | Stress-induced transcription of satellite III repeats[END_REF]Valgardsdottir et al. 2008, paragraph 2.3.1. of this Chapter).

Transcription factors

In mouse, the transcription factor Snail1 has been found to trigger epithelial-to-

mesenchymal

Cell cycle

The possibility that centromeric and pericentromeric transcript levels could be cellcycle regulated could explain the inconsistencies in their detection in mammalian cells. In mouse, major satellite transcripts of different sizes are synthesised during different times of the cell cycle in proliferating cells [START_REF] Lu | Proliferation-dependent and cell cycle regulated transcription of mouse pericentric heterochromatin[END_REF]. A heterogeneous set of transcripts larger than 1kb is detected during G1 phase. These transcripts accumulate until they reach a peak in G1/S, after which their transcription starts to decrease. This decrease coincides with the time of chromocenter replication, which starts at the late S phase [START_REF] Wu | Uncoupling global and fine-tuning replication timing determinants for mouse pericentric heterochromatin[END_REF].

Another class of smaller RNA species of a size around 150 nt, were detected in mitotic cells, [START_REF] Meister | Sequence-specific inhibition of microRNA-and siRNA-induced RNA silencing[END_REF][START_REF] Hutvágner | Sequence-specific inhibition of small RNA function[END_REF]).

To further improve the binding of the modified oligonucleotide to its target sequence, the preorganisation of the phosphate backbone was achieved by the restriction of the conformation of the sugar monomer by covalently linking the 2' and 4' positions of the ribose.

This 2'-4' bicyclic nucleic acids such as LNA, have the sugar locked into 3' endo conformation that drastically enhance the properties of the modified oligonucleotides. These oligonucleotides show the highest improvement of the binding affinity to the complementary RNA among the known sugar modifications (Prakash 2011).

Locked nucleic acids

A new type of oligonucleotide termed LNA was first synthesised and used in a hybridization in 1998 and is since been used for a variety of applications [START_REF] Koshkin | LNA (Locked Nucleic Acid): An RNA Mimic Forming Exceedingly Stable LNA:LNA Duplexes[END_REF][START_REF] Obika | Stability and structural features of the duplexes containing nucleoside analogues with a fixed N-type conformation, 2′-O,4′-C-methyleneribonucleosides[END_REF]. LNA oligonucleotide is a synthetic RNA derivative (ribonucleotide analogue) in which a ribose moiety is structurally "locked" by a methylene bridge between 4'carbon and 2'-oxygene from the ribose (Figure 25). This bridge reduces conformational

Thermodynamic properties of LNA

The potential of LNA lies in its thermodynamic properties. Thermostability of the nucleic acid duplex is highly increased with the use of LNA nucleotides. Due to its structure, LNA is able to hybridize to complementary nucleic acids with high affinity, without loosing the required sequence specificity. Incorporation of an LNA nucleotide can raise the melting temperature (Tm) of the duplex for 1-8 °C per LNA against complementary DNA and 2-10 °C per LNA against complementary RNA, in comparison to the unmodified duplex [START_REF] Kurreck | Design of antisense oligonucleotides stabilized by locked nucleic acids[END_REF]. This increase in Tm is the largest increase in thermostability of a nucleic acid analogue. This characteristic of LNA makes it unparalleled for the use in applications that require high affinity to the complementary strand. Other modified oligonucleotides do not

show such an effect. For example, while a 2,4-4° C increase per LNA was observed when the LNA monomer is introduced as an isolated residue into an 18-mer oligonucleotide, 2'-Omethyl modifications increased the Tm for less than 1°C [START_REF] Kurreck | Design of antisense oligonucleotides stabilized by locked nucleic acids[END_REF].

However, this increase in Tm largely depends on the number of modifications and their position in the oligonucleotide sequence. Moreover, as the number of incorporated LNA bases is higher, the increase per LNA base seems to decrease. Indeed, the thermostability of the oligonucleotide per LNA modification is observed to decrease when the substitution with an LNA nucleotide in the LNA:DNA mixmer reaches 50%. Long non-modified oligonucleotides that possess higher thermal stability do not seem to remarkably increase their melting temperature after LNA incorporation. Short modified oligonucleotides with incorporated LNA bases will, however, have the greatest thermal stability and provide high affinity for the complementary strand [START_REF] Braasch | Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA[END_REF]. DNA antisense oligonucleotides often non-specifically target RNA or even proteins [START_REF] Kurreck | Design of antisense oligonucleotides stabilized by locked nucleic acids[END_REF]. Among different modified oligonucleotides, LNA oligonucleotides are good candidates for antisense gene targeting due to their thermostability, high specificity and nuclease resistance, as exemplified in the following text.

Gapmer LNA oligonucleotides have been successfully used to target nuclear transcripts such as intergenic spacers that separate the rRNA genes [START_REF] Mayer | Intergenic transcripts regulate the epigenetic state of rRNA genes[END_REF]. LNA gapmers designed to specifically target both sense and antisense major satellite transcripts in mouse embryo have shown to have an effect on embryo development [START_REF] Probst | A strand-specific burst in transcription of pericentric satellites is required for chromocenter formation and early mouse development[END_REF][START_REF] Casanova | Heterochromatin Reorganization during Early Mouse Development Requires a Single-Stranded Noncoding Transcript[END_REF]. LNA mixmers have also shown to act as inhibitors of telomerase.

Telomerase is a ribonucleoprotein that contains an RNA, which serves as a template for maintaining the telomere length. It is expressed in cancer cells where it is thought to help sustain tumor proliferation. Successful interference with this RNA has been achieved using an LNA antisense acting as a strong inhibitor of telomerase [START_REF] Elayadi | Implications of High-Affinity Hybridization by Locked Nucleic Acid Oligomers for Inhibition of Human Telomerase †[END_REF]. Targeting of Xist lncRNA allows a rapid displacement of the RNA from the X chromosome, without affecting its stability. LNA mixmers designed to target specific domains of Xist proved that LNA could be used as a powerful tool for analysis of ncRNAs [START_REF] Sarma | Locked nucleic acids (LNAs) reveal sequence requirements and kinetics of Xist RNA localization to the X chromosome[END_REF]. LNA oligonucleotides are used as LNA-antagomirs, antisense oligonucleotides designed to silence specific miRNA that are widely used in loss of function experiments [START_REF] Naguibneva | An LNA-based loss-of-function assay for micro-RNAs[END_REF].

The LNA was one of the first modifications to be used to engineer siRNA. Chemical modifications of the siRNA have been shown to significantly improve the siRNA properties, without negatively affecting siRNA activity [START_REF] Braasch | Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA[END_REF].

b) LNA antigenes LNA can also directly target the chromosomal DNA for inhibition of gene expression.

These so-called LNA antigenes designed to bind to gene promoters can bind chromosomal DNA where they interfere with RNA polymerase and associated transcription factors to reduce RNA expression [START_REF] Beane | Recognition of chromosomal DNA inside cells by locked nucleic acids[END_REF]. All these cellular studies point to the usefulness of LNA oligonucleotides to target RNAs with specificity inside the cell. c) LNA as primers for PCR amplification Amplification by PCR requires specific hybridization to complementary DNA sequences. In cases where DNA template is in low quantities or if amplifying a repetitive region, PCR can be improved by the use of modified oligonucleotides. LNA modifications have been reported as efficient for use as primers where the amplification requires higher specificity and sensitivity [START_REF] Lundin | Biological activity and biotechnological aspects of locked nucleic acids[END_REF]. LNA primers have greater mismatch sensitivity and they can therefore be used under high annealing temperatures for selective amplifications against unmodified primers. Since LNA modifications increase the melting temperature of the duplex, the LNA primers can be made shorter than unmodified oligonucleotides. This is advantageous for amplification of highly similar sequences, as shorter oligonucleotides allow correct targeting between sequences that show low levels of variation. Unmodified primers of the same size would have too low melting temperature for a correct hybridization under standard conditions. However, caution must be taken when designing LNA primers as LNA position in a primer is crucial for an improved performance.

Modifications at the 5'end of the primer significantly improve PCR reactions when compared to modifications at the 3'-end positioning or LNA incorporation throughout the primer [START_REF] Levin | Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers[END_REF]. LNA primers have proven excellent for detection of single nucleotide polymorphism in genotyping experiments [START_REF] Latorra | Enhanced allele-specific PCR discrimination in SNP genotyping using 3' locked nucleic acid (LNA) primers[END_REF]. For example, the LNA incorporated at the 3'-end of the primer was used to quantify the methylation level of specific cytosines in a genome [START_REF] Thomassin | MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome[END_REF]).

d) LNA as hybridization probes

Unique properties of LNA oligonucleotides have opened new possibilities for detection by nucleic acid hybridization. DNA oligonucleotides with several LNA bases interspersed throughout the probe have been used in northern blot experiments for a specific detection of miRNA, characterisation of which has been difficult due to their small size and low abundancy [START_REF] Válóczi | Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes[END_REF][START_REF] Várallyay | MicroRNA detection by northern blotting using locked nucleic acid probes[END_REF]. Indeed, LNA modified probes showed 10-fold higher efficiency when compared to traditional DNA probes. This high specificity of the LNA probes was proved by the use of probes that contain an LNA at different positions [START_REF] Válóczi | Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes[END_REF]). Potential of LNA modified oligonucleotides has been extended to the design of fluorescently labelled LNA probes that can be used for fluorescent in situ hybridization (FISH). For example, LNA/DNA mixmers were used to probe a short human satellite II repeat sequence as well as alpha satellite repeats. Comparing to standard DNA probes, the mixmers give a strong signal with minimum signal to noise ratio in a shorter hybridization time [START_REF] Silahtaroglu | LNA-modified oligonucleotides are highly efficient as FISH probes[END_REF]).

The LNA used in northern experiments have shown to be more sensitive for target detection than their DNA counterparts. The incorporation of LNA increases the Tm of the probe, allowing for the use of more stringent hybridization conditions. In this way it is possible to achieve the optimal conditions for the elimination of the mismatched targets.

However, it has been reported that the introduction of LNA modifications for the hybridization probes could result in an increased background signal. This problem can be solved by increasing the hybridization temperature or using more stringent washing conditions [START_REF] Várallyay | Detection of microRNAs by Northern blot analyses using LNA probes[END_REF]).

Effects of the LNA depend on various parameters such as the length of the probe, the number of LNA modifications and their position in the probe, as well as the sequence of the probe. Therefore, to improve the mismatch discrimination, certain rules in the probe design have to be followed. For example, design of shorter probes can be more useful for mismatch discrimination. The discrimination of the mismatch is also more efficient when the mismatch site is located closer to the centre of the probe. Also, fully modified LNA probes display extremely high binding affinity for the complementary strand [START_REF] You | Design of LNA probes that improve mismatch discrimination[END_REF]).

LNA probes for the study of repetitive sequences

The unique hybridization properties of LNA oligonucleotides proved them to be powerful as probes for targeting nucleic acids sequences. This is especially important when detecting sequences that come from repetitive regions in the genome, such as satellite sequences and their transcripts. Satellite derived sequences have been mostly studied by methods such as RT PCR, RNA FISH and northern blot. All of these methods require use of oligonucleotides for their specific detection. The detection of transcripts by enzymatic methods such as RT PCR has been mainly based on random priming oligonucleotides.

Reverse transcription performed by random priming increases the cDNA complexity and lacks the specificity which can be obtained by the use of sequence specific primers. Also, the strand specific analysis is not possible, which is of great interest when dealing with noncoding sequences that can be transcribed both as sense and antisense molecules.

As previously discussed, detection by northern blot is a simple method, but it can be limiting for the detection of low abundant transcripts. The use of chemically modified probes should increase the sensitivity of the transcript detection. Discriminatory properties of LNA made them a powerful tool for use in conditions that require high specificity and mismatch discrimination, which is the case in the detection of repetitive sequences such as major satellites. Use of these probes in northern blot experiments allow for the discrimination between the sense and antisense strand. Moreover, the use of LNA probes would allow the design of short probes comparing to their DNA counterparts, with high specificity for their target in highly stringent conditions, which is extremely important in studies of repetitive sequences when trying to distinguish a single transcript between the transcripts that show a high degree of similarity, where longer probes could not distinguish between the two highly similar fragments [START_REF] You | Design of LNA probes that improve mismatch discrimination[END_REF][START_REF] Kaur | Thermodynamic, counterion, and hydration effects for the incorporation of locked nucleic acid nucleotides into DNA duplexes[END_REF]). Up until now, the most commonly used DNA binding domains were non-mammalian domains. These include the yeast GAL4-UAS system, the prokaryotic Tet-repressor (TetR)-Tet operator (TetO) system, and the Lac-repressor (LacR)-Lac operator (LacO) system. A main limitation of this approach is that the sequences have to be introduced into the host cells and into the region of interest to evaluate the effects of the targeting enzymes and the induced modifications. Moreover, most of these DNA binding domains lack binding specificity or Recently two novel technologies have emerged as a principle tool for specific genome engineering. One is based on Transcription activator-like effectors (TALE) and the other on the RNA guided CRISPR/Cas9 system. Clustered regularly interspaced short palindromic repeats (CRISPR) are natural bacterial and archeal genome loci that act as a bacterial innate immune system. These loci incorporate short fragments of viral DNA, which are then transcribed by the bacteria as small RNA molecules that, linked to the effector complexes, destroy the homologous viral DNA [START_REF] Mojica | Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements[END_REF][START_REF] Bolotin | Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin[END_REF]. The use of such system has been immediately recognised as a potential tool for eukaryotic genome engineering. The Cas9 endonuclease from the S. pyogenes CRISPR system has been mainly used for genome editing, together with the custom designed target guide RNA. The inactive Cas9 can be linked to different effector domains for sequence specific genome manipulations [START_REF] Mali | Cas9 as a versatile tool for engineering biology[END_REF]).

I. 2. Tools for (epi)genetic engineering

TALE as a novel DNA binding domain

TALEs are sequence specific DNA-binding proteins. They were discovered in the plant pathogenic bacteria Xanthomonas, which secretes this protein to infect the host genome and activate transcription of plant genes needed for the bacterial growth. TALE proteins consist of a N-terminal translocation domain, a C-terminal transcriptional activation signal and a central DNA binding domain. The DNA binding domain is specific in that it contains an array of 15.5-19.5 single repeats, each consisting out of 33-35 amino acid residues [START_REF] Boch | Xanthomonas AvrBs3 family-type III effectors: discovery and function[END_REF]. The last single repeat contains only 20 amino acids and is therefore referred to as a half-repeat. The repeats differ between each other by two amino acids at the positions 12 and 13, respectively, which form the so-called repeat variable di-residue (RVD) (Figure 26A). The amino acid pair at this position recognises a single nucleotide in a DNA sequence, following a specific recognition code which determines the binding speceficity of each repeat variable di-residue for a specific nucleotide. The binding specificity toward a single nucleotide varies depending on the RVD. Certain RDVs show a high specificity toward a single specific nucleotide, while others are more permissive to variations [START_REF] Boch | Breaking the code of DNA binding specificity of TAL-type III effectors[END_REF][START_REF] Moscou | A simple cipher governs DNA recognition by TAL effectors[END_REF]) (Figure26B).

The specificity of TALE proteins was recognised as a powerful tool for genome engineering, since it theoretically allows to target almost any DNA sequence of interest. A variety of custom TALE proteins have been generated in different organisms for multiple applications. To exert different functions in the genome, TALE DNA binding domain can be fused to various effector domains to modify the chosen site in the genome. Commonly used effector domains include nucleases, activation domains, repression domains, chromatin modifiers or fluorescent domains.

Designer TALEs

One of the first designer TALEs was constructed by fusing of the TALE DNA binding domain to the FokI nuclease domain to create the TALE nuclease (TALEN) (Figure 27).

FokI is active as a dimer, therefore, to generate a double strand break (DSB), the designer TALEN must be composed out of two monomers that bind two DNA sites separated by a short spacer sequence to exert an efficient dimer formation and the cleavage of the target site.

The double strand break introduced by the two TALENs can be used for either gene knockout or for an insertion of a DNA sequence of interest to the cleaved locus [START_REF] Scott | Targeted genome regulation and modification using transcription activator-like effectors[END_REF]). Gene knock out can be established after the initial DSB due to repair by error-prone nonhomologous end-joining pathway that will introduce insertions and deletions at the targeted gene. Insertion of a desired DNA sequence into the regions of interest can be achieved by sealing the DSB through homology directed repair in presence of a donor DNA molecule [START_REF] Scott | Targeted genome regulation and modification using transcription activator-like effectors[END_REF]. TALENs have been successfully used for genome engineering in different models organisms. Designer TALENs were used to modify genes in human somatic and pluripotent stem cells [START_REF] Hockemeyer | Genetic engineering of human pluripotent cells using TALE nucleases[END_REF][START_REF] Miller | A TALE nuclease architecture for efficient genome editing[END_REF]. They were also efficiently used for the generation of knock out mouse embryos [START_REF] Kato | Production of Sry knockout mouse using TALEN via oocyte injection[END_REF][START_REF] Li | Simultaneous gene editing by injection of mRNAs encoding transcription activator-like effector nucleases into mouse zygotes[END_REF]. The The application of TALEs goes beyond their use in genome editing and gene regulation. For example, TALE binding domain can be fused to fluorescent proteins to visualise endogenous genomic sequences. Nuclear dynamics of major and minor satellite sequences were monitored in living cells by confocal microscopy after transfection with TALE fused to fluorescent proteins [START_REF] Miyanari | Live visualization of chromatin dynamics with fluorescent TALEs[END_REF][START_REF] Miyanari | TAL effector-mediated genome visualization (TGV). Methods[END_REF][START_REF] Thanisch | Targeting and tracing of specific DNA sequences with dTALEs in living cells[END_REF]. The use of TALE-fluorescent proteins overcomes in situ hybridization, in which the experiment have to be performed on fixed cells. TALE-fluorescent proteins are mostly applicable for targeting repetitive genomic regions. However, a single locus detection has yet to be optimised by using a sufficient number of TALE proteins for efficient visualisation [START_REF] Miyanari | Live visualization of chromatin dynamics with fluorescent TALEs[END_REF]. Moreover, TALE binding domains have recently been linked with different chromatin modifiers to target the epigenome and to analyse the effects of chromatin modifications on specific genomic regions (see further text). Another study made use of a novel chromatin in vivo assay system called chemically induced proximity (CIP). This system uses small molecules for a rapid association of two peptide tags fused to the proteins of interest, to selectively add or remove different chromatin and transcriptional activities to an endogenous locus. The authors genetically modified an Oct4 allele to recruit chromatin regulators by CIP [START_REF] Hathaway | Dynamics and memory of heterochromatin in living cells[END_REF]). Using this system, they successfully targeted HP1 to the Oct4 locus (Figure28), induced H3K9me3 and heterochromatin formation that was stably maintained throughout the cell division and were able to measure the kinetics of the H3K9 establishment in the mouse fibroblast and embryonic stem cells. Specific DNA binding domains have been mostly used as a tool for the direct modulation of gene expression and genome editing. A promising possibility is to erase and/or rewrite epigenetic modifications by inducing local changes in chromatin modifications at endogenous loci. One of the first studies that targeted epigenetic modifications directly at an endogenous sequence made use of a zing finger that specifically bind major satellites (MaSat).

Epigenetic engineering for studying the functions of chromatin

The experiment performed was the following. Methyl CpG binding protein 2 (MeCP2) is a member of methyl CpG binding domain family and is involved in the aggregation of pericentromeric heterochromatin. Mutants of this protein found in the neurological disease called the Rett syndrom are unable to bind heterochromatin, which results in the impaired clustering of the pericentromeric repeats in mouse. To force the binding of the mutated proteins and analyse its functional consequences, the authors artificially recruited different MeCP2 Rett mutants to pericentromeric regions using a zing finger protein that specifically binds major satellites. The zing finger was fused to an antibody fragment with a high affinity for GPF tagged protein, which was therefore able to recruit the GFP labelled MeCP2 mutants to pericentromeres, allowing the visualisation of chromatin reorganisation dynamics (Casas-Delucchi et al. 2012) (Figure 29).

TALE for targeting chromatin modifications

The development of custom TALE binding domains that are linked to an effector domain of interest opened a new possibility for targeted modifications of epigenetic marks at the endogenous loci.

Up until now only several groups used TALE proteins to modify epigenetic marks at a desired locus by targeting chromatin modifiers. TALE linked to the ten-eleven translocation domain (TET1) that oxidizes 5-methylcytosine to hydroxymethylcytosine was used to demethylate and induce activation of several human genes including beta globin genes, KLF4

and RHOXF2 [START_REF] Maeder | Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins[END_REF]. Demethylation by TALE-TET1 fusion protein was induced at the target sites with different levels of efficiency, which was probably caused by the inaccessibility of the TET domain to completely catalyse the demethylation or by the different methylation levels at the specific loci.

Another study showed that it is possible to induce a demethylation of the histone H3K4me1 and H3K4me2 marks at active enhancers by targeting TALE fused to lysinespecific histone demethylase LSD1 [START_REF] Mendenhall | Locus-specific editing of histone modifications at endogenous enhancers[END_REF]. The active enhancers were demethylated at a specifically targeted stem cell leukemia locus in a human erythroleukemia cell line. Histone modification levels at the target locus were reduced by threefold relative to the control TALE lacking LSD1, with no effect on the non-target control enhancers. The changes in the chromatin state of the target enhancers frequently led to the downregulation of genes in their close proximity, which allowed the determination of the target genes of the demethylated enhancers.

Similarly, TALE binding domain were recently fused to a spectrum of 32 histone effector domains including histone deacetylases, histone methyltransferases and their recruiting enzymes, as well as histone acetyltransferase inhibitors to target different epigenetic modifications at different target loci in mouse neurons [START_REF] Konermann | Optical control of mammalian endogenous transcription and epigenetic states[END_REF].

For this, the authors developed the light-inducible transcriptional effector system based on the fusion of the TALE DNA binding domain to a light-sensitive cryptochrome protein from A.

thaliana and the fusion of the effector domains to the cryptochrome interacting partner (Figure 30). The illumination with the blue light induces the conformational change in the cryptochrome protein and triggers the recruitment of cryptochrome interacting partner and the effector domain to the target locus, allowing to regulate the timing of the recruitment of the modifiers. TALE DNA binding domain was fused to a small acidic peptide that has the ability to decondense chromatin. The targeting of this fusion construct in the embryonic stem cells induced nuclear repositioning of the target genes, indicating that the chromatin remodelling is responsible for the nuclear reorganisation [START_REF] Therizols | Chromatin decondensation is sufficient to alter nuclear organization in embryonic stem cells[END_REF]).

Genome engineering using custom designed tools that recognise a specific DNA sequence and deliver the effector domains has become a powerful and important tool for variety of applications. New emerging technologies such as TALE have proven to be more adapted for the use in various contexts, from genome to epigenome editing. While the zinc fingers have been mostly used for genome editing as nucleases, their application has encountered difficulties related to specific sequence recognition without off-target artefacts Despite the numerous evidence of transcription from major satellites in different cellular and physiological contexts, the precise characterisation at the molecular level, as well as the exact mechanisms involved in their expression and processing are still lacking. This is mostly due to the repetitive nature of the sequence, which impose limitations for their precise study. Therefore, in the first part of this work we wanted to provide a better characterization of the transcriptional profile of major satellites and to understand the mechanisms that control their transcription. We made use of short LNA modified oligonucleotides specifically designed to target both strands of major satellite sequence in northern blot experiments in order to characterise the transcriptional profile of major satellites in mouse embryonic fibroblasts grown in normal condition, as well as in a specific physiological context such as heat shock. To determine the mechanisms of transcript expression and regulation, we wanted to analyse the transcriptional profile of cells treated with different RNA polymerase inhibitors and investigate the changes in transcript expression after treatment with inhibitors of different chromatin modifiers.

In the second part of this work we focused on the study of the chromatin assembled at the major satellite repeats. To determine the role of the H3K9me3 epigenetic mark on the organisation of chromocenters, we wanted to use the novel experimental approach based on the fusion of artificial TALE protein with a specific chromatin modifier to introduce chromatin changes at the major satellite region. We constructed a TALE protein that is able to bind an 18 bp sequence of major satellites fused to mouse histone demethylase mJMJD2D to specifically demethylate H3K9me3. The use of a software that has recently been developed in the team enables to monitor in 3D imaging experiments and quantify the demethylation of H3K9 after transfection of cells with a construct expressing the fusion protein.

I.1. Methods

Heat shock

Heat shock was performed by immersing the flask with adherent cells in a water bath for one hour at 43 °C. The cells were left or not to recover at 37 °C during one or more hours.

Drug treatments

Trichostatin A 1 µL of 2 mg/mL TSA was diluted in water to obtain a concentration of 60 µM. 25 µL of 60 µM TSA was resuspended in 10 mL cell culture media and added to freshly split cells. Cells were grown in TSA supplemented media for 24 h at 37 °C.

Chaetocin

Stock solution of chaetocin (1 mg/mL) was diluted in water in 1/1400. 100 µL of intermediate solution was added to 10 mL cell media. Cells were grown for 48 h at 37 °C.

Actinomycin D 10 µL or 0,25 µL of actinomycine D stock solution (2 mg/mL) was resuspended in 500 µL cell media. The mixture was added to the cells to a final volume of 10 mL. The cells were grown in the presence of actinomycin D for 3h.

DRB 25 µL of DRB stock solution (10 mg/mL) were resuspended in 500 µL cell media. The mixture was added to the cells and cells were left for 3 h at 37 °C.

Northern blot

Sample preparation

Total RNA was extracted using TRIzol® Reagent (Invitrogen). 10 µg RNA were mixed with an equal volume of 2X formamide loading dye (Ambion) and heated for 10 minutes at 65 °C.

Migration and transfer

Polyacrylamide gel was pre-run for 30 minutes at 40 W in 1X TBE buffer. RNA samples were loaded into the rinsed wells and migrated for 1h at 20 W. After migration, the gel was stained with ethidium bromide for 15 minutes. The transfer was done on a nylon membrane (Hybond-N+, GE Healthcare Lifesciences) in a semi dry transfer unit (Amersham Biosciences) during 1h. Blotted RNAs were crosslinked in a stratalinker at 1200 mJ.

Hybridization

The membrane was hybridized with radiolabeled probes. Prehybridization was performed with 50% formamide (Bio Basic Canada Inc), 5X SSC (pH 5,6), 5X Denhart solution, 0,5% SDS and 1 mg of sheared salmon sperm (Sigma Aldrich) during 30 minutes. The hybridization was performed at 55°C with 50 % formamide (Bio Basic Canada Inc), 5X SSC (pH 5,6), 5X Denhart solution and 0,5% SDS for 1-3h. For LNA probes, membrane was washed in 2X SSC and 0,1% SDS buffer once for 5 minutes, and twice for 15 minutes. The membrane was wrapped in Saran wrap to avoid drying and was exposed to PhosphorImager during two days.

Radioactive labelling

Oligonucleotides were radioactively labelled on their 3' end by an incorporation of radioactive γ-32 P. 1 µM of oligonucleotides (Table 3) was incubated in the presence of T4 polynucleotide kinase (New England BioLabs) and ATP γ-32 P (Perkin-Elmer) for 1h at 37 °C.

Products were cleaned by passing the samples through a mini spin column in order to eliminate the unincorporated nucleotides (Micro Bio Spin with Bio-Gel P-6 in Tris-Buffer, BioRad).

RNA extraction

Cells were washed in PBS 1X, trypsinized and centrifuged for 5 minutes at 200 g. Pellet was washed twice in PBS 1X. 500 uL of TRIzol® solution (Invitrogen) was added per tube containing a maximum 2x10 7 cells. The homogenized mixture was incubated for 5 minutes at room temperature. Next, 0.2 mL of chloroform was added and the mixture was incubated for 2 min 30 sec at room temperature. Samples were centrifuged for 15 minutes at 4 °C at 11000

g. The aqueous phase was transferred to a new tube, washed with 0.5 mL of isopropanol and incubated at room temperature for 10 minutes. Samples were centrifuged at 4° C at 11000 g for 10 minutes. Pellet was washed with 70% ethanol, centrifuged again for 5 minutes and air-dried until the pellet became translucent. The pellet was resuspended in TE buffer (Ambion) and the samples were heated for 10 minutes at 65 °C. RNA was quantified by spectrophotometer. The RNA quality was checked on an 1% agarose gel. The RNA were stored at -80 °C.

1.5. RT PCR 10 µg of RNA were treated in with 2U of Turbo DNase (Ambion) in a total volume of 50 µL during 30 minutes at 37 °C. Turbo DNase was inactivated in the presence of 15 mM EDTA by incubation at 75 °C for 10 minutes. For reverse transcription, the RNA was incubated for 5 minutes at 65 °C in the presence of dNTPs (Invitrogen) and major satellite specific primers.

The reverse transcription was realized by addition of 5X First Strand buffer (Invitrogen), 10 mM DDT (Invitrogen), 40 U of RNAseOut (Promega) and 200 U of SuperScript™ III Reverse Transcriptase (Life Technologies) enzyme by incubation for 50 minutes at 55 °C followed by termination for 15 minutes at 70 °C.

Obtained cDNA was further amplified by PCR. cDNA was mixed with 200 µM dNTPs (Invitrogen), 1.5 mM MgCl 2 , 0,5 µM primers, 10X Taq buffer and 2,5 U of Taq polymerase (New England BioLabs). PCR conditions were as follows: 3 minutes at 94 °C, 30 seconds at 94 °C, 30 seconds at 57 °C, elongation 1 minute at 72 °C and termination for 5 minutes at 72 °C. Amplification was done in 25 cycles. PCR products were analysed by 1% agarose gel electrophoresis.

Major satellite DNA probe preparation

Strand specific RT PCR product obtained by the primer pair fw170/rv 220, was purified from an agarose gel using Micro Elute Gel Extraction Kit (Omega). 4.5 ng of purified product was cloned using TOPO TA Cloning Kit (Life Technologies). Cloning was verified by sequencing of selected clones after bacterial transformation. Sequencing was done by GATC Biotech by using MP13 forward primer. 1 µg of plasmid DNA was digested with EcoRI-HF® (New England BioLabs). 25 ng of DNA was labeled using Prime-It II Random Priming Labeling Kit (Agilent Technologies) with 5 µL of dCTP α-32 P (Perkin-Elmer). Products were passed through a mini spin column in order to eliminate the unincorporated nucleotides (Micro Bio Spin with Bio-Gel P-6 in Tris-Buffer, BioRad).

In vitro transcription

In vitro transcription was performed from pCR®4-TOPO, a vector expressing T7 bacterial promoter, containing a PCR product obtained by RT-PCR using major satellite specific primers. Plasmid was either linearized on the 3' end of the sequence to be transcribed or double digested leaving a linearized product containing the sequence of interest and the T7 priming site. The linearized plasmid was then gel purified (Gel Extraction Kit, Omega).

Transcription was performed using MegaShortScript kit (Ambion) starting with 100 ng of plasmid DNA in the presence of four ribonucleotides and T7 reaction buffer. The reaction was incubated for 2 hours at 37° C. Transcripts were further purified by phenol-chloroform extraction. The quantity of RNA was measured by Nanodrop.

1.8. Small RNA separation 150 µg of RNA was centrifuged in a Nanosep 100K device in a final volume of 50 µL for 5 min at 5000 g. The quantity was measured on Nanodrop.

Cell transfection

Coverslips were placed at the bottom of the each well of a 6-well plate and 2 mL of warm media was added to the wells. Cells transfection was performed using Amaxa Nucleofector (Lonza). 500 000 cells were mixed with the corresponding plasmid, resuspended in 100 µL of the solution R (Lonza) and subjected to electroporation using the programme U-030. After the electroporation, the cells were directly seeded to the 6-well plate containing the warm media and left for 24h at 37°C. After 24 h, the cells were washed in PBS and fixed with 4% PFA during 10 minutes at room temperature.

Immunofluorescence

Immunofluorescence analysis was performed on cells plated on coverslips (22x22 mm, 0,17+/-0,01 mm, Menzel-Gläser) using the following antibodies:

Primary antibodies: anti-H3K9me3 (Diagenode) and anti-HA (Abcam)

Secondary antibodies: chicken Alexa488 and rabbit Cy3 (Jackson Immuno Research)

Cells were washed in PBS 1X, fixed with 4% paraformaldehyde during 10 minutes, then washed 3x 5 minutes in PBS 1X. To facilitate the manipulation, coverslips were glued using rubber cement on the slides. Cells were permeabilized with PBS 1X-0,1% Triton X-100

during 5 minutes at room temperature. After a brief wash in PBS 1X, aspecific sites were blocked by incubation in 1,5% blocking solution (Roche Applied Science) during 30 minutes at 37° C. Cells were subsequently incubated with the primary antibodies diluted in revelation solution during 1h 30min at RT, in a humid chambre. Excess of antibodies was eliminated by washing with PBS 1X-0,05% Triton X-100 three times and subsequently incubated with secondary antibodies diluted in solution for revelation for 30 minutes in a humid chamber at RT. Cells were washed three times in PBS 1X-0,05% Triton X-100. DNA was stained by incubating each coverslip in 4 µM Hoechst 33342 (Thermo Scientific) during 30 minutes.

Coverslips were mounted by carefully turning the cover slip at the middle of the slide, right to the drop of in PPD8 solution (Sigma Aldrich). Coverslips were fixed with nailpolish, left to dry overnight and then stored at 4 °C for a short period or 20 °C for a long period.

1.12. Microscopy Images were acquired using inversed epifluorescent microscope Zeiss Axio Observer Z1. This microscope is equipped with an ApoTome illumination system ensuring the high quality resolution (xy:0,2 µm, z:0,5 µm). Acquisitions were made using the camera OrcaR2 The used computer programme was ZEN 2012.

TANGO analysis

The images were taken as a Z-stack, a set of optical slices of the lateral (x,y) axis of the specimen plane. The images were imported to the TANGO software [START_REF] Ollion | TANGO: a generic tool for high-throughput 3D image analysis for studying nuclear organization[END_REF].

The imported images are called channel images. The channels correspond to a stack of images aquired at a particular wavelenght according to the fluorescent markers, and corresponds to its an associated biological structure. The defined channels were Hoechst and Cy3 and the corresponding structures nucleus and major satellite foci. The selection or segmentation of the defined structures was performed by applying different processing chains to the image (prefilters: median, Laplacian of Gaussian 3D; postfilters: size and edge filter, morphological filter). The nuclei were selected from the Hoechst channel and the same ROI selection was applied to crop all the other fluorescent channels. Following the segmentation of the nucleus, the previously defined structures within the nucleus were segmented. The segmentation of the selected structures and the quality of the image processing were verified. The cells carrying a defect (for example, nucleus cut in the optical axis) and the cells with an imperfect segmentation of the nuclei (stacking nuclei) were eliminated from the analysis. After the segmentation of the selected structures, quantitative measurements were carried out for each of the objects. The following measurements were carried out:

- From these data, the following calculations were made for both Hoechst and H3K9:

-integrated signal ratio inside the foci (integrated signal inside the foci/integrated total signal) -integrated signal ratio outside the foci (integrated total signal-signal inside the foci/integrated total signal)

The statistical analysis was made using the R studio (version 0.97.551). The p-value was calculated using the Mann-Whitney-Wilcoxon test and corrected with Holm-Bonferroni.

To be able to apply the test, we assured to have a similar distribution of the measured values. At the beginning of my thesis, the team has already designed and successfully used short LNA modified oligonucleotides as probes for specific detection of repeats from individual chromosomes in human cells by FISH experiments (unpublished data). These oligonucleotides were able to discriminate between two highly similar target sequences, where the difference between them was only a couple of nucleotides.

Recognizing their potential, similar oligonucleotides were designed, which target the sequence of the mouse major satellite repeat. These are short 18-mer probes with a LNA modification inserted in one out of two oligonucleotides. The probes were designed to target different parts of both strands of major satellite sequence and distinguish transcripts from different subunits of the major satellite repeats. The sequences of the oligonucleotides can be found in Table 6. The LNA oligonucleotides were designed with a hapten (biotin or digoxigenin) on their 3' end, which makes them usable in DNA-or RNA FISH experiments. They can also be radioactively labelled in 5' end for use in northern blot experiments. We were interested to use these oligonucleotides as probes for DNA and RNA FISH and as for probes for northern blot analysis for a more precise characterisation of major satellite-derived transcripts. At my arrival in the laboratory, the specificity of binding of the designed probes to the major satellites had been already verified by DNA FISH experiments, confirming the strong specificity for the chromocenters on NIH-3T3 cells, which are known to exhibit very recognisable chromocenters by optical microscopy.

Characterisation of major satellite transcription by northern blotting of total

RNA from mouse cells 1.2.1. Transcriptional profile of major satellites in growing mouse cells

To characterize the transcriptional profile of major satellites we tested LNA modified oligonucleotides as probes for northern blot experiments. Given the theoretically high mismatch discrimination provided by these probes and their high specificity for the target sequence, we hoped to obtain the information about the abundance and size of the RNA species produced from major satellite loci in mouse cells grown in different conditions. We first focused on the RNA species produced by exponentially growing MEF cells cultured in standard medium conditions (see Materials and methods). The total RNA extracted from MEF cells was separated on 4% (29:1) denaturing polyacrylamide gel, in order to obtain a good resolution with the transcripts ranging from 50 up to 1000 nucleotides. The RNA were visualised by ethidium bromide before they were transferred to a nylon membrane and hybridized with different LNA-modified probes specific for major satellite. For specific details about conditions used for northern blotting see Materials and methods.

We first assessed the signal given by 12 probes encompassing the major satelite repeat sequence, covering both strands. Figure 31A shows the position of the hybridization sites of the used probes we tested. Highly abundant cellular RNA such as tRNA or rRNA (5s and 5.8s), which are strongly labelled by ethidium bromide (Figure 31A) are also lit up by the LNA probes. We therefore decided to focus only on the bands that do not colocalise with these abundant RNA molecules. Hybridization pattern vary depending on the probe, but all probes reveal a complex transcriptional profile, with multiple bands on the blot. The Figure 31B (forward strand) and Figure 31C (reverse strand) show the profile obtained after hybridization with various probes. Comparing results obtained with different probes, interestingly, the transcriptional profile is similar for the probes targeting a given strand. This suggested that the designed probes were indeed targeting the same RNA species, despite their difference in sequence. The profile obtained with the probes targeting the forward strand reveals more bands that also show a more prominent signal. This could signify that either the forward transcripts could be more expressed, which is detected with our antisense probes, or that the antisense probes show the greater affinity for the forward strand than the sense probes for their respective complementary RNA, therefore introducing a nucleotide bias of the RNA to be recognised, resulting in less signal coming from the opposite strand. Highly abundant RNA molecules such as 18S, 5S, 5.8S and tRNA are strongly labelled. On the right. The

Detailed characterization of northern hybridization signals

The short LNA probes were designed to target the all four subrepeats of major satellite sequence (Figure 31 and Chapter II, Introduction). We wanted to use the possibility of distinguishing between the highly similar subrepeats to get an approximation of the localisation of each detected transcript on the major satellite sequence.

As already mentioned, the hybridization pattern vary depending on the probe, but a similar pattern is revealed with all the used probes. The probe R1 differs from the other probes in that it reveals a strong signal at higher molecular weights. Similar yet somewhat less pronounced strong hybridization at higher molecular weights is seen for the probe 3d. The probes R32 and R31 and 12 each strongly reveal one transcript in particular. However, these transcripts are found to be non-specific, 7SK for R32 and tRNA, for R31 and 12 (Figure 32).

Different probes that target different parts of the repeat reveal the same transcript, therefore, if the signal observed is specific, it should be possible to reconstitute the position of the revealed transcripts on the major satellite repeat unit, in the cases where the transcript is smaller than the length of the major satellite sequence. The satellite transcripts bigger than the major satellite unit should theoretically be revealed with all the probes.

If we look at the Figure 32, we can see that this is not always the case. Figure 32 displays examples of hybridizations for each probe, with a detailed analysis of the revealed bands. The size of each of the bands is summarized in Table 7. The bands smaller than 234 nt are revealed either with almost all the probes, or they are revealed with two or more different probes that are localised too far away from each other on the major satellite sequence to be able to reveal the same RNA segment. For example the band at 138 nt is revealed with all the reverse probes except the probe R34. This band is also revealed with the probes targeting the opposite strand, 12 and 33. The reverse probes that reveal this transcript encompass together a size bigger than the 138 nucleotides. Another example is the band at 165 nt, revealed with all the probes except the R14 and 33.

The transcript at 180 nt is revealed with the probes R11, R31 and R34, as well as 3d

which could indicate that this transcript, if it is a major satellite transcript, is transcribed starting from the end of one repeat, reaching to the beginning of the other. However, it is surprising that it is not revealed with the probe R21, which localise between the probes R11 and R21, or with the probe 31 (Table 7).

Transcript at 120 nt is revealed with only one probe that targets the sense strand (R21), and with three probes targeting the opposite strand, 31, 13 and 3d. The revelation with these three probes is compatible with the size of the revealed transcript, however, the revelation with only one probe from the opposite strand suggests that there is either a problem of non specificity or as already mentioned, a problem of affinity toward certain transcripts. The same problem is found for the transcript at 135 nt, revealed with probes R14 and 3d and 31. The transcript at 96 and 130 nt are the only ones revealed exclusively with the probes targeting the forward strand. However, the specificity problem is again encountered with the 96-nt transcript revealed with the probes that encompass more than 96 bp. The 130 nt transcript is revealed with the probes R32 and very weakly with the probe R31, suggesting the different affinity of these probes toward this transcript.

In addition, the bands longer than the major satellite repeat are not always revealed with all the probes as for example the bands at 235, 275 and 295 or even at 520. If these bands are indeed major satellite transcripts, this could again suggest that the probes have a different level of affinity for a specific transcript. The transcript at 390 nt is revealed with all the probes targeting the forward strand, which could indicate its major satellite origin. This strand is however revealed only with two probes from the opposite strand, suggesting that the probes targeting the reverse strand show less affinity for these transcripts.

In summary, these results cast doubt on the fact that all bands revealed by the probes are specifically major satellite RNA, complicating our analysis of major satellite transcription by this approach. We tried to circumvent this problem by various approaches, testing different probe chemistry, as further described. In order to test if a different chemistry for the probes would help with the specificity of northern blot hybridization, we tested a 2'-O-methyl-RNA probe designed to recognise the sequence 5'AGTTTTCCTCGCCATATT3' within the major satellite sequence (antisense strand). This probe was previously successfully used for FISH experiments. We wanted to compare the signal obtained with the LNA probe to that obtained within the 2'-O-methyl. The membrane was hybridized with the probe R125, dehybridized and hybridized again with the isosequential 2'-O-Me probe MSM12. The hybridization with the LNA probe was performed at 55°C. Hybridization at the same temperature with the 2'-O-Me probe gave no detectable signal. Therefore, the membrane was rehybridized with the 2'-O-Me probe at 42°C. In these conditions, both probes reveal the same pattern (Figure 33). These two isosequential probes strongly reveal two transcripts of the length of 122 nucleotides (5S RNA) and 165 nucleotides, as well as bands at 390, 510, 880 nt only somewhat weaker. The band at 165 nt was revealed with all the probes except R14 and 33. Looking at only the forward probes, we could imagine that the transcript encompassing the range of tha major satellite targeted by the probes 31, 12, and 3d indicate the good size and therefore the major satellite origin. However, the transcript is equally revealed with almost all the reverse probes. It is therefore unlikely that this band represents a major satellite specific signal, since all the reverse probes that reveal this transcript encompass together more than 165 nt. One possibility could be that the some probes, such as R1 give a non-specific signal. We can see also that the transcript at 390 nt which is revealed with all the reverse LNA probes, but not all the forward probes is also revealed with the 2'-O-Me probe. This reaffirms the hypothesis which suggests that the forward LNA probes show less affinity toward certain transcripts. 1.2.4. High stringency washing LNA modified probes allow for the use of highly stringent conditions of hybridization and washing. The hybridization was performed at 55°C for all the LNA probes. The membranes were washed with 2X SSC. In order to increase the specificity of the hybridization and to try to eliminate the signal corresponding to the highly abundant cellular RNA such as 5S RNA or tRNA, we changed the stringency of the hybridization wash to 0,4X SSC. After the wash with 0,4X SSC, the hybridization profile changed, sometimes drastically, depending on the used probe. The most striking difference is the elimination of the long transcripts with the probe R1 (Figure 34). The majority of the observed bands disappeared when the high stringency wash was applied for the probe R11. However, this did not eliminate hybridization to the 5.8S RNA and the tRNAs, which were still revealed except with the probe R11.

RNA and DNA probes

We produced a 308 nucleotide long major satellite DNA probe from the RT-PCR fragment obtained using specific major satellite primers (see page 120 and Materials and methods) to compare the transcriptional profile obtained with this probe to the transcriptional profile obtain by hybridization with major satellite specific LNA probes. Equally, we prepared two major satellite RNA probes that target either the forward or the reverse strand of major satellites, by in vitro transcription of the same RT-PCR fragment. The probes were 32 Plabelled and used in northern experiment as described above. Total RNA, as well as a 308 bp major satellite DNA fragment, used as a positive control, were hybridized with major satellite specific LNA, DNA and RNA probes. As can be seen in the Figure 35, the LNA probe (R32) successfully reveals the major satellite fragment. The transcriptional profile differs greatly between the LNA and both the RNA and DNA probes. We can observe that the DNA probe used at an hybridization temperature of 42°C gives a weak signal at the high molecular weights. RNA probes give somewhat of a stronger signal at the higher molecular weights mostly in a form of a smear. Hardly any signal is observed however, corresponding to the short transcripts (<300 nt). In conclusion, the results obtained with the LNA probes designed to specifically target major satellites do not allow us to determine with certainty which, if any of the transcripts does indeed come from the major satellites. As major satellite specific DNA and RNA probes do not reveal any of the detected short transcripts observed with the LNA probes, nor is there a clear distinction of the longer transcripts, there is no positive control that could allow us to say with certainty which of the bands revealed with the LNA probes are indeed major satellite derived.

t o t a l R N A t o t a l R N A t o t a l R N A t o t a l R N A m a
If we take into consideration the possibility of the non specific hybridization of the probe on a different but highly similar region of the major satellites, or if we consider that there are differences in the affinity of certain probes for the same transcript, we could highlight several transcripts as potential major satellite derived. These are the 96, 120, 130, 135, 180 and 390 nt. In spite of these somewhat ambigous results we have decided to continue our analysis using LNA probes designed to target the major satellites in different growth conditions and following different treatments.

Strand specific RT PCR confirms transcription from major satellites

Reverse transcription (RT) is used to detect RNA expression by converting the cellular RNA to DNA that will further be amplified by PCR. To verify the transcription from major satellites in MEF cells we performed reverse transcription on total RNA. The RT can be performed using random priming method that relies on the random hexamers for the reverse transcription (see Chapter 3, Introduction). We opted for the use of sequence specific primers to select for specific RNA already at the reverse transcription level. This was aimed to further reducing cDNA complexity and at increasing the specificity and sensitivity of the RT PCR.

The primers were designed to correspond in sequence to the LNA antisense oligonucleotides, including one primer pair frequently used in the literature (Bulut-Karslioglu et al. 2012a;[START_REF] Probst | A strand-specific burst in transcription of pericentric satellites is required for chromocenter formation and early mouse development[END_REF]. The list of primers can be found in Materials and methods. All combinations of primer pairs were tested in separate reactions. One sense and one antisense specific primer was used per reverse transcription reaction to generate strand specific cDNA.

Following reverse transcription, the cDNA was submitted to a standard PCR amplification with the same major satellite specific primers.

The major satellite region contains high number of 234 bp monomer units. This repetitive nature of major satellites makes the PCR amplification products to appear on a gel in a form of a ladder. The primers hybridize at the same position on multiple repeats of the major satellite repeat unit, producing fragments of multiple sizes. The distance between the two fragments on a gel corresponds to the size of major satellite monomer (234 bp).

According to the primer binding site on the sequence it is easy to calculate the size of the expected PCR product for a specific primer pair. After testing all combinations of primers in our disposition, two pairs gave the PCR products of the expected length (Figure 36, Table 8). Figure 36 shows the results of the RT-PCR performed on total RNA from MEF cells.

PCR amplification was performed on the cDNA template obtained by RT performed using either indicated forward (fw) or reverse (rv) primer. For the primer pair fw60/rv170, a PCR fragment of the size of 138 bp is obtained for both sense and antisense amplification (Figure 36A). This amplification product corresponds in size to the expected products for this primer pair (Table 8). The Figure 36B shows the PCR amplification with the second primer pair, fw 170/rv 220. The PCR products obtained using this primer pair after the reverse transcription performed with fw170 correspond to the expected lengths (Table 8). These are the bands at 74, 308 and 542 bp. The PCR products obtained after the reverse transcription using the primer rv220 show the same PCR products but in different quantities. The band at 74 bp is more abundant than the band at 308 nt, while the product at 542 bp is barely detected.

The sequencing of the bands at the position 74 bp and 308 bp, as well as the sequencing of the band at 138 bp confirmed that these amplified DNA products correspond to major satellite sequence in both orientations. We can therefore conclude that the expected band at the 542 bp is equally derived from major satellites.

Besides the product of 74, 308 and 542 bp, the amplification of the cDNA obtained with the primer rv220 gives two bands at 150 and 200 bp. These PCR products most likely represent hybridization of the primer to the highly similar region of the major satellite sequence.

The results suggest that both strands of major satellites are transcribed, but probably not in the same abundance. The existence of both short and long PCR products (74 and 542 bp) suggests that the transcripts exist in various sizes. Since more longer PCR products are obtained after the amplification of the reverse transcription with the primer fw170, we can assume that more longer transcripts are produced from the reverse strand of the major satellites.

I.2. Expression and regulation mechanisms implicated in major satellite transcription

2.1. Influence of the inhibitors of chromatin modifiers on major satellite transcription As already described in the Chapter I of the introduction, histone acetylation is an activating heterochromatin mark that correlates with transcriptionally active region. Major satellites, as a heterochromatin region, are mostly hypoacetylated. Hypoacetylation at major satellites is maintained by histone deacetylases (HDAC) and deacetylation of histone H3 is a prerequisite for its methylation [START_REF] Rea | Regulation of chromatin structure by site-specific histone H3 methyltransferases[END_REF]. Previously it was shown that treating the cells with the histone deacetylase inhibitor Trichostatin A (TSA) lead to loss of H3K9 and HP1 from mouse major satellites, loss of centromere function, but seemed to have no effect on major satellite transcription [START_REF] Taddei | Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases[END_REF][START_REF] Maison | Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component[END_REF]. On the other hand, cells depleted from Suv39h show increase in major satellite transcription [START_REF] Lehnertz | Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin[END_REF][START_REF] Martens | The profile of repeat-associated histone lysine methylation states in the mouse epigenome[END_REF].

In order to define epigenetic modifications that may have an influence on the expression of major satellites in MEF cells, we used inhibitors of chromatin modifiers to induce changes in the epigenetic status of heterochromatin. MEF cells were treated with TSA, an inhibitor of histone deacetylases (HDAC), which suppresses its activity leading to an increase in histone acetylation. Cells were also treated with chaetocin, a lysine-specific histone methyltransferase inhibitor that specifically inhibits Su(var)3-9 of D. melanogaster and its human ortholog, Suv39h1 (Table 9). The analysis of RNA from treated cells was made by northern blot (Figure 37). Hybridization was performed using different LNA-modified probes specific for major satellite sequence. The Figure 37 shows two hybridizations with two different oligonucleotides. There was no change in the transcriptional profile of major satellites for the cells treated with either of the inhibitors comparing to the control RNA from non-treated cells. The observation that histone deacetylation has no effect on transcription is in accordance with previously published results, indicating that transcription of major satellites occurs independently of acetylation state of major satellite region. Treatment with the Suv39h inhibitor does not have a high impact on the Suv39h and is not sufficient enough to provoke an increase of major satellite transcription, as previously observed in Suv39dn cells [START_REF] Lehnertz | Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin[END_REF]Bulut-Karslioglu et al. 2012). Even though we cannot observe a modification of expression of major satellite transcripts, we cannot exclude the possibility that interference with other chromatin modifiers could influence major satellite transcription. Another possibility is that non of the revealed bands are indeed major satellite transcripts. In absence of a positive control for these experiments, we cannot rule out this hypothesis.

Changes in major satellite transcription upon thermal stress

In human cells, pericentromeric regions are expressed upon stress conditions such as heat shock, osmotic shock or exposure to certain chemicals [START_REF] Valgardsdottir | Transcription of Satellite III non-coding RNAs is a general stress response in human cells[END_REF][START_REF] Eymery | Heat shock factor 1 binds to and transcribes satellite II and III sequences at several pericentromeric regions in heat-shocked cells[END_REF]. In mouse, the only RNA to be transcribed from the repetitive sequence is B2 RNA. This LINE-derived transcript is overexpressed upon heat shock, where it binds RNA pol II and represses mRNA transcription during heat shock [START_REF] Allen | The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock[END_REF]).

We wanted to investigate if stress conditions would effect the transcription from major satellites, and is this condition could give information on specific major RNA transcript signal in our northern blot experiments. For this, a cell culture flask containing growing MEF cells was immersed in a water bath at 43°C for one hour. Control cells were immersed in a water bath at 37°C. After the treatment, cells were left to recover for one hour at 37°C. RNAs extracted from both treated and control cells were analysed by northern blot with strand specific oligonucleotides. The pattern of expression of one particular transcript of a calculated length of 96 nucleotides is modified in response to heat shock (Figure 38B). The increase in expression was detected by four different oligonucleotides targeting different positions of the forward strand of major satellite. The position of these oligonucleotides can be seen in Figure 38A. The transcript level normalized to the tRNA level shows at least 2,5 fold increase in expression compared to the non-heat shocked cells, when measured for the probe R1 which gives the most prominent signal. However, as already described in the part I.1, the revelation of this transcript is made by five different probes in different intensities, that are located too far away from each other on the satellite sequence. Therefore, we cannot conclude that this signal is major satellite specific. There is, however, no doubt that the level of expression of this RNA is elevated upon heat shock.

We performed a kinetics experiment to monitor the increase in the transcript level at the different temperatures of the water bath, time of the heat shock duration and time of heat shock recovery (Figure 38C). The heat shock performed at the 43°C temperature with different duration and the time of recovery change from 2,5 to 5 fold increase. This increase falls into the range previously measured for the heat shock performed at 43°C during 1h with 1h recovery (Figure 38C). There is a remarkable difference between the heat shock performed at 45°C with different duration and recovery time. Heat shock at 45°C during 1h following 1h recovery shows 7 fold increase while the duration of two hours shows a low signal increase.

The experiment was performed once and requires further confirmation. This suggests transcription from major satellites is mostly mediated by RNA polymerase II.

However, as transcripts do not completely disappear upon this treatment suggests that although important, the RNA pol II is not the only RNA polymerase to act upon major satellites. RNA polymerase III could be a possible candidate, in particular for the synthesis of small RNA transcripts.

We tested the influence of different RNA polymerase inhibitors on major satellite transcription. We used two different RNA pol inhibitors in different concentrations, DRB and actinomycin D. DRB was used as an inhibitor of RNA pol II at 25 µg/mL. Actinomycin D inhibits the transcription by all three RNA polymerases depending on its concentration. The most sensitive to actinomycin D treatment is RNA pol I, which is inhibited at low concentrations (0,05 µg/mL), followed by RNA pol II transcription (0,5 µg/mL). RNA polymerase III is inhibited exclusively at high concentrations of actinomycin D (5 µg/mL) (Bensaude 2011). Cells were treated with both inhibitors during 3 hours ( Treatments with DRB or with low levels of actinomycin D does not change the transcriptional profile obtain by the hybridization with the LNA probes. On the other hand, treatment with high levels of actinomycin D completely eliminated a small transcript corresponding to the size of approximately 96 nucleotides, which is transcribed in growing cells and overexpressed upon heat shock (Figure 39). While this transcript disappears after treatment of cells with high (2µg/mL) dose of actinomycin D, it does not change at the low concentration of actinomycin D (0,05 µg/mL) or DRB. This result suggests that this specific transcript is generated by RNA polymerase III. RNA polymerase III transcribes 5S RNA in eukaryotes. As can be seen from the gel visualised with ethidium bromide, actinomycin D in high concentration do not change the level of either 5S RNA that is a polymerase III transcript.

Higher concentrations may be needed to achieve a strong inhibition of this RNA. We decided to expand the use of specific primers for the identification of the sequences of major satellite transcripts. Of our specific interest is the 96 nt transcript, which is found to be overexpressed in the heat-shocked cells. We decided to combine the ligation of the known adapter sequences to the short cellular RNA, followed by RT PCR using the primers that hybridize to the ligated adapter or the use of major satellite specific primers (see further text).

Small RNA separation

Since northern hybridizations showed non-specific hybridization of our probes to the long RNA, to minimise the non-specific molecules in our sample and avoid the non-specific amplification with our primers, we eliminated the long species before adapter ligation. Short RNA have been separated from the total RNA by ultrafiltration using NanoSep100K device that acts as a molecular sieve. Using this procedure the molecules are separated on the basis of their length, theoretically not allowing sequences larger than 450 bp to pass through. Our experience reveals that there is however a slight recuperation of long molecules. However, their yield is negligible comparing to the yield of short RNA (Figure 40). The yield of recuperation of bands of different sizes can be compared on a gel. As can be seen in Figure 40, the percentage of recuperation varies depending on the length of the transcript. Smaller the transcript is, higher is its yield of recuperation. Adapter ligation to cellular RNA It is still unclear whether all non-coding RNA are post-transcriptionally modified, but majority of them probably do not go through the same maturation steps as mRNA. We have therefore considered that the major satellite derived transcripts could have a nonpolyadenylated and therefore free 3' end. With this in mind we wanted to ligate an adapter on the 3' end of the RNA. A 5' adapter could equally be used for the ligation at the 5' end of the molecules of interest that contains a 5' phosphate. Once ligated, the adapters serve for the fixation of complementary primers and are used to convert a ligated RNA into the complementary DNA.

The 3' adapter molecule was designed to obtain optimal ligation efficiency. This 19mer oligonucleotide has a dideoxycytidine on its 3' end to prevent self-circularization and circularization of ligated RNA molecule. Phosphate at its 5' ensures the adapter is ligated at the 3' end of the RNA molecule. A 5' adapter is a a 17-mer oligonucleotide with 5'OH and 3'OH group. These two adapters can be ligated on the 3'OH or 5'p of the small RNA, respectively.

Both adapters was ligated on the totality of small RNA molecules. The ligation was verified on a polyacrylamide gel (Figure 41). The abundant cellular RNA that ligated the adapter show a shift in their position on the gel. Transfer RNA, as well as 5S and 5.8S RNA ligated the 3' adapter and showed the shift of 19-nt to the higher molecular weights. On the contrary, no shift was observed after the 5' adapter ligation on these RNA. The possibility for this adapter to bind to the RNA was tested on the small 85 nt long synthetic 5' phosphate RNA to which the 5' adapter was efficiently ligated. In order to verify the 3'ligation on a specific cellular RNA, we performed the 3' RACE on a small ncRNA U5. Non-coding U5 RNA is a 118-nt long non-coding RNA that is a component of small nuclear ribonucleic particles. The 3'adapter was ligated on the small RNA after the separation of the long molecules. Ligation to the U5 RNA was verified by northern blot. LNA probe targeting U5 RNA confirmed the 3' ligation on the U5 RNA (Figure 42A). Reverse transcription was performed on the total of the ligated RNA molecules with a primer complementary to the adapter. PCR amplification was performed using two different primers complementary to U5 RNA. As shown in Figure 42B, the PCR amplification gave two different PCR products corresponding in size to the expected lengths of the ligated U5

RNA. The PCR products were cloned into a vector using TopoTA cloning and sequenced.

Sequencing results show that the method works on the control RNA. These results suggested that the technique could be used to characterize transcripts from major satellite DNA.

3' RACE using major satellite specific primers

We decided to proceed with the 3' RACE on the short RNA using primers specific for major satellites. After the 3' ligation, the PCR was performed using the primer corresponding to the 3'adapter and another, major satellite specific primers (for the list of primers used see Materials and methods). The PCR products were mostly of a size of 90 nt. Several primers gave products at the size of 200 and 400 base pairs. First sequencing results gave information only about the two longer bands, which were found to correspond to a partial sequence of 45S rRNA. We decided to focus on the small PCR product obtained by using one of the primers targeting major satellite. The sequencing of this PCR product showed the existence of an artifact sequence that could not be targeted to major satellites. We have therefore decided to abandon this method for the determination of the major satellite sequence.

I. 4. Conclusion and discussion

Transcriptional profile obtained by the hybridization with major satellite specific LNA oligonucleotides

We used LNA modified oligonucleotides designed to specifically target major satellites, hoping to provide better sensibility and specificity toward the transcripts coming from this highly repetitive region. We combined the use of short LNA modified oligonucleotides, where the LNA modification is present in one out of two nucleotides in combination with 4% polyacrylamide gel electrophoresis to obtain a good resolution of the transcripts of different sizes. Different 18-mer probes were used, which target different parts of both strands of the repeat. We have shown that northern hybridization using these probes reveal a complex transcriptional pattern. Multiple bands are visible after the hybridization with different probes. Among the different transcripts present on the membrane, we detected the abundant cellular RNA: tRNA and rRNA (5S, 5.8S and 18S), which were revealed with almost all of our probes. The revelation of the tRNA and rRNA molecules showed that the probes designed to specifically target major satellites are able to hybridize to other than their specific target. Beside these known cellular RNA, other bands are revealed on the membrane, for which we made an thorough analysis trying to situate each of the transcripts on the major satellite repeat, depending on its detection with different LNA probes. Even though the profiles change depending on the probe used, the profiles are somewhat similar for all the probes targeting a given strand, which suggests that we target the same group of transcripts with almost all of our probes.

However, certain inconsistencies were observed that complicated the interpretation of the results. First, the bands with a size bigger than the major satellite repeat (234 bp) were not revealed with all the probes targeting the same strand. Equally, the bands smaller than the major satellite repeat are sometimes revealed with all the probes or, the probes revealing a certain transcript are positioned too far away on the major satellite sequence, covering a size bigger than the transcript in question. If the transcripts that we detect are indeed derived from major satellites, the inconsistencies we encounter, such as the revelation of the same transcripts with different probes that are located to far from each other on the sequence, could be due to the extremely high similarity sequence within satellite repeats. Indeed, the major satellite repeat can be separated into four subrepeats that have a high level of similarity. The LNA probes were designed to target the part of the sequence that show the greatest polymorphism. In our hands, this strategy was however not discriminating enough for northern analysis. It is however possible that we did not found the proper hybridization conditions, or that the LNA modifications were not positioned correctly in a probe for the achievement of the highest mismatch discrimination.

Complicating the reading of the hybridization signal, the majority of the detected transcripts are revealed with several probes, however the signal of the given transcript is not equal for all the probes. One possibility is that a probe complementary to the middle of the transcript hybridize more strongly than the probe hybridizing to one of the ends of the transcript. Post-transcriptional modification of the transcripts could also interfere with the quality of the hybridization signal depending on the probe used.

Change of the washing conditions also changed the hybridization profile of the LNA probes. The short probes with LNA modifications display higher Tm, so it was possible to try used hybridization temperature and highly stringent conditions. The disappearance of the smear at the higher molecular weight in these conditions suggest that some non-specific hybridization is occurring and can be eliminated by changing the stringency of the experiment. However, the change in conditions did not eliminate the hybridization on the ribosomal RNA.

LNA probes for northern blot detection of transcripts from a highly repetitive region

Hybridization of the LNA probes on the highly abundant RNA could be explained by the northern hybridization itself. The hybridization between the probe and the RNA in northern blot occurs on a membrane, where RNA are localised in a small, restricted space on the membrane. Hybridization under these conditions could lead to the retention of the probe by the interactions with these highly abundant RNA in a restricted area of the membrane, which would not occur if these RNA were present in a solution. This could explain why, when the probes used in northern experiments are used in DNA FISH, they specifically reveal the DAPI dense spots in the cells, which represent the clustered major satellites. If we adopt this explanation, we are able to eliminate the abundant, known RNA which are hybridized with the LNA probes and are left with the list of transcripts that appear with the hybridization with more or less all of the probes, as argued in the previous paragraph.

Moreover, the small amounts of the major satellite transcripts could contribute to difficulties in their detection. This is however not in accordance with previously shown application of LNA probes, which were successfully used for the detection of the low abundant miRNA in northern hybridizations [START_REF] Válóczi | Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes[END_REF]). However, the hybridization with LNA modified probes designed to target the U5, U6 and 7SK RNA revealed correctly these target RNA, without any non-specific hybridization on other transcripts (not shown).

The repetitive, AT rich sequences of major satellites that LNA probes are expected to detect could truly be the reason for which it is difficult to obtain a specific hybridization signal.

Comparison between the LNA probes with their sequence equivalent DNA and RNA probes in hybridization experiments

The control hybridizations with DNA and RNA probes did not reveal the same transcriptional patterns. The hybridization profiles revealed with these two probes were rather surprising, since only a smear was detected at higher molecular weights. The revelation of bands at lower molecular weights could have comforted us in the fact that LNA probes were helping reveal short major satellite transcripts. It is possible that the low abundance of these transcripts prevents their detection by standard DNA and RNA probes. On the other hand, the use of the probe carrying another chemical modification, the 2'-O-Me probe revealed the same transcriptional profile as its isosequential LNA probe. This shows that the non-specific hybridization is not limited only to the LNA probes. It would be interesting to perform the hybridization with a negative control containing no specific binding site in the mouse genome such as the probe targeting human alpha satellites. Ideally, in the absence of the target in the major satellites, we should expect this probe not to bind to any of the transcripts, which are detected with the major satellite specific LNA probes.

Although the LNA probes designed to specifically target major satellites showed specificity problems, we can not exclude the possibility that some of the detected transcripts are major satellites derived.

Detection of transcripts upregulated upon heat shock

Northern hybridization of the RNA from mouse cells submitted to a heat shock of 1h at 42°C, using different LNA probes targeting the forward strand of the major satellites showed that a 96 nt transcript is overexpressed. As the LNA probes designed to target the major satellite repeats showed non-specific binding, and as we had no positive control confirming that the revealed transcripts are derived from the major satellite repeat, we can not certify that the observed overexpressed transcript really is major satellite RNA. This transcript was revealed with five LNA probes that target different regions of the major satellites. The signal is found to be most intense with the use of the probe R1, while the hybridization with other probes showed a less prominent signal, suggesting a variable affinity of different probes for this transcript. These probes are positioned too far away from each other on the major satellite subrepeat to give any precise indication of its sequence, casting doubt on the origin of this transcript. Regardless, there is no doubt that the heat shock in mouse cells causes an overexpression of a short transcript.

Up until now, no increase in the RNA of centromeric or pericentromeric origin upon stress has been detected in mouse cells. The only two found RNA upregulated upon heat shock in mouse cells are SINE B1 RNA (136 nt) and B2 RNA (178 nt), that was found to be involved in the regulation of the RNA pol II transcription during stress [START_REF] Allen | The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock[END_REF]). On the other hand, it is well established that in human cells, transcripts from pericentromeric satellite III are upregulated during heat shock [START_REF] Jolly | Stress-induced transcription of satellite III repeats[END_REF]). These transcripts accumulate at their site of transcription and together with the heat shock protein 1 form nuclear stress bodies, of a yet unclear function. Even though this transcript is revealed with several of our LNA probes, we cannot conclude that it is indeed transcribed from the major satellites.

Nevertheless, upregulation of this transcript upon heat shock and its complete disappearance after the treatment with RNA polymerase III inhibitor suggests that the B2 RNA is not the only repeat-associated transcripts that has a possible role in cellular stress. A parallel with the accumulation of the human satellite III transcripts could be imagined. However, we have tried to determine the accumulation of the major satellite transcripts with the heat shock protein, but found no such association in mouse cells (data not shown). This does not completely exclude the possibility of such an interaction. It would therefore be interesting to identify the sequence of this transcript. As our initial experiments of major satellite sequence characterization using adapter ligation failed, we wanted to proceed in the characterization of this specific transcript starting with gel purification of the small region of a gel where this transcript migrates. Following adapter ligation on both ends of the purified pool of transcripts and sequencing of the obtained products, keeping in mind the possible chemical modification that could interfere with the ligations, we wanted to obtain the sequence of the overexpressed transcript. Due to a lack of time, these experiments were not completed, therefore the origin of the short RNA overexpressed upon heat shock in mouse cells still remains unknown. It would be interesting to see if we can detect the B1 or B2 RNA with the LNA probes designed to target these transcripts, and to verify if these transcripts are overexpressed as well in our heat shock conditions.

The 96 transcript was the only transcript that disappeared completely upon the treatment with the high level of actinomycin D, but not upon the treatment with low levels of actinomycin or DRB. This suggested that this is an RNA polymerase III transcript.

Interestingly, the B1 and B2 RNA, that are equally overexpressed upon heat shock are also RNA pol III transcripts [START_REF] Allen | The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock[END_REF]). The RNA polymerase III is the least sensible RNA polymerase, and can be inhibited only with high levels of actinomycin D. We wanted to confirm our experiment by using directly a selective RNA polymerase III drug, the tagetitoxin. Unfortunately, production of the drug has stopped. Despite the disappearance of the 96 nt transcript, we observed no impact of any of the other inhibitors on RNA (for example 5S RNA should be inhibited by the low levels of actinomycin D). This suggests that the 3h treatment is not enough to induce a visible effect on these RNA. On the contrary, the short half life of the 96 transcript proves that it is very sensible to actinomycin D treatment.

Northern hybridization profiles in presence of inhibitors of chromatin modifiers

It has previously been shown that transcription within major satellite is elevated in the cells that do not have functional Suv39h histone methyltransferase [START_REF] Lehnertz | Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin[END_REF]. We have therefore treated the cells with the chaetocin, a drug that inhibits the histone methyltransferase Suv39h, hoping to provoke changes in major satellite transcription. We also used TSA, an inhibitor of histone deacetylase (HDAC), as a negative control, since TSA treatment of mouse cells does not interfere with the satellite transcription as observed with RT PCR [START_REF] Terranova | The reorganisation of constitutive heterochromatin in differentiating muscle requires HDAC activity[END_REF]. Despite the unclear results obtained in the northern blots with the LNA modified probes, we decided to investigate if we could observe a change for some of the detected transcripts after the treatment of cells with these two inhibitors of the chromatin modifiers.

We could not observe any difference in the hybridization experiments of RNA from non treated cells compared to the RNA from treated cells with both drugs. As we are not sure to be detecting the major satellite transcripts, we cannot conclude that the absence of the visible effect on the revealed transcripts is due to the absence of the effect of the treatment with the inhibitors of chromatin modifiers on major satellites. If we assume that some of our detected transcripts have a major satellite origin, these results would suggest that the treatment with the indicated inhibitors has no impact on the major satellites. Our treatment with chaetocin therefore did not help us to determine RNA from major satellite origin in our hybridization signals.

II.1. Targeting mouse major satellites using TALE fused to histone demethylase mJMJD2D

1.1. Context of the study Eventhought there is no doubt about the involvement of chromatin structures in the functional role of the centromeres, the chromatin code for all the cellular processes associated to centromeric repeats is still elusive. In this project, we propose to study the role of chromatin assembled around centromeric repeats by targeting to the natural major satellite repeats a TALE protein fused to a chromatin modifier.

In mouse cells, the pericentromeric heterochromatin, characterised by major satellite repeats and a specific epigenetic signature, the trimethylation of the histone H3 at lysine 9, H3K9me3, is organised in particular nuclear structures called chromocenters (see Introduction). To determine the role of the H3K9me3 epigenetic mark on chromocenter organisation, we established a new experimental approach using TALE protein fused to mouse histone demethylase mJMJD2D, to introduce chromatin changes at the major satellite region. mJMJD2D is able to remove one or two methyl groups from the H3K9me3, leaving the histone H3 either dimethylated (H3K9me2) or monomethylated (H3K9me1) at this position. Having the opportunity to use TACGENE (TAL effectors CRISPR for GENome Engineering) platform in our laboratory, we were able to design and synthesise TALE protein with DNA binding site that targets the major satellite repeats.

Three different TALE fusion proteins have been produced (Table 11, Figure 43). TALE N212 which targets an 18 nucleotide sequence at the major satellites was fused to either GFP or mouse histone demethylase mJMJD2D. TALE 394, previously used in the team to bind alpha satellite repeats of the centromere of the human chromosome 7, was fused to mouse histone demethylase mJMJD2D. The TALE 394 has no potential binding sites in the mouse genome. The constructs were expressed from a vector carrying the TALE DNA binding domain fused to either cDNA of mouse histone demethylase mJMJD2D or to GFP at the C-terminal of the TALE. TALE N-terminal domain was fused to an HA-tag (Figure 43).

The TALE target sites have been chosen from the sequences of the LNA oligonucleotides, which were previously used in DNA FISH experiments and have proved to successfully target their binding sites. The high copy number of the major satellites facilitates TALE binding and its visualisation in the cells.

Visualisation of the specificity of TALE recruitment on major satellites

To validate the specificity of TALE N212 and its correct recruitment at the major satellites, the NIH-3T3 cells (kind gift of C. Francastel) were transiently transfected with the vector pJL156, which carries TALE N212 fused to GFP. This fibroblast cell line is polyploid and possesses chromocenters which are easily visualised under the fluorescence microscope. 24 h after transfection, the cells were fixed and an immunofluorescence labelling was performed to visualise the TALE foci and assess the binding specificity to the region of interest. The major satellite foci are easily visible after the staining with Hoechst dye, revealing chromocenter structures (Figure 44). The H3K9me3 labelling reveals heterochromatin forming large foci. The Hoechst and H3K9me3 staining colocalise as expected at chromocenters. As can be seen from the Figure 44, the TALE N212-GFP is expressed in the cells and localises to the major satellite foci. The GFP signal is colocalised with the H3K9me3 signal at the major satellites. These results validate that the TALE N212 protein is correctly expressed and recruited at major satellite repeats in NIH-3T3 transfected cells.

Loss of H3K9me3 upon transfection with TALE fused to histone demethylase mJMJD2D

Having confirmed the specific binding of TALE N212, we wanted to verify that the TALE N212 fused to histone demethylase is equally localised to the chromocenters and whether the histone demethylase is active in our system. The cells were transiently transfected with the plasmid pJL172 that contains TALE N212 fused to mouse histone demethylase mJMJD2D. As a control, the plasmid pJL191 expressing the TALE 394 and fused to the same mouse histone demethylase was also introduced into the cells. The TALE 394 is a negative control that is not expected to bind the major satellites, therefore leaving the methylation status of H3K9me3 at the chromocenters intact. The cells were also transfected with TALE N212-GFP as a control. 24h after transfection, the cells were fixed and an immunofluorescence analysis was performed. The TALE fusion proteins were revealed with the anti-HA antibody. The major satellite foci are visualised after Hoechst staining. The histone H3 methylation status was verified using H3K9me3 specific antibody.

First, we observe that the TALE N212-GPF and TALE N212-mJMJD2D are both correctly expressed and recruited at the major satellite repeats, as indicated by the colocalisation of the Hoechst and the HA staining. This result shows that the fusion of the histone demethylase at the C-terminal part of the TALE does not abolish the correct recruitment of the TALE protein to its DNA binding sites. The TALE N394-mJMJD2D shows a punctuate staining all over the nucleus, without a specific localisation to the major satellites (Figure 45A).

Second, we observe that the transfection with TALE N212-mJMJD2D reveals the existence of three populations of transfected nuclei (Figure 45A). The first population of cells has received the TALE fused to the histone demethylase and H3K9me3 signal is still visible in chromocenters despite the correct recruitment of TALE demethylase. The second population shows the localisation of the TALE protein at the chromocenters concomitant with the disappearance of the H3K9me3 from the major satellite foci. In the third population of cells, the H3K9me3 staining is also lost from the major satellites. Most strikingly, however, the major satellite foci show an aberrant form. Most of the Hoechst spots lost their round shape (Figure 45). This suggests that the effect of the TALE demethylase on the H3K9me3 is variable and this variability has a gradual effect on chromatin organisation.

In the cells transfected with the TALE N212-GPF, the TALE protein localises to the major satellite foci (Figure 45B) and the heterochromatin foci are still present at the major satellites. Likewise, in all the cells transfected by the TALE N394-mJMJD2D, the H3K9me3 staining is similar to that observed with the TALE N212-GFP (Figure 45C). These results suggest that the absence of H3K9me3 foci observed in populations 2 and 3 and the associated disorganisation of chromocenters in population 3 is connected to the recruitment of the TALE-demethylase to major satellites, even if in population 1, the correct recruitment of the TALE-demethylase is not sufficient to induce observable H3K9me3 demethylation. This latter result may be explained by a different expression of the TALE fusion protein in these three populations of cells. II.2. Analysis of the effect of histone demethylation upon transfection with TALEs fused to histone demethylase mJMJD2D 2.1. Tools for Analysis of Nuclear Genome Organisation (TANGO)

We made use of the image analysis program recently developed in the team to draw statistical data from 3D fluorescent imaging of NIH-3T3 cells transfected with different TALE fusion proteins [START_REF] Ollion | TANGO: a generic tool for high-throughput 3D image analysis for studying nuclear organization[END_REF]. The quantitative image analysis was performed with TANGO after immunofluorescent staining of four different transfections of NIH-3T3 cells:

-cells transfected with no DNA, assimilated into non-transfected with a TALE fusion protein -cells transfected with the TALE N212-GFP (illustrated in Figure 45B) -cells transfected with the TALE 394-mJMJD2D (illustrated in Figure 45C) -cells transfected with the TALE N212-mJMJD2D (illustrated in Figure 45A)

The first three transfections correspond to the different control experiments. I will describe here briefly the process of analysis, a more detailed description can be found in the Materials and methods. The images of the cells were taken as a multichannel Zstack positions and imported into the TANGO software. The segmentation of the selected cellular structures (nucleus, TALE foci, major satellite foci) was performed on a large number of nuclei (>50). After the segmentation of the structures, quantitative measurements were carried out for each of the objects corresponding to these structures.

For a more precise segmentation and visualisation of major satellite foci, we wanted to combine the immunofluorescence with DNA FISH. On the Figure 46, it can be seen that the DNA FISH signal revealed by the LNA probe R4Y that hybridizes to the major satellite repeats, colocalises with the intense Hoechst-stained foci, confirming that the Hoechst foci represent well the major satellites. The TALE N212-mJMJD2D localises to the same region. However, the combination of the DNA FISH treatment with the immunofluorescence was incompatible with the labelling using anti-H3K9me3 antibody. As the major satellite foci are strongly stained by the DNA dyes as DAPI or Hoechst, we have therefore relied on Hoechst signal to achieve the segmentation of the major satellite foci (Figure 47). 2.2. Quantitative analysis of the demethylation of H3K9me3 at the major satellite foci upon transfection with the TALE N212-mJMJD2D

To assess if the histone demethylase is able to specifically demethylate the H3K9me3 epigenetic mark present at the major satellites, we first investigated the signal of the H3K9me3 measured in the interior of the major satellite foci. The total signal of H3K9me3 inside the segmented Hoechst foci that correspond to major satellites and inside the whole nucleus was measured for each nucleus for the four different types of transfected cells. The ratio of H3K9me3 was calculated with these two values (signal H3K9me3 in major satellite foci/ total H3K9me3 signal in the nucleus). This ratio corresponds to the integrated ratio of H3K9me3 signal inside the major satellite foci. Similarly, the ratio was calculated for the H3K9me3 signal present outside the segmented major satellite foci. We also assessed the total Hoechst signal present inside the segmented foci and in the totality of the nucleus, from which the integrated ratio of the Hoechst signal inside the major satellite foci was calculated. For more information about the measurements see Materials and methods.

For each analysed parameter we compared the data from the non-transfected cells (nt)

and from the cells positive for the different TALE fusion proteins. The cells positive for the TALE fusion proteins were determined by the presence of the TALE signal and correspond to 55-60% of the transfected cells. The cells where no signal of the TALE fusion protein is visible were not included in the analysis.

As can be seen in the Figure 48A, the ratio of the H3K9me3 signal inside the major satellite foci of the cells positive for TALE N212-mJMJD2D decreases 34,4 % in comparison with the non-transfected cells. This decrease is not observed in the cells transfected with TALE N212-GFP or TALE 394-mJMJD2D, which have the same ratio of H3K9me3 signal as the control non-transfected cells (Figure 48A). To confirm that the decrease in the H3K9me3 signal was specific for the major satellite foci, we checked the integrated signal of the H3K9me3 outside the foci in different transfected cells. The Figure 48B shows that the integrated ratio of H3K9me3 signal outside the major satellite foci does not change for the cells transfected with the TALE N212-GPF and the TALE 394-mJMJD2D when compared to the non-transfected cells. However, a modest increase in the signal ratio of H3K9me3 outside the major satellite foci is observed for the TALE N212-mJMJD2D.

The absence of variation of integrated ratios of H3K9me3 inside the major satellite foci, as well as outside the major satellite foci for the cells transfected with TALE 394-mJMJD2D suggests that the presence of histone demethylase mJMJD2D in the nucleus is not sufficient to induce demethylation of the pericentromeric heterochromatin.

In conclusion, this quantitative analysis of H3K9me3 signal in major satellite foci shows that the decrease of H3K9me3 ratio observed only in cells transfected with TALE N212-mJMJD2D is due to the recruitment of the histone demethylase to the major satellites.

This decrease is not associated with a decrease of the Hoechst signal, suggesting that the TALE-demethylase affects the H3K9me3 epigenetic mark but not the presence of major satellite foci (Figure 48C). As previously mentioned, the cells which have received the TALE N212-mJMJD2D

show two different patterns of H3K9me3 signal, and some of the transfected nuclei show an aberrant form of major satellite foci (Figure 45A). We have therefore decided to separate the nuclei on the basis of this observation and analyse the effects of the TALE N212-mJMJD2D on the H3K9me3 signal on these populations. Two criteria were taken into consideration: the existence of the H3K9me3 signal in the cells and the appearance of the Hoechst foci. Three populations were distinguished among the cells transfected with the TALE N212-mJMJD2D (Figure 45A). These three populations were all positive for the TALE N212-mJMJD2D signal and were as follow: cells positive for H3K9me3 signal (population 1), cells without H3K9me3 signal (population 2) and cells without H3K9me3 signal showing aberrant major satellite foci (population 3). These populations were compared to the cells negative for the TALE N212-mJMJD2D signal (nt).

The determination of the cells negative for the TALE signal was made on the basis of the observation of the microscopy images. This selection was made for all different transfections and it was verified that the values measured (integrated ratios of H3K9me3 and of Hoechst signal) correspond to the values obtained for the group of non-transfected control cells (cells that were submitted to electroporation without DNA). No significant difference was found between the population we determined as negative for the TALE signal and the cells which were not transfected with the TALE construct (data not shown).

We calculated the integrated ratio of H3K9me3 signal inside the major satellite foci for each population of the cells. The integrated ratio shows a decrease for each of the populations that received the TALE N212-mJMJD2D compared to the control nt cells (Figure 49A). Population 1 shows a small decrease (15%) of the H3K9me3 in the major satellite foci that was not detected by the simple observation of the microscopy images (Figure 45A). The population 2, which shows a loss of H3K9me3 on the fluorescence images (Figure 45A), shows a decrease of the H3K9me3 signal of 50%. The population 3 shows the strongest decrease when compared to the non transfected nuclei, with 60% decrease of the H3K9me3 signal (Figure 49A). Interestingly, the signal ratio of the TALE inside the major satellite foci strongly correlate with the quantified decrease of the H3K9me3 ratio in different populations (Figure 49B). The highest ratio of TALEs measured at major satellite foci is associated with the highest decrease of H3K9me3 ratio. This result permits to validate the distinction of the 3 populations made only on the basis of visual criteria observed from microscopy images (Figure 45A).

The ratio of H3K9me3 signal outside of the major satellite foci shows a minor increase of 4% for the cells from the population 2 and the 5% for the cells from the population 3 (Figure 49C). However, the raw signal data show that the H3K9me3 outside the foci is modestly decreased, which is probably due to the imperfect segmentation of the Hoechst foci (Figure 47). No loss of the Hoechst signal is observed for the cells positive for the TALE N212-mJMJD2D (Figure 49D). is no significant effect on the Hoechst signal ratio for either population compared to the TALE-negative cells.

The statistical analysis was made using Mann-Whitney-Wilcoxon test, ***p value <0.001, compared to nt.

Effect of the demethylation of H3K9me3 on major satellite foci

Since the H3K9me3 decrease in the major satellite foci is associated with an abnormal form of chromocenters in the cells expressing the highest level of TALE N212-mJMJD2D

(population 3, Figure 45A), we wanted to quantify more precisely this morphological changes of the major satellite foci. For this, we measured the number, volume and the elongation of TALE N212-mJMJD2D.

Compared to the non-transfected cells, the volume of the major satellite foci does not change for the two control cells transfected with TALE N212-GFP and TALE 394-mJMJD2D.

However, the volume of the major satellite foci is visibly increased in the cells transfected with the TALE N212-mJMJD2D. This increase of 17,5% and 14,5% is measured in comparison with the volume of the non-transfected cells and cells transfected with the TALE N212-GFP (Figure 50A), respectively, suggesting that this effect is due to the recruitment of the demethylase to the major satellite foci.

If we look at the three populations of the cells transfected with the TALE N212-mJMJD2D, we can see that the volume of the major satellite foci does not vary in population 1. However, the change in the volume is statistically significant for the populations 2 and 3.

The increase in the volume for the population 2 is 22%, while for the population 3 the volume shows a 24% increase (Figure 50B). shown no change in the elongation when compared to the non-transfected cells. A change can be observed for the population 2, which shows a significative increase of 4% in comparison with the non-transfected cells. The strongest increase is measured for the cells from the population 3, where the elongation increased for 21% comparing to the cells that have not received the TALE. This confirmes our observation of the microscopy images that showed the change in the morphology of the major satellite foci (Figure 45A).

Equally, the change in the volume and elongation correlates with the decrease in the number of major satellite foci. The cells transfected with TALE N212-GFP show the same major satellite foci number as non-transfected cells. A minimal decrease was observed for the control cells transfected with TALE 394-mJMJD2D (5%). However, the cells negative for the TALE 394-mJMJD2D show the same decrease compared to the non-transfected cells, suggesting that this decrease is not a consequence of the transfection with the TALE fused to histone demethylase (not shown). For the cells transfected with the TALE N212-mJMJD2D

we observe a decrease in the major satellite foci number (10%) comparing to the nontransfected cells (Figure 51A). The population 1 of the cells transfected with the TALE N212-mJMJD2D does not show change in the number of major satellite foci (Figure 51B). A significative decrease is observed for the populations 2 and 3, which correlates with the observed change in the volume of the major satellite foci for the same populations. The decrease of 15,6% is measured for the population 2, while a 30,5% decrease is measured for the population 3 relative to non-transfected cells (Figure 51B). of CRISPR/Cas9 would be our first choice for the targeted engineering at the major satellites today.

The Cas9 nuclease inactivated by site directed mutagenesis can be efficiently fused to other effector domains, similar to the fusion of TALE DNA binding domains [START_REF] Scott | Targeted genome regulation and modification using transcription activator-like effectors[END_REF]. It is therefore possible to fuse a chromatin modifier such as histone demethylase mJMJD2D to an inactivated Cas9. The complicated cloning strategies required for the design of specific TALE proteins could be easily replaced by the simple RNA guide for the Cas9.

Possible difficulties may be encountered concerning the accessibility to an environment such as heterochromatin. It is considered that the repeat variable domains of the TALE protein are more successful in binding to DNA. It would therefore be interesting to compare the efficiency and the effects obtained by the use of the Cas9 fused to mJMJD2D to the ones obtained with the TALE fusion protein.

The use of the TANGO software

The quantitative high throughput image analysis software, TANGO, allowed us to quickly and efficiently analyse the effects of the targeted chromatin modifications on the major satellite repeats. The values obtained with TANGO were used to calculate the signal ratio for the measured data. The use of ratio was preferred over the use of the directly measured data in order to correct for the false values that could be obtained due to the different background noise between each of the compared conditions.

Distinction of three populations of the cells that received the TALE N212-mJMJD2D

The analysis of the data has been performed by comparing the cells positive for the TALE N212-mJMJD2D with the non-transfected cells and also by comparing the three populations of the cells transfected with the TALE N212-mJMJD2D. The separation of the three populations was made on the observation of 1) the existence or not of the H3K9me3 signal on the major satellite foci and 2) the morphology of the chromocenters. The separation of the different nuclei to each of the populations was made visually. The population 1 was easily distinguishable from the rest of the transfected nuclei due to the presence of the H3K9me3 despite being positive for the TALE N212-mJMJD2D. The populations 2 and 3 even though both showed the abolishment of the H3K9me3 signal from the major satellite foci, showed differences in the morphology of the chromocenters. The distinction of these two populations was difficult because the effect on the morphology is gradual, making the classification of the nuclei displaying a certain profile to either of the populations rather difficult.

Specific recruitment of the TALE N212-mJMJD2D to the major satellite foci cause the demethylation of the H3K9me3

Comparing the cells positive for the TALE N212-mJMJD2D with the non transfected cells, we found a 34% decrease of the ratio of H3K9me3 signal for the cells positive for TALE N212-mJMJD2D. As no decrease was observed with the TALE control fusion proteins (TALE N212-GFP and TALE 394-mJMJD2D), nor there is an effect on the quantity of the Hoechst signal at the major satellites, we conclude that the demethylation is caused by the specific recruitment of the TALE fusion protein TALE N212-mJMJD2D and the demethylation activity of the demethylase mJMJD2D at the major satellite foci.

Indeed, the ratio of H3K9me3 signal outside the major satellite of the cells transfected with the TALE N212-mJMJD2D is comparable with the same ratio obtained for the cells transfected with the control TALEs. A minimal increase in the signal ratio outside the major satellites of 5% has been measured for the population 3. This population also shows the greatest decrease in the H3K9me3 signal ratio inside the major satellites. This minimal increase outside the major satellite foci is due to the imperfect segmentation of the major satellite foci made by TANGO. During the segmentation, some major satellite foci that were supposed to be segmented, were left out of the selection. The application of prefilters to our processing chains made the segmentation more stringent in order to avoid the segmentation of the objects that do not belong to major satellite foci. In this way we assured that the measurements obtained for the segmented objects belong exclusively to the major satellites, even if this restriction could result in the loss of certain foci that were left out of the selection.

Because of this, the decrease of the H3K9me3 in the major satellite foci that were not segmented will contribute to the measured decrease in the exterior of the foci, increasing the H3K9me3 signal ratio outside the major satellites.

For the cells from population 1, the measurement shows a 15% decrease in comparison with the non-transfected cells, even thought we can observe no impact on the H3K9me3 by a mere observation of the microscopy images. The populations 2 and 3 both show the loss of H3K9me3 on the microscopy images, which is confirmed with TANGO quantitative measurements as 50% and 60% decrease, respectively. It is interesting to notice that this percentage of signal decrease is observed on the microscopy images as the complete abolishment of the H3K9me3 signal for the major satellite foci on these two populations.

These results reveal the high sensitivity of the TANGO quantitative image analysis software.

Variable effect of the histone demethylase mJMJD2D

The difference in the decrease in the H3K9me3 signal ratio between the population 1 and populations 2 and 3 indicates that the effect of the histone demethylase is variable. The variable effect of the demethylase leads to different effects observed for the H3K9me3 and chromocenter organisation. These effects strongly correlate with the levels of expression of TALE protein in each of the populations. The strongest decrease in the H3K9me3 is observed for the major satellite foci with the highest TALE signal ratio. It is necessary to notice, however, that the difference between the populations 2 and 3 is not as important as comparing to the population 1. Indeed, the difference between the H3K9me3 signal ratio and the TALE signal ratio is modest for these two populations. As already mentioned, the separation between these two populations (2 and 3) was made visually upon observation of the integrity of the major satellite foci where we observe the changes in the chromocenter appearance for the population 3 and not for the population 2. The TANGO measurements confirm that there are morphological changes that happen on chromocenters at the population 3 upon transfection with the TALE N212-mJMJD2D. The fact that the smallest decrease of the H3K9me3 is measured for the population 1, and that the populations 2 and 3 can be differentiated on the basis of the chromocenter appearance, generally validate the distinction of these three populations made only on the basis of the visual criteria.

The histone demethylase mJMJD2D belongs to the Jumonji domain 2 lysine demethylase (KDM) family of proteins. Four members of this family in mammalian cells have been found to catalyse demethylation of H3K9me3/me2 and/or H3K36me3/me2. These four lysine demethylases, mJMJD2A, mJMJD2B, mJMJD2C and mJMJD2D differ in their specificity for the substrate [START_REF] Pedersen | Histone demethylases in development and disease[END_REF]. mJMJD2D catalyse demethylation of both H3K9me3 and H3K9me2, but has no activity on the monomethylated residues, nor it has an activity on H3K36me3 [START_REF] Whetstine | Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases[END_REF]. The different levels of expression of the TALE N212-mJMJD2D could impact the degree of demethylation of the major satellites. As the mJMJD2D can catalyse the demethylation leaving either H3K9me2 and H3K9me1, it could be checked using specific H3K9me2 and H3K9me1 antibodies whether the different populations we observed correspond to one of the demethylated profiles.

The mere presence of TALE fused to mJMJD2D in the nucleus is not sufficient to induce the demethylation of the major satellite foci

The control TALE N394 fused to mJMJD2D is as expected, not recruited to the major satellites and no decrease could be observed for the signal ratio of H3K9me3 at major satellites despite the existence of the punctate nuclear TALE staining in the cells transfected with this TALE fusion protein. This suggests that the sole presence of TALE fused to histone demethylase mJMJD2D in the nucleus is not sufficient to induce demethylation of the pericentromeric heterochromatin. These results are not in accordance with the results obtained by Slee et al. In their study, the overexpression of histone demethylase JMJD2B from a myctagged expression construct in human cells showed the loss of nuclear H3K9me3 [START_REF] Slee | Cancer-associated alteration of pericentromeric heterochromatin may contribute to chromosome instability[END_REF]. We could assume 1) either that the TALE 394-mJMJD2D is not functional, and we should test the activity of this TALE in human cells where it has a binding site at the alpha satellite region or 2) either that the difference observed between the two studies reside in the level of expression of the histone demethylase, as we showed that it can highly influence the H3K9me3 nuclear staining.

Moreover, the cells transfected with the JMJD2B show an increase in the chromosome missegregation [START_REF] Slee | Cancer-associated alteration of pericentromeric heterochromatin may contribute to chromosome instability[END_REF]). The similar effect could therefore be expected for the cells transfected with TALE N212-mJMJD2D. It would be necessary to look at the effects of the demethylation with this TALE fusion protein on the mitosis, specifically chromatid cohesion and chromosome segregation.

Effect of the demethylation of H3K9me3 on the organisation of major satellite foci

As already discussed in the previous chapters, the Suv39h histone methyltransferase converts the H3K9me1 to H3K9me3, inducing the binding of HP1, which is the hallmark of heterochromatin [START_REF] Rea | Regulation of chromatin structure by site-specific histone H3 methyltransferases[END_REF]Lachner et al. 2001b). The loss of Suv39h histone methyltransferase disrupts the H3K9me3 from major satellites, leaving the H3K9 in a monomethylated state and abolishing the HP1 binding to the major satellites, but still preserving the organisation of the major satellite foci [START_REF] Peters | Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability[END_REF].

We showed that the demethylation of the H3K9me3 with the TALE N212-mJMJD2D triggers morphological changes of the chromocenters. The major satellite foci show an increase in their volume and the decrease in their number in the cells transfected with the TALE N212-mJMJD2D comparing to control cells. The measure of the elongation allowed us to verify the form of the major satellite foci for each of the populations. The 20% change in the elongation was measured for the the population 3 comparing to non transfected cells, which further confirmed that the minority of the cells (17/126) display an aberrant form of chromocenters. These results suggest that the demethylation of the H3K9me3 epigenetic mark is associated with a disorganisation of major satellite foci, that change their form and become larger, which may be a result of the merging of several chromocenters.

The monomethylation of H3K9 is catalysed by two histone methyltransferases, Prdm3 and Prdm16. It has been shown that the double knock down of these two methyltransferases abolishes the H3K9me3 and disintegrates the major satellite foci [START_REF] Pinheiro | Prdm3 and Prdm16 are H3K9me1 methyltransferases required for mammalian heterochromatin integrity[END_REF]. The effect on the major satellite foci is even more pronounced in cells both double null for Suv39h and knock down for Prdm3/Prdm16. Moreover, these cells display the unstructured nuclear lamina that probably results in the breaking of the heterochromatin anchor in the nuclear lamina. This suggests that the H3K9me1 is important for the subsequent conversion of H3K9me1 to H3K9me3 by Suv39h and that there is a yet unknown factor required for the integrity of the chromocenters. The HDAC inhibitor TSA, which also interferes with the H3K9me3 and H3K9me1 has been shown to partially dissolve the major satellite foci which relocate toward the nuclear periphery with, however, no effect on the nuclear lamina [START_REF] Taddei | Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases[END_REF][START_REF] Pinheiro | Prdm3 and Prdm16 are H3K9me1 methyltransferases required for mammalian heterochromatin integrity[END_REF].

While the loss of H3K9me3 in Suv39h double null cells alone is not sufficient to induce changes on chromocenter organisation, the demethylation induced by mJMJD2D affects the major satellite clustering. The effect of the TALE fused to mJMJD2D shows however only a minor effect on the major satellites. This suggests possible other mechanisms that conserve the chromocenter organisation in Suv39h double null cells that are affected by the mJMJD2D induced demethylation of H3K9me3.

The HP1 binding to heterochromatin is dependent on the H3K9me3. It is probable that the HP1 binding is compromised in the cells transfected with the TALE N212-mJMJD2D.

The HP1 recruitment could be verified by ChIP qPCR in cells treated with TALE N212-mJMJD2D. The changes introduced to the heterochromatin might also have a strong impact on the transcription from major satellites. Derepression of major satellite transcripts has been observed in the cells depleted from Suv39h histone methyltransferase and in Prdm3/Prdm16 double knock down [START_REF] Lehnertz | Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin[END_REF][START_REF] Pinheiro | Prdm3 and Prdm16 are H3K9me1 methyltransferases required for mammalian heterochromatin integrity[END_REF]. We could therefore expect the derepression of the major satellites in the cells transfected with TALE N212-mJMJD2D. The level of transcription of major satellites, but also minor satellites, especially in the cells with the high expression of TALE proteins could be monitored by strand specific qRT PCR with the use of sequence specific primers (see Chapter I, Results). The major satellite transcription could also be investigated by RNA FISH using specific LNA probes that targeting major satellites. With the use of the TANGO software it would also be interesting to verify if the affected chromocenters show a change their in position in the nucleus.

General conclusion and perspectives

Mouse major satellites are formed of repetitive DNA that surrounds the centromeres.

These sequences display characteristic epigenetic marks and show transcriptional activity. To answer some of this questions, this work was divided in two parts. In the first part, we used LNA modified oligonucleotides to characterise the transcripts from major satellites in normal growing conditions and upon stress. In the other part of the work, we use a novel technique that is based on the fusion of TALE DNA binding domain to induce the demethylation of H3K9me3 at major satellites specifically, in order to study its effects on the organisation of chromocenters.

Our use of LNA modified oligonucleotides as probes for northern blot did not allow us to correctly characterise the transcriptional profile of major satellites. Even thought LNA modified probes were already used in northern blot experiments for characterization of low abundant miRNA molecules [START_REF] Válóczi | Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes[END_REF], they were never used for characterisation of non-coding transcripts from the repetitive regions such as major satellite. Previous studies that investigated the transcription from major satellite on northern blot experiments used mostly DNA or RNA probes, that do not highlight distinct bands but mostly show the existence of transcriptional activity in the form of a smear [START_REF] Lu | Proliferation-dependent and cell cycle regulated transcription of mouse pericentric heterochromatin[END_REF][START_REF] Hsieh | WDHD1 modulates the post-transcriptional step of the centromeric silencing pathway[END_REF]Bulut-Karslioglu et al. 2012b). These experiences pointed to the existence of major satellite transcripts of various sizes that accumulate during different stages of the cell cycle [START_REF] Lu | Proliferation-dependent and cell cycle regulated transcription of mouse pericentric heterochromatin[END_REF] or show an enrichment of longer species upon down regulation of a heterochromatin-associated protein WDHD1 [START_REF] Hsieh | WDHD1 modulates the post-transcriptional step of the centromeric silencing pathway[END_REF], with no indication of the precise size or strand of origin of the RNA. LNA modified probes were successfully used in RNA FISH experiments to monitor major satellite transcription in mouse embryos [START_REF] Probst | A strand-specific burst in transcription of pericentric satellites is required for chromocenter formation and early mouse development[END_REF]. A recent study made use of LNA gapmer oligonucleotides for a specific knock down of major satellite transcripts [START_REF] Casanova | Heterochromatin Reorganization during Early Mouse Development Requires a Single-Stranded Noncoding Transcript[END_REF]. We showed that the use of LNA modified oligonucleotides allows the detection of single transcripts on the blot. However, because of the hybridization on various known RNA molecules and the lack of strategies for the elimination of this non-specific signal prevented us from determining which of the transcripts are indeed of major satellite origin. These experiences question the relevance for the use of these oligonucleotides for a specific detection of transcripts from repetitive sequences by northern blotting.

It is known that the variations in major satellite transcription correlate with changes in the epigenetic status of these sequences. Elevated transcription has been observed for cells deficient for the Suv39h histone methyltransferase. Some studies point to the correlation between the major satellite transcription and the organisation of major satellites into chromocenters. For example, elevated levels of major satellite transcripts correlate with enhanced H4K20me3 and H3K9me3 marks during muscle cell differentiation [START_REF] Terranova | The reorganisation of constitutive heterochromatin in differentiating muscle requires HDAC activity[END_REF]). The peak of major satellite transcription and their subsequent downregulation coincide with the time of chromocenter formation during embryo development. Moreover, knock down of reverse major satellite transcripts has been shown to prevent chromocenter organisation during embryo development [START_REF] Casanova | Heterochromatin Reorganization during Early Mouse Development Requires a Single-Stranded Noncoding Transcript[END_REF]. These studies clearly point to the role of these transcripts in major satellite clustering. Indeed, a single stranded major satellite transcript associates with HP1 at heterochromatin. This indicates the possible structural role for major satellite transcripts [START_REF] Maison | SUMOylation promotes de novo targeting of HP1α to pericentric heterochromatin[END_REF]).

There have been several studies that used TALE binding domain fused to different chromatin modifiers to directly interfere with epigenetic marks on different genomic loci [START_REF] Konermann | Optical control of mammalian endogenous transcription and epigenetic states[END_REF][START_REF] Mendenhall | Locus-specific editing of histone modifications at endogenous enhancers[END_REF]. We have shown that the targeting of the histone demethylase mJMJD2D fused to a TALE binding domain designed to target major satellites is an efficient way to specifically modify H3K9me3 epigenetic mark on the major satellite region. The use of TALE DNA binding domain provides new means to simply and efficiently target histone modifications. We have shown that by demethylating the H3K9me3

1. 9 .

 9 Cell transfection .................................................................................................... 1.10. Immunofluorescence ........................................................................................... 1.12. Microscopy ........................................................................................................ 1.13. TANGO analysis ............................................................................................... I.2. Materials .................................................................................................................. 2.1. Oligonucleotides .................................................................................................. 2.2. Primers ................................................................................................................ Results ................................................................................................................................. Characterization of major satellite transcripts using LNA oligonucleotides ............

  4. High stringency washing .............................................................................. 1.2.5. RNA and DNA probes ................................................................................. 2.2. Strand specific RT PCR confirms transcription from major satellites ................ I.2. Expression and regulation mechanisms implicated in major satellite transcription 2.1. Influence of the inhibitors of chromatin modifiers on major satellite transcription .................................................................................................................................... 2.2. Changes in major satellite transcription upon thermal stress .............................. 2.3. Influence of different RNA polymerase inhibitors on major satellite transcription .................................................................................................................................... I.3. Sequence characterization ........................................................................................ 3.1. Technical details/methods for major satellite sequence characterization ........... I. 4. Conclusion and discussion ...................................................................................... CHAPTER II .................................................................................................................... TALE fused to histone demethylase mJMJD2D for epigenetic engineering at pericentromeric regions of mouse cells .......................................................................... II.1. Targeting mouse major satellites using TALE fused to histone demethylase mJMJD2D ...................................................................................................................... 1.1. Context of the study ............................................................................................ 1.2. Visualisation of the specificity of TALE recruitment on major satellites ........... 1.3. Loss of H3K9me3 upon transfection with TALE fused to histone demethylase mJMJD2D .................................................................................................................. II.2. Analysis of the effect of histone demethylation upon transfection with TALEs fused to histone demethylase mJMJD2D ................................................................................. 2.1. Tools for Analysis of Nuclear Genome Organisation (TANGO) ....................... 2.2. Quantitative analysis of the demethylation of H3K9me3 at the major satellite foci upon transfection with the TALE N212-mJMJD2D .................................................. 2.3. Effect of the demethylation of H3K9me3 on major satellite foci ....................... II. 3. Conclusion and discussion ..................................................................................... General conclusion and perspectives .................................................................................. Appendix ............................................................................................................................... Bibliography .........................................................................................................................

Figure 46 .

 46 Figure 46. Immuno-FISH detection of NIH-3T3 cells transfected with the TALE N212-mJMJD2D. .................

Figure 47 .

 47 Figure47. Segmentation of different structures byTANGO. ...............................................................................

Figure 50 .

 50 Figure 50. Volume of the major satellite foci upon transfection with the different TALE fusion proteins.. ........

Figure 51 .

 51 Figure 51. Number of major satellite foci upon transfection with the different TALE fusion proteins. ..............

Figure 1 .

 1 Figure 1. Historical drawings by Walter Flemming (1843-1905) showing cell division. The drawings show segregation of chromosomes or "threads" as Flemming calls them, during mitosis. The drawings represent the green algae Spirogyra (Figs. 47 -60), the plants Lilium corceum (Figs. 61-68), Iris sibirica (Fig. 69), and Lilium tigrinum (Fig. 70), the human cornea (Figs. 71 -73), testes of salamander (Figs. 74, 75) and the egg of the sea urchin Toxopneustes lividus (Fig. 75). Images are reproduced from Flemming's book Zellsubstanz, Kern und Zelltheilung, 1882 (Gonçalves Dos Santos Silva et al. 2008).

Figure 2 .

 2 Figure 2. Organisation of centromeric and pericentromeric region on a chromosome. The centromere (green) can be observed cytologically as a primary constriction on a metaphase chromosome. The centromere is surrounded by pericentromeric regions (red).

Figure 3 .

 3 Figure 3. Classification of repetitive DNA in the human genome. Human genome contains 55% of repetitive sequences. Repetitive DNA can be interspersed throughout the genome or appear as large blocks of tandem repeats. Interspersed sequences include DNA and RNA transposons. DNA transposons propagate through the socalled "cut and paste" mechanism, while RNA transposons are able to make copies of themselves that will be inserted in the genome ("copy and paste"). RNA retrotransposons are usually classified into long terminal repeats (LTR) and non-LTR, which are further classified to long and short interspersed nuclear elements (LINE and SINE). Tandem repeats differ by the length of the repeat unit and are classified into satellites, minisatellites and microsatellites (adapted from Jasinska & Krzyzosiak 2004).

Figure 4 .

 4 Figure 4. Centromeric and pericentromeric region in different organisms. Centromeric and pericentromeric regions show differences in the DNA sequence and length between organisms. a) Three DNA elements build the single point centromere of the S. cerevisiae. The AT-rich, non-repetitive DNA serves as a recognition site for protein recruitment and formation of a functional centromere. S. cerevisiae lacks the flanking pericentromeric heterochromatin. b) In S. pombe centromeres, the non-repetitive, AT-rich central core domain (cen) bordered by repetitive innermost repeats (imr) contains CENP-A homologs. Pericentromeric region is formed at the so-called outer repeats (otr) that flank the centromere. Outer repeats are composed out of two kinds of tandem repeat sequences, dg and dh. They are common to three chromosomes of S. pombe, while the central core together with the inner repeats differ between them. c) Drosophila centromeres are formed on the blocks of simple AATAT and AAGAG repeats, interrupted by transposons. Pericentromeres are composed out of different complex satellites with no known consensus sequence. d) In mouse, major and minor satellites distinguish the pericentromeric from centromeric region, respectively. e) Human α-satellite repeats span the centromeric region of all chromosomes. The 171 bp motif forms higher order units at the centromere. Flanking pericentromeric region also contains different alpha satellite families that are interrupted by satellite I, II, III as well as satellites beta and gamma (adapted from Allshire & Karpen 2009).

Figure 5 .

 5 Figure 5. Dicentric chromosome formation. Dicentric chromosomes arise after the fusion of two pieces of chromosomes that each contain a centromere. The fused chromosome contain two centromeres and is therefore called dicentric (adapted from the US National Library of Medicine).

Figure 6 .

 6 Figure 6. Histone modifications on different histone tails. DNA is wrapped around a nucleosome composed out of an octamere of two molecules of histones H2A, H2B, H3 and H4. Histones contain a flexible N-terminus know as a histone tail that extends from the nucleosome core. Each histone tail carries a different set of posttranslational modifications such as metylation (Me), acetylation (Ac), phosphorylation (P) and ubiquitinylation (Ub). These epigenetic modifications dictate the state of the chromatin and influence gene expression.

Figure 7 .

 7 Figure 7. Euchromatin and heterochromatin histone tail modifications. Different chromatin state is characterized by different histone modification patterns. N-terminal tails of histones H3 and H4 are shown. In heterochromatin, histone H3 is characterized by methylation of lysine (K) 9 and lysine 27. Histone H4 is methylated at lysine 20. Euchromatin s characterized by hyperacetylation of both H3 and H4. Histone H3 is methylated at lysine 4 (adapted from Dunleavy et al. 2005).

Figure 8 .

 8 Figure 8. The trilaminar kinetochore structure. Kinetochore appears as a trilaminated structure under electron microscope. The outer kinetochore is composed out of KNL1, Mis12 and Ndc80 complexes that interact with microtubules (pale blue). This so called KMN network associates with the inner kinetochore composed out of centromere specific group of proteins that form constitutive centromere associated network (orange). The CCAN assembly on the centromere is directed by the deposition of the centromere specific histone H3 variant CENP-A.

Figure 9 .

 9 Figure 9. CENP-A is interspersed with H3 on extended chromatin fibres. CENP-A nucleosomes are not adjacent along the DNA fibre but rather scattered between the blocks of H3 containing nucleosomes. Core histones were detected by immunofluorescence on extended chromatin fibres (Black & Bassett 2008).

Figure 10 .

 10 Figure 10. Organisation of centromeric chromatin. A three-dimensional structure of centromeric chromatin suggests that the blocks of CENP-A and H3 nucleosomes form a cylindrical structure. CENP-A nucleosomes are oriented toward the outer kinetochore plate, where they facilitate the assembly of other kinetochore components.Canonical H3 containing nucleosomes are epigenetically modified. They are dimetylathed on lysine 4 (H3K4me2) and oriented toward the sister chromatids. Flanking the centromere core, pericentromeric heterochromatin carries H3K9me2 and H3K9me3 marks and recruits other proteins such as HP1 and cohesin[START_REF] Sullivan | Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin[END_REF].

Figure 11 .

 11 Figure 11. HP1 domains and interacting partners. HP1 proteins contain three distinct domains, chromodomain, chromoshadow and hinge domain. The N-terminal chromodomain binds methylated lysine 9 of histone 3. The central hinge domain can interact with both DNA and RNA. Chromoshadow domain has multiple interaction partners, including other chromoshadow domain proteins, including HP1 itself, which ensures selfassociation of HP1. Chromoshadow domain binds methyltransferase Suv39h and number of other chromosomal proteins such as Dnmt3a and Dnmt3b and CAF-1 (adapted from Maison & Almouzni 2004).

Figure 12 .

 12 Figure 12. Formation of pericentromeric heterochromatin. Histone methyltransferase Suv39h methylates histone H3 at lysine 9. At the same time, Suv39h interacts with HP1 which is guided to heterochromatin by a non-coding RNA (see Chapter 2, Introduction) and binds to metylated H3K9. Modification of histone H4 is mediated by histone methyltransferase Suv4-20h2 in a Suv39h dependent manner.

Figure 13 .

 13 Figure 13. Heterochromatin formation in S. pombe. Small RNA complementary to the yeast centromeric repeats are transcribed and processed by the RNAi machinery. The dsRNA is cleaved by Dicer1 to generate double stranded siRNA, which are bound by Ago1. Ago1, Tas3 and Chp1 form an RNA interference transcriptional silencing complex (RITS). RITS localizes to heterochromatin, where it associates with RNAdependent RNA polymerase complex (RDRC). The siRNA (red) guides RITS to the complementary sequence on heterochromatin (blue). The histone methyltransferase Clr4 catalyses methylation of H3K9, creating new binding sites for chromodomain proteins Swi6 and Chp1. Chp1, as component of RITS, stabilises RITS by binding to methylated H3K9me nucleosomes. RDRC then generates new dsRNA using nascent RNA as a template, contributing to the self reinforcing loop of heterochromatin assembly (adapted from Castel & Martienssen 2013).

Figure 14 .

 14 Figure 14. Organisation of mouse centromeric and pericentromeric region. Mouse telocentric chromosome.

Figure 15 .

 15 Figure 15. Association of pericentromeric region in mouse cells. Pericentromeric heterochromatin (major satellites) associates during interphase and forms chromocenters. Centromeric regions (minor satellite) are

II. 1

 1 Non-coding RNA 1.1. The RNA world, old and new 1.1.1. A new perspective on RNA Postulated by Crick, the central "dogma" of molecular biology had established the view of RNA molecules as simple intermediaries between the DNA and the protein.

Figure 16 .

 16 Figure 16. The percentage of non-coding to total genomic DNA per haploid genome across species. The amount of non-coding RNA increases with organismal complexity. Prokaryotes contain less than 25% noncoding DNA, simple eukaryotes between 25-50%, while this number rises to more than 50% in fungi, plants and animals, reaching approximately 98,5% in humans. Different colours present prokaryotes (bacteria and archea) (blue), single cell eukaryotes (black), N.crassa (gray), plants (green), non-chordate invertebrates (nematodes, insects) (purple), urochordate C.intestinalis (yellow), vertebrates (red) (Mattick 2004).

(Figure 17 .

 17 Figure 17. piRNA pathway in D.melanogaster. piRNA are amplified via the so-called ping-pong cycle in D.melanogaster germ line. Primary precursors (long orange lines) antisense to transposons are transcribed from the piRNA clusters. These antisense piRNA are sliced by AGO3, which is loaded with a complementary sense piRNA (short purple line). Mature antisense piRNA (short orange lines) are subsequently loaded to Aubergine (Aub) or PIWI that recognises the matching transposon transcript (long purple line) and cleaves it to generate the sense piRNA that are loaded to AGO to produce additional antisense piRNA completing the cycle (adapted from Bourc'his & Voinnet 2010).

  . Despite their abundance, a systematic classification of long non-coding transcripts is still lacking. Most authors define them as non-coding RNA of more than 200 nt in length, with an open reading frame containing less than 100 nucleotides. Long ncRNA have been found to be involved in different functions in the cell where they are implicated in various epigenetic processes. They participate to the regulation of gene expression by chromatin remodelling, acting at the transcriptional and post transcriptional level, by interacting with transcription factors or by being involved in RNA splicing mechanism. They can also participate to the formation of cellular structures and act as scaffolds for miRNA (Figure 18).

Figure 18 .

 18 Figure 18. Functions of long ncRNA. A) LncRNA could be processed to produce small endogenous siRNA that might target other RNA molecules leading to their degradation. B) LncRNA can sequester miRNA acting as miRNA scaffold, affecting the miRNA targeting of mRNA. C) They can also act as protein scaffolds. D) LncRNA can interact with protein to modulate their localisation. For example, lncRNA NRON (long nc repressor of NFAT) bind transcription factor NFAT (nuclear factor of activated T cells), influencing its cytoplasmic export and leading to the repression of NFAT target expression. E) LncRNA can recruit transcription factors to gene promoters to activate gene expression or can block their binding by the formation of RNA-DNA triplex. F) LncRNA can regulate alternative splicing. G) LncRNA can bind to and recruit chromatinremodelling complexes to chromatin thereby inducing changes in the chromatin state. H) LncRNA can participate to the formation of cellular structures such as paraspeckles[START_REF] Gutschner | The hallmarks of cancer: a long non-coding RNA point of view[END_REF]

Figure 19 .

 19 Figure 19. LncRNA recruit chromatin remodelling complexes to chromatin. Long ncRNA HOTAIR, Xist/RepA and Kcnq1ot1 bind Polycomb repressive complex to HOXD locus, X chromosome and Kcnq1 locus, respectively, to trimethylate H3K27 and represses gene expression (Mercer et al. 2009).

Figure 20 .

 20 Figure 20. Xist lncRNA coats the inactive X chromosome. RNA fluorescent in situ hybridization on condensed chromosomes of differentiated mouse cells. Xist RNA (red) covers the inactive X chromosome from which it is derived (Ng et al. 2007).

  the CENP-A to the centromere. A long transcript from the centromeric alpha satellite in human cells have been found to associate with CENP-A and HJURP. The sequence specific knockdown of the transcript by the shRNA resulted in the formation of multipolar spindles and appearance of lagging chromosomes (chromosomes that fails to connect to the mitotic spindle and are left out of the newly formed daughter cell). This is caused by the abrogation of HJURP and CENP-A from the chromosomes in the absence of the centromeric transcript, pointing to the implication of centromeric RNA in targeting these proteins to the centromere[START_REF] Quénet | A long non-coding RNA is required for targeting centromeric protein A to the human centromere[END_REF].Centromeric transcripts are found to interact with the proteins of the Chromosomal passenger complex (CPC), Aurora B kinase, INCENP and Survivin. The Aurora B protein kinase, the principal enzyme of CPC, is involved in spindle formation, regulation of kinetochore-microtubule attachment and checkpoint and sister chromatid cohesion[START_REF] Lampson | Sensing centromere tension: Aurora B and the regulation of kinetochore function[END_REF]. Accumulation of centromeric transcripts results in mislocalization of Aurora B from the centromeres of the mitotic chromosomes and results in impaired targeting of Suv39h histone methyltransferase. Metaphase chromosomes are misaligned, showing altered shape and a defects in sister chromatid cohesion suggesting that the kinase activity of Aurora B and the interaction of Aurora B with centromeric chromatin could be controlled by centromeric transcripts[START_REF] Bouzinba-Segard | Accumulation of small murine minor satellite transcripts leads to impaired centromeric architecture and function[END_REF]. Moreover, mouse minor satellite transcripts accumulate during G2/M phase, coinciding with the association of CPC at the centromere[START_REF] Ferri | Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase[END_REF]).Accumulation of centromeric transcripts was also reported in mouse and human during differentiation and stress conditions such as lack of nutrients or heat shock, suggesting they might be associated with changes in chromatin that occur during this time[START_REF] Bouzinba-Segard | Accumulation of small murine minor satellite transcripts leads to impaired centromeric architecture and function[END_REF][START_REF] Valgardsdottir | Transcription of Satellite III non-coding RNAs is a general stress response in human cells[END_REF]. Centromeric transcripts accumulate as small species of around 100-500 nucleotides that could represent multiples of 120 nt minor satellite units. They might be produced by the cleavage of longer RNA molecules transcribed through contiguous repeats in exponentially growing, undifferentiated cells. For example, in mouse, an overexpression of 120-nt centromeric transcripts corresponding to minor satellites leads to chromosome misalignment and defects in sister chromatin cohesion[START_REF] Bouzinba-Segard | Accumulation of small murine minor satellite transcripts leads to impaired centromeric architecture and function[END_REF]).2.2.2. Centromeric transcription stabilise CENP-C binding to the centromeresStudies have shown that centromeric RNA interacts with the kinetochore proteins. In human cells, INCENP, a component of chromosomal passenger complex and CENP-C were found to localise to the nucleolus during interphase. This localisation seem to be dependent on the centromeric RNA as the RNAse treatment results in the loss of both proteins from the nucleolus. Delocalisation of alpha satellite RNA from the nucleolus seem to be sensitive to inhibition of RNA polymerase I, since actinomycin treatment in conditions required to inhibit RNA polymerase I delocalise centromeric transcripts and both CENP-C and INCENP from the nucleolus[START_REF] Wong | Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere[END_REF]). The significance of RNA pol I involvement is still not clear, since no reduction in expression level of centromeric transcripts could be detected. Moreover, specifically degrading single stranded RNA but not double stranded RNAs, results in the partial delocalisation of CENP-C and complete delocalisation of INCENP and survivin from the mitotic chromosomes. This suggests that their localisation is primarily dependent on the single stranded RNA. CENP-C and INCENP are re-established to both nucleolus and chromosomes after reintroduction of centromeric RNA to the cells.Nucleolus is an organelle that primarily functions as a production site of rRNA but it could be engaged in other cellular functions. Both CENP-C and INCENP contain nucleolus localisation peptides. Localisation of centromere components to the nucleolus could be explained by the similar mechanisms described for telomeric binding protein, which is sequestered in the nucleolus until the time for its release during telomere elongation in the late S phase. Similarly, nucleolus might function in sequestration of the centromeric proteins and RNA, or even act as a place of assembly of nucleoprotein complexes, until the time for mitosis when they are released to the nucleus and serve to the establishment of a functional kinetochore[START_REF] Wong | Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere[END_REF]. It has been therefore suggested that RNA might facilitate the assembly of the nucleoprotein components at the centromere in order to stabilise these centromere-associated complexes. CENP-C was first characterized as a DNA binding protein.CENP-C however has a domain similar to HP1 hinge region and is able to bind RNA in both in vitro and in vivo. In maize, the binding of CENP-C to the DNA is stabilised by its interaction with centromeric RNA[START_REF] Du | DNA binding of centromere protein C (CENPC) is stabilized by single-stranded RNA[END_REF]. Inhibition of RNA polymerase II activity has been shown to deplete centromeric transcription and reduce the CENP-C binding to the chromosomes, resulting in chromosome missegregation[START_REF] Chan | Transcription in the maintenance of centromere chromatin identity[END_REF]. Centromeric transcripts could thus safeguard kinetochore formation by stabilising CENP-C binding at mitosis[START_REF] Chan | Transcription in the maintenance of centromere chromatin identity[END_REF], while the transcription at pericentromere at this stage of the cell cycle could help the reestablishment of HP1 and heterochromatin structure at the end of mitosis[START_REF] Lu | Proliferation-dependent and cell cycle regulated transcription of mouse pericentric heterochromatin[END_REF]. 2.3. Transcription from the pericentromeric region 2.3.1. Pericentromeric transcription during stress

Figure 21 .

 21 Figure 21. Mechanism of transcriptional repression by non-coding RNA. In mouse cells, B2 RNA, an polymerase III transcript, binds to RNA pol II upon heat shock to repress mRNA transcription (adapted from Allen et al. 2004).

Figure 22 .

 22 Figure 22. SUMO-ylated HP1 targeting to pericentromeric heterochromatin. HP1 (red) becomes SUMOylated and associates with non-coding sense RNA (green), which guides HP1 to heterochromatin domains. HP1is further stabilised by its binding to Suv39-directed H3K9me3 (blue). Further accumulation of HP1 at heterochromatin is established in a self-enforcing loop where HP1 multimerise with other HP1 already present at the heterochromatin or bind to H3K9me3[START_REF] Maison | SUMOylation promotes de novo targeting of HP1α to pericentric heterochromatin[END_REF]).

Figure 23 .

 23 Figure 23. Chromatin organisation at major and minor satellites requires centromeric RNA. Minor satellites are characterized by alternating histone variant CENP-A and methylated H3K4. Minor satellite transcripts assist the association of centromeric proteins such as CENP-C, INCENP, Survivin and Aurora B (AUBK) to the centromere. Major satellite transcripts at pericentomeric heterochromatin mediate HP1 deposition to trimethylated H3K9 nucleosomes (Bierhoff et al. 2014).

  Satellite repeats are differentially expressed depending on the tissue, or the developmental and cell cycle stage. Their expression is accompanied by changes in chromatin organisation. Several transcription factor binding sites have been found to reside at the satellite sequences but their relation to satellite transcription has been established only for some of them. Not much is know about how transcription from centromeric and pericentromeric sequences is regulated or whether their transcription involves regulation by transcription factors or require promoters in centromeric chromatin.

  transition during embryogenesis and in pathological situations by interacting with LOXL2, a histone modifying enzyme that demethylates H3K4me3 by deamination, catalysing the formation of oxidised H3. During this transition, a global heterochromatin reorganisation takes place, including the release of HP1 from heterochromatin and is accompanied by the transcription of major satellites, all of which is triggered by Snail1 activation. Depletion of either Snail1 or LOXL2 has been shown to cause a repression of major satellite transcription and an increase in demethylted histone H3 (Millanes-Romero et al. 2013). Transcription factors Pax3 and Pax9 have a conserved binding sites at mouse major satellites sequence. In accordance to that, they localise at these regions in vivo and repress pericentromeric transcription. Cells deficient for both of these factors produce fivefold more transcripts from both strands of major satellite sequence compared to the wild type cells, while at the same time show a reduced H3K9me3 and H4K20me3 marks and mitotic defects (Bulut-Karslioglu et al. 2012a). Major satellite repeats contain binding site for numerous other transcription factors including YY1, Ikaros, Gfi1b, Sall1, Zeb1[START_REF] Brown | Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin[END_REF][START_REF] Shestakova | Transcription factor YY1 associates with pericentromeric gamma-satellite DNA in cycling but not in quiescent (G0) cells[END_REF] Bulut-Karslioglu et al. 2012a). In many other organisms, many transcription factor sites can be found at heterochromatin formed on repetitive elements. This observation led to the idea that intact, randomly distributed transcription factor binding sites on repetitive regions is a conserved mechanisms required for the formation of heterochromatin (Figure24). Transcriptional repression by multiple transcription factors would result in the silencing of the domains and the establishment of heterochromatin by the recruitment of other factors such as histone methyltransferases and histone deacetylases(Bulut-Karslioglu et al. 2012a).

Figure 24 .

 24 Figure 24. Transcription factor based mechanism for heterochromatin formation. Transcription factor binding sites are non-randomly distributed to regulatory elements such as promoters and enhancers at euchromatin. Heterochromatin lacks this synergy of regulation and transcription factor sites are more randomly distributed throughout the heterochromatin, making in this way a distinction between these two chromatin states (from Bulut-Karslioglu et al. 2012)

  even though mitosis is considered to be a transcriptionally silent phase, because of the dissociation of transcription factors from chromatin. Moreover, HP1 delocalises from heterochromatin during mitosis. Mitotic transcription has also been observed from centromeric alpha satellite regions in human cells. Inhibition of RNA polymerase II activity in mitotic cells results in a decrease in transcription from centromeres and causes chromosome missegregation[START_REF] Chan | Transcription in the maintenance of centromere chromatin identity[END_REF].I.1. Oligonucleotides for detection of nucleic acids 1.1. Hybridization properties of nucleic acidsThe discovery of DNA and its structural properties, notably the complementary base pair binding, soon led to the development of new experimental techniques for the study of nucleic acids[START_REF] Noyes | Nucleic acid hybridization using DNA covalently coupled to cellulose[END_REF][START_REF] Southern | Detection of specific sequences among DNA fragments separated by gel electrophoresis[END_REF]). These techniques are mostly based on the process of hybridization between a nucleic acid and its complementary oligonucleotide sequence. Oligonucleotide is a short chemically synthesized, single stranded piece of nucleic acid, that can hybridize to a specific part of the target sequence. The ability of hybridization of the short complementary sequence to any piece of nucleic acid of interest opened the possibilities for the multiple applications of this principle.The oligonucleotides can be used for a specific hybridization-based methods for detection of nucleic acids such as Southern and northern blot. For example, northern blot technique (especially in combination with polyacrylamide gel electrophoresis) is a widely used technique for direct detection of RNA molecules that allows to determine both the size of the RNA of interest and quantify its expression level. They are size fractionated by gel electrophoresis and then transferred or "blotted" to a membrane and then hybridized with the labelled oligonucleotide probes. To be detectable after hybridization, the labelling of the probe can be achieved either by the incorporation of the label into the probe or its direct attachment to the oligonucleotide.The principle of hybridization is also used in enzymatic reactions such as reverse transcription and PCR. These are sensitive methods that allow the detection and amplification of small amounts of nucleic acid material, which are based on a specific hybridization of oligonucleotide primers to the template molecule. Random priming is widely used for the reverse transcription reaction. It is based on the use of short single stranded nucleotide sequences, usually hexamers that are synthesised in each base combination, giving a mixture of oligos able to bind to any piece of RNA to produce a large pool of complementary DNA.On the other hand, oligo dT primers represent mixtures of thymidines of different sizes designed to bind exclusively to the sequences containing a polyA tail, restricting the use of these primers to polyadenylated RNAs, such as mRNAs, and making them impossible to use in hybridization to ribosomal and transfer RNA and to non-polyadenylated non-coding RNA.The use of sequence specific primers are designed according to the known sequence of interest and they allow for the greatest specificity in the detection of the RNA, especially when dealing with the low abundant molecules.Oligonucleotides are also used as antisense molecules. Antisense technology is based on the sequence complementarity between the oligonucleotide and the RNA to specifically inhibit mRNA translation or to degrade a target molecule.Oligonucleotides composed entirely out of DNA or RNA bases are less stable in a cellular environment, being quickly recognised and degraded by nucleases. They also have a limiting efficiency of cellular delivery and often show off-target effects. For this reasons, chemically synthesized nucleic acid analogues have been developed.1.2. Oligonucleotides 1.2.1 A brief history of oligonucleotidesThe history of oligonucleotides synthesis goes back to the beginning of the 20 th century and the studies of the structure of nucleic acids. The first oligonucleotide synthesized was a dinucleotide, created in 1955, by[START_REF] Michelson | Nucleotides part XXXII. Synthesis of a dithymidine dinucleotide containing a 3?: 5?-internucleotidic linkage[END_REF]. This dinucleotide provided the first chemical confirmation of the 3'-5' phosphodiester linkage between the nucleotides. For his study on nucleotides, Todd was awarded the Nobel Prize in Chemistry in 1957. At around the same time, biochemist Har Gobind Khorana brought revolutionary improvements in the field of oligonucleotide development by introducing new method of synthesis[START_REF] Schaller | Studies on Polynucleotides. XXIV. 1 The Stepwise Synthesis of Specific Deoxyribopolynucleotides (4). 2 Protected Derivatives of Deoxyribonucleosides and New Syntheses of Deoxyribonucleoside-3″ Phosphates 3[END_REF]. His oligonucleotides were extended by DNA polymerase and converted into RNA transcripts. These RNAs were then used for in vitro protein synthesis and served as a confirmation of the genetic code, for which Khorana was awarded the Nobel Prize inPhysiology and Medicine in 1968 (Yury E. Khudyakov 2002). Since these early beginnings, new breakthroughs in chemistry made the development of oligonucleotides a quick and standardised procedure. Today, each oligo is custom made and used as a tool in a wide range of molecular biology techniques. 1.2.2. Oligos with chemical modifications: 2'-O-Me and LNAThe broad use of oligonucleotides led to the development of new forms of modifications that provide the oligonucleotides with an increased affinity, enhanced thermal stability and increased nuclease resistance, making them optimal for specific applications.These new nucleic acid analogues such as 2-O-methyl oligonucleotides, locked nucleic acid (LNA), peptide nucleic acid (PNA) and morpholinos outperform the standard DNA probes in nucleic acid detection[START_REF] Karkare | Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino[END_REF].The main chemical difference between the DNA and RNA lies in the 2' position of the sugar moiety. The additional OH group at the 2' carbon induces a change toward the 3' endo conformation of the ribose, leading to the more stable RNA-RNA duplexes. The increase in the thermal stability of the 3' endo conformation of the RNA prompted the development of nucleic acid analogs carrying modifications on the 2'-position of the sugar in order to enhance the binding properties of oligonucleotides to their target sequences[START_REF] Prakash | An overview of sugar-modified oligonucleotides for antisense therapeutics[END_REF].Furthermore, the proximity of the 2'-sugar modification to the 3' phosphate group increases the nuclease resistance of the modified oligonucleotide. One of the limitations of the modifications at the 2'-position is that their introduction inhibit the RNAse H activity of the oligonucleotide. This has been successfully overcome by the use of gapmer oligonucleotides, where the segment of DNA region in the centre of the oligonucleotide is able to induce the RNAse H cleavage, while allowing the modifications at the flanking nucleotides[START_REF] Bennett | RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform[END_REF]. The introduction of the methyl group was one of the first modifications of the 2'-position of the ribose. This modification improved the thermal stability of the oligonucleotide bound to the target sequence and improved its nuclease resistance compared to phosphodiester or phosphothioate oligonucleotides. The 2'-O-methyl modified oligonucleotides had soon been established as tools for inhibition of miRNA and the study of miRNA function

  flexibility of the ribose and increases the local organisation of the phosphate backbone (base stacking interactions). This structure of LNA leads to the high binding affinity of the LNA for the complementary strand (see further text). The phosphate backbone in an LNA oligonucleotide is the same as the one in DNA and RNA, which enables the introduction of LNA monomers at the desired positions amongst DNA or RNA bases. Also, LNA oligonucleotides are soluble in water, which enables them to be labelled and used in hybridization experiments. LNA residues also show an increased resistance to nucleases[START_REF] Braasch | Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA[END_REF].

Figure 25 .

 25 Figure 25. Structure of the locked nucleic acid (LNA) monomer (Kauppinen et al. 2005).

  1.3.2. Use of LNA oligonucleotidesDifferent LNA oligonucleotide designs have been used as a tool for diverse biological applications. LNA oligonucleotides can be used either in the form of a mixmer, where LNA nucleotides are dispersed between the DNA bases, or as a gapmer, in which two LNA segments at the two ends of the oligonucleotide are separated by a stretch of 7-8 DNA bases.Oligonucleotide can also be entirely composed out of LNA nucleotides. These different LNA designs exert different modes of action. For example, the DNA segment in a gapmer oligonucleotide is able to recruit RNAse H enzyme when the oligonucleotide is hybridised to the RNA, leading to a specific cleavage of the DNA: RNA duplex. The LNA mixmers on the other hand are able to bind to the target RNA and induce steric blockage[START_REF] Braasch | Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA[END_REF]. a) LNA nucleotides for antisense targeting of cellular RNAs LNA modified oligonucleotides have a broad application as antisense molecules. The unmodified DNA oligonucleotide has several disadvantages as an antisense molecule. DNA is quite unstable in biological media and is quickly degraded by nucleases. Most importantly,

2. 1 .

 1 Tools for targeted genome manipulation 2.1.1. Targeting specific DNA loci in living cells An attractive means to directly modulate the genome function is via the use of fusion proteins, made out of sequence specific binding domains linked to different effector domains.

  have multiple binding sites (reviewed in de[START_REF] De Groote | Epigenetic Editing: targeted rewriting of epigenetic marks to modulate expression of selected target genes[END_REF]). To overcome these limitations various artificial DNA binding domains such as triple helix forming oligonucleotides and synthetic polyamides have been designed and used for specific targeting of genomic sequences[START_REF] Uil | Therapeutic modulation of endogenous gene function by agents with designed DNA-sequence specificities[END_REF].Lately, the discovery and the use of proteins with DNA binding domains significantly improved the targeted genome engineering. One of the first used natural DNA binding domain was the zinc finger binding domain. Zinc finger proteins are composed of tandem repeats of DNA binding zinc finger motif Cys2His2. Each motif is able to recognize a 3 base pair sequence, allowing the design of a zing finger protein which binds to a specific DNA sequence. Designer zinc finger proteins have been made to modulate the genome in different ways by the fusion of the zinc finger DNA binding domain to various effector domains. One of the main applications of zinc finger proteins is gene editing by zinc finger nucleases. The zinc finger nuclease (ZFN) contains the zinc finger DNA binding domain and a nuclease domain of FokI restriction enzyme.

Figure 26 .

 26 Figure 26. Schematic representation of the TALE protein. A) Transcription activator-like effector (TALE) consists out of a N-terminal translocation domain, a C-terminal transcriptional activation domain and 15.5-19.5 central repeat units, that mediate specific DNA binding. Each repeat module contains a conserved amino acid sequence with the exception of the repeat variable domain (RVD) at the position 12 and 13. B) The combination of two amino acids from the RVD recognise a single nucleotide, providing the binding specificity of each repeat module (one repeat= one nucleotide) (adapted from Biofutur 361, Jan 2015).

Figure 27 .

 27 Figure 27. Designer TALE proteins. Different effector domains can be fused to the TALE DNA binding domain to induce modifications at the genome, transcriptome and epigenome. For example, a nuclease domain can be fused to the TALE domain to create DNA breaks. TALE will guide the transcriptional activator or repressor domain to the regions of interest to specifically control the transcription of the target gene. Different chromatin modifiers such are methyltransferases (MT), histone acetyltransferases (HAT) or histone deacetylases (HDAC) can be fused to TALE DNA binding domain to induce chromatin modifications at the target region (adapted from Mussolino & Cathomen 2012).

  modifications 2.2.1. Targeting chromatin modifications in living cells The functional relevance of the complexity of chromatin modifications on the gene regulation and chromatin dynamics has been recognised. The challenge is still a precise determination of the roles of specific modifications in a specific genomic loci and in different physiological contexts. Numerous studies already began to address the question of functionality of different epigenetic marks and the associated chromatin modifiers. For example, by performing knock out experiments of different chromatin modifiers it was possible to obtain information about changes in transcription level of repetitive sequences.However, as knock out experiments could lead to genome-wide changes, a beter alternative is to target specific genomic loci[START_REF] Voigt | Epigenome editing[END_REF].Human artificial chromosome (HAC) containing alpha satellite DNA sequence arrays have been widely used to study chromatin modifications and CENP-A recruitment at the kinetochore. HAC contains a synthetic array of alpha satellite repeats containing a CENP-B, which is proven sufficient to sustain centromere assembly and function. In a construction where CENP-B containing alpha satellite monomers alternate with Tet operator containing monomers, the effect of the manipulation of chromatin and the kinetochore by targeting various TetR fusion proteins has been evaluated. Cells transfected with an expression construct encoding TetR-EYFP (enhanced yellow fluorescent protein) fused to the lysine specific demethylase 1 (LSD1) was used to deplete H3K4me2 mark from the HAC kinetochore. The loss of H3K4me2 was accompanied by a loss of transcription from the HAC centromeres[START_REF] Bergmann | Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore[END_REF]. The depletion of H3K4me2 correlated with the gradual loss of CENP-A and kinetochore function, caused by a reduced recruitment of the histone chaperon HJURP, confirming the importance of H3K4me2 as an epigenetic mark for the long-term kinetochore maintenance and function. Similarly, fusion of the herpes virus VP16 activation domain to the TetR resulted in elevated transcription and the loss of CENP-A at the kinetochore[START_REF] Bergmann | Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function[END_REF]).

Figure 28 .

 28 Figure 28. Chromatin in vivo assay system employs chemically induced proximity (CIP) to induce chromatin modifications in living cells. CIP uses small membrane-permeable molecules such as rapamycin to target chromatin modifications on the endogenous locus in vivo. Two sets of chimeric proteins bind the CIP molecule. The DNA binding domain such as ZFHD1 is fused to a CIP anchoring partner FKBP. The other protein set contains HP1α fused to CIP recruitment partner FRB. The addition of a small molecule such as rapamycin induces the reversible binding of CIP recruitment partner to the CIP anchor, tethering the HP1 protein to the modified Oct4 allele (Hathaway et al. 2012).

Figure 29 .

 29 Figure 29. Polydactyl zing finger protein fusion. The major satellite binding zing finger protein MaSat fused to GFP-binding protein (GBP) recruits the GFP-labelled proteins (Protein X-GFP) to major satellite region (Casas-Delucchi et al. 2012).

Figure 30 .

 30 Figure 30. Schematic representation of the LITE system. In the absence of light, TALE bound to light sensitive CRY2 bind the specific genomic loci. Stimulation with the blue light induces conformational changes in CRY2 and recruits the CIB1 molecule and its bound effector to the target loci (Konermann et al. 2013).

(

  [START_REF] Beumer | Comparing zinc finger nucleases and transcription activator-like effector nucleases for gene targeting in Drosophila[END_REF]. The TALE offers advantages over zinc fingers such as easier design and construction, simpler optimisation and an ability to recognise any DNA sequence. The fusion of TALE binding domain to specific chromatin modifiers now allow to directly modulate chromatin modification on various endogenous loci and presents a new powerful tool for study of epigenetics.ObjectivesCentromeric regions have a fundamental role during mitosis and meïosis being a place for kinetochore formation and sister chromatid cohesion. This function is evolutionary conserved throughout eukaryotic species, as well as the underlying DNA sequence organisation, repetitive in almost all eukaryotes. Although long considered as transcriptionally inactive, these sequences are transcribed as non-coding RNAs in many organisms. Mouse has a particular organisation at the centromere and presents an interesting model for the study of centromere biology. Centromeric regions in mouse are organized around two well-defined types of repetitive sequences. The centromeric constriction is characterized by the presence of the so-called minor satellite sequences (120 pb, more than 2500 repeats per chromosome), while the more abundant major satellite sequences (234 bp, about 25 000 repeats per chromosome) are located next to the centromere toward the chromosome arm, where they constitute the underlying substrate of what is called pericentromeric heterochromatin. Pericentromeric heterochromatin, which has been suggested to play a role in genome stability and gene regulation, is characterized by the presence of specific epigenetic marks, showing high levels of hypoacetylation, H3K9-and H4K20 trimethylation is organised in particular nuclear structures called chromocenters. Interestingly, major satellites are differentially expressed in mouse cells depending on the developmental stage, cell cycle and the physiological context. These changes in expression level correlate with changes in chromatin marks and with the changes in the nuclear organisation of pericentromeric heterochromatin. It is therefore tempting to assume that these transcripts are a part of physiologically relevant regulatory mechanisms. The experiments in which these transcripts were up-or downregulated affected the cellular division and differentiation and also affected the nuclear organisation of the pericentromeric heterochromatin.

  Probes for detection of major satellite repeatsThere is evidence for the transcription from the major satellite repeats but the precise characterization of the RNA remain elusive and has up until now been a challenge because of the repetitive nature of the sequence. This makes it difficult to determine the exact size of the transcript or its strand of origin. The problems encountered vis a vis the characterization of repeated sequences are mostly methodological. Conventional DNA or RNA oligonucleotides used for hybridization methods or PCR lack the specificity needed in dealing with repetitive sequences of a high degree of similarity. These limitations could theoretically be overcome by the use of chemically modified oligonucleotides such as LNA. The modified nucleotides allow the design of short probes while still retaining high annealing temperatures (and therefore annealing specificity, see Introduction, Chapter III), facilitating the discrimination between the highly similar regions of the repetitive sequence.

Figure 31

 31 Figure 31 Transcriptional profile of major satellites revealed by LNA modified oligonucleotides. A) schematic representation of the localization of the probes at the major satellite sequence. The reverse probes are indicated in blue and the forward in violet. The vertical lines separate the 4 homologous subrepeats on the major satellite repeat unit. B) On the left. RNA were separated on acrylamide gel and stained with ethidium bromide.

  with the probes targeting the forward strand of major satellites. The highly abundant molecules stained with ethidium bromide are equally hybridized with the LNA probes and are framed in red. C) The transcriptional profile revealed by the LNA probes targeting the reverse strand of the major satellites. The abundant cellular RNA are framed in red. RNA were separated on the 4% polyacrylamide gel and transferred to a nylon membrane. All hybridizations were performed at 55° C. Washing was performed with 2xSSC.

Figure 32 .

 32 Figure 32. Complex transcriptional profile revealed by LNA probes. The RNA was separated on 4% polyacrylamide gel and transferred to a nylon membrane. The hybridization was performed with different LNA probes targeting both major satellite strands (+ or -) at 55°C during 1h, washing was performed at 2X SSC.

Figure 33 .

 33 Figure 33. Comparison of the transcriptional profile of MEF cells revealed with isosequential LNA and 2'-O-Me probe targeting major satellites. The membranes were hybridized with either LNA or 2'-O-Me probe at 55° C and 42° C, respectively.

Figure 34 .

 34 Figure 34. Transcriptional profile revealed by northern blot after different stringency washes. The membranes were hybridized with the indicated LNA probes at 55°C. Following hybridization, a washing step was performed with either 2X SSC or 0,4X SSS.

Figure 35 .

 35 Figure 35. Transcriptional profile obtained with LNA, DNA and RNA probe targeting major satellite. The total RNA as well as the major satellite DNA fragment (majsat308) were separated on the 4% polyacrylamide

  of the expected PCR products after RT-PCR amplification of major satellite transcripts using the indicated primer pairs.

Figure 36 .

 36 Figure 36. Strand specific RT PCR analysis of total RNA isolated from MEF cells. After reverse transcription with forward or reverse major satellite specific primers, cDNA synthesis was performed in the presence (+RT) or absence (-RT) of reverse transcriptase as control. A) After strand specific RT-PCR amplification, a PCR product of the size of 138 bp was obtained with the primer pair fw60/rv170. B) Strand specific RT-PCR analysis with the pair fw170/rv220 shows an amplification of three specific PCR fragments (74, 308, 542 bp) for the cDNA obtained with either forward or the reverse primer. The bands indicated with an asterisk represent the non-specific amplification products.

Figure 37 .

 37 Figure 37. Northern analysis of RNA from cells treated with TSA and chaetocin. The RNA were hybridized with two different LNA probes (R21 and R31). No change is observed for the indicated bands when comparing to the lanes containing the RNA from cells treated with TSA (TSA) or chaetocin (C) to the RNA from the control cells (crtl). 5S RNA represent the loading control.

Figure 38 .

 38 Figure 38. Northern analysis of RNA from heat shocked MEF cells. MEF cells were heat shocked for 1h at 43 °C, followed by 1h recovery at 37 °C. Extracted RNA were subjected to northern hybridization using probe for major satellite. A) Schematic representation of the binding sites on major satellite of the five different LNA probes which reveal the increase in the level of the 96-nt transcript. B) The increase in the transcription of the 96-nt transcript revealed by three different LNA probes. The strongest hybridization signal is obtained with the probe R1. C) Quantification of the signal intensity of the 96-nt band in control (NHS) and heat shocked (HS) MEF cells. The signal was normalized to tRNA. D) Increase in the level of transcription of the 96-nt transcript was measured in different conditions, as indicated. The graph on the right shows the highest increase in the hybridization signal when the cells are subjected to heat shock at 45 °C during 1 hour following 1 hour of recovery at 37 °C. The signal was measured for the R1 probe and normalized to 5S RNA.

Figure 39 .

 39 Figure 39. Analysis of RNA from cells treated with different RNA polymerase inhibitors. A) Northern hybridization with three different LNA probes. The 96-nt transcript is downregulated after the treatment of cells with high dose of actinomycin D. B) Quantification of the level of intensity of the 96-nt transcript from MEF heat shock , DRB and actinomycin D treated cells. Level of intensity was normalized to tRNA.

Figure 40 .

 40 Figure 40. Comparison between total RNA and recovered short RNA. The RNA was separated on 4% polyacrylamide gel and stained with ethidium bromide.

Figure 41 .

 41 Figure 41. Northern blot after the ligation of adapters on the short RNA. The RNA were separated on 4%polyacrylamide gel. Following the 3' ligation, the abundant cellular RNA, visible after staining with ethidium bromide, which have ligated the adapter show a shift towards the higher molecular weight. On the contrary, no shift was observed after the 5' ligation.

  Figure 42. 3' adapter ligation on the U5 RNA. A) Northern hybridization of the total short RNA after the adapter ligation with the U5 probe. The 3' adapter was ligated on the 3'end of the U5 RNA (118 bp) which canbe observed as a shift of the ligated molecules toward the higher molecular weight (137 bp). The 5' adapter was not ligated on the U5. B) The PCR on the short RNA was performed using the prmer complementary to the 3' adpater and two U5 specific primers. The PCR products correspond to the size to the expected fragment.

Figure 43 .

 43 Figure 43. Schematic representation of the plasmid pJL156 and different TALE fusion proteins. A) Plasmid expressing TALE N212 fused to GFP and carrying an HA tag. The plasmid contains the TALE domaine to which a GFP is fused on the C-ter, followed by a poly A sequence. The plasmid contains a gene for the antibiotic resistance, viral origin of replication, CMV promoter and a viral NLS. Three HA tags are located at the N-ter of the TALE motif. B) Three different TALE fusion proteins used in the study.

Figure 44 .

 44 Figure 44. Immunofluorescence detection of the TALE N212-GFP binding to major satellites in mouse NIH-3T3 cells. The major satellite foci are easily visible after DNA staining with Hoechst (blue). The H3K9me3 is revealed by anti-H3K9me3 antibody (orange). TALE N212-GFP (green) colocalises with the H3K9me3 signal and chromocenters.

Figure 46 .

 46 Figure 46. Immuno-FISH detection of NIH-3T3 cells transfected with the TALE N212-mJMJD2D. Major satellites are revealed with DNA FISH using fluorescent LNA probe R4Y (red). The TALE N212-mJMJD2D is revealed with the anti-HA antibody (green). The LNA probe targeting major satellites (red) colocalises with the major satellite foci stained with Hoechst (purple) and the TALE N212-mJMJD2D (yellow).

Figure 47 .

 47 Figure 47. Segmentation of different structures by TANGO. A) The major satellite foci are brightly stained with Hoechst. The major satellites are segmented (yellow lines) after the application of the processing chains to the image. B) The segmented structures are labelled with a colour that is assigned to each object. The segmented nucleus is indicated with the yellow line.

Figure 48 .

 48 Figure 48. Effects of the demethylation with TALE N212-mJMJD2D on H3K9me3 signal in NIH-3T3 cells.A) The integrated ratio of the H3K9me3 signal inside the major satellite foci decreases for the cells transfected with the TALE N212-mJMJD2D. B) The ratio of the H3K9me3 signal outside the major satellite foci does not show any change between the transfected and the non-transfected cells. C) There is no effect on the Hoechst signal ratio for the any of the cells, transfected or not. The statistical analysis was made using Mann-Whitney-Wilcoxon test, ***p value <0.001, compared to nt.

Figure 49 .

 49 Figure 49. Quantification of H3K9me3, TALE signal and Hoechst signal in cells transfected with TALE N212-mJMJD2D and in non-transfected cells. A) The integrated ratio of the H3K9me3 signal inside the major satellite foci is measured for the three populations of the cells transfected with TALE N212-mJMJD2D. B) The quantification of the TALE signal ratio in the different populations of cells and in non-transfected cells shows a progressive increase of the TALE signal. C) The ratio of the H3K9me3 signal outside the major satellite foci does not change for any of the populations in comparison with the cells negative for the TALE signal. D) There

Figure 50 .

 50 Figure 50. Volume of the major satellite foci upon transfection with the different TALE fusion proteins.A) The increase in the major satellite foci volume is observed for the cells transfected with the TALE N212-mJMJD2D (17,5%). B) The populations 2 and 3 show an increase in the volume of the major satellite foci when compared to the cells from the population 1 and to the cells negative for the TALE signal. C) Elongation of the major satellite foci shows a progressive increase for the populations 2 and 3. The statistical analysis was made using Mann-Whitney-Wilcoxon test, **p value <0.01,***p value <0.001, compared to nt.

Figure 51 .

 51 Figure 51. Number of major satellite foci upon transfection with the different TALE fusion proteins. A)The decrease in the major satellite foci number is observed for the cells transfected with the TALE N212-mJMJD2D. B) The population 2 and population 3 show the decrease in the major satellite foci number when compared to the cells from the population 1 and to the cells negative for the TALE signal. The statistical analysis was made using Mann-Whitney-Wilcoxon test, **p value <0.01,***p value <0.001, compared to nt.

  Also, in the nucleus, major satellites are organised in distinct nuclear structures, chromocenters, during interphase. There are numerous questions that remain unanswered concerning this specific chromatin region: i) what is the role of the non-coding transcripts, if any, in centromere formation and maintenance? ii) what are the mechanisms that drive the expression of these transcripts? iii) is there an equal contribution of the transcripts from both strands? iv) what is the contribution of short vs long transcripts? v) are the transcripts posttranscriptionally processed? vi) how is their transcription modulated in response to physiological changes? vii) what is the correlation of transcription with epigenetic marks found on heterochromatin? viii) what is the correlation between chromocenter organisation, transcription and epigenetic marks? ix) how does the change in epigenetic marks and transcriptional activity impact genomic stability?

  1.1. Probes for detection of major satellite repeats .................................................... 1.2. Characterisation of major satellite transcription by northern blotting of total RNA from mouse cells ...

..................................................................................................... 1.2.1. Transcriptional profile of major satellites in growing mouse cells .............. 1.2.2. Detailed characterization of northern hybridization signals ........................ 1.2.3. Comparison of transcriptional profile revealed by isosequential LNA and 2'-O-Me oligo ............................................................................................................. 1.2.

Table 2 . Cells lines and culture conditions used in this study.

 2

	1.1. Cell culture
	Cell lines
	Cells were cultivated in Dulbecco's modified Eagle's medium (DMEM, with 4.5 g/L D-
	Glucose, L-Glutamine and 110 mg/L Sodium pyruvate; Gibco) in a humified atmosphere at
	37 °C and 5% CO 2 in a 75 cm 2 tissue flasks, supplemented with 10% foetal bovine serum
				Media (Gibco, Life
	Cell line	Tissue origin	Species		Supplement
				Technologies)
	NIH 3T3	Embryonic fibroblast	Mouse	DMEM	10% FBS
	MEF	Embryonic fibroblast	Mouse	DMEM	10% FBS + glutamine

(FBS). Supplements were added when it was necessary (Table

2

). After reaching confluency, cell monolayers were detached by two washes of PBS 1X (Phosphate Buffer Saline), incubated in 0.05% trypsin/0.5 mM EDTA (Gibco) for 5 minutes, resuspended in an appropriate volume of growth medium and dispensed into sterile flasks in an appropriate dilution.

Table 3 . Complete list of oligonucleotides used in this study.

 3 Solution was vortexed and again briefly centrifuged at 2000 g. Final concentration was verified on Nanodrop or spectrophotometer.

	I.2. Materials
	2.1. Oligonucleotides
	Oligonucleotides were synthesised by Eurogentec. Oligonucleotide powder was briefly
	centrifuged at 5000 g and resuspended in water in order to obtain a concentration superior to
	200 µM. Name	Sequence (5'→ 3')	Size	3'-modification	Strand
	majsat R11	AgTtTtCTCGCCAtAtTCcA	20	3'-biot	antisense
	majsat R21	CaTtTtCcGtGaTtTtCa	18	3'-dig	antisense
	majsat R31	CcTaAaGtGtGTaTtTcT	18	3'-biot	antisense
	majsat R32	CCtAcAgTgGAcAtTtCt	18	3'-dig	antisense
	majsat R1	TtTcTtGcCaTaTtCcAc	18	3'-biot	antisense
	majsat R14	GAtTtCgTcAtTtTtCaA	18	3'-dig	antisense
	majsat R34	CctTcAgTgTGcAtTtCt	18	3'-dig	antisense
	majsat R125	AgTtTtCCtCgCcAtAtT	18	3'-dig	antisense
	majsat R4Y	gGaTgTtTcTcAtTtTcC	18	5'-Cy3	antisense
	majsat 31	AgAaAtACaCaCtTtAgG	18	3'-texas red	sense
	majsat 12	GtgAAaTaTgGcGaGGaA	18	3'-biot	sense
	majsat 33	GaGaAaCaTCcAcTtGaC	18	3'-biot	sense
	majsat 3d	GcAcAcTgAaAgAcCtGg	18	3'-dig	sense

Table 4 . Primers used in the study. Name Sequence

 4

	Ad3Eco	TTTAACCGCGAATTCCAGC
	Ad5Sac	ACGGAATTCCTCACTrArArA

Table 5 . Adapter sequences used for RACE experiments

 5

Table 6 . List of oligonucleotides used in the study

 6

	Name	Sequence (5'→ 3')	Size	3'-modification	Strand
	majsat R11	AgTtTtCTCGCCAtAtTCcA	20	3'-biot	antisense
	majsat R21	CaTtTtCcGtGaTtTtCa	18	3'-dig	antisense
	majsat R31	CcTaAaGtGtGTaTtTcT	18	3'-biot	antisense
	majsat R32	CCtAcAgTgGAcAtTtCt	18	3'-dig	antisense
	majsat R1	TtTcTtGcCaTaTtCcAc	18	3'-biot	antisense
	majsat R14	GAtTtCgTcAtTtTtCaA	18	3'-dig	antisense
	majsat R34	CctTcAgTgTGcAtTtCt	18	3'-dig	antisense
	majsat 31	AgAaAtACaCaCtTtAgG	18	3'-texas red	sense
	majsat 12	GtgAAaTaTgGcGaGGaA	18	3'-biot	sense
	majsat 33	GaGaAaCaTCcAcTtGaC	18	3'-biot	sense
	majsat 3d	GcAcAcTgAaAgAcCtGg	18	3'-dig	sense

. In all the nucleotides, the LNA modification is introduced in one out of two nucleotides. The probe R11 is a gapmer oligonucleotide containing the window of 7 DNA nucleotides in the middle of the sequence. The LNAs are in indicated in lower letters, while the DNA is in upper case. The 3' modifications and the strand of origin are indicated.

Table 10 )

 10 and analysed by northern blot.

	Drug	Target	Duration	Concentration
	Actinomycine D	RNA polymerase I	3h	0,05 µg/mL
	Actinomycine D	RNA polymerase III	3h	5 µg/mL
	DRB	RNA polymerase II	3h	25 µg/mL

Table 10 . Conditions of treatment with different RNA polymerase inhibitors.

 10

Table 12 . Different measured values for non-transcfected cell and cells transfected with different TALE fusion proteins obtained after TANGO analysis.

 12

		Non-transfected	Population 1	Population 2	Population 3
	integrated H3K9 signal ratio inside the foci	0,08039137	0,06795847	0,03978223	0,03152161
	sem	0,001776686	0,00224239	0,002273911	0,003128102
	integrated H3K9 signal ratio outside the foci	0,9196086	0,9320415	0,9602178	0,9684784
	sem	0,001776686	0,00224239	0,002273911	0,003128102
	integrated Hoechst signal ratio	0,07403458	0,07221614	0,07689287	0,07972719
	sem	0,001368793	0,001568758	0,00171489	0,002852586
	mean H3K9 foci volume	278,2803	275,6856	339,426	346,6147
	sem	9,901069	9,648013	14,744851	23,079065
	elongation	1,382845	1,373815	1,440389	1,667597
	sem	0,01659601	0,01489947	0,01744075	0,04891749
	number of Hoechst foci	20,57143	20,66038	17,35714	14,29412
	sem	0,6072616	0,6509697	0,562594	0,7110716

Table 13 . Different measured values for non-transfected cell and three population of cells transfected with TALE N212-mJMJD2D obtained after TANGO analysis

 13

Remerciements

Chapter III Tools for study of repetitive sequences

Results

The world and the universe is an extremely beautiful place, and the more we understand about it the more beautiful does it appear. Concerning the organisation of the major satellite foci, we can conclude that first, the ratio of Hoechst signal inside the major satellite foci does not vary in non-transfected neither in cells transfected with the different TALE fusion proteins (Figure 48C and 49D), suggesting that the total quantity of Hoechst signal inside all major satellite foci is not affected by the TALE proteins; second, the recruitment of the TALE N212-mJMJD2D on major satellite repeats induced the increase in the volume and the form of the major satellite foci that is more accentuate in the populations 2 and 3 and correlated with the quantity of the TALE proteins in the cells (Figure 50); third, the increased level of TALE N212-mJMJD2D in transfected cells is associated with a decrease of the number of Hoechst foci (Figure 51).

Richard

II. 3. Conclusion and discussion

We have shown that the TALE fusion proteins, TALE N212-GFP and TALE N212-