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

上级获奖项目总结 哺乳动物的基因表达是一个随机的过程，以短时间(分钟)的强烈活动或 在长时间(30分钟到几小时)的不活动之间穿插着转录的爆发。许多基因也 显示转录记忆(TM)，其中初始刺激启动启动子，随后允许更快 以及更强的基因表达的重新激活。TM既可以发生在长(细胞分裂/LTTM)上，也可以发生在短上 (小时/STTM)时间标尺。哺乳动物STTM的调控机制及其与转录的关系 人们对爆裂知之甚少。这一知识对于理解这个过程是如何变得至关重要的 在癌症等疾病中调节失调。我们的活细胞成像系统将调查 多个ON状态的突发和转录噪声抑制导致大量基因的STTM。 初步数据显示1)内源性p21和ACTB基因座的转录呈现多时相爆发 (MPTB)与不同的突发持续时间和POL II起始率相关。多期爆裂模式 展示STTM，它可以通过不同的刺激上下调节。3.)噪声与噪声成反比 STTM，并通过组蛋白甲基化被抑制。我们的长期目标是了解基因是如何表达的 由染色质协调控制，以限制或准许转录因子进入目标启动子。 根据我们的初步数据和以前的研究，我们假设MPTB和STTM是由 协调招募替代的预起爆复合体。 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.)明确多相转录爆发(MPTB)的分子起源。活细胞成像将 确定可选的预起爆复合体与不同的爆发状态之间的关系。2.)定义 短时转录记忆(STTM)与MPTB和DNA损伤反应的关系。活细胞 成像将确定STTM在一大群基因中的流行率以及STTM的分子机制 MPTB和STTM在DNA损伤反应过程中受到调控。3.)定义以下关系： 转录噪声、MPTB和STTM。将使用活细胞成像来确定噪声调制器如何影响 MPTB和STTM。这些研究将提供对转录猝发和STTM如何的关键见解 受激活剂、预启动因子和POL II之间动态相互作用的调节。

PARENT AWARD 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 stimulus 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.