Site-specific Acetylation of the Fetal Globin Activator NF-E4 Prevents Its Ubiquitination and Regulates Its Interaction with the Histone Deacetylase, HDAC1

Acetylation provides one mechanism by which the functional diversity of individual transcription factors can be expanded. This is valuable in the setting of complex multigene loci that are regulated by a limited number of proteins, such as the human β-globin locus. We have studied the role of acetylation in the regulation of the transcription factor NF-E4, a component of a protein complex that facilitates the preferential expression of the human γ-globin genes in fetal erythroid cells. We have shown that NF-E4 interacts directly with, and serves as a substrate for, the acetyltransferase co-activator PCAF. Acetylation of NF-E4 is restricted to a single residue (Lys43) in the amino-terminal domain of the protein and results in two important functional consequences. Acetylation of NF-E4 prolongs the protein half-life by preventing ubiquitin-mediated degradation. This stabilization is PCAF-dependent, since enforced expression in fetal/erythroid cells of a mutant form of PCAF lacking the histone acetyltransferase domain (PCAFΔHAT) decreases NF-E4 stability. Acetylation of Lys43 also reduces the interaction between NF-E4 and HDAC1, potentially maximizing the activating ability of the factor at the γ-promoter. These results provide further demonstration that co-activators, such as PCAF, can influence individual transcription factor properties at multiple levels to alter their effects on gene expression. Acetylation provides one mechanism by which the functional diversity of individual transcription factors can be expanded. This is valuable in the setting of complex multigene loci that are regulated by a limited number of proteins, such as the human β-globin locus. We have studied the role of acetylation in the regulation of the transcription factor NF-E4, a component of a protein complex that facilitates the preferential expression of the human γ-globin genes in fetal erythroid cells. We have shown that NF-E4 interacts directly with, and serves as a substrate for, the acetyltransferase co-activator PCAF. Acetylation of NF-E4 is restricted to a single residue (Lys43) in the amino-terminal domain of the protein and results in two important functional consequences. Acetylation of NF-E4 prolongs the protein half-life by preventing ubiquitin-mediated degradation. This stabilization is PCAF-dependent, since enforced expression in fetal/erythroid cells of a mutant form of PCAF lacking the histone acetyltransferase domain (PCAFΔHAT) decreases NF-E4 stability. Acetylation of Lys43 also reduces the interaction between NF-E4 and HDAC1, potentially maximizing the activating ability of the factor at the γ-promoter. These results provide further demonstration that co-activators, such as PCAF, can influence individual transcription factor properties at multiple levels to alter their effects on gene expression. Proteins with intrinsic histone acetyltransferase (HAT) 1The abbreviations used are: HAT, histone acetyltransferase; EKLF, erythroid Kruppel-like factor; SSP, stage selector protein complex; SSE, stage selector element; GFP, green fluorescent protein; GST, glutathione S-transferase; TSA, trichostatin A; HA, hemagglutinin; Ub, ubiquitin; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein.1The abbreviations used are: HAT, histone acetyltransferase; EKLF, erythroid Kruppel-like factor; SSP, stage selector protein complex; SSE, stage selector element; GFP, green fluorescent protein; GST, glutathione S-transferase; TSA, trichostatin A; HA, hemagglutinin; Ub, ubiquitin; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein. activity function as co-activators of transcription through direct acetylation of specific lysine residues within the N-terminal tails of core histones (1Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1277) Google Scholar). This modification leads to destabilization of local nucleosomal structure with induction of an open chromatin configuration, allowing access of the transcriptional machinery to core promoters (reviewed in Refs. 2Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1578) Google Scholar, 3Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1535) Google Scholar, 4Workman J.L. Kingston R.E. Annu. Rev. Biochem. 1998; 67: 545-579Crossref PubMed Scopus (959) Google Scholar). The covalent modification of histones by acetylation also provides recognition sites for factors involved in gene activation or repression (5Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar). Two of the most widely studied protein families with acetylase activity are the GCN5/PCAF (1Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1277) Google Scholar, 6Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1310) Google Scholar) and p300/CBP proteins (7Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1523) Google Scholar, 8Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatini Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2368) Google Scholar). The substrates for these factors are far more diverse than the histone proteins alone and include transcription factors such as p53 (9Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2152) Google Scholar), GATA-1 (10Boyes J. Byfield P. Nakatani Y. Ogryzko V. Nature. 1998; 396: 594-598Crossref PubMed Scopus (632) Google Scholar, 11Hung H.L. Lau J. Kim A.Y. Weiss M.J. Blobel G.A. Mol. Cell. Biol. 1999; 19: 3496-3505Crossref PubMed Scopus (218) Google Scholar), erythroid Kruppel-like factor (EKLF) (12Zhang W. Bieker J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9855-9860Crossref PubMed Scopus (326) Google Scholar), SCL (13Huang S. Qiu Y. Shi Y. Xu Z. Brandt S.J. EMBO J. 2000; 19: 6792-6803Crossref PubMed Scopus (71) Google Scholar), E2F1 (14Martinez-Balbas M.A. Bauer U.M. Nielsen S.J. Brehm A. Kouzarides T. EMBO J. 2000; 19: 662-671Crossref PubMed Scopus (565) Google Scholar), transcriptional co-regulators (15Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar), DNA-binding proteins (16Munshi N. Merika M. Yie J. Senger K. Chen G. Thanos D. Mol. Cell. 1998; 2: 457-467Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), retroviral proteins (17Kiernan R.E. Vanhulle C. Schiltz L. Adam E. Xiao H. Maudoux F. Calomme C. Burny A. Nakatani Y. Jeang K.T. Benkirane M. Van Lint C. EMBO J. 1999; 18: 6106-6118Crossref PubMed Scopus (360) Google Scholar), and nonnuclear proteins (18L'Hernault S.W. Rosenbaum J.L. Biochemistry. 1985; 24: 473-478Crossref PubMed Scopus (342) Google Scholar). The sequelae of acetylation of these proteins range from altered DNA binding or cellular localization to changes in protein stability or protein-protein interactions (reviewed in Ref. 19Kouzarides T. EMBO J. 2000; 19: 1176-1179Crossref PubMed Scopus (998) Google Scholar). These significant changes in function occur in the context of modification of a small number of lysine residues within an individual protein and thus provide mechanisms to expand the roles for single factors in the regulation of complex multigenic loci.One such locus is the β-like globin cluster in which a linear array of five genes is expressed in a highly regulated tissue-specific and developmentally specific manner (20Stamatoyannopoulos G. Nienhuis A.W. Majerus P.W. Varmus H. The Molecular Basis of Blood Diseases. 2nd Ed. W.B. Saunders, Philadelphia, PA1994Google Scholar). To date, very few transcription factors that specifically influence the temporal profile of globin gene expression have been identified. One of these, EKLF is a red cell-specific activator that is critical for switching on high level adult β-globin expression (21Miller I.J. Bieker J.J. Mol. Cell. Biol. 1993; 13: 2776-2786Crossref PubMed Scopus (646) Google Scholar, 22Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (476) Google Scholar, 23Perkins A.C. Sharpe A.H. Orkin S.H. Nature. 1995; 375: 318-322Crossref PubMed Scopus (524) Google Scholar). EKLF binds to the CACCC element in the proximal β-promoter, leading to chromatin remodeling and transcriptional activation (24Armstrong J.A. Bieker J.J. Emerson B.M. Cell. 1998; 95: 93-104Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 25Brown R.C. Pattison S. van Ree J. Coghill E. Perkins A. Jane S.M. Cunningham J.M. Mol. Cell. Biol. 2002; 22: 161-170Crossref PubMed Scopus (49) Google Scholar, 26Donze D. Townes T.M. Bieker J.J. J. Biol. Chem. 1995; 270: 1955-1959Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 27Feng W.C. Southwood C.M. Bieker J.J. J. Biol. Chem. 1994; 269: 1493-1500Abstract Full Text PDF PubMed Google Scholar, 28Wijgerde M. Gribnau J Trimborn T Nuez B. Philipsen S. Grosveld F. Fraser P. Genes Dev. 1996; 10: 2894-2902Crossref PubMed Scopus (181) Google Scholar). The transcriptional activity of EKLF is regulated by site-specific acetylation by p300 and CBP that results in an enhanced activation potential of the protein and increased binding to the SWI/SNF chromatin remodeling complex (12Zhang W. Bieker J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9855-9860Crossref PubMed Scopus (326) Google Scholar, 29Zhang W. Kadam S. Emerson B.M. Bieker J.J. Mol. Cell. Biol. 2001; 21: 2413-2422Crossref PubMed Scopus (157) Google Scholar).NF-E4 is another globin-specific transcription factor (30Zhou W.-L. Clouston D.R. Wang X. Cerruti L. Cunningham J.M. Jane S.M. Mol. Cell. Biol. 2000; 20: 7662-7672Crossref PubMed Scopus (66) Google Scholar). This protein forms the stage selector protein complex (SSP) with the ubiquitous transcription factor CP2 (31Jane S.M. Nienhuis A.W. Cunningham J.M. EMBO J. 1995; 14: 97-105Crossref PubMed Scopus (103) Google Scholar). The SSP facilitates the interaction of the γ-globin genes with the powerful enhancer elements contained in the locus control region in fetal erythroid cells through binding to the stage selector element (SSE) in the proximal γ-promoter (32Jane S.M. Ney P.A. Vanin E.F. Gumucio D.L. Nienhuis A.W. EMBO J. 1992; 11: 2961-2969Crossref PubMed Scopus (118) Google Scholar). Enforced expression of NF-E4 in the human fetal/erythroid K562 cell line and human cord blood progenitors leads to increased fetal globin gene expression (30Zhou W.-L. Clouston D.R. Wang X. Cerruti L. Cunningham J.M. Jane S.M. Mol. Cell. Biol. 2000; 20: 7662-7672Crossref PubMed Scopus (66) Google Scholar). In cord blood progenitors, where active competition between fetal and adult globin genes occurs, enforced expression of NF-E4 also leads to a reduction in β-globin gene expression (30Zhou W.-L. Clouston D.R. Wang X. Cerruti L. Cunningham J.M. Jane S.M. Mol. Cell. Biol. 2000; 20: 7662-7672Crossref PubMed Scopus (66) Google Scholar).In this study, we demonstrate that NF-E4 is a direct target of the co-activator PCAF. The resultant acetylation of Lys-43 in NF-E4 results in both an increase in the stability of the protein due to diminished targeting by ubiquitination and an alteration in protein-protein interactions that favor transcriptional activation. Thus, a single amino acid modification results in complementary functional changes that enhance the ability of NF-E4 to positively regulate fetal globin gene expression.EXPERIMENTAL PROCEDURESCell Culture—K562 cells were grown in RPMI medium 1640 supplemented with 10% fetal bovine serum. 293T cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. All cells were grown at 37 °C and in 5% CO2 supplemented with 50 units of penicillin/ml and 50 μg of streptomycin/ml.Reagents and Antibodies—[14C]Acetyl-CoA (59 mCi/mmol) and sodium [3H]acetate (5 Ci/mmol) were purchased from Amersham Biosciences and PerkinElmer Life Sciences, respectively. Tran35S-label was obtained from ICN (Costa Mesa, CA). Trichostatin A (TSA), sodium butyrate, acetyl-CoA, ubiquitin, MG132, and cycloheximide were from Sigma. Peroxidase-conjugated goat anti-mouse and monoclonal antirabbit immunoglobulin G, monoclonal anti-FLAG (M2), and anti-ubiquitin antibodies were from Sigma. Monoclonal anti-HA (12CA5) antibody was from Roche Applied Science. Monoclonal anti-acetyl lysine and anti-HDAC1 antibodies were from Upstate (Waltham, MA). Anti-PCAF and anti-tubulin antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-NF-E4-specific antibody was generated by immunizing rabbits with a C-terminal synthetic peptide with the amino acid sequence LKTDSALEQTPQQLPSLHLS coupled to keyhole limpet hemocyanin.Plasmids, Transfections, Luciferase, and β-Galactosidase Assays— The expression vectors GST-NF-E4 and GST-CP2 in pGEX were described previously (30Zhou W.-L. Clouston D.R. Wang X. Cerruti L. Cunningham J.M. Jane S.M. Mol. Cell. Biol. 2000; 20: 7662-7672Crossref PubMed Scopus (66) Google Scholar, 31Jane S.M. Nienhuis A.W. Cunningham J.M. EMBO J. 1995; 14: 97-105Crossref PubMed Scopus (103) Google Scholar). For the GST-NF-E4 truncation constructs, PCR fragments corresponding to the shortened coding sequences were generated with BamHI and XhoI ends and were cloned into pGEX vectors. The plasmids pCX-PCAF, pCX-PCAFΔHAT, MSCV-IRES-PCAF-GFP, and MSCV-IRES-PCAFΔHAT-GFP were kindly provided by Dr. Steven Brandt (13Huang S. Qiu Y. Shi Y. Xu Z. Brandt S.J. EMBO J. 2000; 19: 6792-6803Crossref PubMed Scopus (71) Google Scholar). Plasmids expressing GST-PCAF-(352–832) and GST-CBP-(1098–1877) were kindly provided by Dr. Tony Kouzarides (14Martinez-Balbas M.A. Bauer U.M. Nielsen S.J. Brehm A. Kouzarides T. EMBO J. 2000; 19: 662-671Crossref PubMed Scopus (565) Google Scholar). The plasmid pCDNA3.1-FLAG-ubiquitin was kindly provided by Dr. Ivan Dikic. The in vitro transcription/translation plasmid for PCAF-(1–832) was cloned by inserting an EcoRI/XhoI fragment from MSCV-IRES-PCAF-GFP into vector pSP72. Mutation of GST-NF-E4 to GST-K43R was accomplished with the QuikChange mutagenesis system (Stratagene, La Jolla, CA). Retroviral vectors expressing HA-NF-E4 and HA-K43R were constructed by cloning fragments obtained by PCR amplification of coding sequences of pGEX-NF-E4 and pGEX-K43R into MSCV-IRES-GFP. The integrity of all constructs that generated fusion proteins was confirmed by DNA sequencing.Stable K562 cell lines overexpressing HA-NF-E4, HA-K43R, PCAF, and PCAFΔHAT were generated according to the protocols described previously, except that only the top 10% of GFP-positive cells were collected (30Zhou W.-L. Clouston D.R. Wang X. Cerruti L. Cunningham J.M. Jane S.M. Mol. Cell. Biol. 2000; 20: 7662-7672Crossref PubMed Scopus (66) Google Scholar). For the co-immunoprecipitation experiment, 293T cells were transiently transfected with various plasmids by a calcium phosphate method (Invitrogen). For the reporter gene assays, native K562 cells or stable K562 cell lines overexpressing HA-NF-E4 and HA-K43R were co-transfected with HS2–53γ-Luciferase, containing hypersensitivity site 2 of the β-globin locus control region linked to the –53 γ-promoter relative to the CAP site and the firefly luciferase reporter gene (32Jane S.M. Ney P.A. Vanin E.F. Gumucio D.L. Nienhuis A.W. EMBO J. 1992; 11: 2961-2969Crossref PubMed Scopus (118) Google Scholar), and pCH110, containing the β-galactosidase gene driven by SV40 promoter as an internal transfection efficiency control by electroporation. For the TSA induction experiments, the transfected cells were divided into two aliquots, one of which was cultured in various concentrations of TSA for 20 h and the other in standard medium alone. For all other transfection experiments, cells were harvested after 36 h and lysed, and luciferase activity was determined on a Monolight 2001 luminometer using the luciferase assay kit (Promega). β-Galactosidase activity was measured in a spectrophotometer using the enzyme assay system (Promega), and the relative luciferase activities were calculated by dividing the luciferase activity by the β-galactosidase activity to correct for transfection efficiency. Three independent transfections were performed in duplicate.Metabolic Labeling—For 35S labeling, K562 cells were grown in RPMI supplemented with 5% dialyzed fetal bovine serum containing Tran35S-label (20 μCi/ml; a mixture of cysteine and methionine) for 4 h. For 3H labeling, K562 cells were cultured in RPMI containing sodium [3H]acetate (1 mCi/ml) and 2 μm TSA for 2 h. Extracts were prepared and boiled after adding 2× sample buffer (125 mm Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 2% β-mercaptoethanol) to disrupt interactions between proteins before being subjected to immunoprecipitation with specific anti-NF-E4 antibody. The immunoprecipitated samples on beads were extensively washed with stringent washing buffer (500 mm NaCl, 50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 1% Triton X-100, 0.1% SDS), resolved on SDS-PAGE, and analyzed by autoradiography.Immunoprecipitation and Immunoblotting—Cells were lysed in ice-cold lysis buffer (150 mm NaCl, 50 mm Tris-HCl, pH 8.0, 1 mm EDTA, 1% Nonidet P-40, 10 mm sodium butyrate) containing a protease inhibitor mixture (Roche Applied Science) and cleared by centrifugation. Immunoprecipitations were carried out by adding appropriate antibodies plus protein G-Sepharose beads, followed by incubation at 4 °C. The immunoprecipitates were washed extensively, subjected to SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were incubated with various specific antibodies and then washed extensively prior to incubation with peroxidase-conjugated anti-rabbit or antimouse immunoglobulin G. After further extensive washes, the blots were visualized by using ECL reagents (Amersham Biosciences). All immunoprecipitations were performed in duplicate.Recombinant Protein Expression and GST Pull-down Assay—GST-fusion proteins were produced in BL21 Escherichia coli as described previously (30Zhou W.-L. Clouston D.R. Wang X. Cerruti L. Cunningham J.M. Jane S.M. Mol. Cell. Biol. 2000; 20: 7662-7672Crossref PubMed Scopus (66) Google Scholar). 35S-Labeled NF-E4 and PCAF synthesized using the T7 TNT kit (Promega) and Tran35S-label (ICN) were incubated with GST fusion proteins prebound to glutathione beads at 4 °C overnight. The samples were washed extensively and subjected to SDS-PAGE. The gels were dried and analyzed by autoradiography.Determination of Protein Half-life—K562 cells stably expressing HA-NF-E4, HA-K43R, PCAF, and PCAFΔHAT were treated with cycloheximide (100 μm) to stop protein synthesis and incubated for the indicated times. Cells were lysed, and lysates were subjected to SDS-PAGE and Western blotting probed with the indicated specific antibodies. The protein signals were scanned and quantitated by PhosphorImager analysis software (Amersham Biosciences).In Vitro Protein Acetylation Assay—2–3 μg of the indicated GST fusion protein or SCL and 200 ng of acetyltransferase protein were incubated in a reaction containing 50 mm Tris-HCl, pH 8.0, 10% glycerol, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 mm sodium butyrate, and 30 μm acetyl-coenzyme A or 1 μl of [14C]acetyl-CoA for 1 h at 30 °C. The reaction mixture was subjected to SDS-PAGE and analyzed by autoradiography of dried gels or by Western blotting with anti-acetyl lysine antibody.In Vitro Ubiquitination Assay—Recombinant protein was used as the substrate in a ubiquitin reaction containing 20 mm Hepes, pH 7.5, 5 mm MgCl2, 2 mm dithiothreitol, 2 mm ATP, 5 μg of ubiquitin, 20 μm MG132, and 5 μl of crude rabbit reticulocyte (Promega) for 1 h at 30 °C. In some cases, GST fusion proteins were acetylated prior to the ubiquitination reaction by PCAF in the presence or absence of unlabeled acetyl-CoA. The samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes followed by determination of ubiquitination by Western blotting analysis.In Vivo Ubiquitination Assay—293T cells were transfected with pCDNA3.1-FLAG-ubiquitin, and cell lysate was prepared 36 h later. For immunoprecipitation, 1 mg of protein was incubated with α-HA antibody at 4 °C overnight before protein G beads were added for 2 h. The beads were washed twice with NaCl (1 m) in Tris-buffered saline supplemented with Nonidet P-40 (1%), β-mercaptoethanol (0.05%), and EDTA (1 mm). Proteins were loaded onto SDS-PAGE followed by immunoblot analysis with the indicated antibodies and enhanced chemiluminescence detection.Electrophoretic Mobility Shift Assay—The electrophoretic mobility shift assay was performed as previously described (31Jane S.M. Nienhuis A.W. Cunningham J.M. EMBO J. 1995; 14: 97-105Crossref PubMed Scopus (103) Google Scholar) using recombinant proteins. In some cases, recombinant NF-E4 was acetylated in vitro prior to binding DNA. Specific antibodies against NF-E4 or CP2 or specific or nonspecific cold oligonucleotide competitors in molar excess were added to the reaction mixture for 30 min on ice before the addition of the labeled SSE oligonucleotides.RESULTSAcetylation of NF-E4 in Vitro and in Vivo—Distinct functional parallels exist between the globin regulatory transcription factors EKLF and NF-E4. Both factors are expressed throughout erythroid ontogeny and yet exert their predominant effects at distinct developmental time points. Acetylation of EKLF is critical for its role as an adult globin gene activator, and we postulated that a similar posttranslational modification might also regulate NF-E4 function. We therefore examined the ability of NF-E4 to serve as a substrate for two acetyltransferases, PCAF and CBP. A purified GST-NF-E4 fusion protein was incubated with recombinant CBP or PCAF in the presence of [14C]acetyl-CoA and the incorporation of [14C]acetate determined by SDS-PAGE and autoradiography. Recombinant SCL (TAL1), which is acetylated in vitro by both acetyltransferases (13Huang S. Qiu Y. Shi Y. Xu Z. Brandt S.J. EMBO J. 2000; 19: 6792-6803Crossref PubMed Scopus (71) Google Scholar), served as the positive control, and GST alone served as the negative control. As shown in Fig. 1A, acetylation of GST-NF-E4 was observed in the presence of PCAF (lane 3) but not CBP (lane 2). Incorporation of [14C]acetate was dependent on the presence of NF-E4, since GST alone remained unlabeled (lane 1). SCL was acetylated in the presence of both co-activators (lanes 4 and 5). To determine whether NF-E4 was acetylated in vivo in a fetal/erythroid environment, human K562 cells were pulse-labeled with either [35S]methionine/cysteine or sodium [3H]acetate in the presence of TSA. Cellular extract was subjected to immunoprecipitation with anti-NF-E4 antibody, and precipitates were washed under stringent conditions. As a control, extracts were also immunoprecipitated with preimmune sera. As shown in Fig. 1B, NF-E4-specific antisera detected both the 35S-labeled NF-E4 (left panel) and acetylated NF-E4 (right panel). Neither species was detected with preimmune sera. To directly examine the role of PCAF in the acetylation of NF-E4 in a cellular context, we co-transfected the human cell line 293T with mammalian expression vectors containing a hemagglutinin (HA) epitope-tagged NF-E4 (HA-NF-E4), and either the wild-type PCAF cDNA (PCAF) tagged with a FLAG epitope or a mutant PCAF that lacked the histone acetyltransferase domain (PCAFΔHAT) tagged with a FLAG epitope, or vector alone (13Huang S. Qiu Y. Shi Y. Xu Z. Brandt S.J. EMBO J. 2000; 19: 6792-6803Crossref PubMed Scopus (71) Google Scholar, 14Martinez-Balbas M.A. Bauer U.M. Nielsen S.J. Brehm A. Kouzarides T. EMBO J. 2000; 19: 662-671Crossref PubMed Scopus (565) Google Scholar). Cellular extracts from the transfected cells were immunoprecipitated with preimmune sera (lane 1) or anti-HA antibody (lanes 2–4) and immunoblotted with either anti-HA antibody (α-HA) or antibody specific for acetylated lysine (α-AcK) (Fig. 1C). An increase in the level of acetylated NF-E4 was observed in the presence of PCAF compared with vector alone (compare lanes 2 and 3). The level of acetylated NF-E4 was decreased in cells transfected with PCAFΔHAT, consistent with its dominant negative role (lane 4). The expression of HA-NF-E4 and FLAG-PCAF/PCAFΔHAT were similar in the transfected lines (lower panels). These findings indicate that NF-E4 serves as a substrate for the acetyltransferase, PCAF.NF-E4 Interacts with PCAF in Vitro and in Vivo—Acetylation of transcription factors is usually mediated by a direct interaction between the factor and the specific acetyltransferase. To determine whether NF-E4 interacted directly with PCAF, we performed glutathione S-transferase (GST)-chromatography assays (Fig. 2A). In vitro transcribed/translated [35S]methionine-labeled NF-E4 (lane 1) was applied to glutathione-Sepharose beads adsorbed with GST alone (lane 2); GST-PCAF-(352–832) (lane 3), which contains key regulatory domains including the HAT domain; and a positive control, GST-CP2 (lane 4). The labeled protein was retained on both the GST-PCAF and GST-CP2 matrices but not on GST alone. To further localize the region of NF-E4 that interacted with PCAF, we generated a series of truncation deletions of NF-E4 as GST fusion proteins and examined their ability to retain [35S]methionine-labeled PCAF (Fig. 2B). Only NF-E4 fusion proteins containing the first 25 amino acids were capable of interacting with PCAF (lanes 3–5). In contrast, fusion proteins that lacked only the N-terminal 17 amino acids did not interact (lane 6), and no PCAF was retained on the GST alone matrix (lane 2). To confirm the interaction in a cellular context, we transfected 293T cells with mammalian expression vectors containing the NF-E4 cDNA tagged with a hemagglutinin epitope (HA-NF-E4) and the PCAF cDNA tagged with a FLAG epitope (FLAG-PCAF) (Fig. 2C). Cellular extract was immunoprecipitated with an unrelated antibody (mock) or anti-FLAG antibody and immunoblotted with either anti-HA antibody (top panel) or anti-FLAG antibody (bottom panel). Both PCAF and NF-E4 were immunoprecipitated with the anti-FLAG antibody, but not the unrelated antibody, indicating that the two proteins interact in a cellular context. Immunoprecipitation of lysate from nontransfected cells with the anti-FLAG antibody failed to bring down NF-E4 (data not shown). We then examined whether this interaction occurred in the absence of enforced expression of the proteins. K562 cell extract was immunoprecipitated with either preimmune sera or antibody to NF-E4 or PCAF, and the precipitates were electrophoresed and immunoblotted with anti-PCAF antibody. As shown in Fig. 2D, PCAF co-immunoprecipitated with NF-E4 (lane 2), indicating that these two factors form a complex in native cells. No PCAF was observed with preimmune sera (lane 1).Fig. 2NF-E4 and PCAF interact in vitro and in cellular extracts.A, purified GST and GST fusion proteins containing amino acids 352–832 of PCAF (GST-PCAF-(352–832)) or full-length CP2 (GST-CP2) preadsorbed to glutathione-Sepharose beads were incubated with 35S-labeled in vitro transcribed/translated (IVT) NF-E4. Specifically bound protein was eluted from washed beads and visualized by autoradiography after SDS-PAGE. Input represents 20% of the in vitro translated NF-E4 used in the assay. Each experiment was performed in duplicate. B, purified GST and GST fusion proteins containing amino acids 1–25, 1–48, 49–179, and 17–179 (top panel) of NF-E4 preadsorbed to glutathione-Sepharose beads were incubated with 35S-labeled PCAF. Specifically bound protein was eluted from washed beads and visualized by autoradiography (bottom panel) after SDS-PAGE. Input represents 5% of the in vitro translated PCAF used in the assay. C, 293T cells were transfected with expression vectors for HA-tagged NF-E4 and FLAG-tagged PCAF. Cellular extracts were immunoprecipitated (IP) with antibody to the FLAG epitope (α-Flag) or an unrelated antibody (mock) and analyzed by immunoblotting (IB) with anti-HA and anti-PCAF antibodies. NF-E4 protein was identified only in precipitates using anti-FLAG antisera. D, K562 cell extract was immunoprecipitated with either anti-NF-E4 or anti-PCAF antibodies. Preimmune sera served as the control. Immunoprecipitates were fractionated by SDS-PAGE and immunoblotted with anti-PCAF antibody. PCAF protein was detected in precipitates from both anti-NF-E4 and anti-PCAF antibodies but not preimmune sera.View Large Image Figure ViewerDownload (PPT)Identifying Acetylation Sites in NF-E4—To identify the residue(s) in NF-E4 that are acetylated by PCAF, we utilized our series of GST-NF-E4 truncation mutants (Fig. 3A, left panel). The proteins were subjected to a PCAF-dependent in vitro acetylation assay using unlabeled acetyl-CoA, and protein acetylation was detected using an antibody specific for acetylated lysine (α-AcK) (Fig. 3A, top right panel). Full-length NF-E4 (amino acids 1–179) (top panel, lane 5) and NF-E4 (amino acids 1–48) (lane 3) were both acetylated in vitro. In contrast, NF-E4 lacking the first 48 amino acids (lane 4), NF-E4 (amino acids 1–25) (lane 2), and GST alone (lane 1) remained unacetylated, despite significant levels of GST fusion protein expression (Fig. 3A, bottom right panel). This acetylation pattern was duplicated when acetylated lysine was detected by incorporation of [14C]acetyl-CoA in the in vitro assay (data not shown). These results localized the site of acetylation of NF-E4 to the residues between 26 and 48 and indicated that the acetylation status of the NF-E4 protein was accurately reflected by Western analysis