Le complexe de la cohésine est requis pour de nombreuses transactions chromosomiques, la cohésion des chromatides soeurs, la réparation des dommages à l'ADN, la régulation de la transcription et le contrôle de l'architecture de la chromatine en 3D. La manière dont la cohésine engage la chromatine est restée une question majeure. Les sous-unités de base de cohesin, Smc1, Smc3, Scc1 assemblent un complexe en forme d’anneau via la connexion des domaines SMC «charnières» hétérodimères fournis par Smc1 et Smc3, et par la liaison des domaines SMC ATPase par Scc1. D'autres facteurs jouent un rôle dans différents aspects de la fonction de la cohésine, tels que Scc3, qui favorise l'association de la cohésine à l'ADN, les complexes de chargement et de déchargement, Scc2-Scc4 et Pds5-Wapl, respectivement responsables du chargement de la cohésine et de sa dissociation de la chromatine. . Au cours de la phase S, une acétyltransférase appelée Eco1 acétyle le domaine ATPase de Smc3 et déclenche l’établissement de la cohésion. Pour augmenter davantage la cohésion, un facteur métazoaire supplémentaire, la sororine forme un complexe avec Pds5 pour empêcher la liaison de Wapl. Pendant la métaphase, la cohésine centromérique est protégée par shugoshin-PP2A. Chez les métazoaires, la cohésine est libérée des chromosomes en deux étapes. La première nécessite la phosphorylation de la cohésine et permet à Wapl de se lier à nouveau à Pds5 afin d'assurer la médiation de la libération de cohésine indépendante du clivage à partir des bras des chromosomes. La seconde se produit lors de la réalisation de l'assemblage de la broche et nécessite l'activation d'une protéase appelée séparase, ce qui entraîne le clivage Scc1, libérant ainsi des chromatides soeurs à séparer dans des cellules filles. Au-delà de la cohésion, il devient également évident que la cohésine joue des rôles plus divers en interagissant avec une multitude d'autres facteurs, notamment la CTCF, une protéine connue comme un isolant, dont il a été rapporté qu'elle collabore à la détermination du génome 3D. structure.Pour comprendre comment la cohesine engage l'ADN, j'ai étudié les propriétés de liaison à l'ADN de sous-complexes précédemment identifiés. En déterminant une structure cristalline de la levure Scc3 liée à un fragment de la sous-unité Scc1 kleisin et de l'ADN, j'ai pu démontrer que Scc3 et Scc1 forment un module d'interaction composite de l'ADN. Le sous-complexe Scc3-Scc1 engage un ADN double brin à travers une surface conservée, chargée positivement. Nous démontrons que ce domaine est requis pour la liaison à l'ADN par Scc3-Scc1 in vitro, ainsi que pour l'enrichissement de la cohésine sur des chromosomes et pour la viabilité cellulaire. Ces résultats suggèrent que l'interface de liaison à l'ADN Scc3-Scc1 joue un rôle central dans le recrutement des complexes de la cohésine sur les chromosomes et donc que cette dernière exécute fidèlement ses fonctions lors de la division cellulaire.Pour étudier les bases moléculaires de la collaboration fonctionnelle signalée entre la cohésine et le CTCF dans la définition de la structure chromosomique 3D, j'ai identifié et déterminé la structure d'un complexe ternaire composé de SA2 humain (un orthologue de Scc3), de Scc1 et de CTCF. La structure révélait un motif de liaison SA2-Scc1 très répandu qui était présent non seulement dans le CTCF, mais aussi dans d’autres facteurs connexes fonctionellement, tels que shugoshin et Wapl. Les tests de compétition déroulants ont indiqué que la liaison de ces facteurs à SA2-Scc1 était mutuellement exclusive, ce qui suggère fortement qu'ils interagissent avec la cohésine via des mécanismes similaires. Pour démontrer ce principe, j'ai pu déterminer une structure de shugoshin en complexe avec SA2-Scc1, ce qui a confirmé que tant le shugoshin que le CTCF se lient à la même surface conservée sur la cohésine. / The cohesin complex is required for numerous chromosomal transactions including sister chromatid cohesion, DNA damage repair, transcriptional regulation and control of 3D chromatin architecture. How cohesin engages chromatin has remained a major question. The basic subunits of cohesin, Smc1, Smc3, Scc1 assemble a ring-shaped complex via connection of the heterodimeric SMC ‘hinge’ domains contributed by of Smc1 and Smc3, and through linkage of the SMC ATPase domains by Scc1. Additional accessory factors play important roles in different aspects of cohesin function, such as Scc3, which promotes the association of cohesin with DNA, the loading and unloading complexes, Scc2-Scc4 and Pds5-Wapl respectively, responsible for cohesin loading and its disassociation from chromatin. During S phase, an acetyltransferase called Eco1 acetylates the ATPase domain of Smc3 and triggers the stabilization, or establishment, of cohesion. To further augment cohesion, an additional metazoan factor, sororin forms a complex with Pds5 to prevent Wapl binding. During metaphase, centromeric cohesin is protected by the shugoshin-PP2A complex. In metazoans, cohesin is released from chromosomes in two major steps. The first requires cohesin phosphorylation and allows Wapl to bind Pds5 again to mediate cleavage-independent release of cohesin from chromosome arms. The second transpires upon fulfilment of spindle assembly and requires activation of a protease called separase, resulting in Scc1 cleavage, thus releasing sister chromatids to be segregated into daughter cells. Beyond cohesion, it is also becoming apparent that cohesin plays more diverse roles by interacting with a plethora of other factors, most notably CTCF, a zinc finger protein that is known as an insulator, which has been reported to collaborate with cohesin in determining 3D genome structure.To understand how cohesin engages DNA, I investigated the DNA binding properties of previously identified globular sub-complexes. By determining a crystal structure of the budding yeast Scc3 bound to a fragment of the Scc1 kleisin subunit and DNA, I could demonstrate that Scc3 and Scc1 form a composite DNA interaction module. The Scc3-Scc1 subcomplex engages double-stranded DNA through a conserved, positively charged surface. We demonstrate that this conserved domain is required for DNA binding by Scc3-Scc1 in vitro, as well as for the enrichment of cohesin on chromosomes and for cell viability. These findings suggest that the Scc3-Scc1 DNA-binding interface plays a central role in the recruitment of cohesin complexes to chromosomes and therefore for cohesin to faithfully execute its functions during cell division.To investigate the molecular basis of the reported functional collaboration between cohesin and CTCF in defining 3D chromosome structure, I identified and determined the structure of a ternary complex composed of human SA2 (an orthologue of Scc3), Scc1 and CTCF. The structure revealed a wide-spread SA2-Scc1 binding motif which was found to be present not only in CTCF, but also other functionally related factors, including shugoshin and Wapl. Competition pulldown assays indicated that binding of these factors to SA2-Scc1 was mutually exclusive, which strongly suggested that they interact with cohesin via similar mechanisms. To demonstrate this principle, I was able to determine a structure of shugoshin in complex with SA2-Scc1, which confirmed that both shugoshin and CTCF bind the same conserved surface on cohesin.

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.