Novel differentiation repressor module in human ES cells

描述(申请人提供)：尽管在破译胚胎干细胞多能性方面取得了显著进展，特别是在小鼠模型中，但在理解人类胚胎干细胞(HESCs)如何调节多能性状态方面仍存在根本差距。我们的长期目标是破译允许不受限制的hESC增殖的调控网络的架构，同时保留它们形成人体内发现的全部细胞类型的潜力。为了确定该网络的组成和功能，我们首先通过基于shRNA的功能筛选寻找hESCs的转录调控因子。已确定了维持多能性状态所需的几个因素，其中包括三个新的基因BCOR、ZFP42和ZNF649，它们具有抑制hESC分化的功能。虽然不同的hESC对ZFP42和ZNF649的需求不同，但BCOR的缺失导致了所有受试细胞株的快速分化，表明BCOR是启动的多能性状态的核心成分。由于只确定了几个核心因素，因此研究BCOR是一个高度优先的问题。本应用的目的是(1)阐明BCOR抑制分化的机制，(2)在全基因组范围内识别特定的hESC调控机制。我们的中心假设是基于初步数据和之前在其他细胞系统中关于BCOR的工作，即BCOR通过招募Kdm2b和RING1A/B模块在靶点产生抑制染色质足迹来抑制hESCs的分化。这一假说将通过以下具体目标进行验证：1)确定BCOR复合体如何在hESCs中抑制其靶基因；2)确定BCOR复合体如何被招募到靶基因。此外，我们建议3)全面确定调控hESCs多能性的途径。在第一个目标下，将结合生化、功能和基因组学方法来确定(1)BCOR靶基因和hESCs中BCOR复合体的性质；(2)Kdm2b和RING1A/B对复杂功能的相对贡献；以及(3)在BCOR靶点保持独特表观遗传特征的机制。在第二个目标下，将从hESCs中纯化BCOR复合体，并使用质谱仪进行测序，以确定与BCOR物理相互作用的DNA/染色质结合蛋白，然后将测试它们将复合体招募到靶点的能力。此外，还将调查通过规范的PRC依赖机制和通过长的非编码RNA进行的招募。在第三个目标下，将使用无偏见的全基因组shRNA筛选来识别hESC多能性的正负调控因子。我们的方法是创新的，因为它利用新的最先进的技术来获得对HESCs监管复杂性的新见解。这项拟议的研究意义重大，因为它有望在纵向上推进和扩大对hESCs细胞如何控制多能性状态的理解。这些知识有可能加强人类胚胎干细胞的维持和分化--这是治疗各种疾病的新的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.