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Mutation of A677 in Histone Methyltransferase EZH2 in Human B-Cell Lymphoma Promotes Hypertrimethylation of Histone H3 on Lysine 27 (H3K27)

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Mutation of A677 in Histone Methyltransferase EZH2 in Human B-Cell Lymphoma Promotes Hypertrimethylation of Histone H3 on Lysine 27 (H3K27) Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 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 Epigenetics 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 (H3K27me3) 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 lymphomas, 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 GENETICS (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 tyrosine 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- methylation 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 genes 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 histones. 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 demethylase 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 transcription factors and chromatin- Author contributions: M.T.M., G.G., E. Diaz, W.S.H., R.G.K., M.B., B.S., and C.L.C. designed 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 gene 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 Early Edition | 1of6 Downloaded by guest on September 28, 2021 (4, 20, 21) and overexpression of EZH2 attributable to multiple FLs, and myelodysplastic syndrome (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.
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