This study examined the DNA replication timing patterns of endothelial cell (EC)-enriched genes. We especially focused on a unique set of EC-enriched mRNA transcripts that possess differentially methylated regions (DMRs) within proximal promoters. It was previously shown that this DNA methylation plays an important functional role in regulating EC-enriched patterns of gene expression. Since the maintenance of these silencing marks is necessary for the inheritance of cell identity, the cell should ensure the proper transmission of such marks during mitotic cell cycle. Here we show that EC-enriched genes with DMRs replicate early during S phase in both expressing and non-expressing cell types. EC-enriched genes that do not have DMRs followed the expected trend, being early replicating in expressing cell types and late in non-expressing cell types. The relationship between DNA replication and DNA methylation was also investigated. A delay between DNA replication and DNA methylation was observed.

This study examined the DNA replication timing patterns of endothelial cell (EC)-enriched genes. We especially focused on a unique set of EC-enriched mRNA transcripts that possess differentially methylated regions (DMRs) within proximal promoters. It was previously shown that this DNA methylation plays an important functional role in regulating EC-enriched patterns of gene expression. Since the maintenance of these silencing marks is necessary for the inheritance of cell identity, the cell should ensure the proper transmission of such marks during mitotic cell cycle. Here we show that EC-enriched genes with DMRs replicate early during S phase in both expressing and non-expressing cell types. EC-enriched genes that do not have DMRs followed the expected trend, being early replicating in expressing cell types and late in non-expressing cell types. The relationship between DNA replication and DNA methylation was also investigated. A delay between DNA replication and DNA methylation was observed.

<p>To ensure genomic integrity, the genome must be accurately duplicated once and only once per cell division. DNA replication is tightly regulated by replication licensing mechanisms which ensure that origins only initiate replication once per cell cycle. Disruption of replication licensing mechanisms may lead to re-replication and genomic instability. </p><p>DNA licensing involves two steps including the assembly of the pre-replicative compelx at origins in G1 and the activation of pre-RC in S-phase. Cdt1, also known as Double-parked (Dup) in <italic> Drosophila Menalogaster </italic>, is a key regulator of the assembly of pre-RC and its activity is strictly limited to G1 by multiple mechanisms including Cul4<super>Ddb1</super> mediated proteolysis and inhibitory binding by geminin. Previous studies have indicated that when the balance between Cdt1 and geminin is disrupted, re-replication occurs but the genome is only partially re-replicated. The exact sequences that are re-replicated and the mechanisms contributing to partial re-replication are unknown. To address these two questions, I assayed the genomic consequences of deregulating the replication licensing mechanisms by either RNAi depletion of geminin or Dup over-expression in cultured Drosophila Kc167 cells. In agreement with previously reported re-replication studies, I found that not all sequences were sensitive to geminin depletion or Dup over-expression. Microarray analysis and quantitative PCR revealed that heterochromatic sequences were preferentially re-replicated when Dup was deregulated either by geminin depletion or Dup over-expression. The preferential re-activation of heterochromatic replication origins was unexpected because these origins are typically the last sequences to be duplicated during normal S-phase. </p><p>In the case of geminin depletion, immunofluorescence studies indicated that the re-replication of heterochromatin was regulated not at the level of pre-RC activation, but rather due to the restricted formation of the pre-RC to the heterochromatin. Unlike the global assembly of the pre-RC that occurs throughout the genome in G1, in the absence of geminin, limited pre-RC assembly was restricted to the heterochromatin. Elevated cyclin A-CDK activity during S-phase could be one mechanism that prevents pre-RC reassembly at euchromatin when geminin is absent. These results suggest that there are chromatin and cell cycle specific controls that regulate the re-assembly of the pre-RC outside of G1.</p><p>In contrast to the specific re-replication of heterochromatin when geminin is absent, re-replication induced by Dup over-expression is not restricted to heterochromatin but rather includes re-activation of origins throughout the genome, although there is a slight preference for heterochromatin when re-replication is initiated. Surprisingly, Dup over-expression in G2 arrested cells result in a complete endoreduplication. In contrast to the ordered replication of euchromatin and heterochromatin during early and late S-phase respectively, endoreduplication induced by Dup over-expression does not exhibit any temporal order of replication initiation from these two types of chromatin, suggesting replication timing program may be uncoupled from local chromatin environment. Taken together, these findings suggest that the maintenance of proper levels of Dup protein is critical for genome integrity.</p> / Dissertation

<p>Abstract</p><p>DNA replication is an integral part of the cell cycle. Every time a cell divides, the entire genome has to be copied once and only once in a timely manner. In order to accomplish this, DNA replication begins at many points throughout the genome. These start sites are called origins of replication, and they are initiated in a temporal manner throughout S phase. How these origins are selected and regulated is poorly understood. Saccharomyces cerevisiae and Schizosaccharomyces pombe have autonomously replicating sequences (ARS) that can replicate plasmids extrachromosomally and function as origins in the genome. Metazoans, however, have shown no evidence of ARS activity.</p><p>DNA replication is a multistep process with several opportunities for regulation. Potential origins are marked with the origin recognition complex (ORC), a six subunit complex. In S. cerevisiae, ORC binds to the ARS consensus sequence (ACS), but no sequence specificity is seen in S. pombe or in metazoans. Therefore, factors other than sequence play a role in origin selection.</p><p>In G1, the pre-replicative (pre-RC) complex assembles at potential origins. This involves the recruitment of Cdc6 and Cdt1 to ORC, which then recruits MCM2-7 to the origin. In S phase, a subset of these pre-RC marked origins are initiated for replication. These origins are not fired simultaneously; instead, origins are fired in a temporal manner, with some firing early, some firing late, and some not firing at all.</p><p>The temporal firing of origins leads to wide regions of the genome being copied at different times during S phase. , which makes up the replication timing profile of the genome. These regions are not random, and several correlations between replication timing and both transcriptional activity and chromosomal landscape. Regions of the genome with high transcriptional activity tend to replicate earlier in S phase, and it is well know that the gene rich euchromatin replicates earlier than the gene poor heterochromatin. Additionally, areas of the genome with activating chromatin marks also replicate earlier than regions with repressive marks. Though many correlations have been observed, no single mark or transcriptional player has been shown to directly influence replication timing.</p><p>We mapped the replication timing profiles of three cell lines derived from Drosophila melanogaster by pulsing cells with the nucleotide analog bromodeoxyuridine (BrdU), enriching for actively replicating DNA labeled with BrdU, sequencing with high throughput sequencing and mapping the sequences back to the genome. We found that the X chromosome of the male cell lines replicated earlier than the X chromosome in the female cell line or the autosomes. We were then able to compare the replication timing profiles to data sets for chromatin marks acquired through the modENCODE (model organism Encyclopedia Of DNA Elements). We found that the early replicating regions of the male X chromosomes correlates with acetylation of lysine 16 on histone 4 (H4K16).</p><p>Hyperacetylation of H4K16 on the X chromosome in males is a consequence of dosage compensation in D. melanogaster. Like many organisms, D. melanogaster females have two X chromosomes while males have one. To compensate for this difference, males upregulate the genes on the X chromosome two-fold. This upregulation is regulated by the dosage compensation complex (DCC), which is restricted to the X chromosome. This complex includes a histone acetyl transferase, MOF, which acetylates H4K16. This hyperacetylation allows for increased transcription of the X chromosome. </p><p>We hypothesized that the activities of the DCC and the hyperacetylation of H4K16 also influences DNA replication timing. To test this, I knocked down components of the DCC (MSL2 and MOF) using RNAi. Cells were arrested in early S phase with hydroxyurea, released, and pulsed with the nucleotide analog EdU. The cells were arrested in metaphase and labeled for H4K16 acetylation and EdU. We found that male cells were preferentially labeled with EdU on the X chromosome, which corresponded with H4k16 acetylation. When the DCC was knocked down, H4K16 acetylation was lost along with preferential EdU labeling on the X chromosome. These results suggest that the DCC and H4K16 acetylation are necessary for early replication of the X chromosome. Additionally, early origin mapping of different cell lines showed that while ORC density does not differ between male and female cell lines, early origin usage is increased on the X chromosome of males, suggesting that this phenomenon is regulated at the level of activation, not pre-RC formation. Other experiments in female cell lines have been unclear about whether the DCC and subsequent H4K16Ac is sufficient for early X replication. However, these results are exciting because this is, to our knowledge, the first mark that has been found to directly influence replication timing.</p><p>In addition to these timing studies, I attempted to design a new way to map origins. A consequence of unidirectional replication with bidirectional replication fork movement is Okazaki fragments. These are short nascent strands on the lagging strand of replicating DNA. Because these fragments are small, we can isolate them by size and map them back to the genome. Okazaki density could tell us about origin usage and any directional preferences of origins. The process proved to be tedious, and although they mapped back with a higher density around ORC binding sites than randomly sheared DNA, little information about origin usage was garnered from the data. Additionally, the process proved difficult to repeat.</p><p>In these studies, we examined the replication timing program in D. melanogaster. We found that the male X chromosome replicates earlier in S phase, and this early replication is regulated by the DCC. However, it is unclear if the change in chromatin landscape directly influences replication or if the replication program is responding to other dosage compensation cues on the X chromosome. Regardless, we have found one the first conditions in which a mark directly influences the DNA replication timing program.&#8195;</p> / Dissertation

The genetic information required for the existence of a living cell of any kind is encoded in the sequence information scripted in the double helix DNA. A modern trend in biology struggles to come to grip with the amazing fact that there are so many different cell types in our body and that they are directed from the same genomic blueprint. It is clear, that the key to this feature is provided by epigenetic information that dictates how, where and when genes should be expressed. Epigenetic states “dress up” the genome by packaging it in chromatin conformations that differentially regulate accessibility for key nuclear factors and in coordination with differential localizations within the nucleus will dictate the ultimate task, expression. In the imprinted Igf2/H19 domain, this feature is determined by the interaction between the chromatin insulator protein CTCF and the unmethylated H19 imprinting control region. Here I show that CTCF interacts with many sites genome-wide and that these sites are generally protected from DNA methylation, suggesting that CTCF function has been recruited to manifest novel imprinted states during mammalian development. This thesis also describes the discovery of an epigenetically regulated network of intra and interchromosomal complexes, identified by the invented 4C method. Importantly, the disruption of CTCF binding sites at the H19 imprinting control region not only disconnects this network, but also leads to significant changes in expression patterns in the interacting partners. Interestingly, CTCF plays an important role in the regulation of the replication timing not only of the Igf2 gene, but also of all other sequences binding this factor potentially by a cell cycle-specific relocation of CTCF-DNA complexes to subnuclear compartments. Finally, I show that epigenetic marks signifying active or inactive states can be gained and lost, respectively, upon exposure to stress. As many genes belonging to the apoptotic pathway are upregulated we propose that stress-induced epigenetic lesions represent a surveillance system marking the affected cells for death to the benefit of the individual. This important observation opens our minds to the view of new intrinsic mechanisms that the cell has in order to maintain proper gene expression, and in the case of misleads there are several check points that direct the cell to towards important survival decisions.

Les translocations chromosomiques sont associées à un grand nombre de cancers. Les translocations chromosomiques sont impliquées dans la tumorigenèse par différents mécanismes : elles conduisent soit à une dérégulation d’un oncogène, soit à la formation d’un nouvel oncogène de fusion. Cependant, le lien direct entre l'apparition d'une translocation chromosomique et la formation d'une tumeur n'est pas totalement établi. Par exemple, plusieurs translocations associées au cancer ont été détectées dans le sang d’individus sains voire dans le sang de cordon des bébés avec une prévalence bien supérieure à celle de la maladie. Ceci suggère que la seule formation de la translocation ne suffit pas toujours à induire l’oncogenèse. La plupart des travaux de recherche antérieurs reposaient sur la surexpression de la protéine de fusion, oncogène supposé. Ces approches présentent de nombreuses limites, la translocation chromosomique est alors absente de même que le contexte chromosomique natif du gène de fusion (promoteur endogène, statut de la chromatine, etc.) ou les éventuels effets d’haplo-insuffisance qui ne sont pas récapitulés. La molécule d’ADN étant organisée de manière non aléatoire dans le noyau, les réarrangements chromosomiques sont également susceptibles d’affecter le statut épigénétique, la réplication et la transcription du chromosome dérivatif entier, en plus des segments d’ADN nouvellement juxtaposés. Or la technologie CRISPR/Cas9, permet de reproduire la translocation chromosomique in situ, après avoir induit deux cassures double-brin simultanées. Ce travail de thèse a porté spécifiquement sur la translocation t(2,5) (p23, q35) qui induit l’expression de la protéine de fusion NPM1-ALK fréquemment rencontrée dans le lymphome anaplasique à grandes cellules (ALCL). Nous avons reproduit la t(2,5) à la fois dans des lignées cellulaires mais aussi dans des cellules T primaires à la fin de ma thèse. Nous avons pu montrer des modifications significatives du timing de réplication des cellules qui portent la translocation en comparaison des cellules isogéniques de départ (par la méthode du Répli-seq) pouvant avoir un impact sur l’homéostasie des cellules tumorales. En parallèle, nous avons mis en évidence la formation d'ARN circulaires de fusion spécifiques, exprimés à partir du gène de fusion, spécifiques des lignées tumorales. Ces ARN circulaires pourraient donner naissance à de nouveaux biomarqueurs diagnostic/pronostic dans le futur. Ces travaux permettront de mieux comprendre les conséquences des translocations chromosomiques oncogéniques dans les cellules humaines et pourraient mener vers de nouvelles orientations thérapeutiques à l’avenir. / Chromosomal translocations are associated with a wide range of cancers. These chromosomal rearrangements are implicated in tumorigenesis by different mechanisms: either they lead to oncogene upregulation or tumor suppressor downregulation. However, the direct link between the appearance of one chromosomal translocation and tumor formation is not always clear. For example, several cancer translocations have been found in PBMCs or in cord blood cells from healthy individuals, suggesting that translocation formation alone is not always sufficient to drive oncogenesis. Most of previous research works on cancer translocation relied on studies using overexpression of the fusion protein. These approaches do not reproduce the chromosome arm translocation nor the chromosomal context of the fusion gene (endogenous promotor, chromatin status etc…) or do not recapitulate a potential haplo-insufficiency of the translocated cells. Because the DNA molecule is organized non-randomly in the nucleus, chromosomal rearrangements are also likely to impact the epigenetic, replication and transcriptional status of the whole rearranged chromosome in addition to the newly juxtaposed gene segments. Using CRISPR/Cas9 technology, we can recapitulate chromosomal translocation in situ, after inducing 2 concurrent double-strand breaks. In this work, we focus on t(2,5)(p23,q35) leading to NPM1-ALK fusion protein frequently found in Anaplasic Large Cell Lymphoma (ALCL). We could recapitulate t(2;5) in cell lines but more importantly in human primary T cells from healthy donors. We showed significant modifications on Replication Timing in model cell lines compare to isogenic non-translocated cells (using Repli-seq analysis). Importantly, these changes might have a direct impact on tumor cell homeostasis. In parallel, we also highlighted the formation of specific fusion circular RNAs expressed from the fusion gene also found in tumor cells. These circular RNAs could give rise to new diagnostic/prognostic biomarkers in the future. This work will lead to a better understanding of the consequences of cancer translocation in human cells and could give new directions for therapeutics in future.

La synthèse translésionnelle (TLS) est un processus important pour franchir des lésions de l’ADN au cours de la duplication du génome dans les cellules humaines. Le modèle « d’échange de polymérases » suggère que la polymérase réplicative est transitoirement remplacée par une polymérase spécialisée, qui va franchir le dommage et permettre de continuer la synthèse d’ADN. Ces ADN polymérases spécialisées appelées Pol êta (η), iota (ι), kappa (κ), zêta (ζ), et Rev1 ont été bien caractérisées pour leur capacité à franchir différents types de lésions in vitro. Un concept en émergence est que ces enzymes pourraient également être requises pour répliquer des zones spécifiques du génome qui sont « difficiles à répliquer ». Polζ est constituée d’au moins 2 sous-unités : Rev3 qui est la sous-unité catalytique et Rev7 sous-unité augmentant l’activité de Rev3L. Jusqu'ici, la fonction la mieux caractérisée de Polζ était de sa capacité à catalyser l'extension d'un mésappariement en face d'une lésion d'ADN. Cependant, il a été montré que la sous unité catalytique Rev3 de levure et humaine interagissent avec les deux sous-unités accessoires de Polδ que sont pol31 et pol32 chez la levure et p50 et p66 chez l’humain. Il a aussi été mis en évidence que Rev3L est importante pour la réplication des sites fragiles (SFCs) dans les cellules humaines, zones connues pour être à l’origine d’une instabilité génétique et pour être répliquées de manière tardive (en G2/M). Tout ceci suggère que Polζ pourrait jouer un rôle dans la réplication du génome non endommagé, et plus spécifiquement lorsque des barrières naturelles (e.g. ADN non-B) entravent la progression normale des fourches de réplication.Chez la levure S. cerevisiae, l’inactivation du gène rev3 est viable et conduit à une diminution de la mutagenèse spontanée ou induite par des agents génotoxiques suggérant que Polζ est impliquée dans le franchissement mutagène des lésions endogènes ou induite. En revanche, l’inactivation du gène Rev3L chez la souris est embryonnaire létale alors que la plupart des autres ADN polymérases spécialisées ne sont pas vitales. Ceci suggère que Polζ a acquis des fonctions essentielles au cours de l’évolution qui restent inconnues à ce jour. Les fibroblastes embryonnaires murins (MEF) Rev3L-/- présente une grande instabilité génétique spontanée associée une forte augmentation de cassures et de translocations chromosomiques indiquant que Polζ est directement impliquée dans le maintien de la stabilité du génome. Afin de clarifier le rôle de cette polymérase spécialisée au cours de la réplication du génome, nous avons entrepris de procéder à une étude sur les relations structure/fonction/localisation de la protéine Rev3. Notre étude met en évidence que la progression en phase S des cellules Rev3L-/- est fortement perturbée, notamment en fin de phase S. Dans ces cellules invalidées pour Rev3L, on constate des changements dans le programme de réplication et plus particulièrement dans des régions de transition (TTR) répliquées à partir du milieu de la phase S. Nous montrons aussi un enrichissement global en marques épigénétiques répressives (marques associées à l’hétérochromatine et méthylation de l’ADN) suggérant qu’un ralentissement de la progression de la fourche de réplication à des loci particuliers peut promouvoir une hétérochromatinisation lorsque Rev3L est invalidé. De manière intéressante, nous constatons une diminution de l’expression de plusieurs gènes impliqués dans le développement qui pourrait peut-être expliquer la létalité embryonnaire constatée en absence de Rev3L. Enfin, nous mettons en évidence une interaction directe entre la protéine d’organisation de l’hétérochromatine HP1α et Rev3L via un motif PxVxL. Tout ceci nous suggère fortement que Polζ pourrait assister les ADN polymérases réplicatives Polδ et Polε dans la réplication des domaines compactés de la chromatine en milieu et fin de phase S. / DNA polymerase zeta (Polζ) is a key player in Translesion DNA synthesis (TLS). Polζ is unique among TLS polymerases in mammalian cells, because inactivation of the gene encoding its catalytic subunit (Rev3L) leads to embryonic lethality in the mouse. However little is known about its biological functions under normal growth conditions.Here we show that S phase progression is impaired in Rev3L-/- MEFs with a delay in mid and late S phase. Genome-wide profiling of replication timing revealed that Rev3L inactivation induces changes in the temporal replication program, mainly in particular genomic regions in which the replication machinery propagates a slower velocity. We also highlighted a global enrichment of repressive histone modifications as well as hypermethylation of major satellites DNA repeats in Rev3L-deficient cells, suggesting that fork movements can slow down or stall in specific locations, and a delay in restarting forks could promote heterochromatin formation in Rev3L-depleted cells. As a direct or indirect consequence, we found that several genes involved in growth and development are down-regulated in Rev3L-/- MEFs, which might potentially explain the embryonic lethality observed in Rev3L KO mice. Finally we discovered that HP1α directly interacts and recruits Rev3L to pericentromeric heterochromatin. We therefore propose that Polζ has been co-opted by evolution to assist DNA polymerase ε and δ in duplicating condensed chromatin domains during mid and late S phase.

<p>The telomeres are specialized structures at the end of the chromosomes composed of the repeated DNA sequence (TTAGGG)n and specific proteins bound to the DNA. The telomeres protect the chromosomes from degradation and end to end fusions. Due to the end-replication problem, the telomeric DNA shortens every cell division, forcing the cells into senescence at a critical telomere length. This process can be counteracted by activating a specialized enzyme, telomerase, which adds telomeric repeats to the chromosome ends leading to an extended or infinite cellular life span. Telomerase activity is absent in most somatic tissues but is found in germ cells, stem cells, activated lymphocytes and the vast majority of tumor cells and permanent cell lines. Hence, telomerase has been suggested as a target for cancer treatment as malignant cells almost exclusively express the enzyme and in that context telomere length measurements will be of great importance.</p><p>Telomere length is traditionally measured with a Southern blot based technique. A new method for telomere analysis of cells in suspension, called flow-FISH, was developed based on fluorescence in situ hybridization using a telomeric peptide nucleic acid (PNA) probe,</p><p>DNA staining with propidium iodide and quantification by flow cytometry. Flow-FISH had high reproducibility and the telomere length measurements showed good correlation with Southern blotting results. The flow-FISH technique also allows studies of cells in specific phases of the cell cycle and the replication timing of telomeric, centromeric and other repetitive sequences were analyzed in a number of cells. Like previous studies, centromeres were shown to replicate late in S phase while the telomere repeats were found to replicate early in S phase or concomitant with the bulk DNA, which is opposite to the patterns described in yeast.</p><p>In benign immunopurified lymphocytes from tonsils, high telomerase activity was found in germinal center (GC) B cells. This population also had high hTERT mRNA levels and displayed a telomere elongation as shown by flow-FISH and Southern blotting. Combined immunophenotyping and flow-FISH on unpurified tonsil cells confirmed the results.</p><p>Chronic lymphocytic leukemia (CLL), the most common leukemia in adults, can be divided into pre-GC CLL, characterized by unmutated immunoglobulin VH genes and worse prognosis, and post-GC CLL, with mutated VH genes and better prognosis. In 61 cases of CLL, telomere length was measured with Southern blotting and VH gene mutation status was analyzed. A new association was found between VH mutation status and telomere length, where cases with longer telomeres and mutated VH genes (post-GC CLL) had better prognosis</p><p>than CLL with short telomeres and unmutated VH genes (pre-GC CLL). A larger study of 112 CLL cases was performed using flow-FISH. The same correlation between telomere length and VH mutation status was found but gender seemed to be of importance as telomere length was a significant prognostic factor for the male CLL patients but not in the female group. Age of the patients and spread of disease seemed to affect the prognostic value of VH gene mutation status.</p>

The telomeres are specialized structures at the end of the chromosomes composed of the repeated DNA sequence (TTAGGG)n and specific proteins bound to the DNA. The telomeres protect the chromosomes from degradation and end to end fusions. Due to the end-replication problem, the telomeric DNA shortens every cell division, forcing the cells into senescence at a critical telomere length. This process can be counteracted by activating a specialized enzyme, telomerase, which adds telomeric repeats to the chromosome ends leading to an extended or infinite cellular life span. Telomerase activity is absent in most somatic tissues but is found in germ cells, stem cells, activated lymphocytes and the vast majority of tumor cells and permanent cell lines. Hence, telomerase has been suggested as a target for cancer treatment as malignant cells almost exclusively express the enzyme and in that context telomere length measurements will be of great importance. Telomere length is traditionally measured with a Southern blot based technique. A new method for telomere analysis of cells in suspension, called flow-FISH, was developed based on fluorescence in situ hybridization using a telomeric peptide nucleic acid (PNA) probe, DNA staining with propidium iodide and quantification by flow cytometry. Flow-FISH had high reproducibility and the telomere length measurements showed good correlation with Southern blotting results. The flow-FISH technique also allows studies of cells in specific phases of the cell cycle and the replication timing of telomeric, centromeric and other repetitive sequences were analyzed in a number of cells. Like previous studies, centromeres were shown to replicate late in S phase while the telomere repeats were found to replicate early in S phase or concomitant with the bulk DNA, which is opposite to the patterns described in yeast. In benign immunopurified lymphocytes from tonsils, high telomerase activity was found in germinal center (GC) B cells. This population also had high hTERT mRNA levels and displayed a telomere elongation as shown by flow-FISH and Southern blotting. Combined immunophenotyping and flow-FISH on unpurified tonsil cells confirmed the results. Chronic lymphocytic leukemia (CLL), the most common leukemia in adults, can be divided into pre-GC CLL, characterized by unmutated immunoglobulin VH genes and worse prognosis, and post-GC CLL, with mutated VH genes and better prognosis. In 61 cases of CLL, telomere length was measured with Southern blotting and VH gene mutation status was analyzed. A new association was found between VH mutation status and telomere length, where cases with longer telomeres and mutated VH genes (post-GC CLL) had better prognosis than CLL with short telomeres and unmutated VH genes (pre-GC CLL). A larger study of 112 CLL cases was performed using flow-FISH. The same correlation between telomere length and VH mutation status was found but gender seemed to be of importance as telomere length was a significant prognostic factor for the male CLL patients but not in the female group. Age of the patients and spread of disease seemed to affect the prognostic value of VH gene mutation status.

La duplication de l'ADN au cours de la phase S est initiée à partir de l'activation de plusieurs dizaines de milliers d'origines de réplication. La mise en place des origines a lieu au cours de la phase G1 sous la forme de complexe de pré-réplication (pré-RC) et leur activation est orchestrée par un programme spatio-temporel. La régulation spatiale détermine les origines qui seront activées et la régulation temporelle, ou timing de réplication, détermine le moment de leur activation. En effet, toutes ces origines ne sont pas activées en même temps durant la phase S : certaines origines seront activées en début de phase S, d'autre en milieu, ou d'autre à la fin. Ce programme est établi en tout début de phase G1, au " point de décision du timing ". C'est un programme très robuste qui signe l'identité d'une cellule, son état de différenciation et le type cellulaire à laquelle elle appartient. Il a aussi été montré qu'il est altéré dans des situations pathologiques, en particulier le cancer, sans qu'on ne comprenne très bien les raisons mécanistiques. De manière générale, les mécanismes moléculaires qui régulent le timing de réplication sont méconnus. Le premier volet de ma thèse a permis l'identification d'un nouveau régulateur du timing de réplication : il s'agit de l'ADN polymérase spécialisée Thêta. Recrutée à la chromatine très tôt en phase G1, elle interagit avec des composants du pré-RC, et régule le recrutement des hélicases réplicatives à la chromatine. Enfin, sa déplétion ou sa surexpression entraîne une modification du timing de réplication à l'échelle du génome. Dans la deuxième partie de ma thèse, j'ai exploré les mécanismes qui régulent ce programme temporel d'activation des origines suite à un stress réplicatif. J'ai identifié un mécanisme de régulation transgénérationnel inédit : la modification du timing de réplication de domaines chromosomiques ayant subi un stress réplicatif au cycle cellulaire précédent. Des cellules-filles issues d'une cellule ayant subi des problèmes de réplication dans des domaines fragiles (riches en AT, et donc potentiellement structurés, et pauvres en origines) présentent un timing plus précoce de l'activation des origines au niveau de ces domaines. Ce nouveau processus biologique d'adaptation est particulièrement intéressant dans un contexte tumoral de haut stress réplicatif chronique car ce pourrait être un moyen pour la cellule tumorale de survivre à son propre stress réplicatif mais aussi aux thérapies antitumorales qui sont nombreuses à cibler la réplication de l'ADN. / DNA duplication in S phase starts from thousands of initiation sites called DNA replication origins. These replication origins are set in G1 as pre-replication complexes (pre-RC) and fired in S phase following a spatio-temporal program of activation. This program determines which origins will be fired and when. Indeed, all the origins are not fired in the same time and we can distinguish early, middle and late replication origins. This temporal regulation is called "replication timing" and is determined at the "timing decision point" (TDP) in early G1. It's a robust program, which participates to the definition of cell identity, in term of differentiation state or cell type. However, the precise molecular mechanisms involved are poorly understood. Defective timing program has been evidenced in pathological contexts, in particular in cancers, but the mechanisms of this deregulation remain unclear. In the first part of my PhD, I contributed to the discovery of a new regulator of the origin timing program: the specialized DNA polymerase Theta (Pol Theta). Pol Theta is loaded onto chromatin in early G1, coimmunoprecipitates with pre-RC components and modulates the recruitment of Mcm helicases at TDP. Moreover, depletion or overexpression of Pol Theta modifies the timing of replication at a fraction of chromosomal domains. The second part of my work aimed at exploring the mechanisms that regulates replication timing after a replicative stress. I identified a totally new transgenerational adaptive mechanism of DNA replication timing regulation: the modification of the timing of origin activation at chromosomal domains that have suffered from a replicative stress during the previous cell cycle. Daughter cells from a cell that has experienced replication stress at particular domains (late replicating domains, AT rich so they can form structured DNA, and poor in origin density) shows advanced origin activation within these regions. This new biological process in response to replicative stress could be of particular interest in the context of cancer since, tumor cells are characterized by high level of intrinsic chronic replicative stress. This new mechanism may favor cancer cell survival despite replication stress, particularly upon treatments with anti-tumor agents that target DNA.