Oct4 and epigenetic regulation of stem cell pluripotency

描述（由申请人提供）：胚胎干细胞（ESC）是多能的，在再生医学中具有治疗潜力。ESC的多能性是由一组以Oct 4、Sox 2和Nanog为中心的转录因子以及许多表观遗传调节因子共同控制的。为了剖析胚胎干细胞多能性的分子基础，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围绕Nanog的蛋白质相互作用网络，即，纳米互动基因组Nanog相互作用组高度富集了包括Oct 4在内的转录因子，Oct 4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-抑制复合物1（PRC 1）。我们的初步数据证实了Oct 4和PRC 1蛋白之间的内源性相互作用，并揭示了ESCs中包含Rybp但不包含染色体盒蛋白（Cbxs）的独特PRC 1复合物。该研究的总体目标是利用我们成熟的蛋白质组学方法结合质谱法建立以Oct 4为中心的“表观遗传相互作用组”，包括小鼠胚胎干细胞中的PRC 1相互作用组，并阐明PRC 1抑制干细胞维持的分子机制。我们的假设是，Oct 4为中心的“表观遗传相互作用组”链接多个关键的表观遗传途径的Nanog相互作用组，并包括一个独特的PRC 1抑制，以保持ESC的身份。拟议的研究包括：1）剖析干细胞多能性的表观遗传调控的生化基础; 2）建立ESC中的PRC 1相互作用组; 3）破译PRC 1在靶基因调控和干细胞维持中的功能机制。这些研究不仅将导致发现新的多能性因子，而且还阐明了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改变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.