Project Summary Over the past two decades significant effort has been directed to the identification of ligands that target the regulation of epigenetic marks. These post-translational modifications (PTMs) control all aspects of gene expression and are often deregulated in disease, providing attractive vectors for therapeutic intervention. Currently, despite significant investment, marketed drugs in this area have generally arisen from phenotypic screening as opposed to a priori design. One reason for this lack of success is the high degree of complexity within epigenetic regulation, where a protein target may perform multiple contradictory roles based upon cellular context. Additionally, the high homology between epigenetic proteins and their isoforms makes the design of selective inhibitors incredibly challenging. It is therefore critical that both the protein targets of a given ligand and the downstream epigenetic consequences are well characterized before clinical evaluation. This presents a singular challenge as epigenetic states (and therefore epigenetic consequences) differ dramatically between cell types and populations. In this proposal, we will develop proximity proteomics methods to understand how small molecule ligands remodel the chromatin microenvironment over time. We will achieve this through the targeted deployment of iridium catalysts to chromatin via ultrafast split intein splicing. Upon visible light irradiation, these catalysts activate biotin bearing diazirines within a short radius (through a process called Dexter energy transfer) which subsequently release molecular nitrogen and a highly reactive carbene. These carbenes insert into C-H and X-H bonds of biomolecules within ~10 nm, which can be enriched for downstream ‘omics analysis. This method will be used to monitor the biomolecules that associate to and dissociate from chromatin following ligand incubation. At short time points, this will provide target identification as ligand bound proteins no longer interact with chromatin. Following longer incubation, we will measure the functional effect of inhibition of epigenetic modulators as chromatin PTMs reach a new steady state. We will apply this method to two important areas of chromatin regulation that have been the target of intense drug development with limited success, lysine demethylation and c-myc based transcription. We hope to use this method to shed light on these important vectors for gene regulation, identifying new protein targets and off-targets of established inhibitor classes. Broadly, this project will provide a valuable tool to study ligands acting at chromatin that can be applied to many aspects of nuclear biology and drug development, paving the way for better drug candidates, and ultimately improving human health.