新型组蛋白翻译后修饰在人胚胎干细胞定向分化过程中的表观遗传机制

Histone lysine butyrylatino and crotonylation are two new types of histone modifications identified recently and evidences showed that they have unique epigenetic behaviours.Though it is well documented that histone modifications pay key roles in hESCs differentiation, nothing is known about the roles of the two new types of histone modifications in the process of hESCs directed differentiation, hindering the deep understanding to hESCs biology.In this proposal,using hESCs and directed differentiated hepatocytes as research subjects, with quantitive proteomics, epigenetic analysis with ChIP-Seq and bioinformatic analysis, and protein function investigation, we aim to identify the lysine butyrylation and crotonylation sites in histones and quantify the two modifications during hESCs directed differentiation.By investigating the enrichment pattern of the two modifications in whole genome, we will reveal the epigenetic regulative mechanism of hisone lysine butyrylation and crotonylation in hESCs differentiation.With peptide pull-down assay to screen the specific binding proteins to histone lysine butyrylation and crotonylation, hESCs differentiation process will be further understood by exploring the functions of the specific binding proteins.Collectively,studies in this proposal will expand our understanding of epigenetic mechanism in stem cell biology, which may provide new theory evidences to regulate hESCs directed differentiation.

组蛋白赖氨酸丁酰化与巴豆酰化修饰是最近鉴定的两种新型组蛋白修饰类型，初步的研究表明它们有着独特的表观遗传学行为。已知组蛋白修饰在干细胞分化过程中起着关键的作用，但对组蛋白赖氨酸丁酰化与巴豆酰化修饰在人胚胎干细胞定向分化过程中的功能仍一无所知，从而大大局限了对人胚胎干细胞定向分化过程的深入了解。本项目以人胚胎干细胞与定向分化的肝细胞为研究对象，通过定量蛋白质组学、结合ChIP-Seq及生物信息学的表观遗传分析与蛋白质功能研究，系统地鉴定人胚胎干细胞中组蛋白赖氨酸丁酰化与巴豆酰化修饰位点、定量分析人胚胎干细胞定向分化过程中这两种新型组蛋白修饰水平的变化、研究它们在干细胞定向分化过程中全基因组表观遗传调控机制以及特异性修饰结合蛋白在人胚胎干细胞分化过程中的功能。本研究将深化对人胚胎干细胞定向分化表观遗传机理的认识，从而为干细胞定向分化的人工干预提供新的理论依据。

我们首先建立了胚胎干细胞向肝细胞分化的实验体系和检测分化程度的实验体系；随后运用建立了定量蛋白质组学的技术路线和数据分析流程，对干细胞定向分化成肝细胞的过程中，建立了高通量的蛋白质丰度表达、组蛋白三种表观遗传密码（组蛋白乙酰化、组蛋白琥珀酰化、组蛋白巴豆酰化）与非组蛋白修饰（赖氨酸乙酰化、赖氨酸琥珀酰化、赖氨酸巴豆酰化）的定量图谱。定量蛋白质组学数据的生物信息学与生化分析表明，在干细胞分化过程中，TGF、JAK-STAT 、RAS 的信号通路被激活，而ECM相关的生物学过程被抑制；定量蛋白质修饰组学的数据分析表明，在分化过程中，整体上乙酰化与琥珀酰化修饰的水平都显著下调，但琥珀酰化修饰更多地富集在ER通路和能量代谢的通路中；并且琥珀酰化修饰调节了干细胞分化过程中的Warburg效应。作为更新型的赖氨酸巴豆酰化修饰，我们揭示出在干细胞分化过程中的组蛋白N端特异性修饰位点及修饰水平的改变，并揭示出巴豆酰化修饰在维持干细胞分化过程中基因组稳定性等全新的机制。上述研究成果在多维组学的层面上（蛋白质表达与蛋白质修饰）揭示出干细胞分化调整的一种全新机制，加深了对干细胞定向分化的机制认识，并为后继干细胞分化的人工干预乃至实际应用提供了全新的思路。