Fig 2: Oncohistone-mimic, EZHIP blocks H3K27me3 spreading by inhibiting allosterically-stimulated PRC2. Schematic depicting the molecular mechanism by which EZHIP expression leads to the loss of H3K27me3 spreading. In cells lacking EZHIP expression (in blue), PRC2 is recruited to CpG islands and catalyzes H3K27me3 at recruitment sites. H3K27me3-marked nucleosomes proximal to the recruitment site allosterically activate PRC2 and promote spreading in cis to form broad H3K27me3 domains. Similarly, in cells expressing EZHIP (red), PRC2 is recruited to CpG islands and can catalyze H3K27me3 at proximal nucleosomes due the weaker inhibition potential of EZHIP for unstimulated PRC2. However, spreading of H3K27me3 is blunted due to enhanced binding of EZHIP to allosterically stimulated PRC2. The formation of H3K27me3-PRC2-EZHIP ternary complex inhibits PRC2 spreading and provides a mechanism to explain the formation of narrow H3K27me3 peaks found in PFA ependymomas

Fig 3: PFA tumors expressing EZHIP display lowered expression of CDKN2A by increasing local H3K27me3. a Genome browser representation of RPKM normalized H3K27me3 ChIP-Seq profile in Posterior Fossa Group-A ependymomas expressing EZHIP and supratentorial ependymomas. Blue and red boxes represent intergenic and residual (retained) H3 K27me3 respectively. b Boxplot displaying the genomic region occupied by H3K27me3 peaks in supratentorial and PFA ependymomas. p-value was determined for the two groups of tumors using Wilcoxon rank sum test. Center line in the boxplot represents the median, bottom and top of the box represents 25th and 75th quartiles; whiskers extend to 1.5× interquartile range. n represents the number of H3K27me3 peaks in that sample. c Heatmap displaying the normalized H3K27me3 enrichment at peaks retained in PFA ependymomas expressing EZHIP in PFA (left) and DIPG cell lines containing H3K27M mutations (right). d Scatter plot displaying the expression of all genes in PFA and supratentorial ependymomas. Red and blue points represent upregulated and downregulated genes in PFA tumors relative to supratentorial tumors. Encircled, green point represents the expression of CDKN2A gene. e Genome browser representation of H3K27me3 ChIP-Seq profile at the CDKN2A locus in PFA and supratentorial tumors (top); and H3.3WT or H3.3K27M DIPG lines (bottom). f Expression of CDKN2A gene in ependymomas with low (blue, n = 7) or high (red, n = 71) expression of EZHIP, as measured by FPKM values. Ependymomas were grouped based on their expression of EZHIP. n represents the number of individual tumor sample within that group. p-value was calculated using non-parametric t-test

Fig 4: EZHIP is a competitive inhibitor of PRC2. a Silver stained SDS-PAGE gel showing the components of native PRC2 purified from HeLa cells. b In vitro methyltransferase reactions with PRC2 and oligonucleosome substrate. Full length recombinant EZHIP WT or R405E mutant purified from E. coli was titrated into the reaction mixture as shown. Half of the reaction was subjected to SDS-PAGE followed by fluorography and the other half was used for quantification by scintillation counting (Supplementary Fig. 2A). c Sequence alignment showing the similarity between lysine(27)-to-methionine mutated optimal PRC2 substrate and K27M-like EZHIP peptide (top), and H3K27M and K27M-like EZHIP peptide (bottom). d Formation of salt bridges between EZHIP R405 (or H3 R24) with EZH2 D652 (2.4 Å) and Q648 (3.5 Å), while R407 residue is exposed to solvent. e Increasing concentrations of oligonucleosome substrates were incubated with PRC2, SAM, and varying concentrations of EZHIP inhibitor. Ki was determined by fitting Michaelis-Menton and Lineweaver-Burk curves with competitive mode of inhibition. Error bars represent the standard deviation. ± represents standard error. f 0.3 µM EZHIP M406A or M406K mutant proteins were incubated with increasing concentrations of PRC2 with 1 µM 3H-SAM and 25 µM H3K27me3 peptide. Reactions were subjected to SDS-PAGE followed by fluorography. g Immunoblots of 293T expressing EZHIP WT or M406E/I/K/R mutants. Source data for Immunoblots and PRC2 assays are provided in source data file

Fig 5: EZHIP forms a stable complex with PRC2 and lowers H3K27me3 in vivo. a Immunoblots of whole cell lysates generated from 293T cells expressing HA-FLAG-tagged H3.3 WT or K27M/R or FLAG-tagged EZHIP WT or R405E mutant. b Schematic showing the strategy for purification of EZHIP-associated proteins from 293T cells. c Silver stain of FLAG-tagged EZHIP association with PRC2. d Immunoprecipitated material from c were subjected to mass-spectrometry for protein identification and quantification. Proteins identified with at least 2 unique peptides and a log2 fold-change over the mock negative control of greater than 4 were considered hits. Protein abundances were calculated using ProteomeDiscoverer software. Complete list is provided as Supplementary Dataset 2. e Silver stained SDS-PAGE gel of mono-Q column fractions of M2 eluate from c. f, g Immunoblots displaying co-fractionation of EZHIP and PRC2 subunits on mono-Q and Superdex 200 columns, respectively. h Sequence alignment displaying the conserved 12 amino acid sequence in the EZHIP C-terminus. Red and blue domains represent the EZHIP conserved sequence and the site of hotspot mutations in PFA ependymomas respectively. i Immunoblots of lysates generated from mouse embryonic fibroblasts with EED knockout or expressing human EZHIP WT, H3K27M, or H3K27R. j Immunoblots of lysates generated from human 293T cell lines expressing human EZHIP WT or D81Y or mouse EZHIP. k Immunoblots of 293T expressing EZHIP WT, R405E, R407E or R405A;R407A mutants. Source images for Immunoblots are provided in a source data file