SUMMARY: The known histone H3K36-directed methyltransferases are essential for normal animal development and are frequently dysregulated in human cancers. We have found that high-frequency histone H3 mutations (oncohistones) exploit normal chromatin-based regulatory mechanisms, including H3K36 methylation, to drive tumorigenesis. Previously, we found that H3.3 G34 oncohistones selectively block SETD2-mediated H3K36 methylation and affect the activity of gene enhancers that results in aberrant cellular differentiation and proliferation. Moreover, we found that the H3 K36M oncohistone promotes genome-wide changes in histone and DNA methylation through competitive inhibition of NSD1/2 and SETD2 enzymes. We will leverage and extend our preliminary findings to define the mechanisms by H3.3 G34 and H3 K36M oncohistones achieve pro-tumorigenic gene expression programs through misregulation of H3K36 methylation. We will employ a multi-disciplinary approach that integrates biochemical, genomic, and molecular methods to enhance our understanding of K36M and G34 oncohistones and apply our understanding toward diagnostic and therapeutic applications. Specifically, we will: i) identify the changes in chromatin landscape by histone mutations using cell-based systems and patient tumor samples; and ii) characterize misregulated developmental programs that help establish tumorigenesis. These studies will provide guidance for the development of therapeutic strategies designed to ameliorate the pathogenic effects of NSD1/2 and histone H3 mutations in human cancers. We will also extend our findings to elucidate the mechanisms by which NSD1 and H3 K36M mutations alter the chromatin landscape in squamous cell carcinomas (Aim 1). We will define the mechanisms of by which G34 mutations alter chromatin modifications and gene expression, and we will define the role of H3.3 chaperone pathways in mediating G34 phenotypes (Aim 2). Additionally, we have found that H3K36 methylation opposes PRC2 activity, thus preventing Polycomb-mediated gene repression. We will now leverage these biochemical findings to determine the function of the EZH2 H3K36-binding pocket in different tumorigenesis models (Aim 3). Expected results from our study will lead us formulate novel theories and provide crucial mechanistic insights of these oncohistones which can be readily tested in in vivo cancer models (Project 1,2) and in vitro chemistry platforms (Project 3). To accomplish these aims, Project 4 also requires close interactions with both Cores.