O-GlcNAcylation regulates the stability and enzymaticactivity of the histone methyltransferase EZH2Pei-Wen Loa, Jiun-Jie Shieb, Chein-Hung Chena, Chung-Yi Wua, Tsui-Ling Hsua, and Chi-Huey Wonga,1

aGenomics Research Center, Academia Sinica, Taipei 115, Taiwan; and bInstitute of Chemistry, Academia Sinica, Taipei 115, Taiwan

Contributed by Chi-Huey Wong, May 16, 2018 (sent for review February 1, 2018; reviewed by Michael D. Burkart, Benjamin G. Davis, and Gerald W. Hart)

Protein O-glycosylation by attachment of β-N-acetylglucosamine(GlcNAc) to the Ser or Thr residue is a major posttranslationalglycosylation event and is often associated with protein folding,stability, and activity. The methylation of histone H3 at Lys-27catalyzed by the methyltransferase EZH2 was known to suppressgene expression and cancer development, and we previouslyreported that the O-GlcNAcylation of EZH2 at S76 stabilizedEZH2 and facilitated the formation of H3K27me3 to inhibit tumorsuppression. In this study, we employed a fluorescence-basedmethodof sugar labeling combined with mass spectrometry to investigateEZH2 glycosylation and identified five O-GlcNAcylation sites. We alsofind that mutation of one or more of the O-GlcNAcylation sites S73A,S76A, S84A, and T313A in the N-terminal region decreases the stabil-ity of EZH2, but does not affect its association with the PRC2 compo-nents SUZ12 and EED. Mutation of the C-terminal O-GlcNAcylationsite (S729A) in the catalytic domain of EZH2 abolishes the di- andtrimethylation activities, but not the monomethylation of H3K27,nor the integrity of the PRC2/EZH2 core complex. Our results showthe effect of individual O-GlcNAcylation sites on the function of EZH2and suggest an alternative approach to tumor suppression throughselective inhibition of EZH2 O-GlcNAcylation.

O-GlcNAcylation | methyltransferase EZH2 | H3K27me3 | cancer

Protein glycosylation is an important posttranslational modi-fication, of which the addition ofN-acetylglucosamine (GlcNAc) to

the Ser or Thr residue (O-GlcNAcylation) without further glycosylationis commonly found in animals and plants (1). The addition and re-moval of O-GlcNAc by O-linked N-acetylglucosaminyltransferase(OGT) and O-linked N-acetylglucosaminidase (OGA) on nu-clear or cytosolic proteins are keys to maintain the normalfunctions of many proteins, including nuclear pore complexes,transcription factors, dosage compensation complexes, protea-somes, kinases, neuronal proteins, and mitochondria proteins,etc. (1). Changes in the status of protein O-GlcNAcylation caninfluence their downstream biological processes and thus mayaffect the onset of chronic diseases and cancer progression(2, 3).The polycomb-group proteins (PcGs) are a series of proteins

related to embryonic development, including OGT, PRC1, andPRC2. PRC1 is the ubiquitin ligase of H2AK119, and PRC2containing the methyltransferase EZH2 is responsible for themethylation of H3K27. PcGs are recruited to the polycomb-group response elements (PREs) to regulate the expression ofhomeotic genes (HOX) which encode a set of transcription fac-tors that specify the anterior–posterior axis and segment identityin the embryonic development of Drosophila (4–6). PRC1 andPRC2 are conserved in mammalian species and involved in theprogression of several types of cancer (7, 8). In Drosophila, PRC1is composed of Polycomb (Pc), Posterior sex combs (Psc), Dro-sophila RING (dRING), and Polyhomeotic (Ph) (7, 8). In-terestingly, Super sex combs (sxc), one of the PcG genes, encodesDrosophila OGT (9, 10) and is necessary for the repression ofmultiple HOX genes in Drosophila larvae (11, 12). A genome-wide profiling reveals that the PREs bound by OGT are highlyassociated with the regions targeted by PRC1 (9, 13). The sub-units of PRC1, Ph and RING, are found to be O-GlcNAcylated toprevent Ph from aggregation and also to affect pluripotency

maintenance and differentiation in embryonic stem cells (14, 15).It was suggested that O-GlcNAcylation might play an importantrole in the regulation of PRC1-mediated gene expression, andalong this line the O-GlcNAcylation of EZH2 at S76 in the PRC2complex was reported to stablize EZH2 in our previous study (16).The PRC2 complex is composed of Enhancer of zeste 2 (EZH2),Suppressor of Zeste 12 (Suz12), Extraembryonic endoderm (EED),AE binding protein 2 (AEBP2), and retinoblastoma binding protein4/7 (RBBP4/7) (17, 18). Within the PRC2 complex, EZH2 catalyzesthe di- and trimethylation of histone H3 at lysine 27 (K27) to formH3K27me2/3 to regulate embryonic and cancer development(19–23). In contrast to H3K27me2/3, histone H3 with mono-methylation at K27 (H3K27me1) contributes to the promotionof gene transcription (24), but the mechanism of H3K27me1formation in vivo is still ambiguous. In this study, we identifiedfive more O-GlcNAcylation sites on EZH2, using a method offluorescence labeling and mass spectrometry, and revealedthat O-GlcNAcylation mediates EZH2 function in a glycosite-dependent manner.

ResultsAdditional O-GlcNAcyaltion Sites on EZH2 Other than S76. We pre-viously found that the O-GlcNAcyaltion of EZH2 occurred atS76 (equivalent to S75 if ignoring the first amino acid Met) andthe glycosylation increased the protein stability (16). However,the S76A mutant of EZH2 still showed the O-GlcNAcyaltionsignal as detected by Western blot. To enhance the signal, welabeled the O-GlcNAcylation sites of EZH2 expressed in 293Tcells using a peracetylated alkyne-modified GlcNAc analog (Ac4Glc-NAc) as a substrate, followed by copper(I)-catalyzed azide-alkyne cy-cloaddition (CuAAC) of the pulled-down EZH2 using azido-biotin,

Significance

Glycosylation is considered to be a major posttranslationalmodification, and O-GlcNAcylation is known to affect proteinfolding and function. In this study, we show that the methyl-transferase EZH2, which catalyzes the methylation of histone 3at lysine 27 to form H3K27m3, requires O-GlcNAcylation toenhance its stability and enzymatic activity to promote tumorprogression. We further show that the O-GlcNAcylation in theN-terminal region of EZH2 stabilizes the enzyme and the O-GlcNAcylation at S729 in the catalytic domain is essential forits activity of di- and trimethylation. This study indicates thatselective inhibition of EZH2 O-GlcNAcylation may suppress themethylation of H3K27 and thus inhibit tumor progression.

Author contributions: P.-W.L., J.-J.S., T.-L.H., and C.-H.W. designed research; P.-W.L. per-formed research; C.-Y.W. contributed new reagents/analytic tools; P.-W.L. and C.-H.C.analyzed data; J.-J.S. contributed compounds; and P.-W.L., J.-J.S., T.-L.H., and C.-H.W.wrote the paper.

Reviewers: M.D.B., University of California, San Diego; B.G.D., University of Oxford; andG.W.H., Johns Hopkins University.

The authors declare no conflict of interest.

Published under the PNAS license.1To whom correspondence should be addressed. Email: chwong@gate.sinica.edu.tw.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1801850115/-/DCSupplemental.

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azido-TAMRA, or azido BODIPY dye (Az2) (25) (Fig. 1A andB andSI Appendix, Fig. S1A andB). Using these azido probes to analyze andcharacterize the fluorescent triazole adduct through in-gel analysis wasvery convenient compared with Western blot, and of these probes,Az2 was found to be better in terms of fluorescent stability andintensity, and was therefore further exploited in the followingexperiments. Based on the result of metabolic labeling (Fig. 1C),we found that the S76A mutation only caused a slight reduction ofO-GlcNAcylation on EZH2, and the same was observed withOGT overexpression to increase the protein level of both EZH2wild type (WT) and the mutant EZH2 S76A (SI Appendix, Fig.S2). These data suggest the presence of other O-GlcNAcylationsites on EZH2 besides S76.

O-GlcNAcyaltion Distributes over the Whole Protein of EZH2. Toidentify other unknown O-GlcNAcylation sites, we prepared fiveEZH2 truncated fragments based on the domain structure. Itwas found that the wild-type EZH2 and the truncated fragmentsincluding the N-terminal, the middle, and the C-terminal frag-ments exhibited the O-GlcNAcylation signals (SI Appendix, Fig.S3). However, the expression level of fragment 612–746 was toolow to be immunoprecipitated. Likewise, the O-GlcNAcylationlevel of the middle and the C-terminal fragments was higher thanthat of the N-terminal fragment which contained the S76 residue(SI Appendix, Fig. S3). This result indicates that EZH2 hasmultiple O-GlcNAcylation sites.

O-GlcNAcylation Occurs at S73, S84, S87, T313, and S729 of EZH2.Next, we determined the O-GlcNAcylation sites of EZH2 byliquid chromatography-electrospray ionization-mass spectrome-try (LC-ESI-MS). In the beginning, we used the O-GlcNAcylatedpeptide enrichment method to detect the O-GlcNAcylation siteson EZH2 using the azido-biotin probe as described in SIAppendix, Fig. S4A. The MS spectrum revealed that theO-GlcNAcylation occurred at S76 (SI Appendix, Fig. S4B), whichis consistent with our previous findings. Since EZH2 S76A stillcontained other O-GlcNAcylation sites as shown in Fig. 1C, wedecided to identify the other O-GlcNAcylation sites of EZH2from the products of Az2-CuAAC reaction to generate Az2-GlcNAl-EZH2 and two other O-GlcNAcylation–related modi-fications, GlcNAc- and GlcNAl-EZH2 (SI Appendix, Fig. S5A).The MS analysis of Az2-labeled EZH2 indicated an O-GlcNAcylationat T313, shown as Az2-GlcNAl signal (SI Appendix, Fig. S5B).Interestingly, either EZH2 S76A or T313A, or the double mutant

had a similar O-GlcNAcylation level (SI Appendix, Fig. S6), con-sistent with our speculation that O-GlcNAcylation on EZH2occurred transiently and dynamically, and therefore the MSanalysis might not reveal all of the O-GlcNAcylation sitessimultaneously (SI Appendix, Fig. S3).We then overexpressed EZH2 in 293T cells for further de-

tection of other possible O-GlcNAcylation sites because theprotein level of endogenous EZH2 was too low for MS analysis.The O-GlcNAcylation of endogenous EZH2 can be detectedwhen cells are treated with the OGA inhibitor PUGNAC (16),but we could not detect the O-GlcNAcylation on exogenousEZH2 by using Western blot (SI Appendix, Fig. S7). Perhaps thelevel of endogenous OGT within cells was not high enough forthe O-GlcNAcylation of exogenous EZH2 to the level for MSanalysis. Next, we examined whether OGT overexpression couldenhance the O-GlcNAcylation level by introducing a sugar probeto the GlcNAc moiety using the Gal-T1 (Y289L) labeling method(26) (Fig. 2 A and B). The result showed that the O-GlcNAcylationlevel of overexpressed EZH2 was enhanced when OGT was co-overexpressed (Fig. 2C), as shown in the Western blot analysis (SIAppendix, Fig. S7). Next, we evaluated the O-GlcNAcylation siteson EZH2 co-overexpressed with OGT by MS and found threepeptides showing the O-GlcNAcylation signal (SI Appendix, Figs.S8A, S9A, and S10). In addition, overexpression of OGT increasedthe O-GlcNAcylation on the IQPVHILTSVSSLR fragment to25.99%, and the ECSVTSDLDFPTQVIPLK fragment and theS729 glycosite to 42.19% and 0.75%, respectively (Fig. 2D). Inaddition, there was a tiny proportion (about 0.061%) of the peptideECSVTSDLDFPTQVIPLK that possessed two GlcNAc moieties.Since the O-GlcNAc moiety was labile in higher-energy collisionaldissociation (HCD)-MS/MS analysis (27, 28) (SI Appendix, Figs.S8A and S9A), the O-GlcNAcylation sites at S73 and S84 weredetermined by electron-transfer dissociation (ETD)-MS/MS (SIAppendix, Figs. S8B and S9B), and the O-GlcNAcylation at S87 wasdetermined by ETD-MS/MS (SI Appendix, Fig. S9C). On the basis ofthese results, we found six O-GlcNAcylation sites with differentlevels of signal in EZH2 (Fig. 2E).

O-GlcNAcylation in the N-Terminal Region of EZH2 Contributes toProtein Stability. Next, we evaluated whether these newly discov-ered O-GlcNAcylation sites were related to EZH2 stability, sinceit has been known that O-GlcNAcylation contributed to the sta-bility of EZH2 in our previous study (16). We excluded the ex-amination on S87, since the content of O-GlcNAcylation at S87was very low (∼0.061%). Both S76A and T313A were found toreduce the stability of EZH2 compared with the wild type (Fig. 3Aand SI Appendix, Fig. S11A), but there was no statistical differencein the EZH2 half-life between S76A and T313A mutants (Fig.3A). On the other hand, the single, double, and triple mutants ofEZH2 at S73, S84, and S729 were all found to reduce the proteinstability compared to the wild type (Fig. 3B and SI Appendix, Figs.S11B and S12A). Moreover, we found that the single or doublemutation on S73 and S84 had more impact on the half-life ofEZH2 S729A (SI Appendix, Fig. S12B), and the effect of S73A wasequivalent to S84A (SI Appendix, Fig. S12 C and D). This resultindicates that the stability of EZH2 is mainly regulated by theO-GlcNAcylation at S73 and S84, rather than at S729.

O-GlcNAcylation Stabilizes Isolated EZH2 but Not EZH2 in the PRC2Complex. SUZ12 has been known to contribute to the stabilityof EZH2 (18). We found that overexpression of SUZ12, EED, orOGT increased the protein level of EZH2 (SI Appendix, Fig.S13). Furthermore, overexpression of OGT augmented theprotein level of isolated EZH2 to 7.7- to 9.5-fold (Fig. 3C).However, overexpression of OGT had less effect on EZH2 whenco-overexpressed with EED (2.2-fold) (Fig. 3C and SI Appendix,Fig. S14A), SUZ12 (1.4-fold), or both EED and SUZ12 (nosignificant difference) (Fig. 3C and SI Appendix, Fig. S14B). Thisresult indicated that the O-GlcNAcylation contributed mainly tothe stability of isolated EZH2 but not the EZH2 in the complex.Further, we evaluated whether ubiquitin-proteasome degradation

Fig. 1. EZH2 has other O-GlcNAcylation sites in addition to S76. (A) Theflowchart of GlcNAl metabolic incorporation detected by probe Az2. (B) Thechemical structure of Azido-BODIPY dye (AZ2) reporter used in A. (C) Thereare other O-GlcNAcylation sites residing on EZH2 besides S76. The EZH2proteins were purified from 293T cells overexpressed with EZH2 wild type orS76A. The cells were treated with Ac4GlcNAl overnight before protein ex-traction. Then the O-GlcNAcylation level was examined by in-gel fluorescentassay using Az2 as shown in A. WT, wild type. Band intensities were mea-sured by ImageJ. The quantity was determined by dividing the fluorescentsignal to the signal of individual protein stain.

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was associated with the impaired stability of EZH2 N-terminalmutants. The treatment of proteasome inhibitor MG132 was ableto rescue the reduced EZH2 protein level caused by the S73A/S84Amutation (Fig. 3D). In addition, the polyubiquitylation level ofEZH2 S73A/S84A was also increased compared with the wild type(SI Appendix, Fig. S15), all indicating that O-GlcNAcylation inthe N-terminal region stabilized EZH2 by preventing it fromproteasomal degradation.

O-GlcNAcylation on EZH2 Does Not Affect Its Association with thePRC2 Complex. S73 is conserved in mammalian species, and S76and S84 are conserved in vertebrates (SI Appendix, Fig. S16A).Although the ratio of O-GlcNAcylation at S87 is very low, thissite is highly conserved in chordate (SI Appendix, Fig. S16A). Thefour O-GlcNAcylation sites are located in the β-addition motif(BAM), which is composed of three β-strands packed against theside of the β-propeller fold of the WD40 repeats of EED (29) (SIAppendix, Fig. S16C). Another N-terminal O-GlcNAcylation siterelated to the protein stability is T313, which is conserved inmammalian species (SI Appendix, Fig. S16B) and located in thedomain named “Motif Connecting SANT1L and SANT2L”(MCSS) that bundles with the N-terminal loop of the VEFSdomain of SUZ12 (SI Appendix, Fig. S16D) to hold the EED andthe SET domain together (29). On the basis of the structure, wespeculated that the O-GlcNAcylation in the N-terminal region ofEZH2 might be related to its association with the other two

components of PRC2 complex, SUZ12 or EED. Nevertheless,the EZH2 single, double, or triple mutants containing S73A,S76A, S84A, T313A, and/or S729A did not affect the formationof the PRC2 core complex composed of EZH2, SUZ12, EED,and RBBP4/7, nor did these mutated O-GlcNAcylation sitesaffect EZH2 interacting with SUZ12 or EED (SI Appendix, Fig.S17 A and B). In addition, co-overexpression of OGT did notinfluence the interaction of EZH2 wild type or S73A/S84Amutants with SUZ12 or EED (SI Appendix, Fig. S18 A and B).Overall, these results suggest that the O-GlcNAcylation onEZH2 does not affect the integrity of the PRC2 complex.

S729A Mutation Diminishes the Methyltransferase Activity of EZH2 toForm H3K27me2/3 but Has No Effect on the Formation of H3K27me1.S729 is located at the SET domain, the methyltransferase do-main, of EZH2 as shown in Fig. 2E. Therefore, we speculatedthat the O-GlcNAcylation at S729 might interfere with themethyltransferase activity of EZH2. In the PRC2 complex,EZH2, SUZ12, EED, and RBBP4/7 form the core complex toexhibit the minimal enzymatic activity toward the mono-, di-, ortrimethylation of H3K27, while AEBP2 and RBBP4/7 are re-quired for the optimal methyltransferase activity (17, 18). Wethen investigated the methyltransferase activity of the PRC2 corecomplex with EZH2 wild type and mutants (SI Appendix, Fig.S17 A and B) and found that the core complex containing theEZH2 mutant S729A lost the di- and trimethylation activities on

Fig. 2. O-GlcNAcylation sites at S73, S84, S87, and S729 of EZH2 were determined by MS. (A) The flowchart of detection of the O-GlcNAcylation level of EZH2by GalT1 Y289L labeling method using UDP-GalNAz. (B) The chemical structure of azido-biotin probe used in A. (C) OGT overexpression increases the O-GlcNAcylation level of EZH2. The overexpressed EZH2-FLAG was purified from 293T cells with or without OGT co-overexpression, followed by GalT1 Y289Llabeling using UDP-GalNAz as shown in A. (D) OGT overexpression enhances the O-GlcNAcylation level of three peptides containing S73, S84, and S729 intodifferent ratio. The signal was quantitatively determined with LC-MS by dividing the signal of indicated O-GlcNAcylated peptide to the signal of total in-dicated ones, n = 3. (E) The O-GlcNAcylation sites of EZH2, including S73, S76, S84, S87, T313, and S729. DNMT, DNA methyltransferase; EBD, EED bindingdomain; NLS, nuclear location signal.

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H3K27 (Fig. 4 B and C and SI Appendix, Fig. S19C), but stillretained a reduced monomethylation activity (Fig. 4A and SIAppendix, Fig. S19C). Furthermore, we evaluated the mutationsof the other two O-GlcNAcylation sites, S76 and T313, and aspredicted, the core complex containing EZH2 S76A or S76A/T313A mutants did not show changes in the enzymatic activity,while the T313A mutation enhanced the enzymatic activityslightly (SI Appendix, Fig. S19 A and B). This result suggests thatonly the O-GlcNAcylation occurring in the SET domain is re-lated to the methyltransferase activity, and the O-GlcNAcylationat S729 in the EZH2 SET domain is associated with the meth-yltransferase activity to form H3K27me2/3.

DiscussionThe O-GlcNAcylation of EZH2 is a dynamic and transientprocess and it is difficult to detect all glycosites constantly. Wehave used the sensitive glycosylation probes together with theoverexpression and mass spectrometry techniques to identify fiveO-GlcNAcylation sites in EZH2 and elucidated the role of in-dividual glycosites. We have found four O-GlcNAcylation sites,S73, S76, S84, and S87 (Fig. 2E and SI Appendix, Fig. S16C) inthe N-terminal region of EZH2 and the O-GlcNAcylation in thisregion seems to stabilize EZH2 (Fig. 3B and SI Appendix, Fig.S11B) from ubiquitin-proteasome degradation (Fig. 3 D and E).In addition, our results show that O-GlcNAcylation in the BAMregion do not affect EZH2’s association with EED or SUZ12 (SIAppendix, Fig. S18 A and B), and OGT-mediated protein stabilitymerely contributes to the isolated EZH2 but not the EZH2within the PRC2 complex (Fig. 3C). These results suggest that O-GlcNAcylation in the BAM domain of EZH2 is important forthe stabilization of isolated EZH2 before the formation ofPRC2 complex.Although PRC2/EZH2 is known to catalyze the mono-, di-,

and trimethylation of H3K27 in vitro (19), the role of PRC2/EZH2 in the formation of H3K27me1 in vivo is still ambiguous.

Previous study indicated that the formation of H3K27me1 wascatalyzed by G9a, a well-known histone methyltransferase ofH3K9 (30, 31). However, no difference in di- and trimethylationof H3K27 was observed between wild-type and G9a knockoutcells even if the extent of monomethylation was significantlydecreased in G9a knockout cells. Thus, G9a is thought to com-pensate the loss of EZH2 for the formation of H3K27me1 in vivo(32). In this study, we found that the methyltransferase activity ofthe S729A mutant for the formation of H3K27me2/3 was sig-nificantly reduced (Fig. 4), suggesting that the S729 of EZH2 orits O-GlcNAcylation would assist PRC2/EZH2 in the formationof H3K27me2/3 from H3K27me1. The S729 residue of EZH2 ishighly conserved in chordate (Fig. 5A) and is located in the post-SET region (residue 726–729) (Fig. 5B). EZH2-catalyzed for-mation of H3K27me3 is a process critical to many types of cancer(19, 33–36). Therefore, some inhibitors have been developed totarget the post-SET region of EZH2, including Y726, R727, andY728 (37), but the significance of the S729 residue has not beenaddressed. To further investigate whether the O-GlcNAcylationat S729 affects the formation of di- and trimethylation,we aligned the structures of the post-SET region of the isolatedEZH2 [EZH2 520–729, Protein Data Bank (PDB) ID code4MI0] and the complex form of EZH2 (comprising EZH2,EED, VEFS domain of SUZ12, H3 peptide 22–30, andS-adenosylhomocysteine, PDB ID code 5HYN) (SI Appendix,Fig. S20). The chain from residue 726 in the complex forms acanonical post-SET structure, leading to a translocation of resi-dues Y726 and Y728 to the lysine-accessible channel (29). S729is also translocated to the opposite position when the isolatedform transitions to the complex form (SI Appendix, Fig. S20).Next, we aligned the post-SET domain of the complex form withthe inhibitor [comprising EZH2, EED, VEFS domain of SUZ12,and EZH2 inhibitor CPI-1205 (38), PDB ID code 5LS6] andwithout the inhibitor (PDB ID code 5HYN) (Fig. 5B), andfound that Y726, R727, and Y728 were translocated (Fig. 5B).

Fig. 3. O-GlcNAcylation at S73 and S84 may in-crease EZH2 stability. (A and B) Mutations of O-GlcNAcylation sites reduce the half-life of EZH2.The half-life of EZH2-FLAG wild type (WT) or mu-tants is shown in A (S76A, T313A, S76A/T313A, andwild type) or (B) (S73A, S84A, S729A, and wild type).EZH2 WT and mutants were transfected to 293T cellsfor 2 d and subsequently treated with cycloheximideat the final concentration of 50 μg/mL. The proteinlysates were harvested at the indicated time pointsfor Western blot using proper antibodies. (C) OGToverexpression increases the protein level of isolatedEZH2. EZH2 was co-overexpressed with/without OGT,EED, and SUZ12 in 293T. The protein lysates weresubjected to Western blot using proper antibodies.(D) MG132 treatment rescues the decreased proteinlevel of EZH2 S73A/S84A. EZH2 WT and S73A/S84Awere transfected to 293T for 2 d and subsequentlytreated with MG132 at the final concentration of25 μg/mL. Then the lysates were harvested at theindicated time points for Western blot using the in-dicated antibodies. Band intensities were measuredby ImageJ. The protein quantity was determined bydividing the signal of EZH2-FLAG to the signal ofβ-actin. The results are represented as mean ± SD. *Pvalue <0.05, **P value <0.01. n.s., no significantdifference. n = 5 in A and B; n = 3 in C.

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Although S729 was not represented in the complex form withinhibitor, we speculated that it would be in an alternate confor-mation (Fig. 5B). These observations suggest that the O-GlcNAcylation may regulate the enzymatic activity of EZH2 byaltering the subconformation of the EZH2 SET domain.

The ratio of O-GlcNAcylation at S729 was relatively low(0.75%) even if OGT was overexpressed, thus S729A is difficultto represent S729 without O-GlcNAcylation. Further study willbe needed to understand the differential role of O-GlcNAcylatedEZH2-S729, EZH2-S729, and EZH2-S729A. Although we didnot find any phosphorylation signal at S729, the phosphorylationat S734 of EZH2 isoform a (the same site as S729 of EZH2isoform c used in this study), catalyzed by ataxia-telangiectasiamutated (ATM) kinase, was reported to reduce the half-life ofEZH2 (39). Thus, we cannot rule out that O-GlcNAcylation maycoregulate EZH2 with phosphorylation at S729.There are 16 genes coregulated by the OGT–EZH2 axis (16),

suggesting that O-GlcNAcylation may regulate other functions inaddition to gene expression, and our results show that O-GlcNAcylation influences the stability and the methyltransfer-ase activity of EZH2 in a glycosite-dependent manner (Fig. 5C),suggesting that the O-GlcNAcylation of EZH2 may be used as atarget for anticancer drug discovery.

Materials and MethodsSI Appendix, Materials and Methods provides information on cell culture,transfection, drug treatment, antibodies and reagents, plasmids, Westernblotting, in vivo ubiquitylation assay, and in vitro histone methyltransferase(HMT) assay.

Galactosyltransferase-Catalyzed Incorporation of GalNAz to GlcNAc on EZH2 forClick Reaction with the Alkynyl-Biotin Reporter. The N-acetylglucosamine(GlcNAc) moieties were detected by using the Click-iT kit (Invitrogen)according to the manufacturer’s instructions. EZH2-FLAG was purified byanti-FLAG beads and washed by TBS once and TBST three times. EZH2 onbeads was incubated with galactosyltransferase (Gal-T1 Y289L) and 25 μMUDP-GalNAz in a mixture containing 20 mM Hepes (pH 7.9), 50 mM NaCl, 2%Nonidet P-40, and 7.5 mM MnCl2 at 4 °C overnight. The reactions wereterminated by adding an appropriate volume of 4× SDS loading dye con-taining 10% β-mercaptoethanol and subjected to SDS/PAGE. The gel wastransferred onto a PVDF membrane, followed by on-membrane CuAAC re-action using the alkynyl-biotin reporter after blocking with 5% BSA in PBSTfor 1 h. The membrane was then incubated with HRP-streptavidin in 5%BSA/PBST for 1 h. After washing three times with PBST, the membrane wasexposed with ECL (Millipore) and detected by LAS 4000 (Fujifilm).

Protein Labeling with Azido Probes. To probe EZH2 O-GlcNAcylation, EZH2-FLAG was overexpressed in 293T cells treated with Ac4GlcNAc or Ac4GlcNAlovernight. EZH2-FLAG was then pulled down by anti-FLAG beads and thenincubated in a mixture for Cu(I)-catalyzed azide-alkyne cycloaddition(CuAAC) reaction in the presence of 0.1 μM of Az2, 100 μM of Tris-triazoleligand, 1 mM of CuSO4, and 2 mM of sodium ascorbate at room temperaturefor 1 h in the dark. The azido probes used include Az2, azido-biotin, orTAMRA (Invitrogen). Each sample was mixed with an appropriate volume of4× SDS loading dye containing 10% β-mercaptoethanol, and graduallyloaded onto 4–12% Bis-Tris gel. The gel was imaged by a Typhoon 9400Variable Mode Imager (Amersham Biosciences) (λex = 532 nm; λem = 555 nm)and stained with Imperial stain (Invitrogen). The result was detected by

Fig. 4. EZH2 S729A diminishes the methyltransferase activity to formH3K27me2/3. (A–C) EZH2 S729A showed a reduced methyltransferase ac-tivity for the formation of H3K27me2/3 but had no effect on the formationof H3K27me1. In vitro histone methyltransferase (HMT) assays of the PRC2core complexes containing EZH2 wild type (WT) or mutants were performedfor quantification as indicated. The results of HMT assay were evaluated byWestern blot using antibodies against H3K27me1 (A), H3K27me2 (B), orH3K27me3 (C). Band intensities were measured by ImageJ. The quantity wasdetermined by dividing the signal of H3K27me1, -2, or -3 to the signal of H3.n = 3. *P value <0.05, **P value <0.01.

Fig. 5. O-GlcNAcylation may regulate EZH2 in aglycosite-dependent manner. (A) Partial sequencealignment of the EZH2 SET domain. (B) S729 in thepost-SET region is translocated in the complex (PDBID code 5HYN) (purple). Orange shows the complexform of EZH2 with inhibitor (PDB ID code 5LS6);cyan, H3 peptide (26–28); and M27 are highlightedby side chain. The image was captured by PyMOLMolecular Graphics System (version 2.1.0, Schrö-dinger, LLC). (C) O-GlcNAcylation at S73 and S84 maycontribute to the protein stability of EZH2, and O-GlcNAcylation at S729 may promote the methyl-transferase activity for the formation H3K27me2/3.

Lo et al. PNAS Latest Articles | 5 of 6

BIOCH

EMISTR

YCH

EMISTR

Y

HRP-streptavidin when azido-biotin was used. The immunoprecipitation ofproteins was performed with Imperial stain.

In-Gel Digest, Chemoenzymatic Tagging, and Chemical Derivatization. Theprotein EZH2-FLAG obtained from 293T cells after treating with Ac4GlcNAcovernight was resolved by SDS/PAGE and stained with the Imperial stain(Invitrogen). The protein bands of EZH2-FLAG were excised and digestedbased on a standard in-gel digestion protocol (40). The azido-containingUDP-N-azidoacetylgalactosamine (UDP-GalNAz) (Invitrogen) was added (2×in excess) to EZH2-FLAG and the mixture was incubated overnight with Gal-T1 (Y289L) as described. After the reaction, the excess of UDP-GalNAz wasremoved by passing the mixture through a C18 spin column (Thermo Fisher).Peptides were eluted in 70% acetonitrile and dried up by centrifugalevaporator. The peptides were resuspended in PBS by sonication for 10 min.The CuAAC reaction was performed in a solution of 20 μL containing 0.1 μMof alkynyl-biotin, 100 μM of azidopeptide, 1 mM of CuSO4, and 2 mM ofsodium ascorbate in PBS buffer at room temperature for 1 h. After the re-action was completed, the solution was allowed to bind to streptavidin-agarose beads (Pierce) in an IP buffer containing 1% BSA for 2 h at roomtemperature, followed by extensive washing. β-Elimination and Michaeladdition with DTT (BEMAD) directly on the bead was performed using theprotocol previously described (40). The biotin-binding streptavidin beadswere incubated in a 500 μL of BEMAD solution composed of 0.1% (vol/vol)NaOH, 1% (vol/vol) triethylamine, and 10 mM of DTT (made fresh) (pH ad-justed to 12.0–12.5 with triethylamine) for 2.5 h at 50 °C. The reaction wasstopped by addition of TFA to a final concentration of 1% (vol/vol). Peptides

in the supernatant were cleaned up by C18 spin column (Thermo Fisher). Thepeptides containing DTT were detected by MS.

Mass Spectrometry and Data Analysis. Samples were detected by LC-ESI-MS onan Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) equippedwith the Ultimate 3000 RSLC system from Dionex (Dionex Corporation) andnanoelectrospray ion source (New Objective, Inc.). The digestion solution wasinjected (6 nL) at a flow rate of 10 μL/min to a self-packed precolumn (150 μmi.d. × 30 mm, 5 μm, 200 Å). Chromatographic separation was performed on aself-packed reversed phase C18 nanocolumn (75 μm i.d. × 200 mm, 2.5 μm,100 Å) using 0.1% formic acid in water as mobile phase A and 0.1% formicacid in 80% acetonitrile as mobile phase B, operated at 300 nL/min flow rate.The full-scan MS condition was: mass range m/z 200–2,000 (AGC target 4E5)with easy ion chromatography, resolution 120,000 atm/z 200, and maximuminjection time of 50 ms. The 20 most intense ions were sequentially isolatedfor HCD and detected (AGC target 1E4) with maximum injection time of200 ms. The inclusion list m/z was isolated for ETD (reaction time based oncharge) with maximum injection time of 250 ms. Both HCD and ETD wereperformed together with tandem mass (MS2) analysis to elucidate theglycosylation site and peptide sequence.

ACKNOWLEDGMENTS. We thank Dr. Ying-Chih Liu for technology guidanceand the Mass Spectrometry Core Facility at the Genomics Research Center,Academia Sinica, for analysis of glycan profiles. This work was supported bythe Summit Program of the Genomics Research Center, Academia Sinica,Taiwan.

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