Molecular control of pluripotency in humans

项目说明 尽管在破译小鼠模型的多能性调节方面取得了重大进展，但仍有乐趣-- 我们对人类胚胎干细胞如何维持多能性的理解存在着根本的差距，这是我们的长期目标 破译监管网络的架构，允许不受限制的hESC扩散，同时 保存它们的潜力，以形成人体内发现的全部细胞类型。要全面 确定与维持多能性有关的基因我们已经进行了全基因组shRNA筛选 HESC，并开始深入分析最重要的命中。我们重点介绍了BCOR，它是 具有强多能丧失表型的非规范多梳抑制复合体1.1(PRC1.1) 在多个hESC系中一致观察到。HESCs中BCOR的耗竭导致抑制染色质的丢失 关键发育基因和分化的启动。我们发现BCOR定义了一个新的子类型 Prc1复合体具有不同的招募和抑制机制。这种新型的BCOR-PRC1.1络合物 补充前面描述的Kdm2b-PRC1.1复合体，以有效地沉默分化程序 HESCS。我们的中心假设，是基于初步数据和之前在老鼠和人类身上的工作提出的 ESCs，是通过联合作用建立的发育调节剂的多梳抑制 Kdm2b-PRC1.1和BCOR-PRC1.1络合物。Kdm2b调解将PRC1.1招募到可访问的CpG 当BCOR负责募集到高密度染色质时，BCOR则赋予额外的阻遏功能 有利于H3K27me3在靶位的沉积。在目标1中，我们将定义目标确定机制 和非规范的PRC1.1复合体的抑制：(1A)鉴定辅助因子和/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 靶向BCOR-PRC1.1复合体。(1C)定义通过N-的中介的抑制机制 BCOR的末端，和(1D)描述Kdm2b-PRC1.1和BCOR-PRC1.1复合体的特定作用- ES在从幼稚到启动的多能性状态转变过程中的多梳结构域组装。此外，在 目的2，我们将使用我们的shrna筛选数据来重建维持Primed的基因调控网络 人类的多能性。我们将使用CRISPR-I技术敲除130个转录因子，这些转录因子 耗尽会导致多能性丧失。我们将使用单细胞RNA测序和我们的新计算 工具，MAGIC和PHATE，从扰动中推断调控基因模块。我们将使用CHIP-SEQ来解压 精细的关键调控模块的基因组足迹，并将这些数据与hESC表观遗传图谱整合，以 发现转录控制的新机制。我们的方法是创新的，因为我们利用了新奇 最先进的技术，以获得对HESCs监管复杂性的新见解。建议的重行- 研究具有重要意义，因为它有望扩大对细胞命运调控的理解，定义关键差异。 HESCs和mESCs之间的参考，并指导细胞重新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.