Fig. 3

Shared H3K27me3-enriched genes in PFAs and H3K27M DMGs exhibit heterogeneity corresponding to tumor anatomic location. a Workflow of H3K27me3 ChIP-seq analysis. Genes with promoter (2 kb upstream) or gene-body H3K27me3 peaks in more than two-thirds of PFAs (≥ 7/9 samples) and H3K27M DMGs (≥ 5/6) were identified as H3K27me3-retaining genes in each tumor type. The resulting gene sets were then compared to identify shared and subtype-specific H3K27me3-retaining genes. b Heterogeneity in expression of H3K27me3-retaining genes was identified by plotting the expression standard deviations of each gene identified from 3a in H3K27M DMGs (X-axis) and PFAs (Y-axis). Genes, including HOXD3, for which expression data was unavailable for both subtypes were excluded from plotting. c Histogram of HOXA2 expression values per tumor (X-axis = binned expression scores, Y-axis = number of tumors) in PFAs. d Comparison of HOXA2-high (expression > 5 units) vs. -low (expression < 5.5 units) groups based on the local minimum between modes in DNA-methylation defined, anatomically distinct PFA1 (n = 49) and PFA2 (n = 29) subtypes of PFAs. e Histogram of HOXA2 expression values per tumor (X-axis = binned expression scores, Y-axis = number of tumors) in H3K27M DMGs. f Comparison of HOXA2-high (expression > 4.5 units) vs. -low (expression < 4.5 units) groups based on the local minimum between modes with anatomic location of H3K27M DMGs (pons = 65; thalamus = 26; midline not otherwise specified (NOS) = 5; cerebellum = 1, and other = 2). g H3K27me3 ChIP-seq tracks for PFAs (black, n = 9, from Mack et al. [33], and Bayliss et al. [31]) and H3K27M DMGs (red, middle, n = 6, from Bender et al. [11] and Harutyunyan et al. [32]) tumors and patient-derived H3K27M cell lines (red, bottom, n = 2 from Wang et al. [63]) at the HOXA and HOXD clusters. Positions of proximal HOX genes associated with variable expression (HOXA2, HOXA4, HOXD3) indicated with arrowheads. Regions identified with differential patterns in H3K27me3 enrichment are labeled as variable (orange) and constant (green). Anatomic site for DMGs and PFA1/2 status were not available. h Expression patterns of HOXA2 and HOXD3 in single-cell RNA-sequencing (scRNA-seq) experiments (Filbin et al. and Gojo et al.) [4, 6]. Tumors were grouped by anatomic site (pons vs. thalamus for H3K27M DMG) or methylation-based subtype (PFA1 vs PFA2 for PFAs). i H3K27me3 ChIP-seq tracks at the HOXD locus from isogenic patient-derived H3K27M DIPG XIII and BT425 cell lines with or without H3K27M-knockdown