PROJECT SUMMARY/ABSTRACT Human genome is peppered with an estimated one million enhancers that can regulate their specific target gene(s) from a distance up to megabases away, and in an orientation-independent manner. The broad goal of this project is to define the fundamental architecture and function of human enhancers in a in-depth manner and to better understand the specificity of their interplay with different classes of promoters that use different transcription factors and/or are regulated at distinct steps in transcription. We have shown that enhancers can be identified and precisely mapped using our GRO/PRO-cap assay that map transcription start sites of nascent RNA with highest sensitivity of all available methods. This assay has shown that both enhancers and promoters share a common architecture whereby both are delimited by two divergent core promoters (CPs) and a central cluster of transcription factor (TF) binding motifs. The roles and required organization of the multiple sequence motifs that constitute enhancers and the two CPs need to be fully dissected to understand how active enhancers function. Additionally, enhancers can interact productively with specific promoters, and the basis of this specificity especially at long range remains ill-defined. Finally, we know that promoter and enhancer elements have sequence motifs that can act at distinct regulatory steps of the transcription cycle, but how these activities coordinate gene regulation has yet to be examined. In Aim 1, we will test the activity of all PRO-cap identified enhancer candidates in K562 from representative human Chromosomes 8 and 11 with a carefully chosen set of promoters harboring distinct regulatory features. A set of active enhancer-promoter combinations will then be subjected to a comprehensive motif mutagenesis of the central clusters of TF binding motifs and each of the two core promoters. These studies test our fundamental enhancer unit hypothesis and assess the relationships of enhancers to targeted promoters, and the role of specific motifs and sequence features in enhancer function. In Aim 2, we examine quantitatively enhancers and key mutants identified in Aim 1 by barcoding and integrating WT and mutant enhancers 5 kb upstream of their normally responsive promoter in a chromosomal context at the AAVS1 safe harbor locus. These assays will rigorously test function of enhancer motifs, core promoters, and the overall architecture of enhancers in a constant chromosomal background. In addition, we will also test the regulatory code underlying enhancer specificity for promoters and evaluate effects of mutant TF motifs on TF binding and on nascent transcription using targeted genomic assays. Finally, in Aim 3, we explore the ability of enhancers to act over long distances, using the NMU enhancer (eNMU), which resides 94 kb upstream of the NMU promoter and stimulates its transcription by 10,000-fold. We will utilize this robust model enhancer, which is not co...