Principles of the spatiotemporal control of meiotic recombination initiation in mice

小鼠减数分裂重组起始的时空控制原理

• 批准号: 458959104
• 负责人: Professor Steffen Rulands
• 金额: --
• 依托单位: Institut für Physiologische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2021
• 资助国家: 德国
• 起止时间: 2020-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/458959104?language=en
• 关键词: Principles; spatiotemporal; control; meiotic; recombination

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.

在减数分裂中，数百个DNA双链断裂（DSB）的程序性形成是必不可少的，因为DSB导致启动同源减数分裂重组的单链DNA（ssDNA）末端。双链断裂促进同源染色体（同系物）之间的配对和联会，重组介导的DSB修复产生交叉，这对减数分裂中正确的染色体分离至关重要。此外，重组会产生新的等位基因组合，选择会对其产生作用。重组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，DSB的数量、时间和基因组位置受多种因素的影响，从而保证同源物之间的突触和交换。特别地，两种不同的负反馈被认为控制DSB活性：（1）DSB限制其附近的进一步DSB形成，以及（2）同源物的突触区域性地关闭DSB活性。除了这些反馈，细胞周期阶段和DSB促进蛋白复合物和染色质之间的相互作用也影响DSB的位置。减数分裂特异性组蛋白甲基转移酶PRDM 9以序列和减数分裂阶段特异性方式在哺乳动物基因组中的数千个位点产生“开放”染色质位点。DSB机制优先靶向这些PRDM 9相关的“开放”位点，因此它们作为DSB热点，大多数减数分裂重组起始发生。使用时间分辨实验和生物物理建模相结合的跨学科方法，该项目旨在揭示DSB活性的位置如何受到反馈机制、减数分裂阶段和DSB机制的两个关键组件ANKRD 31和IHO 1的影响。其在重组起始中具有不同的作用。我们将首次测量小鼠减数分裂过程中热点活动的时空动态和潜在的染色质特征。具体而言，我们将使用ChIP-seq监测减数分裂时间过程中ssDNA（DMC 1），热点相关开放染色质（组蛋白H3中Lys 4的三甲基化）和非突触区域（HORMAD 1）的基因组分布。为了分离反馈和减数分裂阶段依赖热点动态的影响，我们将不仅在野生型，而且在减数分裂DSB和突触形成缺陷的突变体进行测量。这些测量结果将用于生成热点使用动态的定量模型。下一步，该模型将用于解释IHO 1和ANKRD 31缺陷对SC动力学、DSB数量、时间和定位的多效性影响，以了解IHO 1和ANKRD 31在热点使用中的不同作用。最终，上述测量及其建模将产生对DSB形成机制的全面理解，并使基因组成功传递到下一代。