Epigenetic control of meiotic recombination in mammals.

哺乳动物减数分裂重组的表观遗传控制。

基本信息

批准号：
10194541
负责人：
Roberto Jose Pezza
金额：
$ 34.96万
依托单位：
OKLAHOMA MEDICAL RESEARCH FOUNDATION
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-05 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10194541
关键词：
Address Aneuploidy Architecture Binding Biochemical Cells ChIP-seq Characteristics Chimeric Proteins Chromatin Chromatin Modeling Chromatin Remodeling Factor Chromatin Structure Chromosome Segregation Chromosomes Congenital Abnormality DNA Damage DNA Double Strand Break DNA Repair DNA Sequence Enzymes Epigenetic Process Event Frequencies Gametogenesis Genetic Genetic Recombination Genome engineering Genomic Segment Germ Cells Gonadal structure Histones Human Image Infertility Knock-out Knockout Mice Lead Link Location Mammals Maps Measures Mediating Meiosis Meiotic Recombination Methyltransferase Mitotic Modeling Molecular Monitor Mus Outcome Pathway interactions Physical condensation Positioning Attribute Post-Translational Protein Processing Pregnancy loss Process Protein Subunits Proteins Recruitment Activity Resolution Role Site Spermatocytes Spontaneous abortion Techniques Testing Testis Time Work base chromatin modification chromatin remodeling ds-DNA experimental study genetic approach genome-wide histone modification homologous recombination in vivo next generation prevent recombinational repair recruit repaired response tool

项目摘要

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.

概括减数分裂过程中同源染色体分离的错误是出生缺陷的主要原因，自然流产，并导致不孕。正确的染色体分离需要成对通过交叉产生的母本同源染色体和父本同源染色体的关联重组 (HR) 介导的双链 DNA 断裂 (DSB) 修复。染色质调节 DSB 的可访问性以及 DSB 修复。修复 DSB 需要染色质重塑复合物减数分裂和有丝分裂细胞，但它们如何控制 HR 修复机制的募集和活动 DSB 尚不清楚。 PBAF 和 BAF 染色质重塑因子与发生的组蛋白修饰有关减数分裂 DSB，并在向 DSB 募集 HR 修复因子方面发挥作用。我们的中心假设是 PBAF 和 BAF 将 DSB 周围的组蛋白标记与减数分裂 HR 机制的招募和激活联系起来。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 如何作用调节 HR 介导的 DSB 修复，进而调节 HR 中间体的数量和位置，交叉？ (ii) 特定组蛋白标记是否控制 PBAF/BAF 和 HR 机制向 HR 的招募减数分裂染色体上的热点位点？ (iii) 改变 DSB 周围的染色质结构（即冷凝）影响DSB修复效率？第一个目标将调查 PBAF 的角色- 特定亚基 Baf200 和 BAF 特定亚基 Baf250A，参与减数分裂 DSB 修复、交叉形成，以及同源染色体的结合和分离。我们将使用现有的工具，例如成像和转基因小鼠，以辨别 PBAF 和 BAF 作为减数分裂调节剂的功能。瞄准 2、我们将通过生成高分辨率基因组来阐明PBAF/BAF与HR修复之间的关系- 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 PBAF/BAF 是否存在以及 HR 热点形成所需，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) 至野生型精母细胞。调查 PBAF/BAF 是否足以影响局部 DSB 为了修复和交叉形成，我们开发了一种 lacO-lacI 方法来靶向 lacI 融合蛋白（例如 lacI- Brg1) 到异位 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 和人力资源机构。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 招募，并阐明 PBAF/BAF 如何与 HR途径影响DSB修复效率。目标 1 和 2 中的实验也将告知分子染色质结构的变化如何影响减数分裂重组的首次。结果预计将具有重要意义，因为它们将揭示正确 DNA 背后的表观遗传机制减数分裂过程中的修复和同源染色体分离，保护下一代。