Neurog1介导H3K4me3/H3K27me3表观遗传修饰对小鼠海马神经干细胞调控作用及机制研究

Histone modification, as one of the epigenetic regulations, participates in orchestrating multiple biological processes, like mammalian embryo development and DNA repair. Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) are reported to play an important role in regulating the bio-processes of different species of stem cells. However, little is known about their effects on neural stem cells (NSCs). Our previous research showed that H3K4me3 and H3K27me3 bared cell-type specific distributions, respectively. Robust enrichment of H3K4me3 was observed in the subgranular zone (SGZ), while high levels of H3K27me3 were mainly presented in the granule cell layer (GCL). Generally, in mouse dentate gyrus (DG), neurogenesis occurs in SGZ while GCL mainly contains granule cells which are a type of mature neuron. We also found that the number of cells with high levels of H3K4me3 was decreased during mouse hippocampus development; on the contrary, cells with high levels of H3K27me3 were gradually increased with the development. Furthermore, both H3K4me3 and H3K27me3 have impacts on proliferation and differentiation of NSCs. Moreover, H3K4me3 and H3K27me3 also can affect the expression of Neurog1 which is the key gene of regulation and control in NSCs proliferation and differentiation. These phenomena prompted that epigenetic H3K4me3 and H3K27me3 might act as molecular “switch” to regulate proliferation and differentiation of NSCs through an effect on the Neurog1 gene activation in the mouse hippocampus. In this study, we plan to modulate the levels of H3K4me3 or H3K27me3 by editing the expressions of lysine demethylases (KDM), to detect the proliferation and differentiation changes of NSCs, and then clearly understand the function of H3K4me3 and H3K27me3 in NSCs. Previously, direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from a small amount of cells. Thus, colleagues of our research group successfully developed a micro-scale chromatin immunoprecipitation and sequencing (μChIP-seq) method, which could be used to measure histone modifications from a small amount of cells. The technique will be further applied in systematically profiling genome-wide dynamic changes of H3K4me3 and H3K27me3 at different stages of hippocampus development in SGZ and GCL. Screen out and validate that H3K4me3/H3K27me3 affect proliferation and differentiation of NSCs by regulating the expression of Neurog1. Our study may illustrate a novel epigenetic regulatory mechanism in the regulation of NSCs and provide a new strategy and a novel potential target for the treatment of nervous system disorders.

三甲基化修饰的H3组蛋白第4和第27位赖氨酸（H3K4me3/H3K27me3）调控多种干细胞生物进程，但其在神经干细胞（NSCs）中的作用及机制缺乏研究。预实验发现H3K4me3/H3K27me3调控小鼠海马NSCs增殖分化，并可影响神经发生关键调节基因Neurog1表达。提示H3K4me3/H3K27me3可能通过影响Neurog1活性，进而调控NSCs。本研究通过改变H3K4me3或H3K27me3修饰水平，观察细胞增殖分化变化，明确其在NSCs中的作用。应用μChIP-seq结合激光捕获显微切割，测序分析H3K4me3/H3K27me3，筛选确认Neurog1为靶基因，并进行验证，明确“H3K4me3/H3K27me3-Neurog1-NSCs调控”关系网络，阐明其作用机制。通过本研究实施有望揭示一种新的小鼠海马神经发生表观遗传调控机制，为神经系统疾病治疗提供新思路及潜在治疗靶点。

海马在高级神经活动中发挥关键作用，其结构和功能异常是许多重大神经精神类疾病的病理及病理生理学基础。海马齿状回颗粒细胞下层终生存在神经干细胞（NSCs），多种病理刺激可干扰这些NSCs正常生物学行为，引起海马结构和功能异常，这是多种神经系统疾病的发病机制之一。因此，阐明NSCs增殖分化调控机制具有极为重要的科学和现实意义。既往研究表明，组蛋白甲基化修饰在个体神经系统发育调控中发挥关键作用，但H3K4me3/H3K27me3在NSCs中的作用及机制尚未可知。本研究表明小鼠海马神经发育过程中，H3K4me3和H3K27me3存在动态变化，NSCs展现出高H3K4me3修饰水平，而神经元则具有高H3K27me3修饰水平。提示H3K4me3/H3K27me3参与调控NSCs增殖分化。随后我们通过抑制海马NSCs中组蛋白赖氨酸去甲基酶5B（KDM5B）表达，升高H3K4me3水平；抑制组蛋白赖氨酸去甲基酶6A（KDM6A）表达，升高H3K27me3水平。实验结果显示，升高H3K4me3水平可促进NSCs增殖和神经分化。而升高H3K27me3水平则作用相反，即抑制NSCs增殖和神经分化。但是无论改变H3K4me3或H3K27me3均不影响NSCs胶质分化。RNA-seq表明受H3K4me3/H3K27me3调控的基因与神经系统发育密切相关。综合生信分析、qRT-PCR和ChIP-qPCR结果提示Neurog1为H3K4me3/H3K27me3调控NSCs增殖分化的关键靶基因。功能拯救实验表明Neurog1可消除由于H3K4me3/H3K27me3修饰水平改变所导致的NSCs增殖分化变化。本研究成功建立“H3K4me3/H3K27me3-Neurog1-NSCs调控”关系网络，展现了一种全新的小鼠海马NSCs表观遗传修饰调控机制，对研究NSCs调控和神经系统损伤修复研究，都具有一定价值。