Dissecting the Dynamic Interplay Between, p53, Chromatin and Transcriptional Bursting in Single Cells

项目概要 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 转录爆发散布在长时间不活动的时间间隔（> 30 分钟到几小时）之间。许多基因 还显示转录记忆（TM），其中初始刺激启动启动子，随后允许 更快更强的基因表达重新激活。 TM 可以发生在长（细胞分裂/LTTM）和 短（小时/STTM）时间尺度。哺乳动物 STTM 的调节机制及其与 人们对转录爆发知之甚少。这些知识对于理解这个过程如何至关重要 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 爆发和转录噪声抑制的多个 ON 状态的起源，这些状态在很大程度上导致了 STTM 基因组。 初步数据显示 1.) 内源性 p21 和 ACTB 基因座的转录表现出多相爆发 (MPTB) 与不同突发持续时间和 Pol II 起始速率相关 2.) 多相突发模式 表现出 STTM 可以通过不同的刺激进行上下调节。 3.) 噪声与 STTM 并通过组蛋白甲基化受到抑制。我们的长期目标是了解基因表达是如何进行的 由染色质协调控制，以限制或允许转录因子接近目标启动子。 根据我们的初步数据和之前的研究，我们假设 MPTB 和 STTM 源于 协调招募替代性启动前复合物。 该假设将使用尖端单分子活细胞显微镜在以下 3 个具体方面进行检验 目标： 1.) 定义多相转录爆发 (MPTB) 的分子起源。活细胞成像将 确定替代预引发复合物与不同突发状态之间的关系。 2.) 定义 短期转录记忆 (STTM) 与 MPTB 和 DNA 损伤反应的关系。活细胞 成像将确定大量基因中 STTM 的患病率及其分子机制 MPTB 和 STTM 在 DNA 损伤反应过程中如何受到调节。 3.) 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 如何发挥作用提供重要见解 由激活剂、预引发因子和 Pol II 之间的动态相互作用调节。

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.