组蛋白H3K4甲基化在鸡ESC向雄性生殖干细胞分化中的调控机制

The plasticity of spermatogonial stem cells makes it possible to replace ESCs for therapeutic and genetic modification without involving ethical and immunological rejection, which has aroused great concern in biology. However, two difficulties in stem cell differentiation has not yet broken which is "low differentiation efficiency and induce cell function immature". It is difficult to obtain a large number of such cells in vitro to meet the needs, because the mechanism is poorly understood. People in the process of iPS encountered the same problem, why the same genetic material of the cell group, cell lineage fate is different, some of cells will be directed into differentiated cells? The preliminary study found that the formation of male stem cells in the process does not change the DNA sequence under the occurrence of gene expression differences. Indicating that the apparent genetic modification involved in the regulation, then what is the mechanism? To this end, the project is intended to use modern genomics and proteomics techniques to explore the histone methylation and its modified enzyme regulation of ESC to male reproductive stem cell differentiation mechanism; On the basis of this, look for the relationship between histone methylation and differential gene response; According to the results of the induction agent screening, the establishment of appropriate induction system, in order to clarify the mechanism of germ cell differentiation to provide a theoretical basis and provide a practical technical support for improving cell induction efficiency.

精原干细胞的可塑性使其能替代ESCs用于治疗和遗传修饰，而不涉及伦理和免疫排斥问题，故引起生物学界极大关注。然由于干细胞定向分化遇到了“分化效率低和诱导细胞功能不成熟”两大难点尚未突破，故体外难以获得大量该类细胞来满足需要。原因在于对相关机制知之甚少。人们在进行iPS的过程中遇到了同样的问题。为什么具有相同遗传物质的细胞群，细胞谱系命运不同，其中一些细胞会定向分化成生殖细胞？本课题前期研究发现，雄性干细胞形成过程中在不改变DNA序列情况下发生了基因表达的差异。说明表观遗传修饰参与了调控，那么机理是怎样的？为此，本项目拟将利用现代基因组学和蛋白质组学技术，探悉组蛋白甲基化及其修饰酶调控ESC向雄性生殖干细胞分化选择的机制；在此基础上寻找组蛋白甲基化与差异基因的呼应关系；依此结果对诱导剂进行筛选，建立适宜的诱导体系，为弄清生殖细胞分化机制提供理论依据，为提高细胞诱导效率提供实践技术支撑

为了解析组蛋白H3K4甲基化修饰在生殖细胞形成过程中的分子机制，本项目通过Western Blot以及免疫组化等方法确定H3K4me2为生殖细胞形成过程中的主要表观遗传修饰调控因子，并通过CHIP-seq分析发现H3K4me2在生殖细胞形成过程中经历先擦除后重建的过程，且重建的H3K4me2主要富集于基因的启动子区域，推测H3K4me2可通过调节基因的转录参与生殖细胞的形成过程。随后本项目通过qRT-PCR以及Western Blot等方法确定MLL2和LSD1为生殖细胞形成过程中的关键修饰酶，并通过体内外功能验证试验发现高水平的H3K4me2可以促进生殖细胞的形成。为了进一步明确H3K4me2调控生殖细胞形成的分子机制，本项目联合分析了前期转录组和CHIP-seq数据，结果显示在生殖细胞形成过程中H3K4me2对Wnt5A介导的Wnt信号具有潜在的调控作用，功能验证试验亦表明H3K4me2可通过Wnt信号调控生殖细胞的形成。在机制上，我们发现H3K4me2可通过Wnt信号激活二者共同的靶基因Lin28促进PGCs的形成。同时H3K4me2与Wnt信号可协同调控TDRD1调节SSCs的形成。