PROJECT SUMMARY Members of the bromodomain and extra-terminal domain (BET) family (Brd2, Brd3, Brd4, Brdt) each contain two bromodomains that bind acetyl-lysines on histones and transcription factors. The importance of BET- regulated transcription in human disease is well appreciated with pan-BET bromodomain inhibitors in phase I/II clinical trials for multiple cancers and phase III trials for type 2 diabetes subjects with coronary artery disease. Despite these achievements, several critical questions remain. For example, BET proteins are localized disproportionately at super-enhancers, genomic regions with large clusters of elements that enhance gene transcription. The basis of this localization is unknown but important given that super-enhancers are enriched at loci with oncogenic potential. Our unpublished data support the hypothesis that tandem bromodomains act as a scaffold for acetylation-dependent reorganization of chromatin; for instance, joining promotors with their corresponding distal enhancers to drive transcription (Focus 1). However, the ability of tandem bromodomains to scaffold nucleosomes and transcription factors in an acetylation-dependent manner has not been shown. We take an innovative structural and biophysical approach to investigate the role of Brd4 in maintaining chromatin conformations that facilitate enhancer-driven oncogenic gene transcription. This mechanism of chromatin reorganization, if true, is paradigm shifting and would have broad impact on studies of tandem histone-binding domains. We also hypothesize that metabolic changes induce distinct post-translational modifications on histones that are “read” by bromodomains. Yet, the broader acylation and protein binding specificity of bromodomains is poorly understood. We have begun to address this knowledge gap in our recent publication that highlights how metabolically-derived acylations and neighboring modifications tune BET bromodomain binding to histones. To continue to address this broad metabolic question, we are using biophysical, structural biology, and proteomic techniques to investigate BET bromodomain acylation and protein selectivity in linking acyl-CoA metabolism with transcription (Focus 2). To aid our mechanistic inquiries, we are removing a critical barrier in the study of BET bromodomain biology: the lack of inhibitors and chemical probes that selectively target individual BET proteins. Currently, all existing BET inhibitors target Brd2, Brd3, Brd4, and Brdt with equal nanomolar potency. This lack of selectivity may be responsible for the side effects of memory loss and lymphoid toxicity recently associated with existing pan-BET inhibitors. We are overcoming these barriers with a novel fragment-based ligand discovery and chemical biology strategy to discover selective Brd4 inhibitors by covalently targeting a unique cysteine within Brd4 (Focus 3). These chemical tools will be necessary to distinguish the differential activities of BET proteins in cell...