Reaction Coupling between Wild-Type and Disease-Associated

Aug 25, 2014 - Epizyme, Inc. 400 Technology Square, Fourth Floor, Cambridge, Massachusetts 02139, United States. •S Supporting Information. ABSTRACT...
0 downloads 0 Views 1MB Size
Letters pubs.acs.org/acschemicalbiology

Reaction Coupling between Wild-Type and Disease-Associated Mutant EZH2 Brooke M. Swalm, Sarah K. Knutson, Natalie M. Warholic, Lei Jin, Kevin W. Kuntz, Heike Keilhack, Jesse J. Smith, Roy M. Pollock, Mikel P. Moyer, Margaret Porter Scott, Robert A. Copeland, and Tim J. Wigle* Epizyme, Inc. 400 Technology Square, Fourth Floor, Cambridge, Massachusetts 02139, United States S Supporting Information *

ABSTRACT: EZH2 and EZH1 are protein methyltransferases (PMTs) responsible for histone H3, lysine 27 (H3K27) methylation. Trimethylation of H3K27 (H3K27me3) is a hallmark of many cancers, including non-Hodgkin lymphoma (NHL). Heterozygous EZH2 point mutations at Tyr641, Ala677, and Ala687 have been observed in NHL. The Tyr641 mutations enhance activity on H3K27me2 but have weak or no activity on unmethylated H3K27, whereas the Ala677 and Ala687 mutations use substrates of all methylation states effectively. It has been proposed that enzymatic coupling of the wildtype and mutant enzymes leads to the oncogenic H3K27me3 mark in mutant-bearing NHL. We show that coupling with the wild-type enzyme is needed to achieve H3K27me3 for several mutants, but that others are capable of achieving H3K27me3 on their own. All forms of PRC2 (wild-type and mutants) display kinetic signatures that are consistent with a distributive mechanism of catalysis.

P

H3K27me3).7−9 In addition, two other EZH2 point mutations seen in lymphoma, A677G and A687V, have been characterized and shown to be efficient at methylating all methylation states of H3K27,10,11 distinguishing them from wild-type EZH2 and the Tyr641 EZH2 mutants. Importantly, several groups have now reported potent, selective, active site-directed small molecule inhibitors of EZH2 that display selective cell killing activity for mutant-bearing NHL cells in culture and tumor growth regressions in animal models of disease and that have minimal impact on homozygous EZH2 wild-type NHL cells12−16 An interesting aspect of the NHL-associated mutations in EZH2 is that patients bearing these mutations are always found to be heterozygous, expressing both the wild-type and mutant EZH2 forms. We have hypothesized that this heterozygosity reflects a requirement for coupled enzymatic activity between the wild-type and mutant enzymes to effect hypertrimethylation of H3K27 and the attendant hyperproliferative phenotype of NHL seen in these cells.7 Particularly in the case of the Tyr641 mutants, we suggest that in a cellular context, the wild-type enzyme most effectively catalyzes the monomethylation reaction, both wild-type and mutant enzymes catalyze the dimethylation reaction, and the mutant enzyme most effectively catalyzes the final trimethylation reaction. It is unclear whether lymphoma bearing A677G and A687V mutations would require coordination of activity with the wild-type enzyme to effect

olycomb repressive complex 2 (PRC2) is a multiprotein complex that catalyzes the mono- through trimethylation of histone H3 on lysine 27 (H3K27me1, H3K27me2, and H3K27me3), a chromatin modification that is associated with the repression of gene transcription. The methyltransferase activity associated with PRC2 is attributed to the SET domaincontaining subunit EZH2 (or the closely related EZH1 protein).1,2 In humans, H3K27 methylation is performed exclusively by PRC2; therefore this complex constitutes a master regulator of an epigenetic mark with tremendously complex and critical biological outcomes. EZH2 and other PRC2 complex components are found to be genetically altered in a broad range of hematologic and solid human cancers. Hence, there is significant interest in EZH2 inhibition as a therapeutic strategy in oncology. For example, point mutations within the catalytic SET domain of EZH2 have been identified in patients with germinal center subtypes of non-Hodgkin lymphoma (NHL).3−6 These point mutations have been shown to alter the catalytic efficiency (measured as kcat/KM) of EZH2 for substrates representing different states of H3K27 methylation. Wild-type EZH2 is most efficient at catalyzing monomethylation of the unmethylated H3K27 site, and its catalytic efficiency wanes progressively with increasing methylation state of the target amino acid. The same pattern of catalysis is also observed for EZH1. In stark contrast, the disease-associated Tyr641 EZH2 mutants display the opposite pattern of substrate utilization: they are largely incapable of catalyzing the first methylation reaction (H3K27 to H3K27me1) but are effective at catalyzing the dimethylation reaction (H3K27me1 to H3K27me2) and are very effective at catalyzing the trimethylation reaction (H3K27me2 to © 2014 American Chemical Society

Received: February 3, 2014 Accepted: August 25, 2014 Published: August 25, 2014 2459

dx.doi.org/10.1021/cb500548b | ACS Chem. Biol. 2014, 9, 2459−2464

ACS Chemical Biology

Letters

Figure 1. Predicted effect of EZH2 mutation status on H3K27 trimethylation. Homozygous wild-type or Y641 mutant EZH2 are not expected to lead to hypertrimethylated H3K27; however, heterozygous wild-type + Y641 mutant EZH2 are expected to lead to high levels of H3K27me3. A677G or A687V EZH2 mutations are expected to lead high trimethylation irrespective of the presence of wild-type EZH2.

hypertrimethylation of H3K27, owing to their ability to utilize peptides of all methylation states of H3K27 as substrates. The reaction coupling suggested for wild-type and mutant EZH2, within heterozygous NHL cells, requires that the EZH2 enzyme functions through a distributive mechanism of catalysis. This requires that product is released from the enzyme molecule after each round of lysine methylation, and enzyme rebinding of the previous product molecule must occur prior to the next round of methylation. Early studies of other SET domain-containing protein methyltransferases had suggested that these enzymes perform multiple rounds of site-specific amino acid methylation through a processive mechanism, by which the enzyme binds the unmethylated target amino acid and performs mono-, di-, and trimethylation reactions before release of the final product.17−19 As just described, such a fully processive mechanism of catalysis is incompatible with our hypothesis of requisite coupling of wild-type and mutant EZH2 in NHL. We have therefore sought to test this hypothesis by experimentally measuring the degree of coupling between purified, recombinant forms of PRC2 containing wild-type and mutant EZH2 in vitro. Mass spectrometry was used to follow the methylation state of peptide substrates as a function of time and enables a detailed understanding of the kinetic aspects of H3K27 methylation by the wild-type and mutant enzymes. Figure 1 schematically diagrams the expected effects of enzymatic coupling between wild-type and mutant EZH2 on the level of cellular H3K27 trimethylation assuming that within heterozygous, mutant-bearing NHL cells the wild-type and mutant EZH2 enzymes act independently but in concert to effect H3K27 methylation. We predicted that homozygous wild-type EZH2 cells would display a low level of H3K27me3 at steady state, whereas when both wild-type and Y641 mutant enzyme are present in heterozygous cells, the hypothesized coupling between the two enzyme forms results in a significant elevation of H3K27me3 levels, relative to the homozygous wild-type cells. Homozygous Y641 mutant EZH2 cells would be incapable of producing the initial H3K27me1 state to any significant concentration; as the H3K27me1 state is the required progenitor of the subsequent H3K27me2 and H3K27me3 states, the limited ability of homozygous mutant EZH2 cells to form H3K27me1 results in a very limited steadystate production of H3K27me3. Limiting amounts of

H3K27me2 and H3K27me3 may be inconsistent with cellular proliferation and may account for the complete absence of homozygous mutant EZH2 in observed in cell lines and primary tumor samples. In the case of the A677G and A687V mutations, the enzymes have been shown to be quite active on peptides containing all methylation states of H3K27, and it is possible that the presence of the mutant enzyme alone would be sufficient to achieve H3K27me3. We and others have previously reported that heterozygous, EZH2 mutant-bearing NHL cells indeed display increased H3K27me3 and decreased H3K27me2,7,10 consistent with predictions based on our coupling hypothesis. Additionally, a study of melanoma showed that 3% of tumor samples contained Y641 mutations of EZH2; however, the methylation status of these cells was not examined.20 We have extended these observations to a broader set of homozygous wild-type and heterozygous EZH2 mutant-bearing NHL and melanoma cells (Figure 2). With this additional set of cell lines, we continue to see a distinct pattern of elevated H3K27me3 and diminished H3K27me2 for all of the mutant-bearing cells, relative to their wild-type counterparts.

Figure 2. Lymphoma and melanoma cell lines containing EZH2 with an activating mutation have increased H3K27me3 and decreased H3K27me2 levels as compared to wild-type cell lines. Western blot analysis of H3K27 methylation status was performed on acid-extracted histones from a panel of EZH2 wild-type and mutant cell lines. The cell lines bearing a heterozygous EZH2 mutation are denoted with the type of mutation in brackets, e.g., Pfeiffer (A677G). All cell lines are lymphoma lines with the exception of A375 and IGR1, which are melanoma lines. 2460

dx.doi.org/10.1021/cb500548b | ACS Chem. Biol. 2014, 9, 2459−2464

ACS Chemical Biology

Letters

Figure 3. Investigating the need for enzymatic coupling of wild-type EZH2 or EZH1 + mutant EZH2 to achieve trimethylated H3K27 product beginning with unmethylated substrate. Reactions containing 10 nM total enzyme (10 nM wild-type (WT) only, 10 nM mutant only, or combination of 5 nM wild-type + 5 nM mutant), 500 nM unmethylated H3K27 peptide (histone H3, residues 21−44), and 20 μM SAM were followed for 6.5 h using mass spectrometry to quantitate the relative amounts of each product. The data shown here is the average ± standard deviation of 3 replicates.

To test directly the hypothesis of enzymatic coupling in this system, we performed in vitro experiments with purified, recombinant versions of PRC2 containing wild-type and NHLassociated mutants of EZH2. In each instance we mixed equimolar amounts of wild-type, wild-type plus mutant, and mutant enzyme (total enzyme concentration in all cases was nominally 10 nM) and followed the depletion and accumulation of H3K27, H3K27me1, H3K27me2, and H3K27me3 as a function of time using mass spectrometry. The results of these experiments for all EZH2 mutants seen in NHL patients are illustrated (Figure 3), and the relative rates of formation of H3K27me3 are summarized in Table 1. For each mutant enzyme, we see limited product formation for the mutant enzyme alone, relative to the wild-type enzyme alone, and a dramatic enhancement of product formation when equal amounts of wild-type and mutant enzyme are combined.

Table 1. Steady-State Kinetic Parameters for Trimethylation Reactions Catalyzed by PRC2 Containing Mutant EZH2 in the Presence or Absence of Wild-Type EZH2 EZH2 mutation

turnover no.a (h−1) (−) wild-type EZH2

Y641C Y641F Y641H Y641N Y641S A677G A687V

NDb 8.5 ± 0.4 ND ND ND 19.9 ± 0.9 16.0 ± 0.6

turnover no. (h−1) (+) wild-type EZH2 6.8 12.2 6.7 11.7 10.7 14.0 10.0

± ± ± ± ± ± ±

0.3 0.6 0.2 0.3 0.4 0.3 0.4

a

The turnover number was calculated from the linear portion of the product vs time progress curves corresponding to the appearance of H3K27me3. bND denotes the activity was not detectable.

2461

dx.doi.org/10.1021/cb500548b | ACS Chem. Biol. 2014, 9, 2459−2464

ACS Chemical Biology

Letters

and Tyr641 mutant EZH2 in effecting trimethylation of H3K27. These data are fully consistent with our hypothesis of enzymatic coupling as a driver of disease in the case of heterozygous Tyr641 mutant EZH2-bearing NHL. The relationship between the enzymatic activity and biology surrounding the A687V and A677G mutations is also clarified, as these appear to be “super-EZH2” enzymes capable of effectively catalyzing trimethylation of H3K27 on their own, without coupling to the wild-type enzyme. While these mutations are also observed to be heterozygous, one might expect that they could be dominant-acting and support pathogenesis without the need for an active, wild-type EZH2 allele. The Y641F EZH2 mutant is also capable of achieving a modest amount of H3K27me3 on its own, however, unlike the A677G and A687V mutants, it has reduced ability to perform the H3K27me0 to H3K27me1 reaction and we do not consider it a “super-EZH2”. It should be noted that peptide substrates were used in these experiments and do not recapitulate all of the interactions that occur when PRC2 methylates nucleosome substrates in the cellular milieu, therefore we cannot completely rule out that in the context of nucleosomes EZH2 may act processively. Unfortunately, while advances are being made in the detection of methylation on more complex substrates, these techniques are not widespread and readily accessible. However, we can infer from the Western blots of homozygous wild-type and heterozygous mutant-bearing cells in Figure 2 that enzymatic coupling is most likely occurring on the physiologically relevant nucleosome substrates. Overall, these results support a model whereby EZH2 methylates H3K27 using a distributive mechanism, rather than a processive mechanism as has been reported for other SET domain protein methyltransferase enzymes. A distributive mechanism has also been documented for other non-SET domain PMTs, most notably for the lysine methyltransferase DOT1L23 and for several protein arginine methyltransferases.24 The enzyme’s use of a distributive mechanism has implications for drug design and utilization as well. Since product must be released from the enzyme after each round of methylation, the present data suggest that active site-directed inhibitors would be capable of binding to and inhibiting the enzyme at each point along the reaction coordinate, thus equally affecting formation of all protein methylation products.

For the Tyr641 mutations, in all cases, we observe that the level of product formation seen in the mixture of wild-type and mutant enzyme greatly exceeds that which would occur from a mere summing of the wild-type only and mutant only levels of product formation. Only the Y641F mutation is capable of achieving any significant level of H3K27me3 on its own. Were the Tyr641 mutant and wild-type enzymes acting through independent, processive mechanisms, one would expect that the level of product formation in the mixed enzyme sample would reflect the sum of the product levels for the wild-type only and Tyr641 mutant only experiments. Additionally, for the wild-type enzyme, the A677G and A687V mutants and for the combinations of wild-type and Tyr641 mutants, we observe transient accumulation of intermediate H3K27me1 and/or H3K27me2 species to levels in excess of 200 nM (Figure 3 and Supplemental Tables S1−S15). This implies accumulation of intermediate species during multiple turnover events, leading to levels in great excess of the total enzyme concentration present in the assay mixture (10 nM). The accumulation of intermediate species levels in great excess of the enzyme concentration is inconsistent with a fully processive mechanism of catalysis and is instead most consistent with the alternative distributive mechanism.21 In addition, inhibition of all methylation states by the EZH2 tool compound EPZ00568712 was observed for a reaction containing equal amounts of wild-type and Y641N mutant enzymes that was initiated with unmethylated peptide (Supplemental Figures 1, 2, and 3). The simplest conclusion to be drawn from these data is that both the wild-type and Tyr641 mutant EZH2 enzymes function through a distributive mechanism. This is consistent with the observation that cells treated with EPZ005687 show a reduction of H3K27me2 and H3K27me3, but not with the same cells maintaining robust H3K27me1 upon EPZ005687 treatment.12 However, this can be rationalized by considering that EPZ005687 is over 50-fold selective for EZH2 over EZH1, and the persistence of the H3K27me1 mark can be attributed to the activity of EZH1. Also apparent from Figure 3 and Table 1 is that the A677G and A687V mutations are clearly capable of producing significant H3K27me3 on their own. In fact, when these mutant EZH2 enzymes are mixed with equimolar amounts of the wild-type enzyme, the rate at which H3K27me3 is actually reduced. This latter outcome of the in vitro experiments is consistent with the relative kcat/KM values of the mutant and wild-type EZH2 for utilizing all of the H3K27, H3K27me1, and H3K27me2 methylation states as substrates. While there are no analogous mutations of EZH1 observed in lymphoma, and there is little or no evidence that EZH1 plays a prominent role in lymphoma progression, it is conceivable that that enzymatic coupling between wild-type EZH1 and mutant EZH2 could occur. To investigate this, we performed an identical experiment to the one described above using wildtype EZH1 and Y641N EZH2 and observed clear enzymatic coupling (Figure 3). Therefore, we report the possibility for EZH1 to couple with mutant EZH2 in the hypertrimethylation of H3K27. Nevertheless, it has been reported that EZH1 and EZH2 expression can be markedly different across rapidly proliferating vs nonproliferating tissues,22 and detailed expression analysis and genetic studies would be needed to definitively determine if this coupling was relevant to lymphoma biology. We report here the results of these studies that provide unequivocal evidence of significant synergy between wild-type



METHODS

Enzymatic Assays. Wild-type or EZH2 mutant enzymes were expressed as a component of the PRC2 complex consisting of 4 fulllength subunits. EZH2 (NM_004456.4), SUZ12 (NM_015355), RbAp48 (NM_005601), and EED (NM_003797) with an N-terminal FLAG tag were cloned in parallel into a pFastbac1 insect cell expression vector. EZH2, SUZ12, RbAp48, and EED were coexpressed in the Sf21 insect cell line. Cells were resuspended in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 4 mM MgCl2, 20% glycerol, 0.4 mM EDTA, pH 7.9 and 1 tablet protease inhibitor cocktail (Roche)) and lysed by sonication. The PRC2 complex protein was purified by anti-FLAG M2 affinity gel chromatography (SigmaAldrich) and stored in 20 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl2, 20% glycerol, 0.4 mM EDTA, pH 7.9. Histone H3 peptides spanning residues 21−44 and containing a C-terminal biotin group appended through a lysine were synthesized and HPLC-purified to >95% purity by 21st Century Biochemicals. Enzyme assay buffer was 20 mM Bicine (pH = 7.6), 0.002% Tween 20, 0.005% Bovine Skin Gelatin, and 0.5 mM dithiothreitol. Reactions (50 μL) were carried out at 25 °C and contained 10 nM total enzyme (10 nM wild-type or 5 nM wild-type + 5 nM mutant or 10 nM 2462

dx.doi.org/10.1021/cb500548b | ACS Chem. Biol. 2014, 9, 2459−2464

ACS Chemical Biology

Letters

mutant), 500 nM peptide substrate, and 20 μM SAM. Reactions were terminated by the addition of 0.1% formic acid, and a 2 μL sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, IL) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry.25 Western Blotting for H3K27 Methylation Status in Lymphoma Cell Lines. Relative amounts of methylation were measured by probing with rabbit anti-H3K27me3 (Cell Signaling Technology 9733; 1:20000) or rabbit anti-H3K27me2 (CST 9755; 1:10000) with mouse anti-total H3 (Cell Signaling Technology 3638; 1:20000) used as a loading control.



Shipp, M. A., Getz, G., and Golub, T. R. (2012) Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proc. Natl. Acad. Sci. U.S.A. 109, 3879−3884. (6) Bodor, C., Grossmann, V., Popov, N., Okosun, J., O’Riain, C., Tan, K., Marzec, J., Araf, S., Wang, J., Lee, A. M., Clear, A., Montoto, S., Matthews, J., Iqbal, S., Rajnai, H., Rosenwald, A., Ott, G., Campo, E., Rimsza, L. M., Smeland, E. B., Chan, W. C., Braziel, R. M., Staudt, L. M., Wright, G., Lister, T. A., Elemento, O., Hills, R., Gribben, J. G., Chelala, C., Matolcsy, A., Kohlmann, A., Haferlach, T., Gascoyne, R. D., and Fitzgibbon, J. (2013) EZH2 mutations are frequent and represent an early event in follicular lymphoma. Blood 122, 3165− 3168. (7) Sneeringer, C. J., Scott, M. P., Kuntz, K. W., Knutson, S. K., Pollock, R. M., Richon, V. M., and Copeland, R. A. (2010) Coordinated activities of wild-type plus mutant EZH2 drive tumorassociated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas. Proc. Natl. Acad. Sci. U.S.A. 107, 20980− 20985. (8) Yap, D. B., Chu, J., Berg, T., Schapira, M., Cheng, S. W., Moradian, A., Morin, R. D., Mungall, A. J., Meissner, B., Boyle, M., Marquez, V. E., Marra, M. A., Gascoyne, R. D., Humphries, R. K., Arrowsmith, C. H., Morin, G. B., and Aparicio, S. A. (2011) Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation. Blood 117, 2451−2459. (9) Wigle, T. J., Knutson, S. K., Jin, L., Kuntz, K. W., Pollock, R. M., Richon, V. M., Copeland, R. A., and Scott, M. P. (2011) The Y641C mutation of EZH2 alters substrate specificity for histone H3 lysine 27 methylation states. FEBS Lett. 585, 3011−3014. (10) McCabe, M. T., Graves, A. P., Ganji, G., Diaz, E., Halsey, W. S., Jiang, Y., Smitheman, K. N., Ott, H. M., Pappalardi, M. B., Allen, K. E., Chen, S. B., Della Pietra, A., 3rd, Dul, E., Hughes, A. M., Gilbert, S. A., Thrall, S. H., Tummino, P. J., Kruger, R. G., Brandt, M., Schwartz, B., and Creasy, C. L. (2012) Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27). Proc. Natl. Acad. Sci. U.S.A. 109, 2989−2994. (11) Majer, C. R., Jin, L., Scott, M. P., Knutson, S. K., Kuntz, K. W., Keilhack, H., Smith, J. J., Moyer, M. P., Richon, V. M., Copeland, R. A., and Wigle, T. J. (2012) A687V EZH2 is a gain-of-function mutation found in lymphoma patients. FEBS Lett. 586, 3448−3451. (12) Knutson, S. K., Wigle, T. J., Warholic, N. M., Sneeringer, C. J., Allain, C. J., Klaus, C. R., Sacks, J. D., Raimondi, A., Majer, C. R., Song, J., Scott, M. P., Jin, L., Smith, J. J., Olhava, E. J., Chesworth, R., Moyer, M. P., Richon, V. M., Copeland, R. A., Keilhack, H., Pollock, R. M., and Kuntz, K. W. (2012) A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat. Chem. Biol. 8, 890− 896. (13) McCabe, M. T., Ott, H. M., Ganji, G., Korenchuk, S., Thompson, C., Van Aller, G. S., Liu, Y., Graves, A. P., Della Pietra, A., 3rd, Diaz, E., LaFrance, L. V., Mellinger, M., Duquenne, C., Tian, X., Kruger, R. G., McHugh, C. F., Brandt, M., Miller, W. H., Dhanak, D., Verma, S. K., Tummino, P. J., and Creasy, C. L. (2012) EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2activating mutations. Nature 492, 108−112. (14) Qi, W., Chan, H., Teng, L., Li, L., Chuai, S., Zhang, R., Zeng, J., Li, M., Fan, H., Lin, Y., Gu, J., Ardayfio, O., Zhang, J. H., Yan, X., Fang, J., Mi, Y., Zhang, M., Zhou, T., Feng, G., Chen, Z., Li, G., Yang, T., Zhao, K., Liu, X., Yu, Z., Lu, C. X., Atadja, P., and Li, E. (2012) Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation. Proc. Natl. Acad. Sci. U.S.A. 109, 21360−21365. (15) Garapaty-Rao, S., Nasveschuk, C., Gagnon, A., Chan, E. Y., Sandy, P., Busby, J., Balasubramanian, S., Campbell, R., Zhao, F., Bergeron, L., Audia, J. E., Albrecht, B. K., Harmange, J. C., Cummings, R., and Trojer, P. (2013) Identification of EZH2 and EZH1 small molecule inhibitors with selective impact on diffuse large B cell lymphoma cell Growth. Chem. Biol. 20, 1329−1339.

ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare the following competing financial interest(s): All authors except M.P.S., M.P.M., and L.J. are currently employees and shareholders of Epizyme.

■ ■

ACKNOWLEDGMENTS We thank M. Scholle at SAMDI Tech for assistance with mass spectrometry. REFERENCES

(1) Cao, R., Wang, L., Wang, H., Xia, L., Erdjument-Bromage, H., Tempst, P., Jones, R. S., and Zhang, Y. (2002) Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298, 1039− 1043. (2) Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P., and Reinberg, D. (2002) Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein. Genes Dev. 16, 2893−2905. (3) Morin, R. D., Johnson, N. A., Severson, T. M., Mungall, A. J., An, J., Goya, R., Paul, J. E., Boyle, M., Woolcock, B. W., Kuchenbauer, F., Yap, D., Humphries, R. K., Griffith, O. L., Shah, S., Zhu, H., Kimbara, M., Shashkin, P., Charlot, J. F., Tcherpakov, M., Corbett, R., Tam, A., Varhol, R., Smailus, D., Moksa, M., Zhao, Y., Delaney, A., Qian, H., Birol, I., Schein, J., Moore, R., Holt, R., Horsman, D. E., Connors, J. M., Jones, S., Aparicio, S., Hirst, M., Gascoyne, R. D., and Marra, M. A. (2010) Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nat. Genet. 42, 181−185. (4) Morin, R. D., Mendez-Lago, M., Mungall, A. J., Goya, R., Mungall, K. L., Corbett, R. D., Johnson, N. A., Severson, T. M., Chiu, R., Field, M., Jackman, S., Krzywinski, M., Scott, D. W., Trinh, D. L., Tamura-Wells, J., Li, S., Firme, M. R., Rogic, S., Griffith, M., Chan, S., Yakovenko, O., Meyer, I. M., Zhao, E. Y., Smailus, D., Moksa, M., Chittaranjan, S., Rimsza, L., Brooks-Wilson, A., Spinelli, J. J., BenNeriah, S., Meissner, B., Woolcock, B., Boyle, M., McDonald, H., Tam, A., Zhao, Y., Delaney, A., Zeng, T., Tse, K., Butterfield, Y., Birol, I., Holt, R., Schein, J., Horsman, D. E., Moore, R., Jones, S. J., Connors, J. M., Hirst, M., Gascoyne, R. D., and Marra, M. A. (2011) Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature 476, 298−303. (5) Lohr, J. G., Stojanov, P., Lawrence, M. S., Auclair, D., Chapuy, B., Sougnez, C., Cruz-Gordillo, P., Knoechel, B., Asmann, Y. W., Slager, S. L., Novak, A. J., Dogan, A., Ansell, S. M., Link, B. K., Zou, L., Gould, J., Saksena, G., Stransky, N., Rangel-Escareno, C., Fernandez-Lopez, J. C., Hidalgo-Miranda, A., Melendez-Zajgla, J., Hernandez-Lemus, E., Schwarz-Cruz y Celis, A., Imaz-Rosshandler, I., Ojesina, A. I., Jung, J., Pedamallu, C. S., Lander, E. S., Habermann, T. M., Cerhan, J. R., 2463

dx.doi.org/10.1021/cb500548b | ACS Chem. Biol. 2014, 9, 2459−2464

ACS Chemical Biology

Letters

(16) Konze, K. D., Ma, A., Li, F., Barsyte-Lovejoy, D., Parton, T., Macnevin, C. J., Liu, F., Gao, C., Huang, X. P., Kuznetsova, E., Rougie, M., Jiang, A., Pattenden, S. G., Norris, J. L., James, L. I., Roth, B. L., Brown, P. J., Frye, S. V., Arrowsmith, C. H., Hahn, K. M., Wang, G. G., Vedadi, M., and Jin, J. (2013) An orally bioavailable chemical probe of the lysine methyltransferases EZH2 and EZH1. ACS Chem. Biol. 8, 1324−1334. (17) Patnaik, D., Chin, H. G., Esteve, P. O., Benner, J., Jacobsen, S. E., and Pradhan, S. (2004) Substrate specificity and kinetic mechanism of mammalian G9a histone H3 methyltransferase. J. Biol. Chem. 279, 53248−53258. (18) Shahbazian, M. D., Zhang, K., and Grunstein, M. (2005) Histone H2B ubiquitylation controls processive methylation but not monomethylation by Dot1 and Set1. Mol. Cell 19, 271−277. (19) Dirk, L. M., Flynn, E. M., Dietzel, K., Couture, J. F., Trievel, R. C., and Houtz, R. L. (2007) Kinetic manifestation of processivity during multiple methylations catalyzed by SET domain protein methyltransferases. Biochemistry 46, 3905−3915. (20) Hodis, E., Watson, I. R., Kryukov, G. V., Arold, S. T., Imielinski, M., Theurillat, J. P., Nickerson, E., Auclair, D., Li, L., Place, C., Dicara, D., Ramos, A. H., Lawrence, M. S., Cibulskis, K., Sivachenko, A., Voet, D., Saksena, G., Stransky, N., Onofrio, R. C., Winckler, W., Ardlie, K., Wagle, N., Wargo, J., Chong, K., Morton, D. L., Stemke-Hale, K., Chen, G., Noble, M., Meyerson, M., Ladbury, J. E., Davies, M. A., Gershenwald, J. E., Wagner, S. N., Hoon, D. S., Schadendorf, D., Lander, E. S., Gabriel, S. B., Getz, G., Garraway, L. A., and Chin, L. (2012) A landscape of driver mutations in melanoma. Cell 150, 251− 263. (21) Copeland, R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery, 2nd ed., John Wiley and Sons, Hoboken, NJ. (22) Margueron, R., Li, G., Sarma, K., Blais, A., Zavadil, J., Woodcock, C. L., Dynlacht, B. D., and Reinberg, D. (2008) Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms. Mol. Cell 32, 503−518. (23) McGinty, R. K., Kohn, M., Chatterjee, C., Chiang, K. P., Pratt, M. R., and Muir, T. W. (2009) Structure-activity analysis of semisynthetic nucleosomes: mechanistic insights into the stimulation of Dot1L by ubiquitylated histone H2B. ACS Chem. Biol. 4, 958−968. (24) Wang, M., Xu, R. M., and Thompson, P. R. (2013) Substrate specificity, processivity, and kinetic mechanism of protein arginine methyltransferase 5. Biochemistry 52, 5430−5440. (25) Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7− 18.

2464

dx.doi.org/10.1021/cb500548b | ACS Chem. Biol. 2014, 9, 2459−2464