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

• 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
• 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

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 哺乳动物的基因表达是通过一系列的转录爆发发生的，其中4-20个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。之间 在这些爆发中，基因启动子在1.5至1.8的时间尺度上变得不允许转录。 30分钟许多基因也产生反义转录物，可以调节正义转录爆发。 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- 调节这些转录爆发和转录因子的募集。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中转录爆发的理想系统。我们的活细胞 成像系统将检测p53依赖的组蛋白乙酰化酶（TIP 60），脱乙酰化酶（HDAC 1）， 甲基化酶（EHMT 1）和去甲基化酶（PHF 2）以及正义和反义转录爆发。 初步数据显示1.）内源性p21基因座的转录在低p53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 在转录爆发期间在p21基因座上振荡，和4.）在体外，p53动态结合靶DNA p21启动子上包裹在核小体中的位点。我们的长期目标是了解基因 表达由染色质协调控制，以调节转录因子对靶DNA的接近 网站.我们的目标是将正义和反义转录爆发和转录因子结合与 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以前的 研究中，我们假设肿瘤抑制基因的正义转录爆发谱受到调控， 通过组蛋白乙酰化酶/脱乙酰化酶和甲基化酶/脱甲基化酶的振荡性p53依赖性募集。 这一假设将在以下3个具体的实验中使用最先进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的DNA结合活性和Pol变化联系起来的系统 II体内招募。活细胞成像将有义和反义转录爆发与p53和p54相关。 Pol II结合。2.）的情况。定义转录爆发、p53：DNA结合和Pol II募集如何受 在活细胞中启动子处的组蛋白乙酰化。活细胞成像将转录爆发与TIP 60相关联， 启动子上的HDAC 1、p53和Pol II占用/活性。3.）第三章定义如何转录爆发，p53：DNA 结合和Pol II募集受活细胞中启动子处的组蛋白甲基化调节。活细胞成像 将有义和反义转录爆发与EHMT 1、PHF 2、p53和Pol II占据相关， 启动子这些研究将提供关键的见解，如何转录突发调制的动态 细胞中p53、染色质修饰酶和Pol II之间的相互作用。