Investigating regulatory mechanisms of in vivo transcriptional dynamics

About This Grant

ABSTRACT Essentially all transcription occurs in stochastic and episodic bursts, conferring flexibility, adaptability, and diversity to otherwise identical cells. Regulating this `bursty' transcription in a timely and context-appropriate manner is key to proper development and homeostasis. Its misregulation causes an imbalance between dynamically counteracting genes and improper gene dosage compensation, often leading to various human diseases, including cancer, cardiovascular disease, metastasis, and infertility. However, molecular mechanisms underlying transcriptional burst regulation remain elusive due to the lack of proper in vivo models and precise long-term assays. Also, the results from previous studies often conflict with each other, hampering our precise understanding and therapeutic advancements. Our overarching goal is to elucidate the molecular mechanisms underpinning spatiotemporal regulation of in vivo transcriptional bursting during development, homeostasis, and disease, and discover new factors controlling its context-specificity and adaptability. Recent studies, including our work monitoring transcriptional dynamics of endogenous Notch target genes in live adult C. elegans, contradict the previous findings: the burst duration is the major parameter regulated in vivo, whereas burst frequency is the major target of regulation in vitro. What causes these discrepancies? What modulates the burst behaviors in a context-specific manner and how? To address these questions, we will use the C. elegans gonad as an in vivo transcriptional burst study model with our innovative approach, combining long- term single-molecule live RNA imaging, machine learning-based analysis and modeling, and bioinformatics to analyze burst dynamics regulation in vivo. Focusing on the burst dynamics of powerful and well-characterized Notch pathway, we will determine the precise roles of core Notch cis- and trans-regulatory elements (CREs and TREs) like promoters, enhancers, and mediators in transcriptional burst regulation both in in vivo and in vitro contexts. We will also define the novel functions of the transcriptional co-activator LAG-3 (MAML in humans) for context-specific regulation of transcriptional dynamics, focusing on its functions for biocondensate formation and chromatin modifications. Our results will fill the critical gap in knowledge about in vivo transcriptional bursting and greatly advance our understanding of transcriptional regulation and stem cell control, with the potential to discover new therapeutic targets and strategies for Notch-related diseases and infertility.