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Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. In male and female meiotic cells, DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identified their conserved interacting domains by structural analysis. We then analysed the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity was strongly reduced genome-wide in oocytes, but only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity was delayed in autosomes. These results provide evidence that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity by integrating specific chromosomal features.

International audience; The programmed formation of hundreds of DNA double-strand breaks (DSBs) is essential for proper meiosis and fertility. In mice and humans, the location of these breaks is determined by the meiosis-specific protein PRDM9, through the DNA-binding specificity of its zinc-finger domain. PRDM9 also has methyltransferase activity. Here, we show that this activity is required for H3K4me3 and H3K36me3 deposition and for DSB formation at PRDM9-binding sites. By analyzing mice that express two PRDM9 variants with distinct DNA-binding specificities, we show that each variant generates its own set of H3K4me3 marks independently from the other variant. Altogether, we reveal several basic principles of PRDM9-dependent DSB site determination, in which an excess of sites are designated through PRDM9 binding and subsequent histone methylation, from which a subset is selected for DSB formation.

International audience; Long non‐coding RNAs (lncRNAs) regulating gene expression at the chromatin level are widespread among eukaryotes. However, their functions and the mechanisms by which they act are not fully understood. Here, we identify new fission yeast regulatory lncRNAs that are targeted, at their site of transcription, by the YTH domain of the RNA‐binding protein Mmi1 and degraded by the nuclear exosome. We uncover that one of them, nam1, regulates entry into sexual differentiation. Importantly, we demonstrate that Mmi1 binding to this lncRNA not only triggers its degradation but also mediates its transcription termination, thus preventing lncRNA transcription from invading and repressing the downstream gene encoding a mitogen‐activated protein kinase kinase kinase (MAPKKK) essential to sexual differentiation. In addition, we show that Mmi1‐mediated termination of lncRNA transcription also takes place at pericentromeric regions where it contributes to heterochromatin gene silencing together with RNA interference (RNAi). These findings reveal an important role for selective termination of lncRNA transcription in both euchromatic and heterochromatic lncRNA‐based gene silencing processes.

Germline-specific Piwi-interacting RNAs (piRNAs) protect animal genomes against transposons and are essential for fertility. piRNAs targeting active transposons are amplified by the ping-pong cycle, which couples Piwi endonucleolytic slicing of target RNAs to biogenesis of new piRNAs. Here, we describe the identification of a transient Amplifier complex that mediates biogenesis of secondary piRNAs in insect cells. Amplifier is nucleated by the DEAD box RNA helicase Vasa and contains the two Piwi proteins participating in the ping-pong loop, the Tudor protein Qin/Kumo and antisense piRNA guides. These components assemble on the surface of Vasa's helicase domain, which functions as an RNA clamp to anchor Amplifier onto transposon transcripts. We show that ATP-dependent RNP remodeling by Vasa facilitates transfer of 5′ sliced piRNA precursors between ping-pong partners, and loss of this activity causes sterility in Drosophila. Our results reveal the molecular basis for the small RNA amplification that confers adaptive immunity against transposons. Refereed/Peer-reviewed

Meiosis is a specialized cell division program that allows the formation of haploid gametes from diploid cells in sexually reproducing organisms. The halving of this genetic content is a result of the cell division process where only one round of DNA replication is followed by two divisions (Meiosis I and Meiosis II). Chromosome reduction occurs during meiosis I where maternal and paternal homologous chromosomes are separated. This process involves the formation of DNA double-strand breaks that are repaired by homologous recombination, also called meiotic recombination. This mechanism allows the formation of physical links, the crossing-overs (CO), between homologues through reciprocal exchanges. The formation of at least one CO per homologue pair, called obligatory CO, is required for faithful meiotic recombination. As such, meiosis generates genetic diversity in a population; however, meiotic errors are also the major causes of aneuploidy, congenital anomalies and infertility.DSB formation is catalyzed by the SPO11-TOPOVIBL complex and a set of partially conserved DSB formation-regulating proteins. In mammals, there are at least seven proteins that are essential for DSB formation: REC114, MEI4, IHO1, HORMAD1/2, MEI1, ANKRD31, but the exact role of these proteins and the molecular mechanism of their action remain unclear. My work focused on REC114, MEI4, and IHO1 proteins. The aim of my thesis was to perform a biophysical and structural analysis of these protein factors and characterize the molecular details of their interactions.Constructs of IHO1 and the REC114-MEI4 sub-complex were produced in bacteria and then purified separately. Protein samples were characterized by several techniques (SEC-MALS, mass spectrometry and AUC) to determine their oligomeric states. The different analyses established that the IHO1 protein was tetrameric while the REC114-MEI4 sub-complex is present mainly in heterotrimeric form (2REC114/1MEI4). During this thesis, the interaction between the REC114-MEI4 sub-complex and the IHO1 protein was initially identified by size exclusion chromatography and then confirmed by ITC analyses. This work revealed the role of the N-terminal part of IHO1 and the PH domain of REC114 in this interaction. The structural characterization of the REC114-MEI4 complex and of the IHO1 protein alone or in a complex with REC114 was initiated by several techniques combining crystallography, nuclear magnetic resonance and electronic microscopy but still requires optimizations. Nevertheless, the modelling of the three-dimensional structure of the proteins by AlphaFold2 allowed us to obtain structural insight into the dimerization of REC114 and its interactions with MEI4 and IHOI. I identified residues involved in these interactions and their mutagenesis was used to validate the structural models in vitro. The in vivo characterization of the effect of these mutations is been performed in collaboration with the team of Dr. Bernard De Massy (IGH Montpellier). The results obtained during this work will be followed up with further structural and functional studies on these meiotic proteins, either alone or in the presence of additional partners.; La méiose est une division cellulaire spécialisée des cellules germinales qui permet la production des gamètes haploïdes en réduisant de moitié le nombre de chromosomes des cellules mères. Ce processus est constitué d’un cycle de réplication suivi de deux cycles de division : méiose I et méiose II. La réduction des chromosomes a lieu pendant la méiose I où les chromosomes homologues d’origine maternelle et paternelle sont séparés. Cette ségrégation réductionnelle nécessite la formation de plusieurs cassures double-brin de l'ADN (DSB) et leur réparation par la recombinaison homologue, appelée également recombinaison méiotique. Ce mécanisme permet la création de liens physiques entre les chromosomes via des échanges réciproques d’informations génétiques entre les homologues, ou crossing-over (CO). La formation d'au moins un CO, dit obligatoire, entre chaque paire d'homologues est requise pour une ségrégation réductionnelle appropriée. De plus, ce brassage génétique permet de générer des diversités génétiques au sein des populations. Cependant, les erreurs de la méiose sont les principales causes d'aneuploïdie, de malformations congénitales et d’infertilité.Lors de la recombinaison méiotique, la formation des DSB est catalysée par un complexe du type topoisomérase, composé de l'enzyme SPO11 et de son partenaire de liaison TOPOVIBL. Toutefois, l’activité de ce complexe nécessite un ensemble partiellement conservé de protéines régulatrices, qui sont sept chez les mammifères : REC114, MEI4, IHO1, HORMAD1/2, MEI1, ANKRD31. Malgré leurs importances lors de la formation des DSB, le rôle exact de ces protéines et le mécanisme moléculaire de leurs actions ne sont pas bien caractérisés. Mon travail s’est porté sur les protéines REC114, MEI4, et IHO1. Le but de mon projet de thèse était de réaliser une étude biophysique et structurale de ces facteurs protéiques afin de comprendre leurs organisations et de caractériser les détails moléculaires de leurs interactions.Les constructions protéiques d’IHO1 et du sous-complexe REC114-MEI4 ont été produites en bactérie puis purifiées séparément. Les échantillons protéiques ont été caractérisés par plusieurs techniques (SEC-MALS, spectrométrie de masse et AUC) pour déterminer leurs états oligomériques. Les différentes analyses ont établi que la protéine IHO1 est tétramérique alors que le sous-complexe REC114-MEI4 est présent principalement sous forme hétérotrimérique en solution (2REC114/1MEI4). Durant cette thèse, l’interaction entre le sous-complexe REC114-MEI4 et la protéine IHO1 a été identifiée initialement par la chromatographie d’exclusion de taille, puis elle a été confirmée et son affinité a été caractérisée par des analyses ITC. Ce travail a permis de mettre en évidence le rôle de la partie N-terminale d’IHO1 et du domaine PH de REC114 pour cette interaction. La caractérisation structurale du complexe REC114-MEI4 et de la protéine IHO1 seule ou en complexe a été initiée par plusieurs techniques combinant la cristallographie, la résonance magnétique nucléaire et microscopie électronique. Actuellement, des cristaux sont obtenus et sont en cours d’optimisation pour le fragment structuré d’IHO1, tandis que la caractérisation du complexe REC114-MEI4 par RMN et du complexe REC114-IHO1-MEI4 par la microscopie électronique nécessite encore des optimisations. Par ailleurs, la modélisation de la structure tridimensionnelle des protéines par AlphaFold2 nous a permis d’identifier les résidus impliqués dans la dimérisation de REC114 et dans son interaction avec MEI4 et IHO1. Ceux-ci ont été mutés et analysés in vitro. Un projet de test in vivo de l’effet de ces mutations est en cours en collaboration avec l’équipe du Dr. Bernard De Massy (IGH Montpelier). Les résultats obtenus durant ce travail seront poursuivis par des études structurales et fonctionnelles plus approfondies sur ces protéines méiotiques, seules ou en présence de partenaires supplémentaires.