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Physical and Functional Interactions among AP-2 Transcription Factors, p300/CREB-binding Protein, and CITED2

2003; Elsevier BV; Volume: 278; Issue: 18 Linguagem: Inglês

10.1074/jbc.m208144200

ISSN

1083-351X

Autores

José Bragança, Jyrki J. Eloranta, Simon D. Bamforth, J. Claire Ibbitt, Helen C. Hurst, Shoumo Bhattacharya,

Tópico(s)

Congenital limb and hand anomalies

Resumo

The transcriptional co-activators and histone acetyltransferases p300/CREB-binding protein (CBP) interact with CITED2, a transcription factor AP-2 (TFAP2) co-activator. p300/CBP, CITED2, and TFAP2A are essential for normal neural tube and cardiac development. Here we show that p300 and CBP co-activate TFAP2A in the presence of CITED2. TFAP2A transcriptional activity was modestly impaired in p300+/− and CBP+/− mouse embryonic fibroblasts; this was rescued by ectopic expression of p300/CBP. p300, TFAP2A, and endogenous CITED2 could be co-immunoprecipitated from transfected U2-OS cells indicating that they can interact physically in vivo. CITED2 interacted with the dimerization domain of TFAP2C, which is highly conserved in TFAP2A/B. In mammalian two-hybrid experiments, full-length p300 and TFAP2A interacted only when CITED2 was co-transfected. N-terminal residues of TFAP2A, containing the transactivation domain, are both necessary and sufficient for interaction with p300, and this interaction was independent of CITED2. Consistent with this, N-terminal residues of TFAP2A were required for p300- and CITED2-dependent co-activation. A histone acetyltransferase-deficient p300 mutant (D1399Y) did not co-activate TFAP2A and did not affect the expression or cellular localization of TFAP2A or CITED2. In mammalian two-hybrid experiments p300D1399Y failed to interact with TFAP2A, explaining, at least in part, its failure to function as a co-activator. Our results suggest a model wherein interactions among TFAP2A, CITED2, and p300/CBP are necessary for TFAP2A-mediated transcriptional activation and for normal neural tube and cardiac development. The transcriptional co-activators and histone acetyltransferases p300/CREB-binding protein (CBP) interact with CITED2, a transcription factor AP-2 (TFAP2) co-activator. p300/CBP, CITED2, and TFAP2A are essential for normal neural tube and cardiac development. Here we show that p300 and CBP co-activate TFAP2A in the presence of CITED2. TFAP2A transcriptional activity was modestly impaired in p300+/− and CBP+/− mouse embryonic fibroblasts; this was rescued by ectopic expression of p300/CBP. p300, TFAP2A, and endogenous CITED2 could be co-immunoprecipitated from transfected U2-OS cells indicating that they can interact physically in vivo. CITED2 interacted with the dimerization domain of TFAP2C, which is highly conserved in TFAP2A/B. In mammalian two-hybrid experiments, full-length p300 and TFAP2A interacted only when CITED2 was co-transfected. N-terminal residues of TFAP2A, containing the transactivation domain, are both necessary and sufficient for interaction with p300, and this interaction was independent of CITED2. Consistent with this, N-terminal residues of TFAP2A were required for p300- and CITED2-dependent co-activation. A histone acetyltransferase-deficient p300 mutant (D1399Y) did not co-activate TFAP2A and did not affect the expression or cellular localization of TFAP2A or CITED2. In mammalian two-hybrid experiments p300D1399Y failed to interact with TFAP2A, explaining, at least in part, its failure to function as a co-activator. Our results suggest a model wherein interactions among TFAP2A, CITED2, and p300/CBP are necessary for TFAP2A-mediated transcriptional activation and for normal neural tube and cardiac development. cAMP-response element-binding protein activation domain CREB-binding protein cysteine-histidine-rich domain of p300 cytomegalovirus DNA binding domain Far Western histone acetyltransferase firefly luciferase gene mouse embryonic fibroblasts transcription factor AP-2 Rous sarcoma virus dithiothreitol Recruitment of the transcriptional co-activators p300 and CREB1-binding protein (CBP) to gene promoters by DNA-bound transcription factors activates gene transcription (reviewed in Ref. 1Chan H.M. La Thangue N.B. J. Cell Sci. 2001; 114: 2363-2373Crossref PubMed Google Scholar). 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Cell. 1998; 93: 361-372Abstract Full Text Full Text PDF PubMed Scopus (810) Google Scholar). Mutation of a single allele of CBP in humans causes Rubinstein-Taybi syndrome, characterized by mental retardation and cardiac, cranio-facial, and skeletal malformations (6Petrij F. Giles R.H. Dauwerse H.G. Saris J.J. Hennekam R.C.M. Masuno M. Tommerup N. Ommen G.B. Goodman R.H. Peters D.J.M. Breuning M.H. Nature. 1995; 376: 348-351Crossref PubMed Scopus (1011) Google Scholar). p300 and CBP have overlapping functions, as indicated by the fact that mice lacking one allele of p300 and one allele of CBP (double heterozygotes) show early embryonic lethality, with defects in neural tube closure. However, they also have clearly distinct functions, as well. For instance, retinoid receptor function requires p300 rather than CBP, whereas CREB function requires CBP rather than p300 (5Yao T.P. Oh S.P. Fuchs M. Zhou N.D. Ch'ng L.E. Newsome D. Bronson R.T. Li E. Livingston D.M. Eckner R. 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Mice lacking CITED2 have abnormal cardiac, adrenal, neural tube, and cranial ganglia (19Bamforth S.D. Braganca J. Eloranta J.J. Murdoch J.N. Marques F.I. Kranc K.R. Farza H. Henderson D.J. Hurst H.C. Bhattacharya S. Nat. Genet. 2001; 29: 469-474Crossref PubMed Scopus (255) Google Scholar, 20Barbera J.P. Rodriguez T.A. Greene N.D. Weninger W.J. Simeone A. Copp A.J. Beddington R.S. Dunwoodie S. Hum. Mol. Genet. 2002; 11: 283-293Crossref PubMed Google Scholar, 21Yin Z. Haynie J. Yang X. Han B. Kiatchoosakun S. Restivo J. Yuan S. Prabhakar N.R. Herrup K. Conlon R.A. Hoit B.D. Watanabe M. Yang Y.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10488-10493Crossref PubMed Scopus (166) Google Scholar). CITED2 (and CITED4) interact physically with and co-activate isoforms A, B, and C of the DNA binding transcription factor AP-2 (TFAP2) (16Braganca J. Swingler T. Marques F.I. Jones T. Eloranta J.J. Hurst H.C. Shioda T. Bhattacharya S. J. Biol. Chem. 2002; 277: 8559-8565Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 19Bamforth S.D. Braganca J. Eloranta J.J. Murdoch J.N. Marques F.I. Kranc K.R. Farza H. Henderson D.J. Hurst H.C. Bhattacharya S. Nat. Genet. 2001; 29: 469-474Crossref PubMed Scopus (255) Google Scholar). CITED2 is necessary for TFAP2 transactivation, and Cited2−/− embryos have reduced expression of the TFAP2 target gene ErbB3 (22Skinner A. Hurst H.C. Oncogene. 1993; 8: 3393-3401PubMed Google Scholar) in the neural crest (19Bamforth S.D. Braganca J. Eloranta J.J. Murdoch J.N. Marques F.I. Kranc K.R. Farza H. Henderson D.J. Hurst H.C. Bhattacharya S. Nat. Genet. 2001; 29: 469-474Crossref PubMed Scopus (255) Google Scholar). As TFAP2A and TFAP2B mutations also result in neural tube, cardiac, and cranial ganglia defects (23Zhang J. Hagopian-Donaldson S. Serbedzija G. Elsemore J. Plehn-Dujowich D. McMahon A.P. Flavell R.A. Williams T. Nature. 1996; 381: 238-241Crossref PubMed Scopus (527) Google Scholar, 24Schorle H. Meier P. Buchert M. Jaenisch R. Mitchell P.J. Nature. 1996; 381: 235-238Crossref PubMed Scopus (512) Google Scholar, 25Satoda M. Zhao F. Diaz G.A. Burn J. Goodship J. Davidson H.R. Pierpont M.E. Gelb B.D. Nat. Genet. 2000; 25: 42-46Crossref PubMed Scopus (215) Google Scholar, 26Zhao F. Weismann C.G. Satoda M. Pierpont M.E. Sweeney E. Thompson E.M. Gelb B.D. Am. J. Hum. Genet. 2001; 69: 695-703Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 27Brewer S. Jiang X. Donaldson S. Williams T. Sucov H.M. Mech. Dev. 2002; 110: 139-149Crossref PubMed Scopus (78) Google Scholar), it is likely that abnormal embryonic development in Cited2−/− mice results, at least in part, from defective TFAP2 function (19Bamforth S.D. Braganca J. Eloranta J.J. Murdoch J.N. Marques F.I. Kranc K.R. Farza H. Henderson D.J. Hurst H.C. Bhattacharya S. Nat. Genet. 2001; 29: 469-474Crossref PubMed Scopus (255) Google Scholar). The similar phenotypes observed in mice lacking p300/CBP and TFAP2A/B led us to hypothesize that p300 and CBP may function as TFAP2 co-activators. Here we describe, for the first time, physical and functional interactions between TFAP2 isoforms and p300/CBP. Our results suggest a model wherein interactions among TFAP2A, CITED2, and p300/CBP are necessary for TFAP2A-mediated transcriptional activation and for normal neural tube and cardiac development. Standard molecular biology protocols were used for all procedures (28Ausubel F. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Short Protocols in Molecular Biology. 3rd Ed. John Wiley & Sons, Inc., New York1995Google Scholar). Reagents were from Sigma unless otherwise indicated. CMV-TFAP2A, CMV-TFAP2B, and CMV-TFAP2C and CMV-CITED2 were described previously (19Bamforth S.D. Braganca J. Eloranta J.J. Murdoch J.N. Marques F.I. Kranc K.R. Farza H. Henderson D.J. Hurst H.C. Bhattacharya S. Nat. Genet. 2001; 29: 469-474Crossref PubMed Scopus (255) Google Scholar). RSV-TFAP2A and RSV-TFAP2AΔN165 were gifts from Trevor Williams (University of Colorado, Denver, CO). p3xAP2-Bluc was derived from plasmid A2BCAT (29Williams T. Tjian R. Genes Dev. 1991; 5: 670-682Crossref PubMed Scopus (440) Google Scholar) and contains three copies of the AP-2 binding site in the human metallothionein IIa promoter. CMV-p300, CMV-CBP, CMV-p300-E2, CMV-p300ΔCH1-E2, and the E2-luc reporter were gifts from Richard Eckner (University of Zurich, Zurich, Switzerland) (30Arany Z. Newsome D. Oldread E. Livingston D.M. Eckner R. Nature. 1995; 374: 81-84Crossref PubMed Scopus (489) Google Scholar). CMV-p300ΔCH1-E2 lacks residues 346–510 of p300. CMV-p300-myc and CMV-p300(D1399Y)-myc (containing a point mutation that abolishes HAT activity (31Ito A. Lai C.H. Zhao X. Saito S. Hamilton M.H. Appella E. Yao T.P. EMBO J. 2001; 20: 1331-1340Crossref PubMed Scopus (433) Google Scholar)) were gifts from Tso-Pang Yao (Duke University, Durham, NC). An XbaI-XmaI fragment from p300(D1399Y)-myc was subcloned into CMV-p300-E2 to generate CMV-p300(D1399Y)-E2. CMV-p300(D1399Y)-E2 was sequenced to confirm the presence of the point mutation. CMV-VP16-TFAP2A, CMV-VP16-TFAP2A (1–165), and CMV-VP16-TFAP2A (202–437) were gifts from Stefan Gaubatz (University of Marburg, Marburg, Germany). CMV-VP16-CITED2 has been described previously (9Bhattacharya S. Michels C.L. Leung M.K. Arany Z.P. Kung A.L. Livingston D.M. Genes Dev. 1999; 13: 64-75Crossref PubMed Scopus (316) Google Scholar). CMV-GAL4 and CMV-lacZ were gifts from Ronald Evans (Salk Institute, San Diego, CA). GAL4-p300 fusions (32Yuan W. Condorelli G. Caruso M. Felsani A. Giordano A. J. Biol. Chem. 1996; 271: 9009-9013Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) were gifts from Antonio Giordano (Thomas Jefferson University, Philadelphia, PA). pRL-CMV (Renilla luciferase gene attached to CMV promoter) was from Promega. CMV-GAL4-TFAP2A was generated by PCR and sequenced to confirm the absence of artifacts. The yeast vectors expressing GAL4-DBD fused to TFAP2C full-length protein or subdomains of TFAP2C and the GAL4-AD-CITED2 have been described previously (19Bamforth S.D. Braganca J. Eloranta J.J. Murdoch J.N. Marques F.I. Kranc K.R. Farza H. Henderson D.J. Hurst H.C. Bhattacharya S. Nat. Genet. 2001; 29: 469-474Crossref PubMed Scopus (255) Google Scholar, 33Eloranta J.J. Hurst H.C. J. Biol. Chem. 2002; 277: 30798-30804Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). RK5C1 (Santa Cruz Biotechnology, Inc.) is a monoclonal antibody against GAL4-DBD. Anti-CITED2 polyclonal antibody has been described previously (9Bhattacharya S. Michels C.L. Leung M.K. Arany Z.P. Kung A.L. Livingston D.M. Genes Dev. 1999; 13: 64-75Crossref PubMed Scopus (316) Google Scholar). PAB419 and 9E10 are monoclonal antibodies against SV40 Tag and the myc epitope, respectively, and were gifts from Jim DeCaprio (Dana-Farber Cancer Institute, Boston, MA). Mouse monoclonal 12CA5 antibody was used to detect hemagglutinin-tagged proteins. Anti-tubulin antibody was obtained from Sigma (T-5293). Anti-TFAP2A (sc-184X) and anti-proliferating cell nuclear antigen (sc-56) antibodies were obtained from Santa Cruz Biotechnology, Inc. Western blots were performed as described previously (9Bhattacharya S. Michels C.L. Leung M.K. Arany Z.P. Kung A.L. Livingston D.M. Genes Dev. 1999; 13: 64-75Crossref PubMed Scopus (316) Google Scholar). p300 and CBP heterozygote mice (5Yao T.P. Oh S.P. Fuchs M. Zhou N.D. Ch'ng L.E. Newsome D. Bronson R.T. Li E. Livingston D.M. Eckner R. Cell. 1998; 93: 361-372Abstract Full Text Full Text PDF PubMed Scopus (810) Google Scholar) were gifts from David Livingston (Dana-Farber Cancer Institute, Boston, MA). Mouse embryonic fibroblasts (MEFs) from 15.5-day-old p300 heterozygote or CBP heterozygote embryos, and their wild-type littermate controls were generated as described (34Loo D. Rawson C. Ernst T. Shirahata S. Barnes D. Baserga R. Cell Growth and Division: A Practical Approach. Oxford University Press, Oxford1989: 17-35Google Scholar) and genotyped using allele-specific primers. Hep3B, HepG2, and U2-OS cells were obtained from ATCC. U2-OS cells were plated at 2 × 106 cells per p100 plate and transfected the following day with 10 μg of the indicated plasmids using FuGENE 6 (Roche Molecular Biochemicals). Cells were lysed, and immunoprecipitations were performed as described (9Bhattacharya S. Michels C.L. Leung M.K. Arany Z.P. Kung A.L. Livingston D.M. Genes Dev. 1999; 13: 64-75Crossref PubMed Scopus (316) Google Scholar) in buffer containing 50 mmTris-HCl, pH 8.0, 150 mm NaCl, 0.5% Nonidet P-40, 0.5 mm EDTA, protease and phosphatase inhibitors (Complete protease inhibitor (Roche Molecular Biochemicals), 1 mmphenylmethylsulfonyl fluoride, 0.5 mm sodium orthovanadate, 5 mm sodium fluoride), and 1 mm DTT. Hep3B cells were plated at 2 × 105 cells per six-well plate and transfected the following day with 2 μg of CMV-p300-myc or CMV-p300(D1399Y)-myc and 0.5 μg of CMV-CITED2 or control vector using FuGENE 6 (Roche Molecular Biochemicals). 0.5 μg of CMV-TFAP2A or the control vector were also co-transfected. Immunostaining was performed 48 h after the transfection using anti-TFAP2A and anti-CITED2 polyclonal antibodies at 1:150 dilution, essentially as described (9Bhattacharya S. Michels C.L. Leung M.K. Arany Z.P. Kung A.L. Livingston D.M. Genes Dev. 1999; 13: 64-75Crossref PubMed Scopus (316) Google Scholar). The anti-myc 9E10 monoclonal antibody was used as neat culture supernatant. Nuclei were counterstained with TOPRO (Molecular Probes). Cells were mounted in Vectamount (Vector) and were visualized using a Bio-Rad MRC 1024 confocal microscope. Data from TOPRO (blue), rhodamine (red), and fluorescein isothiocyanate (green) channels were accumulated sequentially. Cells were plated in 24-well plates at 2.5 × 104 cells per well and were transfected in duplicate the following day using FuGENE 6 (Roche Molecular Biochemicals) or Transfast reagent (Promega). CMV-lacZ (100 ng) or pRL-CMV were co-transfected in all experiments. Firefly luciferase and β-galactosidase (lacZ) activities were measured as described (28Ausubel F. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Short Protocols in Molecular Biology. 3rd Ed. John Wiley & Sons, Inc., New York1995Google Scholar). Renilla luciferase activity was measured using the DUAL-luciferase reporter assay kit (Promega). The ratio of firefly luciferase to lacZ or to Renilla luciferase activity (relative luciferase activity) was calculated to correct for variations in transfection efficiency. All expression plasmids were driven by the CMV or the RSV promoter, and an appropriate amount of CMV or RSV control vector was added to each transfection mix so that the total plasmid amount and CMV or RSV promoter per transfection were constant for each experiment. Amounts of DNA used per transfection refer to the amounts added per well of a 24-well plate. The TFAP2 variants used as probes in Far Western (FW) assays were translated in vitro using the TNT coupled reticulocyte lysate system (Promega). The proteins were labeled radioactively in 100-μl reactions usingl-[35S]methionine/l-[35S]cysteine Pro-mix (Amersham Biosciences). Unincorporated amino acids were removed using Microcon-MWCO 3000 (Amicon) centrifugal filter devices, according to the manufacturer's instructions, and the buffer was changed simultaneously to FW buffer (25 mm Tris-HCl, pH 7.5, 100 mm KCl, 5 mm MgCl2, 0.1% Tween 20, 5% glycerol, 1 mm DTT, Complete protease inhibitor mix (Roche Molecular Biochemicals). The successful synthesis and purification of the probes were verified by SDS-PAGE and autoradiography; all the protein probes used were synthesized to a comparable efficiency. Assays were performed against a λTriplEx phage clone encoding the C-terminal residues 234–270 of the human CITED2. The Escherichia coli strain XL1-Blue was transduced with the λTriplEx-CITED2 phage and plated at ∼100 plaque-forming unit per 10-cm Petri dish, according to the λTriplEx library manual (Clontech PT3003–1). The plates were incubated at 42 °C for 3–4 h until the plaques were just visible and then overlaid with Hybond-C Extra (Amersham Biosciences) membranes, prewetted in 10 mmisopropyl-β-d-thiogalactopyranoside, and further incubated at 37 °C for another 4 h. The membranes were then removed from the Petri dishes and placed in blocking solution (FW buffer containing 5% dry milk) for 2 h, shaking gently at room temperature. After blocking, the membranes were placed in FW buffer, supplemented with an in vitro translated,35S-labeled protein probe, and incubated overnight at 4 °C, shaking gently. The membranes were washed several times in FW buffer, and each wash was performed for 30 min at room temperature, shaking gently. The washed membranes were air-dried and exposed to BioMax MR-1 (Eastman Kodak Co.) film for several days. SFY526 yeast were transformed simultaneously with full-length CITED2 cloned into pGAD424 and TFAP2C constructs cloned into pGBT9 as described previously (33Eloranta J.J. Hurst H.C. J. Biol. Chem. 2002; 277: 30798-30804Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Liquid β-galactosidase assays were performed according to the Clontech Matchmaker manual. To verify that TFAP2C deletion mutants containing the dimerization domain were still able to self-associate, additional clones were made in pGAD424 to allow this to be tested in the yeast two-hybrid system (data not shown). For those mutants that also contained the DNA binding domain, electrophoretic mobility shift assays were also performed using unlabeled in vitro translated protein to confirm DNA binding and hence dimerization (data not shown). Hep3B cells were plated at 2 × 106cells per p100 plate and transfected the following day with 15 μg of CMV-p300-myc or CMV-p300(D1399Y)-myc and 5 μg of CMV-CITED2 using FuGENE 6 (Roche Molecular Biochemicals). 5 μg of CMV-TFAP2A or the control vector were also co-transfected. 48 h post-transfection the cell pellet was lysed for 5 min on ice by adding 5–10 volumes of lysis buffer (20 mm Tris-HCl, pH 8.0, 20 mmNaCl, 1 mm DTT, 0.5% Nonidet P-40, 320 mmsucrose, and Complete protease inhibitor mixture (Roche Molecular Biochemicals)). The nuclei were then pelleted by centrifugation and lysed for 30 min on ice by adding 5–10 volumes of nuclei lysis buffer (20 mm HEPES-KOH, pH 8.0, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 1 mm DTT, 25% glycerol, and Complete protease inhibitor mixture (Roche Molecular Biochemicals)). Before the binding assay, the nuclear extracts contained in the supernatant were diluted with two volumes of 20 mm HEPES-KOH, pH 8.0 containing the Complete protease inhibitor mixture (Roche Molecular Biochemicals). The synthetic oligonucleotide 5′-GATCGAACTGACCGCCCGCGGCCCGT-3′ corresponding to the consensus TFAP2 binding site of the human metallothionein IIa promoter was end-labeled with [γ-32P]ATP and T4 polynucleotide kinase and annealed to a slight excess of the unlabeled complementary strand. Unincorporated nucleotides were removed using a MicrospinTM G-25 column (Amersham Biosciences). Unlabeled oligonucleotide was annealed in a similar manner and used for competition experiments. The DNA binding reaction was performed in a volume of 25 μl containing 1.5 μg of nuclear extract, 1 μg of poly(dI-dC)·poly(dI-dC), 25 mm HEPES-KOH, pH 7.9, 150 mm NaCl, 1 mm EDTA, 5 mm DTT, and 10% glycerol. This mixture was incubated for 10 min on ice before adding 10–15 fmol of radiolabeled probe. The binding reaction was then carried out for 30 min at room temperature before loading the samples onto a 5% polyacrylamide gel (37.5:1, acrylamide:bisacrylamide) pre-run for at least 2 h in 25 mm Tris, pH 8.3, 25 mmboric acid, 0.5 mm EDTA. Electrophoresis was performed for 2–3 h at 200 V at 4 °C. The gel was dried out and autoradiographed. For competition experiments, a 50-fold molar excess of unlabeled oligonucleotide was mixed with the probe before adding nuclear extract. To determine whether p300 and CBP act as TFAP2A co-activators, we transiently co-transfected Hep3B cells (a human hepatocellular carcinoma cell line that has low levels of endogenous TFAP2 and CITED2) with a luciferase reporter cloned downstream of TFAP2 binding elements (p3xAP2-Bluc) and vectors expressing TFAP2A, CITED2, CBP, or p300 (Fig.1 A). Under these conditions, the reporter was mildly activated by the transfection of TFAP2A expression vector alone. Consistent with our previous results (19Bamforth S.D. Braganca J. Eloranta J.J. Murdoch J.N. Marques F.I. Kranc K.R. Farza H. Henderson D.J. Hurst H.C. Bhattacharya S. Nat. Genet. 2001; 29: 469-474Crossref PubMed Scopus (255) Google Scholar), co-transfection of CMV-CITED2 resulted in further activation of the reporter, which was dependent on the presence of co-transfected CMV-TFAP2A. A further increase of the reporter activity (in a dose-responsive manner) was observed when CBP or p300 expressing plasmids were co-transfected with both CMV-TFAP2A and CMV-CITED2 (Fig.1 A). CMV-CBP or CMV-p300 showed no effect on reporter plasmid activity in the absence of co-transfected CMV-TFAP2A or CMV-CITED2. These results indicate that p300/CBP can co-activate TFAP2A but only in the presence of CITED2. Similar results were observed in HepG2 cells, another human hepatocellular carcinoma cell line (Fig. 1 B), indicating that the CITED2-dependent co-activation of TFAP2A by CBP and p300 is not restricted to one particular cell line. To determine whether endogenous p300 and CBP are necessary for TFAP2 transactivation, we examined the transcriptional activity of TFAP2 isoforms in MEFs. We isolated MEFs from embryos lacking either one allele of p300 (p300+/−) or one allele of CBP (CBP+/−). We co-transfected the TFAP2 reporter gene and plasmids expressing TFAP2 isoforms into these MEFs and into wild-type control MEFs isolated from littermate embryos (Fig. 1, C andD). As MEFs express CITED2 (9Bhattacharya S. Michels C.L. Leung M.K. Arany Z.P. Kung A.L. Livingston D.M. Genes Dev. 1999; 13: 64-75Crossref PubMed Scopus (316) Google Scholar), it was not necessary to also transfect CITED2 in these experiments. In p300+/− and CBP+/− MEFs we observed reduced transcriptional activities of TFAP2A and TFAP2C isoforms (Fig. 1, C and D). TFAP2B transcriptional activity was also reduced in CBP+/−MEFs (Fig. 1 D), whereas only a weak effect was observed in p300+/− MEFs (Fig. 1 C). Co-transfection of p300 and CBP expressing plasmids in p300+/− and CBP+/− MEFs, respectively, successfully rescued the defective TFAP2 co-activation (Fig. 1, C and D). These results indicate that normal levels of endogenous p300 and/or CBP are necessary for full transcriptional activation by TFAP2. We next determined whether p300 and TFAP2A can physically interact in mammalian cells. We initially attempted to detect TFAP2A in anti-p300 immunoprecipitates. TFAP2A migrates at ∼50 kDa, very close to the immunoglobulin heavy chain from the immunoprecipitating antibody, and this interfered with its detection by Western blotting (data not shown). To overcome this, we transfected U2-OS cells with CMV-p300-myc and CMV-GAL4-TFAP2A. GAL4-TFAP2A (a fusion of the yeast GAL4 DNA binding domain with TFAP2A) migrates at 70 kDa, away from the immunoglobulin heavy chain (Fi

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