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：
9903386
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 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9903386
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.

项目摘要哺乳动物基因表达通过一系列转录爆发发生，其中4-20 RNA的车队在约1分钟的时间内，将聚合酶II（POL II）分子加载到启动子上。介于两者之间这些爆发，基因启动子对从1.5到1.5的时间尺度上的转录变得不可授课 30分钟。许多基因还产生反义转录本，可能调节感官转录爆发。然而，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细化调整这些转录爆发和转录因子募集。这些知识对了解该过程如何在癌症等疾病中失调。 p53肿瘤抑制应力反应系统是研究活细胞中转录爆发的理想选择。我们的活细胞成像系统将调查组蛋白乙酰酶（TIP60），脱乙酰基酶（HDAC1），依赖P53依赖性募集。甲基酶（EHMT1）和脱甲基酶（PHF2）以及感觉和反义转录爆发。初步数据显示1.）内源性P21基因座的转录发生在低p53处的零星爆发中级别2.）p53诱导导致更频繁且更长的转录爆发，3。）p53占用率迅速转录爆发过程中的p21基因座振荡，4.）在体外，p53动态结合目标DNA 包裹在p21启动子上的核小体中的位点。我们的长期目标是了解基因表达通过染色质协调控制以调节转录因子靶向DNA的访问站点。我们旨在将意义和反义转录爆发和转录因子与结合活细胞中单基因基因座的组蛋白修饰的变化。根据我们的初步数据和以前研究，我们假设在肿瘤抑制基因处的感官转录爆发曲线受到调节通过振荡，p53依赖性组蛋白乙酰酶/脱乙酰基酶和甲基化酶/脱甲基酶的募集。该假设将在以下3个特定中使用尖端的单分子活细胞显微镜进行检验目的：1。）建立一个将转录动力学与p53 DNA结合活性和POL的变化联系起来的系统 II体内招募。活细胞成像将与p53和反义转录爆发相关联 pol II结合。 2.）定义转录爆发的方式，p53：DNA结合和POL II募集受到调节活细胞中启动子的组蛋白乙酰化。活细胞成像将将转录爆发与tip60相关联，启动子上的HDAC1，p53和Pol II占用率/活动。 3.）定义转录如何破裂，p53：DNA 结合和POL II募集受活细胞启动子的组蛋白甲基化调节。活细胞成像将与EHMT1，PHF2，p53和Pol II占用率相关的感官和反义转录爆发发起人。这些研究将提供有关转录爆发如何通过动态调节的关键见解细胞中p53，染色质调节酶和POL II之间的相互作用。