Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma

Abstract

Sequencing of pediatric gliomas has identified missense mutations Lys27Met (K27M) and Gly34Arg/Val (G34R/V) in genes encoding histone H3.3 (H3F3A) and H3.1 (HIST3H1B). We report that human diffuse intrinsic pontine gliomas (DIPGs) containing the K27M mutation display significantly lower overall amounts of H3 with trimethylated lysine 27 (H3K27me3) and that histone H3K27M transgenes are sufficient to reduce the amounts of H3K27me3 in vitro and in vivo. We find that H3K27M inhibits the enzymatic activity of the Polycomb repressive complex 2 through interaction with the EZH2 subunit. In addition, transgenes containing lysine-to-methionine substitutions at other known methylated lysines (H3K9 and H3K36) are sufficient to cause specific reduction in methylation through inhibition of SET-domain enzymes. We propose that K-to-M substitutions may represent a mechanism to alter epigenetic states in a variety of pathologies.

Fig. 1. Histones extracted from human DIPG containing H3F3A K27M (H3.3) or HIST3H1B K27M (H3.1) mutations exhibit decreased H3K27me3

(A) Immunoblots of acid-extracted histones from DIPGs of different indicated H3 genotypes. (B) Immunoblots of histones from PDGF-induced gliomas with and without the H3.3K27M transgene. (C) Immunohistochemistry of H3K27me3 in human or murine gliomas containing wild-type H3.3 or H3.3K27M. (D) Immunohistochemistry quantification of nuclei staining positive for H3K27me3 (P=0.01 for human DIPG, and P = 0.0001 for murine gliomas with the unpaired t test).

Fig. 2. H3K27M transgenes cause a global decrease of H3K27me3

(A) Immunoblots of whole-cell extract from lentivirus-transduced 293T cells expressing the indicated H3.3 transgenes. (B) Immunoblots of whole extract from H3.3 K27-to-X transduced 293T cells. The antibody against HA (anti-HA) blot shows the relative H3.3 transgene amounts. (C) Immunoblots of anti-HA–immunoprecipitated heterotypic H3.3 K27M or G34R/V mononucleosomes from the indicated H3.3 transgenic 293T cell lines. (D) Immunoblots of anti-HA–immunoprecipitated oligonucleosomes (>95%of four to five nucleosomes in length, with a median of two to three nucleosomes) from the indicated H3.3 transgenic 293T cell lines (vertical black lines indicate where control lanes were removed for clarity).

Fig. 3. H3K27M inhibits PRC2 methyltransferase activity in cis and trans

(A) Fluorography of PRC2-mediated methylation of wild-type H3.3 or heterotypic H3.3K27M nucleosome substrates. (B) Fluorography of PRC2-mediated methylation of heterotypic mononucleosomes containing the indicated amino acid substitution. (C) Fluorography of PRC2-mediated methylation of wild-type mononucleosomes in the presence of 10 or 100 µM of the indicated peptide. (D) Quantification of PRC2 methyltransferase activity with varying concentrations of wild-type oligonucleosomes in the presence of 45 µM of H3K27ac or H3K27M peptides. Error bars represent the standard deviation for three repeats of the experiment. (E) Fluorography of PRC2-mediated nucleosome methylation with titration of K27M or K27ac peptides.

Fig. 4. H3K27M-mediated inhibition of PRC2 occurs through interaction with EZH2

(A) Coomassie gel of purified PRC2 cross-linked to peptide 1 (fig. S4F) and immunoblots with streptavidin–horseradish peroxidase (SA-HRP) or antibody against EZH2. (B) IC₅₀ measurement for methionine or norleucine substitution at K27. Titration reactions of H3.3K27M or K3.3K27Nle peptides with 70 ng PRC2 and 0.8 µg wild-type oligonucleosomes. Error bars represent the standard deviation of five repeats. (C) Quantification of PRC2 methyltransferase activity in the presence of 50 µM of H3 peptides (18 to 37) containing K27acetyl, K27Leu, K27Ile, K27Met, or K27Nle. Error bars represent standard deviation of three repeats. (D) Fluorography of methyltransferase reactions with wild-type or EZH2-Y641N PRC2 on wild-type H3.3 nucleosomes in the presence of 50 µM H3 peptides (18 to 37) with K27acetyl, K27Met, or K27Nle. Error bars represent the standard deviation of three repeats. (E) Quantification of methyltransferase activity of reactions in (D). Error bars represent the standard deviation of five repeats. (F) Immunoblots of 293T cells expressing the indicated EZH2 and H3.3 transgenes. The anti-HA blot shows relative H3.3 transgene amounts, and the anti-FLAG shows the relative EZH2 or Y641N amounts. (G) Immunoblots of whole-cell extract from transduced 293T cells expressing the indicated H3.3 transgenes. The arrows to the right of the total H3 blot point to the transgenic histone (red) or endogenous wild-type histone (black). (H) Quantification of histone methyltransferase activity on H3 peptides (1 to 20) by SUV39H1 or G9a in the presence of 10 µM H3K9me3 or H3K9M or no peptide. Error bars represent the standard deviation for five repeats.