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Molecular control of pluripotency in humans

人类多能性的分子控制

基本信息

批准号：
10682996
负责人：
Natalia B Ivanova
金额：
$ 5.92万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10682996
关键词：
Adult Affect Alzheimer&apos s Disease Architecture Autoimmune Diseases BCL6 gene Behavior Cell Fate Control Cells ChIP-seq Chromatin Chromatin Structure Clustered Regularly Interspaced Short Palindromic Repeats Complement Complex Congenital Abnormality CpG Islands Data Deposition Development Epiblast Epigenetic Process Genes Genetic Transcription Genomics Goals Grant Histones Human Human body Individual Inner Cell Mass Knowledge Laboratories Maintenance Malignant Neoplasms Maps Mediating Mission Molecular Mus PRC1 Protein Parkinson Disease Patients Phenotype Play Polycomb Proteins Public Health Regulation Regulator Genes Repression Research Role Signal Transduction Somatic Cell Spinal cord injury System Technology Testing Therapeutic Human Experimentation Transcriptional Regulation United States National Institutes of Health Work base burden of illness cell behavior cell type computerized tools disability embryonic stem cell foot gene regulatory network genetic corepressor genome-wide human disease human embryonic stem cell implantation innovation insight knock-down loss of function member mouse model natural Blastocyst Implantation novel novel strategies pluripotency preimplantation preservation programs recruit regenerative therapy self-renewal single-cell RNA sequencing small hairpin RNA stem cells transcription factor ubiquitin-protein ligase

项目摘要

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.

项目描述尽管在破译小鼠模型多能性的调控方面取得了重大进展，但仍然存在一些有趣的问题我们对人类 (h)ESC 如何维持多能状态的理解存在根本差距我们的长期目标的目的是破译允许hESC不受限制增殖的调控网络的架构，同时保留它们形成人体中发现的完整细胞类型的潜力。为全面为了鉴定参与多能性维持的基因，我们进行了全基因组 shRNA 筛选 hESC 并开始对最重要的命中进行深入分析。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 con- 在多个 hESC 系中持续观察到。 hESC 中的 BCOR 耗竭导致抑制性染色质丧失关键发育位点和分化起始。我们发现 BCOR 定义了一个新的子类型 PRC1 复合物具有独特的招募和抑制机制。这种新型 BCOR-PRC1.1 复合物补充先前描述的 KDM2B-PRC1.1 复合物，以有效沉默分化程序人类胚胎干细胞。我们的中心假设是根据小鼠和人类的初步数据和先前的工作制定的 ESCs，是 Polycomb 对发育调节因子的抑制是通过 KDM2B-PRC1.1 和 BCOR-PRC1.1 复合物。 KDM2B 介导 PRC1.1 募集至可及的 CpG 岛，而 BCOR 负责招募致密染色质，赋予额外的阻遏功能并促进 H3K27me3 在目标位点的沉积。在目标 1 中，我们将定义目标机制和非典型 PRC1.1 复合体的抑制：(1A) 识别辅助因素和/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-PRC1.1 复合物的靶向。 (1C) 定义通过 N-介导的抑制机制 BCOR 的末端，以及 (1D) 描述 KDM2B-PRC1.1 和 BCOR-PRC1.1 复合体的具体作用- 在从幼稚状态到引发多能状态的转变过程中，Polycomb 结构域组装中存在 es。此外，在目标 2，我们将使用 shRNA 筛选数据来重建维持启动状态的基因调控网络人类的多能性。我们将利用 CRISPR-i 技术敲低 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 测序和我们新颖的计算技术 MAGIC 和 PHATE 工具，用于从扰动推断调控基因模块。我们将使用 ChIP-Seq 来解析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转录控制的新机制。我们的方法是创新的，因为我们利用新颖的最先进的技术，以获得对 hESC 监管复杂性的新见解。建议重新研究意义重大，因为它有望扩大对细胞命运调控、关键差异的定义的理解 hESC 和 mESC 之间的参考并指导细胞重编程新方法的开发。