Polycomb group proteins are essential for normal development and are frequently dysregulated in human cancers. Several mechanisms have evolved to fine-tune PRC2 activity, and we have found that high- frequency histone H3 mutations (oncohistones) and the oncohistone-mimic EZHIP exploit these regulatory mechanisms to drive tumorigenesis. We found that H3.3 G34 oncohistones selectively promote PRC2 repression of gene enhancers, and that H3 K27M and EZHIP directly inhibit PRC2 function to promote dysregulated gene expression, aberrant cellular differentiation and cell proliferation. We will leverage and extend our preliminary findings to define the mechanisms by which H3 K27M and H3.3 G34X oncohistones and the oncohistone-mimic EZHIP achieve pro-tumorigenic gene expression programs through misregulation of Polycomb activity. Despite a massive reduction in H3K27 methylation levels caused by EZHIP or H3 K27M, our work has revealed residual PRC2 and H3K27me3 at CpG islands in gliomas containing these oncohistones. Furthermore, evidence suggests that this residual PRC2 activity is important for tumor proliferation. We will also extend our findings to elucidate the mechanisms by which PRC2 is targeted to genomic loci for repression in K27M and EZHIP-containing tumors (Aim 1). While we have found that K27M and EZHIP preferentially inhibit the allosterically activated form of PRC2, the exact molecular determinants necessary for interaction between K27M/EZHIP and PRC2 remain unknown. Therefore, we now seek to determine the critical molecular events necessary for EZHIP and K27M inhibition of PRC2 using a combination of cell-based and in vitro biochemical assays (Aim 2). Additionally, we will explore the regulation of PRC2 and H3K27 methylation spreading by H3K36 methylation. We have found that H3K36 methylation opposes PRC2 activity, thus preventing Polycomb-mediated gene repression. We have made progress on understanding the interplay between H3K36 methylation and PRC2 by identifying a highly conserved binding pocket on the surface of EZH2 for H3K36. We will now leverage our biochemical findings to determine the function of the EZH2 H3K36-binding pocket in different tumorigenesis models (Aim 3). Expected results will help us formulate novel theories and provide crucial mechanistic basis underlying the pathogenesis by 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.
Recently specific mutations in our DNA packaging proteins known as histones are found in a variety of cancers. This Project aims to understand how mutant histones affect the activity of Polycomb group proteins in order to promote tumorigenesis. Expected results from proposed studies will provide novel insights into molecular targets for disease diagnosis and advance therapeutic intervention.
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