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Abstract
A high percentage of pediatric gliomas and bone tumors reportedly harbor missense mutations at glycine 34 in genes encoding histone variant H3.3. We find that these H3.3 G34 mutations directly alter the enhancer chromatin landscape of mesenchymal stem cells by impeding methylation at lysine 36 on histone H3 (H3K36) by SETD2, but not by the NSD1/2 enzymes. The reduction of H3K36 methylation by G34 mutations promotes an aberrant gain of PRC2-mediated H3K27me2/3 and loss of H3K27ac at active enhancers containing SETD2 activity. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumor development in vivo. Our findings are mirrored in G34W-containing giant cell tumors of bone where patient-derived stromal cells exhibit gene expression profiles associated with early osteoblastic differentiation. Overall, we demonstrate that H3.3 G34 oncohistones selectively promote PRC2 activity by interfering with SETD2-mediated H3K36 methylation. We propose that PRC2-mediated silencing of enhancers involved in cell differentiation represents a potential mechanism by which H3.3 G34 mutations drive these tumors.
G34 mutated oncohistones exhibit increased H3K27me2/3. ( A ) Schematic displaying the histone…
Fig. 1. G34 mutated oncohistones exhibit increased H3K27me2/3. (A) Schematic displaying the histone H3.3 mutant transgenes used to assess PTM changes in cis using the purification strategy depicted in B. (C) Mass spectrometry data displaying the abundance of H3K36me3 on the mutated H3.3 peptides. (D and E) SETD2 and NSD2 in vitro methyltransferase assays using recombinant mononucleosome substrates containing point mutations on H3.3 as labeled. CPM: counts per minute. P value for reduced counts on the G34 mutated substrates was determined using t test. n.s.: not significant. (F) Mass spectrometry data displaying the abundance of H3K27me3 on the mutated peptides. (G) Immunoblots of in vitro PRC2 methyltransferase reactions using recombinant mononucleosome substrates containing point mutations in H3.3. (H and I) Schematic for sequential methyltransferase reactions using recombinant mononucleosomes containing WT or G34-mutated H3.3. (I) Immunoblots of reactions preincubated with WT or catalytically dead SETD2 and 50 µM S-adenosyl-methionine depicted in H. PRC2 and additional 50 µM SAM were added to the reaction and incubated for 3 h. (J) Model depicting the effect of G34 mutations. Missense mutations on H3.3 G33-K37 block SETD2 activity in cis. However, only mutations at G34 position permit PRC2 activity at K27 and hence, uniquely increase H3K27me2/3 in cis by decreasing H3K36me3. Error bars represent SD.
Fig. 2.
Local loss of H3K36me3 and…
Fig. 2.
Local loss of H3K36me3 and gain of H3K27me3 in cells expressing H3.3G34W. ( …
Fig. 2. Local loss of H3K36me3 and gain of H3K27me3 in cells expressing H3.3G34W. (A) Strategy used to identify local changes in H3K27me3 and H3K36me3 in cells expressing H3.3 WT or G34W in vivo. (B) Scatterplot displaying the correlation between loss of H3.3X-K36me3 and gain of H3.3X-K27me3 at H3K36me3-enriched peaks. Each point represents a H3K36me3 peak, and the difference in the number of normalized ChIP-Seq reads was plotted. (C) Heatmap displaying the enrichment of H3.3-K36me3 and H3.3-K27me3 reChIP enrichment at H3K36me3 peaks in cells expressing H3.3 WT or G34W. (D) Genome browser view of the Sox6 gene displaying the loss of H3.3X-K36me3 and gain of H3.3X-K27me3. (E) Metagene analysis showing the average profile of H3.3X-K36me3 and H3.3X-K27me3 reChIP at active enhancers (promoter-distal H3K27ac peaks) in cells expressing H3.3 WT or G34W.
Fig. 3.
Aberrant reduction of H3K27ac at…
Fig. 3.
Aberrant reduction of H3K27ac at enhancers in cells expressing H3.3G34W. ( A )…
Fig. 3. Aberrant reduction of H3K27ac at enhancers in cells expressing H3.3G34W. (A) Immunoblots of whole cell extracts from EEDf/f and EED−/− cells (Left). Heatmap displaying the H3K27ac ChIP enrichment at promoter-distal sites (n = 14,420) that gained H3K27ac in EED−/− cells (Right). (B) Heatmaps displaying the ChIP enrichment profiles of H3K27ac and HA-tagged H3.3 or H3.1 at all promoter-distal active enhancers in mMSCs (n = 15,198). The color scale of H3.1* is modified from 0 to 5. (C) Model depicting the effect of H3.3 G34W mutation at enhancers. Increased PRC2-mediated H3K27me1/2/3 on H3.3 G34W oncohistones antagonizes H3K27ac by providing a placeholder methyl group(s). (D) Scatterplot displaying the correlation between reference-normalized H3K27ac intensities at active enhancers in cells expressing H3.3 WT or G34W. (E) Metaplot displaying the normalized H3K27ac ChIP, H3.3-K36me3 reChIP, H3.3-K27me3 reChIP enrichments at a 10-kb region around enhancers with reduced H3K27ac as defined in B. (F) Genome browser view displaying enrichment of H3.3X-K36me3, H3.3X-K27me3 reChIPs, and H3K27ac ChIPs in cells expressing H3.3WT or G34W. (G) Boxplot displaying the reference-normalized H3K36me2 enrichment at unchanged enhancers (0.9 ≤ ∆K27ac ≤ 1.1, n = 7,458) and enhancers with decreased H3K27ac (∆K27ac ≤ 0.5, n = 3,440). (H) Boxplots displaying the fold change in reference-normalized H3K27ac intensities at enhancers containing low (1st to 10th quantile), medium (45th to 55th quantiles), and high levels of H3K36me2 (90th to 100th quantile). P values for loss of H3K27ac were determined using Wilcoxon’s rank sum test. Dashed line represent a twofold loss. (I) Boxplots displaying the H3K27ac ChIP enrichment at enhancers with reduced H3K27ac in cells expressing H3.3 WT or H3.3 G34W or SETD2−/− cells. P value was determined using Wilcoxon’s rank sum test. (J) Boxplots displaying the H3K27ac ChIP enrichment at enhancers with reduced H3K27ac in SETD2−/− cells overexpressing H3.3 WT or G34W. P values for loss of H3K27ac were determined using Wilcoxon’s rank sum test. n.s.: not significant.
Fig. 4.
Reduced gene enhancer activity in…
Fig. 4.
Reduced gene enhancer activity in cells expressing H3.3 G34W. ( A ) Pie…
Fig. 4. Reduced gene enhancer activity in cells expressing H3.3 G34W. (A) Pie chart displaying the proportion of enhancers with reduced H3K27ac (∆K27ac ≤ 2), unchanged (0.9 ≤ ∆K27ac ≥ 1.1) and increased H3K27ac (∆K27ac ≥ 2) in H3.3 G34W-expressing cells. (Right) Pie chart displaying the changes in Med1/Brd4 occupancy at enhancers with reduced H3K27ac and expression of genes associated with them. Gene-enhancer associations were identified using GREAT and genes associated with at least three enhancers with reduced H3K27ac (n = 99) were used for the analyses. (B) Average profiles of Med1 (Left) and Brd4 ChIP-Seq (Right) displaying their average profiles at enhancers with decreased H3K27ac (Top, n = 3,440) and unchanged enhancers (Bottom, n = 7,458). (C) Boxplot displaying the change in the expression of H3.3 G34W down-regulated genes (fold change ≤ 0.3, n = 279) in SETD2−/− cells. Dashed line displays a twofold change. (D) Boxplot displaying the change in the expression of genes down-regulated by SETD2 knockout (fold change ≤ 0.3, n = 132) in cells expressing H3.3G34W. Dashed line displays a twofold change. (E) Boxplot displaying the H3K27ac enrichment at enhancers with reduced H3K27ac (as defined in Fig. 3A) in cells expressing H3.3 WT or G34R. (F) Boxplot displaying the expression of H3.3 G34W down-regulated genes (fold change ≤ 0.3, n = 279). TPM, transcripts per million. (G) Immunoblots of whole cell extracts from cells expressing H3.3G34W treated with 0 µM, 1 µM, or 5 µM tazemetostat. (H) Fold change in the expression of genes down-regulated by the expression of H3.3G34W. Samples represent G34W/WT (red), cells expressing H3.3G34W treated with tazemetostat 1 µM/0 µM (light green) and 5 µM/0 µM (dark green) as measured by RT-qPCR. (I) Boxplots displaying the fold change in the expression of genes down-regulated by the expression of H3.3G34W (FC ≤ 0.3, n = 279) in the same samples as shown in H. Center line in the boxplots represents the median; Bottom and Top of the box represent 25th and 75th quartiles; whiskers extend to 1.5× interquartile range. Background includes all genes with posterior probability of differential expression (PPDE) ≥ 0.95. P value was determined using Wilcoxon’s rank sum test.
Fig. 5.
H3.3G34W generates a unique gene…
Fig. 5.
H3.3G34W generates a unique gene expression profile in a PRC2-dependent manner. ( A …
Fig. 5. H3.3G34W generates a unique gene expression profile in a PRC2-dependent manner. (A) Heatmap displaying the expression profiles of 180 differentially expressed genes (PPDE > 0.95; fold change > 3) in mMSCs expressing H3.3 G34W relative to H3.3 WT. Unguided hierarchical clustering was used to plot the divergence between samples. Data displays two independent biological replicates for each samples. (B) Schematic displaying the effect of K36R and K27R mutations in cis with the G34W mutation. G34W mutation allows increased PRC2 activity by blocking SETD2. PRC2 does not tolerate arginine substitution at the 36th position in vitro and in vivo; therefore, G34W-K36R is unable to increase G34W-dependent PRC2 activity in cis. K27R-G34W substitution disrupts K27ac/K27me3 on G34W oncohistone, without affecting G34W-dependent changes at K36 methylation. (C) Gene set enrichment analysis of ostoblastic differentiation signature in mMSCs expressing H3.3 G34W compared to H3.3 WT. (D) Fold change in the expression of genes associated with osteoblastic (green) and adipogenic differentiation (blue) in mMSCs expressing H3.3G34W. Genes implicated in promoting cell motility and chemotaxis are shown in red. (E) Heatmap displaying the expression profiles of genes associated with adipogenic and osteoblastic differentiation in human GCTB-derived cells. (F and G) Kaplan–Meier survival curve displaying the survival of mice injected with mesenchymal stem cells overexpressing H3.3WT (n = 9, 10), H3.3G34W (n = 10, 9), or H3.3G34W;K27R (n = 5, 10) double mutant. P values for significant difference for H3.3G34W samples were determined using the log rank test with respect to WT (in blue) or G34W;K27R double mutant (in green).
Fig. 6.
H3.3 G34W alters enhancer landscapes…
Fig. 6.
H3.3 G34W alters enhancer landscapes by boosting the “hit-and-run” mechanism of PRC2 recruitment.…
Fig. 6. H3.3 G34W alters enhancer landscapes by boosting the “hit-and-run” mechanism of PRC2 recruitment. A balance between the activities of K36 methyltransferases, SETD2 and NSD2, and PRC2 exists at enhancers. “Robust” enhancers have high levels of NSD2-mediated H3K36me2 while other enhancers contain low NSD2 activity. Likewise, robust enhancers have little H3K27me2/3 while low-H3K36me2 enhancers exhibit higher PRC2 activity. The H3.3 G34W oncohistone disrupts the balance by decreasing SETD2 activity and promoting spreading of PRC2-mediated H3K27 methylation at low-H3K36me2 “G34W-sensitive” enhancers. However, the high NSD2 activity at robust enhancers prevents additional PRC2 activity. As a result of the increased H3K27 methylation, H3.3 G34W decreases H3K27ac specifically at enhancers containing low NSD2 activity. H3.3 G34W suppresses enhancers associated with genes involved in the maintenance of cell identity. See this image and copyright information in PMC
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