Les trois complexes SMC Cohésine, Condensine et SMC5/6 sont principalement étudiés pour leurs rôles mitotiques, cependant tous trois sont localisés à proximité des fourches de réplication en condition de stress réplicatif. Au cours de cette thèse, nous nous sommes particulièrement intéressés aux complexes Cohésine et Condensine. Dans une première partie, nous décrivons un nouveau rôle des condensines dans la progression des fourches de réplication en condition de stress réplicatif à l’hydroxyurée (HU) et au Méthyl-Méthane-Sulfonate (MMS). Nos données montrent que dans ces conditions, les condensines limitent l’accumulation de la protéine de liaison à l’ADN simple-brin RPA (Replication Protein A) à proximité des fourches de réplication. Ces résultats révèlent que les condensines limitent l’exposition d’ADN simple brin et pourraient ainsi protéger l’intégrité des fourches de réplication et la stabilité du génome. Dans une seconde partie nous décrivons le mécanisme de recrutement du complexe Cohésine aux fourches de réplication en condition de stress réplicatif. Dans ces conditions, les cohésines renforcent la cohésion des chromatides sœurs afin de faciliter le redémarrage des fourches de réplication par recombinaison homologue. Nous montrons que le complexe SMC-like MRX (Mre11-Rad50-Xrs2), l’histone méthyle-transférase Set1 et l’histone acétyle-transférase Gcn5 sont requis pour le recrutement des cohésines aux fourches de réplication. Nos données révèlent qu’en réponse au stress réplicatif, Gcn5, Set1 et MRX modifient la dynamique des histones. Gcn5 et MRX réduisent la densité d’histone sur l’ADN répliqué alors que Set1 maintient la mobilité des nucléosomes. La modification de la dynamique des histones semble importante pour une réponse cellulaire efficace au stress réplicatif et pour le chargement de complexes SMC aux fourches de réplication. / The three SMC complexes Cohesin, Condensin and SMC5/6 are mainly studied for their role in mitosis, nevertheless they all localize at replication forks in replicative stress conditions. During this thesis, we focused on Cohesin and Condensin. In the first part we describe a new role for the condensin complex in response to replicative stress. In the presence of Hydroxyurea (HU) and Methyl-Methan-Sulfonate (MMS), condensin is required for cell growth and replication fork progression. Moreover, our results show that condensin limits the accumulation of the specific single-strand DNA (ssDNA) binding protein RPA (Replication Protein A) in the vicinity of replication forks under HU treatment, revealing that condensin limits ssDNA accumulation during replicative stress. In this way, Condensin could protect replication fork integrity and genome stability in response to replicative stress. In the second part, we decipher the cohesin recruitment mechanisms at replication fork under replicative stress. In that context, cohesin reinforces sister chromatid cohesion and facilitates homologous recombination (HR) dependent replication fork restart pathways. We show here that the SMC-like MRX complex, the histone methyl transferase Set1 and the histone acetyl transferase Gcn5 are required for cohesin recruitment at stalled replication forks. Our results show that these three proteins affect histone H3 dynamics on replicated DNA in response to replicative stress. Gcn5 and MRX reduce H3 density whereas Set1 maintains nucleosome mobility. These two parameters seem to be important for efficient response to replicative stress and for SMC complexes loading close to stressed replication forks.

Les composants cellulaires sont constamment exposés à un stress oxydatif, lié à l’environnement et au métabolisme cellulaire. Les espèces réactives de l’oxygène produites par ce stress induisent de nombreuses lésions dans l’ADN, telles que l’oxydation des bases, la formation de sites abasiques ou la cassure de brins d’ADN. Ces dommages sont corrigés par un panel de systèmes de réparation, qui jouent un rôle critique dans la survie cellulaire et dans la prévention de pathologies telles que les maladies neurodégénératives ou le cancer. La modification de bases est le type de dommage le plus abondant, généré spontanément ou par des agents exogènes. Notre laboratoire s’intéresse ainsi au système de réparation par excision de base (BER), qui élimine les bases nucléotidiques altérées. Des études antérieures ont montré la formation « d’usines de réparation du BER » suite à des traitements induisant l’oxydation des bases dont la forme la plus courante est la 8-oxoguanine (8-oxoG). Dans le cas de cette lésion mutagène, l’assemblage du complexe BER dépend du recrutement d’OGG1 à la chromatine, l’enzyme qui reconnaît et excise la 8-oxoG. Cependant, ce recrutement ne nécessite pas la reconnaissance de la 8-oxoG, indiquant que d’autres signaux interviennent pour initier la réparation de la 8-oxoG par OGG1. Un crible à haut débit a été réalisé dans des cellules humaines pour rechercher des protéines impliquées dans le recrutement d’OGG1. Deux complexes ont été identifiés, les cohésines et le médiateur de la transcription.Dans ce projet de recherche, nous avons exploré le rôle de ces protéines dans la relocalisation d’OGG1 suite à un stress oxydatif. Nos études ont tout d’abord permis d’identifier des protéines essentielles au recrutement d’OGG1 : les protéines formant l’anneau de cohésines (SMC1, SMC3 et RAD21), plusieurs sous-unités du médiateur dont MED14, ainsi que le module CDK (MED12, MED13, Cycline C et CDK8). De plus, ces protéines sont nécessaires pour le recrutement d’OGG1 tout au long du cycle cellulaire. Nos résultats montrent que la relocalisation d’OGG1 sur la chromatine est liée à sa fonction de réparation de la 8-oxoG. Nous avons d’autre part montré que deux sous-unités du médiateur (MED12 et CDK8) sont relocalisées dans l’euchromatine, comme OGG1, de façon dépendante du corps du médiateur et des cohésines. Enfin, l’association d’OGG1 avec ses partenaires a été validée par microscopie FLIM-FRET et co-immunoprécipitation dans des conditions de stress oxydatif.En conclusion, ces résultats montrent pour la première fois un lien entre la réparation des bases oxydées et les complexes du médiateur et des cohésines, tous deux connus pour leur participation à d’autres voies de réparation de l’ADN. L’identification des mécanismes moléculaires et de nouveaux facteurs impliqués dans la réparation des bases oxydées pourrait fournir à terme des éléments essentiels pour la prise en charge de maladies telles que le cancer ou les maladies neurodégénératives. / Our laboratory focuses on the base excision repair (BER) mechanism that is responsible for the removal of damaged bases in DNA. Oxidative DNA damage is generated spontaneously by the endogenous metabolism of the cells or induced exogenously by chemical or physical agents. Our aim is to understand how BER complexes are assembled in the context of the cell nucleus in response to genotoxic stress. We previously found that after treatments generating oxidized bases into cellular DNA BER complexes are assembled on the chromatin. In the case of the 8-oxoguanine (8-oxoG) mutagenic lesion, assembly of the BER complex depends on the recruitment to the chromatin of OGG1, the DNA glycosylase that recognizes and excises the lesion. Surprisingly, characterization of OGG1 mutants that are not able to recognize 8-oxoG showed that the recruitment of this initiator protein does not require the recognition of the damaged base. This suggests that there are other mechanisms that allow recruitment of the enzyme to chromatin and thus initiation of the repair of the 8-oxoG by the BER. We performed a high-throughput siRNA screen in human cells to identify proteins required for the recruitment of OGG1 to chromatin. Among the candidates issued from the screen, two groups of proteins were selected for further study: members of the mediator and cohesin complexes.In this project, we explored the role of these proteins in OGG1 relocalization after an oxidative stress. Our studies confirmed the requirement of essential proteins for OGG1 recruitment: cohesins subunits (SMC1, SMC3 and RAD21), mediator subunits including the central protein MED14, and CDK subunits (MED12, MED13, Cyclin C and CDK8). Requirement of all these proteins is independent of the cell cycle. Furthermore we show that recrutement of OGG1 is essential for its 8-oxoG repair function. Microscopy studies revealed recruitment and colocalization of two mediator subunits (MED12 and CDK8) with OGG1 on euchromatin domains after an oxidative stress. Finally, the association between OGG1 and its partners, specifically after an oxidative stress, was validated by FLIM-FRET microscopy and co-immunoprecipitation.To conclude, these results show for the first time a link between repair of oxidized bases and mediator and cohesin complexes, both of them being already involved in other DNA repair pathways. The identification of molecular mechanisms and new factors involved in the repair of oxidized bases may ultimately provide new elements for the management of diseases such as cancer and neurodegenerative diseases.

No description available.

In the intricate realm of biology, meiosis stands as the remarkable process responsible for generating genetically diverse haploid gametes from diploid cells. In 2000, Pezzi et al., identified STAG3 as a novel meiotic-specific synaptonemal-complex associated protein belonging to the highly conserved family of stromalin nuclear proteins. Later, over the years, research groups characterised the depletion phenotype of STAG3 in mice, where deficiency of STAG3 causes severe chromosomal defects and early meiotic arrest. These studies together collectively highlighted STAG3 as the most important meiotic cohesin. Traditionally, the role of the cohesin complex was understood as maintaining cohesion between chromatids during cell division. However, over the years, this perception has evolved significantly, expanding to include the regulation of dynamic chromosomal configurations during meiosis. With the realisation of STAG3's importance in meiotic progression, the next pressing question becomes: how does STAG3 coordinate this intricate process? This study sought to address that question by examining the STAG3 interactome in male germ cells, aiming to uncover novel pathways through which STAG3 contributes to maintaining meiotic progression. Through successful purification of the STAG3-REC8 complex, its ability to form functional complexes in-vitro was demonstrated. During my PhD thesis, I discovered links between STAG3 and DNA repair mechanisms beyond the well-known homologous recombination /non-homologous end joining pathway in meiotic recombination. By looking at the meiotic-specific protein interactome bound to the STAG3-REC8 complex through Mass Spectroscopic analysis, we identified STAG3 involvement in PARP-1-mediated repair of DNA double-strand breaks occurring outside of the programmed DSB repair during the zygotene stage of prophase I. PARP-1 is an ADP-ribose polymerase which acts as a first responder that detects DNA damage and facilitates the activation of the DNA repair pathway. STAG3 shows a preferential interaction with PARP-1 when spermatocytes are challenged with extensive DNA damage. Furthermore, the interaction of SMC3, another component of the cohesin complex, with PARP-1 during DNA damage suggests that STAG3, as part of the cohesin complex, contributes to DNA damage repair in spermatocytes. To gain deeper insights into the distinctive characteristics of STAG3, an extensive analysis of spermatogenesis in mice expressing a C-terminus truncated form of STAG3 was performed. The C-terminal region of STAG3 is not conserved among the stromalin family members, and hence it was speculated that this region might have unique functions to meiosis. Removal of the C-terminal end comprising 47 amino acids led to an early meiotic arrest, mirroring the phenotype in most cohesin subunit deletion mutants. The phenotype observed mimics the complete STAG3 depletion phenotype to some extent. The truncated STAG3 resulted in an arrest at a late zygotene/early pachytene-like stage during meiotic prophase I. One of the most notable observations was the significant reduction in the length of the axial elements (AE) in this mutant. Despite stable expression of and localisation of STAG3 to the axis, the axis length decreased by over 60%. This mutation compromised synaptonemal complex formation, leading to the early meiotic arrest. Although SYCP1 loads onto the axis and initiate synapsis, the shortened axial elements could not synapse, marked by HORMAD-1, a well-known asynapsis marker. The average number of SYCP3-marked stretches was 35 in this mutant. The increased number of AE and shortened axis length did not result from chromosome fragmentation because most chromosomes/axes had intact telomere and centromeric signals, validated by RAP1 and ACA foci, respectively. Centromeric and telomeric cohesion may be partially affected as some chromosome showed aberrant telomeric and centromeric defects. C terminal truncated STAG3 impairs synapsis between homologous chromosomes, but the sister chromatid cohesion remains largely unaffected. Also, this deletion did not affect the loading of the cohesin complex subunits onto the chromosome axis. The early meiotic arrest resulted in underdeveloped gonads, leading to infertility in otherwise healthy mice. Taken together, these results suggest novel roles for STAG3 in meiosis, and the meiotic-specific C terminal region of STAG3 is critical for proper meiotic progression in mice.

Lors des réponses immunitaires, le répertoire des lymphocytes B est diversifié par l’hypermutation somatique (HMS) et la commutation isotypique (CI), dépendant d’«activation-induced cytidine deaminase» (AID), qui introduit des lésions dans les gènes Ig. Une déficience d’AID cause un défaut d’HMS et de CI; par contre, une délétion du domaine C-terminal d’AID cause un défaut spécifique de la CI, suggérant que ce domaine interagit avec des facteurs spécifiques de la CI. Pour identifier ces facteurs, nous avons étudié une immunodéficience présentant un défaut de la CI non lié à la carence d’AID ni à un défaut d’HMS. De plus, les cassures de l’ADN ne sont pas détectées au niveau des gènes Ig suggérant qu’AID n’est pas correctement ciblée dans ces loci. Nous avons identifié et analysé des candidats : Spt6, les cohésines et le complexe Smc5/6. Dans les cellules B activées, AID interagit avec Spt6, Spt5, l’ARN polymérase II et le complexe PAF. Par contre, les cohésines pourraient réguler la structure du locus IgH lors de la CI et la voie de réparation des cassures de l’ADN générées pendant la CI. Ces résultats contribuent à une meilleure compréhension des étapes de la CI. / During immune responses, B cell repertoire is diversified through somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR require activation-induced cytidine deaminase (AID), which induces DNA damage. While AID deficiency abrogates SHM and CSR, C-terminal truncations impair CSR without affecting SHM and it has been proposed that AID C-terminal domain associates with CSR-specific factor(s). In order to identify these factors, we studied a human CSR-specific immunodeficiency, characterized by normal SHM and AID expression. B cells from these patients do not display DSBs at switch (S) regions, suggesting that they might lack an AID-binding factor(s) required to target AID to S regions during CSR. Through a multi- approach strategy, we identified and analyzed candidate factors, including Spt6, the cohesin complex and the Smc5/6 complex. We show that, in B cells poised to undergo CSR, AID is in a complex with Spt6, Spt5, the RNA polymerase II and the PAF complex while cohesins might regulate the 3D structure of the IgH locus and the pathway of DSBs repair at the Ig S regions. Our work thus contributes to a better understanding of the CSR reaction.