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. 2011 Feb 24;117(8):2451-9.
doi: 10.1182/blood-2010-11-321208. Epub 2010 Dec 29.

Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation

Damian B Yap  1 , Justin Chu, Tobias Berg, Matthieu Schapira, S-W Grace Cheng, Annie Moradian, Ryan D Morin, Andrew J Mungall, Barbara Meissner, Merrill Boyle, Victor E Marquez, Marco A Marra, Randy D Gascoyne, R Keith Humphries, Cheryl H Arrowsmith, Gregg B Morin, Samuel A J R Aparicio
Affiliations

Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation

Damian B Yap et al. Blood. .

Abstract

Next-generation sequencing of follicular lymphoma and diffuse-large B-cell lymphoma has revealed frequent somatic, heterozygous Y641 mutations in the histone methyltransferase EZH2. Heterozygosity and the presence of equal quantities of both mutant and wild-type mRNA and expressed protein suggest a dominant mode of action. Surprisingly, B-cell lymphoma cell lines and lymphoma samples harboring heterozygous EZH2(Y641) mutations have increased levels of histone H3 Lys-27-specific trimethylation (H3K27me3). Expression of EZH2(Y641F/N) mutants in cells with EZH2(WT) resulted in an increase of H3K27me3 levels in vivo. Structural modeling of EZH2(Y641) mutants suggests a "Tyr/Phe switch" model whereby structurally neutral, nontyrosine residues at position 641 would decrease affinity for unmethylated and monomethylated H3K27 substrates and potentially favor trimethylation. We demonstrate, using in vitro enzyme assays of reconstituted PRC2 complexes, that Y641 mutations result in a decrease in monomethylation and an increase in trimethylation activity of the enzyme relative to the wild-type enzyme. This represents the first example of a disease-associated gain-of-function mutation in a histone methyltransferase, whereby somatic EZH2 Y641 mutations in lymphoma act dominantly to increase, rather than decrease, histone methylation. The dominant mode of action suggests that allele-specific EZH2 inhibitors should be a future therapeutic strategy for this disease.

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Figures

Figure 1
Figure 1
Quantitation of EZH2 wild-type and EZH2 Y641F (MutF) protein expression by mass spectrometry. (A) Tandem MS spectra of recombinant EZH2 wild-type and MutF peptides (residues 635-654) used in the MRM assay. Peptide fragments (y and b ions) identified are labeled, and transitions used in the MRM assay are circled in red (supplemental Figure 2). (B) Extracted ion chromatograms of MRM signals for EZH2 from the gel fractions containing total EZH2 from the DOHH2 and WSU-DLCL2 samples. Traces correspond to the wild-type (green), MutF (red), and common (blue, residues 442-456) peptides. For MRM quantification, peak areas for each coeluting transition were summed for each peptide. (C) External standard curve of mixtures of recombinant EZH2WT and EZH2Y641F proteins, in triplicate. The wild-type and MutF peptides show different inherent ionization ability as illustrated by the difference in heights and areas of their specific peaks, thus necessitating the use of an external calibration curve to determine the corresponding percentage of EZH2WT and EZH2Y641F proteins in the sample. The equation fit to the curve used for the calculations is shown. Points A and B on the graph represent 2 biologic replicates of total endogenous EZH2 immunoprecipitated from the WSU-DLCL2 cell line. An independent 50:50 mixture of EZH2WT/EZH2Y641F recombinant proteins was analyzed (labeled as point “1:1”) to validate the accuracy of the procedure.
Figure 2
Figure 2
Y641 mutations in B-cell lines and tumors increase steady state of H3K27me3. (A) Steady-state H3K27me3 levels in DLBCL. Nuclear lysates from DLCBCLs with either wild-type EZH2 (Pfeiffer, MD903, NU-DHL-1, NU-DUL-1, DOHH2, and Toledo) or heterozygous for EZH2 (SU-DHL-6 (+/Y641N), Karpas 422 (+/Y641N), DB (+/Y641N), WSU-DLCL-2 (+/Y641F)) were probed with the respective antibodies to the following epitopes: H4K20me3 (arrowed lower band), H3K27me3, H3K27me2, H3K9me3, or EZH2. Levels of H3 were used as a loading control for histones. (B) Whole cell lysates from frozen tumor sections (each 5 × 20-μm slices) of 10 patients with either wild-type EZH2 (IDs 396, 839, 315, 085, or 900) or heterozygous for EZH2 (IDs 694 (+/Y641F), 178 (+/Y641H), 883 (+/Y641N), 940 (+/Y641F), or 353 (+/Y641S)) were probed with the respective antibodies. This a composite figure assembled to reflect similar levels of H3 from the 2 blots. (C) Nuclear lysates from HEK293T cells stably expressing GFP-tagged EZH2 and Y641 mutants were probed with anti-Ezh2 to assess ectopically expressed levels (top band) or endogenous (lower band) Ezh2 levels and anti-H3K27me3. Anti-H3 was used to assess histones as a loading control. (D) Respective plasmids encoding GFP, EZH2, or mutants (as indicated) were transfected in HEK293T cells; the lysates were probed with the antibodies (anti-FLAG M2, anti-GFP, anti-EZH2 (top band shows GFP-tagged EZH2, lower band shows endogenous EZH2 and FLAG-tagged EZH2), monomethyl, dimethyl, or trimethyl specific H3K27. These antibodies are specific for the respective methylation states (supplemental Figure 3). (E) GFP-tagged proteins from nuclear lysates of HEK293T lines stably expressing GFP-EZH2 wild-type and mutants were immunoprecipitated with GFP-trap (“Western blotting and immunoprecipitation”) and probed with anti-EZH2 to show precipitation of GFP-tagged EZH2 (top band) with copurification of endogenous EZH2 (lower band) and EED and SUZ12 (lower band, arrowed). *Nonspecific band. (F) Mouse bone marrow stem cells were infected with the respective retroviruses expressing wild-type or Y641F EZH2 and differentiated in vitro into B cells before whole cell lysates were probed with the respective antibodies. The top band (arrowed) in the EZH2 blot represents ectopic EZH2-HA and endogenous EZH2. *The lower band indicates degradation products. (G) Densitometry measurements of H3K27me3 to total H3 ratio in wild-type and mutant cell lines. The boxplots represent the distribution of ratios in mutant and wild-type. Significance testing by unpaired Student t test. (H) Densitometry measurements of H3K27me3 to total H3 ratio in wild-type and lymphoma samples. The boxplots represent the distribution of ratios in mutant and wild-type. Significance testing by unpaired Student t test.
Figure 3
Figure 3
Homology model of the active site of EZH2. The model is based on sequence and structure alignment with histone dimethyltransferase G9a/EHMT2 (PDBID 2O8J). This was also structurally aligned with GLP/EHMT1 bound to an H3K9me2 peptide substrate (PDBID 2RFI), and SETD7 (PDBID 1O9S) using ICM software, Version 3.6-1g (Molsoft LLC). See supplemental Figures 6-8 for sequence and structural alignments. The model shows the reaction products dimethyllysine and S-adenosylhomocysteine (SAH) in purple. The black arrow indicates the path of methyl transfer from the sulfur atom (yellow) of the cofactor to the nitrogen atom (blue) of substrate lysine. Hydrogen bonds are shown as dotted blue lines.
Figure 4
Figure 4
In vitro PRC2 activity of EZH2. Histone methyltransferase reaction was performed using 1.2μM biotinylated peptide substrate (ie, peptide mimicking the H3 tail, H3(21-44)), which has been unmethylated (H3K27me0, open bar or me0), monomethylated (H3K27me1, gray bar or me1) or dimethylated (H3K27me2, black bar or me2) at Lys-27. The reactions were incubated with purified 250 ng (A) or various amounts (B) of PRC-2 complex containing wild-type EZH2 (WT), EZH2 mutants (Y641N or Y641F). (A) Error bars represent SD of replicates from 2 independent experiments.
Figure 5
Figure 5
Model depicting pathways to increase H3K27me3 levels that result in cancer. In this schematic diagram: solid arrow represents a direct effect; dotted arrow, multiple steps that lead to an effect.
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