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: 10667607
• 负责人: Robert Coleman
• 金额: $ 34.61万
• 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 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667607
• 关键词: Architecture; Binding; Biology; CDKN1A gene; Cell division; Cells; Chromatin; Complex; DNA; DNA Damage; DNA Polymerase II; DNA Repair; DNA biosynthesis; Data; Disease; Exhibits; Foundations; Future; Gene Activation; Gene Expression; Genes; Genetic Transcription; Goals; Grant; Hour; Image; Kinetics; Knowledge; Lead; Length; Link; Malignant Neoplasms; Measures; Memory; Microscopy; Mission; Modeling; Molecular; Noise; Output; Pathway interactions; Pattern; Peptide Initiation Factors; Phase; Prevalence; Process; Prothrombin; Public Health; Regulation; Research; SAGA; Site; Stimulus; Stochastic Processes; Surveys; System; TAF1 gene; TP53 gene; Testing; Time; Transcriptional Activation; Transcriptional Regulation; United States National Institutes of Health; cell growth; cohort; histone methylation; human disease; imaging approach; imaging system; insight; live cell imaging; live cell microscopy; molecular imaging; novel; promoter; recruit; response; single molecule; therapeutic development; transcription factor

Project Summary Mammalian gene expression is a stochastic process marked by short periods (minutes) of intense activity or bursts of transcription interspersed between long intervals (>30 minutes to hours) of inactivity. Many genes also display transcriptional memory (TM), where an initial stimuli primes a promoter subsequently allowing faster and stronger re-activation of gene expression. TM can occur over both long (cell-divisions/LTTM) and short (hours/STTM) timescales. Mechanisms regulating mammalian STTM and its relationship to transcriptional bursting are poorly understood. This knowledge is critical for understanding how this process becomes dysregulated in diseases such as cancer. Our live cell imaging system will survey the molecular origins of the multiple ON states of bursting and transcriptional noise suppression that lead to STTM at a large cohort of genes. Preliminary data show 1.) transcription from the endogenous p21 and ACTB loci displays multi-phasic bursting (MPTB) associated with different burst durations and Pol II initiation rates 2.) multi-phasic bursting patterns exhibit STTM which can be modulated up and down via different stimuli. 3.) Noise is inversely correlated with STTM and is suppressed via histone methylation. Our long-term goal is to understand how gene expression is coordinately controlled by chromatin to restrict or grant of access of transcription factors to target promoters. Based on our preliminary data and previous studies, we hypothesize that MPTB and STTM arise from the coordinated recruitment of alternative pre-initiation complexes. This hypothesis will be tested using cutting-edge single molecule live-cell microscopy in the following 3 specific aims: 1.) Define the molecular origins of Multi-Phasic Transcriptional Bursting (MPTB). Live cell imaging will determine the relationship between alternative pre-initiation complexes and the different burst states. 2.) Define how Short-term transcriptional memory (STTM) is related to MPTB and the DNA damage response. Live cell imaging will determine the prevalence of STTM at a large cohort of genes and the molecular mechanism of how MPTB and STTM is regulated during the DNA damage response. 3.) Define the relationship between transcriptional noise, MPTB, and STTM. Live-cell imaging will be used to determine how noise modulators impact MPTB and STTM. These studies will provide key insights into how transcriptional bursting and STTM is modulated by the dynamic interplay between activators, pre-initiation factors, and Pol II.

项目摘要 哺乳动物基因表达是一个随机过程，以短期（分钟）的激烈活动或 在不活动的长间隔（> 30分钟至小时）之间散布的转录爆发。许多基因 还显示转录内存（TM），其中初始刺激素启动子随后允许 基因表达的更快，更强的重新激活。 TM可以在长（细胞划分/LTTM）和 短（小时/sttm）时标。调节哺乳动物sttm的机制及其与 转录爆发知之甚少。这些知识对于理解这一过程至关重要 在癌症等疾病中变得失调。我们的活细胞成像系统将调查分子 多个在爆发和转录噪声抑制状态下的多重源的起源 基因队列。 初步数据显示1.）来自内源性P21和ACTB基因座的转录显示多重爆发 （MPTB）与不同的爆发持续时间和pol II的起始率2.）多重爆发模式 展示STTM，可以通过不同的刺激在上下调节。 3.）噪声与 STTM并通过组蛋白甲基化抑制。我们的长期目标是了解基因表达如何 通过染色质协调控制，以限制或授予转录因子对目标启动子的访问。 根据我们的初步数据和以前的研究，我们假设MPTB和STTM来自 协调的替代启动前复合物的募集。 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。）定义多侧转录爆发（MPTB）的分子起源。现场单元成像将 确定替代性生成复合物与不同爆发状态之间的关系。 2.）定义 短期转录记忆（STTM）与MPTB和DNA损伤响应有何关系。活细胞 成像将确定大量基因队列和分子机制在大量基因上的患病率 在DNA损伤响应期间，如何调节MPTB和STTM。 3.）定义 转录噪声，MPTB和STTM。活细胞成像将用于确定噪声调节器 影响MPTB和STTM。这些研究将提供有关转录爆发和sttm的关键见解 由激活因子，发射因子和POL II之间的动态相互作用调节。