Uncovering fundamentals of gene regulation by enhancers

PROJECT SUMMARY/ABSTRACT A class of cis-regulatory elements, called enhancers, plays a central role in orchestrating spatiotemporally precise gene expression programs during development. Perturbations in enhancer sequence or regulation can lead to disease, including congenital malformations and cancer. Furthermore, enhancer sequence divergence is emerging as an important mediator of human phenotypic variation. Key features of enhancers include their role as major sites for transcription factor (TF) and coactivator binding in the genome, as well as their ability to activate transcription over long genomic distances from their target promoters. Indeed, at some genomic loci enhancers can regulate promotes at distances of over a megabase away. Such long-range function raises a question of how is regulatory specificity within chromosomal domains containing many cis-regulatory elements accomplished to allow for precise and robust gene regulation during development and in tissue homeostasis? Proposed research program focuses on addressing fundamental knowledge gaps in enhancer biology and long- range gene regulation. We will utilize novel genomic and computational approaches to systematically probe how enhancer-promoter genomic distance, 3D contacts and inherent features of cis-regulatory elements influence transcriptional outputs, and how a broader genomic and epigenomic environment modulates promoter responsiveness to enhancers. We will complement these studies with live-cell and multiplexed DNA FISH imaging of model loci where enhancers regulate their target genes over ultra-long genomic distances. We will track movement of cis-regulatory elements and visualize nascent transcripts to capture the kinetic behavior of enhancers and promoters and its relationship with the discontinuous nature of transcription. We will introduce genetic perturbations to address how a new class of structural elements identified in our previous studies mediates robust transcriptional regulation. Finally, through precise modulation of TF and coactivator dosage at physiological levels, we will explore features that confer sensitivity or robustness of enhancers and gene expression to changes in trans-activator concentrations. Altogether, the proposed research will yield new concepts and quantitative models of long-range gene control, with broad future implications for understanding and treatment of human disease.