Micromechanical basis of meiotic chromosome condensation and architecture

总结 染色质折叠是将DNA分子包装成10，000倍的生殖细胞的关键步骤，但确切地说， 减数分裂染色质是折叠的，时空折叠如何影响转录，染色体配对， 重组在很大程度上仍然是个谜。同源染色体配对和重组是必需的 精确的染色体分离。同源染色体的错误分离是导致染色体畸变的主要原因。 流产和出生缺陷（例如，唐氏综合症）。最近有三篇论文报道说， 染色质组织/结构域动态地形成重组景观和种系转录组。 这些引人注目的染色质重组依赖于染色质轴，因为结构域边界蛋白 (e.g.,内聚素）位于轴中。目前尚不清楚的是1）染色体轴如何减数分裂 有助于减数分裂基因转录，同源染色体配对，染色体刚度，通过 染色质重组; 2）减数分裂染色体结构动态是否是性别和阶段- 特定.我们的长期目标是破译减数分裂基因组结构及其在转录中的作用， 同源配对和重组。这里提出的工作将专门测试整个假设 减数分裂染色体轴通过控制局部和全局来调节转录组和同源配对 染色质以阶段和性别依赖的方式折叠。为了验证这一假设，我们将追踪三个 具体目标：确定减数分裂染色体轴是否调节1）转录和同源 通过重组局部染色质环进行配对; 2）染色体刚性和通过介导全局 染色质折叠3)揭示减数分裂转录组和染色质的时间和性别差异 organization.方法：在目标1中，空间域中的局部染色质重组将被检测到， 染色体构象捕获（Hi-C）接触图及其相应的转录水平变化 在这些结构域中的分子量可以通过RNAseq测量。染色体相互作用将在当地由Hi- C分析（目标1）和刚度将通过显微操作（目标2）进行全面测量。荧光原位 杂交将用于验证Hi-C图谱中发现的染色体内和染色体间相互作用。高保真，单声道- 细胞RNAseq和显微操作将被引入，以揭示4D减数分裂基因组重组， aim 3中转录组和染色质折叠的两性二态性。这种方法是创新的，因为 Hi-C、单细胞RNAseq、显微操作、纳米牛顿力等多种先进方法 测量和定量免疫细胞学将被整合，以产生一个更完整的图片， 减数分裂染色体拟议的研究是重要的，因为它有望提供更深入的 了解染色体结构以及结构如何在不同阶段和性别中变化。 最终，这些研究的见解将帮助我们开发不孕症，流产， 出生缺陷。

Summary Chromatin folding is a key step to pack DNA molecules 10,000-fold into a germ cell, but exactly how the meiotic chromatin is folded and how spatiotemporal folding impacts transcription, chromosome pairing, and recombination remains largely mysterious. Homologous chromosome pairing and recombination are required for accurate chromosome segregation. Mis-segregation of homologous chromosomes is a major cause of miscarriage and birth defects (e.g., Down Syndrome). Three papers have recently reported that high-order chromatin organizations/domains dynamically shape recombination landscape and germline transcriptomes. These dramatic chromatin reorganizations depend on chromatin axes because the domain boundary proteins (e.g., cohesins) are located in the axes. What remains unknown is 1) how meiotic chromosome axis contributes to meiotic gene transcription, homologous chromosome pairing, and chromosome stiffness via chromatin reorganization; and 2) whether the meiotic chromosome structure dynamics are sex- and stage- specific. Our long-term goal is to decipher the meiotic genome organization and its roles in transcription, homologous pairing, and recombination. The proposed work here will specifically test the overall hypothesis that meiotic chromosome axes regulate transcriptome and homologous pairing via controlling local and global chromatin folding in a stage- and sex- dependent manner. To test this hypothesis, we will pursue three specific aims: Determine whether meiotic chromosome axis regulates 1) transcription and homologous pairing via reorganizing local chromatin loops 2) chromosome stiffness and pairing by mediating global chromatin folding. 3) Uncover the temporal and sexual differences of meiotic transcriptomes and chromatin organization. Method: In aim 1, local chromatin reorganization in spatial domains will be detected by chromosome conformation capture (Hi-C) contact map and the corresponding changes of transcriptional levels within these domains can be measured via RNAseq. Chromosome interactions will be examined locally by Hi- C analysis (aim 1) and stiffness will be measured globally by micromanipulation (aim 2). Fluorescent in situ hybridization will be used to verify the intra- and inter-chromosome interaction found in Hi-C map. Hi-C, single- cell RNAseq, and micromanipulation will be introduced to reveal the 4D meiotic genome reorganization and sexual dimorphism of transcriptome and chromatin folding in aim 3. The approach is innovative because multiple advanced methods including Hi-C, single-cell RNAseq, micromanipulation, nano-newton force measurement, and quantitative immunocytology will be integrated to generate a more complete picture of meiotic chromosomes. The proposed research is significant because it is expected to provide a deeper understanding of chromosome structure and how the structure varies in different stages and genders. Ultimately, insights from these studies will help us develop diagnosis and treatment for infertility, miscarriage, and birth defects.