Extended Data Fig. 9: Uncoupling the effect of histone variants from cell-of-origin chromatin state and cycling rate.
a. Validation of CRISPR removal of ACVR1 in H3.1K27M ACVR1-mutant cell lines by MiSeq (multiple deletions on both alleles (complete KO)). b. Validation of CRISPR removal of H3K27M in H3.1K27M cell lines DIPGIV and DIPG36 (1 bp deletion on K27M allele (frameshift)) and DIPG21 (2 bp deletion on K27M allele) by MiSeq and Western Blot. For Western Blot, G477, an H3.1 WT HGG patient-derived cell line, was used as control. CRISPR removal of H3K27M in H3.3K27M cell lines has been reported previously for BT245 and DIPGXIII in Krug et al., Cancer Cell, 2019 33; and for HSJ019 in Harutyunyan et al., Cell Reports, 2020 35. c. Doubling time of H3.3K27M and H3.1K27M HGG cell lines (DIPGXIII, N = 4 biological replicates; HSJ019, N = 3; DIPG36, N = 9; DIPGIV, N = 12). Error bars represent mean +/− SD. d. Doubling time of H3.1K27M cell line DIPGIV in ACVR1 mutant and ACVR1-KO conditions. Error bars represent mean +/− SD. e. Schematic of experimental design. f. Heatmap showing distribution of Rx-normalized ChIPseq signal for H3K27me3 in DIPGXIII at CpG islands (CGIs), flanked by 20kbp on either side. g. Rx-normalized H3K27me3 tracks in each condition at a representative genomic region. Y-axis limit is indicated in brackets and identical for all tracks. h. Left: Rx-normalized H3K27me2 tracks in each condition at the same region as in (g). Y-axis limit is indicated in brackets and identical for all tracks. Right: genome-wide distribution of H3K27me2 ___domain length in each condition (H3.3K27M, N = 16,630 domains; K27M-KO, N = 3388; H3.1K27M, N = 11,568). i. Heatmap showing distribution of Rx-normalized ChIPseq signal for H3K27me2 in DIPGXIII H3K27me2 domains across the genome in each condition. Domains are scaled to 50 kb and flanked by 50 kb on either side. The maximum of the color scale is set to the 90th percentile value across all data points.
