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（由申请人提供）：干细胞分化过程中染色质状态的变化控制基因表达以及肠道干细胞（ISC）的缺陷是疾病的根源例如结直肠癌和炎症性肠病。尽管最近在 ISC 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。 EZH2 是 Polycomb 抑制复合体 (PRC) 2 的一部分，在许多结直肠癌中过度表达。然而，目前对大多数表观遗传酶（包括 PRC2）的功能和机制的了解都源自癌细胞系或 ES 细胞的体外研究。在充分发挥此类酶作为药物靶点的潜力之前，必须对其作用进行体内验证和理解。 PRC2被认为可以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 PRC2 介导的组蛋白标记 H327me3 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，我将结合三种实验方法：对各种肠道细胞中的 H3K27me3 标记进行 ChIP-seq（目标 1）； Eed 基因删除以消除 PRC2 活性（目标 2）； RNA-seq 对具有完整和破坏的 PRC2 功能的不同肠道细胞进行详细的转录组分析（目标 3）。为了了解 ISC 分化过程中 H3K27me3 修饰的动态模式，我们使用 ChIP 检测在 ISC 和肠绒毛分化细胞中绘制了 H3K27me3 标记基因图谱。为了了解 H3K27me3 标记如何控制基因表达，我们将使用 RNA-seq 在 ISC 和绒毛细胞中进行详细的转录组分析。通过关联干细胞和分化细胞之间的差异 H3k27me3 标记和差异基因表达，我们将获得有关 PRC2 在控制肠道基因中的作用的原始且详细的信息。为了评估 ISC 功能和肠道疾病中的 PRC2 需求，我们使用细胞特异性敲除小鼠模型，删除了整个肠上皮，特别是 ISC 中的核心 PRC2 组成基因 Eed。 Eed-null 肠道显示出严重的缺陷，对此表型的深入分析，再加上野生型和 Eed-null 细胞的基因表达分析，将使我们能够识别参与 ISC 维持和分化的特定基因、信号通路和调节机制。我们的研究有望揭示分化过程中基因调控的新见解以及 PRC2 介导的染色质修饰的机制细节。