A gain-of-function mutation in histone H3, specifically K27M, found in pediatric glioblastoma, inhibits the activity of the Polycomb repressive complex 2 (PRC2). This mutation reduces the levels of trimethylated lysine 27 (H3K27me3) on histone H3, which is crucial for maintaining epigenetic gene silencing. The study shows that H3K27M interacts with the EZH2 subunit of PRC2, thereby inhibiting its enzymatic activity. Additionally, other lysine-to-methionine substitutions in histone H3 also inhibit SET-domain enzymes, leading to specific reductions in methylation. These findings suggest that K-to-M substitutions may be a mechanism for altering epigenetic states in various pathologies. The K27M mutation is prevalent in diffuse intrinsic pontine gliomas (DIPGs) and is associated with reduced H3K27me3 levels. This mutation leads to a decrease in H3K27me3 and an increase in H3K27ac. In vivo, transgenic expression of H3.3K27M increases H3K27ac and reduces H3K27me3. The study also shows that H3K27M inhibits PRC2 activity by interacting with the EZH2 subunit. This inhibition is specific to the K27M mutation, as other substitutions do not have the same effect. The study further demonstrates that the K27M mutation can inhibit PRC2 activity by interfering with the normal stimulation of PRC2 by H3K27me3. The K27M peptide reduces PRC2-dependent methylation by 6.2-fold at 45 μM. The inhibition is dose-dependent and is mediated by the methionine side chain interacting with aromatic residues in the EZH2 active site. The study also shows that K-to-M mutations in other histone residues, such as K9 and K36, can inhibit SET-domain methyltransferases, leading to reduced methylation at those sites. The findings suggest that K-to-M mutations can target the active sites of various SET-domain methyltransferases, thereby competing with substrate binding and turnover. These mutations, even when present in low abundance, can significantly affect global methylation patterns. The study proposes a model where the aberrant epigenetic silencing caused by H3K27M-mediated inhibition of PRC2 activity promotes gliomagenesis. The results highlight the potential of K-to-M mutations as a mechanism for altering epigenetic states and suggest that similar mutations may exist in other histone-related pathologies.A gain-of-function mutation in histone H3, specifically K27M, found in pediatric glioblastoma, inhibits the activity of the Polycomb repressive complex 2 (PRC2). This mutation reduces the levels of trimethylated lysine 27 (H3K27me3) on histone H3, which is crucial for maintaining epigenetic gene silencing. The study shows that H3K27M interacts with the EZH2 subunit of PRC2, thereby inhibiting its enzymatic activity. Additionally, other lysine-to-methionine substitutions in histone H3 also inhibit SET-domain enzymes, leading to specific reductions in methylation. These findings suggest that K-to-M substitutions may be a mechanism for altering epigenetic states in various pathologies. The K27M mutation is prevalent in diffuse intrinsic pontine gliomas (DIPGs) and is associated with reduced H3K27me3 levels. This mutation leads to a decrease in H3K27me3 and an increase in H3K27ac. In vivo, transgenic expression of H3.3K27M increases H3K27ac and reduces H3K27me3. The study also shows that H3K27M inhibits PRC2 activity by interacting with the EZH2 subunit. This inhibition is specific to the K27M mutation, as other substitutions do not have the same effect. The study further demonstrates that the K27M mutation can inhibit PRC2 activity by interfering with the normal stimulation of PRC2 by H3K27me3. The K27M peptide reduces PRC2-dependent methylation by 6.2-fold at 45 μM. The inhibition is dose-dependent and is mediated by the methionine side chain interacting with aromatic residues in the EZH2 active site. The study also shows that K-to-M mutations in other histone residues, such as K9 and K36, can inhibit SET-domain methyltransferases, leading to reduced methylation at those sites. The findings suggest that K-to-M mutations can target the active sites of various SET-domain methyltransferases, thereby competing with substrate binding and turnover. These mutations, even when present in low abundance, can significantly affect global methylation patterns. The study proposes a model where the aberrant epigenetic silencing caused by H3K27M-mediated inhibition of PRC2 activity promotes gliomagenesis. The results highlight the potential of K-to-M mutations as a mechanism for altering epigenetic states and suggest that similar mutations may exist in other histone-related pathologies.