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

DNA双链断裂（DSB）是导致基因组不稳定、有害突变和癌症的主要因素。然而，数百个dsb的程序化形成是减数分裂的重要组成部分，因为dsb可以启动同源减数分裂重组。重组介导的dsb修复产生交叉，这对于同源染色体的正确分离和单倍体配子的产生是必不可少的。交叉形成的异常导致人类非整倍体和不孕症，持续的dsb具有潜在的基因毒性。因此，减数分裂DSB的形成受到严格的时空控制。哺乳动物基因组中有几千个位点在减数分裂过程中容易频繁形成DSB。这些DSB热点被认为依赖于“开放”的染色质标记，如组蛋白H3中赖氨酸4 （H3K4me3）和赖氨酸36 （H3K36me3）的三甲基化。奇怪的是，携带这些标记的活跃启动子和基因在野生型中不充当热点。因此，如何区分热点与活性启动子和基因是一个关键问题。DSB形成活性在X和Y染色体的同源假常染色体区域（PARs）特别强，热点依赖于PARs和基因组其余部分的不同因素（非par热点）。非par热点的位置由PRDM9确定，PRDM9结合热点位点，局部催化H3K4me3和H3K36me3修饰。这些组蛋白修饰和prdm9结合的组合被认为可以招募dsb形成机制，但其潜在机制尚不清楚。指定par相关热点的蛋白质是未知的；PAR dsb不需要PRDM9。我们发现了一个迄今为止未知的减数分裂特异性蛋白ANKRD31，它与dsb促进蛋白的染色质结合复合物共定位。我们发现ankrd31缺陷小鼠DSB形成严重延迟，热点分布异常。dsb既可以在传统的非par热点形成，也可以反常地在活性启动子处形成。独特的是，在par区DSB的形成似乎严重减少。这可能是在缺乏ankrd31的精母细胞中，性染色体之间特异性交叉丢失和染色体分离失败的原因。我们的主要假设是ANKRD31在PAR和非PAR区域DSB形成的时空控制中起核心作用。我们的目标是分析这一角色。在其他实验中，我们将研究PRDM9和ANKRD31之间的功能关系，并验证ANKRD31通过调节DSB热点的染色质状态来控制DSB形成的关键假设。为了实现这些目标，我们将使用小鼠表型分析，染色质免疫沉淀法分析染色质，以及涉及生物化学和酵母两种杂交分析的蛋白质相互作用研究。解剖ankrd31缺陷小鼠的独特表型对于阐明哺乳动物DSB形成的正确时空控制机制至关重要。

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.