Oct4 and epigenetic regulation of stem cell pluripotency

描述（由申请人提供）：胚胎干细胞（ESCs）具有多能性，在再生医学中具有治疗潜力。ESC的多能性是由以Oct4、Sox2和Nanog为中心的一系列转录因子以及一些表观遗传调控因子控制的。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，用于蛋白质复合物的亲和纯化（称为bioSAIP）和染色质免疫沉淀（称为bioChIP）。这些方法使我们能够描绘出Nanog周围的蛋白质相互作用网络，即Nanog相互作用组。Nanog相互作用组富含包括Oct4在内的转录因子，这些转录因子在干细胞多能性、体细胞重编程和早期发育中起着关键作用。它还包括几种表观遗传调控途径，包括polycomb - suppression complex 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，并发现了ESCs中包含Rybp而不包含色盒蛋白（Cbxs）的独特PRC1复合物。本研究的总体目标是利用我们完善的蛋白质组学方法结合质谱法，在小鼠ESCs中建立以oct4为中心的包含PRC1相互作用组的“表观遗传相互作用组”，并阐明PRC1抑制对干细胞维持的分子机制。我们的假设是，以oct4为中心的“表观遗传相互作用组”将多个关键的表观遗传途径与Nanog相互作用组联系起来，并包含一个独特的PRC1抑制来维持ESC的身份。拟开展的研究包括：1)剖析干细胞多能性表观遗传调控的生化基础；2)在ESCs中建立PRC1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，而且将阐明干细胞多能性的基本特性和体细胞重编程的过程。此外，它将为探索干细胞多能性的表观遗传机制和寻找干扰细胞命运变化的表观遗传途径提供框架。

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.