PROJECT ABSTRACT Enhancers play critical roles in determining lineage-specific gene expression and cellular identities, and are enriched in noncoding disease mutations and risk loci. There is an urgent need to elucidate the complete mechanisms of enhancer functions and malfunctions to better understand disease etiology and to develop new, enhancer-targeting therapies. The recent finding that active enhancers often generate noncoding enhancer RNAs (eRNAs) has provided a new perspective to interpret enhancer activity in the context of RNA-mediated regulation. This proposal aims to establish a unique research direction by developing new approaches to address the molecular mechanisms underlying eRNA functions in gene/genome regulation. Our overall hypothesis is that eRNAs assemble specific ribonucleoprotein complexes (RNPs) to organize high-order enhancer structures in the three-dimensional nucleus to control gene transcription. We have three specific aims in this proposal. We plan to use a robust RNA capture method that we have developed to systematically identify eRNA:protein interactomes in human cells. For the first aim, we plan to focus on two novel eRNA- binding proteins (eRBPs) to characterize their functions in gene transcription and 3D genome interaction in mammalian cells. Subsequently, we have two aims to study the mechanisms of each of the two eRBPs to fully characterize the biochemical basis permitting eRNA:protein interaction and their possible involvement in mediating the formation of transcriptionally-associated sub-nuclear condensates. The expected results from this proposal will provide novel mechanistic insights into gene transcriptional regulation and RNA functions in mammalian cells. As enhancers become important drivers in disease development, these mechanistic insights may offer new therapeutic strategies to treat enhancer-driven diseases.