PROJECT SUMMARY Chromatin accessibility plays a critical role in determining the gene expression programs that underlie cellular fates and functions in development and disease. Conserved chromatin modifying complexes, such as Polycomb-Group (PcG) and Trithorax-Group (trxG) proteins, are believed to engage in a competitive process to set either repressed or accessible chromatin states, respectively. Mutations in these multiprotein complexes are known to cause a wide range of diseases, including developmental disorders and cancers. However, the precise molecular mechanisms that govern the competition between repressive and activating complexes for the establishment, maintenance, or alteration of chromatin accessibility remain largely unexplored. This K99/R00 Pathway to Independence Award proposal seeks to define the mechanisms and dynamics that shape the competition between the Polycomb Repressive Complex 1 (PRC1) and the activating chromatin remodeler mSWI/SNF for chromatin accessibility in health and disease contexts. The candidate for this award, Dr. Stefan Niekamp, is a biophysicist with experience in microscopy and biochemistry. He trained in the laboratory of Dr. Ronald Vale at the University of California – San Francisco, where he determined the conformational dynamics of the microtubule-based motor protein dynein. During his postdoctoral studies in Dr. Robert Kingston’s laboratory at the Massachusetts General Hospital, he has established a tractable single-molecule assay which provides a unique perspective with unprecedented spatiotemporal resolution to derive kinetic models and to determine the sequence of events during transitions between chromatin states. Combining this reconstitution approach with cellular super-resolution microscopy and cutting-edge genomic approaches, that the candidate will learn during the mentored phase, this will provide a powerful system to discern the molecular basis of chromatin accessibility regulation by the competition of chromatin modifying complexes. To obtain these skill sets, Dr. Niekamp will work with world renowned experts in the fields of transcriptional regulation, genomics, and super-resolution imaging at Harvard University. In addition, the candidate will take a suite of courses to advance his professional development. Together, this project will (i) define the roles of PRC1 composition in organizing chromatin and regulating gene repression (Aim 1), determine how repressive and activating complexes compete to control chromatin structure transitions (Aim 2), and identify the molecular mechanisms of cancer mutations in disrupting the balance between repressive and activating complexes (Aim 3). The successful execution of this proposal will equip the candidate with a suite of interdisciplinary skill sets in biophysics, cell biology, and genetics which will provide a robust platform to launch his career as an independent researcher to decipher the molecular basis of transcription regulation.