PROJECT SUMMARY The goals of this research program are to understand how bromodomain ‘reading’ of the epigenetic histone language is regulated by metabolism (Direction 1) and disease-associated missense variants (Direction 2). Our approach is multidisciplinary, focusing on mechanisms and developing novel chemical tools as starting points for drug development (Direction 3). Bromodomains bind acyl-lysines on histones and other nuclear proteins to modulate transcription. The importance of bromodomain-mediated transcription in human disease is well- established. Bromodomain inhibitors are in clinical trials for multiple indications, including cardiovascular disease and cancer. Despite these achievements, several critical questions remain. For example, histone and transcription factor lysine residues are modified by an array of non-acetyl acylations. These acylations are derived from acyl-CoA metabolites, indicating an intricate interplay between the cellular energy states that modulate acyl-CoA levels and bromodomain-mediated transcriptional regulation. Where, when, and how these acylations recruit bromodomains to chromatin for transcriptional regulation is poorly understood. Therefore, we are testing the hypothesis that changes in metabolic flux induce distinct histone acylations that are ‘read’ by specific bromodomains to regulate transcription and inflammation (Direction 1). Bromodomains are also hot spots for cancer-associated missense variants. However, the impacts of these variants on bromodomain- mediated transcriptional regulation are largely unknown. Elucidating the mechanisms through which bromodomain missense variants transcriptionally regulate key cancer signaling pathways will spur the development of novel precision medicine therapeutic approaches. Accordingly, we are testing the hypothesis that bromodomain missense variants with disrupted stability, structure, and dynamics attenuate bromodomain- mediated transcriptional regulation of key cancer signaling pathways (Direction 2). Here, we employ the integrated computational and biophysical pipeline that we developed in the Medical College of Wisconsin Structural Genomics Unit to interrogate disease-associated missense variants from protein structure to integrative transcriptomics. Another critical barrier to progress in bromodomain biology is the lack of inhibitors and chemical probes specifically targeting individual bromodomains. We are overcoming this barrier by developing selective bromodomain inhibitors using a novel fragment NMR screening pipeline we pioneered in the Medical College of Wisconsin Program in Chemical Biology (Direction 3). In line with the NIGMS mission to increase understanding of biological processes and advance knowledge of disease, our mechanistic inquiries into bromodomain-mediated transcriptional regulation, coupled with our development of inhibitors and chemical probes, will distinguish the differential activities of bromodomains in cell and animal models of he...