Novel differentiation repressor module in human ES cells

描述（由申请人提供）：尽管在解读胚胎干细胞多能性方面取得了显著进展，特别是在鼠模型中，但在理解人胚胎干细胞（hESC）如何调节多能性状态方面存在根本性差距。我们的长期目标是破译允许不受限制的hESC增殖的调控网络的结构，同时保留它们形成人体中发现的细胞类型的完整库的潜力。为了确定这个网络的组成和功能，我们首先通过基于shRNA的功能筛选来寻找hESCs的转录调控因子。鉴定了维持多能状态所需的几个因子，包括三个新基因，BCOR，ZFP 42和ZNF 649，其功能是抑制hESC分化。虽然不同hESC系对ZFP 42和ZNF 649的需求不同，但BCOR的消耗导致所有测试细胞系的快速分化，表明 BCOR是“启动”多能状态的核心组成部分。由于只确定了几个核心因素，因此研究变革和组织振兴巴塞尔公约是一个高度优先事项。本申请的目的是（1）阐明BCOR抑制分化的机制方面和（2）在全基因组范围内鉴定特异性hESC调控机制。我们的中心假设，制定的基础上的初步数据和其他细胞系统中的BCOR的工作，是BCOR抑制hESC的分化，招募KDM 2 B和RING 1A/B模块，在靶位点产生抑制性染色质足迹。该假设将通过以下具体目标进行检验：1）确定BCOR复合物如何在hESC中抑制其靶基因，以及2）确定BCOR复合物如何被募集至靶基因。此外，我们提出3）全面鉴定调节hESC中多能性的调控途径。在第一个目标下，将结合生物化学、功能和基因组学方法来确定（1）BCOR靶基因和hESC中BCOR复合物的性质;（2）KDM 2 B和RING 1A/B对复合物功能的相对贡献;以及（3）在BCOR靶位点维持独特表观遗传特征的机制。在第二个目标下，将从hESC中纯化BCOR复合物并使用质谱法进行测序，以确定与BCOR物理相互作用的DNA-/染色质结合蛋白，然后测试其将复合物募集到靶标的能力。此外，将研究通过典型的PRC依赖性机制和通过长非编码RNA的募集。在第三个目标下，将使用无偏的全基因组shRNA筛选来鉴定hESC多能性的正和负调节因子。我们的方法是创新的，因为它利用最先进的新技术来获得对hESC调控复杂性的新见解。所提出的研究是重要的，因为它有望垂直推进和扩展对hESC细胞如何控制多能状态的理解。这些知识有可能提高人胚胎干细胞的维持和分化，这是治疗各种疾病的新方法和创新方法的关键步骤。

DESCRIPTION (provided by applicant): Despite the remarkable progress made in deciphering embryonic stem cell pluripotency, particularly in murine models, there is a fundamental gap in understanding how human embryonic stem cells (hESCs) regulate the pluripotent state. Our long-term goal is to decipher the architecture of the regulatory network that allows for un- restricted hESC proliferation while preserving their potential to form the full repertoire of cell types found in the human body. To define the composition and function of this network, we first searched for transcriptional regulators of hESCs through shRNA-based functional screen. Several factors were identified that are required for the maintenance of the pluripotent state including three novel genes, BCOR, ZFP42 and ZNF649, that function to repress hESC differentiation. While the requirements for ZFP42 and ZNF649 varied among different hESC lines, depletion of BCOR resulted in rapid differentiation in all cell lines tested indicating that BCOR is a core component of the "primed" pluripotent state. As only a few core factors have been identified, studying BCOR is a high priority. The objectives in this application are (1) to elucidate the mechanistic aspects of differentiation repression by BCOR and (2) to identify-specific hESC regulatory mechanisms genome-wide. Our central hypothesis, formulated based on the preliminary data and prior work on BCOR in other cell systems, is that BCOR suppresses differentiation of hESCs by recruiting KDM2B and RING1A/B modules to generate repressive chromatin footprints at the target sites. This hypothesis will be tested through the following specific aims: 1) Determine how the BCOR complex represses its target genes in hESCs and 2) Determine how the BCOR complex is recruited to target genes. In addition, we propose to 3) comprehensively identify regulatory path- ways that modulate pluripotency in hESCs. Under the first aim, biochemical, functional and genomics approaches will be combined to determine (1) BCOR target genes and the nature of the BCOR complex(es) in hESCs; (2) relative contributions of KDM2B and RING1A/B to the complex function; and (3) the mechanism that maintains unique epigenetic signatures at BCOR target sites. Under the second aim, the BCOR complex will be purified from hESCs and sequenced using mass spectrometry in order to define DNA-/ chromatin binding proteins that physically interact with BCOR which then will be tested for their ability to recruit the complex to targets. In addition, recruitment via the canonical PRC-dependent mechanism and via long non-coding RNAs will be investigated. Under the third aim, positive and negative regulators of hESC pluripotency will be identified using an unbiased whole-genome shRNA screen. Our approach is innovative, because it utilizes novel state-of-art technologies to obtain new insights into the regulatory complexity of hESCs. The proposed re- search is significant, because it is expected to vertically advance and expand understanding of how hESCs cells control the pluripotent state. Such knowledge has the potential to enhance hESC maintenance and differentiation - critical steps for new and innovative approaches to treatment of a variety of diseases.