PROJECT ABSTRACT/SUMMARY Cell type-specific gene expression, the basis for complex multicellular life, is achieved through packaging a common, invariant genome into higher-order chromatin structures with myriad levels of regulation. Covalent post- translational modifications (PTMs) at specific histone residues are major points of regulation that are dynamically modified throughout development to ensure proper tissue lineage specification, cell fate transitions, and maintenance of cell identity. Mutations in histone modifiers, as well as the histone substrates themselves, underlie a variety of human diseases including cancer. Recurrent lysine-to-methionine mutations at residue 27 of histone H3 (H3 K27M) are found in >80% of diffuse intrinsic pontine gliomas (DIPG), a high-grade pediatric brain cancer with dismal prognosis. The H3 K27M oncohistone drives gliomagenesis in part through its inhibition of polycomb repressive complex 2 (PRC2), the methyltransferase complex responsible for methylating H3K27, resulting in genome-wide depletion of the repressive mark H3K27me3. This proposal endeavors to address a long-observed yet poorly characterized aspect of H3K27M-driven PRC2 dysregulation: residual PRC2 activity at focal sites of H3K27me3. Leveraging biochemical, genomic, and cell- based approaches, the experiments described in this study seek to define the PRC2 subunits necessary for targeting to, and catalytic activity at, sites of PRC2 recruitment in DIPG cells. These results stand to significantly deepen our understanding of H3K27M-dysregulated chromatin and may reveal exploitable vulnerabilities for H3 K27M diffuse gliomas. The training plan outlined in this proposal will be performed at the University of Wisconsin-Madison within an excellent institutional research environment boasting cutting-edge resources, facilities, and dynamic opportunities for collaboration. Execution of this training plan, under the guidance of expert mentors and collaborators in fields of cancer biology, chromatin biochemistry, proteomics, and genomics, will provide an invaluable foundation meant to launch a successful career as a physician-scientist cancer researcher.