Friday, April 4, 2025
4/4/2025
Dynamics and mechanisms of long-range enhancer activity - Project Summary/Abstract Proper spatiotemporal gene expression is fundamental to all biological processes. Cell-type-specific gene expression programs are driven in large part by enhancers, which are cis-regulatory sequences that determine when and where genes are activated in a cell-type-specific manner. Enhancers often have to regulate genes across large genomic distances, but the molecular mechanisms underlying long-range enhancer-promoter (E- P) interactions remain unclear. Cell-type-specific regulators are also often regulated by enhancer clusters, which have been proposed to activate target genes via a condensate mechanism. The goal of this proposal is to understand the mechanistic basis of individual and clustered enhancers in human cells. The MYC locus is uniquely suited to address questions about enhancer function because it is regulated by different enhancers in different cell types. In several cancers including lung and endometrial cancers, the respective MYC enhancers are focally amplified to form enhancer clusters, which is thought to drive MYC overexpression. In Aim 1, super-resolution live-cell imaging (SRLCI) will be employed to simultaneously visualize MYC E-P interactions and nascent transcription over time in living lung and endometrial cells, which utilize different enhancers located ~450kb and ~800kb downstream respectively. To further validate that the inferred models from SRLCI are accurate, perturbations to key molecular players will be performed followed by SRLCI. By integrating 3D genomics and polymer simulations, the proposed research will generate quantitative models of how different enhancers regulate the same promoter. In Aim 2, the hypothesis that enhancer clusters activate transcription via a condensate mechanism will be tested. Additional enhancers will be introduced in both lung and endometrial cell lines to generate enhancer clusters, and SRLCI will be performed to determine the mechanism underlying enhancer clusters. Similar to Aim 1, 3D genomics data will be incorporated to generate polymer models of how enhancer clusters impact chromatin structure and E-P interactions to drive transcription. Together, these aims will provide a comprehensive and quantitative mechanistic understanding of how dynamic E-P interactions regulate transcription in two different cell types. The proposed research will be conducted in parallel with career development training to develop the necessary skillsets required to achieve the applicant’s goals of becoming an independent PI at a research institution. In addition to experimental and computational training, the applicant will receive mentorship from both sponsors on oral and written scientific communication, leadership, lab management and responsible conduct of research. This will be complemented by the world-class academic environment in MIT’s Department of Biological Engineering, Department of Physics and the Broad Institute of MIT and Harvard, where the applicant will be able to take advantage of the wealth of resources to expand her conceptual understanding of transcriptional regulation and learn to approach biological questions from an interdisciplinary, holistic viewpoint.
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