Dissecting the dynamic interplay between p53, chromatin and transcriptional bursting in single cells

剖析单细胞中 p53、染色质和转录爆发之间的动态相互作用

基本信息

批准号：
10133087
负责人：
Robert Coleman
金额：
$ 33.4万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-01 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10133087
关键词：
Binding Biological Assay Biology Cell division Cells Cellular Stress Chromatin Chromatin Structure DNA DNA Binding DNA Polymerase II DNA Repair DNA biosynthesis DNA-Directed RNA Polymerase Data Deacetylase Disease Enzymes Foundations Frequencies Gene Expression Genes Genetic Transcription Goals HDAC1 gene HTATIP gene Histone Acetylation Histone Deacetylase Histones Homeostasis Hour Image In Vitro Knowledge Link Malignant Neoplasms Measures Methyltransferase Mission Mutation Nucleosomes Pathway interactions Process Public Health Puma Research Series Site Surveys System TP53 gene Techniques Testing Transcript Transcriptional Regulation Tumor Suppression United States National Institutes of Health base biological adaptation to stress cell growth chromatin modification epigenetic regulation experimental study genomic locus histone acetyltransferase histone methylation histone modification human disease imaging approach imaging system in vivo insight live cell imaging live cell microscopy molecular imaging novel promoter recruit single molecule therapeutic development transcription factor

项目摘要

Project Summary Mammalian gene expression occurs via a series of transcriptional bursts, where convoys of 4-20 RNA Polymerase II (Pol II) molecules are loaded onto the promoter over the span of about 1 minute. In between these bursts, the gene promoter becomes non-permissive for transcription on timescales ranging from 1.5 to 30 minutes. Many genes also produce antisense transcripts that may regulate sense transcriptional bursting. However, it is not known how transient chromatin states involving histone acetylation and methylation can fine- tune these transcriptional bursts and transcription factor recruitment. This knowledge is critical for understanding how this process becomes dysregulated in diseases such as cancer. The p53 tumor suppression stress response system is ideal for studying transcriptional bursting in live cells. Our live cell imaging system will survey p53-dependent recruitment of histone acetylases (TIP60), deacetylases (HDAC1), methylases (EHMT1) and demethylases (PHF2) alongside sense and antisense transcriptional bursting. Preliminary data show 1.) transcription from the endogenous p21 locus occurs in sporadic bursts at low p53 levels, 2.) p53 induction leads to more frequent and longer transcriptional bursts, 3.) p53 occupancy rapidly oscillates on the p21 locus during transcriptional bursting, and 4.) in vitro, p53 dynamically binds target DNA sites that are wrapped in a nucleosome on the p21 promoter. Our long-term goal is to understand how gene expression is coordinately controlled by chromatin to regulate access of transcription factors to target DNA sites. We aim to correlate sense and antisense transcriptional bursting and transcription factor binding with changes in histone modification at a single-gene locus in live cells. Based on our preliminary data and previous studies, we hypothesize that sense transcriptional bursting profiles at tumor suppression genes are regulated via oscillatory, p53-dependent recruitment of histone acetylases/deacetylases and methylases/demethylases. This hypothesis will be tested using cutting-edge single molecule live-cell microscopy in the following 3 specific aims: 1.) Establish a system to link transcription dynamics with changes in p53's DNA binding activity and Pol II recruitment in vivo. Live cell imaging will correlate sense and antisense transcriptional bursting with p53 and Pol II binding. 2.) Define how transcription bursting, p53:DNA binding and Pol II recruitment is regulated by histone acetylation at promoters in live cells. Live cell imaging will correlate transcriptional bursting with TIP60, HDAC1, p53 and Pol II occupancy/activity on a promoter. 3.) Define how transcription bursting, p53:DNA binding and Pol II recruitment is regulated by histone methylation at promoters in live cells. Live cell imaging will correlate sense and antisense transcriptional bursting with EHMT1, PHF2, p53 and Pol II occupancy on a promoter. These studies will provide key insights into how transcriptional bursting is modulated by the dynamic interplay between p53, chromatin-modification enzymes and Pol II in cells.

项目概要哺乳动物基因表达通过一系列转录爆发发生，其中 4-20 个 RNA 组成的车队聚合酶 II (Pol II) 分子在大约 1 分钟的时间内加载到启动子上。介于两者之间这些爆发，基因启动子在 1.5 到 1.5 的时间尺度上变得不允许转录 30分钟。许多基因还产生反义转录本，可以调节有义转录爆发。然而，目前尚不清楚涉及组蛋白乙酰化和甲基化的瞬时染色质状态如何精细化。调整这些转录爆发和转录因子招募。这些知识对于了解这一过程如何在癌症等疾病中失调。 p53肿瘤抑制应激反应系统是研究活细胞转录爆发的理想选择。我们的活细胞成像系统将调查 p53 依赖性组蛋白乙酰酶 (TIP60)、脱乙酰酶 (HDAC1)、甲基化酶 (EHMT1) 和去甲基化酶 (PHF2) 以及正义和反义转录爆发。初步数据显示 1.) 来自内源性 p21 位点的转录在低 p53 条件下以零星爆发的方式发生水平，2.) p53 诱导导致更频繁和更长的转录爆发，3.) p53 迅速占据转录爆发期间在 p21 位点上振荡，4.) 在体外，p53 动态结合靶 DNA 包裹在 p21 启动子上核小体中的位点。我们的长期目标是了解基因如何表达由染色质协调控制，以调节转录因子接近目标 DNA 网站。我们的目标是将正义和反义转录爆发以及转录因子结合与活细胞中单基因位点组蛋白修饰的变化。根据我们的初步数据和之前的数据研究中，我们假设肿瘤抑制基因的感知转录爆发谱受到调节通过振荡、依赖于 p53 的组蛋白乙酰酶/脱乙酰酶和甲基化酶/去甲基化酶的募集。该假设将使用尖端单分子活细胞显微镜在以下 3 个具体方面进行检验目标： 1.) 建立一个系统，将转录动态与 p53 DNA 结合活性和 Pol 的变化联系起来 II体内募集。活细胞成像将正义和反义转录爆发与 p53 和 Pol II 结合。 2.) 定义转录爆发、p53:DNA 结合和 Pol II 募集是如何调节的活细胞启动子处的组蛋白乙酰化。活细胞成像将转录爆发与 TIP60 关联起来， HDAC1、p53 和 Pol II 在启动子上的占据/活性。 3.) 定义转录爆发的方式，p53:DNA 结合和 Pol II 招募受活细胞启动子处组蛋白甲基化的调节。活细胞成像将有义和反义转录爆发与 EHMT1、PHF2、p53 和 Pol II 占用相关联发起人。这些研究将为转录爆发如何被动态调节提供重要的见解。细胞中 p53、染色质修饰酶和 Pol II 之间的相互作用。