Le variant d'histone CENP-A est le facteur responsable de la détermination épigéné- tique du centromère. Il permet le recrutement de nombreuses protéines centromériques, et constitue ainsi la brique fondatrice du kinétochore. Il possède un domaine amino-terminal non structuré dont la fonction précise reste encore à élucider, bien qu'il soit déjà établi chez certaines espèces que ce domaine est requis pour le bon fonctionnement du cen- tromère et conséquemment le bon déroulement de la mitose. Nous avons construit des lignées cellulaires humaines exprimant stablement diverses formes mutantes de CENP-A, qui nous ont permis de réaliser des expériences de pseudogénétique en supprimant l'ex- pression de la protéine CENP-A endogène. Nous observons une augmentation drastique du taux de défauts de ségrégation des chromosomes et de cellules plurinucléées dans des cellules exprimant uniquement le domaine globulaire de CENP-A, ce qui est en accord avec les données de la littérature et confirme l'importance du domaine amino-terminal. Un phénotype similaire est observé dans des cellules exprimant une protéine CENP-A entière mais dont le domaine amino-terminal n'est pas phosphorylable. Nos résultats montrent l'implication de la phosphorylation de la sérine de CENP-A dans le bon déroulement de la mitose, et suggèrent que la fonction mitotique du domaine amino-terminal est centrée sur cette seule phosphorylation. / The histone variant CENP-A is the epigenetic factor responsible for centromere deter- mination. It allows the recruitment of a handful of centromeric proteins, and thus acts as the primary foundation for the kinetochore. It comprises an unstructured amino-terminal domain to which no precise function has yet been assigned, although it is established in some species that the mere presence of that domain is required for proper centromere func- tion and thus successful completion of mitosis. We have established several human cell lines stably expressing GFP-tagged CENP-A constructs, allowing us to perform pseudoge- netic experiments by siRNA-mediated silencing of the endogenous CENP-A. Our results show a dramatic increase of mitotic defects and plurinuclear cells when cells express only the globular domain of CENP-A; this is in accordance with the litterature and confirms the importance of the amino-terminal tail. More importantly, a similar increase of mitotic defects is observed when cells express a full-length, but non-phosphatable, CENP-A. Our results show the involvement of the phosphatable serine 7 of CENP-A in the successful completion of mitosis, and may suggest that the role of the whole amino-terminal tail of CENP-A could be reduced to this single phosphorylation event.

Histone H2A.Z is a highly conserved replication-independent histone variant that is essential for survival in diverse organisms including Tetrahymena thermophila, Drosophila melanogaster, Xenopus laevis, and Mus musculus. H2A.Z has been shown to play a role in many cellular processes including, but not limited to, gene expression, chromosome segregation, cell cycle progression, heterochromatin maintenance and epigenetic transcriptional memory. However, the mechanism by which H2A.Z and its post-translationally modified forms participate in these diverse cellular events and their subsequent effects on chromatin structure and function are not entirely clear. A thorough review of H2A.Z is provided in Chapter 1. We have isolated native non-acetylated and acetylated forms of H2A.Z and characterized nucleosome core particles (NCPs) reconstituted with these proteins using the analytical ultracentrifuge (Chapter 2). We report that NCPs reconstituted with native non-acetylated H2A.Z exhibit a slightly more compact conformation compared to those reconstituted with H2A. Furthermore, we show that acetylation of H2A.Z in conjunction with acetylation of the histone complement, results in NCPs that are less compact and less stable than H2A.Z-containing NCPs reconstituted with non-acetylated histones. Acetylated H2A.Z NCPs are nevertheless more compact and stable than acetylated H2A-containing NCPs. We have also identified the presence of two H2A.Z protein isoforms in vertebrates, H2A.Z-1 and H2A.Z-2, and characterized the sites and abundances of their N-terminal peptide acetylation. Further characterization of the human H2A.Z isoforms is presented in Chapter 3 and indicates that they are expressed across a broad range of human tissues, and that they exhibit a similar but non-identical distribution within chromatin. Our results suggest that H2A.Z-2 preferentially associates with H3 trimethylated at lysine 4 compared to H2A.Z-1, and the phylogenetic analysis of the promoter regions of H2A.Z-1 and H2A.Z-2 indicate that they have evolved separately during vertebrate evolution. Overall, these data suggest that the two isoforms of H2A.Z present in vertebrates may have acquired a degree of functional independence. In Chapter 4, we show that H2A.Z and an N-terminally acetylated form of H2A.Z associate with the prostate specific antigen (PSA) gene promoter and the levels of these proteins are reduced upon induction of the gene with androgen. Furthermore, H2A.Z protein levels increase in response to treatment with androgen which correlates with an increase in the mRNA expression levels of the H2A.Z-1 gene. Preliminary Western Blot and quantitative PCR analysis of H2A.Z (-1 and -2) levels in a tumor progression model of prostate cancer indicate that increased H2A.Z expression may be involved in the development of androgen independent prostate cancer. Collectively, our results contribute to our understanding of H2A.Z biology in vertebrates and support a role for this protein and its acetylated forms in poising promoter chromatin for subsequent gene transcription.

Eukaryotes organize their genomes by wrapping DNA around positively charged proteins called histones to form a structure known as chromatin. This structure is ideal for keeping the genome safe from damage, but also becomes an obstacle for the transcriptional machinery to access information stored in the DNA. To facilitate a balance between storage and accessibility, eukaryotes utilize a family of enzymes known as ATP-dependent chromatin remodelers to directly manipulate chromatin structure. The diverse activities of these chromatin remodeling enzymes range from simply sliding nucleosomes to reveal transcription start sites, to editing the composition of a nucleosome by exchanging canonical histones for histone variants. Chromatin remodeling enzymes recognize features of the nucleosome that activate their ATPase domains and enable proper remodeling function. One nuclear epitope that has been extensively studied is the nucleosomal acidic patch. This negatively charged region on the face of the nucleosome has been shown to be essential for remodeling enzymes like Chd1, ISWI, and INO80C. The chromatin remodeler SWR1C edits nucleosomes by removing the canonical histone H2A from nucleosomes and exchanges it for the histone variant H2A.Z, but the role of the acidic patch in this process has not been investigated. In this work, I showed that SWR1C has normal binding affinity to acidic patch mutant nucleosomes and retains ATPase stimulation but can no longer exchange dimers on this substrate. This work also identified a novel arginine anchor on the essential SWR1C subunit, Swc5, that binds specifically to the nucleosomal acidic patch. The data in this work suggest a mechanism where SWR1C engages nucleosomes and uses the Swc5 subunit to recognize the nucleosomal acidic patch to couple ATPase activity to histone dimer exchange.

The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues. The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin. The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those in the frog erythrocytes. Characterization of the H1° in frog erythrocytes proved it to be the H1°-2 subvariant. Other experiments demonstrated that normal human B lymphocytes expressed the linker histone variants H1.2, H1.3, H1.4, and H1.5, and that B cells from patients with B-CLL expressed the same variants although in different amounts. The most striking dissimilarity was that amounts of H1.3 in the cells were decreased or undetectable in some samples. Sequencing did not discern any defects in the H1.3 gene, and thus the absence of H1.3 is probably regulated at the post-translational level. It was also observed that the levels of linker histone phosphorylation in EBV-transformed B lymphocytes were already increased in the G1 phase of the cell cycle, which is earlier than previously thought. This increase in phosphorylation is probably responsible for the lower affinity of linker histones for chromatin in EBV-transformed cells in the G1 phase of the cell cycle.

H2A.Z is an essential histone variant that has multiple chromosomal functions. One such role is transcriptional regulation. However, its role in this process is complex since it has been reported to function both as a repressor and activator. Earlier work in our lab showed that H2A.Z can be post-translationally modified with monoubiquitin (H2A.Zub1) and this form of H2A.Z is linked to transcriptional silencing. We further predicted that changes in the H2A.Z ubiquitylation status directly modulated its function in transcription. Furthermore, H2A.Z-containing nucleosomes possess a unique set of post-translational modifications (PTMs), compared to H2A nucleosomes, many of which are linked to transcriptional activation. The central aim of this thesis was to characterize the role of PTMs on H2A.Z nucleosomes in transcriptional regulation. To this end, I have provided the first evidence linking H2A.Z deubiquitylation to transcriptional activation. I demonstrated that ubiquitin specific protease 10 (USP10) is a deubiquitylase that targets H2A.Z in vitro and in vivo. Moreover, I found that both H2A.Z and USP10 are required for activation of androgen-receptor (AR)-regulated genes, and that USP10 regulates the levels of H2A.Zub1 at these genes. To understand how H2A.Z engages downstream effector proteins, in the nucleosome context, we used a mass spectrometry approach to identify H2A.Z-nucleosome-interacting proteins. Many of the identified proteins contained conserved structural motifs that bind post-translationally modified histones. For example, we found that Brd2 contains tandem bromodomains that engage H2A.Z nucleosomes through acetylated H4 residues. To investigate the biological relevance of this interaction, I present evidence that Brd2 is recruited to AR-regulated genes in a manner dependent on H2A.Z and the bromodomains of Brd2. Consistent with this observation, chemical inhibition of Brd2 recruitment greatly inhibited AR-regulated gene expression. Collectively, these studies have defined how H2A.Z mediates transcriptional regulation through multiple mechanisms and pathways.

<p>Transcription initiation is a key component in the regulation of gene expression. Recent high-throughput sequencing techniques have enhanced our understanding of mammalian transcription by revealing narrow and broad patterns of transcription start sites (TSSs). Transcription initiation is central to the determination of condition specificity, as distinct repertoires of transcription factors (TFs) that assist in the recruitment of the RNA polymerase II to the DNA are present under different conditions. However, our understanding of the presence and spatiotemporal architecture of the promoter patterns in the fruit fly remains in its infancy. Nucleosome organization and transcription initiation have been considered hallmarks of gene expression, but their cooperative regulation is also not yet understood.</p> <p>In this work, we applied a hierarchical clustering strategy on available 5' expressed sequence tags (ESTs), and developed an improved paired-end sequencing strategy to explore the transcription initiation landscape of the D.melanogaster genome. We distinguished three initiation patterns: 'Peaked or Narrow Peak TSSs&#8219;, 'Broad Peak TSSs&#8219;, and 'Broad TSS cluster groups or Weak Peak TSSs&#8219;. The promoters of peaked TSSs contained the location specific sequence elements, and were bound by TATA Binding Protein (TBP), while the promoters of broad TSS cluster groups were associated with non-location-specific elements, and were bound by the TATA-box related Factor 2 (TRF2).</p> <p>Available ESTs and a tiling array time series enabled us to show that TSSs had distinct associations to conditions, and temporal patterns of embryonic activity differed across the majority of alternative promoters. Peaked promoters had an association to maternally inherited transcripts, and broad TSS cluster group promoters were more highly associated to zygotic utilization. The paired-end sequencing strategy identified a large number of 5' capped transcripts originating from coding exons that were unlikely the result of alternative TSSs, but rather the product of post-transcriptional modifications.</p> <p>We applied an innovative search program called FREE to embryo, head, and testes specific core promoter sequences and identified 123 motifs: 16 novel and 107 supported by other motif sources. Motifs in the embryo specific core promoters were found at location hotspots from the TSS. A family of oligos was discovered that matched the Pause Button motif that is associated with RNA pol II stalling.</p> <p>Lastly, we analyzed nucleosome organization, chromatin structure, and insulators across the three promoter patterns in the fruit fly and human genomes. The WP promoters showed higher associations with H2A.Z, DNase Hypersensitivity Sites (DHS), H3K4 methylations, and Class I insulators CTCF/BEAF32/CP190. Conversely, NP promoters had higher associations with polII and GAF binding. BP promoters exhibited a combination of features from both promoter patterns. Our study provides a comprehensive map of initiation sites and the conditions under which they are utilized in D. melanogaster. The presence of promoter specific histone replacements, chromatin modifications, and insulator elements support the existence of two divergent strategies of transcriptional regulation in higher eukaryotes. Together, these data illustrate the complex regulatory code of transcription initiation.</p> / Dissertation

H2A.Z is an essential histone variant that has multiple chromosomal functions. One such role is transcriptional regulation. However, its role in this process is complex since it has been reported to function both as a repressor and activator. Earlier work in our lab showed that H2A.Z can be post-translationally modified with monoubiquitin (H2A.Zub1) and this form of H2A.Z is linked to transcriptional silencing. We further predicted that changes in the H2A.Z ubiquitylation status directly modulated its function in transcription. Furthermore, H2A.Z-containing nucleosomes possess a unique set of post-translational modifications (PTMs), compared to H2A nucleosomes, many of which are linked to transcriptional activation. The central aim of this thesis was to characterize the role of PTMs on H2A.Z nucleosomes in transcriptional regulation. To this end, I have provided the first evidence linking H2A.Z deubiquitylation to transcriptional activation. I demonstrated that ubiquitin specific protease 10 (USP10) is a deubiquitylase that targets H2A.Z in vitro and in vivo. Moreover, I found that both H2A.Z and USP10 are required for activation of androgen-receptor (AR)-regulated genes, and that USP10 regulates the levels of H2A.Zub1 at these genes. To understand how H2A.Z engages downstream effector proteins, in the nucleosome context, we used a mass spectrometry approach to identify H2A.Z-nucleosome-interacting proteins. Many of the identified proteins contained conserved structural motifs that bind post-translationally modified histones. For example, we found that Brd2 contains tandem bromodomains that engage H2A.Z nucleosomes through acetylated H4 residues. To investigate the biological relevance of this interaction, I present evidence that Brd2 is recruited to AR-regulated genes in a manner dependent on H2A.Z and the bromodomains of Brd2. Consistent with this observation, chemical inhibition of Brd2 recruitment greatly inhibited AR-regulated gene expression. Collectively, these studies have defined how H2A.Z mediates transcriptional regulation through multiple mechanisms and pathways.

CENP-A est le variant d’histone qui remplace spécifiquement l’histone H3 au niveau des centromères et confère ses propriétés uniques à la chromatine centromérique. La cristallographie aux rayon X, ainsi que la digestion à la MNase des nucléosomes contenant CENP-A suggèrent une flexibilité de l’ADN entrant et sortant de ce nucléosome. Néanmoins ces déductions restent aujourd’hui au stade hypothétique, en particulier, rien n’est connu sur le rôle éventuelle de cette particularité dans la fonction du nucléosome CENP-A. L’utilisation de la cryo-électromicroscopie nous a permis de déterminer les caractéristiques de la dynamique de l’ADN sortant du nucléosome CENP-A. Nos analyses biochimiques, de protéomiques et de pseudo-génétiques révèlent que la flexibilité élevée de l’ADN du nucléosome CENP-A ne permet pas l’interaction avec l’histone de liaison H1. In vitro, remplacer les 2 tours de l’hélice aN de CENP-A avec les 3 tours de l’hélice aN de H3 permet de restaurer l’interaction de l’histone H1. In vivo, le replacement des nucléosomes CENP-A par des nucléosomes contenant ce même nucléosome hybride aN-CENP-A permet également le recrutement de H1, mais cela conduit également à la délocalisation d’un certain nombre de protéines du kinétochore. Ce kinétochore ne permet pas une ségrégation correcte des chromosomes et il conduit à des phases de mitose et de cytokinèse défectueuses. L’ensemble de ces données montre que la conservation au cours de l’évolution de la flexibilité de l’ADN dans le nucléosome CENP-A est essentielle pour l’accomplissement de la division cellulaire. / CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure and MNase digestion of CENP-A nucleosome suggests flexible nucleosomal DNA ends but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic and genetic data reveal that the high flexibility of the DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn aN-helix of CENP-A with the 3-turn N-helix of H3 results in particles able to bind histone H1. In vivo replacement of CENP-A nucleosomes with the same NH3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins and significant mitotic and cytokinesis defects. Put together, ourdata reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway.

L’histone variante H2A.Z, histone de la famille H2A est enrichie dans certaines régions non transcrites de la chromatine, telles que la chromatine péricentromérique, centromérique et télomérique. Elle existe sous la forme de deux isoformes, H2A.Z-1 et H2A.Z-2, qui diffèrent par seulement 3 acides aminés et sont codés par deux gènes distincts, H2afz et H2afv. L’histone H2A.Z apparait impliquée dans divers évènements cellulaires tels que la transcription, la réparation de l’ADN ainsi que la prolifération et la différenciation cellulaire. La fonction de H2A.Z a été, jusqu’ici, analysée surtout grâce à la culture cellulaire in-vitro. Peu d’informations sont disponibles concernant le rôle de H2A.Z in-vivo dans différents organes, reflétant le manque de modèles animaux permettant l’invalidation génique de H2A.Z. Nous avons créé un modèle de souris transgénique permettant de réaliser in-vivo le double knock-out conditionnel (KI/cKO) des gènes H2afz et H2afv de manière tissu-spécifique dans les kératinocytes de la peau. Ce modèle d’étude in-vivo est unique car le seul à ce jour permettant d’éliminer complètement l’expression de H2A.Z. L’histone variante est physiologiquement présente dans toutes les cellules wild-type. Si les deux gènes codant pour H2A.Z sont délétés, la concentration de l’histone diminue au fur et à mesure des mitoses successives et finit par disparaître.L’épiderme en constante prolifération (tissu mitotique) mais aussi en constante différenciation (tissu post-mitotique), ainsi que le follicule pileux où ces deux processus intervenent de manière cyclique lors de la formation du poil, constituent un excellent modèle afin de disséquer le rôle spécifique de H2A.Z dans les processus de prolifération et de différenciation.L’induction par le tamoxifène du transgène K14CreERT2 invalidant les gènes H2A.Z (H2afz et H2afv) dans les kératinocytes, a tout d’abord été réalisée chez la souris adulte âgée de 6-8 mois. Elle entraine progressivement la perte totale de l’histone variante H2A.Z dans les cellules d’amplification transitoire (TA) qui se multiplient activement : au niveau du follicule pileux en phase de croissance (anagène) et au niveau des cellules situées de place en place au niveau de l’assise basale de l’épiderme. Le blocage en phase G2/M de ces cellules, perturbe l’homéostasie de la peau et appelle en retour une migration des cellules souches du follicule pileux vers l’épiderme, entrainant un épaississement de l’épiderme et une alopécie dans la région ventrale thoracique.Lors de la mise en place de la peau embryonnaire la délétion des deux gènes H2afz et H2afv, par l’induction du transgène K14CreERT2 suite à l’injection de tamoxifène ou l’utilisation du transgène K5Cre dont l’activité est constitutive, entraine la perte progressive de l’histone H2A.Z dans toutes les cellules épidermiques et les cellules des bourgeons pileux, qui toutes ont un fort indice de prolifération. La perte de H2A.Z entrainant le blocage des cellules en phase G2/M, ces cellules se différencient et s’accumulent dans la couche cornée.En conclusion, les différents phénotypes développés après le knock-out de H2A.Z dans les kératinocytes chez l’adulte ainsi qu’au cours de l’embryogénèse de la peau, nous ont permis de montrer l’implication de H2A.Z dans la progression de la mitose, et par la même directement son implication dans la régulation de l’homéostasie de l’épiderme. / Histone variant H2A.Z replaces the canonical histone H2A and is particularly enriched at non-transcribed chromatin regions as pericentromeric, centromeric and telomeric. Histone variant H2A.Z exists in two isoforms H2A.Z-1 and H2A.Z-2 coded by 2 distinct genes, H2afz and H2afv, that differ only by 3 amino acids. H2A.Z seems to be involved in several cellular events as transcription, DNA repair as well as proliferation and cellular differentiation. The function of H2A.Z has been, until now, mostly studied by the in-vitro cell culture. Few data are available concerning the role of H2A.Z in-vivo, regarding different organs, reflecting the lack of animal models to follow the genetic invalidation of H2A.Z. Histone variant H2A.Z is present in wild-type cells and when the 2 genes coding for H2A.Z are deleted, its concentration decreases progressively with succeeding mitosis until it disappeared.We have created a new and unique transgenic mouse model enabling to achieve, in-vivo, a double conditional knock-out of H2afz and H2afv genes, in a specific tissue, the skin epidermis. Constantly proliferating (mitotic tissue) and differentiating (post-mitotic tissue), the epidermis and hair follicles are excellent models to address the role of H2A.Z in cell proliferation and differentiation.In adult 6-8 months mice, the induction of the transgene K14CreERT2 by tamoxifen invalidates H2A.Z genes (H2afz and H2afv) and leads to the progressive loss of H2A.Z in transient amplifying cells (TA) that actively proliferate: in growing hair follicles (anagen) and in epidermal basal layer. The blocking of cells in G2/M phase, affects skin homeostasis calling in return the migration of stem cells from the hair follicle and epidermis, resulting in further epidermis thickening and alopecia of ventral thoracic regions.During skin embryogenesis, the deletion of both H2A.Z genes, activating K14CreERT2 transgene by tamoxifen or by using the constitutively activated K5Cre transgene, leads to a progressive loss of histone variant H2A.Z in all epidermal cells and hair bud cells, which both have a high proliferation index. The loss of H2A.Z results in cell block in G2/M phase, leads to cell differentiation and finally a build-up of dead skin cells in corneum layer.To conclude skin phenotypes obtained H2A.Z knock-out in the adult or during skin embryogenesis, show that H2A.Z plays an essential role in mitosis and appears directly involved in the regulation of epidermis homeostasis.

In multicellular organisms, all the cells are genetically identical but turn genes on or off at the right time to promote differentiation into specific cell types. The regulation of higher-order chromatin structure is essential for genome-wide reprogramming and for tissue-specific patterns of gene expression. The complexity of the genome is regulated by epigenetic mechanisms, which act at the level of DNA, histones, and nucleosomes. Epigenetic machinery is involved in many biological processes, including genomic imprinting, X-chromosome inactivation, heterochromatin formation, and transcriptional regulation, as well as DNA damage repair. In this review, we summarize the recent understanding of DNA methylation, cytosine derivatives, active and passive demethylation pathways as well as histone variants. DNA methylation is one of the well-characterized epigenetic signaling tools. Cytosine methylation of promoter regions usually represses transcription but methylation in the gene body may have a positive correlation with gene expression. The attachment of a methyl group to cytosine residue in the DNA sequence is catalyzed by enzymes of the DNA methyltransferase family. Recent studies have shown that the Ten-Eleven translocation family enzymes are involved in stepwise oxidation of 5-methylcytosine, creating new cytosine derivatives including 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Additionally, histone variants into nucleosomes create another strategy to regulate the structure and function of chromatin. The replacement of canonical histones with specialized histone variants regulates accessibility of DNA, and thus may affect multiple biological processes, such as replication, transcription, DNA repair, and play a role in various disorders such as cancer.