Common fragile sites are a set of genomic locations with a propensity to form lesions, breaks and gaps on mitotic chromosomes upon induction of replication stress. While the exact reasons for their fragility are unknown, CFS display instability in a cell-type specific manner, suggesting a substantial contribution from an epigenetic component. CFSs also overlap with sites of increased breakage and deletions in tumour cells, as well as evolutionary breakpoints, implying that their features shape genome stability in vivo. Previously, factors such as delays in replication timing, low origin density and transcription of long genes have been implicated in instability at CFS locations but comprehensive molecular studies are lacking. Chromatin structure, an important factor that fits the profile of cell-type specific contributor, has also not been investigated yet. Throughout their efforts to determine the factors that lead to the appearance of CFS lesions, investigators have focused on a single component at a time, potentially missing out complex interactions between cellular processes that could underlie fragility. Additional difficulties come from the cell-type specificity of CFS breakage: it indicates that only cell type-matched data would be informative, limiting the scope for studies using publicly available data. To perform a comprehensive study defining the role of different factors in determining CFS fragility, I explored replication timing, transcriptional landscapes and chromatin environment across a number of CFSs in two cell types exhibiting differential CFS breakage. Initially, I characterised the patterns of CFS fragility in the two cell types on both the cytogenetic and the molecular level. I then used a FISH-based technique to investigate the process of mitotic compaction at active CFS sites and found that the cytogenetically fragile core of these sites sits within larger regions which display a tendency to mis-fold in mitosis. The aberrant compaction of these regions could be observed on cytogenetically normal metaphase chromosomes, suggesting that finer scale abnormalities in chromosome structure underlie the cytogenetically visible breaks at fragile sites. I also investigated the links between transcription of long genes and CFS fragility using two approaches: I quantified levels of expression across all fragile sites using RNA-seq and modified transcription at a single active CFS using the CRISPR genome engineering methodology. My results indicate a complex interplay between transcription and CFS fragility: no simple linear correlation can be observed, but an increase of transcriptional levels at the active CFS led to a corresponding increase in fragility. To investigate the influence of the cell type specific replication programme and replication stress on CFS instability, I mapped replication timing genome-wide in unperturbed cells and under conditions of replication stress in both cell types. I found that replication stress induces bi-directional changes in replication timing throughout the genome as well as at CFS regions. Surprisingly, the genomic regions showing the most extreme replication timing alterations under replication stress do not overlap with CFS, implying that CFS instability is not fully explained by replication delays as previously suggested. Instead, I observed a range of replication-stress induced timing changes across CFS regions: while some CFSs appear under-replicated, others display switches to both earlier and later replication as well as differential recruitment of both early and late origins, implying that dis-regulation of replication timing and origin firing, rather than simply delays, underlie the sensitivity to CFS regions to replication stress. Finally, I investigated large-scale chromatin states at two active CFSs throughout S phase and into G2, the cell cycle stages most relevant stage for CFS breakage. I found that changes in large-scale chromatin architecture accompany the replication timing shifts triggered by replication stress, raising the possibility that such alterations contribute to instability. In conclusion, I assessed the influence of multiple relevant factors on CFS fragility. I found that bi-directional replication timing changes and alterations in interphase chromatin structure are likely to play a role, converging to promote mitotic folding problems which ultimately result in the well-described cytogenetic lesions on metaphase chromosomes and genomic instability.

Les Sites Fragiles Communs (SFC) sont des loci incluant des grands gènes où des cassures chromosomiques apparaissent chez des cellules soumises à un stress réplicatif. Il est couramment admis que ces sites sont des régions dont la réplication n’est pas terminée lorsque les cellules entrent en mitose mais le mécanisme sous-jacent restait mal compris. Notre laboratoire a montré par la technique du peignage moléculaire que l’instabilité du site FRA3B chez les lymphocytes est due à la présence d’une grande région pauvre en origines de réplication actives (cœur) dont la réplication ne peut pas se terminer en cas de stress réplicatif. Dans le but de généraliser ces conclusions, j’ai étudié par la technique du « Répli-Seq », le timing de réplication à l’échelle du génome entier chez des cellules lymphoblastoïdes humaines traitées ou non à l’aphidicoline. Les résultats confirment clairement que les SFC connus dans ce type cellulaire correspondent à des régions incluant un grand gène et qui sont déficitaires en réplication sous un stress réplicatif. D’autre part, j’ai montré que retarder l’entrée des cellules en mitose par l’utilisation du RO-3306, un inhibiteur de CDK1, abolit complètement la fragilité des SFC induite par l’aphidicoline chez des lymphocytes et des fibroblastes humains. De plus, j’ai montré que cette suppression des cassures au niveau de FRA3B et FRA16D chez les lymphocytes et de FRA1L chez les fibroblastes ne s’accompagne pas d’une diminution de la transcription de FHIT, WWOX et NEGR1, les trois grands gènes contenus respectivement dans ces trois SFC. Par ailleurs, j’ai corrélé l’expression des gènes FHIT, WWOX, NEGR1 et LSAMP à la fragilité des sites FRA3B, FRA16D, FRA1L et FRA3L dans 7 lignées de fibroblastes et 7 lignées de lymphocytes. J’ai aussi mis en évidence une expression atypique de ces gènes dans certaines de ces lignées et montré que cette expression atypique s’accompagne d’une fragilité atypique du SFC correspondant. Cependant, j’ai montré que l’inhibition de la transcription par deux inhibiteurs différents, le triptolide et l’α-amanitine, ne diminue pas la fragilité induite par l’aphidicoline. Je propose un modèle pour expliquer ces résultats apparemment contradictoires. Enfin, j’ai corrélé la disparition des cassures au niveau de FRA3B dans les cellules traitées à l’aphidicoline et au RO-3306 à une augmentation des événements d’initiation dans la région cœur. J’ai montré que l’inhibition de CDK1 par le RO-3306 permet l’accumulation à la chromatine des facteurs CDC6, CDT1, ORC1 et MCM7 dans les cellules bloquées en phase G2 et que l’accumulation de CDT1 et de CDC6 est indispensable à la diminution de la fragilité sous RO-3306. Je propose un modèle permettant de rendre compte de ces observations qui postule l’existence d’origines de réplication latentes partiellement chargées en complexes de pré-réplication. / Common Fragile Sites (CFS) are loci harboring large genes where chromosome breaks occur in cells under replication stress. It is widely accepted that these sites are regions whose replication is incomplete when cells enter mitosis, but the underlying mechanism remains poorly understood. Our laboratory has shown by the technique of molecular combing, that the instability of FRA3B in lymphocytes is due to the presence of a large region poor in active replication origins (core) whose replication can not be completed under replicative stress. In order to generalize these findings, I studied by the technique of "Repli-Seq", the replication timing across the entire genome in human lymphoblastoid cells treated or not with aphidicolin. The results clearly confirm that CFS known in this cell type correspond to regions including large genes and which are underreplicated under replication stress conditions. Moreover, I have shown that delaying entry of cells into mitosis by using RO-3306, an inhibitor of CDK1, completely abolishes the fragility of SFC induced by aphidicolin in human lymphocytes and fibroblasts. I also have shown that abolition of the breaks at FRA3B and FRA16D in lymphocytes and at FRA1L in fibroblasts is not accompanied by a decreased transcription of FHIT, WWOX and NEGR1, respectively, the three large genes including these three SFC. Moreover, I have correlated the expression of FHIT, WWOX, NEGR1 and LSAMP genes to the fragility of FRA3B, FRA16D, FRA1L and FRA3L sites in 7 lines of fibroblasts and 7 lines of lymphocytes. I also highlighted an atypical expression of these genes in some of these lines and showed that this atypical expression is accompanied by an unusual fragility of the corresponding CFS. However, I have also shown that inhibition of transcription by two different inhibitors, triptolide and α-amanitine, does not diminish the fragility induced by aphidicolin. I propose a model to explain these apparently contradictory results.Finally, I have correlated the disappearance of breaks at FRA3B in cells treated with aphidicolin and RO-3306 with an increase in initiation events in the core region. I have shown that inhibition of CDK1 by the RO-3306 allows the accumulation of factors CDC6 CDT1, ORC1 and MCM7 on the chromatin in cells blocked in G2 phase, and that the accumulation of CDT1 and CDC6 is essential to reducing fragility under RO-3306. I propose a model to explain these observations that postulates the existence of latent replication origins partially loaded with the pre-replication complex.

Les sites fragiles communs (SFC) sont des loci instables en cas de stress réplicatif et des lieux préférentiels de réarrangements dans les tumeurs. Les SFC sont associés aux plus grands gènes du génome et il a été proposé que la transcription de ces gènes soit à l'origine de cassures de l'ADN. Cependant, de nombreux grands gènes transcrits ne sont pas fragiles. Un second modèle, celui de notre laboratoire, associe la fragilité des SFC à un programme de réplication particulier, combinant pauvreté en événements d'initiation et réplication tardive. Il serait alors possible que la transcription soit liée à leur programme de réplication, ce qui conduirait à la fragilité. Pour mieux comprendre ces relations, j'ai modifié la transcription de deux grands gènes et analysé les conséquences sur leur réplication et leur fragilité. Ces manipulations génétiques sont réalisées dans le modèle aviaire DT40, qui permet la modification ciblée d'ADN par recombinaison homologue avec une grande efficacité. De façon surprenante, j'ai observé que la fragilité d'un grand gène est diminuée aussi bien en abolissant sa transcription qu'en l'augmentant. J'ai étudié la densité en événements d'initiation par peignage moléculaire au locus et le programme temporel de réplication dans des clones présentant des niveaux différents de transcription. J'ai ainsi pu montrer que la surexpression massive de deux grands gènes avance le programme temporel de la réplication, les préservant de la fragilité. Au cours de ma thèse, j'ai donc montré que la transcription exerce des effets antagonistes sur la stabilité du génome, bénéfiques ou délétères, selon le niveau d'expression des grands gènes associés aux SFC. / Common Fragile Sites (CFSs) are loci displaying instability upon replicative stress, which localization correlates with chromosomal rearrangements in tumours. CFSs are associated with the largest genes of the genome and it has been proposed that their transcription leads to DNA breaks. However, many transcribed large genes are not fragile. Our laboratory proposed an alternative model in which CFS instability results from a specific replication program, combining late replication with paucity in initiation events. To reconcile the two models, we hypothesized that transcription impacts the replication programs. In order to characterize those potential relationships, I manipulated the transcription of two large genes associated with CFSs and determined the consequences of these manipulations on replication and fragility. I used chicken DT40 cells to perform these analyses because this cellular model allows efficient engineering of specific DNA sequences by homologous recombination. Surprisingly, I observed that increasing or suppressing transcription of large gene both lead to a decrease fragility. I then analyzed clones displaying variable transcription levels. I determined the distribution and density of initiation event, using molecular combing at two loci, as well as the profiles of replication timing along the genes. I showed that a massive overexpression of two large genes led to an earlier replication timing. Overall, my results highlight the opposite effects of transcription on genome stability, which range from beneficial to deleterious depending on the expression level of large genes associated with CFSs.

Les sites fragiles communs (SFCs) sont des régions génomiques particulièrement sensibles au stress réplicatif et sont impliqués dans l’initiation et la progression du cancer. L’Anémie de Fanconi (AF) est une maladie génétique rare qui se caractérise principalement par une aplasie médullaire, des malformations congénitales ainsi qu’une forte prédisposition au cancer chez les patients (leucémies myéloïdes et tumeurs solides de la tête et du coup). L’instabilité génomique a été identifiée comme étant une source majeure de prédisposition des patients AF au cancer et les SFCs sont particulièrement sensibles dans cette maladie. L’AF est causée par la mutation de gènes codant des protéines participant à une voie moléculaire appelée voie FANC qui a été décrite dans la réparation des ponts inter-brins. Malgré l’importance de la voie FANC dans le maintien de la stabilité des SFCs, les mécanismes sous-jacents restent à élucider. Au cours de ma thèse, nous avons identifié un nouveau rôle de FANCD2 dans le maintien des SFCs. En effet, nous montrons que FANCD2 atténue l’expression des gènes présents au sein des SFCs maintenant leur stabilité. De plus, nous montrons que la transcription de ces gènes est nécessaire au recrutement et au rôle de FANCD2 au sein de ces régions. Enfin, nous avons identifié le stress métabolique comme étant un signal induisant l’expression des gènes des SFCs et que FANCD2 module cette réponse. La réduction de ce stress pourrait être une piste thérapeutique intéressante afin de prévenir l’instabilité des SFCs dans l’AF. / Common fragile sites (CFSs) are genomic regions prone to form breaks and gaps on metaphase chromosomes after replicative stress and promote genomic instability in the earliest steps of tumor development. Proteins involved in replication/repair of CFSs are necessary to prevent their instability. Among them is FANCD2, a key protein of the FANC pathway necessary to resolve inter-strand crosslinks and defective in Fanconi Anemia (FA). FA is a rare genomic instability disorder characterized by bone marrow failure, congenital abnormalities and predisposition to acute myeloid leukemia and epithelial cancer. Genomic instability in FA is supposed to predispose patients to cancers. Importantly, CFSs are more unstable in FA and chromosome breaks observed in FA cells occur preferentially at CFSs. During my PhD, we identified a new role of FANCD2 in CFS stability maintenance. We show that FANCD2 attenuates transcription of the large genes present at CFSs, preventing their instability. Moreover, we demonstrate that transcription is necessary for FANCD2 recruitment and function at CFSs. Importantly, we identified the metabolic stress as a signal triggering CFS gene expression and FANCD2 is necessary to modulate this response. Reducing this stress is a promising therapeutic issue to prevent CFS and genomic instability in FA.

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