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Role of histone H3K27me3 mark in intestinal homeostasis

组蛋白 H3K27me3 标记在肠道稳态中的作用

基本信息

批准号：
9125811
负责人：
Unmesh Jadhav
金额：
$ 6.2万
依托单位：
DANA-FARBER CANCER INST
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：
2014-07-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9125811
关键词：
Adult Affect American Apoptosis Apoptotic Bioinformatics Cancer cell line Cell Differentiation process Cell Maintenance Cell physiology Cells ChIP-seq Chromatin Colorectal Cancer Complex Data Defect Development Disease Drug Targeting EZH2 gene Ectopic Expression Electron Microscopy Enzymes Epigenetic Process Epithelial Cells Gene Components Gene Expression Gene Expression Profiling Gene Expression Regulation Genes Genetic Genetic Enhancer Element Health Histones Human In Vitro Individual Inflammatory Bowel Diseases Intestinal Diseases Intestinal Mucosa Intestines Knockout Mice Knowledge Lead Length Maintenance Malignant Neoplasms Mammalian Cell Maps Mass Spectrum Analysis Measures Mediating Modification Mus Mutate Natural regeneration Null Lymphocytes Pattern Pharmaceutical Preparations Phenotype Play Polycomb Population Positioning Attribute Process Proliferating Role Signal Pathway Signaling Protein Stem cells Time Tissue-Specific Gene Expression Tissues Validation Villus Work base cell type chromatin immunoprecipitation chromatin modification crypt cell drug development effective therapy embryonic stem cell genome-wide analysis histone modification in vivo insight intestinal crypt intestinal epithelium intestinal homeostasis intestinal villi mouse model mutant novel diagnostics overexpression progenitor programs promoter protein complex rapid growth self-renewal stem stem cell differentiation tool transcriptome transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): Changes in chromatin states control gene expression during differentiation of stem cells and defects in intestinal stem cells (ISCs) underlie disorders such as colorectal cancer and inflammatory bowel diseases. In spite of recent advances in identification of ISCs, the role of epigenetic modifications in ISC gene expression, function and intestinal disease remains unknown. EZH2, which is part of the Polycomb repressive complex (PRC) 2, is overexpressed in many colorectal cancers. However, the current knowledge of the functions and mechanisms of most epigenetic enzymes (including PRC2) is derived from in vitro studies in cancer cell lines or ES cells. An in vivo validation and understanding of their working is imperative before the potential of such enzymes as drug targets can be fully realized. PRC2 is thought to repress gene expression in stem cells and work with other histone modifications to control stem cell differentiation. We propose to study the role of the PRC2-mediated histone mark H327me3 in Intestinal stem cell gene expression, function and differentiation. To achieve this, I will combine three experimental approaches: ChIP-seq for the H3K27me3 mark in various intestinal cells (Aim 1); genetic deletion of Eed to eliminate PRC2 activity (Aim 2); and RNA-seq for detailed transcriptome analysis in different intestinal cells with intact and disrupted PRC2 function (Aim 3). To understand the dynamic pattern of H3K27me3 modification during ISC differentiation, we have mapped H3K27me3-marked genes using ChIP assays in ISC and differentiated cells from intestinal villi. In order to understand how the H3K27me3 mark controls gene expression, we will perform detailed transcriptome analysis using RNA-seq in ISC and villus cells. By correlating the differential H3k27me3 mark and differential gene expression between stem and differentiated cells, we will obtain original and detailed information about the role of PRC2 in control of intestinal genes. To evaluate PRC2 requirements in ISC function and intestinal diseases, we have deleted the core PRC2 component gene Eed in the whole intestinal epithelium and also specifically in ISCs, using cell specific knock-out mouse models. Eed-null intestines show severe defects and in-depth analysis of this phenotype, together with gene expression analysis of wild type and Eed-null cells will allow us to identify specific genes, signaling pathways, and regulatory mechanisms involved in ISC maintenance and differentiation. Our studies hold promise to reveal new insights into gene regulation during differentiation and mechanistic details of PRC2-mediated chromatin modifications.

描述（申请人提供）：在干细胞分化过程中，染色质状态的变化控制基因表达，肠道干细胞（ISCs）缺陷是疾病的基础例如结肠直肠癌和炎症性肠病。尽管最近在ISC的鉴定方面取得了进展，但表观遗传修饰在ISC基因表达、功能和肠道疾病中的作用仍然未知。EZH 2是Polycomb抑制复合物（PRC）2的一部分，在许多结直肠癌中过表达。然而，目前对大多数表观遗传酶（包括PRC 2）的功能和机制的了解来自癌细胞系或ES细胞的体外研究。在这些酶作为药物靶点的潜力完全实现之前，必须对其工作进行体内验证和理解。PRC 2被认为抑制干细胞中的基因表达，并与其他组蛋白修饰一起控制干细胞分化。我们拟研究PRC 2介导的组蛋白标记H327 me 3在肠干细胞基因表达、功能和分化中的作用。为了实现这一目标，我将结合联合收割机三种实验方法：ChIP-seq用于各种肠细胞中的H3 K27 me 3标记（目标1）; Eed的遗传缺失以消除PRC 2活性（目标2）;以及RNA-seq用于在具有完整和破坏的PRC 2功能的不同肠细胞中进行详细的转录组分析（目标3）。为了了解ISC分化过程中H3 K27 me 3修饰的动态模式，我们使用ChIP测定在ISC和来自肠绒毛的分化细胞中绘制了H3 K27 me 3标记的基因。为了了解H3 K27 me 3标记如何控制基因表达，我们将在ISC和绒毛细胞中使用RNA-seq进行详细的转录组分析。通过将干细胞和分化细胞之间的差异H3 k27 me 3标记和差异基因表达相关联，我们将获得关于PRC 2在控制肠道基因中的作用的原始和详细的信息。为了评估PRC 2在ISC功能和肠道疾病中的需求，我们使用细胞特异性敲除小鼠模型，在整个肠上皮中以及特别是在ISC中删除了核心PRC 2组分基因Eed。Eed基因缺失的肠道表现出严重的缺陷，对这种表型的深入分析，以及野生型和Eed基因缺失细胞的基因表达分析，将使我们能够鉴定出参与ISC维持和分化的特定基因、信号通路和调控机制。我们的研究有望揭示分化过程中基因调控的新见解和PRC 2介导的染色质修饰的机制细节。