Spatiotemporal control of DNA double strand break formation in mammalian germ cells by a newly discovered meiosis-specific protein, ANKRD31

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DNA double-strand-breaks (DSB) are major contributors to genome instability, deleterious mutations and cancer. Yet, programmed formation of several hundred DSBs is an essential part of meiosis, as DSBs serve to initiate homologous meiotic recombination. Recombination-mediated repair of DSBs generates crossovers, which are indispensable for correct segregation of homologous chromosomes and thus the generation of haploid gametes. Anomalies in crossover formation cause aneuploidies and infertility in humans, and persistent DSBs are potentially genotoxic. Hence, meiotic DSB formation is under tight spatiotemporal control. Mammalian genomes have several thousand sites which are prone to frequent DSB formation during meiosis. These DSB hotspots are thought to depend on "open" chromatin marks such as tri-methylation of lysine4 (H3K4me3) and lysine 36 (H3K36me3) in histone H3. Curiously, active promoters and genes, which carry these marks, do not act as hotspots in wild-type. Thus, it is a key question what distinguishes hotspots from active promoters and genes. DSB forming activity is uniquely strong at homologous pseudoautosomal regions (PARs) of X and Y chromosomes, and hotspots depend on distinct factors in PARs and the rest of the genome (non-PAR hotspots). The positions of non-PAR hotspots are defined by PRDM9, which binds hotspot sites and locally catalyses H3K4me3 and H3K36me3 modifications. The combination of these histone modifications and PRDM9-binding is thought to recruit the DSB-forming machinery, but the underlying mechanism remains unclear. Proteins that designate PAR-associated hotspots are unknown; PRDM9 is not needed for PAR DSBs. We identified a hitherto unknown meiosis-specific protein, ANKRD31, that colocalizes with chromatin-bound complexes of DSB-promoting proteins. We found that ANKRD31-deficient mice have a severe delay in DSB formation and an abnormal hotspot distribution. DSBs form both at conventional non-PAR hotspots and also, aberrantly, at active promoters. Uniquely, DSB formation seems to be severely reduced at PARs. This is the likely reason for a specific loss of crossovers between sex chromosomes and a chromosome segregation failure in ANKRD31-deficient spermatocytes. Our overriding hypothesis is that ANKRD31 plays a central role in the spatiotemporal control of DSB formation both in PAR and non-PAR regions. We aim to analyse this role. Among other experiments, we will examine the functional relationship between PRDM9 and ANKRD31, and test the key hypothesis that ANKRD31 controls DSB formation by modulating chromatin status at DSB hotspots. To achieve these aims we will use phenotypic analysis in mice, chromatin-analysis by chromatin immunoprecipitations, and protein interaction studies involving biochemistry and yeast two hybrid assays. Dissecting the unique phenotype of ANKRD31-deficient mice will be essential for elucidating the mechanisms that underpin correct spatiotemporal control of DSB formation in mammals.