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

项目摘要 哺乳动物的基因表达是通过一系列转录爆发发生的，其中4-20个RNA车队 聚合酶II(POL II)分子在大约1分钟的跨度内被加载到启动子上。介于两者之间 这些爆发，基因启动子变得不允许转录在时间尺度上从1.5到 30分钟。许多基因也产生反义转录本，可能调节正义转录的爆发。 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能够改善- 调整这些转录突发和转录因子招募。这方面的知识对 了解这一过程是如何在癌症等疾病中变得失调的。P53肿瘤 抑制应激反应系统是研究活细胞转录爆发的理想系统。我们的活细胞 成像系统将检测P53依赖的组蛋白乙酰酶(Tip60)、脱乙酰酶(HDAC1)、 甲基酶(EHMT1)和去甲基酶(PHF2)以及正义和反义转录爆发。 初步数据显示1)内源性p21基因的转录发生在低p53的零星突发区 级别，2。)P53的诱导导致更频繁和更长的转录突发。P53迅速被占用 在转录爆发过程中在p21基因座上振荡，以及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是如何 染色质协调调控转录因子对靶DNA的访问 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上组蛋白修饰的变化。基于我们的初步数据和之前 研究中，我们假设肿瘤抑制基因的正义转录爆发模式是受调控的 通过振荡的、依赖于P53的组蛋白乙酰基酶/去乙酰基酶和甲基酶/去甲基酶的募集。 这一假设将用尖端的单分子活细胞显微镜在以下三个特定的方面进行验证 目标：1.)建立转录动力学与P53‘S DNA结合活性和POL变化的关联系统 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.)确定转录爆发、P53：DNA结合和POL II募集是如何通过 活细胞中启动子上的组蛋白乙酰化。活细胞成像将转录爆发与TIP60相关联， HDAC1、P53和POL II在启动子上的占有率/活性。3.)定义转录猝发，P53：DNA 在活细胞中，结合和Pol II的募集是由启动子上的组蛋白甲基化调节的。活细胞成像 正义和反义转录爆发与EHMT1、PHF2、P53和Pol II在 推动者。这些研究将为转录猝发如何受动态调控提供关键见解 细胞中P53、染色质修饰酶和POL II的相互作用。

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.