O-GlcNAcylation regulates the stability and enzymatic activity of the EZH2

Pei-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 maintenance and differentiation in embryonic stem cells (14, 15). (GlcNAc) to the Ser or Thr residue is a major posttranslational It was suggested that O-GlcNAcylation might play an important glycosylation event and is often associated with folding, role in the regulation of PRC1-mediated expression, and stability, and activity. The methylation of at Lys-27 along this line the O-GlcNAcylation of EZH2 at S76 in the PRC2 catalyzed by the methyltransferase EZH2 was known to suppress complex was reported to stablize EZH2 in our previous study (16). gene expression and development, and we previously The PRC2 complex is composed of Enhancer of zeste 2 (EZH2), reported that the O-GlcNAcylation of EZH2 at S76 stabilized Suppressor of Zeste 12 (Suz12), Extraembryonic endoderm (EED), EZH2 and facilitated the formation of to inhibit tumor AE binding protein 2 (AEBP2), and retinoblastoma binding protein suppression. In this study, we employed a fluorescence-based method 4/7 (RBBP4/7) (17, 18). Within the PRC2 complex, EZH2 catalyzes the di- and trimethylation of histone H3 at lysine 27 (K27) to form of sugar labeling combined with mass spectrometry to investigate H3K27me2/3 to regulate embryonic and cancer development EZH2 glycosylation and identified five O-GlcNAcylation sites. We also – O (19 23). In contrast to H3K27me2/3, histone H3 with mono- find that mutation of one or more of the -GlcNAcylation sites S73A, methylation at K27 (H3K27me1) contributes to the promotion S76A, S84A, and T313A in the N-terminal region decreases the stabil- of gene transcription (24), but the mechanism of H3K27me1 ity of EZH2, but does not affect its association with the PRC2 compo- formation in vivo is still ambiguous. In this study, we identified nents SUZ12 and EED. Mutation of the C-terminal O-GlcNAcylation five more O-GlcNAcylation sites on EZH2, using a method of site (S729A) in the catalytic domain of EZH2 abolishes the di- and fluorescence labeling and mass spectrometry, and revealed trimethylation activities, but not the monomethylation of H3K27, that O-GlcNAcylation mediates EZH2 function in a glycosite- BIOCHEMISTRY nor the integrity of the PRC2/EZH2 core complex. Our results show dependent manner. the effect of individual O-GlcNAcylation sites on the function of EZH2 and suggest an alternative approach to tumor suppression through Results selective inhibition of EZH2 O-GlcNAcylation. Additional O-GlcNAcyaltion Sites on EZH2 Other than S76. We pre- viously found that the O-GlcNAcyaltion of EZH2 occurred at O-GlcNAcylation | methyltransferase EZH2 | H3K27me3 | cancer S76 (equivalent to S75 if ignoring the first amino acid Met) and the glycosylation increased the protein stability (16). However,

O CHEMISTRY rotein glycosylation is an important posttranslational modi- the S76A mutant of EZH2 still showed the -GlcNAcyaltion N signal as detected by Western blot. To enhance the signal, we Pfication, of which the addition of -acetylglucosamine (GlcNAc) to O the Ser or Thr residue (O-GlcNAcylation) without further glycosylation labeled the -GlcNAcylation sites of EZH2 expressed in 293T cells using a peracetylated alkyne-modified GlcNAc analog (Ac Glc- is commonly found in animals and plants (1). The addition and re- 4 O O N NAc) as a substrate, followed by copper(I)-catalyzed azide-alkyne cy- moval of -GlcNAc by -linked -acetylglucosaminyltransferase cloaddition (CuAAC) of the pulled-down EZH2 using azido-biotin, (OGT) and O-linked N-acetylglucosaminidase (OGA) on nu- clear or cytosolic are keys to maintain the normal functions of many proteins, including nuclear pore complexes, Significance transcription factors, dosage compensation complexes, protea- somes, kinases, neuronal proteins, and mitochondria proteins, Glycosylation is considered to be a major posttranslational etc. (1). Changes in the status of protein O-GlcNAcylation can modification, and O-GlcNAcylation is known to affect protein influence their downstream biological processes and thus may folding and function. In this study, we show that the methyl- affect the onset of chronic diseases and cancer progression transferase EZH2, which catalyzes the methylation of histone 3 O (2, 3). at lysine 27 to form H3K27m3, requires -GlcNAcylation to The polycomb-group proteins (PcGs) are a series of proteins enhance its stability and enzymatic activity to promote tumor related to embryonic development, including OGT, PRC1, and progression. We further show that the O-GlcNAcylation in the PRC2. PRC1 is the ubiquitin ligase of H2AK119, and PRC2 N-terminal region of EZH2 stabilizes the enzyme and the O- containing the methyltransferase EZH2 is responsible for the GlcNAcylation at S729 in the catalytic domain is essential for methylation of H3K27. PcGs are recruited to the polycomb- its activity of di- and trimethylation. This study indicates that group response elements (PREs) to regulate the expression of selective inhibition of EZH2 O-GlcNAcylation may suppress the homeotic (HOX) which a set of transcription fac- methylation of H3K27 and thus inhibit tumor progression. tors that specify the anterior–posterior axis and segment identity in the embryonic development of Drosophila (4–6). PRC1 and Author contributions: P.-W.L., J.-J.S., T.-L.H., and C.-H.W. designed research; P.-W.L. per- PRC2 are conserved in mammalian species and involved in the formed research; C.-Y.W. contributed new reagents/analytic tools; P.-W.L. and C.-H.C. progression of several types of cancer (7, 8). In Drosophila, PRC1 analyzed data; J.-J.S. contributed compounds; and P.-W.L., J.-J.S., T.-L.H., and C.-H.W. is composed of Polycomb (Pc), Posterior sex combs (Psc), Dro- wrote the paper. sophila RING (dRING), and Polyhomeotic (Ph) (7, 8). In- Reviewers: M.D.B., University of California, San Diego; B.G.D., University of Oxford; and G.W.H., Johns Hopkins University. terestingly, Super sex combs (sxc), one of the PcG genes, encodes Drosophila OGT (9, 10) and is necessary for the repression of The authors declare no conflict of interest. multiple HOX genes in Drosophila larvae (11, 12). A genome- Published under the PNAS license. wide profiling reveals that the PREs bound by OGT are highly 1To whom correspondence should be addressed. Email: [email protected]. associated with the regions targeted by PRC1 (9, 13). The sub- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. units of PRC1, Ph and RING, are found to be O-GlcNAcylated to 1073/pnas.1801850115/-/DCSupplemental. prevent Ph from aggregation and also to affect pluripotency

www.pnas.org/cgi/doi/10.1073/pnas.1801850115 PNAS Latest Articles | 1of6 Downloaded by guest on September 28, 2021 had a similar O-GlcNAcylation level (SI Appendix,Fig.S6), con- sistent with our speculation that O-GlcNAcylation on EZH2 occurred transiently and dynamically, and therefore the MS analysis might not reveal all of the O-GlcNAcylation sites simultaneously (SI Appendix, Fig. S3). We then overexpressed EZH2 in 293T cells for further de- tection of other possible O-GlcNAcylation sites because the protein level of endogenous EZH2 was too low for MS analysis. The O-GlcNAcylation of endogenous EZH2 can be detected when cells are treated with the OGA inhibitor PUGNAC (16), but we could not detect the O-GlcNAcylation on exogenous EZH2 by using Western blot (SI Appendix, Fig. S7). Perhaps the level of endogenous OGT within cells was not high enough for the O-GlcNAcylation of exogenous EZH2 to the level for MS analysis. Next, we examined whether OGT overexpression could enhance the O-GlcNAcylation level by introducing a sugar probe Fig. 1. EZH2 has other O-GlcNAcylation sites in addition to S76. (A) The to the GlcNAc moiety using the Gal-T1 (Y289L) labeling method flowchart of GlcNAl metabolic incorporation detected by probe Az2. (B) The (26) (Fig. 2 A and B). The result showed that the O-GlcNAcylation chemical structure of Azido-BODIPY dye (AZ2) reporter used in A.(C) There level of overexpressed EZH2 was enhanced when OGT was co- are other O-GlcNAcylation sites residing on EZH2 besides S76. The EZH2 overexpressed (Fig. 2C), as shown in the Western blot analysis (SI proteins were purified from 293T cells overexpressed with EZH2 wild type or Appendix,Fig.S7). Next, we evaluated the O-GlcNAcylation sites S76A. The cells were treated with Ac4GlcNAl overnight before protein ex- on EZH2 co-overexpressed with OGT by MS and found three traction. Then the O-GlcNAcylation level was examined by in-gel fluorescent peptides showing the O-GlcNAcylation signal (SI Appendix,Figs. assay using Az2 as shown in A. WT, wild type. Band intensities were mea- S8A,S9A, and S10). In addition, overexpression of OGT increased sured by ImageJ. The quantity was determined by dividing the fluorescent O signal to the signal of individual protein stain. the -GlcNAcylation on the IQPVHILTSVSSLR fragment to 25.99%, and the ECSVTSDLDFPTQVIPLK fragment and the S729 glycosite to 42.19% and 0.75%, respectively (Fig. 2D). In azido-TAMRA, or azido BODIPY dye (Az2) (25) (Fig. 1 A and B and addition, there was a tiny proportion (about 0.061%) of the peptide SI Appendix,Fig.S1A and B). Using these azido probes to analyze and ECSVTSDLDFPTQVIPLK that possessed two GlcNAc moieties. O characterize the fluorescent triazole adduct through in-gel analysis was Since the -GlcNAc moiety was labile in higher-energy collisional SI Appendix very convenient compared with Western blot, and of these probes, dissociation (HCD)-MS/MS analysis (27, 28) ( , Figs. A A O Az2 was found to be better in terms of fluorescent stability and S8 and S9 ), the -GlcNAcylation sites at S73 and S84 were SI intensity, and was therefore further exploited in the following determined by electron-transfer dissociation (ETD)-MS/MS ( Appendix B B O experiments. Based on the result of metabolic labeling (Fig. 1C), ,Figs.S8 and S9 ), and the -GlcNAcylation at S87 was SI Appendix C we found that the S76A mutation only caused a slight reduction of determined by ETD-MS/MS ( ,Fig.S9 ). On the basis of O-GlcNAcylation on EZH2, and the same was observed with these results, we found six O-GlcNAcylation sites with different OGT overexpression to increase the protein level of both EZH2 levels of signal in EZH2 (Fig. 2E). wild type (WT) and the mutant EZH2 S76A (SI Appendix, Fig. S2). These data suggest the presence of other O-GlcNAcylation O-GlcNAcylation in the N-Terminal Region of EZH2 Contributes to sites on EZH2 besides S76. Protein Stability. Next, we evaluated whether these newly discov- ered O-GlcNAcylation sites were related to EZH2 stability, since O-GlcNAcyaltion Distributes over the Whole Protein of EZH2. To it has been known that O-GlcNAcylation contributed to the sta- identify other unknown O-GlcNAcylation sites, we prepared five bility of EZH2 in our previous study (16). We excluded the ex- EZH2 truncated fragments based on the domain structure. It amination on S87, since the content of O-GlcNAcylation at S87 was found that the wild-type EZH2 and the truncated fragments was very low (∼0.061%). Both S76A and T313A were found to including the N-terminal, the middle, and the C-terminal frag- reduce the stability of EZH2 compared with the wild type (Fig. 3A ments exhibited the O-GlcNAcylation signals (SI Appendix, Fig. and SI Appendix,Fig.S11A), but there was no statistical difference S3). However, the expression level of fragment 612–746 was too in the EZH2 half-life between S76A and T313A mutants (Fig. low to be immunoprecipitated. Likewise, the O-GlcNAcylation 3A). On the other hand, the single, double, and triple mutants of level of the middle and the C-terminal fragments was higher than EZH2 at S73, S84, and S729 were all found to reduce the protein that of the N-terminal fragment which contained the S76 residue stability compared to the wild type (Fig. 3B and SI Appendix,Figs. (SI Appendix, Fig. S3). This result indicates that EZH2 has S11B and S12A). Moreover, we found that the single or double multiple O-GlcNAcylation sites. mutation on S73 and S84 had more impact on the half-life of EZH2 S729A (SI Appendix,Fig.S12B), and the effect of S73A was O-GlcNAcylation Occurs at S73, S84, S87, T313, and S729 of EZH2. equivalent to S84A (SI Appendix,Fig.S12C and D). This result Next, we determined the O-GlcNAcylation sites of EZH2 by indicates that the stability of EZH2 is mainly regulated by the liquid chromatography-electrospray ionization-mass spectrome- O-GlcNAcylation at S73 and S84, rather than at S729. try (LC-ESI-MS). In the beginning, we used the O-GlcNAcylated peptide enrichment method to detect the O-GlcNAcylation sites O-GlcNAcylation Stabilizes Isolated EZH2 but Not EZH2 in the PRC2 on EZH2 using the azido-biotin probe as described in SI Complex. SUZ12 has been known to contribute to the stability Appendix, Fig. S4A. The MS spectrum revealed that the of EZH2 (18). We found that overexpression of SUZ12, EED, or O-GlcNAcylation occurred at S76 (SI Appendix, Fig. S4B), which OGT increased the protein level of EZH2 (SI Appendix, Fig. is consistent with our previous findings. Since EZH2 S76A still S13). Furthermore, overexpression of OGT augmented the contained other O-GlcNAcylation sites as shown in Fig. 1C,we protein level of isolated EZH2 to 7.7- to 9.5-fold (Fig. 3C). decided to identify the other O-GlcNAcylation sites of EZH2 However, overexpression of OGT had less effect on EZH2 when from the products of Az2-CuAAC reaction to generate Az2- co-overexpressed with EED (2.2-fold) (Fig. 3C and SI Appendix, GlcNAl-EZH2 and two other O-GlcNAcylation–related modi- Fig. S14A), SUZ12 (1.4-fold), or both EED and SUZ12 (no fications, GlcNAc- and GlcNAl-EZH2 (SI Appendix, Fig. S5A). significant difference) (Fig. 3C and SI Appendix, Fig. S14B). This The MS analysis of Az2-labeled EZH2 indicated an O-GlcNAcylation result indicated that the O-GlcNAcylation contributed mainly to at T313, shown as Az2-GlcNAl signal (SI Appendix, Fig. S5B). the stability of isolated EZH2 but not the EZH2 in the complex. Interestingly, either EZH2 S76A or T313A, or the double mutant Further, we evaluated whether ubiquitin-proteasome degradation

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1801850115 Lo et al. Downloaded by guest on September 28, 2021 BIOCHEMISTRY

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 EZH2 CHEMISTRY by 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 Y289L labeling using UDP-GalNAz as shown in A.(D) OGT overexpression enhances the O-GlcNAcylation level of three peptides containing S73, S84, and S729 into different 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 binding domain; NLS, nuclear location signal.

was associated with the impaired stability of EZH2 N-terminal components of PRC2 complex, SUZ12 or EED. Nevertheless, mutants. The treatment of proteasome inhibitor MG132 was able the EZH2 single, double, or triple mutants containing S73A, to rescue the reduced EZH2 protein level caused by the S73A/S84A S76A, S84A, T313A, and/or S729A did not affect the formation mutation (Fig. 3D). In addition, the polyubiquitylation level of of the PRC2 core complex composed of EZH2, SUZ12, EED, EZH2 S73A/S84A was also increased compared with the wild type and RBBP4/7, nor did these mutated O-GlcNAcylation sites (SI Appendix,Fig.S15), all indicating that O-GlcNAcylation in affect EZH2 interacting with SUZ12 or EED (SI Appendix, Fig. the N-terminal region stabilized EZH2 by preventing it from S17 A and B). In addition, co-overexpression of OGT did not proteasomal degradation. influence the interaction of EZH2 wild type or S73A/S84A mutants with SUZ12 or EED (SI Appendix, Fig. S18 A and B). O-GlcNAcylation on EZH2 Does Not Affect Its Association with the Overall, these results suggest that the O-GlcNAcylation on PRC2 Complex. S73 is conserved in mammalian species, and S76 EZH2 does not affect the integrity of the PRC2 complex. and S84 are conserved in vertebrates (SI Appendix, Fig. S16A). Although the ratio of O-GlcNAcylation at S87 is very low, this S729A Mutation Diminishes the Methyltransferase Activity of EZH2 to site is highly conserved in chordate (SI Appendix, Fig. S16A). The Form H3K27me2/3 but Has No Effect on the Formation of H3K27me1. four O-GlcNAcylation sites are located in the β-addition motif S729 is located at the SET domain, the methyltransferase do- (BAM), which is composed of three β-strands packed against the main, of EZH2 as shown in Fig. 2E. Therefore, we speculated side of the β-propeller fold of the WD40 repeats of EED (29) (SI that the O-GlcNAcylation at S729 might interfere with the Appendix, Fig. S16C). Another N-terminal O-GlcNAcylation site methyltransferase activity of EZH2. In the PRC2 complex, related to the protein stability is T313, which is conserved in EZH2, SUZ12, EED, and RBBP4/7 form the core complex to mammalian species (SI Appendix, Fig. S16B) and located in the exhibit the minimal enzymatic activity toward the mono-, di-, or domain named “Motif Connecting SANT1L and SANT2L” trimethylation of H3K27, while AEBP2 and RBBP4/7 are re- (MCSS) that bundles with the N-terminal loop of the VEFS quired for the optimal methyltransferase activity (17, 18). We domain of SUZ12 (SI Appendix, Fig. S16D) to hold the EED and then investigated the methyltransferase activity of the PRC2 core the SET domain together (29). On the basis of the structure, we complex with EZH2 wild type and mutants (SI Appendix, Fig. speculated that the O-GlcNAcylation in the N-terminal region of S17 A and B) and found that the core complex containing the EZH2 might be related to its association with the other two EZH2 mutant S729A lost the di- and trimethylation activities on

Lo et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 28, 2021 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, and wild type) or (B) (S73A, S84A, S729A, and wild type). EZH2 WT and mutants were transfected to 293T cells for 2 d and subsequently treated with cycloheximide at the final concentration of 50 μg/mL. The protein lysates were harvested at the indicated time points for Western blot using proper antibodies. (C) OGT overexpression increases the protein level of isolated EZH2. EZH2 was co-overexpressed with/without OGT, EED, and SUZ12 in 293T. The protein lysates were subjected to Western blot using proper antibodies. (D) MG132 treatment rescues the decreased protein level of EZH2 S73A/S84A. EZH2 WT and S73A/S84A were transfected to 293T for 2 d and subsequently treated with MG132 at the final concentration of 25 μg/mL. Then the lysates were harvested at the indicated time points for Western blot using the in- dicated antibodies. Band intensities were measured by ImageJ. The protein quantity was determined by dividing the signal of EZH2-FLAG to the signal of β-actin. The results are represented as mean ± SD. *P value <0.05, **P value <0.01. n.s., no significant difference. n = 5inA and B; n = 3inC.

H3K27 (Fig. 4 B and C and SI Appendix, Fig. S19C), but still Previous study indicated that the formation of H3K27me1 was retained a reduced monomethylation activity (Fig. 4A and SI catalyzed by G9a, a well-known histone methyltransferase of Appendix, Fig. S19C). Furthermore, we evaluated the mutations H3K9 (30, 31). However, no difference in di- and trimethylation of the other two O-GlcNAcylation sites, S76 and T313, and as of H3K27 was observed between wild-type and G9a knockout predicted, the core complex containing EZH2 S76A or S76A/ cells even if the extent of monomethylation was significantly T313A mutants did not show changes in the enzymatic activity, decreased in G9a knockout cells. Thus, G9a is thought to com- while the T313A mutation enhanced the enzymatic activity pensate the loss of EZH2 for the formation of H3K27me1 in vivo slightly (SI Appendix, Fig. S19 A and B). This result suggests that (32). In this study, we found that the methyltransferase activity of only the O-GlcNAcylation occurring in the SET domain is re- the S729A mutant for the formation of H3K27me2/3 was sig- lated to the methyltransferase activity, and the O-GlcNAcylation nificantly reduced (Fig. 4), suggesting that the S729 of EZH2 or at S729 in the EZH2 SET domain is associated with the meth- its O-GlcNAcylation would assist PRC2/EZH2 in the formation yltransferase activity to form H3K27me2/3. of H3K27me2/3 from H3K27me1. The S729 residue of EZH2 is highly conserved in chordate (Fig. 5A) and is located in the post- Discussion SET region (residue 726–729) (Fig. 5B). EZH2-catalyzed for- The O-GlcNAcylation of EZH2 is a dynamic and transient mation of H3K27me3 is a process critical to many types of cancer process and it is difficult to detect all glycosites constantly. We (19, 33–36). Therefore, some inhibitors have been developed to have used the sensitive glycosylation probes together with the target the post-SET region of EZH2, including Y726, R727, and overexpression and mass spectrometry techniques to identify five Y728 (37), but the significance of the S729 residue has not been O-GlcNAcylation sites in EZH2 and elucidated the role of in- addressed. To further investigate whether the O-GlcNAcylation dividual glycosites. We have found four O-GlcNAcylation sites, at S729 affects the formation of di- and trimethylation, S73, S76, S84, and S87 (Fig. 2E and SI Appendix, Fig. S16C)in we aligned the structures of the post-SET region of the isolated the N-terminal region of EZH2 and the O-GlcNAcylation in this EZH2 [EZH2 520–729, (PDB) ID code region seems to stabilize EZH2 (Fig. 3B and SI Appendix, Fig. 4MI0] and the complex form of EZH2 (comprising EZH2, S11B) from ubiquitin-proteasome degradation (Fig. 3 D and E). EED, VEFS domain of SUZ12, H3 peptide 22–30, and In addition, our results show that O-GlcNAcylation in the BAM S-adenosylhomocysteine, PDB ID code 5HYN) (SI Appendix, region do not affect EZH2’s association with EED or SUZ12 (SI Fig. S20). The chain from residue 726 in the complex forms a Appendix, Fig. S18 A and B), and OGT-mediated protein stability canonical post-SET structure, leading to a translocation of resi- merely contributes to the isolated EZH2 but not the EZH2 dues Y726 and Y728 to the lysine-accessible channel (29). S729 within the PRC2 complex (Fig. 3C). These results suggest that O- is also translocated to the opposite position when the isolated GlcNAcylation in the BAM domain of EZH2 is important for form transitions to the complex form (SI Appendix,Fig.S20). the stabilization of isolated EZH2 before the formation of Next, we aligned the post-SET domain of the complex form with PRC2 complex. the inhibitor [comprising EZH2, EED, VEFS domain of SUZ12, Although PRC2/EZH2 is known to catalyze the mono-, di-, and EZH2 inhibitor CPI-1205 (38), PDB ID code 5LS6] and and trimethylation of H3K27 in vitro (19), the role of PRC2/ without the inhibitor (PDB ID code 5HYN) (Fig. 5B), and EZH2 in the formation of H3K27me1 in vivo is still ambiguous. found that Y726, R727, and Y728 were translocated (Fig. 5B).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1801850115 Lo et al. Downloaded by guest on September 28, 2021 The ratio of O-GlcNAcylation at S729 was relatively low (0.75%) even if OGT was overexpressed, thus S729A is difficult to represent S729 without O-GlcNAcylation. Further study will be needed to understand the differential role of O-GlcNAcylated EZH2-S729, EZH2-S729, and EZH2-S729A. Although we did not find any phosphorylation signal at S729, the phosphorylation at S734 of EZH2 isoform a (the same site as S729 of EZH2 isoform c used in this study), catalyzed by ataxia-telangiectasia mutated (ATM) kinase, was reported to reduce the half-life of EZH2 (39). Thus, we cannot rule out that O-GlcNAcylation may coregulate EZH2 with phosphorylation at S729. There are 16 genes coregulated by the OGT–EZH2 axis (16), suggesting that O-GlcNAcylation may regulate other functions in addition 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 a target for anticancer drug discovery. Materials and Methods SI Appendix, Materials and Methods provides information on cell culture, transfection, drug treatment, antibodies and reagents, plasmids, Western blotting, in vivo ubiquitylation assay, and in vitro histone methyltransferase (HMT) assay.

Galactosyltransferase-Catalyzed Incorporation of GalNAz to GlcNAc on EZH2 for Click 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 by BIOCHEMISTRY anti-FLAG beads and washed by TBS once and TBST three times. EZH2 on beads was incubated with galactosyltransferase (Gal-T1 Y289L) and 25 μM UDP-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 were terminated by adding an appropriate volume of 4× SDS loading dye con- taining 10% β-mercaptoethanol and subjected to SDS/PAGE. The gel was transferred onto a PVDF membrane, followed by on-membrane CuAAC re- Fig. 4. EZH2 S729A diminishes the methyltransferase activity to form

action using the alkynyl-biotin reporter after blocking with 5% BSA in PBST CHEMISTRY H3K27me2/3. (A–C) EZH2 S729A showed a reduced methyltransferase ac- for 1 h. The membrane was then incubated with HRP-streptavidin in 5% tivity for the formation of H3K27me2/3 but had no effect on the formation BSA/PBST for 1 h. After washing three times with PBST, the membrane was of H3K27me1. In vitro histone methyltransferase (HMT) assays of the PRC2 exposed with ECL (Millipore) and detected by LAS 4000 (Fujifilm). core complexes containing EZH2 wild type (WT) or mutants were performed for quantification as indicated. The results of HMT assay were evaluated by Protein Labeling with Azido Probes. To probe EZH2 O-GlcNAcylation, EZH2- Western blot using antibodies against H3K27me1 (A), H3K27me2 (B), or FLAG was overexpressed in 293T cells treated with Ac GlcNAc or Ac GlcNAl H3K27me3 (C). Band intensities were measured by ImageJ. The quantity was 4 4 overnight. EZH2-FLAG was then pulled down by anti-FLAG beads and then determined by dividing the signal of H3K27me1, -2, or -3 to the signal of H3. incubated in a mixture for Cu(I)-catalyzed azide-alkyne cycloaddition n = 3. *P value <0.05, **P value <0.01. (CuAAC) reaction in the presence of 0.1 μM of Az2, 100 μM of Tris-triazole

ligand, 1 mM of CuSO4, and 2 mM of sodium ascorbate at room temperature Although S729 was not represented in the complex form with for 1 h in the dark. The azido probes used include Az2, azido-biotin, or TAMRA (Invitrogen). Each sample was mixed with an appropriate volume of inhibitor, we speculated that it would be in an alternate confor- × β B O 4 SDS loading dye containing 10% -mercaptoethanol, and gradually mation (Fig. 5 ). These observations suggest that the - loaded onto 4–12% Bis-Tris gel. The gel was imaged by a Typhoon 9400

GlcNAcylation may regulate the enzymatic activity of EZH2 by Variable Mode Imager (Amersham Biosciences) (λex = 532 nm; λem = 555 nm) altering the subconformation of the EZH2 SET domain. and stained with Imperial stain (Invitrogen). The result was detected by

Fig. 5. O-GlcNAcylation may regulate EZH2 in a glycosite-dependent manner. (A) Partial sequence alignment of the EZH2 SET domain. (B) S729 in the post-SET region is translocated in the complex (PDB ID code 5HYN) (purple). Orange shows the complex form of EZH2 with inhibitor (PDB ID code 5LS6); cyan, H3 peptide (26–28); and M27 are highlighted by side chain. The image was captured by PyMOL Molecular Graphics System (version 2.1.0, Schrö- dinger, LLC). (C) O-GlcNAcylation at S73 and S84 may contribute 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 | 5of6 Downloaded by guest on September 28, 2021 HRP-streptavidin when azido-biotin was used. The immunoprecipitation of in the supernatant were cleaned up by C18 spin column (Thermo Fisher). The proteins was performed with Imperial stain. peptides containing DTT were detected by MS.

In-Gel Digest, Chemoenzymatic Tagging, and Chemical Derivatization. The Mass Spectrometry and Data Analysis. Samples were detected by LC-ESI-MS on

protein EZH2-FLAG obtained from 293T cells after treating with Ac4GlcNAc an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) equipped overnight was resolved by SDS/PAGE and stained with the Imperial stain with the Ultimate 3000 RSLC system from Dionex (Dionex Corporation) and (Invitrogen). The protein bands of EZH2-FLAG were excised and digested nanoelectrospray ion source (New Objective, Inc.). The digestion solution was based on a standard in-gel digestion protocol (40). The azido-containing injected (6 nL) at a flow rate of 10 μL/min to a self-packed precolumn (150 μm UDP-N-azidoacetylgalactosamine (UDP-GalNAz) (Invitrogen) was added (2× i.d. × 30 mm, 5 μm, 200 Å). Chromatographic separation was performed on a in excess) to EZH2-FLAG and the mixture was incubated overnight with Gal- self-packed reversed phase C18 nanocolumn (75 μm i.d. × 200 mm, 2.5 μm, T1 (Y289L) as described. After the reaction, the excess of UDP-GalNAz was 100 Å) using 0.1% formic acid in water as mobile phase A and 0.1% formic removed by passing the mixture through a C18 spin column (Thermo Fisher). acid in 80% acetonitrile as mobile phase B, operated at 300 nL/min flow rate. Peptides were eluted in 70% acetonitrile and dried up by centrifugal The full-scan MS condition was: mass range m/z 200–2,000 (AGC target 4E5) evaporator. The peptides were resuspended in PBS by sonication for 10 min. with easy ion chromatography, resolution 120,000 at m/z 200, and maximum The CuAAC reaction was performed in a solution of 20 μL containing 0.1 μM injection time of 50 ms. The 20 most intense ions were sequentially isolated μ of alkynyl-biotin, 100 M of azidopeptide, 1 mM of CuSO4, and 2 mM of for HCD and detected (AGC target 1E4) with maximum injection time of sodium ascorbate in PBS buffer at room temperature for 1 h. After the re- 200 ms. The inclusion list m/z was isolated for ETD (reaction time based on action was completed, the solution was allowed to bind to streptavidin- charge) with maximum injection time of 250 ms. Both HCD and ETD were agarose beads (Pierce) in an IP buffer containing 1% BSA for 2 h at room performed together with tandem mass (MS2) analysis to elucidate the β temperature, followed by extensive washing. -Elimination and Michael glycosylation site and peptide sequence. addition with DTT (BEMAD) directly on the bead was performed using the protocol previously described (40). The biotin-binding streptavidin beads ACKNOWLEDGMENTS. We thank Dr. Ying-Chih Liu for technology guidance μ were incubated in a 500 L of BEMAD solution composed of 0.1% (vol/vol) and the Mass Spectrometry Core Facility at the Genomics Research Center, NaOH, 1% (vol/vol) triethylamine, and 10 mM of DTT (made fresh) (pH ad- Academia Sinica, for analysis of glycan profiles. This work was supported by justed to 12.0–12.5 with triethylamine) for 2.5 h at 50 °C. The reaction was the Summit Program of the Genomics Research Center, Academia Sinica, stopped by addition of TFA to a final concentration of 1% (vol/vol). Peptides Taiwan.

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