Fig. 3
From: Pediatric glioma histone H3.3 K27M/G34R mutations drive abnormalities in PML nuclear bodies
PML and H3.3 profiles in H3.3 K27M mutant patient-derived glioma cells. Immunofluorescence of A PML (red) and DAXX (green) and B PML (red) and ATRX (green), in H3.3 K27M in the DIPG XIII patient-derived glioma cell line compared to an H3.3 WT (K27M KO) isogenic control. Scale bar 2 µM. C PML-NBs per cell in H3.3 K27M and H3.3 WT (K27M KO) DIPG XIII cells (n = 103). P-values were calculated using two-tailed Student’s t test (*P < 0.0001). 25th, median, and 75th percentiles are shown. D ChIP-seq read count density heatmaps (normalized for total read counts) of H3.3 ChIP-seq at TSS ± 3 kb in H3.3 WT (K27M KO) and H3.3 K27M DIPG XIII cells. E ChIP-seq read counts at H3K27ac enriched promoters (TSS ± 1 kb) in H3.3 WT (K27M KO) and H3.3 K27M DIPG XIII cells (n = 5108). P-values were calculated using two-tailed Student’s t test (*P < 0.0001). 25th, median, and 75th percentiles are shown. F Composite profile of H3.3 at H3K27ac enriched promoters, centered on transcriptional start sites in H3.3 K27M DIPG XIII glioma cells compared to H3.3 WT (K27M KO) isogenic control. G Representative UCSC genome browser profile of H3.3 ChIP-seq in H3.3 K27M DIPG XIII and H3.3 WT (K27M KO) isogenic control (Ak3; hg38; chr9:4,635,308 – 4,827,283). H–N Experiments repeated across an independent (BT245) H3.3 K27M patient-derived glioma cell line. H Immunofluorescence of PML (red) and DAXX (green) and I PML (red) and ATRX (green), in H3.3 K27M BT245 cells compared to an H3.3 WT (K27M KO) isogenic control. Scale bar 2 µM. J PML-NBs per cell in H3.3 K27M BT245 and H3.3 WT (K27M KO) cells (n = 53). P-values were calculated using two-tailed Student’s t test (*P < 0.0001). K ChIP-seq read count density heatmaps of H3.3 ChIP-seq at TSS ± 3 kb in H3.3 WT (K27M KO) and H3.3 K27M BT245 cells. L ChIP-seq read counts at H3K27ac enriched promoters (TSS ± 1 kb) in H3.3 WT (K27M KO) and H3.3 K27M BT245 cells. P-values were calculated using two-tailed Student’s t test (*P < 0.0001). 25th, median, and 75th percentiles are shown. M Composite profile of H3.3 at promoters, centered on transcriptional start sites in H3.3 K27M BT245 ells compared to H3.3 WT (K27M KO) isogenic control. N Representative UCSC genome browser profile of H3.3 ChIP-seq in H3.3 K27M BT245 and H3.3 WT (K27M KO) isogenic control. Rims2; hg38; chr8:103,455,749 – 103,545,748