Diffuse midline gliomas (DMGs) carrying the H3 K27M inhibitor histone exhibit global H3K27me3 reduction but retain focal enrichment at PRC2-bound CpG islands. Understanding how this residual methylation is maintained and its functional role is a critical unresolved question. 2/13

Recent studies show non-core (accessory) subunits are necessary for PRC2 targeting in mammalian cells. These subunits, interacting via SUZ12, guide PRC2 precisely to CpG islands, ensuring accurate H3K27me3 deposition and gene silencing. 3/13

To investigate PRC2 targeting, we disrupted PRC2 subcomplexes using SUZ12 mutants. VEFS truncation removes all accessory subunits, while the non-core mutant (ncMUT) selectively disrupts PRC2.1 and PRC2.2 subcomplexes, preserving core PRC2 integrity. 4/13

ChIP-seq analyses reveal PRC2.1 and PRC2.2 subcomplexes act collectively to recruit PRC2 to critical genomic loci in K27M DMGs. Mutants lacking these accessory subunits (ncMUT, VEFS) show severely impaired PRC2 localization at key target genes, including CDKN2A. 5/13

RNA-seq data show that disrupting PRC2 subcomplexes (ncMUT, VEFS) in DMG cells significantly derepresses genes critical for neurodevelopment. PRC2.1/2.2 complexes thus maintain silencing of developmental regulators essential for tumor cell identity. 6/13

EZH2 inhibition (Tazemetostat) or disruption of PRC2 subcomplexes (shSUZ12, ncMUT, VEFS) derepresses CDKN2A, causing significant growth defects. Knockout of CDKN2A rescues DMG cells from these proliferation defects, highlighting its critical role downstream of PRC2. 7/13

In vitro and in vivo data confirm PRC2 subcomplexes collectively support K27M-DMG proliferation. Disrupting PRC2 recruitment (ncMUT, VEFS) dramatically reduces tumor growth, and remarkably, VEFS expression alone leads to 100% survival in orthotopic xenograft models. 8/13

Expression of the VEFS truncation globally elevates H3K27me3 independently of non-core accessory subunits. This increase occurs across multiple cell types, including both K27M-mutant DMG cells and wild-type H3 cells, highlighting intrinsic SUZ12 regulatory mechanisms. 9/13

Widespread, diffuse H3K27me3 deposition induced by VEFS expression titrates canonical PRC1 away from key genomic targets. This dispersal of PRC1 disrupts Polycomb domains, resulting in transcriptional derepression and upregulation of genes normally silenced by PRC1. 10/13

Domain analysis identifies a discrete nucleic acid-binding domain (NBD) within SUZ12 responsible for constraining PRC2 activity. CryoEM studies showed the NBD in close proximity to nucleic acids. Loss of this region (NEC, VEFS) causes global H3K27me3 hypermethylation. 11/13

PRC2 complexes containing the SUZ12-NBD strongly bind diverse forms of nucleic acids, including structured RNA and double-stranded DNA, significantly inhibiting enzymatic activity. PRC2 lacking the NBD (VEFS) is resistant to nucleic acid-mediated inhibition. 12/13