减数分裂同源重组时期染色质三维空间结构的高分辨研究

The successful analyses of three-dimensional spatial chromatin structures in nuclei of multiple species revealed that high order chromatin organization is closely related with many key biological processes such as transcription regulation, mitosis, cellular senescence, etc. Homologous recombination during meiotic prophase is highly conserved and is of great significance for genetic diversity of gametes and progeny’s adaptability. However, the three-dimensional spatial chromatin structure during meiotic homologous recombination phase still remains as a mystery. The applicant’s group has successfully isolated pachytene spermatocytes of Mus musculus and crescent micronuclei of Tetrahymena thermophila, and also obtained genome-wide chromatin interaction map of crescent micronuclei of Tetrahymena thermophila through Hi-C, the genome-wide chromatin conformation capture method. Based on these preliminary data, the application will use these two model systems which are evolutionary far apart but both are during homologous recombination phases, to analysis three-dimensional spatial chromatin structure of meiotic homologous recombination phase of both mammal and protozoan, utilizing indirect method - (Hi-C) for capturing chromatin spatial conformation and direct method - super resolution imaging (STORM) for observing three-dimensional spatial chromatin structure, and the active or repressive epigenetic states of chromatin, in high resolution. We are also going to study the conservation and diversity of the three-dimensional spatial chromatin structure from evolutionary viewpoint. Our project is of great significance for understanding the molecular mechanism of meiotic homologous recombination, and may also provide new strategy into intervention of meiosis related diseases.

多个物种染色质三维空间结构的成功解析揭示其与转录调控、有丝分裂、细胞衰老等生物学过程密切相关。减数分裂前期同源重组过程高度保守且对遗传多样性意义重大，但此时期的染色质三维结构亟待解析。申请人已成功分离处于减数分裂同源重组时期的小鼠粗线期精母细胞和嗜热四膜虫新月期小核，并已通过高通量染色质构象捕获技术(Hi-C)获得后者的全基因组染色质相互作用图谱。在此基础上，本申请拟以两种进化上较远却又同时处于减数分裂同源重组时期的模型—哺乳动物小鼠粗线期精母细胞和原生动物嗜热四膜虫新月期小核为研究对象，利用Hi-C间接检测三维染色质构象和利用超高分辨率显微成像技术(STORM)直接观察染色质三维结构相结合，深度解析减数分裂同源重组时期染色质三维空间结构和活化/抑制等染色质表观遗传状态，从进化上研究其保守性和多样性。本项目对理解减数分裂同源重组时期的分子机制意义重大，并可为干预减数分裂相关疾病提供新策略。

减数分裂前期同源重组过程高度保守且对遗传多样性意义重大，但此时期的染色质三维结构亟待解析。我们通过Hi-C等技术对小鼠精子发生过程中处于减数分裂阶段的粗线期精母细胞（pacSC）以及减数分裂前后其他的几种精子细胞进行了染色质高级结构的解析。我们也通过 HiChIP 技术对处于减数分裂过程中的嗜热四膜虫小核的染色质高级结构进行了分析。结果表明小鼠的粗线期精母细胞中基本不存在哺乳动物分裂间期细胞中典型的染色质高级结构TADs 和 loops，另一典型结构 A/B compartments 仍然存在，但强度也有所减弱。结合ChIP-seq， ATAC-seq 以及 RNA-seq 等数据，我们发现TADs 和 loops 在粗线期的丢失不依赖于 CTCF 或 cohesin 在染色体上结合状态的改变，提示粗线期细胞中存在非 TADs和loops 依赖的基因转录调控机制。对嗜热四膜虫减数分裂小核染色质高级结构的研究发现其新月期小核中存在染色体内以及染色体间的‘X’型染色质相互作用，提示其在减数分裂过程中亦发生了类似小鼠中TADs结构丢失的过程。此外，利用超高分辨率显微镜STORM，我们对小鼠减数分裂粗线期的精细胞进行了免疫荧光染色及成像。目前我们获得了转录活化（H3K4me3）及转录抑制（H3K9me3）的染色质标记以及SYCP3（用来标记粗线期染色体）的超高分辨率的空间定位图像，其他的染色质标记物还在进行中。本项目利用Hi-C间接检测三维染色质构象和利用超高分辨率显微成像技术(STORM)直接观察染色质三维结构相结合，深度解析减数分裂同源重组时期染色质三维空间结构和活化/抑制等染色质表观遗传状态，从进化上研究其保守性和多样性，对理解减数分裂同源重组时期的分子机制意义重大，并可为干预减数分裂相关疾病提供新策略。