组蛋白甲基化调控ESC向雄性生殖干细胞分化选择过程中的作用机制

Spermatogonial stem cells could be used to replace embryonic stem cells for disease therapy and genetic modification since it has the merits of pluripotency and do not involve in ethical and immune rejection problem, therefore, it caused great concern in the biological community. However, "low efficiency of differentiation and functions immature" is the two major difficulties for the direct differentiation of embryonic stem cell; it is difficult to get a large number of such cells to meet the requirement in vitro. The reason is that little is known about the mechanisms. The people encountered the same problem when they make iPS. Why the cell populations have the same genetic material, but the cell lineage fate was such a different, some of the cells will be directed to differentiate into germ cells? The preliminary study of our lab found that male stem cells does not change DNA sequences during the formation of male germ cells, but the differences gene expression occurred. Which indicated the epigenetic modification was involved in the regulation process, but what is the mechanism? To this end, the project intends to ascertain histone methylation and its modified enzyme regulation for ES cell into male germ stem cell differentiation mechanism by genomic and proteomics technology, and analyze the relationship between the histone methylation and targeted gene. Finaly, the inducer was screened base on the above results and establishes the suitable induction system, so as to provide a theoretical basis for the clarifying the mechanism of germ cell differentiation and provide technical support for improving the cell induction efficiency.

精原干细胞的可塑性使其能替代ESCs用于遗传修饰和治疗疾病，而不涉及伦理和免疫排斥问题，故引起生物学界极大关注。然而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。

按照项目申请书及合同书的计划，2016年度-2017年度该课题的研究计划要点是：⑴．ESCs、PGCs、SSCs 的分离培养及流式细胞分选；⑵．ESCs、PGCs、SSCs的ChIP-seq测序分析；⑶．组蛋白H3K4me2 peak关联的靶基因和信号通路富集分析；⑷．SSCs形成过程中关键组蛋白甲基化修饰酶的筛选及功能验证；⑸．组蛋白H3K4me2在SSCs形成过程中的变化及功能验证；⑹．组蛋白H3K4me2 peak富集的关键信号通路在SSCs形成过程中的功能验证。该课题的研究按照合同书计划内容正常有序地进行，并提前完成全部研究计划。