Oct4 and epigenetic regulation of stem cell pluripotency

描述(申请人提供)：胚胎干细胞(ESCs)是多能的，在再生医学中具有治疗潜力。ESC的多能性由一组以Oct4、Sox2和Nanog为核心的转录因子以及一些表观遗传调控因子共同调控。为了分析ESCs多能性的分子基础，我们开发了一种体内生物素化策略，用于亲和纯化蛋白质复合体(称为BioSAIP)和染色质免疫沉淀(称为生物芯片)。这些方法使我们能够描绘出围绕Nanog的蛋白质相互作用网络，即Nanog相互作用组。Nanog相互作用组高度富含包括Oct4在内的转录因子，Oct4在干细胞多能性、体细胞重新编程和早期发育中发挥关键作用。它还包括几个表观遗传调控通路，包括多梳抑制复合体1(PRC1)。我们的初步数据证实了Oct4和PRC1蛋白之间的内源性相互作用，并在ESCs中发现了独特的PRC1复合体，这些复合体包含RYBP，但不包括染色框蛋白(CBX)。这项研究的总体目标是使用我们成熟的蛋白质组学方法结合质谱仪建立以Oct4为中心的“表观遗传相互作用组”，包括小鼠ESCs中的PRC1相互作用组，并阐明PRC1抑制干细胞维持的分子机制。我们的假设是，以Oct4为中心的“表观遗传交互作用组”将多条关键的表观遗传途径连接到Nanog交互作用组，并包含一个独特的PRC1抑制物，以维持ESC的身份。拟开展的研究包括：1)剖析干细胞多能性的表观遗传调控的生化基础；2)建立胚胎干细胞中PRC1的相互作用组；3)破译PRC1在靶基因调控和干细胞维持中的作用机制。这些研究不仅将导致新的多能性因子的发现，而且还将阐明干细胞多能性的基本特性和体细胞重新编程的过程。此外，它将为探索干细胞多能性的表观遗传机制和找到扰乱细胞命运变化的表观遗传途径的方法提供一个框架。

DESCRIPTION (provided by applicant): Embryonic stem cells (ESCs) are pluripotent and have therapeutic potential in regenerative medicine. The pluripotent ESC identity is governed by a set of transcription factors centered on Oct4, Sox2 and Nanog together with a number of epigenetic regulators. To dissect the molecular basis for the pluripotency of ESCs, we have developed an in vivo biotinylation strategy for affinity purification of protein complexes (dubbed bioSAIP) and chromatin immunoprecipitation (dubbed bioChIP). These approaches have allowed us to delineate a protein interaction network surrounding Nanog, i.e., the Nanog interactome. The Nanog interactome is highly enriched for transcription factors including Oct4, which plays critical roles in stem cell pluripotency, somatic cell reprogramming and early development. It also encompasses several epigenetic regulatory pathways including Polycomb-repressive complex 1 (PRC1). Our preliminary data confirmed endogenous interaction between Oct4 and PRC1 proteins and uncovered unique PRC1 complexes in ESCs that comprise Rybp but not chromobox proteins (Cbxs). The overall objective of the proposed research is to use our well-established proteomics approaches coupled with mass spectrometry to establish the Oct4-centered "epigenetic interactome" encompassing the PRC1 interactome in mouse ESCs, and to elucidate molecular mechanism of PRC1 repression for stem cell maintenance. Our hypothesis is that the Oct4-centered "epigenetic interactome" links multiple critical epigenetic pathways to the Nanog interactome and comprises a unique PRC1 repression to maintain ESC identity. The proposed studies are: 1) to dissect the biochemical basis for epigenetic regulation of stem cell pluripotency; 2) to establish the PRC1 interactome in ESCs; and 3) to decipher the mechanism of PRC1 function for target gene regulation and stem cell maintenance. These studies will not only lead to the discovery of novel pluripotency factors, but also illuminate the fundamental properties of stem cell pluripotency and the process of somatic cell reprogramming. Furthermore, it will provide a framework for exploring epigenetic mechanisms for stem cell pluripotency and finding ways to perturb epigenetic pathways for cell fate changes.