Biochem. J. (2015) 466, 587–599 (Printed in Great Britain) doi:10.1042/BJ20141072 587

O-GlcNAcylation of co-activator-associated 1 regulates its specificity Purin Charoensuksai*, Peter Kuhn*†, Lu Wang*, Nathan Sherer* and Wei Xu*1 *McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, U.S.A. †Biological Sciences Department, Edgewood College, Madison, WI 53711, U.S.A.

Co-activator-associated arginine methyltransferase 1 (CARM1) (CARM1QM)] markedly decreased O-GlcNAcylation, but did asymmetrically di-methylates on arginine residues. not affect protein stability, dimerization or cellular localization CARM1 was previously known to be modified through O-linked- of CARM1. Moreover, CARM1QM elicits similar co-activator β-N-acetylglucosaminidation (O-GlcNAcylation). However, the activity as CARM1 wild-type (CARM1WT) on a few site(s) of O-GlcNAcylation were not mapped and the effects factors known to be activated by CARM1. However, O-GlcNAc- of O-GlcNAcylation on biological functions of CARM1 depleted CARM1 generated by wheat germ agglutinin (WGA) were undetermined. In the present study, we describe enrichment, O-GlcNAcase (OGA) treatment and mutation of the comprehensive mapping of CARM1 post-translational putative O-GlcNAcylation sites displays different substrate modification (PTM) using top-down MS. We found that all specificity from that of CARM1WT. Our findings suggest that detectable recombinant CARM1 expressed in human embryonic O-GlcNAcylation of CARM1 at its C-terminus is an important kidney (HEK293T) cells is automethylated as we previously determinant for CARM1 substrate specificity. reported and that about 50% of this automethylated CARM1 contains a single O-linked-β-N-acetylglucosamine (O-GlcNAc) Key words: co-activator-associated arginine methyltransferase 1 moiety [31]. The O-GlcNAc moiety was mapped by MS to four (CARM1), co-activator, mass spectrometry (MS), protein methyl- possible sites (Ser595,Ser598,Thr601 and Thr603) in the C-terminus ation, O-linked-β-N-acetylglucosaminidation (O-GlcNAcyla- of CARM1. Mutation of all four sites [CARM1 quadruple mutant tion), transcription.

INTRODUCTION methylate diversified substrates allows it to orchestrate a broad spectrum of biological processes. However, little is known about Co-activator-associated arginine methyltransferase 1 (CARM1), how the methyltransferase activity and substrate specificity of also known as protein arginine methyltransferase4 (PRMT4), is a CARM1 are regulated. type I PRMT that asymmetrically dimethylates arginine on target Proteins in the PRMT family share a common domain proteins. CARM1 was first identified as an interacting partner organization. The core region contains a methyltransferase of the glucocorticoid receptor interacting protein 1 (GRIP1) co- catalytic site, methyl donor S-adenosyl (SAM)- activator protein that enhanced transcriptional activity of several binding pocket and dimerization arm. Although the catalytic steroid hormone receptors [1]. Subsequent studies showed that cores are highly conserved among PRMTs, CARM1 possesses CARM1 activates many cancer-relevant transcription factors a unique C-terminus [9]. This C-terminal region is not essential including PPARγ (peroxisome proliferator-activated receptor for CARM1’s methyltransferase activity in vitro;however, γ ), p53, NF-κB (nuclear factor kappa-light-chain-enhancer of it exhibits an autonomous transcriptional activation function activated B-cells), E2F1 (E2F transcription factor 1) and β- whose mechanism remains unclear. The C-terminal region can catenin [1–5]. The methyltransferase activity of CARM1 has also mediate protein–protein interaction. For example, TIF1α been extensively studied. CARM1 methylates and modulates the (transcription intermediary factor 1 α)/TRIM24 (tripartite motif- functions of a plethora of proteins. The first CARM1 substrate containing 24) was reported to interact with this domain [10]. identified was H3 [1]. CARM1-specific histone H3 O-GlcNAcylation (O-linked-β-N-acetylglucosaminidation), at Arg17 has been linked to gene activation and is found on >1000 nuclear and cytoplasmic proteins, is considered part of the ‘’ [6]. Other substrates of characterized by the addition of N-acetyl-D-glucosamine to CARM1 include BAF155 (BRG1-associated factor 155), SmB and residues of target proteins [11–14]. O- (small nuclear ribonucleoprotein polypeptide B), SAP49 (splicing GlcNAcylation is a reversible post-translational modification factor 3B subunit 4), U1C (U1 small nuclear RNP specific C), (PTM) governed by two : O-GlcNAc (O-linked-β-N- CA150 (co-activator of 150 kDa), HuR (Hu antigen R), HuD (Hu acetylglucosamine) transferase (OGT) [15,16] and O-GlcNAcase antigen D), TARPP (thymocyte cAMP-regulated phosphoprotein) (OGA) [17] (reviewed in [18–21]). UDP–GlcNAc, the GlcNAc and PABP1 [poly(A)-binding protein 1], many of which are donor molecule, is generated in the hexosamine involved in mRNA processing and transcription elongation [7,8]. pathway. Protein O-GlcNAcylation has been shown to regulate The ability of CARM1 to activate many transcription factors and diverse protein functions including stability, localization,

Abbreviations: CAD, collisionally-activated dissociation; CARM1, co-activator-associated arginine methyltransferase 1; CARM1QM, CARM1 quadruple mutant; CARM1WT, CARM1 wild-type; CHX, cycloheximide; DMEM, Dulbecco’s modified Eagle’s medium; E2, oestradiol; ECD, electron-capture dissociation; ERα, oestrogen receptor α; GRIP1, glucocorticoid receptor interacting protein 1; HEK, human embryonic kidney; OGA, O-GlcNAcase; O- GlcNAc, O-linked-β-N-acetylglucosamine; O-GlcNAcylation, O-linked-β-N-acetylglucosaminidation; OGT, O-GlcNAc transferase; PABP1, poly(A)-binding protein 1; PBST, phosphate-buffered saline with Tween 20; PPARγ, peroxisome proliferator-activated receptor γ; PRMT, protein arginine methyltransferase; PTM, post-translational modification; TIF1α, transcription intermediary factor 1 α; WGA, wheat germ agglutinin; ZFN, finger . 1 To whom correspondence should be addressed (email [email protected]).

c The Authors Journal compilation c 2015 Biochemical Society 588 P. Charoensuksai and others protein–protein interaction, transcriptional activity, enzymatic 6 h. The reaction was then resolved by SDS/PAGE and activity and substrate specificity (reviewed in [18,22–24]). stained with Coomassie Brilliant Blue [0.05 (w/v):50:10:40;

Using top-down MS, we found that nearly 100% of CARM1 Coomassie Brilliant Blue R-250/methanol/acetic acid/H2O] is automethylated in vivo and that 50% of this automethylated overnight followed by destaining with dye-free buffer for 2– CARM1 exhibits a 203 Da mass shift, indicative of mono- 4 h. Gels were then incubated with Amplify scintillation fluid O-GlcNAcylation. We mapped Ser595,Ser598,Thr601 and Thr603 (GE Healthcare Life Sciences) for 20 min prior to drying as major O-GlcNAcylation sites located in the C-terminus of on a Whatman paper and exposing to an X-ray film. For CARM1 protein, implying that CARM1 could exist as a mixture 2D electrophoresis, 2.5 μg of purified CARM1 was incubated −/− of mono-O-GlcNAcylated forms. O-GlcNAcylation of CARM1 with 500 μgofCal51CARM1 cell lysates. The reaction was did not appear to alter stability, nuclear–cytoplasmic distribution, resolved using isoelectric focusing pH 4–8. 2D electrophoresis dimerization capability and co-activator activity of CARM1 on was performed by Kendrick Labs. a few tested transcription factors. However, O-GlcNAcylation of CARM1 affects its substrate specificity. Our findings reveal that O-GlcNAcylation of the CARM1 C-terminus regulates substrate Enrichment of O-GlcNAcylated CARM1 with wheat germ agglutinin specificity and thus may affect a variety of CARM1 functions resin depending on substrate methylation. Enrichment of O-GlcNAcylated CARM1 was adapted from the protocol described by Zachara et al. [27]. Purified CARM1 EXPERIMENTAL (20 μg) was incubated with 100 μl of WGA (wheat germ agglutinin) bead (Vector Labs) for 1 h on ice. After The expression and purification of recombinant CARM1 centrifugation, the flow-through fraction was collected. The Full-length mouse CARM1 cDNA was cloned into Halo– column was then washed with WGA wash buffer (25 mM Tris, tag vector, pFC14K (Promega), as described previously 300 mM NaCl, 5 mM CaCl2 and 1 mM MgCl2) followed by [25]. CARM1S595A, CARM1S598A, CARM1T601A, CARM1T603A and elution with WGA elution buffer (0.2% v/v Nonidet P40 and CARM1QM (CARM1 quadruple mutant) were generated by site- 1MN-acetyl-D-glucosamine). directed mutagenesis using pFC14K–CARM1 construct as a template. CARM1CTD (C-terminal domain deleted CARM1) was generated by PCR cloning of mouse CARM1 encoding Generation of CARM1 (WT or QM) stably expressing cell lines 1–553 into a pFC14K Flexi vector. The expression and purification of Halo–tagged recombinant CARM1 were CARM1-null human embryonic kidney (HEK293T), MCF7 (ERa performed as previously described [25]. positive breast cancer cell line) and MDA-MB-231 (triple- negative breast cancer cell line) cell lines were described in [28]. CARM1-null Cal51 cell lines were generated using Top-down MS the same method as described in [28]. Flag–tagged mouse The high-resolution FT-ICR (Fourier transform-ion cyclotron CARM1WT (CARM1 wild-type) or CARM1QM was cloned into resonance) MS analysis of Halo–tag-purified CARM1 was pCDH-lentiviral vector and pBABE retroviral vector. pCDH performed as described previously [26]. viral vector was used to transduce recombinant CARM1 into −/− MDA − MB − 231CARM1 cells, whereas pBABE viral vector −/− Middle-down MS was used to transduce CARM1 into MCF7CARM1 cells. Cells were then selected in medium containing 2 μg/ml puromycin for Purified CARM1 protein was digested with endoprotease Lys- 1 week and pooled cells were used in the experiments. C (Promega) for FT-ICR MS analysis. Purified recombinant CARM1 (20 μg) was incubated with 200 ng of Lys-C for 1 h at ◦ 37 Cin50mMNH4HCO3 and 10% acetonitrile solution at pH 8. Subsequently, the mixture was desalted using a 10 kDa NMWL Measurement of transcriptional activation by reporter assay Amicon Ultra (Millipore). Samples were injected using nano-ESI For CARM1-mediated activation of Sertad, NFκBIB (nuclear in vehicle containing 50% methanol and 1% acetic acid through a factor of kappa light polypeptide gene enhancer in B-cells −/− Triversa Nanomate injector (Advion). For fragmentation analysis, inhibitor, beta) and p53, HEK293TCARM1 cells, seeded on a single charged state was isolated and dissociated by CAD a 48-well plate, were transiently transfected with GAL4–Luc (collisionally-activated dissociation) using 12–16% collision (luciferase reporter vector driven by a GAL4 responsive element) energy or ECD (electron-capture dissociation) at 3% activation reporter plasmid (20 ng), Renilla luciferase plasmid (20 ng), energy for 50 ms. GAL4-fused designated transcription factor in pCMX vector (20 ng) and Flag–tagged CARM1WT or Flag–tagged CARM1QM OGA treatment in pCMX vector. For autonomous activation activity of full-length CARM1, A total of 45 ng of purified OGA (PRO-E0255, lot 2012–0255, −/− CARM1 Prozomix) was used per 5 μg of purified CARM1. The reaction HEK293T cells, seeded on a 24-well plate, were was incubated at 37 ◦Cfor4h. transiently transfected with GAL4–Luc reporter plasmid (50 ng), Renilla luciferase plasmid (50 ng) and pM vector encoding Gal4- fused CARM1WT or Gal4-fused CARM1QM (200 ng). In vitro methylation reaction Empty vectors of the respected constructs were used to adjust Recombinant CARM1 (200 ng) was incubated with substrates total amounts of plasmids. Proteins were allowed to express for (500 ng of purified substrates or 10 μg of cell lysate) in 2 days. Cells were then lysed with buffer containing 100 mM buffer containing 5 mM MgCl2, 20 mM HEPES, pH 7.9, 1 mM potassium phosphate and 0.2% Triton X-100. All experiments EDTA, 1 mM DTT, 10% glycerol containing 2 μlof[3H] were performed in triplicate. Luciferase signal was normalized to S-adenosylmethionine (10 Ci/mmol, Perkin Elmer) for 1– Renilla luciferase internal control.

c The Authors Journal compilation c 2015 Biochemical Society O-GlcNAcylation regulates CARM1 substrate specificity 589 qPCR analysis of antibody (Life Technologies) diluted 1:1000 in 3% BSA for −/− WT QM CARM1 1 h at room temperature. Slides were prepared with Prolong CARM1 or CARM1 stably expressed MCF7 was gold mounting reagent with DAPI (Life Technologies), enabling maintained in Phenol Red-free Dulbecco’s modified Eagle’s visualization of cell nuclei. All images were collected using a medium (DMEM) supplemented with 5% charcoal-stripped Nikon Ti Eclipse inverted fluorescence microscope using a ×60 FBS for 3 days. Cells were then treated with DMSO or 10 nM objective. oestradiol (E2) for 4 or 12 h. RNA was then extracted using EZNA kit (Omega Bio-Tek) according to manufacturer’s protocol. RNA (2 μg) was added to Superscript II (Life Technologies) reverse transcriptase with random primers. Reversed transcribed Quantification of fluorescence intensity reactions were diluted 100-fold and used as template for qPCR Images of >50 cells under each condition were processed using analyses. Reactions were performed in triplicate. Cycle threshold NIS Elements software, with DAPI and CARM1 intensity profiles (CT) values were compared with standard curves prepared from determined across the nuclear and cytoplasmic compartments serially diluted cDNA of the same cell type, then normalized to using a transect method. Briefly, the DAPI profile was used internal control RPL13A. to define the nuclear–cytoplasmic boundary, subsequent to determining the average CARM1 intensity in the nucleus compared with the cytoplasm. Western blotting analysis Purified proteins or cell lysates were resolved by SDS/PAGE and transferred to nitrocellulose membrane. Membranes were RESULTS blocked with 5% milk in PBS with 0.1% Tween 20 for conventional Western blot or Odyssey Blocking Buffer (Licor) Identification of CARM1 O-GlcNAcylation sites for quantitative Western blot analysis. Membranes were probed A study by Cheung et al. [30] showed that CARM1 expressed in with mouse anti-CARM1 (ab110024, Abcam) at 1:2000 dilution, murine neuroblastoma cell lines was O-GlcNAcylated; however, mouse anti-O-GlcNAc RL2 (ab2739, Abcam) at 1:100 dilu- the O-GlcNAcylation site(s) of CARM1 was unknown. To tion, mouse anti-p21 (05-345, Millipore) at 1:1000 dilution, rabbit examine whether CARM1 is O-GlcNAcylated in breast cancer anti-R551me2a CARM1 at 1:2000 dilution [29] or mouse anti- cells, we immunoprecipitated CARM1 from MCF7 cells and β- (A5441, Sigma–Aldrich) at 1:10000 dilution as primary probed with an anti-O-GlcNAc antibody. In accordance with the antibodies. Horseradish peroxidase-conjugated goat anti-mouse, previous report [30], endogenous CARM1 was O-GlcNAcylated horseradish peroxidase-conjugated goat anti-rabbit or IRDye 680 as detected by Western blot (Figure 1A). We next purified CARM1 LT goat anti-mouse (Licor) were used as secondary antibody at from HEK293T cells using Halo–tag technology, as previously 1:10000 dilution. described [25]. Halo–tag was cleaved off, yielding highly pure CARM1 protein (Figure 1B) which was subjected to top-down MS. Nearly 100% of CARM1 was found to be di-methylated, Immunoprecipitation as we previously reported [31]. Approximately 50% of this automethylated CARM1 contains a 203 Da mass shift, indicative For the detection of O-GlcNAcylation on endogeneous β CARM1 in MCF7, MCF7 cells were maintained in DMEM of a single O-linked -N-acetylglucosamine (O-GlcNAc) moiety (Figure 1C). supplemented with 10% FBS. Cells were lysed with Triton lysis To identify O-GlcNAcylation site(s), we performed MS with buffer (50 mM Tris, 150 mM NaCl, 10% glycerol and 0.5% Triton X-100). Cell lysates were incubated with rabbit polyclonal a limited Lys-C digestion (i.e. middle-down MS) of purified CARM1 antibody or IgG overnight at 4 ◦C. The mixture was CARM1. Peaks matching the expected mass of a peptide then incubated with Dynabead® protein A (Life Technologies) corresponding to CARM1 residues 552–608 were observed at two charge states: 5 + (A, experimental mass 7128.28 Da) and for 1 h at room temperature. The immunoprecipitated proteins + were dissolved in SDS sample buffer heated at 95 ◦Cfor5min. 4 charge state (B, experimental mass 7128.33 Da) (Figure 2A). For the effect of loss of O-GlcNAcylation on CARM1 A second peak 203 Da larger than the expected mass of the −/− unmodified CARM1 fragment was observed for both charge states dimerization, HEK293TCARM1 cells were co-transfected with WT QM (A’, experimental mass 7331.36 Da and B’, experimental mass CARM1 and CARM1 containing either Halo–tag or Flag– 7331.37 Da for 5 + and 4 + charge state respectively). This mass tag. After 2 days, cells were harvested and lysed with Triton lysis shift is consistent with the addition of an O-GlcNAc moiety to buffer. Cell lysates were incubated with Anti-FLAG® M2 Beads ◦ a subpopulation of the C-terminal fragment of CARM1. Amino (Sigma–Aldrich) overnight at 4 C. Immunoprecipitated proteins acid sequence analysis using MS/MS coupled with CAD of peaks were then dissolved in SDS sample buffer. A, A’, B and B’ confirmed that amino acid sequence of these four peaks match residues 552–608 of CARM1 (Supplementary Figure S1). Confocal immunofluorescence To further refine O-GlcNAcylation region of CARM1, the A’ Cells were seeded on a coverslip in a six-well plate. At 24 peak which exhibited 203 Da increase in mass was isolated for h after seeding, cells were washed with PBS then fixed with MS/MS using ECD, a low-energy dissociation method that should 4% paraformaldehyde. Fixed cells were incubated with 0.2% retain O-GlcNAc modifications. In the present study, peptide Triton X-100 in PBS for 3 min for permeabilization, then blocked fragments consistent with O-GlcNAcylation of residues 594–606 with 3% BSA in PBST (phosphate-buffered saline with Tween of the CARM1 C-terminus were detected. This region of CARM1 20) for 1 h at room temperature. Cells were then washed with contains four possible O-GlcNAcylation sites: Ser595,Ser598,Thr601 PBST followed by incubation with mouse anti-CARM1 antibody and Thr603 (Figure 2B). (ab110024, Abcam) diluted 1:500 in 3% BSA for 1 h at room The C-terminus of CARM1 was predicted to be disordered temperature. Subsequently, cells were washed with PBST, then [32]. Using PONDR-FIT algorithm [33], we also predict that incubated with Alexa Fluor 594 conjugated goat-anti-mouse the putative O-GlcNAcylation region of CARM1 (amino acids)

c The Authors Journal compilation c 2015 Biochemical Society 590 P. Charoensuksai and others

Figure 1 Both endogenous and recombinant CARM1 are O-GlcNAcylated

(A) Endogenous CARM1 in MCF7 cells was immunoprecipitated and detected using anti-O-GlcNAc antibody. (B) Coomassie Brilliant Blue staining of purified CARM1 from HEK293T cells using Halo–tag expression system. (C) Top-down MS analysis of PTMs on purified recombinant CARM1. Nearly 100% of CARM1 is dimethylated, of which approximately 50% exhibits a 203 Da mass increase, indicative of a single O-GlcNAc moiety. The predicted isotope distribution of methylated CARM1 is represented by red circles and the predicted isotope distribution of methylated and O-GlcNAcylated CARM1 is represented by yellow circles. Listed masses are the most abundant isotope.

595–603) resides in a disordered region (0.7P1). This echoes with our MS data, O-GlcNAcylation of a CARM1 variant the previous report that the likelihood of O-GlcNAcylation lacking amino acids 554–608 (CARM1CTD) was undetectable. to occur in the disordered region is 6-fold higher than the CARM1 variants lacking one of the putative O-GlcNAc structured area [14]. Next we mutated O-GlcNAcylation sites sites (CARM1S595A, CARM1S598A, CARM1T601A andCARM1T603A) to alanine individually or in combination and expressed mutant exhibited a decrease in O-GlcNAcylation level to various degrees. CARM1 proteins in HEK293T cells using the Halo–tag system When all four sites were mutated to alanine (CARM1QM), a (Figure 2C). The O-GlcNAcylation levels of the purified drastic decrease in O-GlcNAcylation was observed to a level recombinant CARM1 proteins were analysed by quantitative comparable with the negative control, implying that these four Western blot (Figure 2D), where recombinant CARM1 sites are the major sites of CARM1 O-GlcNAcylation. Moreover, purified from Escherichia coli served as a negative control alignment of CARM1 sequence across species revealed that the because O-GlcNAcylation is absent from E. coli. Consistent four O-GlcNAcylation sites were conserved among vertebrates

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Figure 2 CARM1 has four putative site of O-GlcNAcylation in its C-terminus

(A) High resolution middle-down MS reveals a mass of the 5 + charge state (A and A’) and the 4 + charge state (B and B’) precursor ions exhibiting 203 Da mass shift, indicative of O-GlcNAcylation. CAD fragmentation of the populations A, A’, B and B’ mapped these peptides to amino acid 552–608 of CARM1 (Figure S1). (B) ECD fragmentation map of CARM1 population A’. Residues 552–608

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(Figure 2E), suggesting that CARM1 O-GlcNAcylation sites, that are preferentially methylated by non-GlcNAcylated CARM1 which were preserved through the course of evolution, might and those that are methylated regardless of O-GlcNAc status of possess significant biological functions. CARM1, band isolation and subsequent protein identification by MS are likely to yield many false-positive hits, due to the limited resolution of 1D SDS/PAGE to resolve proteins within CARM1 O-GlcNAcylation determines its substrate specificity a complex cell lysate. To increase resolution and to determine if O-GlcNAcylation of CARM1 affects methylation of other Next, we sought to investigate whether O-GlcNAcylation affects cellular substrates, we performed a large-scale in vitro methylation the methyltransferase activity of CARM1. Our MS analysis −/− assay using lysates obtained from Cal51CARM1 cells as a indicated that the recombinant protein expressed from HEK293 source of methylation substrates and resolved the proteins by cells is an approximately 1:1 mixture of CARM1 with and 2D gel (Figure 3E). Some substrates were found preferentially without the O-GlcNAc moiety (Figure 1C). In order to enrich O- methylated by O-GlcNAc-depleted CARM1 (Figure 3E, white GlcNAcylated CARM1, we incubated recombinant CARM1 with arrows), whereas others were found preferentially methylated by WGA, a plant-derived lectin that had a high affinity for N-acetyl- O-GlcNAc-enriched CARM1 (Figure 3E, black arrows). There D-glucosamine. CARM1 in the flow-through, later referred to as were proteins methylated similarly by both forms of CARM1 the O-GlcNAc-depleted fraction, was largely unmodified. The O- (Figure 3E, red arrows). GlcNAcylated CARM1 was concentrated on the beads, washed Spots which exhibited a difference in autoradiographic pattern and eluted using a saturated solution of N-acetyl-D-glucosamine were excised. Proteins were retrieved by in-gel digestion and (referred to as the O-GlcNAc enriched fraction). A diagram subjected to protein identification by MS. As a result, 840 illustrating the O-GlcNAcylated protein enrichment procedure proteins were identified (Supplementary Table S1). Further using WGA is shown in Figure 3(A). Additionally, we incubated substrate validation is needed to determine which protein CARM1input with the purified OGA to achieve partial removal is a bona fide substrate for CARM1 and which protein is of the O-GlcNAc moiety on CARM1. The O-GlcNAc level of differentially methylated by O-GlcNAcylated and non-O- CARM1input treated with OGA, O-GlcNAc enriched and depleted GlcNAcylated CARM1. fractions were validated using the anti-O-GlcNAc antibody by We previously reported automethylation of CARM1 [31]. Western blot (Figure 3B). Since automethylation and O-GlcNAcylation both occur at the Next we investigated the effect of O-GlcNAcylation on C-terminus, we ask whether the two PTMs exhibit cross-talk. the methyltransferase activity of CARM1 using an in vitro −/− CARM1 In the present study, O-GlcNAc and automethylation level of methylation assay. Total cell lysates of Cal51 was used CARM1 mutants defective of O-GlcNAcylation (CARM1QM)and as a source of protein substrates. Since CARM1 is not expressed 551 automethylation (CARM1Arg K ) were determined using anti- in this cell line, all the methylation sites on CARM1 substrates O-GlcNAc and site-specific anti-asymmetrically dimethylated are available for incorporation of [3H] CH by recombinant 3 Arg551 (anti-R551me2a CARM1) [29] antibodies by Western CARM1 in vitro. With short exposure, only two major bands blot. Purified CARM1WT served as a positive control. We found were shown to be strongly methylated by CARM1. CARM1input that automethylation and O-GlcNAcylation of CARM1, although preferentially methylated a substrate at ∼50 kDa. Depletion of both occur at the C-terminus, are probably independent events CARM1 O-GlcNAcylation, either by WGA resin or by OGA- (Supplementary Figure S2). mediated O-GlcNAc removal, shifted the substrate specificity of CARM1 toward a substrate at ∼60 kDa, whereas the O- GlcNAc-enriched CARM1 preferentially methylated a substrate ∼ at 50 kDa (Figure 3C). Further, the methylation pattern of cell O-GlcNAcylation does not affect cellular localization, stability or QM lysate by CARM1 resembled that of the O-GlcNAc-depleted dimerization capability of CARM1 CARM1, implying that CARM1QM carries the biochemical We have previously reported CARM1 knockout MDA-MB-231 property of the non-O-GlcNAcylated CARM1. For subsequent −/− −/− biochemical studies, we substituted O-GlcNAc-depleted CARM1 (MDA − MB − 231CARM1 )andMCF7(MCF7CARM1 ) cell with CARM1QM. We next examined whether methylation of lines generated by zinc finger nuclease (ZFN) technology [28]. histone H3 and PABP1, two CARM1 substrates [8], was affected Flag–tagged CARM1WT and CARM1QM were restored in these by CARM1 O-GlcNAcylation. Our results showed that O- cell lines, which allow direct comparison with CARM1WT and GlcNAc enriched and O-GlcNAc-depleted CARM1 methylated CARM1QM with no interference from endogenous CARM1 in histone H3 and PABP1 to a similar extent, suggesting that O- these cells. GlcNAcylation did not affect the ability of CARM1 to methylate To examine whether the subcellular localization of these two substrates (Figure 3D). CARM1 is affected by O-GlcNAcylation, we performed Although the result of our 1D analyses implies that substrates immunofluorescence using the MDA-MB-231 parental cell −/− of CARM1 can be classified into three classes, e.g. those that line and MDA − MB − 231CARM1 restored with CARM1WT, are preferentially methylated by O-GlcNAcylated CARM1, those CARM1QM or empty vector control. Western blotting results

was a part of the CARM1 C-terminus, whereas 609–621 was the Halo–tag linker after TEV (tobacco etch virus nuclear inclusion in endopeptidase) cleavage. C- and z-ions predicted from the amino acid sequence of CARM1 with the added mass of O-GlcNAc matched the observed ECD peak masses are denoted by  and . The putative site(s) of O-GlcNAcylation were mapped to a region between amino acid residues 594–606. Four possible O-GlcNAcylation sites are present in this fragment and are marked by arrows. Grey, black and striped boxes depict the N-terminal domain, core region and C-terminal domain of CARM1 respectively. (C) Schematic illustration of CARM1 mutants generated by site-directed mutagenesis. Stars denote mutation sites disrupting O-GlcNAcylation. CARM1CTD denotes a truncated CARM1 with deletion of amino acids 554–608. CARM1S595A, CARM1S598A, CARM1T601A andCARM1T603A are single site mutants and CARM1QM has mutations at all four putative O-GlcNAcylation sites. (D) Quantitative Western blotting analysis of O-GlcNAcylation levels of the recombinant CARM1 mutants expressed and purified using Halo–tag system. Bar graph (top) shows the relative band intensity of O-GlcNAc–CARM1 quantified using Odyssey Imaging System. A representative Western blot image of a triplicate biological sample is shown. (E) Partial ClustalW alignment of known CARM1 sequence from different species indicates high conservation of the O-GlcNAcylation sites within vertebrates (highlighted).

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Figure 3 The substrate specificity of CARM1 is affected by O-GlcNAcylation status

c The Authors Journal compilation c 2015 Biochemical Society 594 P. Charoensuksai and others shows that the stable cell lines express CARM1WT and CARMQM to be indistinguishable (Figure 5B), further supporting the notion at comparable levels (Figure 4A). In the MDA-MB-231 parental that the co-activator function of CARM1 is not regulated by O- cells, endogeneous CARM1 is localized primarily in the GlcNAcylation. cytoplasm. CARM1WT and CARM1QM are indistinguishable from The C-terminus of CARM1 mediates its interaction with the that of endogeneous CARM1 (Figure 4B). Quantification of co-activator protein TIF1α [10]. Since O-GlcNAcylation sites CARM1 immunofluorescence intensity revealed no detectable of CARM1 reside at the C-terminus, we determined if O- difference in nuclear–cytoplasmic distribution of CARM1WT and GlcNAcylation affects the interaction of these two proteins in a CARM1QM (Figure 4C), indicating that loss of O-GlcNAcylation mammalian two-hybrid assay. CARM1WT and CARM1QM were does not affect cellular localization of CARM1. fused to the GAL4 DNA-binding domain whereas TIF1α Next we examined the effect of O-GlcNAcylation on was fused to herpes simplex virus (VP16). Our result showed CARM1 protein stability. MDA-MB-231 parental cells that CARM1WT and CARM1QM interacted with TIF1α similarly, −/− and CARM1WT or CARM1QM expressing MCF7CARM1 indicating that O-GlcNAcylation does not affect the interaction −/− α MDA − MB − 231CARM1 were treated with cycloheximide between CARM1 and TIF1 (Supplementary Figure S4A). (CHX) to inhibit protein synthesis. CARM1 protein in MDA-MB- CARM1 was previously shown to form a ternary complex α 231 cells appeared to be very stable, as even after 24 h treatment with GRIP1 and TIF1 [10]. Together, these three cofactor with CHX CARM1 protein level remained unchanged, whereas proteins synergistically activated transcription of a reporter gene p21 was completely undetectable after 6 h CHX treatment [10]. To investigate whether O-GlcNAc affects the synergistic (Supplementary Figure S3A). The stability of CARM1 protein in activity of this complex, we performed reporter assays by co- −/− α WT QM CARM1WT- and CARM1QM-restored MDA − MB − 231CARM1 transfecting GAL4–GRIP1, TIF1 and CARM1 or CARM1 cells was similar to that of the parental cells. No discernible with GAL4–Luc. In agreement with the previous report, we WT observed a synergistic increase in reporter activity when GAL4– difference was observed in the protein stability of CARM1 α and CARM1QM (Supplementary Figure S3B), indicating that O- GRIP1, TIF1 and CARM1 were transfected with the GAL4–Luc GlcNAcylation does not affect CARM1 protein stability. reporter (Supplementary Figure S4B) [10]. A dose-dependent Next we asked whether O-GlcNAcylation could affect increase in reporter activity was observed as the amount of transfected CARM1 construct increased. However, CARM1WT dimerization of CARM1. In the present study, Halo–tag and QM Flag–tag CARM1 (WT and QM) were expressed in HEK293T and CARM1 displayed a similar ability to activate the GAL4– −/− CARM1 Luc reporter, indicating that O-GlcNAcylation of CARM1 does cells with CARM1 knocked out by ZFN (HEK293T ) not affect the synergistic interactions of these co-activators. [28]. Immunoprecipitation was performed using Anti-FLAG® QM Finally, we investigated whether O-GlcNAc regulates the M2 Beads. We found that CARM1 could form dimers with α α WT QM activity of CARM1 on oestrogen receptor (ER ) target genes. −/− both CARM1 and CARM1 (Supplementary Figure S3C), WT QM CARM1 indicating that dimerization of CARM1 was not obviously Either CARM1 or CARM1 was restored in MCF7 cells to comparable levels (Figure 5C) and qPCR of previously affected by O-GlcNAcylation under these conditions. These α results were consistent with the previous observation that the reported ER –CARM1 co-regulated genes including pS2, c-Myc, dimerization domain resides in the central catalytic region [32,34]. PTGES (prostaglandin E synthase), EGR3 (early growth response factor 3) and IGFBP (-like growth factor-binding protein) [31,35] was performed. As expected, the expression levels of these genes are E2-responsive. However, no statistical difference was O-GlcNAcylation does not affect the co-activator function of CARM1 observed between their expression levels in MCF7–CARM1WT QM Because both the O-GlcNAcylation sites of CARM1 and the co- and MCF7–CARM1 cells, indicating that O-GlcNAcylation activator activity reside in the C-terminus [9], we asked whether status of CARM1 did not influence the expression levels of these CARM1 O-GlcNAcylation affects co-activator activity. We have genes (Figure 5D). previously shown that CARM1 increases the transcriptional activity of Sertad, NFκBIB and p53 [31]. Using a mammalian one-hybrid approach, we examined the co-activator activity of DISCUSSION CARM1QM with these transcription factors. Plasmids encoding Sertad, NFκBIB and p53 fused to the DNA-binding domain of Recombinant CARM1 is 50% mono-O-GlcNAcylated at the GAL4 were co-transfected with CARM1WT or CARM1QM and a C-terminus luciferase reporter driven by a GAL4-responsive element (GAL4– Although CARM1 was previously reported to be O-GlcNAcylated Luc). CARM1WT and CARM1QM exhibited similar activities to [30], neither the extent nor the sites of O-GlcNAcylation activate these transcription factors (Figure 5A), suggesting that were known. Using high-resolution top-down MS, we have loss of O-GlcNAcylation does not compromise the ability of discovered that ∼50% of recombinant CARM1 expressed from CARM1 to activate these transcription factors. Moreover, the HEK293T cells is mono-O-GlcNAcylated. We did not detect activities of GAL4–CARM1WT or GAL4–CARM1QM to activate a higher glycoforms (i.e. di- or tri-glycosylated) of CARM1, GAL4-response element fused luciferase reporter [9] were found suggesting that they may not exist in the condition we investigated

(A) The schematic illustration of O-GlcNAcylated CARM1 enrichment procedure using WGA. (B) Western blotting of CARM1input, CARM1input treated with OGA, CARM1OGN-enriched, CARM1OGN-depleted and CARM1QM. OGA was visualized by Coomassie Blue staining (*). Arrows denote CARM1 in Coomassie Blue staining. (C) Differential substrate methylation patterns by CARM1input treated with OGA, −/− CARM1OGN-enriched and CARM1OGN-depleted using Cal51CARM1 lysates as substrate in in vitro methylation reaction. CARM1QM resembles the activity of CARM1OGN-depleted and CARM1input treated with OGA. Reaction without CARM1 served as a negative control and CARM1input was used as a positive control. (D) CARM1input, CARM1OGN-enriched and CARM1OGN-depleted exhibit no difference in in vitro −/− methylation of PABP1 or histone H3. Reaction without CARM1 served as a negative control. (E) The Cal51CARM1 cell lysates were in vitro methylated by CARM1OGN-enriched or CARM1OGN-depleted and resolved by 2D-electrophoresis. Autoradiograph shows the differential methylation patterns by two forms of CARM1. Red arrows denote proteins methylated by both OGN-enriched and -depleted CARM1. White arrows denote proteins only methylated by CARM1OGN-depleted whereas black arrows denote proteins only methylated by CARM1OGN-enriched.

c The Authors Journal compilation c 2015 Biochemical Society O-GlcNAcylation regulates CARM1 substrate specificity 595

Figure 4 Mutations of CARM1 at O-GlcNAcylation sites do not affect the subcellular localization of CARM1

(A) Western blotting shows the expression level of CARM1 in MDA-MB-231 parental cells (lane 1), CARM1 knockout (KO) cells expressing empty vector (lane 2), CARM1 KO cells restored with CARM1WT (lane 3) or CARM1QM (lane 4). (B) Detection of CARM1 subcellular distribution by immunofluorescence in MDA-MB-231 cells in Figure 5(A). Cells were seeded on coverslip, fixed and stained for CARM1 and DAPI. Representative images are taken under a fluorescent microscope with ×100 magnification. (C) The fluorescence intensity of cytoplasmic and nuclear CARM1 in CARM1WT or CARM1QM expressing MDA-MB-231 cells were quantified and plotted as proportion of total. Error bar: S.D.

c The Authors Journal compilation c 2015 Biochemical Society 596 P. Charoensuksai and others

Figure 5 Mutations of CARM1 at O-GlcNAcylation sites do not affect its co-activator activity

(A) Either CARM1WT or CARM1QM can activate transcription of Sertad, NFκBIB and p53 in a GAL4-reporter assay. (B) CARM1WT and CARM1QM display similar autonomous activation activity in a −/− −/− GAL4-reporter assay in HEK293TCARM1 cells. (C) Western blot analysis depicts the comparable expression levels of CARM1 in MCF7CARM1 cells restored with CARM1WT and CARM1QM.(D) −/− CARM1WT and CARM1QM enhanced the expression of several known ERα–CARM1 co-regulated genes to a similar level. CARM1WT or CARM1QM stably expressing MCF7CARM1 cells were treated + with 10 nM E2 or DMSO for 4 or 12 h. RNA was isolated and endogenous gene expression was analysed by qPCR. Expression levels of target genes were normalized to PRL13A. A mean − S.D. (n=3) was graphically displayed. *P < 0.05 and **P < 0.01 respectively. or are present at a low level that is under the detection limit Ser181 [40]. Thus far, no consensus sequence has been identified by MS. Furthermore, we identified four adjacent putative O- for protein O-GlcNAcylation [41], although unstructured regions GlcNAcylation sites at the C-terminus of CARM1, indicating are preferred for O-GlcNAcylation [14]. It is likely that these that mono-O-GlcNAcylated CARM1 is probably comprised of disordered regions are transiently stabilized upon binding to OGT, a mixture of forms with modification at Ser595,Ser598,Thr601 require stabilization provided by other binding partners or both. or Thr603. Multiple O-GlcNAcylation sites located proximal to each other on a protein is a well-known phenomenon, having been observed on over 30 proteins [36,37]. For example, DRP1 (dynamin-related protein 1), a protein involving in mitochondria O-GlcNAcylation fine-tunes CARM1 function by regulating substrate fission, is O-GlcNAcylated at Thr585 and Thr586 [38]. The C- specificity terminal domain of RNA polymerase II is O-GlcNAcylated at Ser5 The C-terminus of CARM1 was shown to have autonomous and Ser7 [39]. Transcription factor C/EBPβ (CCAAT/enhancer activation function [9]. O-GlcNAcylation has been reported to binding protein (C/EBP), beta) is O-GlcNAcylated at Ser180 and affect diverse functions of protein, including enzymatic activity,

c The Authors Journal compilation c 2015 Biochemical Society O-GlcNAcylation regulates CARM1 substrate specificity 597 protein–protein interaction, DNA binding and transcriptional changes in methylation of histone H3 and PABP1 (Figure 3D). activity of transcription factors, subcellular localization and One possible explanation is that, for some CARM1 substrates, the protein stability (reviewed in [18,22–24]). For example, the C-terminus provides an essential docking platform for efficient stability of Snail1, p53 and plakoglobin protein is regulated by O- methylation. O-GlcNAcylation may alter the affinity of this GlcNAcylation [42–44]. Subcellular localization of actin-binding interaction, thus shifting substrate specificity of CARM1 towards protein cofilin, transcription factor Sp1 and neuroD1 is altered by a subpopulation of substrates. In support of this notion, O- O-GlcNAcylation [45–47]. Subcellular localization of CARM1 GlcNAcylation was shown to alter protein–protein interactions was reported to be cell-type dependent. For example, CARM1 is between many proteins, such as Sp1–Oct1 [58], Sp1–Elf1 [59] primarily localized in the cytoplasm in HEK293 cells, but is found and Stat5–CBP [60]. For more potent CARM1 substrates, such in the nucleus in MCF7 and HeLa cells [48]. Our result indicated as histone H3 and PABP1, the C-terminus of CARM1 may be that CARM1 localizes primarily in the cytoplasm in MDA-MB- dispensable for their methylation. For example, we have shown 231 cells. Further, our results showed that O-GlcNAcylation that even when CARM1 was reduced to 10% of endogenous level of CARM1 does not alter the co-activator activity, stability, in MCF7 cells, CARM1-mediated methylation of PABP1 was localization of CARM1 or the growth of breast cancer cell lines not strikingly affected [61]. Co-crystal structures of full-length (result not shown), yet it does regulate the substrate specificity of CARM1 with some substrates will help to elucidate whether the CARM1. In line with this finding, O-GlcNAcylation of CK2 also role of the C-terminus in regulating CARM1 substrate binding is regulates its substrate specificity [49]. Future work is warranted substrate specific. to identify substrates differently methylated by O-GlcNAcylated and non-GlcNAcylated CARM1 in order to better understand the functional significance of CARM1 O-GlcNAcylation. The CARM1 O-GlcNAcylation may be a sensor of cellular stress and fact that O-GlcNAcylation does not affect many aspects of metabolism status CARM1’s function implies that O-GlcNAcylation is likely to Cellular O-GlcNAcylation is known to be sensitive to various be a mechanism for fine-tuning CARM1 function. Among the stresses including glucose availability, inflammatory stimuli, three events on CARM1, two occur in the α insulin and oxidative stress, among others (reviewed in central catalytic domain, mutation of which abrogates ER - [18,19,62]). UDP–GlcNAc, situated at the nexus of the glucose, mediated transcription [50,51]. Phosphorylation at the third site 448 , nucleotide and amino acid biosynthesis pathways, is Ser enables CARM1 to directly interact with un-liganded believed to be a sensor for cellular metabolites. Moreover, diseases ERα [52]. This direct interaction is important for ligand- α with impaired metabolism such as neurodegenerative disease, independent activation of ER and possibly contributes to diabetes, cardiovascular disease and cancer often exhibit altered tamoxifen resistance. Finally, automethylation of CARM1 at 551 levels of cellular O-GlcNAcylation (reviewed in [12,21,22,62– Arg modulates CARM1-mediated exon skipping [31].Taken 65]). together, functions of CARM1 appear to be regulated by multiple A few studies have implicated CARM1 as a regulator of cellular PTMs, the majority of which are localized to the C-terminus. metabolism [5,66–69]. CARM1 was shown to be a co-activator Competition of O-GlcNAcylation and phosphorylation on serine of PPARγ [5], a key transcription factor regulating glucose and or threonine residues has been reported on some proteins. For lipid metabolism. The expression level of CARM1 in cattle example, Thr58 of c-Myc [53], Ser16 of ERβ [54], Ser452 of heat β 733 κ β [66] and subcutaneous adipose tissue [67] is sensitive to types of shock protein (Hsp90 ) [55] and Ser of I B kinase (IKK ) [56] fatty acid in diet. Moreover, CARM1 regulates expression of can be either O-GlcNAcylated or phosphorylated. However, even key enzymes in gluconeogenesis [68] and glycogen metabolism with the use of non-ergodic dissociation methods such as ECD [69]. We speculate CARM1 O-GlcNAcylation status, like ‘histone in MS, we were unable to detect phosphorylation at these O- codes’, functionally define CARM1 function in cellular stress and GlcNAcylation sites. Although we observed the labile O-GlcNAc metabolism processes (e.g. CARM1 O-GlcNAcylation level may modification on CARM1, no H3PO4 (98 Da) shift was observed respond to cell glucose levels and be part of the central hub of cell even in the presence of phosphatase inhibitor cocktails, suggesting growth signals). Determining CARM1 O-GlcNAcylation levels that phosphorylated CARM1 is below the limit of detection < in response to different cellular stimuli may provide insights into ( 5%) by top-down MS. This result agrees with previous reports this mechanism. that CARM1 phosphorylation occurs mainly in mitosis [50,51,57] or in response to specific stimuli [52] in the central domain and implies that phosphorylation is unlikely to play a significant role in modulating CARM1 O-GlcNAcylation. AUTHOR CONTRIBUTION Purin Charoensuksai and Wei Xu designed the experiments. Purin Charoensuksai The C-terminal domain of CARM1 is involved in regulation of its performed the experiments. Peter Kuhn performed MS. Lu Wang provided CARM1- methyltransferase activity knockout cells. Nathan Sherer supervised confocal immunofluorescent analysis. Purin Charoensuksai, Peter Kuhn and Wei Xu wrote the manuscript. CARM1 O-GlcNAcylation sites are mapped to the C-terminal domain which possesses an autonomous activation activity and is essential for full co-activator activity of CARM1 [9]. Mutation ACKNOWLEDGEMENTS of O-GlcNAcylation sites did not alter the ability of CARM1 to activate a few transcription factors or change the expression We thank Dr Michael R. Stallcup for providing the pM–CARM1, pM–GRIP1 and pSG5– levels of endogenous ERα target genes. We could not exclude the TIF1α plasmids and Dr Richard R. Burgess for critical reading of the manuscript. possibility that O-GlcNAcylation of CARM1 alters the regulation of other genes and other transcription factors that were not tested in our study. Although we found that O-GlcNAcylation of FUNDING CARM1 at its C-terminus altered substrate specificity (Figures 3C This work was supported by the HOPE Scholar Award [grant number W81XWYH-11-1- and 3E), mutation of O-GlcNAcylation residues did not result in 0237 (to W.X.)]; and the Royal Thai Government Scholarship (to P.C.).

c The Authors Journal compilation c 2015 Biochemical Society 598 P. Charoensuksai and others

REFERENCES 23 O’Donnell, N. (2002) Intracellular and development. Biochim. Biophys. Acta 1573, 336–345 CrossRef PubMed 1 Chen, D., Ma, H., Hong, H., Koh, S.S., Huang, S.M., Schurter, B.T., Aswad, D.W. and 24 Ozcan, S., Andrali, S.S. and Cantrell, J.E. (2010) Modulation of transcription factor Stallcup, M.R. (1999) Regulation of transcription by a protein methyltransferase. Science function by O-GlcNAc modification. Biochim. Biophys. Acta 1799, 353–364 284, 2174–2177 CrossRef PubMed CrossRef PubMed 2 Miao, F., Li, S., Chavez, V., Lanting, L. and Natarajan, R. (2006) Coactivator-associated 25 Chumanov, R.S., Kuhn, P.A., Xu, W. and Burgess, R.R. (2011) Expression and purification arginine methyltransferase-1 enhances nuclear factor-kappaB-mediated gene of full-length mouse CARM1 from transiently transfected HEK293T cells using HaloTag transcription through methylation of histone H3 at arginine 17. Mol. Endocrinol. 20, technology. Protein. Expr. Purif. 76, 145–153 CrossRef PubMed 1562–1573 CrossRef PubMed 26 Kuhn, P., Xu, Q., Cline, E., Zhang, D., Ge, Y. and Xu, W. (2009) Delineating Anopheles 3 An, W., Kim, J. and Roeder, R.G. (2004) Ordered cooperative functions of PRMT1, p300, gambiae coactivator associated arginine methyltransferase 1 automethylation using and CARM1 in transcriptional activation by p53. Cell 117, 735–748 CrossRef PubMed top-down high resolution tandem mass spectrometry. Protein Sci. 18, 1272–1280 4 Koh, S.S., Chen, D., Lee, Y.H. and Stallcup, M.R. (2001) Synergistic enhancement of CrossRef PubMed nuclear receptor function by p160 coactivators and two coactivators with protein 27 Zachara, N.E., Vosseller, K. and Hart, G.W. (2011) Detection and analysis of proteins methyltransferase activities. J. Biol. Chem. 276, 1089–1098 CrossRef PubMed modified by O-linked N-acetylglucosamine. Curr. Protoc. Protein Sci. Chapter 12, 5 Yadav, N., Cheng, D., Richard, S., Morel, M., Iyer, V.R., Aldaz, C.M. and Bedford, M.T. Unit12.18 (2008) CARM1 promotes adipocyte differentiation by coactivating PPARgamma. EMBO 28 Wang, L., Zhao, Z., Meyer, M.B., Saha, S., Yu, M., Guo, A., Wisinski, K.B., Huang, W., Rep. 9, 193–198 CrossRef PubMed Cai, W., Pike, J.W. et al. (2014) CARM1 methylates remodeling factor BAF155 6 Bauer, U.M., Daujat, S., Nielsen, S.J., Nightingale, K. and Kouzarides, T. (2002) to enhance tumor progression and metastasis. Cancer Cell 25, 21–36 CrossRef PubMed Methylation at arginine 17 of histone H3 is linked to gene activation. EMBO Rep. 3, 29 Wang, L., Charoensuksai, P., Watson, N.J., Wang, X., Zhao, Z., Coriano, C.G., Kerr, L.R. 39–44 CrossRef PubMed and Xu, W. (2013) CARM1 automethylation is controlled at the level of alternative 7 Cheng, D., Cotˆ e,´ J., Shaaban, S. and Bedford, M.T. (2007) The arginine methyltransferase splicing. Nucleic Acids Res 41, 6870–6880 CrossRef PubMed CARM1 regulates the coupling of transcription and mRNA processing. Mol. Cell 25, 30 Cheung, W.D., Sakabe, K., Housley, M.P., Dias, W.B. and Hart, G.W. (2008) O-linked 71–83 CrossRef PubMed beta-N-acetylglucosaminyltransferase substrate specificity is regulated by myosin 8 Lee, J. and Bedford, M.T. (2002) PABP1 identified as an arginine methyltransferase phosphatase targeting and other interacting proteins. J. Biol. Chem. 283, 33935–33941 substrate using high-density protein arrays. EMBO Rep. 3, 268–273 CrossRef PubMed CrossRef PubMed 9 Teyssier, C., Chen, D. and Stallcup, M.R. (2002) Requirement for multiple domains of the 31 Kuhn, P., Chumanov, R., Wang, Y., Ge, Y., Burgess, R.R. and Xu, W. (2011) protein arginine methyltransferase CARM1 in its transcriptional coactivator function. J. Automethylation of CARM1 allows coupling of transcription and mRNA splicing. Nucleic Biol. Chem. 277, 46066–46072 CrossRef PubMed Acids Res 39, 2717–2726 CrossRef PubMed 10 Teyssier, C., Ou, C.Y., Khetchoumian, K., Losson, R. and Stallcup, M.R. (2006) 32 Troffer-Charlier, N., Cura, V., Hassenboehler, P., Moras, D. and Cavarelli, J. (2007) Transcriptional intermediary factor 1alpha mediates physical interaction and functional Functional insights from structures of coactivator-associated arginine methyltransferase 1 synergy between the coactivator-associated arginine methyltransferase 1 and domains. EMBO J. 26, 4391–4401 CrossRef PubMed glucocorticoid receptor-interacting protein 1 nuclear receptor coactivators. Mol. 33 Xue, B., Dunbrack, R.L., Williams, R.W., Dunker, A.K. and Uversky, V.N. (2010) Endocrinol. 20, 1276–1286 CrossRef PubMed PONDR-FIT: a meta-predictor of intrinsically disordered amino acids. Biochim. Biophys. 11 Nandi, A., Sprung, R., Barma, D.K., Zhao, Y., Kim, S.C. and Falck, J.R. (2006) Global identification of O-GlcNAc-modified proteins. Anal. Chem. 78, 452–458 Acta 1804, 996–1010 CrossRef PubMed CrossRef PubMed 34 Yue, W.W., Hassler, M., Roe, S.M., Thompson-Vale, V. and Pearl, L.H. (2007) Insights 12 Copeland, R.J., Han, G. and Hart, G.W. (2013) O-GlcNAcomics-revealing roles of into histone code syntax from structural and biochemical studies of CARM1 O-GlcNAcylation in disease mechanisms and development of potential diagnostics. methyltransferase. EMBO J. 26, 4402–4412 CrossRef PubMed Clin. Appl., doi: 10.1002/prca.201300001 35 Wu, J. and Xu, W. (2012) Histone H3R17me2a mark recruits human RNA 13 Hahne, H., Sobotzki, N., Nyberg, T., Helm, D., Borodkin, V.S., van Aalten, D.M., Agnew, B. polymerase-associated factor 1 complex to activate transcription. Proc. Natl. Acad. Sci. and Kuster, B. (2013) Proteome wide purification and identification of O-GlcNAc-modified U.S.A. 109, 5675–5680 CrossRef PubMed proteins using click and mass spectrometry. J. Proteome Res. 12, 927–936 36 Myers, S.A., Panning, B. and Burlingame, A.L. (2011) Polycomb repressive complex 2 is CrossRef PubMed necessary for the normal site-specific O-GlcNAc distribution in mouse embryonic stem 14 Trinidad, J.C., Barkan, D.T., Gulledge, B.F., Thalhammer, A., Sali, A., Schoepfer, R. and cells. Proc. Natl. Acad. Sci. U.S.A. 108, 9490–9495 CrossRef PubMed Burlingame, A.L. (2012) Global identification and characterization of both 37 Alfaro, J.F., Gong, C.X., Monroe, M.E., Aldrich, J.T., Clauss, T.R., Purvine, S.O., Wang, Z., O-GlcNAcylation and phosphorylation at the murine synapse. Mol. Cell. Proteomics 11, Camp, D.G., Shabanowitz, J., Stanley, P.et al. (2012) Tandem mass spectrometry identifies 215–229 CrossRef PubMed many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc 15 Haltiwanger, R.S., Blomberg, M.A. and Hart, G.W. (1992) Glycosylation of nuclear and transferase targets. Proc. Natl. Acad. Sci. U.S.A. 109, 7280–7285 CrossRef PubMed cytoplasmic proteins. Purification and characterization of a uridine 38 Gawlowski, T., Suarez, J., Scott, B., Torres-Gonzalez, M., Wang, H., Schwappacher, R., diphospho-N-acetylglucosamine: polypeptide beta-N-acetylglucosaminyltransferase. J. Han, X., Yates, J.R., Hoshijima, M. and Dillmann, W. (2012) Modulation of Biol. Chem. 267, 9005–9013 PubMed dynamin-related protein 1 (DRP1) function by increased 16 Kreppel, L.K., Blomberg, M.A. and Hart, G.W. (1997) Dynamic glycosylation of nuclear O-linked-β-N-acetylglucosamine modification (O-GlcNAc) in cardiac myocytes. J. Biol. and cytosolic proteins. Cloning and characterization of a unique O-GlcNAc transferase Chem. 287, 30024–30034 CrossRef PubMed with multiple tetratricopeptide repeats. J. Biol. Chem. 272, 9308–9315 CrossRef PubMed 39 Ranuncolo, S.M., Ghosh, S., Hanover, J.A., Hart, G.W. and Lewis, B.A. (2012) Evidence of 17 Gao, Y., Wells, L., Comer, F.I., Parker, G.J. and Hart, G.W. (2001) Dynamic the involvement of O-GlcNAc-modified human RNA polymerase II CTD in transcription O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a in vitro and in vivo. J. Biol. Chem. 287, 23549–23561 CrossRef PubMed neutral, cytosolic beta-N-acetylglucosaminidase from human brain. J. Biol. Chem. 276, 40 Li, X., Molina, H., Huang, H., Zhang, Y.Y., Liu, M., Qian, S.W., Slawson, C., Dias, W.B., 9838–9845 CrossRef PubMed Pandey, A., Hart, G.W. et al. (2009) O-linked N-acetylglucosamine modification on 18 Zachara, N.E. and Hart, G.W. (2006) , the essential role of O-GlcNAc!. CCAAT enhancer-binding protein beta: role during adipocyte differentiation. J. Biol. Biochim. Biophys. Acta 1761, 599–617 CrossRef PubMed Chem. 284, 19248–19254 CrossRef PubMed 19 Zachara, N.E. and Hart, G.W. (2004) O-GlcNAc a sensor of cellular state: the role of 41 Jochmann, R., Holz, P., Sticht, H. and Sturzl,¨ M. (2014) Validation of the reliability of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition computational O-GlcNAc prediction. Biochim. Biophys. Acta 1844, 416–421 and stress. Biochim. Biophys. Acta 1673, 13–28 CrossRef PubMed CrossRef PubMed 20 Hanover, J.A., Krause, M.W. and Love, D.C. (2010) The hexosamine signaling pathway: 42 Park, S.Y., Kim, H.S., Kim, N.H., Ji, S., Cha, S.Y., Kang, J.G., Ota, I., Shimada, K., O-GlcNAc cycling in feast or famine. Biochim. Biophys. Acta 1800, 80–95 Konishi, N., Nam, H.W. et al. (2010) Snail1 is stabilized by O-GlcNAc modification in CrossRef PubMed hyperglycaemic condition. EMBO J. 29, 3787–3796 CrossRef PubMed 21 Hart, G.W., Slawson, C., Ramirez-Correa, G. and Lagerlof, O. (2011) Cross talk between 43 Hu, P., Berkowitz, P., Madden, V.J. and Rubenstein, D.S. (2006) Stabilization of O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic plakoglobin and enhanced keratinocyte cell-cell adhesion by intracellular disease. Annu. Rev. Biochem. 80, 825–858 CrossRef PubMed O-glycosylation. J. Biol. Chem. 281, 12786–12791 CrossRef PubMed 22 Hart, G.W., Housley, M.P. and Slawson, C. (2007) Cycling of O-linked 44 Yang, W.H., Kim, J.E., Nam, H.W., Ju, J.W., Kim, H.S., Kim, Y.S. and Cho, J.W. (2006) beta-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 446, 1017–1022 Modification of p53 with O-linked N-acetylglucosamine regulates p53 activity and CrossRef PubMed stability. Nat. Cell Biol. 8, 1074–1083 CrossRef PubMed

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45 Huang, X., Pan, Q., Sun, D., Chen, W., Shen, A., Huang, M., Ding, J. and Geng, M. (2013) 57 Sakabe, K. and Hart, G.W. (2010) O-GlcNAc transferase regulates mitotic chromatin O-GlcNAcylation of cofilin promotes breast cancer cell invasion. J. Biol. Chem. 288, dynamics. J. Biol. Chem. 285, 34460–34468 CrossRef PubMed 36418–36425 CrossRef PubMed 58 Lim, K. and Chang, H.I. (2009) O-GlcNAc modification of Sp1 inhibits the functional 46 Dauphinee, S.M., Ma, M. and Too, C.K. (2005) Role of O-linked interaction between Sp1 and Oct1. FEBS Lett. 583, 512–520 beta-N-acetylglucosamine modification in the subcellular distribution of alpha4 CrossRef PubMed phosphoprotein and Sp1 in rat lymphoma cells. J. Cell Biochem. 96, 579–588 59 Lim, K. and Chang, H.I. (2009) O-GlcNAc inhibits interaction between Sp1 and Elf-1 CrossRef PubMed transcription factors. Biochem. Biophys. Res. Commun. 380, 569–574 47 Andrali, S.S., Qian, Q. and Ozcan, S. (2007) Glucose mediates the translocation of CrossRef PubMed NeuroD1 by O-linked glycosylation. J. Biol. Chem. 282, 15589–15596 CrossRef PubMed 60 Gewinner, C., Hart, G., Zachara, N., Cole, R., Beisenherz-Huss, C. and Groner, B. (2004) 48 Herrmann, F., Pably, P., Eckerich, C., Bedford, M.T. and Fackelmayer, F.O. (2009) Human The coactivator of transcription CREB-binding protein interacts preferentially with the protein arginine in vivo–distinct properties of eight canonical glycosylated form of Stat5. J. Biol. Chem. 279, 3563–3572 CrossRef PubMed members of the PRMT family. J. Cell Sci. 122, 667–677 CrossRef PubMed 61 Zeng, H., Wu, J., Bedford, M.T., Sbardella, G., Hoffmann, F.M., Bi, K. and Xu, W. (2013) A 49 Tarrant, M.K., Rho, H.S., Xie, Z., Jiang, Y.L., Gross, C., Culhane, J.C., Yan, G., Qian, J., TR-FRET-based functional assay for screening activators of CARM1. Chembiochem 14, Ichikawa, Y., Matsuoka, T. et al. (2012) Regulation of CK2 by phosphorylation and 827–835 CrossRef PubMed O-GlcNAcylation revealed by semisynthesis. Nat. Chem. Biol. 8, 262–269 62 Slawson, C., Copeland, R.J. and Hart, G.W. (2010) O-GlcNAc signaling: a metabolic link CrossRef PubMed between diabetes and cancer? Trends Biochem. Sci. 35, 547–555 50 Higashimoto, K., Kuhn, P., Desai, D., Cheng, X. and Xu, W. (2007) CrossRef PubMed Phosphorylation-mediated inactivation of coactivator-associated arginine 63 Slawson, C. and Hart, G.W. (2011) O-GlcNAc signalling: implications for cancer cell methyltransferase 1. Proc. Natl. Acad. Sci. U.S.A. 104, 12318–12323 CrossRef PubMed . Nat. Rev. Cancer 11, 678–684 CrossRef PubMed 51 Feng, Q., He, B., Jung, S.Y., Song, Y., Qin, J., Tsai, S.Y., Tsai, M.J. and O’Malley, B.W. 64 Lynch, T.P. and Reginato, M.J. (2011) O-GlcNAc transferase: a sweet new cancer target. (2009) Biochemical control of CARM1 enzymatic activity by phosphorylation. J. Biol. Cell Cycle 10, 1712–1713 CrossRef PubMed Chem. 284, 36167–36174 CrossRef PubMed 65 Fardini, Y., Dehennaut, V., Lefebvre, T. and Issad, T. (2013) O-GlcNAcylation: a new cancer 52 Carascossa, S., Dudek, P., Cenni, B., Briand, P.A. and Picard, D. (2010) CARM1 mediates hallmark? Front Endocrinol. 4,99CrossRef the ligand-independent and tamoxifen-resistant activation of the estrogen receptor alpha 66 Akbar, H., Schmitt, E., Ballou, M.A., Correa,ˆ M.N., Depeters, E.J. and Loor, J.J. (2013) by cAMP. Genes Dev. 24, 708–719 CrossRef PubMed Dietary lipid during late-pregnancy and early-lactation to manipulate metabolic and 53 Chou, T.Y., Hart, G.W. and Dang, C.V. (1995) c-Myc is glycosylated at threonine 58, a inflammatory gene network expression in dairy cattle liver with a focus on PPARs. Gene known phosphorylation site and a mutational hot spot in lymphomas. J. Biol. Chem. 270, Regul. Syst. Bio. 7, 103–123 PubMed 18961–18965 CrossRef PubMed 67 Schmitt, E., Ballou, M.A., Correa, M.N., DePeters, E.J., Drackley, J.K. and Loor, J.J. 54 Cheng, X., Cole, R.N., Zaia, J. and Hart, G.W. (2000) Alternative (2011) Dietary lipid during the transition period to manipulate subcutaneous adipose O-glycosylation/O-phosphorylation of the murine estrogen receptor beta. tissue peroxisome proliferator-activated receptor-γ co-regulator and target gene 39, 11609–11620 CrossRef PubMed expression. J. Dairy Sci. 94, 5913–5925 CrossRef PubMed 55 Overath, T., Kuckelkorn, U., Henklein, P., Strehl, B., Bonar, D., Kloss, A., Siele, D., 68 Krones-Herzig, A., Mesaros, A., Metzger, D., Ziegler, A., Lemke, U., Bruning,¨ J.C. and Kloetzel, P.M. and Janek, K. (2012) Mapping of O-GlcNAc sites of 20 S proteasome Herzig, S. (2006) Signal-dependent control of gluconeogenic key genes through subunits and Hsp90 by a novel biotin-cystamine tag. Mol. Cell. Proteomics 11, 467–477 coactivator-associated arginine methyltransferase 1. J. Biol. Chem. 281, 3025–3029 CrossRef PubMed CrossRef PubMed 56 Kawauchi, K., Araki, K., Tobiume, K. and Tanaka, N. (2009) Loss of p53 enhances catalytic 69 Wang, S.C., Dowhan, D.H., Eriksson, N.A. and Muscat, G.E. (2012) CARM1/PRMT4 is activity of IKKbeta through O-linked beta-N-acetyl glucosamine modification. Proc. Natl. necessary for the glycogen gene expression programme in skeletal muscle cells. Acad. Sci. U.S.A. 106, 3431–3436 CrossRef PubMed Biochem. J. 444, 323–331 CrossRef PubMed

Received 18 August 2014/5 January 2015; accepted 13 January 2015 Published as BJ Immediate Publication 13 January 2015, doi:10.1042/BJ20141072

c The Authors Journal compilation c 2015 Biochemical Society