Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas

Abstract

EZH2, the catalytic subunit of the PRC2 complex, catalyzes the mono- through trimethylation of lysine 27 on histone H3 (H3K27). Histone H3K27 trimethylation is a mechanism for suppressing transcription of specific genes that are proximal to the site of histone modification. Point mutations of the EZH2 gene (Tyr641) have been reported to be linked to subsets of human B-cell lymphoma. The mutant allele is always found associated with a wild-type allele (heterozygous) in disease cells, and the mutations were reported to ablate the enzymatic activity of the PRC2 complex for methylating an unmodified peptide substrate. Here we demonstrate that the WT enzyme displays greatest catalytic efficiency (k(cat)/K) for the zero to monomethylation reaction of H3K27 and diminished efficiency for subsequent (mono- to di- and di- to trimethylation) reactions. In stark contrast, the disease-associated Y641 mutations display very limited ability to perform the first methylation reaction, but have enhanced catalytic efficiency for the subsequent reactions, relative to the WT enzyme. These results imply that the malignant phenotype of disease requires the combined activities of a H3K27 monomethylating enzyme (PRC2 containing WT EZH2 or EZH1) together with the mutant PRC2s for augmented conversion of H3K27 to the trimethylated form. To our knowledge, this is the first example of a human disease that is dependent on the coordinated activities of normal and disease-associated mutant enzymatic function.

Fig. 1.

B-cell lymphoma-associated mutants of EZH2 are active histone methyltransferases. In vitro methyltransferase activity of PRC2 complexes containing wild-type and various Y641 mutants of EZH2 was measured as (A) methyl transfer reactions using a peptide (H3 21-44) as substrate and (B) methyl transfer reactions using avian nucleosomes as substrate. Symbols: wild type (•), Y641F (○), Y641H (□), Y641N (▪), and Y641S (▴). CPM is counts per minute, referring to scintillation counting as a result of ³H radiation. See Materials and Methods for further information.

Fig. 2.

PRC2 complexes containing mutant EZH2 preferentially catalyze di- and trimethylation of histone H3K27. (A) Methyltransferase activity of mutant and WT complexes on unmethylated peptide (open bars), monomethylated peptide (hatched bars), and dimethylated peptide (closed bars). (B) Affinity for peptide substrates as defined by K_1/2 is similar across all peptide methylation states for PRC2 complexes containing wild-type (○),Y641F (•), Y641H (□), Y641N (▪), and Y641S (▴) EZH2. Note that the K_1/2 values across all substrates and all enzyme forms varies less than 3.5-fold. For any particular methylation state of substrate, the variation in K_1/2 value is less than 2-fold. (C) Enzyme turnover number (k_cat) varies with substrate methylation status in opposing ways for WT and Y641 mutants of EZH2. The k_cat decreases with increasing K27 methylation states for wild-type (○), but increases for Y641F (•), Y641H (□), Y641N (▪), and Y641S (▴) mutants of EZH2. (D) Catalytic efficiency (k_cat/K_1/2) decreases with increasing K27 methylation states for wild-type (○) but increases for Y641F (•), Y641H (□), Y641N (▪), and Y641S (▴) mutants of EZH2. (B–D) The lines drawn to connect the data points are not intended to imply any mathematical relationship; rather, they are intended merely to serve as a visual aid to guide the eye of the reader.

Fig. 3.

Steady-state enzyme kinetics predicts the relative patterns of H3K27 methylation status in EZH2 WT and heterozygous mutant lymphoma cell lines. (A) Predicted relative levels of H3K27me3 (Top), H3K27me2 (Middle), and H3K27me1 (Bottom) for cells containing different EZH2 mutants. The simulations were performed using a coupled enzyme steady-state velocity equation (30) and the steady-state kinetic parameters reported in Table 1. All values are relative to the homozygous WT EZH2-containing cells and assume saturating concentrations of intracellular SAM, relative to K_m and intracellular nucleosome concentrations similar to K_m. Bars shown in red are meant to highlight WT and mutants for which experimental data are available for lymphoma cell lines in B. See text for further details. (B) Western blot analysis of relative patterns of H3K27 methylation status for lymphoma cell lines containing homozygous WT EZH2 or heterozygous for the indicated EZH2 Y641 mutation. (Top to Bottom) Results for probing with specific antibodies for the following: total EZH2 levels, H3K27me3, H3K27me2, H3K27me1, and total histone H3 as a loading control.

Fig. 4.

Proposed mechanisms leading to aberrantly high levels of trimethylation on histone H3K27 in cancer include (A) mutation of Y641 in EZH2 resulting in a change in substrate preference from the nonmethylated to the mono- and dimethylated histone H3K27, (B) overexpression of EZH2, (C) mutations in UTX that inactivate enzyme function, causing a decrease in demethylation of H3K27me3, and (D) overexpression of the PRC2 complex subunit PHF19/PCL3 that leads to increases in recruitment of the PRC2 complex to specific genes and an increase in histone H3K27 trimethylation. In all four models the alteration leads to aberrant histone H3K27 trimethylation in the proximal promoter regions of genes resulting in transcriptional repression of key genes in cancer.