PROJECT SUMMARY The goal of this administrative supplement request is to provide a state-of-the-art crystallization instrument for structural studies of bromodomain binding to metabolically-derived histone acyl-lysines and small-molecule inhibitors covered by the R35 GM128840 parent award. The role of bromodomain-regulated transcription in human disease is well appreciated with bromodomain inhibitors in clinical trials. Despite these achievements, several critical questions remain. For example, bromodomains are localized disproportionately at super- enhancers. The basis of this localization is unknown but important given that super-enhancers are enriched at loci with oncogenic potential. We hypothesize that tandem bromodomains act as a scaffold for acetylation- dependent chromatin reorganization, for instance, joining promotors with enhancers to drive transcription (Focus 1). We are taking a structural and biophysical approach to investigate the role of tandem bromodomains in maintaining chromatin conformations. We also hypothesize that metabolic changes induce post-translational modifications on histones “read” by bromodomains. Yet, the acylation and protein binding specificity of bromodomains are poorly understood. To address this metabolic question, we use biophysical, structural biology, and proteomic techniques to investigate bromodomain acylation selectivity and link acyl-CoA metabolism with transcription (Focus 2). To aid mechanistic inquiries, we are developing inhibitors of bromodomains using a novel fragment-based NMR screening strategy with a current focus on the PBRM1 bromodomains (Focus 3). These chemical tools will distinguish the differential activities of bromodomains in disease models and lead to therapeutics targeting the PBRM1 axis in cancer. To determine optimal conditions toward x-ray structure determination of bromodomains bound to ligands, the proposed instrumentation provides the necessary platform and infrastructure to screen and automate several orders of magnitude greater than a single dispenser can perform. Furthermore, this screening platform will be used for collaborative projects for the Program in Chemical Biology at the Medical College of Wisconsin (MCW) and open to all MCW investigators. The instrumentation will be installed in the shared crystallization instrumentation room controlled by the Department of Biochemistry. As this instrumentation will replace obsolete 14-year-old instrumentation, this room is equipped with the necessary space and infrastructure for the installation and operation. The instrumentation will be maintained by PhD-level research-track faculty and staff. Consistent with its record of significant investments in biophysical research infrastructure and facilities, MCW has committed funds toward the total cost, space to house the requested instrument, 50% of the expenses for its maintenance, and salary support for training and supervision.