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.

Histone H3 K27M mutations occur in 80% of DIPGs and exert a dominant effect driving global loss of H3K27me3. It is unclear how this dramatic change in epigenetic regulation contributes to oncogenic transformation. We used genetically engineered mouse models to show that H3.3 K27M alone enhanced neural stem cell self-renewal. Neonatal induction of H3.3 K27M cooperated with active PDGFRα mutant and p53 inactivation to accelerate DIPG formation. H3.3 K27M and the resulting H3K27me3 loss drove selective regulation of bivalent promoters in tumors, dysregulating neural development genes. These genetically engineered models of spontaneous DIPG recapitulate the most common mutations from human tumors, reveal insights into disease pathogenesis, and provide physiologically relevant immunocompetent models for future mechanistic and preclinical studies.

In this study, we set out to determine the consequence of H3.3 K27M mutations in different brain regions, how H3.3 K27M-mediated depletion of H3K27me3 impacts other aspects of epigenetic regulation, and how this connects with changes in transcription and oncogenic activity.

Experimental systems to study H3 K27M have relied on exogenous overexpression of H3.3 K27M along with different combinations of cooperating mutations to induce knockdown or deletion of Trp53 and/or activation of PDGFR signaling, and most did not target brainstem or midline brain structures. Mutants were virally transduced into neural progenitor cells (NPCs) induced from human embryonic stem cells (ESCs) or neural stem cells (NSCs) isolated from embryonic mouse forebrain and generated low-grade gliomas or HGGs, respectively, when implanted into brain (). In utero electroporation of constructs encoding various mutants into mouse embryos evaluated cooperating effects of H3.3 K27M with other mutations in a limited number of hindbrain tumors, but predominantly targeted the cortex due to technical challenges with hindbrain delivery (). In an alternate approach, overexpressed H3.3 K27M in combination with Trp53 deletion and overexpression of PDGF-B, a PDGFRα ligand not typically mutated in human tumors, were introduced by in vivo retroviral transduction into neonatal brainstem using RCAS-tVA (). The varying levels of expression that can result from viral transductions or electroporation of different constructs contributes heterogeneity to each of these systems. This can be significant, as overexpression of the wild-type (WT) H3.3 was associated with altered neurogenic properties, showing important functional consequences of histone dosage, independent of K27M mutation (). Despite this limitation, all of these models showed that H3.3 K27M was insufficient to drive oncogenic transformation in the absence of other mutations, but cooperated with other mutations to drive tumors. However, none of the models evaluated genomic occupancy of H3K27me3 in tumors or cells from brainstem or midbrain, and minimal information is available about changes in genomic occupancy of other H3 PTMs in response to H3.3 K27M.

In addition to H3 mutations, DIPGs also contain alterations targeting canonical cancer signaling pathways, most frequently p53 loss of function and platelet-derived growth factor receptor α (PDGFRα) activation through gene amplification and/or mutation. Numerous other lower-frequency mutations contribute to significant inter- and intratumoral DIPG heterogeneity (). Diffuse midline gliomas with K27M mutation, including DIPGs, also show distinct DNA methylation patterns when compared with other HGGs (). This highlights the unique biology of K27M mutant gliomas; however, it is difficult to disentangle the effects of H3 K27M mutation from the signatures of midline developmental origin.

The genetic configuration of H3 K27M mutations implies a strongly dominant mode of action. 75% of K27M mutations occur in H3F3A, one of 15 genes encoding histone H3 variants. H3 K27M expression in the context of primary tumors or heterologous cell types, confers a dominant and profound decrease in H3K27me3, a posttranslational modification (PTM) associated with transcriptional repression (). Although the K27M-mediated loss of H3K27me3 is context independent, the high-frequency association of H3 K27M with diffuse midline gliomas of childhood indicates that it only confers a selective advantage in specific developmental settings.

Diffuse intrinsic pontine gliomas (DIPGs) are incurable brainstem tumors arising almost exclusively in children, with peak incidence between 6 and 8 years. These devastating tumors comprise approximately half of all pediatric high-grade gliomas (HGGs) (). Recurrent, somatic mutation in histone H3 is a molecular hallmark distinguishing pathogenesis of HGG in children and adults (). Histone H3 K27M mutations occur in ∼80% of DIPGs, and other HGGs arising in midline brain structures such as the thalamus. Diffuse midline glioma, H3 K27M mutant is now recognized as a distinct entity by the World Health Organization classification system (). In contrast, histone H3 G34R/V mutations are mutually exclusive with H3 K27M and occur in ∼15% of cortical HGGs in older adolescents and young adults ().

Although these apparently bivalent promoters are marked with H3K27me3 and H3K4me3 in H3.3 WT tumors, some genes were clearly expressed in contrast to the expected silent bivalent promoter ( Figure 7 B, gold violin, potentially bivalent genes; and Figure 7 C, black track, RNA-seq in H3.3 WT for three H3K27me3H3K4me3genes). These could represent bivalent promoters associated with variable expression levels, or a mixed cell or allele population in which the same promoters are marked with H3K27me3 in one subpopulation and H3K4me3 in another. To address this possibility, we performed co-occupancy analysis by sequential ChIP (ReChIP). At Pbx3, Eya1, and Meis2 promoters, ChIP analysis for each PTM showed recruitment of both H3K27me3 and H3K4me3 in H3.3 WT DIPGs, while all three gene loci showed substantial loss of H3K27me3 recruitment in H3.3 K27M DIPGs ( Figures 7 C, red arrows and 7 D, upper graphs). ReChIP of H3K4me3 from the chromatin pulled down by an H3K27me3 ChIP showed considerable enrichment compared with immunoglobulin G control at all three loci, confirming that H3K4me3 and H3K27me3 co-occupy the same fragment of DNA ( Figure 7 D, lower graphs). While the expression of these H3K27me3 and H3K4me3 marked genes suggests that they are actively transcribed in a portion of the H3.3 WT tumor cell population, ReChIP indicates that a true bivalent population exists. Together, these data indicate that bivalency release through loss of H3K27me3 is a plausible mechanism for H3.3 K27M-mediated differential gene expression signatures important in DIPG development.

Epigenetic signatures are associated with important effects on gene expression, especially during development. Under normal conditions, H3K27me3 is found primarily at repressed loci, H3K4me3 associates with active promoters, and the combination of H3K27me3 and H3K4me3 marks poised or bivalent promoters. Loss of H3K27me3 from bivalent promoters is associated with increased gene expression in normal developmental transitions (). To determine the effect of H3.3 K27M-mediated H3K27me3 loss on expression of genes with bivalent promoters, we identified all promoters marked with both H3K27me3 and H3K4me3 in H3.3 WT mouse DIPGs ( Figure 7 A, 12%, gold, left bar). There was significant increase in the proportion of apparently bivalent promoters in H3.3 WT tumors among promoters of genes with H3.3 K27M-dependent differential expression, including 57% of upregulated genes (p = 0.031) and 39% of downregulated genes ( Figure 7 A, gold, center, and right bars). Notably, genes that are differentially expressed in H3.3 K27M DIPGs and have H3K27me3H3K4me3at their promoters in H3.3 WT tumors are significant PRC1 and PRC2 targets (Enrichr, p < 10) highly associated with development and neurogenesis (GO, p < 10and 10, respectively) ( Table S7 ), and include genes we identified as differentially upregulated in hindbrain NSCs such as Lgr5, Irx1, and Irx2.

(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.

(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).

(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.

(A) Stacked bar graphs showing peak call status for H3K27me3 and H3K4me3 in the 2-kb surrounding the 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 (H3K27me3H3K4me3).

Globally, H3.3 K27M-dependent changes in PTMs at enhancers resemble those at promoters. Enhancers also show reciprocal shifts of H3K27me3 and -ac, minimal change in H3K4me3, and lack evidence of redistribution of any of these PTMs ( Figures S8 A–S8C). Enhancers associated with H3.3 K27M upregulated genes ( Figure S8 D, red) also demonstrate an H3.3 K27M-dependent increase in H3K27ac consistent with increased activation, compared with downregulated (blue) or unchanged (gray) gene-associated enhancers. As with promoters, most enhancers show a similar H3.3 K27M-dependent loss of H3K27me3 regardless of the H3.3 K27M-dependent expression status of the nearest gene ( Figure S8 D). However, while the promoters of H3.3 K27M upregulated genes usually have a true H3K27me3 peak in H3.3 WT DIPG that is lost in H3.3 K27M DIPG, active enhancers in H3.3 K27M DIPG that are associated with H3.3 K27M upregulated genes usually lack discrete H3K27me3 peaks in H3.3 WT DIPG, suggesting that the change in enhancer activity is an indirect effect of H3.3 K27M-mediated H3K27me3 loss ( Figures S8 E and S8F).

Interestingly, a small number of downregulated genes (n = 38) in H3.3 K27M tumors do not follow global trends for H3K27me3 and H3K27ac. The promoters of this group of genes retain H3K27me3, and show reduced H3K27ac and H3K4me3 ( Figures 6 C, 6E, and 6F, purple) as exemplified by the Six1 locus ( Figure 6 G). Strikingly, this group of genes is significantly enriched for targets of BMI1, a core component of the PRC1 complex involved in gene silencing, as well as targets of the PRC2 components, JARID2 and EZH2 (Enrichr), highly associated with development and gene regulation (GO) ( Table S6 ).

We next integrated analysis of changes in the epigenome and transcriptome to evaluate the mechanisms driving aberrant gene regulation in H3.3 K27M tumors. H3K27me3 is normally associated with transcriptionally silent genes (), but, as observed in H3.3 K27M NSCs, the dramatic genome-wide decrease of H3K27me3 in DIPGs produces relatively modest transcriptome changes. Overall, there were more genes upregulated than downregulated in H3.3 K27M DIPGs ( Figure 6 C, 299 up [red] versus 155 down [blue or purple]) and H3.3 K27M upregulated genes display marked H3K27me3 decrease agreeing with their expression change. Consistent with a role for H3K27me3 in regulation of genes involved in development, cell fate, and differentiation, genes upregulated in mouse H3.3 K27M DIPGs are significantly enriched for association with neural development and differentiation (GO), and with PRC1 and PRC2 targets (Enrichr) ( Figure 6 D; Table S6 ). While decrease in H3K27me3 at H3.3 K27M upregulated gene promoters is similar to genes with unchanged expression ( Figure 6 E, red [H3.3 K27M up] versus gray [unchanged] histograms on top of plot), both activation-associated H3K27ac and H3K4me3 show an average gain consistent with increased gene expression ( Figures 6 E and 6F, right marginal plots; peaks of red histograms are shifted up versus both blue [H3.3 K27M down] and gray). Usp44 is an example of a K27M upregulated gene showing clear loss of H3K27me3 and gain of H3K27ac ( Figure 6 G). The average epigenetic signature for H3.3 K27M downregulated genes is discordant with their expression and reflects the global loss of H3K27me3 and gain of H3K27ac. However, these genes show less H3K27ac and H3K4me3 compared with genes with H3.3 K27M upregulated or unchanged expression ( Figures 6 E and 6F, right marginal plots; peaks of blue histograms are shifted down versus both red and gray). Lif highlights a locus where loss of H3K27me3 and relatively unchanged H3K27ac is nonetheless accompanied by reduced gene expression ( Figure 6 G).

To better understand the epigenetic effects of H3.3 K27M in spontaneous DIPG, we compared the global occupancy of PTMs associated with gene repression (H3K27me3) and activation (H3K27ac and H3K4me3) in H3.3 K27M;PDGFRA;p53and H3.3 WT;PDGFRA;p53DIPGs. H3.3 K27M facilitated a genome-wide reduction of H3K27me3 and a reciprocal increase in H3K27ac, with minimal global changes in H3K4me3 ( Figures S7 A–S7C). Promoter regions recapitulated the global changes in H3K27me3 and H3K27ac ( Figures S7 D–S7F). Notably, while the levels of the H3K27 modifications were dramatically changed by H3.3 K27M, the positioning of the H3K27 PTMs across the promoter region was unaltered ( Figures 6 A and 6B ).

(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.

(E and F) Plots of H3.3 K27M/H3.3 WT log2 ratio in mouse DIPGs for promoter regions comparing H3K27ac with 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 with the gene loci bulk (gray).

(C) Plot of H3.3 K27M/H3.3 WT log2 ratio in mouse DIPGs for promoter regions comparing RNA-seq with 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 with 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.

(A and B) Promoter-based histograms representing counts within 40 bp bins across a 4-kb region centered at transcriptional start site (TSS) for H3K27me3 (A) or H3K27ac (B) in H3.3 K27M (n = 6) and H3.3 WT (n = 5) mouse DIPGs.

DIPGs, regardless of H3.3 K27M status, have distinct expression patterns from cortical pediatric HGGs, as shown by PCA ( Figure S5 A). We previously reported that these expression differences are significantly associated with transcription factors and developmental processes (). To assess transcription effects of H3.3 K27M without the confounding influence of regional expression differences in tumors arising from multiple anatomic locations, we compared DIPGs in mouse and human. The human H3 WT DIPGs used in our comparison were of similar age to the H3.3 K27M DIPGs (median age 8.9 versus 6, respectively, p = 0.9), had expression signatures that group with other DIPGs, and MRI images consistent with typical DIPG ( Figures 5 B and S5 A–S5D). GSEA demonstrated that human H3.3 K27M signatures were significantly enriched in mouse H3.3 K27M DIPGs ( Figure 5 C; Table S5 ). Pbx3, Eya1, and Plag1 are among the most significant ( Figure 5 C) and show a clear expression shift by H3.3 K27M in human DIPG, mouse DIPG, and mouse embryonic NSCs ( Figure 5 D). H3.3 K27M downregulated genes in human DIPGs were not strongly enriched in mouse H3.3 WT compared with H3.3 K27M DIPG. However, a number of transcription factors associated with neural development were consistently downregulated by H3.3 K27M in human and mouse DIPGs as well as mouse hindbrain NSCs ( Figure S6 A). Several differentially expressed genes also show regional differences in NSCs, such as higher basal expression of Pbx3 and En1 in hindbrain, marked H3.3 K27M-dependent differential expression of Six1, En1, and Hoxd8 in hindbrain versus forebrain, and downregulation of Cdkn2a in forebrain ( Figures 5 D, S6 A, and S6B).

Gene expression signatures used to identify molecular subclasses of human HGG reflect the heterogeneity of this disease in adult and pediatric supratentorial HGGs and DIPGs (). We used single-sample gene set enrichment analysis (ssGSEA) to compare the gene expression signatures of the mouse brainstem gliomas with signature gene sets for human HGG subclasses (proneural, proliferative, and mesenchymal) (), and signature gene sets from normal mouse neural cell types () ( Table S4 ). Both mouse DIPGs and human DIPGs, with or without H3.3 K27M mutation, reflected intertumoral heterogeneity with varying enrichment for the different HGG subgroups and normal neural cell types ( Figure 5 A), and intermixing of H3.3 K27M and H3 WT tumors when viewed by principal-component analysis (PCA) ( Figure 5 B). Two main subgroups of mouse DIPG showed predominantly proneural or proliferative signatures from the human HGG subclasses, and GO analysis of the most differentially expressed genes between tumors in these subgroups similarly identified signatures in synaptic transmission (p = 1.9 × 10) or cell cycle (p = 1.75 × 10), consistent with the HGG subgroup categories ( Table S4 ).

(D) Expression of leading edge upregulated genes Pbx3, Eya1, and Plag1. Boxplots depict log2-scale RNA-seq counts per million (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. Boxplots 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.

(C) Gene set enrichment analysis 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).

(A) Heatmaps of single-sample gene set enrichment analysis 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.

H3F3A, TP53, and PDGFRA are the most commonly mutated genes in human DIPGs, and can occur in varying combinations (). To assess the cooperative oncogenic effect of these mutations, we bred the respective engineered mice to Nestin-CreERto generate H3.3 K27M;PDGFRA;p53and H3.3 WT;PDGFRA;p53mice. Mutations were induced at P0 and P1, resulting in highly penetrant brainstem and supratentorial HGGs in H3.3 WT;PDGFRA;p53. Importantly, H3.3 K27M accelerated HGG development and significantly increased the proportion of HGGs arising in the brainstem from 59% to 95% (p < 0.0001) ( Figures 4 A and 4B ). Tumors were diffusely infiltrative HGGs with similar histopathology to human DIPG for both H3.3 WT and K27M. Tumor cells showed robust expression of cytoplasmic PDGFRα, and also expressed Olig2. Expression of the knockin H3.3 WT or H3.3 K27M was detected by FLAG in all tumors tested. While H3.3 WT tumors contained strong H3K27me3 expression, H3.3 K27M tumors consistently displayed marked loss of H3K27me3 ( Figure 4 C). Interestingly, H3K27me3 levels in H3.3 WT mouse DIPGs were noticeably higher than in H3.3 WT mouse NSCs and comparable with the levels found in human H3 WT HGG xenografts ( Figure 4 D), likely reflecting differences in H3K27me3 associated with developmental context.

(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. An 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.

(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 bars, 1 mm (whole brain images), 50 μm (higher-magnification images).

PDGFRα is the most frequently mutated receptor tyrosine kinase in pediatric HGG (). Therefore, to model cooperative effects of pediatric HGG mutations, we generated LSL-PDGFRAtransgenic mice with Cre-inducible expression of a mutant human PDGFRα containing a 15-amino acid duplication in the transmembrane domain (PDGFRA Figures S4 A and S4B). This alteration occurred as a heterozygous mutation in DIPG resulting in ligand-independent activation of PDGFRα (). Nestin-CreER;LSL-PDGFRA(hereafter PDGFRA) mice induced at P0 and P1 to express PDGFRAin neonatal NSCs/NPCs and their subsequent progeny did not exhibit obvious abnormality or reduced lifespan when observed for over 1 year ( Figure 3 A). In contrast, PDGFRA;p53mice developed brain tumors faster than p53mice, and substantially shifted the tumor spectrum to HGG (96%) including a significant increase of tumors involving the brainstem (52%, p < 0.0001) ( Figures 3 A and 3B). The cooperative effects of PDGFRA show that Trp53 loss alone is not sufficient for efficient gliomagenesis from brainstem neonatal NSCs/NPCs compared with the supratentorial compartment. Brainstem HGGs showed moderate nuclear pleomorphism, variable astrocytic differentiation, mitotic activity, extensive infiltration, and strong nuclear Olig2, consistent with the pathology of human DIPG. Tumor cells also expressed robust cytoplasmic PDGFRα. However, consistent detection of H3K27me3 shows that these tumors did not acquire a somatic H3 K27M mutation or employ another genetic or epigenetic mechanism to suppress levels of this PTM ( Figure 3 C).

(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 bars, 1 mm (whole brain image), 50 μm (higher-magnification images).

To model the contribution of H3.3 K27M to gliomagenesis in children, we bred H3f3aor H3f3amice with tamoxifen-inducible Nestin-CreERmice (). The resulting Nestin-CreER;H3f3aor Nestin-CreER;H3f3amice (hereafter H3.3 K27M or H3.3 WT) were induced at post-natal days 0 and 1 (P0 and P1) to activate the knockin alleles in neonatal NSCs/NPCs. H3.3 K27M alone failed to cause brain tumor formation, and induction of either H3.3 K27M or H3.3 WT alleles did not cause obvious abnormalities or premature death within 1 year of age ( Figures 2 A and 2B ). To evaluate cooperative oncogenic activity, H3.3 K27M or H3.3 WT mice were bred with Trp53mice () (hereafter p53). Neonatal deletion of Trp53 induced highly penetrant brain tumors, with mice developing macroscopically visible cerebellar medulloblastomas in 59%, supratentorial HGG in 27%, and both concurrently in the remainder. H3.3 WT expression combined with p53did not significantly alter tumor location, histopathology, or latency ( Figures 2 A and 2C). In contrast, H3.3 K27M combined with p53significantly accelerated brain tumor development ( Figure 2 B) and increased medulloblastoma frequency ( Figure 2 D, p = 0.0004). The histopathology of all evaluated supratentorial tumors was HGG and most developed as large masses with extensive infiltration of adjacent cerebral tissues. Pleomorphic cells occasionally showing astrocytic or oligodendroglial differentiation were associated with brisk mitotic activity and, in rare cases, areas of necrosis ( Figure 2 E). All histologically assessed tumors arising in the cerebellum appeared embryonal and were classified as medulloblastoma, with classic ( Figure 2 F) or large-cell anaplastic morphologies at similar frequencies in all genotypes. Expression of the epitope-tagged knockin H3.3 WT or H3.3 K27M was detected by nuclear FLAG expression, with H3.3 K27M;p53tumors also showing the expected loss of H3K27me3, regardless of tumor histology ( Figures 2 G and 2H).

(G and H) Expression of FLAG-tagged H3.3 (upper images) and H3K27me3 (lower images) is shown by immunohistochemistry (IHC) on sections of representative supratentorial HGG (G) or medulloblastoma (H) for the indicated genotypes. Scale bars, 50 μm.

(E and F) H&E stain of representative supratentorial HGG (E) or medulloblastoma (F) observed in all genotypes. ∗ In upper images indicates tumor. Scale bars, 1 mm (top images), 50 μm (bottom images).

(C and D) Location of macroscopic brain tumors in cohorts shown in (A) and (B), (C) and (D), respectively. Supra, supratentorial; CB, cerebellar; Spinal, spinal cord; BS, brainstem.

(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.

NSC expression signatures differentiating hindbrain and forebrain origin were not dramatically changed by loss of H3K27me3 ( Figure 1 E), and the majority of genes that were differentially upregulated in H3.3 WT H- or F-NSCs were also differentially upregulated in H3.3 K27M NSCs ( Figure S3 A). Gene ontology (GO) analysis of these expression signatures showed similar enrichment in H3.3 WT or H3.3 K27M NSCs for regional development, including telencephalon regionalization in forebrain (p = 1.6 × 10), and nervous system development and homeodomain transcription factors in hindbrain (p = 1.3 × 10and 3.7 × 10, respectively, Table S2 ). For example, in situ hybridization data from the Allen Brain Atlas () showed expression of Foxg1, which is not expressed in hindbrain, or Irx2, which is not expressed in forebrain, remains silenced in H3.3 K27M H- and F-NSCs, respectively, even with substantial loss of H3K27me3 at these loci ( Figures 1 F and 1G). Although global transcription was mostly unaffected by loss of H3K27me3, we detected specific H3.3 K27M-mediated gene expression changes including upregulation of genes involved in neural development and proliferation ( Figure S3 B; Tables S1 and S3 ). The most significant H3.3 K27M-induced changes included increased expression of genes important for regulating NPC proliferation and differentiation, such as Lin28b, Igf2bp2, and Plag1 ( Figure 1 H; Table S1 ). These data indicate that H3.3 K27M contributes to programming enhanced self-renewal and a proliferative, progenitor cell state, while driving only selective changes in the transcriptome.

Consistent with reported effects of H3.3 K27M, chromatin immunoprecipitation with high-throughput chromatin immunoprecipitation sequencing (ChIP-seq) combined with spike-in normalization revealed a profound global H3K27me3 reduction in H3.3 K27M compared with H3.3 WT NSCs ( Figures S2 C and S2D). However, RNA sequencing (RNA-seq) analysis showed differences in the transcriptomes of H3.3 K27M, and WT NSCs were relatively modest and selective rather than global ( Figures 1 C and S2 E; Table S1 ). To assess whether global loss of the transcriptional repression-associated PTM H3K27me3 could result in a global increase in all transcription, we also normalized the RNA-seq to a spike-in control, which confirmed the absence of an overall gain in transcription. Integrated analysis showed that the bulk of both up- and downregulated genes have a similar decrease in H3K27me3 at their promoters ( Figures 1 D and S2 F).

To investigate the role of H3.3 K27M in NSCs, we bred H3f3aor H3f3amice to Nestin-Cre mice (), which constitutively express Cre in neural stem and progenitor cells throughout the CNS beginning at approximately embryonic day 10.5 (E10.5). NSCs were isolated from hindbrain or forebrain (H- or F-NSC, respectively) of the resulting embryos at E15.5. Analysis of these NSCs over multiple passages in neurosphere growth conditions showed that H3.3 K27M promoted increased cell growth compared with H3.3 WT, regardless of origin location ( Figures S2 A and S2B). Clonogenic growth assays with H-NSCs showed H3.3 K27M significantly enhanced self-renewal capacity compared with H3.3 WT, and generated larger spheres, reflecting a modest increase in proliferation ( Figures 1 A and 1B ). Interestingly, while the renewal capacity for H3.3 WT was similar from passages 3 through 9, H3.3 K27M cells displayed progressively enhanced clonogenic growth. Both genotypes began losing self-renewal by passage 11 ( Figure 1 A).

(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.

(F and 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 bars, 1 mm.

(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 with WT, with p < 0.05 and log2 fold change greater than 0.75 or less than −0.75, respectively, compared with the gene loci bulk (gray).

(A and 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.

To study H3.3 K27M in the context of developing brain, we generated conditional knockin mice, H3f3a, in which H3.3 K27M is expressed from the endogenous H3f3a locus following Cre recombinase (Cre)-mediated excision of a loxP-flanked transcriptional STOP cassette (LSL). We included a C-terminal FLAG-HA tandem epitope tag immediately upstream of the termination codon to allow detection of the mutant protein ( Figures S1 A and S1B). A second mouse line, H3f3a, with identical construction but without the K27M mutation, was generated for controlled comparisons.

Discussion

H3 K27M mutations represent a unifying feature of incurable childhood brain tumors that are otherwise molecularly heterogeneous. The selective association of these mutations with pediatric midline diffuse gliomas, especially DIPG, indicates a critical connection between epigenetic dysregulation and developmental context. Elucidating the mechanisms through which these mutations contribute to cancer is essential to improve outcome for DIPG patients. Our results provide a number of insights into the consequences of H3.3 K27M that contribute to DIPG pathogenesis.

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Tabar V. Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation. H3.3 K27M, in the absence of other mutations, caused a transient increase in self-renewal of hindbrain NSCs in vitro without inducing immortality or delaying senescence. This would be predicted to increase the pool of cells with greatest propensity for transformation, but only for a limited duration. A role for this mutation in early stages of tumor initiation is consistent with the clonal incidence of H3.3 K27M mutations in DIPGs and the restricted developmental window of susceptibility during childhood in which DIPGs arise. Strikingly, the genes most differentially induced by H3.3 K27M in NSCs included Lin28b, Plag1, and Igf2bp2; heterochronic genes associated with regulating developmental differences in fetal and adult NSCs (). Upregulation of LIN28B and PLAG1 was also seen with overexpression of H3.3 K27M in NPCs derived from human ESCs; however, increased neurosphere formation in vitro was only seen with the combination of three alterations; H3.3 K27M, mutant PDGFRα and p53 knockdown (). It is possible that the H3.3 K27M-dependent enhancement of self-renewal that we detected in NSCs acutely isolated from embryos was not readily detected in the human NPCs, which require prolonged culturing for in vitro induction and may be less developmentally synchronized.

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et al. Inactivation of Ezh2 upregulates Gfi1 and drives aggressive Myc-driven group 3 medulloblastoma. H3.3 K27M accelerated hindbrain tumorigenesis from neonatal progenitors. Unexpectedly, combined H3.3 K27M expression and Trp53 deletion accelerated medulloblastoma formation. Multiple lines of evidence highlight an emerging role for H3K27me3 loss in pediatric hindbrain tumorigenesis including pediatric posterior fossa group A ependymoma () and group 3 medulloblastoma (). Thus, acceleration of medulloblastoma formation by H3.3 K27M may reflect an increased potency for H3K27me3 loss to contribute to hindbrain tumor development. Combining PDGFRα activation with Trp53 deletion in neonatal NSCs/NPCs shifted the spectrum of tumors to HGGs, including a high proportion involving brainstem. Consistent with the hypothesis that developing hindbrain may have an increased vulnerability to transformation associated with H3K27me3 depletion, H3.3 K27M significantly increased the incidence of diffuse brainstem gliomas driven by combined PDGFRα activation and Trp53 deletion, and further accelerated tumor development.

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et al. H3.3(K27M) cooperates with Trp53 loss and PDGFRA gain in mouse embryonic neural progenitor cells to induce invasive high-grade gliomas. We induced genetically engineered mutations in Nestin-positive cells in neonatal mice to coincide with the developmental period when most gliogenesis occurs, including the period of greatest postnatal growth in the pons, from P0 to P4 (). A recent study using neonatal in vivo electroporation to introduce H3.3 K27M combined with p53 knockdown failed to induce tumors, while in utero electroporation to overexpress H3.3 K27M with CRISPR/Cas9-mediated Trp53 deletion induced gliomas (). In contrast, our results show cooperative effects of H3.3 K27M in generating both medulloblastomas and HGGs when induced in neonatal mice. This may reflect technical differences such as expression from endogenous loci and different targeted cell populations.

The H3.3 K27M;PDGFRA;p53cKO and H3.3 WT;PDGFRA;p53cKO models reported here investigate the most common human DIPG mutation targets, recapitulate the spectrum of gene expression subgroups in human HGGs, and show significant similarity in gene expression signatures to primary human DIPGs with and without H3 K27M mutation, respectively. Many comparisons between primary pediatric gliomas with and without H3.3 K27M mutation include H3 WT cortical gliomas and are confounded by regional developmental epigenetic and expression signatures along with variations in other oncogenic mutations. Our experimental system provides a robust setting to evaluate the direct effects of H3.3 K27M in the context of DIPGs induced at the same developmental time point and with the same oncogenic drivers.

cKO compared with H3.3 WT;PDGFRA;p53cKO DIPGs showed global changes in H3K27 PTMs, but selective changes in gene expression significantly associated with signatures of neural development. Genes with apparently bivalent promoters were significantly enriched among those upregulated with H3.3 K27M mutation. Thus, promoters marked by both H3K27me3 and H3K4me3 in H3 WT tumors would be poised for expression, while loss of H3K27me3 at these promoters in H3.3 K27M tumors would release the bivalent state, resulting in upregulation ( Lu et al., 2018 Lu T.T.

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et al. EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas. Figure 8 H3.3 K27M Impact on Poised Promoters in DIPGs Show full caption 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 with the H3 WT state. Integrated analysis of the epigenome and transcriptome of H3.3 K27M;PDGFRA;p53compared with H3.3 WT;PDGFRA;p53DIPGs showed global changes in H3K27 PTMs, but selective changes in gene expression significantly associated with signatures of neural development. Genes with apparently bivalent promoters were significantly enriched among those upregulated with H3.3 K27M mutation. Thus, promoters marked by both H3K27me3 and H3K4me3 in H3 WT tumors would be poised for expression, while loss of H3K27me3 at these promoters in H3.3 K27M tumors would release the bivalent state, resulting in upregulation ( Figure 8 ). This outcome is consistent with a direct effect of depleted H3K27me3 on selective changes in gene expression, and is reminiscent of altered expression of bivalent genes associated with differentiation or development in response to deletion of Ezh2 or Eed encoding PRC2 components (). This key selectivity has not been previously demonstrated for H3.3 K27M.found that H3K4me3 in promoters remained stable, but H3K27me3 decreased in gene bodies, not promoters, of genes upregulated in human NPCs overexpressing H3.3 K27M. Overexpression of H3.3 K27M or WT H3 in mouse forebrain NSCs showed that the majority of differentially expressed genes were not associated with H3K27me3 in H3 WT NSCs, leading to the suggestion that these expression changes were indirect effects of the mutation (). The clear association of upregulated genes with H3.3 K27M-mediated release of bivalent promoters identified in our mouse model may be attributed to both the regulation of H3.3 K27M at physiological levels from its endogenous promoter and our direct analysis of DIPG tumors rather than NSC cultures in vitro. The low levels of H3K27me3 in NSCs compared with tumors ( Figure 4 D) may explain why the role of bivalency in H3.3 K27M-dependent changes in gene expression was not previously identified in comparisons with NSCs.

While our mouse DIPGs are genetically engineered, the spontaneous development of DIPGs recapitulates the human process and may involve initiation from slightly different developmental states or acquisition of other mutations that could introduce intertumoral heterogeneity. Importantly, an independent study to identify the consequences of H3.3 K27M depletion in DIPG patient-derived xenografts also demonstrated significant enrichment in upregulation of genes with K27M-dependent release of bivalent promoter regulation (Silveira et al., unpublished data).

cKO DIPGs despite global reduction of this PTM. The selective retention, or suggestion of increased deposition of H3K27me3 in the context of H3 K27M-mediated depletion, has been demonstrated in primary human tumors, cell lines, and model systems ( Bender et al., 2013 Bender S.

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et al. Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas. cKO compared with much more significant decreases in homeodomain transcription factors that were already heavily marked with H3K27me3 in H3.3 WT tumors, suggesting tight regulation by strong transcriptional repressors that overcome H3.3 K27M-dependent effects by effectively recruiting residual PRC2 activity. Additional genes that were downregulated despite substantial loss of H3K27me3 may represent indirect H3.3 K27M-independent effects. A small collection of downregulated genes that were very significantly enriched for BMI1 targets and homeobox transcription factors retained H3K27me3 in H3.3 K27M;PDGFRA;p53DIPGs despite global reduction of this PTM. The selective retention, or suggestion of increased deposition of H3K27me3 in the context of H3 K27M-mediated depletion, has been demonstrated in primary human tumors, cell lines, and model systems (). CDKN2A was previously reported as a target for residual PRC2 activity associated with selective downregulation in H3 K27M model systems (), although this was not consistent in all DIPG cell lines (). Cdkn2a was modestly downregulated in H3.3 K27M;PDGFRA;p53compared with much more significant decreases in homeodomain transcription factors that were already heavily marked with H3K27me3 in H3.3 WT tumors, suggesting tight regulation by strong transcriptional repressors that overcome H3.3 K27M-dependent effects by effectively recruiting residual PRC2 activity. Additional genes that were downregulated despite substantial loss of H3K27me3 may represent indirect H3.3 K27M-independent effects.

Our results demonstrate that H3.3 K27M enhances self-renewal of NSCs without inducing immortalization, and accelerates hindbrain tumorigenesis, of either medulloblastoma or HGG from neonatal stem/progenitor cells. Upregulation of genes normally restrained by bivalent promoter PTMs results in transcriptional changes in genes relevant for both development and tumorigenesis, perhaps creating an expanded pool of cells susceptible to transformation that may progress to DIPG if they acquire other critical mutations during a narrow window of development. Because the epigenetic state is strongly interconnected with development and differentiation, it is likely that the specific collection of H3.3 K27M-dependent target genes may vary depending on the age and precise cellular state from which DIPG initiates, contributing to heterogeneity in tumor expression signatures and highlighting the power of the inducible genetically engineered approach for controlled comparisons. Furthermore, these experimental systems provide immune competent, physiologically relevant spontaneous models of DIPG that will be useful for future mechanistic and preclinical studies of DIPG pathogenesis and therapeutic response.