NCOR和SMRT抑制复合物在调控多能干细胞上的机制研究

Pluripotent stem cells have two major states: naïve and primed. Although these two cells share the property of pluripotency, they have their own intrinsic mechanisms of self-renewal with significant differences in differentiation potential. In principal, naïve pluripotent stem cells have a higher differentiation potential compared to the primed cells. In this regard, compared to the well-established human primed pluripotent stem cells, human naïve pluripotent stem cells hold greater prospect in human diseases research and regenerative medicine. Yet, human naïve stem cells remain something of a problem. Whilst mouse naïve cells are well characterized, the human naïve cells derived to date remain heterogeneous and are incompletely naïve when compared to the mouse naïve cells. Additionally, there are many competing protocols, and there remains considerable argument over which protocol is correct and gives genuine naïve human pluripotent cells. Consequently, how to establish and maintain human naïve pluripotent stem cells is still a problem. To this end, understanding the mechanisms of the cell fate conversion from the primed to the naïve state by exploring the obstacles in this process may provide a promising approach. In this proposal, we utilize the well-established mouse epiblast stem cells (EpiSCs) and embryonic stem cells (ESCs) as models of primed and naïve cells respectively, to study the conversion from the primed to the naïve state. We focus on the co-repressors that regulate this transition, especially the role of NCOR and SMRT co-repressors in the regulating EpiSCs and the cell conversion from EpiSCs to ESCs. We also explore the epigenetic barriers induced by NCOR and SMRT co-repressors in the process of cell conversion. The lessons we learn in the mouse system will then be applied to attempt to establish a more homogeneous human naïve pluripotent stem cells by clearing the epigenetic barriers erected by NCOR and SMRT.

多能干细胞可分为原始态和始发态两种状态，虽然这两种状态的干细胞都具有多能性，然而它们在各自的自我更新机制以及分化潜能上都存在明显的差异。一般来讲，原始态的多能干细胞具有更高的分化潜能，在疾病模型研究和再生医学领域，人的原始态多能干细胞也具有更广泛的前景。目前，国际上对于建立原始态人的多能干细胞研究尚处在初级阶段，来自不同实验室尝试建立的人的原始态多能干细胞方法各异，形态参差不齐，且细胞维持的花费也较高。因此，深入理解干细胞始发态向原始态转变的机制，并发掘和克服转变过程中的障碍，可成为解决这一科学命题的重要手段。本项目拟采用小鼠始发态EpiSC细胞和原始态ESC细胞之间的细胞命运转换为模型，研究转录抑制复合物NCOR和SMRT在调控始发态EpiSC细胞自我更新以及促使EpiSC细胞向ESC细胞转化过程中的重要作用，从而为建立人的原始态多能干细胞打下坚实基础。

该项目主要围绕小鼠多能干细胞的两种状态，即始发态（primed）和原始态（naive），进行细胞间命运转换的研究。基于我们的研究成果表明抑制复合物NCOR和SMRT在体细胞重编程为iPS细胞中发挥了重要的作用。但是它在始发态的多能干细胞诱导变成原始态的多能干细胞中的作用依然你不是很清楚。我们首先通过一系列的探索，最终通过添加两个小分子化合物建立了一套高效诱导EpiSC的过程，使携带有Oct4-GFP原始态报告基因的多能干细胞变成GFP-的始发态多能干细胞。然而，我们的结果Ncor2-/-相对Ncor2f/f的细胞的RNA测序结果并没有明显的趋势。而我们利用shRNA敲降的方法发现shRNA敲降NCOR或者SMRT后能有效抑制原始态的ESC细胞向始发态的EpiLC细胞分化。进而我们利用CRISPR/Cas9基因编辑工具在OG2-ESC细胞中重新构建了Ncor1-/-和Ncor2-/-的ESC细胞系，以期有重大发现。NCOR和SMRT作为抑制复合物在调控细胞命运中的主要作用方式是通过调控目的基因的表观遗传学修饰来发挥作用的，而多能干细胞原始态和始发态之间的命运转换依赖的也是表观遗传学重塑，为了更好地研究表观遗传调控多能干细胞原始态和始发态之间的命运转换作用机制，我们设计了一套基于CRISPR/Cas9的sgRNA文库，该文库涵盖了细胞内所有表观遗传相关的修饰酶和调控因子。在原始态向始发态诱导分化的系统中，我们发现20个仍然保持GFP positive的细胞团，后续验证发现Bromodomain Containing 8（BRD8）能够促进EpiSC向ESC的转变，利用shRNA敲降Brd8以及二代测序技术，蛋白相互作用组学发现BRD8能够促进naïve 标志基因的表达，是通过与SMARCA5相互与作用影响基因表达。最后我们推测Brd8与SMARCA5在EpiSC中相互作用来影响基因组的开放性，在ESC中与Sox2相互作用来影响多能性基因的表达。