Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions.
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We thank members of the A.P.B., S.M.P. and G.L.B. laboratories for helpful discussions and critical reading of the manuscript. We are grateful to the Genomics Core at University College Dublin for expertise and help with next-generation sequencing. Work in the A.P.B. laboratory is supported by Worldwide Cancer Research and The Brain Tumour Charity (18-0592), the Irish Research Council Advanced Laureate Award (IRCLA/2019/21), the Health Research Board (HRB-ILP-POR-2017-078), Science Foundation Ireland (SFI) under the SFI Investigators (SFI/16/IA/4562) and BBSRC-SFI (SFI/17/BBSRC/3415) programs, the Irish Cancer Society (CancersUnmetNeeds012) and the St. Vincent’s Foundation. O.D. was supported by a PhD fellowship from the Irish Research Council Government of Ireland Postgraduate Scholarship Programme (GOIPG/2017/2009). D.G. was supported by a PhD fellowship from the Irish Research Council Government of Ireland Postgraduate Scholarship Programme (GOIPG/2019/2084). R.B.B. was supported by a fellowship from the Science Without Borders Program (CAPES, Governo Dilma Rousseff, Brazil) and a postdoctoral fellowship from EMBO. S.M.P. was supported by a Cancer Research UK Senior Research Fellowship (A17368). S.M.P. and R.B.B. were supported by a project grant from Children with Cancer (15/189). Support for cellular models was provided by G. Morrison and the Cancer Research UK-funded Accelerator Award (A21922; http://gcgr.org.uk). Work in the G.L.B. laboratory is supported by an SFI Starting Investigator Research Grant (18/SIRG/5573), an Irish Cancer Society Biomedical Research Fellowship (CRF18BRI) and a Worldwide Cancer Research grant (21-0271).
S.M.P. is a founder and shareholder of, and paid consultant to, Cellinta—a biotechnology start-up that is developing cancer therapeutics. S.M.P. is also an inventor on a University of Edinburgh patent related to NSC culture methods (WO2005121318A3). The other authors declare no competing interests.
Peer review information Nature Genetics thanks Gerald Crabtree and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a. Immunoblots in cell lysates from unmanipulated NSCs and lines expressing WT or K27M-mutant H3.3. Representative example of n = 2 data. b. Absolute quantitative PCR of endogenous/exogenous H3F3A transcripts in NSCs expressing WT or K27M H3F3A, n = 3 independent samples. c. Immunoblots in cell lysates from WT or K27M H3.3 NSC cultures. Representative example of n = 2 data. d. Immunoblots (Left) and quantifications (Right) of total H3 levels in NSCs expressing WT or K27M H3F3A, n = 4 independent samples. Presented as mean values ± SD. e. Immunoblots in NSCs expressing WT or K27M H3F3A or H3K27M mutant DIPG cell lines. Representative example of n = 2 data. f. Relative quantitative PCR analysis of INK4A levels in NSCs expressing WT or K27M H3.3, n = 2 independent samples. g. Growth analysis of H3.3 WT and K27M expressing NSCs, n = 3 independent samples. Presented as mean values ± SD.
a. EdU positivity in PP5W and PP5K cultures derived from embryos GCGR-NS19 and GCGR-NS13, n = 4 independent samples. Presented as mean values ± SD. b. Colony forming activity in PP5W and PP5K cultures, n = 6 independent samples. Data are median values and interquartile ranges (IQR), with whiskers representing quartiles 1/3 ± (1.5 × IQR) respectively. c. Senescence Associated ß-Galactosidase (SA-ß-Gal) positivity in PP5W and PP5K cultures, n = 3 independent samples. Presented as mean values ± SD. d. Mouse survival following stereotactic injection of PP5W or PP5K NSCs, n = 4 PP5W and n = 8 PP5K mice. e. Brightfield and GFP micrographs of wholemount/cross-sectional views of a representative mouse brain injected with PP5K cells. f. Immunohistochemistry of brain samples from mice transplanted with PP5W and PP5K cells. Transplanted V5-tag (H3.3) positive cells display a diffuse pattern in mouse tissue, with no defined margins between human cells and the mouse brain parenchyma. Scale bar: 1.5mm. Representative example of n = 4 data. g. Immunohistochemistry of V5-tag (H3.3) and Ki67 in brain tissue from mice transplanted with PP5W and PP5K cells. Bar plot shows percentage of proliferating transplanted cells (KI67±V5-tag+) in each group. Scale bar: 20mM, n = 4 WT and n = 3 K27M biological samples. Presented as mean values ± SD.
Extended Data Fig. 3 Transcriptional and chromatin mapping in H3.3 WT and K27M expressing hindbrain NSCs.
a. RNA-seq volcano plot presenting the gene expression changes observed between NSC cultures expressing WT or K27M-mutant H3F3A. Red and blue denotes genes significantly up and down-regulated, respectively in K27M cultures. b. Box plots presenting the expression level of the indicated genes in a cohort of H3.3 WT (black) and K27M-mutant (red) DIPG patient samples. This data was downloaded from the pediatric cBioportal database (https://pedcbioportal.kidsfirstdrc.org), n = 118 WT and n = 71 H3.3 K27M independent tumour samples. The box plots present median values and interquartile ranges (IQR), with whiskers representing quartiles 1/3 ± (1.5 × IQR) respectively. c. Gene Set Enrichment Analysis (GSEA) of genes up- and down-regulated in K27M expressing NSC cultures compared to genes up- and down-regulated in K27M-mutant patient samples. d. Genome-wide correlations of H3.3 WT and K27M (V5) ChIP-seq read densities derived from embryo GCGR-NS13. The correlation coefficient for the two conditions is indicated. e. Violin plots representing the normalised abundance of H3.3 (V5) at the indicated genomic locations in embryos GCGR-NS19 and GCGR-NS13, in H3.3 WT (left panel) and K27M (right panel) conditions. An equivalent number of non-PRC2 target, repressed gene promoters (as compared to PRC2 target genes) were randomly selected from RNA-seq results, as an additional control set of genomic loci. Data presented are from each biologically independent embryo sample, n = 1/embryo. f. Meta-plots of average H3.3-WT ChIP-seq enrichment in biological duplicate experiments in genomic windows ±10kb of Active Promoter, Active Enhancer, PRC2 target promoters and non-PRC2 target Repressed gene promoters.
a. Venn diagrams presenting the total numbers and overlap of identified SUZ12 peak sets in WT and K27M-mutant NSC cultures (top panel). Meta-tracks presenting quantitative ChIP-Rx normalized SUZ12 signal at overlapping and distinct peak sets identified by Venn diagram analysis (middle panels). Meta-tracks presenting ChIP-seq normalized V5 H3.3 WT and K27M signal at the genomic regions outlined (bottom panels). b. Genomic tracks showing average SUZ12, H3K27me3 and BMI1 ChIP-Rx and H3.3-K27M ChIP-seq signal at the indicated genomic locus (chr6:125,737,994-125,772,907) in biological duplicate WT and K27M-mutant hindbrain NSC cultures. The chromosome ideograms are displayed above the gene track panels with the relevant regions highlighted. c. Box plots presenting the fold-change in quantitative SUZ12 ChIP-Rx signal at SUZ12 peaks and outside SUZ12 peaks between K27M-mutant and WT hindbrain NSC cultures for embryos GCGR-NS19 (left) and GCGR-NS13 (right). Data presented are from each biologically independent embryo sample, n = 1/embryo. Box plots present median values and interquartile ranges (IQR), with whiskers representing quartiles 1/3 ± (1.5 × IQR) respectively. d. Meta-tracks with density of CpG dinucleotides in a ± 10kb genomic window around the transcriptional start sites of genes gaining, with unchanged or reduced SUZ12 binding. e. Meta-tracks presenting ChIP-Rx normalised H3K4me3 levels at the promoter regions of the same genes as in panel b. f. Meta-tracks presenting ChIP-Rx (H3K27me3, H3K4me1, SUZ12, H3K4me3, H3K27ac) or ChIP-seq (H3.3A) normalised read counts at 263 identified poised enhancer elements in hindbrain NSC cultures.
a Gene ontology analysis of genes with unchanged (±0.1-fold) or reduced (>1.5-fold) SUZ12 binding in K27M-mutant NSC cultures. b Box plots presenting expression values for each of the indicated PRC2 target genes (Gaining SUZ12) in H3.3 WT or K27M mutant NSCs, n = 4 biologically independent samples. Box plots present median values and interquartile ranges (IQR), with whiskers representing quartiles 1/3 ± (1.5 × IQR) respectively.
a. H3K27me3 peak number in WT and K27M NSC cultures. Total number of peaks in each condition are indicated, n = 2 independent samples. b. Number of ChIP-Rx normalised read counts for SUZ12 (left) and H3K27me3 (right) at ‘Targeted’ and ‘Dispersed’ regions in embryo GCGR-NS13. c. Fold-change in H3K27me3 ChIP-Rx signal at ‘Targeted’ and ‘Dispersed’ sites in WT and K27M hindbrain NSCs for embryos GCGR-NS19 (left) and GCGR-NS13 (right), n = 1/embryo. Data are median values and interquartile ranges (IQR), with whiskers representing quartiles 1/3 ± (1.5 × IQR) respectively. d. Tornado plots and meta tracks of ChIP-Rx normalised SUZ12 and H3K27me3 signal ±10kb of all SUZ12 peak regions in duplicate WT and K27M NSCs. e. Genomic tracks showing SUZ12, H3K27me2 and H3K27me3 ChIP-Rx signal at the indicated genomic loci (chr2:175,593,388-176,711,387 and chr7:155,162,247-156,015,446) in WT and K27M-mutant NSC cultures for embryos. f. Scatter density plots correlating change in H3K27me2 (left) and H3K27me3 (right) with H3K27ac in 10kb genomic bins between H3.3 WT and K27M NSCs. g. Rolling average plots presenting fold-change of H3K27 modifications in ChIP-Rx across chromosome 4 in Ezh2 wildtype and heterozygous knockout mouse ESCs. h. Genome-wide correlation of H3K27ac ChIP-Rx signal in WT and K27M conditions in embryo GCGR-NS13 (top panels) and Ezh2 wildtype and heterozygous knockout mouse ESCs (bottom panels). Correlations of H3K27ac ChIP-Rx read densities are also shown for low abundance regions (right panels). i. Violin plots presenting fold-change of H3K27 modifications in ChIP-Rx in 10kb bins grouped into quintiles based on H3K27ac abundance in WT cells (left panels). The number of bins in each quintile is indicated. Right panels show violin plots presenting the abundance H3K27 modifications within quintiles as per the left panels. Data presented are from each independent embryo sample, n = 1/embryo.
a. Ranked Order of Super Enhancer (ROSE) analysis in WT and K27M-mutant NSC cultures for embryo GCGR-NS13. b. Bar charts presenting the relative shift in the number of typical and super enhancer elements in WT and K27M-mutant hindbrain NSC cultures, n = 2 biologically independent samples. c. Tornado plots of averaged ChIP-Rx normalised H3K27ac signal ±10kb of the centre of active promoters and enhancer elements in duplicate WT and K27M-mutant NSC cultures. d. Meta-tracks of ChIP-Rx normalised signal for the indicated antibodies at active promoter and enhancer regions in duplicate WT and K27M-mutant NSC cultures. e. Waterfall plots presenting the log2 fold-change in H3K27ac ChIP-Rx signal at all identified active enhancers (top) and active promoters (bottom) between WT and K27M-mutant NSC cultures. f. Box plots presenting the log2 fold-change in H3K27ac ChIP-Rx signal at active promoters and enhancers separated into their respective top (Q5), and bottom (Q1) quintiles based on H3.3-K27M abundance, n = 2 biologically independent samples. Box plots present median values and interquartile ranges (IQR), with whiskers representing quartiles 1/3 ± (1.5 × IQR) respectively. g. Genomic tracks showing quantitative H3K27ac ChIP-Rx signal around the HOXA gene cluster on chromosome 7 (chr7:26,811,403-27,600,402). h. Meta-tracks of ChIP-seq normalised H3K27M signal at active promoter and enhancer regions in two independent K27M mutant patient derived DMG cell lines. One sample is H3.1-K27M, while the second is H3.3-K27M.
Extended Data Fig. 8 Gene ontology analysis of differentially expressed genes associated with gene enhancer dynamics.
a. Waterfall plots presenting the log2 fold-change in ATAC-seq signal at all identified active enhancers (left) and active promoters (right) between WT and K27M-mutant NSC cultures. b. Gene ontology (GO) analysis of all genes associated with enhancer regions losing both H3K27ac signal and chromatin accessibility in K27M mutant NSCs (left). GO analysis of downregulated genes associated with enhancer regions losing both H3K27ac signal and chromatin accessibility (right). c. GO analysis of all genes associated with enhancer regions losing H3K27ac signal only in K27M mutant NSCs (left). GO analysis of downregulated genes associated with enhancer regions losing H3K27ac (right).
a. Cellular viability dose-response in PP5W (black) or PP5K (red) cultures treated with the indicated small-molecules. Mean ± s.d., n = 3 independent samples. b. GO analysis of genes significantly upregulated (top panels) or downregulated (bottom panels) in PP5W and PP5K cultures following treatment with Tazemetostat. c. RNA-seq volcano plots showing expression changes in H3.3-K27M mutant mouse NSC cells treated with Tazemetostat (left) or a DMG cell line following shRNA SUZ12 knockdown (right). Total numbers of up/down regulated genes are indicated, as are the numbers of up/down regulated genes gaining SUZ12 at their promoters in H3.3-K27M human NSCs. In mouse cells, the homologs of genes gaining SUZ12 are highlighted. d. Plots showing log2 fold-change in expression for the set of non-PRC2 repressed loci in Tazemetostat treated PP5W or PP5K cultures, n = 3 independent samples. Presented are median values and interquartile ranges (IQR), with whiskers representing quartiles 1/3 ± (1.5 × IQR) respectively. e. Genomic tracks showing RNA-seq data at the INK4A-ARF locus (chr9:21,961,091-22,001,052) in PP5W and PP5K cultures treated with DMSO or Tazemetostat. Indicated are the INK4A and ARF specific exons for each transcript. f. Plots showing log2 expression values for the indicated genes in H3.3 WT or K27M NSCs (left panels), n = 4 independent samples or PP5K cells treated with DMSO or Tazemetostat (right panels), n = 3 independent samples. Presented are median values and interquartile ranges (IQR), with whiskers representing quartiles 1/3 ± (1.5 × IQR) respectively. g. Plots presenting the log2 fold-change in gene expression for the set of neurodevelopmental regulatory genes which lose H3K27ac and chromatin accessibility at their promoters in Tazemetostat treated PP5W or PP5K cultures, n = 3 independent samples. Presented are median values and interquartile ranges (IQR), with whiskers representing quartiles 1/3 ± (1.5 × IQR) respectively.
Extended Data Fig. 10 Transcriptional disruption in DIPG cell lines treated with compounds targeting chromatin regulators.
RNA-seq volcano plots presenting the gene expression changes observed in an established H3.3-K27M mutant DIPG cell, DIPGXIII following treatment with Tazemetostat, JQ1, THZ1 or Panobinostat for 24 hours. Red and blue denotes genes significantly up and down-regulated, respectively in drug treated cultures.
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Brien, G.L., Bressan, R.B., Monger, C. et al. Simultaneous disruption of PRC2 and enhancer function underlies histone H3.3-K27M oncogenic activity in human hindbrain neural stem cells. Nat Genet 53, 1221–1232 (2021). https://doi.org/10.1038/s41588-021-00897-w