Project Summary (Overall) The chromatin landscape impacts fundamental cellular processes including gene expression, DNA damage repair, and cell fate and differentiation, all of which are extensively dysregulated in cancer. The collective number of oncogenic mutations in chromatin regulators has led to the emerging view of driver mutations underlying cancer epigenomes. In keeping with theme, over the past several years our current collaborative program members have been critical to the discovery and characterization of ‘classical’ oncohistone mutations in the histone H3.3 N-terminal tail. These mutations globally alter chromatin by inhibiting the activity of chromatin modifying enzymes. While much progress has been made to understand these effects, important questions remain including the nature of the dysregulation of chromatin modifying enzymes by these mutations and how these mutations lead to cancer. In addition, Program members have recently identified an expanded number of cancer-associated somatic histone mutations that occur in as many as 4 % of human cancers and involve both globular and tail domains of all four core histones. These findings generate additional important questions such as if the newly observed histone mutations have functional effects on chromatin and through what mechanisms they rely on. Given that some of the most prevalent mutations are in the globular domains of histones, we hypothesize that these mutations affect nucleosome structure and/or integrity. Lastly, we and others have also identified a novel function of the EZHIP protein, which is overexpressed in posterior fossa A ependymomas, and acts as an oncohistone mimic to directly inhibit the Polycomb Repressive Complex 2 (PRC2) function. The single goal of our Program is to illuminate the molecular mechanisms underlying classical and novel “oncohistone” mutations and oncohistone mimics to advance the diagnosis and exploration of therapeutic avenues for the cancers. Specifically, we will: i) develop and employ novel patient sample-, cell culture- and animal model-based systems to recapitulate oncohistone-associated cancers and investigate the underlying pathogenic mechanisms; ii) evaluate the activity of a comprehensive set of novel cancer-associated histone mutations using a multidisciplinary approach that includes genetics (barcoded oncohistone libraries, mouse models, barcoded-cell lines), epigenetics (ChIP-seq, ATAC-seq, DNA-methylation profiling), transcriptomics (RNA-seq), and chemical biology (“designer chromatin”, small molecule inhibitors); iii) define the mechanisms by which oncohistones, and oncohistone-mimics, dysregulate the Polycomb Repressive Complexes (PRC1 and PRC2) activity to promote gliomas and bone tumors; and iv) identify which of novel histone mutations perturb chromatin states (and by what mechanisms) to subsequently cause cellular phenotype using newly developed high-throughput biochemical and yeast genetic screening tools. These studies wil...