Principles of the spatiotemporal control of meiotic recombination initiation in mice

数百个DNA双链断裂(DSB)的程序性形成在减数分裂中是必不可少的，因为DSB导致单链DNA(SsDNA)末端启动同源减数分裂重组。重组促进同源染色体之间的配对和突触，重组介导的DSB修复产生交叉，这对于减数分裂中正确的染色体分离是必不可少的。此外，重组创造了新的等位基因组合，选择对其起作用。重组中的异常会导致非整倍体和不育，持续的DSB具有潜在的遗传毒性。因此，减数分裂DSB的形成受到严格的时空控制。人们对多种因素的相互作用知之甚少，这些因素调节DSB的数量、时机和基因组位置，以确保DSB之间的突触和交叉。特别是，两个明显的负反馈被认为控制了DSB的活性：(1)DSB限制了其附近DSB的进一步形成，(2)同系物的突触区域性地关闭了DSB的活性。除了这些反馈，细胞周期阶段以及DSB促进蛋白复合体和染色质之间的相互作用也影响DSB的位置。减数分裂特异的组蛋白甲基转移酶，PRDM9，在哺乳动物基因组的数千个位置以序列和减数分裂阶段特有的方式产生“开放的”染色质位点。DSB机制优先以这些与PRDM9相关的“开放”位点为目标，因此它们充当DSB热点，大多数减数分裂重组发生在那里。该项目采用时间分辨实验和生物物理模拟相结合的跨学科方法，旨在揭示DSB活动的位置如何受到反馈机制、减数分裂阶段以及DSB机制的两个关键组件ANKRD31和IHO1的影响，这两个组件在重组启动中具有不同的作用。我们将首次测量小鼠减数分裂过程中热点活动和潜在染色质特征的时空动态。具体地说，我们将使用CHIP-SEQ来监测单链DNA(DMC1)、热点相关的开放染色质(组蛋白H3中Lys 4的三甲基化)和非突触区域(HORMAD1)在减数分裂过程中的基因组分布。为了分离反馈和减数分裂阶段对热点动态的影响，我们将不仅测量野生型，也测量在减数分裂DSB和突触形成方面存在缺陷的突变体。这些测量将被用来生成热点使用动态的量化模型。在下一步，该模型将用于解释IHO1和ANKRD31-缺失对SC动力学、DSB数、时间和定位的多效性效应，以了解IHO1和ANKRD31在热点使用中的不同作用。最终，上述测量及其模型将产生对支配DSB形成格局的机制的全面理解，并使基因组能够成功地传给下一代。

Programmed formation of several hundred DNA double-strand-breaks (DSBs) is essential in meiosis, as DSBs result in single-stranded DNA (ssDNA) ends which initiate homologous meiotic recombination. Recombination promotes pairing and synapsis between homologous chromosomes (homologs), and recombination-mediated repair of DSBs generates crossovers, which are essential for correct chromosome segregation in meiosis. Further, recombination creates new allele combinations on which selection acts. Anomalies in recombination cause aneuploidies and infertility, and persistent DSBs are potentially genotoxic. Hence, meiotic DSB formation is under tight spatiotemporal control.A poorly understood interplay of multiple factors regulates the numbers, the timing and the genomic locations of DSBs to ensure synapsis and crossovers between homologs. In particular, two distinct negative feedbacks are thought to control DSB activity: (1) DSBs limit further DSB formation in their vicinity, and (2) synapsis of homologs regionally shuts down DSB activity. Beyond these feedbacks, the cell cycle stage and interactions between DSB-promoting protein complexes and chromatin also affect the location of DSBs. A meiosis-specific histone methyltransferase, PRDM9, generates "open" chromatin sites in a sequence and meiosis stage-specific manner at several thousand sites in mammalian genomes. The DSB machinery preferentially targets these PRDM9-associated "open" sites, hence they act as DSB hotspots, where most meiotic recombination initiation occurs.Using an interdisciplinary approach combining time-resolved experiments and biophysical modelling the project aims to uncover how the positions of DSB activity are affected by feedback mechanisms, the stage of meiosis, and two key components of the DSB machinery, ANKRD31 and IHO1, which have distinct roles in recombination initiation. We will, for the first time, measure spatiotemporal dynamics of hotspot activity and underlying chromatin features during meiotic progression in mice. Specifically, we will use ChIP-seq to monitor genomic distribution of ssDNAs (DMC1), hotspot-associated open chromatin (tri-methylation of Lys 4 in histone H3) and unsynapsed regions (HORMAD1) in meiotic time-courses. To separate feedback- and meiosis stage-dependent effects on hotspot dynamics we will make measurements not only in wild-type but also in mutants defective in meiotic DSB and synapsis formation. These measurements will be used to generate a quantitative model of hotspot usage dynamics. The model, in the next step, will be used to interpret pleiotropic effects of IHO1- and ANKRD31-deficiencies on SC kinetics, DSB numbers, timing and positioning, to understand the differential roles of IHO1 and ANKRD31 in hotspot usage. Ultimately, the above measurements and their modelling will produce a comprehensive understanding of the mechanisms that govern the landscape of DSB formation and enable a successful passage of the genome to the next generation.