Extended Data Fig. 3: Transcriptional and chromatin mapping in H3.3 WT and K27M expressing hindbrain NSCs.

a. RNA-seq volcano plot presenting the gene expression changes observed between NSC cultures expressing WT or K27M-mutant H3F3A. Red and blue denotes genes significantly up and down-regulated, respectively in K27M cultures. b. Box plots presenting the expression level of the indicated genes in a cohort of H3.3 WT (black) and K27M-mutant (red) DIPG patient samples. This data was downloaded from the pediatric cBioportal database (https://pedcbioportal.kidsfirstdrc.org), n = 118 WT and n = 71 H3.3 K27M independent tumour samples. The box plots present median values and interquartile ranges (IQR), with whiskers representing quartiles 1/3 ± (1.5 × IQR) respectively. c. Gene Set Enrichment Analysis (GSEA) of genes up- and down-regulated in K27M expressing NSC cultures compared to genes up- and down-regulated in K27M-mutant patient samples. d. Genome-wide correlations of H3.3 WT and K27M (V5) ChIP-seq read densities derived from embryo GCGR-NS13. The correlation coefficient for the two conditions is indicated. e. Violin plots representing the normalised abundance of H3.3 (V5) at the indicated genomic locations in embryos GCGR-NS19 and GCGR-NS13, in H3.3 WT (left panel) and K27M (right panel) conditions. An equivalent number of non-PRC2 target, repressed gene promoters (as compared to PRC2 target genes) were randomly selected from RNA-seq results, as an additional control set of genomic loci. Data presented are from each biologically independent embryo sample, n = 1/embryo. f. Meta-plots of average H3.3-WT ChIP-seq enrichment in biological duplicate experiments in genomic windows ±10kb of Active Promoter, Active Enhancer, PRC2 target promoters and non-PRC2 target Repressed gene promoters.