Project Summary/Abstract: Regulatory DNA encodes signals that are recognized by transcriptional regulators to drive development- and stimulus-specific patterns of gene expression. While individual DNA binding sites recognized by transcription factors are often short and promiscuous in the genome, functional regulatory elements usually contain several closely spaced binding sites that specify the cooperative assembly of regulatory factors on DNA. Multiple lines of evidence suggest the configuration of these binding sites, including their relative binding affinity, spacing and orientation, play important roles in the context-dependent recruitment of RNA polymerase II to drive transcription. Furthermore, regulatory elements do not function independently in the genome. Proper gene regulation often depends on multiple regulatory elements scattered across gene loci that work in concert to enhance or silence target promoters. Yet despite extensive research efforts, our understanding of how regulatory DNA is organized is still rudimentary, limiting our ability to accurately model transcriptional networks and interpret the function of genetic variants. My laboratory seeks answers to these fundamental biological questions using a combination of experimental and computational approaches to decode regulatory DNA. Previous efforts to investigate the function of regulatory regions have typically relied on indirect measurements of transcriptional activity, such as the profiling of epigenetic markers, transcription factor binding, chromatin accessibility, or the expression of nearby genes or reporters. We have found that precise measurements of transcription initiation, which record the frequency and base positions where RNAPII initiates transcription, yield novel insights into the roles of transcription factor motifs and other regulatory DNA features in regulating transcription. Transcription initiation profiling also provides sensitive and precise measurements of activation at promoter-distal regulatory elements, enabling us to track the functional interactions between promoter and enhancer elements in the genome in the context of chromatin modifications and changes in 3D genome structure. Using these approaches we will explore how transcription factors exert activating or inhibitory effects on individual transcription start sites (TSS), depending on their spatial location within regulatory elements, and investigate how multiple regulatory elements work together to drive the transcription of target genes. These studies will greatly expand the ruleset to interpret how regulatory DNA and genetic variation affect gene regulation.