Micromechanical basis of meiotic chromosome condensation and architecture

摘要 染色质折叠是将DNA分子折叠10,000倍进入生殖细胞的关键一步，但到底是如何 减数分裂染色质折叠以及时空折叠如何影响转录、染色体配对和 重组在很大程度上仍然是个谜。需要进行同源染色体配对和重组 以实现准确的染色体分离。同源染色体的错误分离是导致 流产和出生缺陷(如唐氏综合症)。最近有三家报纸报道称，高阶 染色质组织/结构域动态地塑造重组景观和生殖系转录本。 这些戏剧性的染色质重组依赖于染色质轴，因为区域边界蛋白 (例如，粘附素)位于轴上。目前尚不清楚的是1)减数分裂染色体轴是如何 有助于减数分裂基因转录、同源染色体配对和染色体僵硬 染色质重组；以及2)减数分裂染色体结构动态是否与性别和阶段有关。 具体的。我们的长期目标是破译减数分裂基因组组织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总体假设 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相关的方式折叠。为了验证这一假设，我们将研究三个问题 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)染色体刚性和通过调节全局 染色质折叠。3)揭示减数分裂转录本和染色质的时间和性别差异 组织。方法：在目标1中，将检测空间域中的局部染色质重组 染色体构象捕捉(Hi-C)接触图及其转录水平的相应变化 可以通过RNAseq来测量。染色体的相互作用将通过Hi- C分析(目标1)和硬度将通过微操作进行全球测量(目标2)。原位荧光 杂交将被用来验证在Hi-C图谱中发现的染色体内和染色体间的相互作用。嗨-C，单人- 细胞RNAseq，以及微操作将被引入以揭示4D减数分裂基因组重组和 Aim 3转录组和染色质折叠的性别二型性。该方法具有创新性，因为 包括Hi-C、单细胞RNAseq、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结合在一起，以产生更完整的图像 减数分裂染色体。这项拟议的研究意义重大，因为它有望提供更深层次的 了解染色体结构以及染色体结构在不同阶段和不同性别中的变化。 最终，来自这些研究的见解将帮助我们开发不孕不育、流产、 和先天缺陷。

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.