O-Glcnacylation Regulates EZH2 Protein Stability and Function

O-GlcNAcylation regulates EZH2 protein stability and function

Chi-Shuen Chua,1, Pei-Wen Loa,b,1, Yi-Hsien Yeha, Pang-Hung Hsuc,d, Shih-Huan Penga,e, Yu-Ching Tenga, Ming-Lun Kanga, Chi-Huey Wonga,2, and Li-Jung Juana,2

aGenomics Research Center, Academia Sinica, Taipei 115, Taiwan; bInstitute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan; cDepartment of Life Science and dInstitute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 202, Taiwan; and eInstitute of Molecular Medicine, National Taiwan University, Taipei 100, Taiwan

Contributed by Chi-Huey Wong, December 16, 2013 (sent for review September 25, 2013)

O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) is the completely understand how OGT is recruited to chromatin, al- only known enzyme that catalyzes the O-GlcNAcylation of pro- though some progress has been made. It is known that OGT can teins at the Ser or Thr side chain hydroxyl group. OGT participates bind to corepressor mSin3A (21) and also associate with the DNA in transcriptional and epigenetic regulation, and dysregulation of demethylase TET family proteins TET2 and TET3 and potentiate OGT has been implicated in diseases such as cancer. However, the TET family protein-mediated gene activation (22–25). Another underlying mechanism is largely unknown. Here we show that critical mediator of OGT is the polycomb repressive complex OGT is required for the trimethylation of histone 3 at K27 to form 2(PRC2). the product H3K27me3, a process catalyzed by the histone meth- PRC2 is a conserved complex that in humans contains the en- yltransferase enhancer of zeste homolog 2 (EZH2) in the polycomb hancer of zeste homolog 2 (EZH2), SUZ12, EED, and RbAp46/48 repressive complex 2 (PRC2). H3K27me3 is one of the most impor- (26, 27). As a Su(var)3-9, enhancer of zeste and trithorax domain- tant histone modifications to mark the transcriptionally silenced containing enzyme, the major function of EZH2 is to catalyze the chromatin. We found that the level of H3K27me3, but not other transfer of methyl groups to the K27 residue of histone H3 to form H3K27me3 and to induce a signal to recruit polycomb repressive H3 methylation products, was greatly reduced upon OGT deple- – tion. OGT knockdown specifically down-regulated the protein sta- complex 1 (PRC1) for establishing the silenced chromatin (26 29). EZH2/PRC2 has been shown to play critical roles in diverse bi- bility of EZH2, without altering the levels of H3K27 demethylases ological processes, such as development, stem cell maintenance, and UTX and JMJD3, and disrupted the integrity of the PRC2 complex. X-chromosome inactivation (30). Most importantly, EZH2 over- Furthermore, the interaction of OGT and EZH2/PRC2 was detected expression in various types of cancers has been linked to onco- by coimmunoprecipitation and cosedimentation experiments. Im- genesis, partly via H3K27me3 in promoters of specific tumor portantly, we identified that serine 75 is the site for EZH2 O- suppressor genes, and thus causes gene silencing (31). Targeting GlcNAcylation, and the EZH2 mutant S75A exhibited reduction in EZH2 is believed to be a promising strategy for cancer therapy stability. Finally, microarray and ChIP analysis have characterized (32, 33). a specific subset of potential tumor suppressor genes subject to Functional association of OGT and EZH2/PRC2 was first – repression via the OGT EZH2 axis. Together these results indicate reported in Drosophila. Sxc/Ogt, the Drosophila homolog of that OGT-mediated O-GlcNAcylation at S75 stabilizes EZH2 and hence mammalian OGT, was identified as a polycomb group (PcG) facilitates the formation of H3K27me3. The study not only uncovers protein involving in polycomb repression during larvae de- a functional posttranslational modification of EZH2 but also reveals velopment (34, 35). The chromosomal location of O-GlcNAc a unique epigenetic role of OGT in regulating histone methylation. coincides with the PcG response elements in both Drosophila (34, 35) and Caenorhabditis elegans (36). Interestingly, mutations rotein glycosylation with β-N-acetyl-D-glucosamine (O- in PRC2 subunits decrease the level of Ogt protein and global PGlcNAcylation) is a widespread and dynamic modification in both cytosol and nucleus (1, 2). It occurs by O-linked N-acetyl- Significance glucosamine (GlcNAc) transferase (OGT)-catalyzed glycosylation at the hydroxyl group of serine or threonine residue of the pro- The present study identifies a cross-talk of two important tein substrate, and removal of the O-GlcNAc group is catalyzed posttranslational modifications, revealing that enhancer of by the glycosidase O-GlcNAcase (OGA) (3–6). How protein zeste homolog 2 (EZH2) O-GlcNAcylation (GlcNac, N-acetylglu- O-GlcNAcylation exerts its effect is largely unknown, but previous cosamine) at serine 75 is required for EZH2 protein stability and studies show that it could induce a conformational change to therefore facilitates the histone H3 trimethylation at K27 to initiate protein folding (7), compete with phosphorylation at the form H3K27me3. The finding is significant because both same or proximal serine or threonine (8), disrupt protein–protein O-linked GlcNAc transferase-mediated O-GlcNAcylation and interaction (9), serve as a protein recruiting signal (10), or regulate EZH2-mediated H3K27me3 formation play a pivotal role in protein stability (11). development, and their up-regulation is believed to participate More than thousands of proteins are modified by O-GlcNAcylation. in tumor malignancy. The identification of O-linked GlcNAc These proteins are involved in a variety of biological and patho- transferase association with the polycomb repressive com- logical processes, including epigenetic regulation, transcription, plex 2 (PRC2) further provides a new approach to regulate translation, signal transduction, cell division, synaptic plasticity, PRC2 function. embryonic stem cell identity, type II diabetes, Alzheimer’s disease, and tumor malignancy (8, 12–15). O-GlcNAcylation regulates Author contributions: C.-S.C., P.-W.L., C.-H.W., and L.-J.J. designed research; C.-S.C., P.-W.L., transcription and epigenetics at least through the following two Y.-H.Y., P.-H.H., S.-H.P., Y.-C.T., and M.-L.K. performed research; C.-S.C., P.-W.L., P.-H.H., mechanisms. First, OGT adds GlcNAc to DNA-binding tran- C.-H.W., and L.-J.J. analyzed data; and C.-S.C., C.-H.W., and L.-J.J. wrote the paper. scription factors, such as p53, c-Myc, etc. (16), and to histone The authors declare no conflict of interest. proteins such as histone H2A at T101, H2B at S36 and S112, H3 1C.-S.C. and P.-W.L. contributed equally to this work. – at S10 and T32, and H4 at S47 (17 20). O-GlcNAcylation of 2To whom correspondence may be addressed. E-mail: [email protected] or H2B at S112 facilitates the ubiquitination of K120, leading to up- [email protected].

regulation of transcriptional elongation (18). The functions of most This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. BIOCHEMISTRY histone O-GlcNAcylations are currently not clear, nor do we 1073/pnas.1323226111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1323226111 PNAS | January 28, 2014 | vol. 111 | no. 4 | 1355–1360 Downloaded by guest on September 26, 2021 O-GlcNAcylation in mouse embryonic stem cells (37). These with scramble control. In contrast, none of H3 methylation at observations indicate that OGT and PRC2 may function de- other sites was significantly altered (Fig. 1A). Subsequently, mass pendently on each other. Nevertheless, it is obscure how OGT spectrometry was performed to further confirm the result. Histo- regulates PcG repression. Three of the PRC1 components, Ph, nes from MCF7 cells transfected with two OGT siRNAs were Pc, and Ring, are likely Sxc/Ogt substrates in Drosophila (34). purified, separated by SDS/PAGE, and analyzed by liquid chro- However, these observations have not been confirmed by mass matography (Agilent Technologies) coupled with mass spectrom- spectrometry analysis, and no function has been reported with etry (LTQ-FT; Thermo Scientific) after in-gel trypsin digestion. regard to the O-GlcNAcylation of these proteins (34). In the Consistently, the H3K27me3 level showed a more than 50% re- present study, using an unbiased small-scale screening, we in- duction when OGT was deficient, with a concomitant increase dependently demonstrate that OGT depletion leads to the down- of H3K27me2 and H3K27me1 (Fig. 1B). This OGT-dependent regulation of H3K27me3. We also found that OGT is able to H3K27me3 was unlikely cell type specific because the same re- associate with EZH2 and the PRC2 complex and that EZH2 is sult was also observed in MDA-MB-231, another breast cancer O-GlcNAcylated at S75 to maintain its stability and activity. cell line (Fig. S2), suggesting a general control mechanism of H3K27me3 by OGT. Results OGT Knockdown Reduces EZH2 Protein Stability. OGT Knockdown Down-Regulates H3 Trimethylation at K27. To ex- The significant loss plore whether OGT not only directly modifies histone proteins of H3K27me3 by OGT knockdown prompted us to investigate with monosaccharides but also indirectly regulates other histone whether OGT depletion alters the expression level of H3K27 methyltransferase EZH2 or demethylases UTX and JMJD3 (38). modifications, we depleted OGT from cells and analyzed the As shown in Fig. 2A, OGT deprivation by siRNA had no sig- histone extracts by Western blot to determine whether any of the nificant effect on the protein levels of UTX and JMJD3; how- histone modifications is altered. Here we focus on H3 methyla- ever, the EZH2 protein level was greatly reduced (Fig. 2A). tion at the sites associated with transcriptional repression, such Importantly, the OGT knockdown-mediated down-regulation of as methylation at K9 and K27, and the sites for transcriptional EZH2 and H3K27me3 could be rescued by ectopic expression of activation, such as methylation at K4, K36, K79, R17, and R26. siRNA-resistant OGT, indicating that it was not an off-target Remarkably, the human breast cancer cell line MCF7 transfected effect (Fig. 2B). Moreover, we found that OGT depletion not with two different OGT siRNAs, separately or together, not only only reduced the protein level of EZH2 but also diminished the completely blocked the synthesis of OGT and the global O- expression of all other PRC2 subunits (Fig. 2C), consistent with GlcNAcylation, but also greatly down-regulated H3K27me3 (Fig. the previous report that disruption of an essential subunit may 1A). The experiments were repeated three times, and the intensity lead to destabilization of the whole PRC2 complex (39–41). It of H3K27me3 bands was quantified. As shown in Fig. S1,more should be noted that OGT knockdown-mediated down-regula- than half of H3K27me3 was lost by OGT knockdown, compared tion of PRC2 expression was at the protein level because OGT deprivation did not alter the mRNA levels of all PRC2 subunits (Fig. S3). These results suggested that OGT knockdown may alter the stability of EZH2/PRC2 complex. To test this hypoth- esis, MCF7 cells with or without OGT knockdown were treated with cycloheximide to inhibit protein synthesis, followed by the chase of the remaining EZH2. As shown in Fig. 2D, 10 h after cycloheximide treatment, the level of EZH2 only dropped to 93% in cells with scramble control (compare lane 6 with lane 1). However, in cells with OGT siRNA, EZH2 was reduced to 54% of its original amount (compare lane 12 with lane 7). Together these experiments demonstrate that OGT is required for the maintenance of EZH2/PRC2 stability and the subsequent H3K27me3 level.

OGT Interacts with EZH2 in the PRC2 Complex. Because OGT is essential to EZH2/PRC2 function, we investigated whether OGT physically interacts with EZH2/PRC2. To test this, 293T cells expressing EZH2-FLAG or OGT-V5 or both were subject to immunoprecipitation with FLAG Ab (Fig. 3A, Left)orV5Ab (Fig. 3A, Right), followed by Western blot with the Abs indicated. Indeed, EZH2-FLAG was coprecipitated with OGT-V5 in the reciprocal experiments (Fig. 3A). Consistently, the endogenous OGT was also co-pulled down by EZH2 Ab (Fig. 3B). It is noted that the signal appearing in the IgG immunoprecipitation (IP) control (Fig. 3B, lane 2) is not at the same position of EZH2. We believe that it is a nonspecific band. Glycerol-sizing gradient sedimentation was further applied to understand whether OGT not only interacts with EZH2 but also associates with the PRC2 complex.AsshowninFig.3C, OGT coeluted with EZH2 and all other PRC2 subunits in fraction nos. 6–9. These results provide evidence that OGT physically associates with EZH2/PRC2 in cells.

EZH2 Is O-GlcNAcylated in Vivo, and the O-GlcNAcylation Site Mutant Fig. 1. OGT knockdown reduces H3 trimethylation at K27. (A) OGT depletion decreases the level of H3K27me3 but not other H3 methylation of EZH2 Shows Reduced Protein Stability. Our data so far indicate products. Total cell lysates or histones purified from MCF7 cells mock trans- that OGT is required for sustaining EZH2 protein stability and fected (NT), transfected with scramble RNA (Scr), or two different siOGT (#1 H3K27me3 formation and that OGT physically interacts with EZH2. and #2) separately or together were subjected to Western blot using in- Next we sought to analyze whether OGT directly modifies EZH2 dicated Abs. (B) OGT knockdown decreases global H3K27me3 measured by with O-GlcNAc, and if yes, whether EZH2 O-GlcNAcylation mass spectrometry. Histones purified from MCF7 cells transfected with in- affects the protein stability of EZH2. To this end, first we treated dicated siRNAs were subjected to SDS/PAGE, followed by LC/MS analysis. MCF7 cells with the OGA inhibitor O-(2-acetamido-2-deoxy-d-

1356 | www.pnas.org/cgi/doi/10.1073/pnas.1323226111 Chu et al. Downloaded by guest on September 26, 2021 chromatography (Agilent Technologies) coupled with mass spectrometry (LTQ-FT) after in-gel trypsin digestion. We found that O-GlcNAcylation occurred on S75 of EZH2 (Fig. 4D). To understand the function of EZH2 O-GlcNAcylation at S75, a mutant EZH2 with S75 substituted with alanine (S75A) was constructed and expressed in 293T cells, followed by a cycloheximide-chase experiment. The result showed that the S75A mutant of EZH2 was more labile compared with WT EZH2 (Fig. 4E). These experiments strongly suggest that EZH2 O-GlcNAcylation at S75 is essential for EZH2 protein stability. Given that OGT is the sole enzyme to carry out O-GlcNAcylation in cells, it is reasonable to propose that the loss of H3K27me3 in cells depleted of OGT likely results from the absence of EZH2 O-GlcNAcylation at S75 and reduced stability of EZH2.

OGT-EZH2 Axis Suppresses Specific Tumor Suppressor Gene Expression. To gain insight into the role of OGT in EZH2-mediated function, microarray analysis was applied to identify genes controlled by both OGT and EZH2. Because overexpression of OGT and EZH2 has been implicated in breast tumorigenesis (43, 44) and EZH2 has been known to repress specific tumor suppressor gene expression in breast cancer cells (45), microarray analysis was carried out using the breast cancer cell line MCF7 treated with scramble siRNA, siOGT, or siEZH2 (Fig. S5A). It was found that a total of 63 genes were coregulated by OGT and EZH2 (Fig. S5B). Among them, we focused on 20 genes whose expressions were increased in both OGT-KD and EZH2-KD cells (1.5-fold Fig. 2. OGT knockdown reduces EZH2 protein stability. (A) OGT knockdown cutoff, P < 0.01) (Fig. S5B). The expressions of this set of genes decreases the protein level of EZH2 but not UTX or JMJD3. (B) Reduction of were further analyzed by real-time RT-PCR, and 16 genes among EZH2 by OGT knockdown can be rescued by adding back resistant OGT. (C) the set were found significantly up-regulated in both OGT-KD OGT knockdown decreases the protein levels of PRC2 components. Total (Fig. 5A) and EZH2-KD (Fig. 5B) cells. Potentially this group of lysates or histone extracts from MCF7 cells transfected with scramble siRNA genes might exert tumor suppressor function under the control of (Scr) or siOGT were subjected to Western blot using indicated Abs. For B, cells the OGT-EZH2 axis. Indeed, IL1R1, SCUBE2, UNC5A, and were treated with scramble (Scr) siRNA or siOGT for 24 h, followed by SPATA17 have been shown negatively correlated with oncogenesis transfection of vector alone (lanes 1 and 2) or the plasmid encoding the – siRNA-resistant OGT (lane 3) for 2 d. (D) OGT knockdown destabilizes EZH2. (46 49). In addition, CSTA can inhibit metastasis in breast cancer MCF7 cells were transfected with scramble siRNA (Scr) or siOGT for 3 d and cells and is negatively correlated with metastasis (50). Importantly, subsequently treated with cycloheximide at the final concentration of 50 μg/mL ChIP assays indicate that the promoter regions of these 16 genes and harvested at the indicated time points for Western blot. Band intensities were bound by OGT (Fig. 5C) and enriched in EZH2 and were measured by ImageJ. Normalization was done by dividing the EZH2 H3K27me3 in an OGT-dependent manner, because depletion of signal to α-tubulin signal. P values were measured by Student’s t test. The results are presented as mean ± SD. *P = 0.031, n = 3.

glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and found that the treatment not only led to increased global O- GlcNAcylation but also enriched H3K27me3 and EZH2 (Fig. 4A), suggesting that the up-regulated EZH2 level might be a result of EZH2 O-GlcNAcylation. Consistent with this notion, in the presence of PUGNAc the endogenous EZH2 pulled down by EZH2 Ab could be recognized by O-GlcNAc Ab (Fig. 4B). This result indicates that EZH2 was most likely modified with this sugar in vivo. However, the experiment cannot exclude the possibility that the O-GlcNAcylation signal was from an EZH2- associated proteins. To rule out this possibility, we applied the following cell labeling experiment using O-GlcNAz. It is known that proteins modified with O-GlcNAz, an O-GlcNAc derivative with azido-acetyl group, can be conjugated with phosphine-FLAG, which then can be detected by FLAG Ab (42) (Fig. S4). As shown in Fig. 4C, in MCF7 cells metabolically labeled with O-GlcNAz, the EZH2 precipitated with EZH2 Ab in a denatured condition (34) could be recognized by FLAG Ab (arrowhead), supporting the same conclusion that EZH2 is O-GlcNAcylated in cells. It Fig. 3. OGT stably interacts with EZH2 in the PRC2 complex. (A)Exoge- should be noted that the denatured condition used in the exper- nous OGT associates with exogenous EZH2. Total lysates from 293T cells – expressing OGT-V5 and/or EZH2-FLAG were subjected to IP with FLAG Ab iment above in theory had disrupted protein protein interaction. (Left)orV5Ab(Right), followed by Western blot using indicated Abs. (B) Therefore, the signal by FLAG Ab was most likely from EZH2 Endogenous OGT interacts with endogenous EZH2. Nuclearextractsfrom conjugated with O-GlcNAz-FLAG but not from any EZH2- MCF7 cells were subjected to IP, followed by Western blot using indicated associated protein. Abs. Asterisk indicates a nonspecific band. (C) OGT cofractions with PRC2 Finally, mass spectrometry was applied to confirm EZH2 complex. Nuclear extracts from MCF7 cells were subjected to 10–50% O-GlcNAcylation and to identify the modification site(s). The glycerol sizing gradient sedimentation. Fractions were collected, pre-

FLAG-tagged EZH2 was precipitated from 293T cells over- cipitated by trichloroacetic acid, and analyzed by Western blot using BIOCHEMISTRY expressing both OGT-V5 and EZH2-FLAG, followed by liquid indicated Abs.

Chu et al. PNAS | January 28, 2014 | vol. 111 | no. 4 | 1357 Downloaded by guest on September 26, 2021 OGT abrogated EZH2 recruitment (Fig. 5D and Fig. S6)and H3K27me3 occupancy (Fig. 5E and Fig. S7). In summary, we identified a set of tumor suppressor genes whose expressions in breast cancer cell are down-regulated by the OGT-EZH2 axis. Discussion Here we provide a unique regulatory mechanism by which OGT facilitates H3K27me3 synthesis. Our results indicate that OGT modifies the H3K27 methyltransferase EZH2 with O-GlcNAc at S75 and by this O-GlcNAcylation maintains EZH2 protein stability and function. Because O-GlcNAc is known to regulate the proteasome (51), we thus cannot exclude the possibility that the decrease in EZH2 levels upon OGT knockdown was partly through proteasomal dysregulation. However, because (i)OGTis the only enzyme to carry out protein O-GlcNAcylation, (ii), EZH2 was O-GlcNAcylated at S75 in cells (Fig. 4), and (iii) the S75A EZH2 mutant showed decreased protein stability (Fig. 4E), we believe that loss of protein stability does contribute to the decrease of EZH2 protein level in OGT-KD cells. Although endogenous WT EZH2 in MCF7 cells treated with siRNA or siOGT looks stable over the experiment time frame (still approximately 100% at 8 h) (Fig. 2D), less than 75% of FLAG-tagged EZH2 content remains at 8 h when analyzed in 293T cells with no treatment of any siRNA (Fig. 4E). With these differences, it is not appropriate to directly compare the turnover rate of the endogenous EZH2 in Fig. 2D with that of overex- pressed FLAG-EZH2 in Fig. 4E. Most importantly, the results in both figures are consistent in that OGT knockdown decreases EZH2 protein stability, and EZH2 O-GlcNAcylation mutant shows decreased protein stability. How S75 O-GlcNAcylation regulates EZH2 protein stability is currently under investigation. As mentioned in the Introduction, O-GlcNAcylation is known to compete with phosphorylation (52). It is likely that S75 O-GlcNAcylation prevents phosphorylation at the same site that serves as a signal for EZH2 degra- dation. Alternatively, it is likely that O-GlcNAcylation at S75 disrupts EZH2 modification(s) at other sites required for deg- radation. A previous study indicates that EZH2 phosphorylated at T345 by cyclin-dependent kinase 1 (CDK1) is unstable during mitosis and prone to be degraded via proteasome-mediated pathway (53). Whether the monosaccharide at S75 disturbs EZH2 phosphorylation at T345 is currently not known. Interestingly, the CDK1 activity can be inhibited by OGT overexpression (54). Together, these studies imply a potential regulatory network among OGT, CDK1, and EZH2. Unlike other hit-and-run enzyme–substrate interactions, OGT is known to bind to some of its substrates tightly (55, 56). Therefore, it is not surprising to find that the binding of OGT to EZH2 is stable enough to be detected by co-IP and cosedimentation (Fig. 3). This and other results of the present study support previous observations that OGT and EZH2/PRC2 are functionally associated with each other (34, 35, 37). Neverthe- less, discrepancy still exists. In contrast to our study, Myers et al. (37) did not observe reduction in EZH2 or H3K27me3 in Ogt- knocked-down mouse ES cells. Gambetta et al. (34) failed to Fig. 4. S75 O-GlcNAcylation of EZH2 is required for EZH2 stability. (A)Ac- detect O-GlcNAcylation of E(z), the Drosophila homolog of cumulation of global O-GlcNAcylation level increases EZH2 and global EZH2, nor did they observe down-regulation of global H3K27me3 H3K27me3. Total lysates or histones from MCF7 treated with DMSO or in Ogt mutants, although their data showed reduction of H3K27me3 PUGNAc for 16 h were subjected to Western blot using indicated Abs. (B) in specific genes (34). Currently we do not know what causes Endogenous EZH2 is O-GlcNAcylated. Nuclear extracts from MCF7 cells with these differences. One possibility is that different species and cell or without PUGNAc were subjected to Western blot with O-GlcNAc Ab or IP types were analyzed. We used human breast and kidney epi- with IgG or EZH2, followed by Western blot using O-GlcNAc or EZH2 Ab. (C) thelium cells, whereas Drosophila and mouse were analyzed in Detection of EZH2-O-GlcNAz-FLAG. Nuclear extracts from MCF7 cells treated previous studies. with O-GlcNAz for 16 h were incubated with phosphine-FLAG overnight at OGT knockdown only reduced the H3K27me3 to half of its 4 °C. Subsequently lysates were subjected to IP in denatured condition, fol- original level (Fig. 1). Therefore, it is unlikely that OGT knock- lowed by Western blot using indicated Abs. Arrowheads indicate the band of down has an overwhelming effect on global gene expression. EZH2. Asterisk indicates a nonspecific band. (D) EZH2 is O-GlcNAcylated at serine 75. An MS/MS spectrum was generated from microliquid chromatography/tandem MS. (E) Serine 75 mutation reduces EZH2 protein stability. 293T cells expressing EZH2-WT-FLAG or EZH2-S75A-FLAG were treated with were measured by ImageJ. Normalization was done by dividing the FLAG cycloheximide at the final concentration of 50 μg/mL and harvested at in- signal to β-tubulin signal. P values were measured by Student’s t test. The dicated time points for Western blot using indicated Abs. Band intensities results in E are presented as mean ± SD. *P < 0.05, n = 3.

1358 | www.pnas.org/cgi/doi/10.1073/pnas.1323226111 Chu et al. Downloaded by guest on September 26, 2021 posttranslational modifications, not all changes in H3K27me3 result in an alteration of DNA transcription level. For example, Hosogane et al. (57) show that, in NIH 3T3 cells, H3K27me3 change caused by depleting SUZ12, Ras, or Raf does not cor- relate with changes in target gene expressions. It is unlikely ei- ther that the small number gene effect is because OGT only affects H3K27me3 level at the repeated sequences. Vella et al. (23) has shown that, in mouse ES cells, 82% of OGT ChIP signals are within promoter-transcription start site and gene body, and only 16% of signals are present in intergenic regions. An- other ChIP-seq analysis indicates that, in human 293T cells, OGT binds to promoter region but not gene body or intergenic region (24). Thus, the majority of OGT does not associate with repeated sequences, excluding the possibility that OGT regulates H3K27me3 at these sequences. Although OGT associated with EZH2 in the PRC2 complex, these two proteins also exist in free form independently of each other (Fig. 3). This is consistent with the microarray analysis in Fig. S5, which indicates that OGT and EZH2 only coregulate a subset of genes. Among the genes most significantly controlled by the OGT–EZH2 axis, two (IL1R1 and UNC5A) have been shown to be negatively associated with tumor malignancy. One is IL1R1 with low expression in colon cancer cell line HCT116 (47), and the other is UNC5A as a downstream target of p53 to induce apoptosis (46). Together these results uncovered a previously unknown OGT–EZH2 regulatory axis that may play a critical role in tumor malignancy. Materials and Methods SI Materials and Methods provides information on cell culture, knockdown by siRNA, antibodies and reagents, plasmids, quantitative real-time RT-PCR (primer sets are listed in Table S1), Western blotting and histone extraction, IP and co-IP, and microarray.

Metabolic Labeling and Denatured IP. Metabolic labeling was performed as previously described (42). Briefly, MCF7 cells were treated with 40 μM

O-GlcNAz overnight at 37 °C with 5% (vol/vol) CO2. Nuclei were prepared and incubated with equal volumes of 500 μM phosphoine-FLAG overnight at 4 °C with rotation. Denatured IP was performed as previously described (34) with modifications. In brief, the nuclear lysates were adjusted to denatured condition by the addition of 10% (wt/vol) SDS (final concentration 0.5%) and 1 M DTT (final concentration 5 mM). The lysates were boiled for 5 min, and SDS was diluted to 0.1% and DTT to 1 mM, followed by proce- dures described in SI Materials and Methods, Immunoprecipitation and Coimmunoprecipitation.

Glycerol Gradient Sedimentation Analysis. The glycerol gradient sedimentation analysis was performed as previously described (58), with modifications. Briefly, nuclear extracts from MCF7 cells were prepared in BC100 buffer

[5 mM Hepes/KOH (pH 7.3), 100 mM NaCl, 1 mM MgCl2, 0.5 mM EGTA, 0.1 mM EDTA, 10% (vol/vol) glycerol, 1 mM DTT, and 0.2 mM PMSF]. Glycerol gradients [10–50% (wt/vol)] were prepared in BC100 buffer using a gradient maker (Hoefer). A 100-μL sample containing 1 mg of MCF7 nuclear extract or 30 mg of individual standards (Sigma, MW-GF-1000) was loaded on top of the gradient. Gradients were kept at 4 °C for 30 min, then spun in a SW41 rotor Fig. 5. The OGT-EZH2 axis suppresses specific tumor suppressor gene ex- (Beckman) at 41,000 rpm for 28 h at 4 °C. Fractions (0.5 mL) were collected and pression. mRNA levels of 16 genes corepressed by OGT and EZH2 were analyzed by 8% SDS/PAGE, followed by Western blot. confirmed with RT-PCR in MCF7 depleted of OGT (A) or EZH2 (B). (C) OGT occupancy at the promoter regions of the 16 genes in A and B. MCF7 cells Identification of O-GlcNAcylation Sites on EZH2 by Mass Spectrometry. EZH2- were subjected to ChIP with control IgG (white bar) or OGT Ab (black bar). (D FLAG and OGT-V5 were coexpressed in 293T cells for 2 d. EZH2-FLAG were and E) EZH2 occupancy (D) and H3K27me3 (E) associated with the promoter immunoprecipitated by anti-FLAG antibody and subjected to SDS/PAGE. After regions of the 16 genes in A and B depend on OGT. MCF7 cells with scramble protein gel electrophoresis, immunoprecipitated EZH2 were excised from gels siRNA (white bar) or siOGT (black bar) were subjected to ChIP with control and subjected to in-gel trypsin digestion. The digested peptides were extracted IgG, EZH2, or H3K27me3 Ab. In D and E the data are shown as fold en- and analyzed by microliquid chromatography/tandem MS. richment relative to IgG. GAPDH gene was used as a negative control. All results from A to E are presented as mean ± SD. P values were measured by ChIP. MCF7 transfected with scramble RNA, siOGT, or siEZH2 for 3 d were Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, n = 3. subjected to ChIP as previously described (59). The precipitated chromatin was washed, and DNAs were purified for measurement by quantitative PCR using LightCycler 480 SYBR Green I Master (04 887 352 001, Roche) with

Indeed, OGT and EZH2 only coregulated 63 genes (Fig. S5B). primers against specific promoter region. The primers used for ChIP real- BIOCHEMISTRY In addition, similar to DNA methylation and other histone time PCR are listed in Table S2.

Chu et al. PNAS | January 28, 2014 | vol. 111 | no. 4 | 1359 Downloaded by guest on September 26, 2021 ACKNOWLEDGMENTS. We thank Drs. W. H. Lee at University of Califor- Sinica for performing the microarray experiments. This study was sup- nia, Irvine, and Y. Zhang at Harvard Medical School for critical sugges- ported by grants from Academia Sinica [to L.-J.J. (career development tions, and Affymetrix Gene Expression Service Laboratory at Academia grant) and C.-H.W.].

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