Fam60a在小鼠胚胎干细胞多潜能性维持和胚胎早期发育过程中的分子机制研究

Mouse embryonic stem cells (mESCs) are pluripotent stem cells isolated from the inner cell mass of a blastocyst, an early-stage preimplantation embryo. The POU transcription factor Oct-4 (encoded by Pou5f1) is essential for the maintainance of mESCs. The precise level of Oct4 governs three distinct fates of mESCs. Loss of the expression of Oct4 induces differentiation into trophectoderm. In contrast, a less than two-fold increase in expression causes differentiation into primitive endoderm and mesoderm. However, little is known how the expression of Oct4 is negatively regulated. Our preliminary data shows that Fam60a interacts with HDAC2 and is required for maintaining self-renewal and pluripotency of mESCs through negative regulating Oct4 expression. In this proposal, we are going to investigate the detail molecular mechanism of how Fam60a regulates genes transcription in mESCs, which includes: 1) The components of the Fam60a-containing protein complex and how they interact with Fam60a; 2) How Fam60a regulates the chromatin status and gene transcription in mESCs; 3) How Fam60a regulates the differentiation of mESCs and development of early-stage mouse embryo. The accomplishment of these works will help us not only get more insight of the molecular mechanism of maintaining mESCs self-renewal and pluripotency but also the development of the early-stage mouse embryo.

小鼠胚胎干细胞(mESCs)是分离自囊胚内细胞团，在体外培养建立得到的多潜能性细胞系。Oct4对mESCs的保持很重要，它精确的表达控制着mESCs分化命运。Oct4表达下调会使mESCs分化为滋养外胚层；相反，Oct4表达升高可诱导mESCs分化成原始内胚层和中胚层。但在mESCs中Oct4的负向调控机制所知甚少。我们的前期工作发现Fam60a是mESCs维持的必需基因，其与HDAC复合物有相互作用，负向调节包括Oct4在内的基因表达。在这个课题中我们将：1)确定在mESCs中Fam60a参与蛋白复合物的组件以及它们与Fam60a相互作用的区域；2)确定Fam60a在mESCs中如何调节染色体组蛋白修饰状态；3)确定Fam60a如何调节mESCs的分化过程以及在小鼠胚胎早期发育过程中的功能。我们的研究不仅可以理解Oct4负向调控机制，而且可以更深入的理解mESC的保持和小鼠胚胎早期发育。

解析新型干性调控因子及机制对细胞干性维持至关重要。先前，我们通过OSN复合物ChIP-seq结果分析出Fam60a是该复合物直接下游，并通过功能缺失实验鉴定其为鼠胚胎干细胞新型干性调控因子。在项目实验过程中，通过蛋白纯化技术与质谱联用，我们鉴定出Fam60a参与Sin3a复合物调控mESC细胞基因表达。通过ChIP-seq与RNA-seq联合分析，我们确认了Fam60a与Sin3a复合物结合在mESC细胞H3K4me3区域，并维持细胞谱系基因与TGF-beta信号通路相关基因的抑制从而维持mESC细胞的干性与自我更新。我们更是制作了Fam60a条件敲除小鼠，发现Fam60a缺失抑制了小鼠肝的再生，机制上Fam60a的缺失抑制了自噬相关基因的激活。通过本项目的实施，阐明了Fam60a在胚胎干细胞中维持干性和自我增殖的机制，其条件敲除小鼠更是可作为肝再生相关模型的研究，为有干性相关的组织器官研究奠定了基础。