At the very center of sexual reproduction is meiosis. During meiosis, the formation of meiotic Double-Strand-Breaks (DBSs) and their repair by homologous recombination are widely conserved events occurring among most eukaryote species. Meiosis-specific DSB formation requires at least nine proteins (Spo11, Ski8, Rec102, Rec104, Mei4, Mer2, Rec114, Mre11/Rad50/Xrs2) in S. cerevisiae, and the resection of the DSB ends requires additional four proteins (Mre11/Rad50/Xrs2, and Sae2). Spo11 has been identified as the catalytic component of this DSB-initiating complex. However, the roles played by the majority of these proteins are not clear. I have purified the recombinant Spo11/Ski8/Rec102/Rec104 complex, characterized its DNA binding ability as well as its cleavage activity on supercoiled plasmid DNA. Sae2 functions in both meiotic and mitotic repair of DNA double-strand breaks (DSBs) in S. cerevisiae. In vivo experiments have shown that Sae2 collaborates with the Mre11/Rad50/Xrs2 (MRX) complex in DNA end processing. Our laboratory previously showed that recombinant Sae2 exhibits endonuclease activity on single-stranded DNA and single-strand/double-strand DNA junctions using purified proteins in vitro. The MRX complex stimulates Sae2 endonuclease activity on single-stranded DNA close to single-strand/double-strand junctions, through its endonucleolytic activity. However, Sae2 contains no conserved typical nuclease domain, and it only shares very limited homology with its human functional counterpart CtIP. To characterize Sae2 and the active sites responsible for its nuclease activity, I used partial proteolysis and site-directed mutagenesis to analyze the protein. Biochemical assays in vitro show that acidic residues in the central domain play an important role in Sae2 endonuclease activity. Sae2 has also been shown to be phosphorylated by CDK (Cyclin-Dependent Kinase) during the S and G2 phases of the cell cycle, as well as by Tel1/Mec1 upon DNA damage. These modifications are essential for the function of Sae2 in DNA repair, but the function of these modifications are not clear. I have demonstrated that, in the presence of MRX, Sae2 (5D/S267E) mimicking constitutive phosphorylation by CDK and Mec1/Tel1 can assist the 5’ to 3’ exonuclease Exo1 significantly in 5’ end resection by suppressing the inhibitory effect of Ku. These results suggest that Sae2 is a critical switching protein which determines the choice between HR and NHEJ in yeast cells upon DNA damage. / text

The ability of oocytes and early cleavage-stage embryos to regulate their volume is essential to avoid developmental arrests at in vivo-osmolarities. This is accomplished primarily via GLYT1-mediated glycine transport into the cells. GLYT1 activity has previously been shown to be absent in freshly isolated oocytes but becomes activated ~3-4 hours after oocyte maturation has been initiated either by isolation from ovarian follicles in vitro or following an ovulatory stimulus in vivo. GLYT1 activity then persists until the 4-cell stage of preimplantation embryo development. GLYT1 has been shown to spontaneously activate in oocytes that are isolated from follicles either as denuded oocytes or as cumulus-oocyte complexes (COCs), this implies that GLYT1 activity is suppressed in intact follicles in the ovary. However, it is not known how GLYT1 activity is suppressed within the ovarian follicle or how initial GLYT1 activation occurs. The activation of independent cell volume regulation in oocytes first involves the release of the strong adhesion between the oocyte and zona pellucida (ZP) followed by secondary GLYT1 activation. These two processes have been shown to occur spontaneously in fully grown oocytes following isolation from ovarian follicles, however, it is not known whether small growing oocytes within ovarian follicles already possess the ability to detach from the ZP and activate GLYT1. An osmotic assay was used to determine when during oogenesis oocytes are first able to detach from the ZP while the ability to activate GLYT1 was determined by measuring [3H]-glycine uptake into oocytes. I found that oocytes acquire the ability to detach from the ZP when they are nearly fully grown and similarly, that high levels of GLYT1 activity first develop in isolated oocytes during the late stages of oogenesis. Furthermore, I showed that SLC6A9 protein (GLYT1 transporter protein) and Slc6a9a transcripts steadily increased during oogenesis with SLC6A9 protein becoming localized to the oocyte plasma membrane during oocyte growth with predominant membrane localization apparent in fully grown oocytes. Taken together, these results suggest that oocytes become able to detach from the ZP and fully activate GLYT1 towards the end of oogenesis but that these processes remain suppressed in the ovarian follicle. Intact and punctured antral follicles were used as a model to examine the potential mechanism(s) mediating GLYT1 suppression before ovulation is triggered. Using these models, I found that GLYT1 activity remains suppressed within preovulatory antral follicles in contrast to the spontaneous GLYT1 activation that occurred in isolated denuded oocytes or within COCs. Recently, the mechanism mediating oocyte maintenance of prophase I arrest within the ovarian follicle was elucidated and was shown to depend on the release of Natriuretic Peptide Precursor C (NPPC) from mural granulosa cells (MGCs) into follicular fluid which binds to NPR2 guanylate cyclases on cumulus cells stimulating the production of cyclic GMP (cGMP) within these cells. Diffusion of cGMP from cumulus granulosa cells to the oocyte via gap junctions is required to maintain meiotic arrest. Although GLYT1 activation and meiotic resumption are both suppressed in antral follicles prior to the ovulatory trigger and these two processes occur simultaneously following oocyte isolation from ovaries, I have shown here that GLYT1 suppression within the preovulatory antral follicle is mediated by a mechanism distinct from the gap junction-dependent NPPC-cGMP pathway controlling meiotic arrest. I also showed for the first time a direct requirement for meiotic arrest of both gap junctions between granulosa cells (composed of connexin-43) and between the inner layer of cumulus granulosa cells and the oocyte (composed of connexin-37). Since I showed that GLYT1 was suppressed in isolated antral follicles but not COCs, I hypothesized that MGCs are required to maintain low GLYT1 activity in antral follicles. I showed here that MGCs isolated from preovulatory antral follicles were sufficient to maintain GLYT1 suppression in co-cultured COCs, but not denuded oocytes. Furthermore, I found that GLYT1 activity was suppressed in COCs cultured in conditioned medium from MGC cultures. Thus, GLYT1 activity appears to be suppressed within the ovary prior to the ovulatory LH-stimulus likely by an unidentified inhibitory signal within the ovarian follicle originating from the MGCs and propagated by a gap junction-independent mechanism involving multiple cell types in the follicle.

<p>PIWI proteins belong to an evolutionary conserved protein family as the sister sub-family of ARGONAUTE (AGO) proteins. While AGO proteins are functionally well-characterized and shown to mediate small-RNA guided gene regulation, the function of PIWI proteins remain elusive. Here we pursued functional characterization of PIWI proteins by studying MILI and MIWI, two PIWI proteins in the mouse.</p><p>We first show that both MIWI and MILI co-immunoprecipitate with a novel class of non-coding small RNAs from the post-natal mouse testis extract, which are named Piwi-interacting RNAs (piRNAs). Our cloning efforts identified thousands of different piRNA sequences, mostly derived from intergenic regions. Interestingly, both MILI and MIWI piRNAs correspond to the same regions on the genome and differ primarily in length. We propose piRNAs in the adult testis are produced by the processing of long, single stranded RNA precursors, based on the observation that piRNAs originate in clusters from a number of sites on the genome in a head-to-tail homology. In support, we bioinformatically predicted putative promoters, and yeast one hybrid analysis on two such regions found out that they interact with Krueppel C2H2 type zinc finger transcription factors. We did not observe the features of the "ping-pong" mechanism in their biogenesis: Both MILI and MIWI piRNAs are biased for 5` Uracil without an Adenine bias on the 10th nucleotide position, and do not significantly consist of sequences complementary to each other along their first 10nt. Moreover, MILI piRNAs are not down-regulated in Miwi-/- testis. These results indicate that the post-natal testicular piRNAs are produced independent of the ping-pong mechanism. </p><p>Although piRNAs are highly complex, PAGE and in situ analyses showed that piRNAs are germ cell-specific with predominant expression in spermatocytes and round spermatids, suggestive of a meiotic function. Correspondingly, we found that Miwi-/-; Mili-/- mice undergo only male infertility with terminal spermatogenic arrest during meiosis. piRNAs show a nucleo-cytoplasmic distribution, with enrichment in the chromatoid and dense bodies, two male germ cell-specific structures. The dense body has been implicated in synapsis and in the heterochromatinization of the sex chromosomes during male meiosis, a process known as meiotic sex chromosome inactivation (MSCI). Our histological analysis on Miwi-/-; Mili-/- testes showed that, while the overall synapsis is not affected, the sex chromosomes retain the euchromatin marker acetyl-H4K16 and lacks the heterochromatin marker H3K9-dimethyl. These observations indicate that murine PIWI proteins are necessary for MSCI. Moreover, we identified piRNA production from the X chromosome before MSCI, and propose PIWI proteins utilize piRNAs to target and silence unpaired chromosomal regions during meiosis.</p> / Dissertation

Meiotic recombination involves the exchange of DNA between two homologous chromosomes, forming cross-overs and gene conversion events. The cross-over process is important for the proper segregation of chromosomes during meiosis, and drives genetic diversity. Human hotspots are enriched for a 13-bp motif, CCNCCNTNNCCNC; a close match to this motif occurs in about 40% of our cross-over hotspots. A DNA binding protein called PRDM9, having histone trimethyltransferase (H3K4me3) activity, binds the motif and is becoming established as a major determinant of recombination hotspots (narrow regions with high cross-over activity). This research aimed to understand the mechanisms involved in promoting PRDM9 binding to its target sites, and subsequently, initiating cross-over hotspot activity. We first explored the relationship between PRDM9 binding and DNA sequence, to directly confirm whether PRDM9 binds to the 13-bp hotspot motif using in-vitro gel-shift assays, and found that it does bind sequence specifically to the canonical 13-mer motif. PRDM9 is able to bind the motif in a highly selective manner, with certain single base pair changes abolishing binding. However, we observe that it is also able to tolerate degeneracy in its binding sites, as demonstrated by strong in-vitro binding to degenerate versions of the 13-bp motif. Hence, these results confirmed that PRDM9 is able to directly bind to the 13-bp hotspot motifs, and given that it can also tolerate degeneracy, this raised the question of why PRDM9 is able to bind only a subset of all such potential binding sites in the genome. To address this, a ChIP-seq analysis was performed to identify genome wide binding sites for PRDM9. This information also helped us to characterise binding sites and investigate if factors such as the local chromatin environment play a role in specifying PRDM9 binding tar- gets and hotspot formation. We were able to identify over 170,000 PRDM9 binding sites in the genome. Surprisingly, these binding sites were also enriched in promoter regions, however, bound sites in these regulatory regions showed low recombination activity. We found that PRDM9 is able to confer the H3K4me3 mark on all bound sites, even those without a pre-existing H3K4me2 mark. We also investigated the role of other chromatin related marks on PRDM9 binding and found that binding occurs in chromatin accessible, but nucleosome rich regions, whereas heterochromatin regions tend to inhibit binding. Further, for hotspot formation, it was seen that less chromatin accessible, nucleosome dense regions away from transcribed sites, are preferred. Hotspots tend to avoid regions marked by transcription activating histone modifications, however, these regions do not appear to inhibit PRDM9 binding itself. These results show how PRDM9 binding in the genome is dependent on both primary DNA sequence and the surrounding epigenetic factors. Together these factors promote binding and, with additional downstream factors, positioning of hotspot locations in the human genome.

Meiotic recombination promotes genetic variation by reciprocal exchange of genetic material producing novel allelic combinations that influence important agronomic traits in crop plants. Therefore, harnessing meiotic recombination has the potential to accelerate crop improvement via classical breeding. Numerous genes involved in crossover formation have been identified in model systems. For example, SPO11 mediates generation of meiotic DNA double-strand breaks (DSBs) across all eukaryotes, which may be repaired as crossovers. However, downstream regulators of recombination remain to be identified, including those with species-specific roles. To isolate crossover frequency modifiers I performed a high-throughput forward genetic screen using EMS mutagenesis of Arabidopsis carrying a fluorescent crossover reporter line called 420. The primary screen isolated nine mutants from ~3,000 scored individuals that showed significantly higher (high crossover rate, hcr) or lower (low crossover rate, lcr) crossover frequency, including a new fancm allele. Four mutants (hcr1, hcr2, hcr3 and lcr1) were mapped by sequencing and candidate genes identified. The hcr1 mutation was confirmed as being located within the PROTEIN PHOSPHATASE X-1 (PPX-1) gene, using isolation of an independent allele and complementation studies. Similarly, the lcr1 mutation was confirmed to be within the gene TBP-ASSOCIATED FACTOR 4B (TAF4B). Using immunocytological staining I observed that hcr1 did not show changes in DSB-associated foci (RAD51), but it did show a significant increase in crossover-associated MLH1 foci. The hcr1 mutation increases crossovers mainly in the sub-telomeric chromosome regions, which remain sensitive to crossover interference. Also the genetic interaction between the hcr1 and fancm mutations is additive. These results support a model where PPX- 1 acts to limit recombination via the Class I interfering CO pathway, downstream of DSB formation. In summary, this genetic screen has led to discovery of novel genes that regulate meiotic recombination and their functional characterization may find utility in crop breeding programs.

Sex chromosomes represent a unique part of the genome in many eukaryotic organisms. They differ significantly from autosomes by their evolution, specific features, and meiotic behaviour. Recent advances in the knowledge of sex chromosomes in non-model organisms have been largely enabled by modern cytogenetic methods. The present study explores several topics related to sex chromosomes in Lepidoptera, the largest group of animals with female heterogamety, using methods of molecular cytogenetics, immunocytogenetics, and molecular biology. These topics include physical mapping of chromosomes by BAC-FISH, molecular differentiation and composition of the W chromosome, differences in the evolution of the W and Z chromosome, and meiotic sex chromosome inactivation. The results obtained brought new information not only about the W and Z chromosomes in Lepidoptera, but also about the evolution and specific features of sex chromosomes in general.

Meiosis is a specialized type of cell division essential for the production of four normal haploid gametes. In early prophase I of meiosis, the intimate synapsis between homologous chromosomes, and the formation of chiasmata, is facilitated by a proteinaceous structure known as the synaptonemal complex (SC). Ultrastructural analysis of germ cells of a number of organisms has disclosed that SC is a specialized tripartite structure composed of two lateral elements, one on each homolog, and a central element, which, in turn, are linked by transverse elements. Genetic studies have revealed that defects in meiotic chromosome alignment and/or segregation result in aneuploidy, which is the leading cause of spontaneous miscarriages in humans, hereditary birth defects such as Down syndrome, and are also, associated with the development and progression of certain forms of cancer. The mechanism(s) underlying the alignment/pairing of chromosomes at meiosis I differ among organisms. These can be divided into at least two broad pathways: one is independent of DNA double-strand breaks (DSB) and other is mediated by DSBs. In the DSB-dependent pathway, SC plays crucial roles in promoting homolog pairing and disjunction. On the other hand, the DSB-independent pathway involves the participation of telomeres, centromeres and non-coding RNAs in the pre-synaptic alignment, pre-meiotic pairing as well as pairing of homologous chromosomes. Although a large body of literature highlights the central role of SC in meiotic recombination, the possible role of SC components in homolog recognition and alignment is poorly understood. Genetic screens for Saccharomyces cerevisiae mutants defective in meiosis and sporulation lead to the isolation of genes required for interhomolog recombination, including those that encode SC components. In S. cerevisiae, ten meiosis-specific proteins viz., Hop1, Red1, Mek1, Hop2, Pch2, Zip1, Zip2, Zip3, Zip4 and Rec8 have been recognized as bona fide constituents of SC or associated with SC function. Mutations in any of these genes result in defective SC formation, thus leading to reduction in the rate of recombination. HOP1 (Homolog Pairing) encodes a ̴ 70 kDa structural protein, which localizes to the lateral elements of SC. It was found to be essential for the progression of meiotic recombination. In hop1Δ mutants, meiosis specific DSBs are reduced to 10% of that of wild type level and it fails to produce viable spores. It also displays relatively high frequency of inter-sister recombination over inter-homolog recombination. Bioinformatics analysis suggests that Hop1 comprises of an N-terminal HORMA domain (Hop1, Rev7 and Mad2), which is conserved among Hop1 homologs from diverse organisms. This domain is also known to be present in proteins involved in processes like chromosome synapsis, repair and sex chromosome inactivation. Additionally, Hop1 harbors a 36-amino acid long zinc finger 348374 motif (CX2CX19CX2C) which is critical for DNA binding and meiotic progression, and a putative nuclear localization signal corresponding to amino acid residues from 588-594. Previous studies suggested that purified Hop1 protein exists in multiple oligomeric states in solution and displays structure specific DNA binding activity. Importantly, Hop1 exhibited higher binding affinity for the Holliday junction (HJ), over other early recombination intermediates. Binding of Hop1 to the HJ at the core resulted in branch migration of the junction, albeit weakly. Intriguingly, Hop1 showed a high binding affinity for G4 DNA, a non-B DNA structure, implicated in homolog synapsis and promotes robust synapsis between double-stranded DNA molecules. Hop1 protein used in the foregoing biochemical studies was purified from mitotically dividing S. cerevisiae cells containing the recombinant plasmid over-expressing the protein where the yields were often found to be in the low-microgram quantities. Therefore, one of the major limitations to the application of high resolution biophysical techniques, such as X-crystallography and spectroscopic analyses for structure-function studies of S. cerevisiae Hop1 has been the non-availability of sufficient quantities of functionally active pure protein. In this study, we have performed expression screening in Escherichia coli host strains, capable of high level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed +2 for expression and purification of S. cerevisiae Hop1 protein, using Ni-NTA and double-stranded DNA-cellulose chromatography. Recombinant S. cerevisiae Hop1 protein thus obtained was >98% pure and exhibited DNA binding activity with high-affinity for Holliday junction. The availability of the bacterial HOP1 expression vector and functionally active Hop1 protein has enabled us to glean and understand several vital biological insights into the structure-function relationships of Hop1 as well as the generation of appropriate truncated mutant proteins. Mutational analyses in S. cerevisiae has shown that sister chromatid cohesion is required for proper chromosome condensation, including the formation of axial elements, SC assembly and recombination. Consistent with these findings, homolog alignment is impaired in red1hop1 strains and associations between homologs are less stable. red1 mutants fail to make any discernible axial elements or SC structures but exhibit normal chromosome condensation, while hop1 mutants form long fragments of axial elements but without any SCs, are defective in chromosome condensation, and produce in-viable spores. Using single molecule and ensemble assays, we found that S. cerevisiae Hop1 organizes DNA into at least four major distinct DNA conformations: (i) a rigid protein filament along DNA that blocks access to nucleases; (ii) bridging of non-contiguous segments of DNA to form stem-loop structures; (iii) intra-and intermolecular long range synapsis between double-stranded DNA molecules; and (iv) folding of DNA into higher order nucleoprotein structures. Consistent with B. McClintock’s proposal that “there is a tendency for chromosomes to associate 2-by-2 in the prophase of meiosis involving long distance recognition of homologs”, these results to our knowledge provide the first evidence that Hop1 mediates the formation of tight DNA-protein-DNA nucleofilaments independent of homology which might help in the synapsis of homologous chromosomes during meiosis. Although the DNA binding properties of Hop1 are relatively well established, comparable knowledge about the protein is lacking. The purification of Hop1 from E. coli, which was functionally indistinguishable from the protein obtained from mitotically dividing S. cerevisiae cells has enabled us to investigate the structure-function relationships of Hop1, which has provided important insights into its role in meiotic recombination. We present several lines of evidence suggesting that Hop1 is a modular protein, consisting of an intrinsically unstructured N-terminal domain and a core C-terminal domain (Hop1CTD), the latter being functionally equivalent to the full-length Hop1 in terms of its in vitro activities. Importantly, however, Hop1CTD was unable to rescue the meiotic recombination defects of hop1null strain, indicating that synergy between the N-terminal and C-terminal domains of Hop1 protein is essential for meiosis and spore formation. Taken together, our findings provide novel insights into the molecular functions of Hop1, which has profound implications for the assembly of mature SC, homolog synapsis and recombination. Several lines of investigations suggest that HORMA domain containing proteins are involved in chromatin binding and, consequently, have been shown to play key roles in processes such as meiotic cell cycle checkpoint, DNA replication, double-strand break repair and chromosome synapsis. S. cerevisiae encodes three HORMA domain containing proteins: Hop1, Rev7 and Mad2 (HORMA) which interact with chromatin during diverse chromosomal processes. The data presented above suggest that Hop1 is a modular protein containing a distinct N-terminal and C-terminal (Hop1CTD) domains. The N-terminal domain of Hop1, which corresponds to the evolutionarily conserved HORMA domain, although, discovered first in Hop1, its precise biochemical functions remain unknown. In this section, we show that Hop1-HORMA domain expressed in and purified from E. coli exhibits preferential binding to the HJ and G4 DNA, over other early recombination intermediates. Detailed functional analyses of Hop1-HORMA domain, using mobility shift assays, DNase I footprinting and FRET, have revealed that HORMA binds at the core of Holliday junction and induces marked changes in its global conformation. Further experimental evidence also suggested that it causes DNA stiffening and condensation. However, like Hop1CTD, HORMA domain alone failed to rescue the meiotic recombination defects of hop1 null strain, indicating that synergy between the N-and C-terminal domains of Hop1 is essential for meiosis as well as for the formation of haploid gametes. Moreover, these results strongly implicate that Hop1 protein harbours a second DNA binding motif, which resides in the HORMA domain at its N-terminal region. To our knowledge, these findings also provide the first insights into the biochemical mechanism underlying HORMA domain activity. In summary, it appears that the C-terminal (CTD) and N-terminal (HORMA) domains of Hop1 may perform biochemical functions similar (albeit less efficiently) to that of the full-length Hop1. However, further research is required to uncover the functional differences between these domains, their respective interacting partners and modulation of the activity of these domains.

La recombinaison homologue au cours de la méiose est un événement essentiel pour la ségrégation fidèle des chromosomes homologues, et contribue à la production de la diversité génétique. La recombinaison méiotique est initiée par l'induction de cassures double brin d'ADN (CDB), catalysée par SPO11, à des régions spécifiques du génome appelés points chauds. Récemment, il a été montré que PRDM9 est un déterminant majeur des points chauds de recombinaison chez la souris et l'homme. PRDM9 contient un domaine PR/SET avec une activité d'histone méthyltransférase, un domaine de liaison à l'ADN constitué d'une série de doigts de zinc en tandem, et des domaines prédit pour être impliqué dans des interactions protéine-protéine. Notre modèle de travail récent place PRDM9 comme un élément clé pour l'initiation de la recombinaison méiotique: PRDM9 se lie à l'ADN via le domaine à doigts de zinc, et modifie localement la structure de la chromatine. Grâce à un processus encore inconnu, SPO11 est recruté à proximité des sites de liaison de PRDM9, où il catalyse la formation de CDB. Le but de ma thèse était de répondre à la question : comment PRDM9 recrute-t-elle la machinerie CDB aux points chauds ? Pour mieux comprendre ce mécanisme, je me suis attaché à la caractérisation des protéines interagissant avec PRDM9. Les protéines interagissant potentiellement avec PRDM9 ont été identifiées, par criblage double hybrides dans la levure avec des banques d'ADNc issues de testicules, et par purification par affinité-spectrométrie de masse des complexes PRDM9. La cartographie par double hybride avec des formes tronquées de PRDM9 a révélé que le domaine KRAB atypique de PRDM9 joue un rôle clé dans les interactions protéine-protéine. Les protéines identifiées comprennent CXXC1, un composant évolutivement conservé du complexe SET1-COMPASS, et HELLS qui est indispensable à la progression de la méiose I chez la souris. J’ai montré que ces deux protéines sont exprimées au cours de la spermatogenèse chez la souris. Puisque Spp1, l'orthologue chez S. cerevisiae de CXXC1, est connu pour servir de médiateur de recrutement de la machinerie de formation des CDB aux sites de CBD, l'interaction entre PRDM9 et CXXC1 pourrait refléter la conservation de la fonction méiotique de Spp1 chez la souris. / Meiotic homologous recombination is an essential event for faithful segregation of homologous chromosomes, and contributes to production of genetic diversity. Meiotic recombination is initiated by the induction of programmed DNA double strand breaks (DSBs), which are catalyzed by SPO11, at specific regions of the genome called hotspots. Recently, PRDM9 was reported as a major determinant of recombination hotspots in mouse and human. PRDM9 contains a PR/SET domain with histone methyltransferase activity, a zinc-finger array, and putative domains for protein-protein interactions. Our recent working model involves PRDM9 as a key component for the initiation of meiotic recombination: PRDM9 binds DNA via the zinc-finger array, and modifies chromatin structure locally. Through an unknown process, SPO11 is recruited and catalyzes DSB formation near PRDM9-bound sites. The aim of my thesis was to address the question: how does PRDM9 recruit DSB machinery to hotspots. To gain insight into this mechanism, I focused on characterization of PRDM9-interacting proteins. Potential interactors of PRDM9 were identified by yeast two hybrid (Y2H) screens with testis cDNA libraries and by affinity purification-mass spectrometry of PRDM9 complexes. Further Y2H assays with truncated derivatives of PRDM9 revealed that the atypical KRAB domain of PRDM9 plays a key role in protein-protein interactions. The identified proteins include CXXC1, a component of the evolutionarily conserved SET1-COMPASS complex, and HELLS, which is indispensable for progression of meiotic prophase I in mouse. Both proteins were found to be expressed during mouse spermatogenesis. Since Spp1, the S.cerevisiae orthologue of CXXC1, is known to mediate tethering of DSB sites to DSB machinery, the interaction between PRDM9 and CXXC1 might imply potential conservation of the Spp1 function in mouse meiosis.

La recombinaison homologue est un processus conservé chez les eucaryotes. Au cours de la méiose,ce mécanisme est essentiel à la formation des crossing-overs, eux-mêmes essentiels à la bonne ségrégation des chromosomes homologues. La recombinaison méiotique est assurée par l’action combinée de facteurs mitotiques et méiotiques. La protéine MEIOB a récemment été identifiée et caractérisée comme étant essentielle à la réparation des cassures double brin de l’ADN au cours de la méiose. MEIOB est un paralogue de RPA1, la grande sous-unité du complexe RPA qui est un complexe de liaison à l’ADN simple brin ubiquitaire et composé de RPA1, RPA2 et RPA3. MEIOB peut interagir avec SPATA22 et RPA2. Cette observation suggère que MEIOB, SPATA22 et RPA pourraient agir ensemble au cours de la recombinaison méiotique. En se basant sur l’homologie de structure entre MEIOB, SPATA22 et les sous-unités de RPA, nous avons caractérisé les modalités et le rôle de leur interaction. Nous avons montré que MEIOB et SPATA22 interagissent grâce à leur domaines OB-folds C-terminaux à l’image de RPA1 et RPA2 et que MEIOB et SPATA22 coopèrent pour interagir avec le complexe RPA. Par microscopie électronique, nous avons mis en évidence que la présence de MEIOB-SPATA22 induit une forte condensation du filament RPA ADN simple brin. Nous avons également montré par immunofluorescence sur chromosomes méiotiques murins que l’hélicase BLM accumule sur les axes chromosomiques et que cette accumulation est corrélée avec l’élimination de la recombinase DMC1 des cassures méiotiques non-réparées, en absence de MEIOB. Enin, nous avons mis en évidence par microscopie à haute résolution que l’absence de MEIOB favorise une distribution anormale des protéines recombinases. Nos résultats suggèrent que MEIOB, SPATA22 et RPA collaborent pour assurer l’intégrité des intermédiaires de recombinaison méiotiques au cours de l’invasion d’un brin homologue. / Homologous recombination is a conserved process among eukaryotes. During meiosis, thismechanism is essential to the formation of crossovers and thus for the proper segregation of chromosomes. Meiotic recombination is ensured by the combined action of mitotic and meiotic factors. MEIOB has been recently identiied and shown to be essential to the repair of meiotic DNA double-strand breaks. MEIOB is aparalog of RPA1, the large subunit of RPA, which is a ubiquitous ssDNA-binding trimeric composed ofRPA1, RPA2 and RPA3. MEIOB has been shown to interact with SPATA22 and RPA2. This observation suggested that MEIOB, SPATA22 and RPA may work together. Based on the homology existing betweenstructural domains of MEIOB, SPATA22 and the RPA subunits, we deciphered the modality and the role oftheir interactions. We show that MEIOB and SPATA22 interact through their C-terminal OB domains like RPA1 and RPA2 and cooperate to interact with the RPA complex. Using Transmission Electron Microscopy,we evidenced that the presence of MEIOB/SPATA22 induces a strong compaction of the RPA/ssDNAilament. Immunofluorescent microscopy performed on murin meiotic chromosomes revealed that in theabsence of MEIOB, the BLM helicase accumulates on chromosomes axis and correlates with the eviction ofthe DMC1 recombinase from unrepaired meiotic breaks. Finally, we show that the absence of MEIOB favorsabnormal recombinase distribution observed by SIM microscopy. Together, our results evidence thatMEIOB, SPATA22 and RPA act together to insure the integrity of recombination intermediates during strandinvasion.

Les distorteurs de ségrégation méiotiques sont des éléments génétiques égoïstes qui favorisent leur propre transmission en manipulant la méiose à leur avantage. La diffusion dans les populations d’un distorteur lié au chromosome X (Sex-Ratio) provoque un excès de femelles et cela conduit à un conflit entre le chromosome X et les autres chromosomes. Ces conflits intra-génomiques sont d’importants moteurs de l’évolution des génomes. Mais, peu de choses sont connues sur la nature moléculaire et la fonction des éléments égoïstes Sex-Ratio. Le premier chapitre de cette thèse présente une synthèse actualisée sur les distorteurs de ségrégation méiotiques liés à un chromosome sexuel. Le second chapitre est consacré à l’identification et la caractérisation d’un élément distorteur du système Sex-Ratio « Paris » de Drosophila simulans, dans lequel deux éléments distorteurs liés au chromosome X provoquent ensemble la misségrégation des chromatides sœurs du chromosome Y lors de la méiose II. J’identifie à travers une cartographie génétique par recombinaison un des loci distorteur et je conduis une validation fonctionnelle de son implication dans la distorsion. Il s’agit d’un jeune gène qui évolue rapidement et appartient à une famille de gènes bien connus, impliquée dans la constitution de l’hétérochromatine. Ce gène a émergé par duplication il y a environ 15-22 millions d’années et a connu de façon indépendante de multiples duplications en cis, pseudogenizations, ou bien directement sa perte tout au long de son histoire évolutive. Cela suggère que ce gène pourrait avoir été impliquée dans de multiples conflits génétiques. Le dernier chapitre est consacré à une étude exploratoire de la diversité structurale des chromosomes Y en relation avec la distorsion de ségrégation méiotique du système « Paris ». Les résultats présentés dans ce manuscrit contribuent à augmenter les connaissances sur l’origine moléculaire des conflits génétiques et sur leur impact évolutif. / Segregation distorters are selfish genetic elements that promote their own transmission by subverting the meiotic process to their advantage. The spread of an X-linked distorter (Sex-Ratio) in populations results in an excess of females, which triggers a genetic conflict between the X chromosome and the rest of the genome. Such conflicts are important drivers of genome evolution, but little is known about the molecular nature and the function of the Sex Ratio selfish elements. The first chapter of this manuscript is a review of the current knowledge about X-linked segregation distorters. Then, I present my work on the « Paris » Sex Ratio system of Drosophila simulans, in which two distorter elements on the X chromosome co-operate to prevent Y chromosome sister chromatids segregation during meiosis II. I mapped a gene in one of the distorter loci and achieved the functional validation of its involvement in sex-ratio distortion. It is a young and rapidly evolving gene that belongs to a well-known gene family involved in chromatin state regulation. It emerged through duplication about 15-22 Myrs ago and has experienced multiple independant cis-duplications, loss or pseudogenization throughout its evolutionary history. This suggests that this gene could have been involved in multiple genetic conflicts. Finally, the last chapter is about an opening study of the strucural diversity of Y chromosomes in relation to « Paris » segregation distorter. These findings should help understanding the molecular basis of genetic conflicts and the evolutionary impact of heterochromatin regulation during meiosis.