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Characterization of the Histone Acetyltransferase (HAT) Domain of a Bifunctional Protein with Activable O-GlcNAcase and HAT Activities

2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês

10.1074/jbc.m410406200

1083-351X

Clifford A. Toleman, Andrew J. Paterson, Thomas R. Whisenhunt, Jeffrey E. Kudlow,

Peptidase Inhibition and Analysis

Histones and transcription factors are regulated by a number of post-translational modifications that in turn regulate the transcriptional activity of genes. These modifications occur in large, multisubunit complexes. We have reported previously that mSin3A can recruit O-GlcNAc transferase (OGT) along with histone deacetylase into such a corepressor complex. This physical association allows OGT to act cooperatively with histone deacetylation in gene repression by catalyzing the O-GlcNAc modification on specific transcription factors to inhibit their activity. For rapid, reversible gene regulation, the enzymes responsible for the converse reactions must be present. Here, we report that O-GlcNAcase, which is responsible for the removal of O-GlcNAc additions on nuclear and cytosolic proteins, possesses intrinsic histone acetyltransferase (HAT) activity in vitro. Free as well as reconstituted nucleosomal histones are substrates of this bifunctional enzyme. This protein, now termed NCOAT (nuclear cytoplasmic O-GlcNAcase and acetyltransferase) has a typical HAT domain that has both active and inactive states. This finding demonstrates that NCOAT may be regulated to reduce the state of glycosylation of transcriptional activators while increasing the acetylation of histones to allow for the concerted activation of eukaryotic gene transcription. Histones and transcription factors are regulated by a number of post-translational modifications that in turn regulate the transcriptional activity of genes. These modifications occur in large, multisubunit complexes. We have reported previously that mSin3A can recruit O-GlcNAc transferase (OGT) along with histone deacetylase into such a corepressor complex. This physical association allows OGT to act cooperatively with histone deacetylation in gene repression by catalyzing the O-GlcNAc modification on specific transcription factors to inhibit their activity. For rapid, reversible gene regulation, the enzymes responsible for the converse reactions must be present. Here, we report that O-GlcNAcase, which is responsible for the removal of O-GlcNAc additions on nuclear and cytosolic proteins, possesses intrinsic histone acetyltransferase (HAT) activity in vitro. Free as well as reconstituted nucleosomal histones are substrates of this bifunctional enzyme. This protein, now termed NCOAT (nuclear cytoplasmic O-GlcNAcase and acetyltransferase) has a typical HAT domain that has both active and inactive states. This finding demonstrates that NCOAT may be regulated to reduce the state of glycosylation of transcriptional activators while increasing the acetylation of histones to allow for the concerted activation of eukaryotic gene transcription. The genomes of eukaryotes are assembled into the highly condensed structure of chromatin. Chromatin is composed of repeating units of nucleosomes that are comprised of DNA coiled around an octameric particle consisting of two molecules of each core histone, H2A, H2B, H3, and H4 (1Wolffe A.P. Chromatin: Structure and Function. Academic Press, London, UK1992Google Scholar, 2Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar). The acetylation state of these histone protein tails has for some time been known to be correlated with gene expression, where transcriptionally competent loci are hyperacetylated and silenced loci hypoacetylated (3Turner B.M. O'Neill L.P. Semin. Cell Biol. 1995; 6: 229-236Crossref PubMed Scopus (134) Google Scholar, 4Tse C. Sera T. Wolffe A.P. Hansen J.C. Mol. Cell. Biol. 1998; 18: 4629-4638Crossref PubMed Scopus (478) Google Scholar, 5Richards E.J. Elgin S.C. Cell. 2002; 108: 489-500Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar). Histone acetyltransferases (HATs) 1The abbreviations used are: HAT, histone acetyltransferase; AT, acetyltransferase; HDAC, histone deacetylase; OGT, O-GlcNAc transferase; CBP, CREB-binding protein; CREB, cAMP-responsive element-binding protein; NCOAT, nuclear cytoplasmic O-GlcNAcase and acetyltransferase; bNCOAT, bacterial NCOAT; mNCOAT, mammalian NCOAT; GST, glutathione S-transferase; TPR, tetratricopeptide; STZ, streptozotocin.1The abbreviations used are: HAT, histone acetyltransferase; AT, acetyltransferase; HDAC, histone deacetylase; OGT, O-GlcNAc transferase; CBP, CREB-binding protein; CREB, cAMP-responsive element-binding protein; NCOAT, nuclear cytoplasmic O-GlcNAcase and acetyltransferase; bNCOAT, bacterial NCOAT; mNCOAT, mammalian NCOAT; GST, glutathione S-transferase; TPR, tetratricopeptide; STZ, streptozotocin. compose a superfamily of enzymes broken into subfamilies based on sequence similarities, active site size, and the presence or absence of other protein domains (6Marmorstein R. J. Mol. Biol. 2001; 311: 433-444Crossref PubMed Scopus (134) Google Scholar). In each HAT protein there exists a structurally homologous region that composes the active site and includes four universally present motifs, designated A-D, that form the scaffold of the catalytic core (6Marmorstein R. J. Mol. Biol. 2001; 311: 433-444Crossref PubMed Scopus (134) Google Scholar). Recently published three-dimensional structures of various acetyltransferases (ATs) has allowed for a comprehensive view of the roles played by each motif as well as the roles of many amino acids structurally conserved therein (6Marmorstein R. J. Mol. Biol. 2001; 311: 433-444Crossref PubMed Scopus (134) Google Scholar, 7Wolf E. Vassilev A. Makino Y. Sali A. Nakatani Y. Burley S.K. Cell. 1998; 94: 439-449Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 8Angus-Hill M.L. Dutnall R.N. Tafrov S.T. Sternglanz R. Ramakrishnan V. J. Mol. Biol. 1999; 294: 1311-1325Crossref PubMed Scopus (100) Google Scholar). HATs and histone deacetylases (HDACs) act competitively within large multiprotein complexes that recruit them to their nucleosomal substrates on DNA and give them the ability to contribute to the activation or repression of gene expression, respectively (9Hassig C.A. Fleischer T.C. Billin A.N. Schreiber S.L. Ayer D.E. Cell. 1997; 89: 341-347Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar).The covalent addition of the monosaccharide, N-acetylglucosamine (GlcNAc) to serine or threonine residues of proteins is catalyzed by the enzyme, O-GlcNAc transferase (OGT) that is encoded by a single gene (10Shafi R. Iyer S.P. Ellies L.G. O'Donnell N. Marek K.W. Chui D. Hart G.W. Marth J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5735-5739Crossref PubMed Scopus (583) Google Scholar). Recently, we reported that the corepressor, mSin3A, known to recruit HDAC (9Hassig C.A. Fleischer T.C. Billin A.N. Schreiber S.L. Ayer D.E. Cell. 1997; 89: 341-347Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, 11Zhang Y. Iratni R. Erdjument-Bromage H. Tempst P. Reinberg D. Cell. 1997; 89: 357-364Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar), also recruits OGT via its TPR domains to specific genes (12Yang X. Zhang F. Kudlow J.E. Cell. 2002; 110: 69-80Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). OGT can thereby contribute along with HDAC to the repression of gene expression through addition of O-GlcNAc modifications on transcriptional activators, inhibiting their activity. Such inhibitory effects have been witnessed on the transactivation domain of Sp1 (13Yang X. Su K. Roos M.D. Chang Q. Paterson A.J. Kudlow J.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6611-6616Crossref PubMed Scopus (233) Google Scholar), the C-terminal tail of RNA polymerase II (14Kelly W.G. Dahmus M.E. Hart G.W. J. Biol. Chem. 1993; 268: 10416-10424Abstract Full Text PDF PubMed Google Scholar) and the TAFII130 recruitment domain of CREB (15Lamarre-Vincent N. Hsieh-Wilson L.C. J. Am. Chem. Soc. 2003; 125: 6612-6613Crossref PubMed Scopus (89) Google Scholar) among others. It has also been documented that the repression of genes is associated with the hyperglycosylation of the proteins bound to their promoters (12Yang X. Zhang F. Kudlow J.E. Cell. 2002; 110: 69-80Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar).Many genes, including those responsive to variable hormone levels, can be activated or repressed to maintain homeostasis. For the repressed gene state to be reversible, the complex responsible for the repressed state must be exchanged with a complex that allows gene activation (16Perissi V. Aggarwal A. Glass C.K. Rose D.W. Rosenfeld M.G. Cell. 2004; 116: 511-526Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 17Li X. Oghi K.A. Zhang J. Krones A. Bush K.T. Glass C.K. Nigam S.K. Aggarwal A.K. Maas R. Rose D.W. Rosenfeld M.G. Nature. 2003; 426: 247-254Crossref PubMed Scopus (501) Google Scholar). As part of the activation process, the action of the enzymes residing in the repression complex would need to be removed, including the inhibitory modification of many transcriptional activators by O-GlcNAc. The enzyme O-GlcNAcase, which is the only enzyme capable of catalyzing the removal of these regulatory O-GlcNAc modifications on proteins in the nucleus and cytosol, must necessarily then play a role in cooperation with HATs in the activation of genes, just as OGT plays its role along with HDAC in repression (12Yang X. Zhang F. Kudlow J.E. Cell. 2002; 110: 69-80Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Of interest in this regard, it has recently been reported that the C terminus of O-GlcNAcase contains a domain with similar composition to eleven different AT active sites, as predicted by SMART computer analysis (18Comtesse N. Maldener E. Meese E. Biochem. Biophys. Res. Commun. 2001; 283: 634-640Crossref PubMed Scopus (137) Google Scholar). While it has been argued that the O-GlcNAcase may have evolved from an AT (19Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (2983) Google Scholar), this protein could potentially play a dual role in the reversibility of corepression by removing O-GlcNAc modification from activators while also adding acetyl groups to histones, allowing a target gene to be expressed. Here we report that O-GlcNAcase does in fact possess acetyltransferase activity in vitro for a synthetic histone substrate tail as well as for free core histones and reconstituted oligonucleosome substrates. However, the HAT activity is regulated and can only be observed when the enzyme is expressed in mammalian cells. The active site for this domain lies in the C terminus of the protein, where it resembles other acetyltransferases both structurally and in catalytic mechanism and has complete functional distinction from the N-terminal O-GlcNAcase domain. Because the enzyme is bifunctional with two important enzymatic domains, we have renamed it nuclear cytoplasmic O-GlcNAcase and acetyl transferase (NCOAT).EXPERIMENTAL PROCEDURESPlasmids and Recombinant NCOAT—A pUC118-pTM hybrid expression vector was designed by removing a 2-kb fragment of pTM containing T7 and GST elements and inserting it into the pUC118 MCS. This construct was used for GST peptide expression. A PCR product containing full-length mouse NCOAT was then cloned into the pUC118-pTM hybrid for N-terminal GST fusions or into pcDNA 3.1 (Invitrogen) for GAL4 fusions. Mouse NCOAT was also inserted into a pGEX vector (Amersham Biosciences) for expression in bacteria (bNCOAT). All recombinant DNA manipulations were performed by standard procedures (20Ausubel J. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 1. Greene Publishing and Wiley Interscience, New York1990: 2.0.1-3.19.8Google Scholar).Splice Variant Identification and Cloning—NCOATs were cloned by reverse transcription-PCR from Goto Kakazaki or Sprague-Dawley rat brains. The full-length and variant isoforms were cloned into pBlue-script and sequenced. The full-length rat NCOAT was very similar to the mouse NCOAT at the nucleotide level, and therefore the 3′-end of the mouse cDNA was replaced with the 3′-end of the respective Goto Kakazaki or Sprague-Dawley rat cDNA to create chimeric NCOAT molecules with the missing exons. These constructs were cloned into the pUC-pTM vector.Truncations and Site-directed Mutagenesis—Full-length pcDNA-NCOAT was digested with EcoRI to excise a fragment that consisted of nucleotides 1-1747 of NCOAT. This digested plasmid was religated onto itself for use as an expression vector for a GAL4 fusion to the NCOAT acetyltransferase domain (NCOAT nucleotides 1748-2771). The AT domain fusion was also placed into the pGEX vector for bacterial expression. For the expression of NCOAT with a deletion of its AT domain, the pcDNA-GAL4-NCOAT vector was transformed into DM-1 cells (Invitrogen) then the plasmid was digested with PmeI and PflMI. The resulting vector, missing NCOAT nucleotides 2086-2771 was blunt-end-ligated. For point mutations, the pUC118-pTM-NCOAT construct was modified by site-directed mutagenesis using a four-primer cassette strategy to introduce the substitution (mutated residues underlined) and sites for selection (bold). Standard PCR amplifications were performed with the following oligonucleotides: for D853N, 5′-GATAT CCATAAAAAAGTGACTGACCCGAGTGTTGCC-3′; for D884N, 5′-CTGTGAAGTAAGACCAGATAATAAAAGGAT CCTGG-3′; for Y891F, 5′-GGATTCTGGAATTTTTCAGCAAG CT TGGCTG-3′. Flanking oligonucleotides were as follows: 5′-CATGGATCCAGTCAACCTGACCTTATTGG-3′ (upstream) and 5′-ACGCCAAGCTCGAAATTAACC-3′ (downstream). The products were separated by electrophoresis, extracted, and purified by QIAquick extraction kit (Qiagen). The purified products were digested with Bsu36I and KpnI and ligated into pUC118-pTM. Each ligation mixture was transformed into DH5α cells (Invitrogen) and screened for the selection sites.Cell Culture and Bacterial Expression—BSC-40 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal bovine serum (Atlanta Biologicals) with 0.2 mg/ml penicillin and 0.4 mg/ml gentamicin according to standard procedures. 20 μg of plasmid DNA were transfected by electroporation, incubated overnight, and then infected with VTF-7 vaccinia virus. Cells were harvested after overnight incubation in 0.5% Nonidet P-40 lysis buffer containing 50 mm Tris-HCl, 0.5 m NaCl, 20% glycerol (v/w), 5 mm MgCl2, 0.2 mm EDTA, 5 mm dithiothreitol, pH 8.0. Bacterial protein expression was achieved by transforming a 500-ml culture of BL-21 Escherichia coli in Terrific Broth (BD Biosciences) with pGEX-mNCOAT and expression induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside (final). After 1 h, 1 mm benzamidine (final) was added, and incubation was continued another 2 h. Cultured cells were spun down and resuspended in Nonidet P-40 lysis buffer with lysozyme. Lysed cells were sonicated and spun down, and supernatant was collected in 500-μl aliquots and stored at -80 °C until use.Affinity Purification and Immunoprecipitation—GST or GST fusion proteins were purified from lysates using glutathione-Sepharose (Amersham Biosciences) and stringently washed three times with 5 volumes of radioimmune precipitation assay buffer. Proteins were eluted for filter binding assays and in assays with oligonucleosomes in gel slices. The fusion proteins were left resin-bound for pretreatment with streptozotocin (STZ) or when the proteins were resolved on SDS-PAGE. Elutions were carried out according to manufacturer's protocol. Immunoprecipitation of GAL4 and GAL4 fusion proteins were performed by preclearing lysates with 20 μl of a 50:50 protein A:protein G-Sepharose bead mixture (Amersham Biosciences). Precleared lysates were then incubated with an N-19 GAL4 DNA binding domain antibody (Santa Cruz Biotechnology) for 1 h at 4 °C and then with a 100-μl mixture of 50:50 bead mixture for an additional 2 h. Immunoprecipitates were collected by centrifugation and washed. An aliquot of each purified enzyme was quantitated by Bio-Rad protein assay to ensure equivalent amounts of protein were used in each assay and were comparable in concentration to the positive controls. Bacterial O-GlcNAcase proteins were also run on SDS-PAGE and Western blotted with α-GST according to standard protocols. Resin-bound incubations with mammalian whole cell lysates were carried out for 1 h at 4 °C and then washed.Oligonucleosome Reconstitution—Histone octamers were assembled as described by Dyer et al. (21Dyer P.N. Edayathumangalam R.S. White C.L. Bao Y. Chakravarthy S. Muthurajan U.M. Luger K. Methods Enzymol. 2004; 375: 23-44Crossref PubMed Scopus (523) Google Scholar) using unfractionated type IIA calf thymus whole histones (Sigma). Briefly, these histones were dissolved in 4 ml of unfolding buffer (6 m guanidine HCl) at a concentration of 2 mg/ml for 2 h at room temperature then dialyzed against 3 changes of refolding buffer (2 m NaCl, 10 mm Tris-HCl, pH 7.5, 1 mm EDTA, 5 mm dithiothreitol) at 4 °C for 6 h, overnight, and 6 h. Precipitated protein was removed, and the histone mixture was concentrated in a YM-10 protein concentrator (Amicon) down to 1 ml. Octamer reconstitution was confirmed by non-denaturing gel electrophoresis. Nucleosomal DNA was prepared using 186-bp HeLa cell α-satellite DNA as described by Tanaka et al. (22Tanaka Y. Tawaramoto-Sasanuma M. Kawaguchi S. Ohta T. Yoda K. Kurumizaka H. Yokoyama S. Methods. 2004; 33: 3-11Crossref PubMed Scopus (134) Google Scholar). Finally, oligonucleosomes were reconstituted using the standard salt dialysis method as described by Dyer et al. (21Dyer P.N. Edayathumangalam R.S. White C.L. Bao Y. Chakravarthy S. Muthurajan U.M. Luger K. Methods Enzymol. 2004; 375: 23-44Crossref PubMed Scopus (523) Google Scholar) using equal concentrations of the histone mixture and DNA. Reconstitution was confirmed by mobility shift assay using 15 μg of nucleosome particles in both 0.7% agarose gel and 5% polyacrylamide gel as described elsewhere (21Dyer P.N. Edayathumangalam R.S. White C.L. Bao Y. Chakravarthy S. Muthurajan U.M. Luger K. Methods Enzymol. 2004; 375: 23-44Crossref PubMed Scopus (523) Google Scholar, 22Tanaka Y. Tawaramoto-Sasanuma M. Kawaguchi S. Ohta T. Yoda K. Kurumizaka H. Yokoyama S. Methods. 2004; 33: 3-11Crossref PubMed Scopus (134) Google Scholar). Shifted bands were excised from these gels to ensure only nucleosomal histones were used. These gel fragments were minced and diluted in HAT buffer for 1 h. These solutions were used in the oligonucleosomal HAT assays.HAT and O-GlcNAcase Assays—Filter binding assays were performed as described elsewhere (23Brownell J.E. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6364-6368Crossref PubMed Scopus (238) Google Scholar, 24Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1523) Google Scholar). 60 μl of purified enzyme were added to 20 μg of synthetic histone H4 peptide (Upstate Biotechnology), 100 μl of 5 mg/ml bovine serum albumin, 29 μl of 10× HAT buffer (50 mm Tris-HCl, pH 8.0, 10% glycerol, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 mm sodium butyrate), and 3 μl of [3H]acetyl coenzyme A (4.7 mCi/mmol). Reactions were carried out for 1 h at room temperature and then spotted onto 0.45-μm nitrocellulose membranes (Whatman), dried, and unincorporated [3H]removed with three 15-min washes using 50 mm Na2CO3, pH 9.2. Dried filters were then suspended in scintillation fluid, and acetyl incorporation was measured by scintillation counter. HAT assays to be resolved on SDS-PAGE were performed as above except purified proteins were left resin-bound in 120 μl of 1× HAT buffer, and 2 μlof[14C]acetyl-CoA (50 mCi/mmol) were used. Calf thymus histones were reconstituted in 1× HAT buffer and 20 μg used per reaction. 40 μl of each reaction were run on a 15% SDS-PAGE and radiolabeling monitored by autoradiography. Where noted, CBP (Pierce) or p300 HAT domain (Upstate Biotechnology) were used as positive controls. Streptozotocin (Sigma) was prepared as a 1 m fresh solution in 1× HAT buffer. 6 μl were added to each 60-μl solution of resin-bound protein and incubated for 1 h at 37 °C. STZ was removed with three washes using excess 1× HAT buffer before addition of HAT assay components. STZ treatment before hexosaminidase assays was performed as described elsewhere (25Roos M.D. Xie W. Su K. Clark J.A. Yang X. Chin E. Paterson A.J. Kudlow J.E. Proc. Assoc. Am. Physicians. 1998; 110: 422-432PubMed Google Scholar). HAT assays on oligonucleosome substrates were performed as described by Tse et al. (4Tse C. Sera T. Wolffe A.P. Hansen J.C. Mol. Cell. Biol. 1998; 18: 4629-4638Crossref PubMed Scopus (478) Google Scholar) with the following exceptions: minced gel fragments (15 μg) were incubated with eluted O-GlcNAcase in the presence of either 10 μm acetyl-CoA or 2 μl of [14C]acetyl-CoA (50 mCi/mmol) in assays to be resolved by Western blot or autoradiograph, respectively. Final assay volumes were brought up to 70 μl in 1× HAT buffer. O-GlcNAcase assays were carried out as described elsewhere (26Dong D.L. Hart G.W. J. Biol. Chem. 1994; 269: 19321-19330Abstract Full Text PDF PubMed Google Scholar). Caspase 3 (R&D Systems) reactions were carried out as described by Hart and co-workers (27Wells L. Gao Y. Mahoney J.A. Vosseller K. Chen C. Rosen A. Hart G.W. J. Biol. Chem. 2002; 277: 1755-1761Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar).Secondary Structure Prediction—Structural Classification of Protein (SCOP) analysis was used to reveal the presence of the mixed beta sheet motif associated with the acyl-CoA N-acyltransferase (Nat) superfamily of proteins within each NCOAT sequence indicated (19Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (2983) Google Scholar). This region was then subjected to secondary structure prediction by Jpred (28Cuff J.A. Clamp M.E. Siddiqui A.S. Finlay M. Barton G.J. Bioinformatics. 1998; 14: 892-893Crossref PubMed Scopus (916) Google Scholar). The presence of each α-helix and β-sheet was confirmed individually using CHOFAS secondary structure prediction (version 2.0u61) (29Chou P.Y. Fasman G.D. Biochemistry. 1974; 13: 222-245Crossref PubMed Scopus (2530) Google Scholar).RESULTSO-GlcNAcase Possesses Intrinsic Histone Acetyltransferase Activity in Vitro—To determine whether O-GlcNAcase possessed acetyltransferase activity, recombinant GST-tagged O-GlcNAcase was expressed in both mammalian BSC-40 cells and E. coli BL-21 cells and affinity-purified. The purity of the protein samples were determined by Coomassie-plus staining (Fig. 1A). GST-O-GlcNAcase was visible as a dominant band at 130 kDa. There were other slightly detectable products copurified, most of which were determined by Western blot to be truncated GST-O-GlcNAcase products. Eluted protein was incubated with a synthetic histone H4 tail and [3H]acetyl-CoA for a filter binding assay or with core histones and [14C]acetyl-CoA for resolution on 15% SDS-PAGE and detection by autoradiography. These results were directly compared with those for the global coactivators CBP or p300. The HAT activity, as measured by scintillation counting, of the O-GlcNAcase expressed in mammalian cells, but not that expressed in E. coli, was comparable with that obtained when using CBP. The activity of equimolar amounts of CBP and O-GlcNAcase were comparable on the histone H4 tail substrate (Fig. 1B) under dose and time conditions determined to be within the linear range for enzyme activity (data not shown). The counts obtained in this assay were determined to be only those for 3H incorporation into the histone substrate, since O-GlcNAcase could neither acetylate itself (Fig. 1B, lane 5) nor the bovine serum albumin in the reaction mixture (lane 6), both of which would adhere as equally as the histone substrate to the nitrocellulose membrane. The contingency that a separate, distinct HAT, which may copurify with O-GlcNAcase, may be responsible for this activity seems unlikely, given the relative purity determined in Fig. 1A. However, to fully ensure that the trace amount of copurified products observable in Fig. 1A were not responsible for HAT activity, we expressed a protein containing a single point mutation that abolishes HAT activity and ran this sample side by side with wild type active O-GlcNAcase. As shown in Fig. 1A, the copurified bands were identical in each case, however the Tyr891 → Phe mutation, a well characterized residue involved in catalysis in other HATs (see below), resulted in an absence of HAT activity. In no known cases has this residue been shown to be involved in protein-protein binding. Because we were in the linear range for enzyme activity, the specific activity can be determined, which would necessitate that the observed small quantities of impurity would have a specific activity much higher, by orders of magnitude, than CBP to accumulate acetates on the histone substrate. Conversely, the O-GlcNAcase protein itself, which possesses similar specific activity to CBP and shares a HAT motif with the protein (see below) must be the source of the HAT activity. The bifunctionality of the protein as an O-GlcNAcase (30Gao Y. Wells L. Comer F.I. Parker G.J. Hart G.W. J. Biol. Chem. 2001; 276: 9838-9845Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar) and an acetyltransferase prompted us to change the name of the protein to NCOAT, giving no priority to either activity.Similar results between NCOAT and p300/CBP are also seen when visualized by SDS-PAGE using free histones as substrates. Labeling of all four core histones is detectable when NCOAT is used in this assay just as seen for p300 (Fig. 1E). NCOAT actually appears to have a greater efficiency in labeling the histones when compared with p300 in this assay. This greater activity may perhaps be due to the ability of NCOAT to acetylate a greater number of sites on the histones or can be a function of the fact that only the HAT domain of p300 was used in these assays, whereas the full-length p300 may possess greater activity. No HAT activity was measured when using NCOAT expressed in bacteria. The results in either assay, when NCOAT was expressed in bacteria, were similar to those seen when histone substrate was incubated with either GST or in the absence of any enzyme, a finding that may be indicative of why O-GlcNAcase/NCOAT may not have been discovered to have HAT activity to date.These results indicate that NCOAT, when expressed in a mammalian system, possesses acetyltransferase activity for both a synthetic histone substrate and for free core histones. The total lack of activity of the bacterially expressed NCOAT is the likely result of the absence of an as yet uncharacterized post-translational modification(s) that is present on the material expressed in mammalian cells. This likelihood for the mammalian enzyme can be demonstrated when the bacterially expressed enzyme is incubated with a mammalian whole cell lysate prior to the activity assay. After such treatment on resin followed by washing in radioimmune precipitation assay buffer, full HAT activity can be observed in this protein (Fig. 1B, lane 8). The hexosaminidase activity of the enzyme was also considerably less when the full-length protein was expressed in bacteria compared with mammalian cells, although it was not completely absent as seen in the HAT assay. The hexosaminidase activity of the bacterial protein similarly increased to the level of mammalian-expressed protein following incubation with mammalian cell extract (Fig. 1D).NCOAT Can Acetylate Oligonucleosomal Substrates—HAT proteins are classically described as being either type A, those that acetylate histones within chromatin, or type B, those that acetylate free histones within the cytoplasm. For NCOAT to act as theorized on gene transcription directly, it would need to act as a type A HAT. As described above, NCOAT, like other type B HATs, can acetylate free core histones. We next tested its ability to act upon nucleosomal histones. For this, histones were reconstitituted into oligonucleosome arrays for use as substrates in the HAT assays. Successful formation of these substrates was determined by gel shift assay (Fig. 2A). The shifted oligonucleosomes were purified from these gels and incubated with p300 or NCOAT in the HAT assay and run on 15% SDS-PAGE. NCOAT acetylated all four core histones even when bound by double-stranded DNA in the context of reconstituted nucleosomes (Fig. 2B). Again, mNCOAT and bNCOAT incubated with mammalian whole cell extract were active, while bNCOAT not preincubated with extract was not active. These HAT assays were also repeated using 10 μm unlabeled acetyl-CoA. Western blots of the subsequent gels were then probed with antibodies for specific acetylated lysine residues to determine some of the modified residues. Lysine 14 on histone 3 was efficiently acetylated by NCOAT in these assays (Fig. 2C). Lysine 8 on histone 4 was also acetylated by NCOAT under the same conditions (Fig. 2D). In both cases, mNCOAT was active, while bNCOAT required exposure to mammalian whole cell extract. When these reactions were probed with an antibody to lysine 16 on histone 4, we were unable to detect any acetylation of this particular residue by NCOAT (data not shown). While other modified lysine residues have not been mapped, these data suggest that NCOAT is a type A HAT capable of modifying the histones within chromatin. While we cannot conclude at this time how promiscuous NCOAT is as an acetyltransferase, it is likely to have specific targets in the context of nucleosomes, as do all other previously characterized HATs. Further selectivity is likely provided for by the complex(es) in which NCOAT is bound at the promoter.Fig. 2NCOAT has the ability to acetylate oligonucleosomal substrates. A, gel mobility shift assay. 186-bp α-sate