PROJECT SUMMARY Distant-acting (“remote”) transcriptional enhancers bound by transcription factors (TFs) drive gene expression patterns in space and time. The importance of this function is underscored by mounting evidence linking mutations affecting remote enhancers to human disease. What remains unclear is how these remote enhancers regulate their target genes over long genomic distances, often skipping intervening genes. Remote enhancers typically interact with target promoters with the support of higher-order chromatin organization, but disruption of this chromatin organization does not abolish enhancer–promoter interactions. What additional factors regulate these long-range regulatory interactions remains largely unexplored. To address this central question, the proposed project will use a novel gain-of-function approach to identify cis-regulatory sequences that mediate long-range enhancer–promoter communication in the context of mammalian development. Traditional methods that assess in vivo enhancer activity rely on enhancer-reporter transgenesis; however, technical limitations preclude this approach from assessing long-range enhancer activity. The proposed work overcomes this limitation by utilizing efficient enhancer replacement technology in mice to systematically characterize in vivo long-range enhancer activity and identify cis-regulatory sequences that are critical for this long-range activity. To determine the mechanism of long-range regulation by these remote control elements and identify regulatory proteins that bind them, this work will use a multidisciplinary approach to map and visualize enhancer–promoter contacts at macromolecular resolution. Functional characterization of remote control elements will expand the repertoire of known cis-regulatory elements, provide a novel general mechanism for long-range enhancer– promoter communication, and substantially advance knowledge of the role of 3D-genome organization in the context of development and disease.

PROJECT NARRATIVE Mutations leading to disease frequently affect distant-acting transcriptional enhancers, non-coding DNA elements that regulate gene expression and that are often located hundreds of thousands of base pairs away from their target genes. This project aims to determine how enhancers regulate their target genes over such long genomic distances using novel genome editing, genomics, and imaging technologies, advancing an essential foundation to understand the pathological consequences of single-nucleotide and structural variation in non- coding regions of the human genome.