H3K36 methylation antagonizes PRC2-mediated H3K27 methylation

Abstract

H3K27 methylation mediated by the histone methyltransferase complex PRC2 is critical for transcriptional regulation, Polycomb silencing, Drosophila segmentation, mammalian X chromosome inactivation, and cancer. PRC2-mediated H3K27 methylation can spread along the chromatin and propagate the repressive chromatin environment; thus, chromatin components that antagonize the activity of PRC2 are important for restraining Polycomb silencing. Here we report that in HeLa cells, H3 histones unmethylated at Lys-36 are mostly methylated at Lys-27, with the exception of newly synthesized H3. In addition, K27me3 rarely co-exists with K36me2 or K36me3 on the same histone H3 polypeptide. Moreover, PRC2 activity is greatly inhibited on nucleosomal substrates with preinstalled H3K36 methylation. These findings collectively identify H3K36 methylation as a chromatin component that restricts the PRC2-mediated spread of H3K27 methylation. Finally, we provide evidence that the controversial histone lysine methyltransferase Ash1, a known Trithorax group protein that antagonizes Polycomb silencing in vivo, is an H3K36-specific dimethylase, not an H3K4 methylase, further supporting the role of H3K36 methylation in antagonizing PRC2-mediated H3K27 methylation.

FIGURE 1.

Except for newly synthesized histone H3, H3 moieties unmodified at both Lys-27 and Lys-36 rarely exist in HeLa cells. A, experimental scheme. B, almost all HeLa cells advanced through S phase 9 h after cell cycle release, as indicated by the Lys-8/Lys-0 ratio of H3 backbone peptides. C, summary table for the Lys-8/Lys-0 ratio of H3 backbone peptides and H3:K27-R40 peptides with various modifications at the 9-h time point. Data for three independent repeat experiments were provided individually. Mean value and standard deviation of the three repeats were also calculated. D, mass spectra of an H3 backbone peptide and an H3K27me0/K36me0 peptide at the 9-h time point. E, mass spectra of an H3 backbone peptide and an H3K27me0/K36me0 peptide at the 3-h time point.

FIGURE 2.

Immunoprecipitation experiments showing that H3K27me3 and H3K36me3 markers rarely co-exist in the same mononucleosomes.

FIGURE 3.

Approximate abundance of each H3:K27-R40 peptide with a distinct modification in HeLa cells at the G₁/S boundary. A, XICs of all detected H3:K27-R40 peptides with various degrees of modifications and their relative abundance. B, summary table of A. Data for three independent repeat experiments were provided individually. Mean value and standard deviation of the three repeats were also calculated.

FIGURE 4.

PRC2 activity is inhibited by premethylated H3K36. A, experimental scheme. B, PRC2 activity is inhibited by premethylated H3K36. C, Dot1L activity is not affected by premethylated H3K36.

FIGURE 5.

Ash1 is an nucleosomal H3K36-specific dimethylase. A, Ash1 prefers nucleosomal H3 as its substrates. B, H3K36A, but not the other mutant nucleosomes, abolishes the enzymatic activity of Ash1. C, enzymatic activity of Ash1 on nucleosomes with H3Kc27me0/1/2/3.