组蛋白甲基化转移酶EZH2在小鼠精原干细胞增殖和分化过程中的作用机制研究

With the aggravation of environmental pollution and lifestyle changes, the prevalence of male infertility in the world is rising. Epigenetic modifications characterized by DNA methylation, histone modifications, and chromatin remodeling are important regulators in a number of biological processes, including spermatogenesis. The epigenetic marks are highly dynamic and can adapt, or be altered in response to the internal or external environment. Researches on epigenetic mechanisms will help us to solve male infertility. In mammalian, spermatogonial stem cells (Spermatogonial Stem Cells, SSCs), through their proliferation and differentiation, support spermatogenesis, which is governed by unique epigenetic marks and processes. However, the epigenetic mechanisms in controlling spermatogenesis remain unknown. Enhancer of Zeste homlog 2 (EZH2), a catalytic subunit of epigenetic regulator Polycomb repressive complex 2 (PRC2), has been shown to be a key regulator in controlling cellular proliferation and differentiation. EZH2 is a histone methyltransferase that not only methylates histone H3 on Lys 27 (H3K27me3) but also interacts with and recruits DNA methyltransferases to methylate CpG at certain EZH2 target genes to establish firm repressive chromatin structures, contributing to tumor progression and the regulation of development and lineage commitment both in embryonic stem cells (ESCs) and adult stem cells. However, spermatogenesis is a very complex process. As a result, an efficient in vitro culture system for producing functional sperm will facilitate the in-depth study of the mechanism of spermatogenesis Our research group has been established the system for SSC induction into functional sperm. We will intend to use this in vitro system combined with the technologies of EZH2 gene knockout, SSC function analysis and high-throughput bioinformatics analysis to ascertain the mechanisms of EZH2 as an epigenetic regulator in controlling spermatogonial proliferation and differentiation. This project will provide the adequate theoretical basis for human translational medicine applications and male infertility treatment.

随着环境污染加重,生活模式的改变，男性不育的患病率在世界范围内呈上升趋势。表观遗传修饰可根据机体所处的内外环境的改变，来调节基因的表达，所以表观遗传学的研究将有助于男性不育问题的解决。精原干细胞（Spermatogonial Stem Cells，SSCs）通过增殖和分化支持着体内精子的发生。但表观遗传在精子发生中的调控机制仍然不清楚，研究表明表观遗传因子EZH2在细胞的增殖和分化中扮演了非常重要的角色。但是，精子发生在体内是一个十分复杂的过程，因此一个高效的精子体外分化模型的建立将有助于精子发生机制的深入研究。本研究组已建立了小鼠SSCs体外分化为功能性精子的分化系统，所以，本项申请拟利用已建立的SSCs分化系统，通过干扰EZH2基因、SSCs功能分析以及高通量生物信息学分析，来探明EZH2在SSCs增殖和分化过程中的调控机理，为男性不育的治疗提供充分的理论依据。

我们证明了在精子发生中，EZH2并不是通过调节组蛋白H3k27e的甲基化数量来发挥作用，而是作为转录因子调节SSCs相关基因（如NGN3, KIT）来调控精原干细胞的增殖和分化。其次，我们发现精原细胞（progenitors）的增殖可以促进细胞分化，促进精子发生。EZH2的敲除可以促进分化精原细胞的增殖并促进其向精子方向分化。在EZH2敲除小鼠中发现，细胞数量受睾丸空间占位的调节，所以EZH2在促进分化细胞数量增多的同时，也会通过体内凋亡机制的激活，来调节精子细胞的数量。综上，EZH2在精子发生中的调节机制中是作为转录因子来调控精子发生，EZH2的敲除可以促进精子发生。与EZH2在肿瘤中的调节机理不同。EZH2的突变或者过表达与多种类型癌症相关，如乳腺癌、前列腺癌、黑色素瘤和膀胱癌等。因为异常激活的EZH2可以抑制抑癌基因的正常表达，所以抑制EZH2的活性可以使肿瘤生长变慢。结合我们的实验结果，SSCs除了可以作为精子发生的模式细胞，在肿瘤发生机制的研究中，也可以作为参考模型进行研究，另外EZH2抑制剂在肿瘤患者或男性不育患者中的应用，有可能保护男性的生育力，值得今后的研究进行进一步的探讨。