Histone H3.3 K27M Accelerates Spontaneous Brainstem Glioma and Drives Restricted Changes in Bivalent Gene Expression

Abstract

Diffuse intrinsic pontine gliomas (DIPGs) are incurable childhood brainstem tumors with frequent histone H3 K27M mutations and recurrent alterations in PDGFRA and TP53. We generated genetically engineered inducible mice and showed that H3.3 K27M enhanced neural stem cell self-renewal while preserving regional identity. Neonatal induction of H3.3 K27M cooperated with activating platelet-derived growth factor receptor α (PDGFRα) mutant and Trp53 loss to accelerate development of diffuse brainstem gliomas that recapitulated human DIPG gene expression signatures and showed global changes in H3K27 posttranslational modifications, but relatively restricted gene expression changes. Genes upregulated in H3.3 K27M tumors were enriched for those associated with neural development where H3K27me3 loss released the poised state of apparently bivalent promoters, whereas downregulated genes were enriched for those encoding homeodomain transcription factors.

Keywords: DIPG; H3K27me3; PDGFRA; bivalent; epigenetic; glioma; histone H3 K27M; knockin; mouse; oncohistone.

Figure 1.. H3.3 K27M Promotes Self-Renewal and Mediates Global H3K27me3 Depletion but Discrete Transcription Changes That Do Not Disrupt Regional Signatures

(A,B) Self-renewal of H3.3 K27M and H3.3 WT H-NSCs was assessed by clonogenic growth in methylcellulose at subsequent passages measuring number (A) and size (B) of spheres. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant. Error bars show +/− SEM. n = 3 per genotype (C) Scatterplot comparing expression in H3.3 K27M and H3.3 WT H-NSCs (RNA-seq; log2(FPKM+1)). (D) Plot of H3.3 K27M/H3.3 WT log2 ratio for RNA-seq versus H3K27me3 in H-NSCs. Colored dots depict genes up (red) and downregulated (blue) in H3.3 K27M compared to WT, with p < 0.05 and log2 fold change greater than 0.75 or less than −0.75, respectively, compared to the gene loci bulk (gray). (E) PCA of H3.3 WT and H3.3 K27M F- and H- NSCs. (F,G) Regional specific expression of Foxg1 (F) and Irx2 (G) shown by in situ hybridization (Allen Brain Atlas, E18.5) and average IGV tracks in H3.3 K27M F- and H-NSCs. *indicates Gm20554 locus near Irx2. Scale bar = 1 mm. (H) Average H-NSC tracks for three H3.3 K27M upregulated genes, Lin28b, Igf2bp2 and Plag1. *indicates Chchd7 locus near Plag1. In (F-H), tracks show H3K27me3, H3K27ac and H3K4me3 enrichment and RNA-seq in H3.3 WT or H3.3 K27M expressing NSCs. For each pair of tracks, n = 3 per genotype, scale is the same for both genotypes. See also Figures S1, S2, S3 and Tables S1–S3.

Figure 2.. H3.3 K27M Accelerates Medulloblastoma Formation Caused by Trp53-Deficiency

(A) Kaplan-Meier survival analysis in mice with induced H3.3 WT (n = 4), p53^cKO (n = 37; gray), or H3.3 WT;p53^cKO (n = 42), ns, p = 0.195. (B) Kaplan-Meier survival analysis with induced H3.3 K27M (n = 5), p53^cKO (n = 46), or H3.3 K27M;p53^cKO (n = 51), *p < 0.0001. Cohorts in A and B bred separately and used littermate controls to compare survival and tumor spectrum. (C,D) Location of macroscopic brain tumors in cohorts shown in the panel A (C) and the panel B (D). Supra, Supratentorial; CB, Cerebellar; Spinal, Spinal cord; BS, Brainstem. (E,F) H&E stain of representative supratentorial HGG (E) or medulloblastoma (F) observed in all genotypes. *in upper images indicates tumor. Scale bar = 1 mm (top images), 50 μm (bottom images). (G,H) Expression of FLAG-tagged H3.3 (upper images) and H3K27me3 (lower images) is shown by IHC on sections of representative supratentorial HGG (G) or medulloblastoma (H) for the indicated genotypes. Scale bar = 50 μm.

Figure 3.. Active PDGFRα Mutant Cooperates with Trp53 Deficiency to Accelerate High-Grade Glioma Formation

(A) Kaplan-Meier survival analysis in mice with induced mutant PDGFRA (n = 10), p53^cKO (n = 46), or PDGFRA;p53^cKO (n = 46), *p < 0.0001. (B) Location of macroscopic brain tumors for cohorts shown in (A): Supra, Supratentorial; CB, Cerebellar; Spinal, Spinal cord; BS, Brainstem. p53^cKO cohort same as in Figure 2B. (C) HGG in PDGFRA;p53^cKO mice. Sagittal section immunostained with anti-human PDGFRα (top image), and higher magnification of the pons for H&E stain and IHC of PDGFRα, Olig2 and H3K27me3 in representative HGG. Scale bar = 1 mm (whole brain image), 50 μm (higher magnification images). See also Figure S4.

Figure 4.. H3.3 K27M Accelerates DIPG Formation from Postnatal Neural Progenitors

(A) Kaplan-Meier survival analysis of cohorts with induced PDGFRA;p53^cKO combined with H3.3 WT (n = 44) or H3.3 K27M (n = 43; green), p < 0.0001. (B) Location of macroscopic brain tumors for cohorts shown in (A): Supra, Supratentorial; CB, Cerebellar; Spinal, Spinal cord; BS, Brainstem. (C) DIPG in H3.3 WT;PDGFRA;p53^cKO and H3.3 K27M;PDGFRA;p53^cKO mice. Sagittal sections immunostained with anti-human PDGFRα. Boxed areas in brainstem are shown at higher magnification for H&E, and IHC for PDGFRα, Olig2, FLAG-tagged H3.3, or H3K27me3 in representative HGG. Scale bar = 1 mm (whole brain images), 50 μm (higher magnification images). (D) Western blot of acid extracted mouse hindbrain NSCs, mouse DIPGs and xenografted human HGGs that express WT H3 (H3 WT) or the H3.3 K27M mutant from the endogenous H3f3a/H3F3A promoter. A H3.3 K27M-specific antibody is used to confirm mutation status. Epitope tagged mouse H3.3 K27M protein is slightly larger than human H3.3 K27M protein. Xenografted human HGG H3 WT is a cerebellar tumor and H3.3 K27M is a DIPG.

Figure 5.. Gene Expression in Mouse H3.3 K27M DIPGs Significantly Resembles That in Human H3.3 K27M DIPG

(A) Heatmaps of ssGSEA scores comparing signatures for human HGG subgroups (PN, proneural; Pro, Proliferative; Mes, Mesenchymal) and normal murine neural cell types (N, Neurons; Astro, Astrocytes; MO, Myelinating oligodendrocytes; NFO, Newly formed oligodendrocytes; OPC, Oligodendrocyte precursor cells) between spontaneous mouse DIPG expressing H3.3 K27M (n = 20) or H3.3 WT (n = 9) or primary human DIPGs with H3.3 K27M (n = 20) or H3 WT (n = 3). For each panel, tumors were first separated by genotype then ordered by hierarchical clustering of gene signatures from human HGG subgroups. (B) PCAs of mouse and human DIPGs in (A). (C) GSEA showing significant enrichment in H3.3 K27M mouse DIPGs of genes upregulated in H3.3 K27M compared with H3.3 WT human DIPGs. Running enrichment score plots (left) and gene expression heatmaps in mouse H3.3 K27M or H3.3 WT DIPGs showing top leading edge genes (right). (D) Expression of leading edge upregulated genes Pbx3, Eya1 and Plag1. Boxplots depict log2-scale RNA-seq CPM values for primary human and mouse DIPGs, and mouse hindbrain (H-NSC) and forebrain (F-NSC) NSCs expressing H3.3 K27M or H3.3 WT. Box plots show the interquartile range (IQR). Median is shown as a horizontal line, highest and lowest values up to 1.5 times the IQR are shown with dotted lines outside box, and outliers greater than 1.5 times the IQR are shown as black squares. See also Figures S5 and S6 and Tables S4 and S5.

Figure 6.. Spontaneous H3.3 K27M DIPGs Exhibit Global Changes in H3K27 Epigenetic State and Selective Expression Changes in PRC1 and PRC2 Targets

(A,B) Promoter based histograms representing counts within 40 bp bins across 4 kb region centered at TSS for H3K27me3 (A) or H3K27ac (B) in H3.3 K27M (n = 6) and H3.3 WT (n = 5) mouse DIPGs. (C) Plot of H3.3 K27M/H3.3 WT log2 ratio in mouse DIPGs for promoter regions comparing RNA-seq versus H3K27me3. Colored data points depict genes up (red) and downregulated (blue and purple) in H3.3 K27M tumors, with p < 0.05 and a log2 fold change greater than 0.75 or less than −0.75, respectively, compared to the gene loci bulk (gray). Purple data points show downregulated genes with H3K27me3 log2 fold change of −0.75 or greater (relative H3K27me3 retention). RNA-seq: H3.3 WT, n = 9; H3.3 K27M, n = 20. (D) Gene ontology and Enrichr ChEA2016 analysis of H3.3 K27M upregulated genes. Length of bar indicates p value. (E,F) Plots of H3.3 K27M/H3.3 WT log2 ratio in mouse DIPGs for promoter regions comparing H3K27ac versus H3K27me3 (E) and H3K4me3 versus H3K27me3 (F). H3K4me3 (H3.3 WT, n = 3; H3.3 K27M, n = 2). Shaded density histograms illustrate relative overlap of PTM changes in promoters of up (red) and downregulated (blue) genes compared to the gene loci bulk (gray). (G) Average tracks (identical scale for each genotype pair) showing H3K27me3, H3K27ac and H3K4me3 enrichment in H3.3 WT or H3.3 K27M expressing mouse DIPGs and average RNA-seq tracks for Usp44, Lif and Six1. See also Figures S7 and S8 and Table S6.

Figure 7.. Epigenetic Release at Bivalent Promoters Associated with Differentially Expressed Genes in H3.3 K27M DIPG Oncogenic Signature

(A) Stacked bar graphs showing peak call status for H3K27me3 and H3K4me3 in the 2 kb surrounding the transcriptional start site (TSS) of all genes, H3.3 K27M up or downregulated genes in H3.3 WT DIPGs. Differential genes defined as in Figure 6. Gold represents proportion of potential bivalent gene targets (H3K27me3⁺ H3K4me3⁺). (B) Violin plot showing the average RNA-seq signal for all genes in H3.3 WT mouse DIPGs for each promoter status. The width of the violin shows how common expression levels are, with the widest part of the violin corresponding to the mode average. (C) Average tracks (identical scale for each genotype pair) showing H3K27me3, H3K27ac and H3K4me3 enrichment in H3.3 WT or H3.3 K27M expressing mouse DIPGs and average RNA-seq tracks for three potentially bivalent genes, Pbx3, Eya1 and Meis2. n = 6 (H3.3 K27M) and n = 5 (H3.3 WT) for H3K27me3 and H3K27ac; n = 2 (H3.3 K27M) and n = 3 (H3.3 WT) for H3K4me3; n = 20 (H3.3 K27M) and n = 9 (H3.3 WT) for RNA-seq. *portion of G630016G05 gene near Meis2. Red arrows, primer locations used for qPCR in (D). (D) ReChIP experiment from Pbx3, Eya1 and Meis2 promoters. Signal for primary ChIPs is shown as percent of the starting ChIP input (top row). The material pulled down with the H3K27me3 primary IP was used for ReChIP with indicated antibodies. ReChIP signal is shown as percent of original chromatin input for primary ChIP (bottom row). n = 2 for each genotype. Error bars show standard deviation. See also Table S7.

Figure 8.. H3.3 K27M Impact on Poised Promoters in DIPGs

In H3 WT DIPGs (top), poised promoters, primed for expression, bear both H3K27me3 (purple) and H3K4me3 (orange) PTMs on the same or nearby nucleosomes (left side). Some genes appear to have both poised and active promoter states represented in different H3 WT cells (or on different alleles within the same cell), as the genes are expressed and bulk analyses show both H3K27me3 and H3K27ac (not shown) present at the promoter (right side). In H3.3 K27M cells (bottom), H3K27me3 is diminished globally and bivalent gene promoters can be converted from poised to active, resulting in increased expression compared to the H3 WT state.