Epigenetic control of meiotic recombination in mammals.

摘要 减数分裂过程中同源染色体分离的错误是导致出生缺陷的主要原因， 自然流产，并导致不孕不育。正确的染色体分离需要两两配对 母系和父系同源染色体的关联通过交叉，由同源染色体产生 重组(HR)介导的双链DNA断裂(DSB)修复。染色质调节 DSB的可访问性，反过来，DSB修复。染色质重塑复合体是修复DSB所必需的 减数分裂和有丝分裂细胞，但它们如何控制HR修复机制的招募和活动 DSB尚不清楚。PBAF和BAF染色质重构体与发生的组蛋白修饰有关 在减数分裂DSB，并在招募HR修复因子到DSB的功能。我们的中心假设是PBAF和 BAF将组蛋白与减数分裂HR机制的招募和激活联系在DSB周围。 我们的具体目标将通过解决以下问题来检验这一假设：(I)PBAF和BAF如何 调节HR介导的DSB修复，进而调节HR中间体的数量和位置 跨界车？(2)特定的组蛋白标记是否控制PBAF/BAF和HR机制到HR的招募 减数分裂染色体上的热点位置？(3)DSB周围染色质结构的变化(即 冷凝)影响DSB修复效率？第一个目标将调查PBAF的角色- 在减数分裂DSB修复、交换形成中， 以及同源染色体的联合和分离。我们将使用成熟的工具，例如 成像和转基因小鼠，以辨别PBAF和BAF作为减数分裂调节因子的功能。在AIM 2，我们将通过产生高分辨率的基因组来阐明PBAF/BAF与HR修复的关系。 小鼠精母细胞中PBAF/BAF和HR修复因子(Dmc1/RAD51)的广泛结合谱。评估 是否存在PBAF/BAF并且是HR热点形成所需的，我们将生成并比较 缺乏BRG1、Baf200(PBAF)或Baf250A的精母细胞中Dmc1/RAD51的全基因组结合谱 (Baf)转化为野生型精母细胞。调查PBAF/BAF是否足以影响当地的DSB 修复和交叉形成，我们开发了一种LACO-LACI方法来靶向LacI融合蛋白(例如LacI- BRG1)连接到异地LACO阵列。在目标3中，我们将确定DSB周围的哪些特定染色质修饰 需要招募PBAF/BAF和人力资源机器。这些实验将测试我们的模型 DSB周围的染色质景观控制PBAF/BAF的招募，并阐明PBAF/BAF如何与 HR途径影响DSB修复效率。目标1和目标2中的实验也将告知分子 染色质结构的改变如何影响减数分裂重组的一些方面首次得到证实。结果是 预计将具有重大意义，因为它们将揭开适当DNA背后的表观遗传机制 在减数分裂过程中修复和同源染色体分离，保护下一代。

Summary Errors in homologous chromosome segregation during meiosis are the leading cause of birth defects, spontaneous abortions, and contribute to infertility. Proper chromosome segregation requires pairwise associations of maternal and paternal homologous chromosomes via crossovers, generated by homologous recombination (HR)-mediated repair of double-strand DNA breaks (DSBs). Chromatin regulates the accessibility to DSBs and, in turn, DSB repair. Chromatin remodeling complexes are required to repair DSBs in meiotic and mitotic dividing cells, but how they control recruitment and activity of the HR repair machinery at DSBs is unclear. The PBAF and BAF chromatin remodelers are connected to histone modifications occurring at meiotic DSBs, and function in recruiting HR repair factors to DSBs. Our central hypothesis is that PBAF and BAF link histone marks surrounding DSBs with the recruitment and activation of the meiotic HR machinery. Our Specific Aims will test this hypothesis by addressing the following questions: (i) How do PBAF and BAF regulate HR-mediated repair of DSB and, in turn, the number and position of HR intermediates and crossovers? (ii) Do specific histone marks control the recruitment of PBAF/BAF and the HR machinery to HR hotspot sites on meiotic chromosomes? (iii) Do changes in chromatin architecture around DSBs (i.e. condensation) influence the efficiency of DSB repair? The first Aim will investigate the roles for the PBAF- specific subunit Baf200, and the BAF-specific subunit Baf250A, in meiotic DSB repair, crossover formation, and the association and disjunction of homologous chromosomes. We will employ established tools, such as imaging and genetically modified mice, to discern the functions of PBAF and BAF as meiotic regulators. In Aim 2, we will elucidate the relationship between PBAF/BAF and HR repair by generating high-resolution genome- wide binding profiles for PBAF/BAF and HR repair factors (Dmc1/Rad51) in mouse spermatocytes. To assess whether PBAF/BAF are present and required for HR hotspot formation, we will generate and compare genome-wide binding profiles of Dmc1/Rad51 in spermatocytes that lack Brg1, Baf200 (PBAF) or Baf250A (BAF) to wild-type spermatocytes. To investigate whether PBAF/BAF are sufficient to influence local DSB repair and crossover formation, we developed a lacO-lacI approach to target lacI fusion proteins (e.g. lacI- Brg1) to ectopic lacO arrays. In Aim 3, we will determine which specific chromatin modifications around DSBs are required to recruit PBAF/BAF and the HR machinery. These experiments will test our model that the chromatin landscape around DSBs control PBAF/BAF recruitment, and illuminate how PBAF/BAF interact with the HR pathway to influence DSB repair efficiency. Experiments in Aim 1 and 2 will also inform the molecular aspects of how changes in chromatin structure influence meiotic recombination for the first time. The outcomes are expected to be significant because they will unravel the epigenetic mechanisms underlying proper DNA repair and homologous chromosome segregation during meiosis, which safeguards the next generation.