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O-Glcnacylation Regulates the Stability and Enzymatic Activity of the Histone Methyltransferase EZH2

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O-Glcnacylation Regulates the Stability and Enzymatic Activity of the Histone Methyltransferase EZH2 O-GlcNAcylation regulates the stability and enzymatic activity of the histone methyltransferase 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 protein folding, role in the regulation of PRC1-mediated gene expression, and stability, and activity. The methylation of histone H3 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 cancer 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 H3K27me3 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 proteins 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 genes (HOX) which encode 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.
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  • A High-Density Map for Navigating the Human Polycomb Complexome
    Resource A High-Density Map for Navigating the Human Polycomb Complexome Graphical Abstract Authors Simon Hauri, Federico Comoglio, Makiko Seimiya, ..., Renato Paro, Matthias Gstaiger, Christian Beisel Correspondence [email protected] (M.G.), [email protected] (C.B.) In Brief Polycomb group (PcG) proteins mediate gene silencing and epigenetic memory in higher eukaryotes. By systematically mapping the human PcG complexome, Hauri et al. resolve Polycomb subcomplexes at high resolution and identify two human PRC2 and two PR- DUB complexes. Furthermore, genomic profiling reveals segregation of PRC1 and PR-DUB target genes. Highlights Accession Numbers d 1,400 high-confidence interactions reveal the modular GSE51673 organization of human PcG proteins d Detailed dissection of PRC1 and PRC2 subcomplexes d Two human PR-DUB complexes contain the glycosyltransferase OGT1 d PR-DUB and PRC1 bind largely distinct sets of target genes Hauri et al., 2016, Cell Reports 17, 583–595 October 4, 2016 ª 2016 The Authors. http://dx.doi.org/10.1016/j.celrep.2016.08.096 Cell Reports Resource A High-Density Map for Navigating the Human Polycomb Complexome Simon Hauri,1,2,7,8 Federico Comoglio,3,7,9 Makiko Seimiya,3 Moritz Gerstung,3,10 Timo Glatter,1,11 Klaus Hansen,4 Ruedi Aebersold,1,5 Renato Paro,3,6 Matthias Gstaiger,1,2,* and Christian Beisel3,12,* 1Department of Biology, Institute of Molecular Systems Biology, ETH Zurich,€ 8093 Zurich,€ Switzerland 2Competence Center Personalized Medicine UZH/ETH, 8044 Zurich,€ Switzerland 3Department
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