Fig. 2 |. PRC2 target DNA methylation is a key switch in the differentiation of malignant GBM cells.

a, Two-dimensional representation of cellular states across seven GBM samples (n = 844 malignant-only cells that passed scRNA-seq quality control). b, Heat map of P values obtained when comparing mean CpG methylation at promoters (TSS ± 1 kb) between GBM cellular states (n = 706, cells in a with matched scDNAme data). c, Volcano plot of differentially methylated promoters between the NPC/OPC-like and AC/MES-like GBM cellular states. Promoters (n = 459) with an absolute mean DNA methylation difference of greater than 5% and a P value below 0.05 were defined as differentially methylated. Genes correlated with the classical TCGA GBM subtype⁴⁵ (blue) and genes corresponding to PRC2 targets⁴⁶ (red) are highlighted (BH FDR-adjusted permutation-based P < 0.05). d, Enrichment score plots (reflecting whether a gene set is over-represented at the top or bottom of the ranked list of genes used in c; n = 15, 218 genes) for gene sets enriched at hypomethylated promoters in NPC/OPC-like (top) or AC/MES-like (bottom) cells. e, Volcano plot of differentially methylated enhancers in comparison of NPC/OPC-like and AC/MES-like GBM cellular states. Putative gene targets⁵¹ of hypomethylated enhancers in stem-like cells that are PRC2 targets⁴⁶ are labeled in red. Key neurodevelopmental genes are highlighted in orange. f, Proportion of chromatin states at hypomethylated promoters in GBM AC/MES-like cells (defined in c), randomly sampled promoters (1,000 promoters sampled) and hypomethylated promoters in GBM stem-like cells (defined in c). g, UMAP plots of scATAC-seq GBM data⁵⁵ (sample SF11956) overlaid with density plots showing chromatin accessibility of genes belonging to NPC/OPC-like gene modules (top) and PRC2 targets⁴⁶ (bottom). h, Comparison of rank (by P value) in the enrichment of open chromatin at transcription factor-binding sites (n = 188) between NPC/OPC-like (top) and AC/MES-like (bottom) cells. PRC2 subunits are highlighted in red. i, Spearman’s rank-order correlation between mean DNA methylation at the promoters of PRC2 targets⁴⁶ and RNA differentiation score (defined as the difference in gene module scores between AC/MES-like and NPC/OPC-like cellular states) for 67 TCGA GBM samples^40,41. A linear regression line (red) with its 95% confidence interval is shown. j, Same as in i, for PRC2 target⁴⁶ gene expression and RNA differentiation score. k, Comparison of performance, as assessed by ROC curve, in correctly classifying bulk TCGA GBM samples^40,41 to DNA methylation glioma subtypes (LGm4 or LGm5)³⁹ using 1,300 previously defined CpG sites³⁹, mean global DNA methylation and mean PRC2 target⁴⁶ DNA methylation. P values were determined by two-sided Mann-Whitney U test (b), generalized linear model (c,e), permutation test (f), BH FDR-adjusted hypergeometric test (h) or Spearman’s rank-order correlation (i,j).