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

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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.