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，其特征是短时间（分钟）的强烈活动或 转录的爆发散布在长时间间隔（>30分钟至数小时）的不活动之间。许多基因 也显示转录记忆（TM），其中初始刺激引发启动子，随后允许 更快更强的基因表达再激活。TM可以在长时间（细胞分裂/LTTM）和 短（小时/STTM）时间尺度。哺乳动物STTM的调控机制及其与 对转录爆发的了解很少。这些知识对于理解这个过程是如何 在癌症等疾病中变得失调。我们的活细胞成像系统将检测 爆发和转录噪声抑制的多个ON状态的起源，导致STTM在很大程度上 一群基因。 初步数据显示1.）内源性p21和ACTB基因座的转录显示多相爆发 （MPTB）与不同的突发持续时间和Pol II起始速率2相关联。多相爆发模式 表现出STTM，其可以通过不同的刺激向上和向下调制。3.）第三章噪声与 STTM通过组蛋白甲基化被抑制。我们的长期目标是了解基因表达是如何 由染色质协调控制以限制或允许转录因子接近靶启动子。 根据我们的初步数据和以前的研究，我们假设MPTB和STTM产生于 替代性前起始复合物的协调募集。 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.）定义多相转录爆发（MPTB）的分子起源。活细胞成像将 确定备选预引发复合物与不同爆发状态之间的关系。2.）的情况。定义 短期转录记忆（STTM）如何与MPTB和DNA损伤反应相关。活细胞 成像将确定STTM在一个大的基因组中的患病率以及STTM的分子机制。 MPTB和STTM在DNA损伤反应中是如何调节的。3.）第三章定义之间的关系 转录噪声、MPTB和STTM。活细胞成像将用于确定噪声调制器如何 影响MPTB和STTM。这些研究将为转录爆发和STTM是如何在 通过激活剂、前起始因子和Pol II之间的动态相互作用进行调节。