PROJECT SUMMARY Our long-term goal is to develop new chemical probes to dissect the molecular mechanisms associated with epigenetic-complexes and transcriptional regulation. Establishing detailed mechanisms for the dynamic regulation of gene expression remains a significant challenge for the field of epigenetics. Our research program is addressing these challenges by developing selective chemical probes, discovery of new protein- protein interactions, and improving structural biology and biophysical approaches to quantify these dynamic multivalent interactions. These approaches are helping to create a detailed picture of diverse molecular assemblies of epigenetic complexes and facilitating development of new epigenetic therapies. Bromodomain-containing proteins are a subset of epigenetic reader protein, and are an emerging protein- drug class for epigenome therapy. My research program is developing chemical biology approaches to study two specific bromodomain-containing proteins, BPTF and BRD4. One innovation behind our approach uses a 19F NMR protein-based methodology for the discovery of isoform selective bromodomain inhibitors. Due to the hyper-responsive nature of fluorine to ligand binding and simplified spectra, this structural biology tool is ideally suited to rapidly screen small molecules, characterize the transient interactions associated with native transcription factor-protein interactions, and to quantify the dynamics of the interactions from micro to millisecond timescales. Over the next five years, we will further develop this approach for enabling small molecule discovery, characterizing large (> 50 kDa) proteins, and quantifying the affinity and dynamics of multidomain proteins associated with synthetic nucleosomes. With their discovery enabled by 19F NMR, our new chemical probes for BPTF reader domains have potential for high impact as chemical tools for inhibiting cancer progression and regulating autophagy, while more specific BRD4 chemical probes will help uncover new biology in cancer and inflammation obscured by less specific tool compounds. We will use these tools to dissect the functional roles of these bromodomains on transcription, including gene regulation, chromatin engagement, and novel-protein-protein interactions. Next generation chemical probes will be further developed including bifunctional molecules as inhibitors of multi-domain proteins, PROTACs with improved selectivity profiles, and synthetic transcription factors through conjugation to DNA-targeting polyamides. This proposal has broad biomedical significance for early lead discovery and studies of epigenetic reader domains important in human health and disease. Given that epigenetic proteins represent a major class of potential drug targets, our new 19F NMR small molecule discovery method and cell-based approaches for epigenetic reader domains described here could significantly increase the repertoire of targets and thereby open up new avenues for drug d...