PFA ependymoma-associated protein EZHIP inhibits PRC2 activity through a H3 K27M-like mechanism

Abstract

Polycomb group (PcG) proteins are essential for development and are frequently misregulated in human cancers. Polycomb Repressive Complexes (PRC1, PRC2) function in a collaborative epigenetic cross-talk with H3K27me3 to initiate and maintain transcriptional silencing. Diffuse intrinsic pontine gliomas (DIPGs) have extremely low H3K27me3 levels mediated by H3 K27M oncohistone. Posterior fossa type A (PFA) ependymomas also exhibit very low H3K27 methylation but lack the K27M oncohistone. Instead, PFA tumors express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). We find that a highly conserved sequence within the C-terminus of EZHIP is necessary and sufficient to inhibit the catalytic activity of PRC2 in vitro and in vivo. Our biochemical experiments indicate that EZHIP directly interacts with the active site of the EZH2 subunit in a mechanism that is remarkably similar to the K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promote similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at the sites of PRC2 recruitment. Importantly, we find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a potential mechanism for loss of H3K27me3 spreading from CpG islands in vivo. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors-the K27M oncohistone and the EZHIP 'oncohistone-mimic'-that dysregulate gene silencing to promote tumorigenesis. Covalent modifications to both DNA and histone proteins allow chromatin to act as a dynamic information hub that integrates diverse biochemical stimuli to regulate genomic DNA access to the transcription machinery and ultimately establish and maintain cellular phenotypes. There is increasing appreciation that aberrant chromatin modifications are involved in the pathogenesis of cancer. Nowhere is this better supported than with the groundbreaking discoveries of highfrequency, somatic mutations in histones that are drivers of oncogenesis.

The interplay between DNA and histone methylation: molecular mechanisms and disease implications

“…Interestingly, oncohistone H3 lysine 27 to methionine (H3K27M) mutation-found in diffuse intrinsic pontine gliomas (DIPGs)-dominantly inhibits PRC2 activity and reduces global H3K27me3 yet results in DNA hypomethylation (Bender et al, 2013;Lewis et al, 2013). Furthermore, EZHIP/ CXORF67, a germline-specific gene, encodes a protein that binds to and blocks PRC2 spreading from H3K27me3-occupied CpG islands through an H3K27M-like mechanism (Jain et al, 2019;Ragazzini et al, 2019). EZHIP is silenced through promoter methylation in adult tissues, and its de-repression upon DNA hypomethylation results in PRC2 inhibition (Bayliss et al, 2016;Piunti et al, 2019).…”

Pediatric CNS tumors and 2021 WHO classification: what do oncologists need from pathologists?

“…In both subtypes, the final result is a loss of H3 K27 trimethylation (H3 K27me3) due to the inhibition of the methyltransferase activity of EZH2, which is the catalytic subunit of PRC2. In the first subtype, this inhibitory effect is a consequence of the H3 mutation (Bender et al, 2013;Lewis et al, 2013), whereas in the second subtype, this inhibitory effect is probably mediated by EZHIP overexpression, acting as an endogenous mimic of mutated H3 genes (Jain et al, 2019). EGFR-mutant subtype commonly shows small in-frame insertions/duplications within exon 20, which encodes the intracellular tyrosine kinase domain (TKD).…”

“…The oncogenesis of PFA is driven by epigenetic alterations, consisting in CpG islands hypermethylation and global DNA hypomethylation, associated with a reduction in the repressive histone mark H3 K27me3 (Mack et al, 2014). This reduction is the consequence of EZHIP overexpression, which mimics the oncohistone H3 K27M by binding to the EZH2 subunit of PRC2 complex and then inhibiting its methyltransferase activity (Hübner et al, 2019;Jain et al, 2019;Ragazzini et al, 2019). Immunohistochemistry is useful to demonstrate the loss of H3 K27me3 and also the presence of EZHIP overexpression (Antin et al, 2020;Nambirajan et al, 2021).…”