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Mutation of A677 in EZH2 in human B-cell promotes hypertrimethylation of on 27 (H3K27)

Michael T. McCabea,1, Alan P. Gravesb, Gopinath Ganjia, Elsie Diazb, Wendy S. Halseyb, Yong Jiangb, Kimberly N. Smithemana, Heidi M. Otta, Melissa B. Pappalardia, Kimberly E. Allenb, Stephanie B. Chenb, Anthony Della Pietra IIIa, Edward Dulb, Ashley M. Hughesb, Seth A. Gilberta, Sara H. Thrallb, Peter J. Tumminoa, Ryan G. Krugera, Martin Brandtb, Benjamin Schwartzb, and Caretha L. Creasya

aCancer Discovery Performance Unit, Cancer Research, Oncology Research and Development and bPlatform Technology and Sciences, GlaxoSmithKline, Collegeville, PA 19426

Edited* by Judith P. Klinman, University of California, Berkeley, CA, and approved January 11, 2012 (received for review October 19, 2011)

Trimethylation of histone H3 on lysine 27 () is a re- B cells, and recirculating B cells (12). EZH2 expression is re- pressive posttranslational modification mediated by the histone quired in the bone marrow for progression of pro-B cells into methyltransferase EZH2. EZH2 is a component of the polycomb pre-B cells and immature B cells, because genetic inactivation of repressive complex 2 and is overexpressed in many cancers. In EZH2 leads to an accumulation of cells at the pro–B-cell stage B-cell , its substrate preference is frequently altered (12). However, if EZH2 is inactivated after the pro–B-cell stage, through somatic mutation of the EZH2 Y641 residue. Herein, we additional maturation steps are not hindered, suggesting that identify mutation of EZH2 A677 to a glycine (A677G) among lym- EZH2 functions early in B-cell differentiation (12). In fact, phoma cell lines and primary tumor specimens. Similar to Y641 multiple groups have shown that EZH2 plays an important role mutant cell lines, an A677G mutant cell line revealed aberrantly in the maintenance of hematopoietic stem cells (HSCs) and pro- elevated H3K27me3 and decreased monomethylated H3K27 genitor cells (13, 14). In particular, EZH2 overexpression in (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 HSCs leads to continued self-renewal capacity in serial trans- possessed catalytic activity with a substrate specificity that was plantation models, suggesting that EZH2 contributes to repo- pulating potential and helps cells resist replicative stress (13). distinct from those of both WT EZH2 and Y641 mutants. Whereas EZH2 is frequently amplified and/or overexpressed in most WT EZH2 displayed a preference for substrates with less methyla- k K solid tumor types (15); however, this does not appear to be the tion [unmethylated H3K27 (H3K27me0):me1:me2 cat/ m ratio = case in lymphomas, perhaps attributable to the high basal ex- 9:6:1] and Y641 mutants preferred substrates with greater meth- k K pression of EZH2 in normal proliferating B cells. Instead, EZH2 ylation (H3K27me0:me1:me2 cat/ m ratio = 1:2:13), the A677G has been reported to harbor recurrent mutations of the fi EZH2 demonstrated nearly equal ef ciency for all three substrates 641 (Y641) residue in 22% of germinal center B-cell (GCB) dif- k K (H3K27me0:me1:me2 cat/ m ratio = 1.1:0.6:1). When transiently fuse large B-cell lymphomas (DLBCLs) and 7% of follicular expressed in cells, A677G EZH2, but not WT EZH2, increased global lymphomas (FLs) (3). Although initially reported to be a loss-of- H3K27me3 and decreased H3K27me2. Structural modeling of WT function mutation (3), subsequent biochemical work demonstrated and mutant EZH2 suggested that the A677G mutation acquires the a unique gain-of-function activity for Y641 mutant EZH2 (16, 17). ability to methylate H3K27me2 through enlargement of the lysine Although WT EZH2 exhibits a strong preference for unmethylated tunnel while preserving activity with H3K27me0/me1 substrates (H3K27me0) and monomethylated H3K27 (H3K27me1) sub- through retention of the Y641 residue that is crucial for orienta- strates, the Y641 mutants observed in lymphomas (Y641F/N/S/H/ tion of these smaller substrates. This mutation highlights the C) exhibit profoundly increased activity for dimethylated H3K27 interplay between Y641 and A677 residues in the substrate spec- (H3K27me2), decreased activity for H3K27me1, and little to no ificity of EZH2 and identifies another lymphoma patient popula- activity for H3K27me0 (16, 17). Through the coordinated activ- tion that harbors an activating mutation of EZH2. ities of WT and mutant EZH2, there is a global increase in tri- of H3K27 (H3K27me3) in Y641 mutant lymphomas ecent genome-wide sequencing studies have revealed several concomitant with a decrease in H3K27me1 and H3K27me2 (16). Rgenes that are frequently altered in non-Hodgkin lympho- These EZH2 Y641 mutations, along with EZH2 overexpression mas, including EZH2, MLL2, MEF2B, CREBBP, and TP53 in many tumors, suggest that dysregulation of H3K27me3 is im- among others (1–3). Many of these mediate, either directly portant in human tumorigenesis. Indeed, H3K27me3 levels corre- or indirectly, through the recruitment of cofactors, the array of late with progression-free survival in renal cell carcinoma (18) and posttranslational modifications observed on the amino-terminal with disease severity and poor tumor differentiation in esophageal tails of . Similar studies have implicated these and other squamous cell carcinoma (19). In addition to mutation of EZH2 epigenetic factors in transitional cell carcinoma of the bladder Y641, alternative mechanisms for dysregulation of H3K27me3 in- (e.g., UTX, ARID1A, MLL, MLL3), head and neck squamous clude inactivating mutations of the H3K27 UTX cell cancers (e.g., EZH2, MLL2), and myeloid malignancies (e.g., IDH1/2, TET2, DNMT3A, EZH2)(4–7). The prevalence of ge-

netic changes affecting factors and - Author contributions: M.T.M., G.G., E. Diaz, W.S.H., R.G.K., M.B., B.S., and C.L.C. designed GENETICS modifying genes highlights the central role of transcriptional research; M.T.M., A.P.G., E. Diaz, W.S.H., K.N.S., H.M.O., M.B.P., K.E.A., A.D.P., A.M.H., and dysregulation in tumorigenesis. S.A.G. performed research; Y.J., S.B.C., and E. Dul contributed new reagents/analytic tools; M.T.M., A.P.G., G.G., E. Diaz, W.S.H., K.N.S., M.B.P., K.E.A., A.M.H., S.H.T., P.J.T., R.G.K., The EZH2 encodes a SET domain-containing lysine M.B., B.S., and C.L.C. analyzed data; and M.T.M., A.P.G., M.B.P., and B.S. wrote the paper. methyltransferase that, along with EED, SUZ12, RbAp48, and fl AEBP2, forms the polycomb repressive complex 2 (PRC2) (8, 9). Con ict of interest statement: All authors are employees of GlaxoSmithKline. EZH2 is responsible for the methylation of histone H3 on lysine *This Direct Submission article had a prearranged editor. 27 (H3K27), which is generally associated with transcriptional Freely available online through the PNAS open access option. repression when present in the di- or trimethylated state (8–11). 1To whom correspondence should be addressed. E-mail: [email protected]. EZH2 is highly expressed in pro-B cells and progressively de- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. creases in expression as cells progress into pre-B cells, immature 1073/pnas.1116418109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1116418109 PNAS | February 21, 2012 | vol. 109 | no. 8 | 2989–2994 Downloaded by guest on September 28, 2021 (4, 20, 21) and overexpression of EZH2 attributable to multiple FLs, and (1–3, 26–28). The end result mechanisms, including decreased miR-101 levels (22, 23), aberrant of these mutations is increased or decreased methylation of E2F activity (24), and chromosomal amplification (25). H3K27 (16, 17, 26, 28). To characterize alterations of H3K27me3 Through the investigation of global H3K27me3 levels in more in human cancer cell lines, we quantified global H3K27me3 and than 100 cancer cell lines, we identified a unique EZH2 mutation total histone H3 levels by ELISA in 111 cell lines from seven at the A677 residue that is capable of increasing global H3K27me3 unique tumor types (Fig. 1A). The methylation-specific antibody levels when exogenously expressed in cells. Characterization of this was confirmed to be highly specific for H3K27me3 under these mutant protein revealed that exchange of A677 for glycine assay conditions through titration of full-length recombinant (A677G) leads to increased activity with H3K27me2 substrates methylated histones and competition of signal from a protein ly- A B similar to the Y641 EZH2 mutations. However, in contrast to the sate with methylated peptides (Fig. S1 and ). Each of the tumor Y641 mutants, which lose activity with H3K27me0 substrates, this types examined exhibited a range of H3K27me3 levels, with several substitution retains critical interactions present in WT EZH2 lymphoma cell lines possessing H3K27me3 levels two- to threefold leading to efficient utilization of all three methylation substrates higher than the highest nonlymphoma cell lines. Western blot analysis of protein lysates from several of these cell lines with (H3K27me0, H3K27me1, and H3K27me2). This mutation pres- fi ents a unique approach for cells to dysregulate H3K27 methylation antibodies speci c for H3K27me3, H3K27me2, and H3K27me1 revealed an apparent imbalance between the methylation states without requiring cooperation with WT EZH2 as is the case for of H3K27 (Fig. 1B and Fig. S2). Overall, lymphoma cell lines with Y641 EZH2 mutants. elevated H3K27me3 appear to have increased trimethylation at Results the expense of dimethylation and monomethylation, because H3K27me2 levels and H3K27me1 levels, to a lesser extent, were Aberrantly Elevated H3K27me3 Levels in Human Lymphoma Cell Lines. reduced in these cell lines relative to those with lower global A variety of activating and inactivating mutations of EZH2 have H3K27me3 levels. been described in primary tumors derived from GCB DLBCLs, Mutation of the A677 Residue of EZH2 to Glycine in a Lymphoma Cell Line with Aberrantly Elevated H3K27me3. Based on recent findings demonstrating an H3K27 hypertrimethylation phenotype in lymphoma cells harboring mutation of the Y641 residue of

Fig. 2. The Pfeiffer lymphoma cell line harbors a heterozygous A677G mutation in EZH2. (A) Chromatograms from Sanger sequencing of EZH2 in a WT control sample, the Pfeiffer DLBCL cell line, and a primary DLBCL pa- tient sample (sample ID CD564308). Heterozygous nonsynonymous missense mutation of C2045C/G (red asterisks) translates to A677A/G. Nucleotide and Fig. 1. A subset of lymphoma cell lines exhibits elevated H3K27me3 levels. (A) amino acid residue numbering is based on the NM_001203247 EZH2 cDNA Global H3K27me3 levels (normalized to total H3) were determined for 111 cancer transcript. (B) EZH2 domain architecture (UniProt Q15910). Sites of non- cell lines from seven different cancer types using H3K27me3 and total H3 ELISAs. synonymous mutations identified in lymphoma cell lines and primary tumors Lymphoma cell lines harboring heterozygous Y641 mutations are indicated by in this study are highlighted. (C) Alignment of human EZH2 with human black tick marks below the graph. MM, multiple myeloma. (B) Western blot EZH1, the fly ortholog E(z), and six other related SET domain-containing analysis was performed with antibodies specific for H3K27me3, H3K27me2, histone lysine showing that Y641 and A677 are highly H3K27me1, total histone H3, EZH2, and actin using protein lysates from a panel conserved. Blue shading represents identical residues, and yellow shading of lymphoma cell lines. Actin serves as a loading control. EZH2 mutation status as represents conserved residues. In order for a column to be shaded, seven of determined from full-length Sanger sequencing is indicated. nine residues must be conserved/identical.

2990 | www.pnas.org/cgi/doi/10.1073/pnas.1116418109 McCabe et al. Downloaded by guest on September 28, 2021 EHZ2 to either (F), asparagine (N), (S), (Fig. 2A and Table S1). These data confirm that the A677G mu- (H), or cysteine (C), we hypothesized that the lym- tation occurs in primary human lymphoma and is not simply an phoma cell lines with the highest levels of H3K27me3 may artifact of cell culture. harbor activating mutations in EZH2. Sanger sequencing of all cell lines for the Y641 codon revealed activating mutations for EZH2 A677G Mutation Confers Biochemical Activity Independent of six of the top seven lymphoma types when ranked by global H3K27 Methylation State. Given the hypertrimethylation pheno- H3K27me3 levels (Fig. 1A). type in cells bearing the A677G mutation and the spatial proximity The one cell line with high H3K27me3 levels that was not of A677 to Y641 in our structural models (vide infra), we tested mutated at Y641 was the Pfeiffer cell line. That cell line was in vitro whether A677G EZH2 displayed an altered substrate established in 1992 from the pleural effusion of a patient in the specificity similar to Y641 mutants (Fig. 3 and Table S2). When leukemic phase of DLBCL (29). Sanger sequencing of genomic comparing turnover and catalytic efficiency with the H3K27 DNA for all EZH2 coding revealed a heterozygous C-to-G peptide substrates (k and k /K , respectively), WT EZH2 loses mutation at nucleotide 2045, leading to a nonsynonymous mu- cat cat m A activity when progressively more methyl groups are incorporated tation of the A677 residue to a glycine (A677G) (Fig. 2 ). Se- into H3K27 (i.e., H3K27me0 > H3K27me1 > H3K27me2), with quence analysis of cDNA revealed that both WT and mutant the ratio of catalytic efficiencies for H3K27me0, H3K27me1, and alleles are expressed (Fig. S3). This residue falls within the cat- H3K27me2 being 9:6:1. In contrast, all Y641 mutant that alytic SET domain of EZH2 and is located in 18 two exons were evaluated (Y641F/N/S/H/C) displayed the opposite trend, downstream of the Y641 residue (Fig. 2B). This residue is highly with the H3K27me2 peptide being used most efficiently conserved across multiple species and multiple histone methyl- (H3K27me0:me1:me2 k /K ratio = 1:2:13). These substrate (Fig. 2C), indicating that it may play an essential cat m role in the function of EZH2. preferences are consistent with recently reported data demon- strating H3K27me0:me1:me2 kcat/Km ratios of 13:4:1 and 1:2:22 Occurrence of the A677G EZH2 Mutation in Primary Lymphoma for WT and Y641F EZH2, respectively (16). The A677G EZH2 fi Samples. To establish whether the mutation identified in the complex, on the other hand, displayed a pro le different from Pfeiffer cell line occurs in primary human lymphomas, this residue both WT and Y641 mutants. A677G EZH2 used all three sub- fi k K was sequenced in a panel of 41 lymphoma specimens. This panel strates with nearly equal ef ciency (H3K27me0:me1:me2 cat/ m consisted of 30 DLBCLs, six FLs, one mantle cell lymphoma, one ratio = 1.1:0.6:1) and at a rate comparable to the highest turnover k −1 extranodal marginal zone B-cell lymphoma of mucosa-associated rate observed with the WT ( cat = 0.84 min for A677G −1 lymphoid tissue, one splenic marginal zone lymphoma, and two with H3K27me0 vs. 1.01 min for WT with H3K27me0). When Waldenström macroglobulinemia or lymphoplasmacytic lympho- purified from HeLa cells were evaluated, the activity mas (Table S1). In addition to four occurrences of the Y641 of A677G EZH2 was slightly higher than WT EZH2 and the −1 −1 mutation (1 Y641N, 1 Y641F, 1 Y641H, and 1Y641C), non- Y641 mutants (kcat = 0.19 min for A677G vs. 0.11 min for − synonymous missense mutations were observed at P488 (P488S) WT and 0.15 min 1 for Y641S), likely attributable to the ability of and A677 (A677G). The A677 mutation was heterozygous and the A677G EZH2 complex to act on a greater proportion of the occurred in a stage IIE DLBCL obtained from a 74-y-old woman HeLa histones, which are heterogeneously modified at H3K27. GENETICS

−1 Fig. 3. A677G EZH2 mutant exhibits a unique substrate specificity. Enzyme turnover number [kcat (min )], the substrate concentration at which the reaction −1 −1 rate was half of Vmax [Km (μM)], and catalytic efficiency [kcat/Km (μM ·min )] were evaluated for recombinant five-member PRC2 complexes containing WT, A677G, or Y641 mutant EZH2 using peptides from histone H3 AA21–44 with no methylation (K27me0), monomethylation (K27me1), or dimethylation (K27me2) at the lysine 27 position. Briefly, EZH2 (20 nM) was combined with varying concentrations of peptide and [3H]-SAM before quenching with un- labeled SAM during the linear portion of their progress curves. Reactions were then captured using filter plates, scintillation mixture was added, and signal was detected with a TopCount liquid scintillation counter (PerkinElmer). Error bars represent the standard deviation from replicate experiments.

McCabe et al. PNAS | February 21, 2012 | vol. 109 | no. 8 | 2991 Downloaded by guest on September 28, 2021 To determine whether the A677G mutation might affect EZH2 specificity toward other histone substrates, we also eval- uated WT and mutant EZH2 complexes using a library of 602 peptides representing sequences within histones H2A, H2B, H3, or H4 and possessing up to five posttranslational modifications, such as lysine and/or methylation; lysine ; or of serine, tyrosine, and/or (Dataset S1). This global analysis revealed that the A677G mutant EZH2 did not enhance activity for any other histone sites compared with WT EZH2 (Fig. S4A). In addition, when comparing the A677G mutant with the Y641N mutant, it was observed that their only common activity was with a peptide containing H3K27me2 (Fig. S4B). Thus, the A677G EZH2 complex is unique in that it has both retained WT activities with unmodified and mono- methylated substrates and acquired the neomorphic activity ob- served in Y641 EZH2 mutants with dimethylated substrates.

Expression of A677G EZH2 Is Sufficient to Drive Hypertrimethylation of H3K27. To explore the effect of the A677G and Y641 EZH2 mutants on levels, WT and mutant versions of EZH2 were transiently expressed in cells before evaluation of global H3K27me3 levels. MCF-7 cells were selected for this analysis because they are WT for EZH2, exhibit relatively low levels of H3K27me3 (26% of Pfeiffer H3K27me3 levels; Fig. 1A), and are easily transfected. Consistent with the biochemical data, exogenous expression of either A677G or Y641 mutant EZH2 increased H3K27me3 levels 1.9- to 2.8-fold relative to the empty vector control (Fig. 4 A and B). H3K27me2, on the other hand, was depleted in cells expressing A677G or Y641 mutant EZH2. H3K27me1 levels were not significantly affected by expression of either WT or mutant EZH2. These observa- tions are consistent with those from EZH2 WT and mutant lymphoma cell lines, where the A677G and Y641 EZH2 mutant cell lines exhibit increased H3K27me3, decreased H3K27me2, and modest effects on H3K27me1 (Fig. 1B).Thesedatadem- onstrate that expression of A677G EZH2 is sufficient to induce a global hypertrimethylation of the H3K27 residue similar to Y641 mutants. Discussion Disruption of the normal patterning of H3K27me3 occurs in hu- man cancers (30–32) and appears to be required for the pro- liferation of some lymphoma cells because knockdown of EZH2 in the SU-DHL-4 DLBCL cell line results in growth arrest at the G1/S transition (33). Recent studies have demonstrated that one com- mon mechanism for the H3K27 hypertrimethylation phenotype in lymphoma is mutation of the EZH2 Y641 residue (1, 3, 16, 17). Herein, we have reported the identification and characterization of a unique EZH2 mutation equally capable of increasing global H3K27me3 in human lymphoma cells. In the absence of an EZH2 crystal structure, a homology model was constructed to understand the distinct substrate specificities of WT, A677G, and Y641 mutant forms of EZH2. The models were based on the protein sequence of EZH2 and a crystal Fig. 4. Exogenous expression of A677G EZH2 stimulates trimethylation of structure of GLP/EHMT1 bound to a histone H3 peptide sub- H3K27 at the expense of H3K27me2. (A) MCF-7 breast cancer cells were strate containing H3K9me2 (Fig. 5A). The homology model of transiently transfected with mammalian expression constructs encoding ei- WT EZH2 is consistent with the biochemical data, demonstrating ther a WT or mutant form of EZH2. In addition, transfection reagent alone that it primarily catalyzes mono- and dimethylation, but not tri- (No DNA) and empty vector treatments were included as controls. Cells were methylation, of H3K27 (Fig. 3 and Table S2). Similar to other lysed 72 h after transfection, and whole-cell protein extracts were assessed for levels of H3K27me3, H3K27me2, H3K27me1, total histone H3, EZH2, and methyltransferases, such as Set7/9, the hydroxyl group of the actin via Western blotting as described in SI Materials and Methods. Actin highly conserved EZH2 Y641 residue appears to optimally orient and total histone H3 were included as loading controls. (B) Average values of the unmethylated and monomethylated forms of the H3K27 H3K27me3, H3K27me2, or H3K27me1 normalized to total histone H3 from substrate for methyl transfer through a hydrogen bond with the at least two replicates. Values are presented as a percentage of the empty ε-amine group of H3K27 (34, 35). However, with ∼3.3 Å between vector control sample. the hydroxyl oxygen of Y641 and the ε-amine group of H3K27me2, the model shows very little room for the dimethy- lated lysine to rotate into position to accept a third methyl group. This model further predicts that the mutation of Y641 to a Therefore, the Y641 residue of EZH2 appears to have a dual significantly smaller residue, such as asparagine (Y641N), would purpose, participating in the orientation of the unmethylated and result in a larger lysine tunnel (H3K27me2 ε-amine–to-N641 side- monomethylated lysine and, at the same time, sterically re- chain distance of at least 4.7 Å) and loss of the critical tyrosine stricting activity with a dimethylated substrate. hydroxyl-to–ε-amine hydrogen bond (Fig. 5B). These changes

2992 | www.pnas.org/cgi/doi/10.1073/pnas.1116418109 McCabe et al. Downloaded by guest on September 28, 2021 Fig. 5. Y641 and A677G EZH2 mutations alter the lysine binding pocket to affect H3K27 substrate specificity. A homology model of WT EZH2 was generated using the crystal structure of GLP/EHMT1 bound to an H3K9me2 peptide substrate as described in SI Materials and Methods. Modeled structures of the region in WT (A), Y641N (B), Y641F (C), and A677G (D) EZH2 are depicted. For the Y641F and A677G mutant models, the lowest energy rotamer for the 641 residue (F or Y, respectively) was selected after rotating the dimethylated lysine into an orientation optimal for trimethylation. In C and D, the WT Y641 orientation is shown in a gray outline to highlight the alternative low-energy conformations adopted by the 641 residue in these mutants. Key heavy atom distances (measurement unit = Å) are indicated with green lines. S-adenosyl- (SAH) is colored with orange carbons, EZH2 residues are colored with magenta carbons, and the dimethylated H3K27 is colored with cyan carbons.

presumably weaken stabilization of the highly flexible unmodified model therefore suggests that the retention of the Y641 residue, or monomethylated lysine, whereas the larger lysine tunnel permits combined with the ability of this residue to adopt an alternative the dimethylated lysine to rotate into position for the third methyl conformation in the A677G mutant, contributes to the efficient transfer. Although the Y641N, Y641S, and Y641C mutations methylation of unmodified, mono- and dimethylated substrates dramatically decrease the size of the residue at position 641, the (Fig. 3 and Table S2). mutation of Y641 to a phenylalanine (Y641F) is relatively con- Although the EZH2 Y641 mutation occurs in 22% of GCB servative, with only loss of the tyrosine hydroxyl group (Fig. 5C). DLBCLs and 7% of FLs (3), our study indicates that mutation of Modeling of the Y641F EZH2 suggests that loss of the hydroxyl the EZH2 A677 to glycine is a fairly rare event. We observed this group may allow the phenylalanine residue to adopt an alternative mutation in 1 of 50 lymphoma cell lines and 1 of 41 primary low-energy conformation in which it rotates away from the lysine lymphoma samples. Morin et al. (1) also recently observed a tunnel toward the A677 residue. This second conformation likely single case of DLBCL (a 63-y-old woman with stage IAE) with generates sufficient space (H3K27me2 ε-amine–to-F641 side-chain an A677G mutation among 127 samples that were assessed by distance of at least 4.0 Å) for the dimethylated lysine to orient RNA-seq, exome-seq, and/or genome-seq. Although a matched optimally for the third methyl transfer. However, with the loss of normal sample was not available for the A677G EZH2 mutant the stabilizing hydrogen bond that occurs between the tyrosine lymphoma specimen reported in this study, the mutation was hydroxyl group and the lysine substrate, the Y641F mutant exhibits reported to be somatic in the case reported by Morin et al. (1), greatly reduced activity with unmodified . This interpre- and it is not reported as a normal sequence variant in the dbSNP tation is consistent with data presented in this report and by others database or the 1000 Genomes project. Thus, although a more (16, 17) demonstrating that Y641 mutants do not efficiently use extensive study with focused genotyping of the A677 codon will unmethylated lysines yet have acquired robust activity with dime- be required to establish the true incidence of this alteration, thylated substrates (Fig. 3 and Table S2). these initial data suggest that the frequency of this mutation is Interestingly, although the structural model for WT EZH2 likely below 2–3%. suggests that the highly conserved A677 residue does not interact Considering that the end result of these two mutations may be directly with either H3K27 or [3H]-S-adenosyl-methionine quite similar (i.e., increased H3K27me3), it is at first somewhat (SAM), it is in close proximity to the hydroxyl group of Y641 surprising that these mutations occur at such different rates.

with a distance of ∼3.3 Å (Fig. 5A). It is therefore predicted that However, this discrepancy might be explained, in part, by the GENETICS mutating A677 to a smaller glycine residue permits Y641 to spectrum of possible activating mutations at each site. To date, adopt an alternative low-energy conformation similar to that mutation of Y641 to any of five different residues (F, N, S, C, or predicted for the phenylalanine in the Y641F mutant (Fig. 5 C H) has been reported to increase activity with an H3K27me2 and D). This alternative conformation for Y641 produces suffi- substrate (refs. 1, 3, 16, 17, 36 and this study). This increased cient space (at least 4.6 Å) between the hydroxyl oxygen of Y641 activity has been attributed to the exchange of Y641 for smaller and the ε-amine group of H3K27me2 to permit the lysine tail to residues, which permit the larger H3K27me2 substrate to rotate rotate into position for trimethylation. However, in contrast to into a position for methyl transfer. The A677G mutation appears the Y641F mutant, the A677G mutant retains the tyrosine hy- to increase the dimensions of the lysine tunnel similarly by per- droxyl group at position 641, thus preserving the stabilizing in- mitting an alternate conformation of Y641 through exchange of teraction between Y641 and unmodified lysine substrates. This alanine for a smaller amino acid; however, because alanine is

McCabe et al. PNAS | February 21, 2012 | vol. 109 | no. 8 | 2993 Downloaded by guest on September 28, 2021 already the second smallest amino acid, it may only be exchanged SET domain methyltransferases, plays an important role in the for a glycine. At the nucleotide level, only one of nine single- regulation of substrate specificity without being in direct contact nucleotide mutations within the A677 codon will result in a gly- with the substrate. This interplay between Y641 and A677 in cine residue. In contrast, five of nine single-nucleotide mutations EZH2 highlights just one of the many important mechanisms within the Y641 codon generate a gain-of-function mutant. Thus, that have likely evolved to regulate the substrate and product the apparently low incidence of the EZH2 A677G mutation may specificities of lysine methyltransferases. be attributable in part to the extremely limited number of pos- sible alterations for this particular site. Materials and Methods The fact that the SET domain is highly conserved across All cell lines were obtained from either the American Type Culture Collection or orthologous and homologous methyltransferases readily per- Deutsche Sammlung von Mikroorganismen und Zellkulturen and maintained mits translation of findings from one methyltransferase to an- in the recommended cell culture media. Primary tissue and DNA samples were other. For example, the effect of changes at the Y641 residue of obtained from OriGene (Table S1). Global histone modification levels were EZH2 is predictable based on mutational analyses of other SET determined with ELISA or Western blot methods using antibodies specificfor domain methyltransferases whose biochemical properties have total histone H3, H3K27me1, H3K27me2, or H3K27me3 (Figs. S1 and S2). The been more extensively studied. The human SET7/9 methyl- full-length EZH2 sequence was determined for genomic DNA and cDNA using normally monomethylates H3K4; however, when primers described in Table S3 and standard Sanger sequencing methods. MCF- the SET7/9 Y245 residue (the equivalent of EZH2 Y641) is 7 cells were transiently transfected with WT and mutant EZH2 expression mutated to alanine, the mutant can no longer modify an constructs using Lipofectamine 2000 (Invitrogen) according to the manu- H3K4me0 substrate but, instead, gains the ability to di- and facturer’s instructions. Biochemical studies were performed with peptide and 3 trimethylate an H3K4me1 peptide substrate (37). Similarly, HeLa nucleosomes using [ H]-SAM and purified recombinant five-member exchange of the Y641 equivalent in the H3K9 methyltransfer- PRC2 complexes containing WT or mutant forms of EZH2 (Fig. S5 and Table ase G9a (Y1067) for phenylalanine converts the enzyme from S4). Structural models of EZH2 were built using GLP/EHMT1 bound to an a mono- and dimethyltransferase to a trimethyltransferase (38). H3K9me2 peptide substrate ( ID code 2RFI) as a primary This study examines the biochemical and cellular activity of template and structurally compared with other related SET domain-contain- the conserved A677 residue of EZH2. Interestingly, the struc- ing histone lysine methyltransferases with determined crystal structures. Ad- ditional detailed information is available in SI Materials and Methods. turally related SET domain-containing DIM-5 from Neurospora crassa has a glycine at the equivalent position (Fig. 2C) and has ACKNOWLEDGMENTS. We acknowledge Daniel Fornwald for the produc- been reported to perform all three methylation events on its tion of baculovirus, Amy Taylor for assistance with protein purification, H3K9 substrate (39, 40). Thus, it appears that the alanine at Harjeet Van Der Keyl for assistance with construct generation, Gilbert Scott residue 677 of EZH2, and likely equivalent residues in other for MS support, and Don Fisher for coordinating reagent generation.

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