Molecular control of pluripotency in humans

项目描述 尽管在破译小鼠模型中多能性的调节方面取得了重大进展，但仍然存在乐趣- 我们对人类胚胎干细胞如何维持多能状态的理解存在重大差距我们的长期目标 是破译允许hESC不受限制增殖的调控网络的结构， 保留它们形成人体中发现的全部细胞类型的潜力。全面 鉴定参与多能性维持的基因，我们已经进行了全基因组的shRNA筛选， hESC，并开始深入分析最重要的命中。我们专注于BCOR，它是 非典型的Polycomb抑制复合物1.1（PRC1.1）具有强烈的多能性丧失表型， 在多个hESC系中观察到了抑制。hESC中BCOR的缺失导致抑制性染色质的丢失， 关键发育位点和分化的起始。我们发现BCOR定义了一个新的亚型， PRC 1复合物具有不同的募集和抑制机制。新的BCOR-PRC 1.1复合物 补充了先前描述的KDM 2B-PRC 1.1复合物以有效地沉默分化程序， 人胚胎干细胞我们的中心假设是基于小鼠和人类的初步数据和先前工作制定的 胚胎干细胞，是Polycomb抑制发育调节剂是建立通过联合行动， KDM 2B-PRC 1.1和BCOR-PRC 1.1复合物。KDM 2B介导PRC1.1募集到可接近的CpG 岛，而BCOR负责募集到致密的染色质，赋予额外的阻遏物功能 并促进H3 K27 me 3在靶位点的沉积。在目标1中，我们将定义靶向机制 和抑制的非典型PRC1.1复合物：（1A）确定辅助因子和/或表观遗传 BCOR介导的PRC1.1靶向所需的标记;（1B）确定局部染色质结构如何影响 BCOR-PRC 1.1复合物的靶向性。(1C)定义通过N- （1D）描述KDM 2B-PRC 1.1和BCOR-PRC 1.1复合物的特定作用。 在Polycomb结构域组装从幼稚到引发多能状态的过渡过程中。更以 目的2，我们将使用我们的shRNA筛选数据来重建维持启动子的基因调控网络， 人类的多能性。我们将采用CRISPR-i技术敲除130种转录因子， 耗尽导致多能性的丧失。我们将使用单细胞RNA测序和我们的新的计算 工具，MAGIC和PHATE，从扰动中推断调控基因模块。我们将使用ChIP-Seq去- 关键调控模块的精细基因组足迹，并将这些数据与hESC表观遗传图谱整合， 发现新的转录控制机制。我们的方法是创新的，因为我们利用新颖的 国家的最先进的技术，以获得新的见解调控的复杂性hESC。拟议的重新- 搜索是重要的，因为它有望扩大对细胞命运调控的理解，定义关键的差异， hESC和mESC之间的联系，并指导细胞重编程的新方法的开发。

PROJECT DESCRIPTION Despite significant progress in deciphering the regulation of pluripotency in mouse models, there remain fun- damental gaps in our understanding of how human (h)ESCs maintain the pluripotent state Our long-term goal is to decipher the architecture of the regulatory network that allows for unrestricted hESC proliferation while preserving their potential to form the full repertoire of cell types found in the human body. To comprehensively identify genes involved in pluripotency maintenance we have conducted genome-wide shRNA screens in hESC and begun in-depth analyses of the most significant hits. We focused on BCOR, a component of the non-canonical Polycomb repressive complex 1.1 (PRC1.1) with a strong loss-of-pluripotency phenotype con- sistently observed in multiple hESC lines. BCOR depletion in hESCs led to a loss of repressive chromatin at key developmental loci and initiation of differentiation. We found that BCOR defines a novel subtype of the PRC1 complexes with distinct recruitment and repression mechanisms. This novel BCOR-PRC1.1 complex complements previously described KDM2B-PRC1.1 complex to efficiently silence differentiation programs in hESCs. Our central hypothesis, formulated based on the preliminary data and prior work in mouse and human ESCs, is that Polycomb repression of developmental regulators is established through combined action of the KDM2B-PRC1.1 and the BCOR-PRC1.1 complexes. KDM2B mediates PRC1.1 recruitment to accessible CpG islands while BCOR is responsible for the recruitment to dense chromatin, confers additional repressor function and facilitates the deposition of H3K27me3 at target loci. In Aim 1, we will define the mechanisms of targeting and repression by the non-canonical PRC1.1 complexes: (1A) Identify accessory factors and/or epigenetic marks required for BCOR-mediated PRC1.1 targeting; (1B) Determine how local chromatin structure affects the targeting of BCOR-PRC1.1 complexes. (1C) Define the repression mechanism mediated through the N- terminus of BCOR, and (1D) Delineate specific roles of the KDM2B-PRC1.1 and the BCOR-PRC1.1 complex- es in Polycomb domain assembly during the transition from naïve to primed pluripotent state. Furthermore, in Aim 2, we will use our shRNA screen data to reconstruct the gene regulatory network that maintains primed pluripotency in humans. We will employ CRISPR-i technology to knock-down 130 transcription factors whose depletion leads to a loss of pluripotency. We will use single cell RNA-sequencing and our novel computational tools, MAGIC and PHATE, to infer regulatory gene modules from perturbations. We will use ChIP-Seq to de- fine genomic footprints of the key regulatory modules and integrate these data with hESC epigenetic map to discover novel mechanisms of transcriptional control. Our approach is innovative, because we utilize 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 expand understanding of cell fate regulation, define of key dif- ferences between hESCs and mESCs and guide development of new approaches for cellular reprogramming.