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• W2083136237 abstract "An intricate array of heterogeneous transcription factors participate in programming tissue-specific gene expression through combinatorial interactions that are unique to a given cell-type. The zinc finger-containing transcription factor GATA4, which is widely expressed in mesodermal and endodermal derived tissues, is thought to regulate cardiac myocyte-specific gene expression through combinatorial interactions with other semi-restricted transcription factors such as myocyte enhancer factor 2, nuclear factor of activated T-cells, serum response factor, and Nkx2.5. Here we determined that GATA4 also interacts with the cardiac-expressed basic helix-loop-helix transcription factor dHAND (also known as HAND2). GATA4 and dHAND synergistically activated expression of cardiac-specific promoters from the atrial natriuretic factor gene, the b-type natriuretic peptide gene, and the α-myosin heavy chain gene. Using artificial reporter constructs this functional synergy was shown to be GATA site-dependent, but E-box site-independent. A mechanism for the transcriptional synergy was suggested by the observation that the bHLH domain of dHAND physically interacted with the C-terminal zinc finger domain of GATA4 forming a higher order complex. This transcriptional synergy observed between GATA4 and dHAND was associated with p300 recruitment, but not with alterations in DNA binding activity of either factor. Moreover, the bHLH domain of dHAND directly interacted with the CH3 domain of p300 suggesting the existence of a higher order complex between GATA4, dHAND, and p300. Taken together with previous observations, these results suggest the existence of an enhanceosome complex comprised of p300 and multiple semi-restricted transcription factors that together specify tissue-specific gene expression in the heart. An intricate array of heterogeneous transcription factors participate in programming tissue-specific gene expression through combinatorial interactions that are unique to a given cell-type. The zinc finger-containing transcription factor GATA4, which is widely expressed in mesodermal and endodermal derived tissues, is thought to regulate cardiac myocyte-specific gene expression through combinatorial interactions with other semi-restricted transcription factors such as myocyte enhancer factor 2, nuclear factor of activated T-cells, serum response factor, and Nkx2.5. Here we determined that GATA4 also interacts with the cardiac-expressed basic helix-loop-helix transcription factor dHAND (also known as HAND2). GATA4 and dHAND synergistically activated expression of cardiac-specific promoters from the atrial natriuretic factor gene, the b-type natriuretic peptide gene, and the α-myosin heavy chain gene. Using artificial reporter constructs this functional synergy was shown to be GATA site-dependent, but E-box site-independent. A mechanism for the transcriptional synergy was suggested by the observation that the bHLH domain of dHAND physically interacted with the C-terminal zinc finger domain of GATA4 forming a higher order complex. This transcriptional synergy observed between GATA4 and dHAND was associated with p300 recruitment, but not with alterations in DNA binding activity of either factor. Moreover, the bHLH domain of dHAND directly interacted with the CH3 domain of p300 suggesting the existence of a higher order complex between GATA4, dHAND, and p300. Taken together with previous observations, these results suggest the existence of an enhanceosome complex comprised of p300 and multiple semi-restricted transcription factors that together specify tissue-specific gene expression in the heart. α-myosin heavy chain atrial natriuretic factor b-type natriuretic peptide myocyte enhancer factor-2 electrophoretic mobility shift assay glutathione S-transferase basic-helix-loop-helix cAMP-response element-binding protein-binding protein cytomegalovirus nuclear factor of activated T-cells serum response factor The zinc finger-containing transcription factor GATA4 is expressed in multiple organs derived from both endodermal and mesodermal origins, where it regulates tissue-specific gene expression through interactions with other semi-restricted transcription factors. In cardiac myocytes, GATA4 is thought to play a particularly important role in regulating expression of most cardiac-expressed genes, including α-myosin heavy chain (α-MHC),1 cardiac troponin-C, atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), cardiac troponin-I, sodium/calcium exchanger, cardiac-restricted ankyrin repeat protein, A1 adenosine receptor, m2 muscarinic receptor, and myosin light chain 1/3 (1Thuerauf D.J. Hanford D.S. Glembotski C.C. J. Biol. Chem. 1994; 269: 17772-17775Abstract Full Text PDF PubMed Google Scholar, 2Molkentin J.D. Kalvakolanu D.V. Markham B.E. Mol. Cell. Biol. 1994; 14: 4947-4957Crossref PubMed Google Scholar, 3Grepin C. Dagnino L. Robitaille L. Haberstroh L. Antakly T. Nemer M. Mol. Cell. Biol. 1994; 14: 3115-3129Crossref PubMed Scopus (245) Google Scholar, 4Ip H.S. Wilson D.B. Heikinheimo M. Tang Z. Ting C.N. Simon M.C. Leiden J.M. Parmacek M.S. Mol. Cell. 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Chem. 1999; 274: 14204-14209Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 12Rosoff M.L. Nathanson N.M. J. Biol. Chem. 1998; 273: 9124-9129Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar–13Kuo H. Chen J. Ruiz-Lozano P. Zou Y. Nemer M. Chien K.R. Development. 1999; 126: 4223-4234PubMed Google Scholar). In addition to directly controlling cardiac structural and regulatory gene expression, cardiac-expressed GATA factors indirectly support tissue-specific gene expression by regulating expression of other transcription factors. For example, GATA factors regulate developmental expression of the homeodomain-containing transcription factor Nkx2.5, myocyte enhancer factor-2 (MEF2), and dHAND in the heart by providing a reinforcing transcriptional regulatory circuit mediated through direct promoter interactions (14Lien C.L., Wu, C. Mercer B. Webb R. Richardson J.A. Olson E.N. Development. 1999; 126: 75-84Crossref PubMed Google Scholar, 15Searcy R.D. Vincent E.B. Liberatore C.M. Yutzey K.E. Development. 1998; 125: 4461-4470Crossref PubMed Google Scholar, 16Gajewski K. Fossett N. Molkentin J.D. Schulz R.A. Development. 1998; 126: 5679-5688Google Scholar, 17McFadden D.G. Charite J. Richardson J.A. Srivastava D. Firulli A.B. Olson E.N. Development. 2000; 127: 5331-5341Crossref PubMed Google Scholar). GATA4 was shown to directly interact with Nkx2.5 through the C-terminal zinc finger domain and the helix III region of the homeodomain present within each factor, respectively (18Durocher D. Charron F. Warren R. Schwartz R.J. Nemer M. EMBO J. 1997; 16: 5687-5696Crossref PubMed Scopus (537) Google Scholar, 19Sepulveda J.L. Belaguli N. Nigam V. Chen C.Y. Nemer M. Schwartz R.J. Mol. Cell. Biol. 1998; 18: 3405-3415Crossref PubMed Scopus (270) Google Scholar, 20Lee Y. Shioi T. Kasahara H. Jobe S.M. Wiese R.J. Markham B.E. Izumo S. Mol. Cell. Biol. 1998; 18: 3120-3129Crossref PubMed Scopus (242) Google Scholar). GATA-4 also physically interacts by way of its C-terminal zinc finger with nuclear factor of activated T-cells (NFAT) and MEF2 (21Molkentin J.D., Lu, J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2182) Google Scholar,22Morin S. Charron F. Robitaille L. Nemer M. EMBO J. 2000; 19: 2046-2055Crossref PubMed Scopus (274) Google Scholar). Finally, GATA4 directly interacts with the MADS box-containing transcription factor serum response factor (SRF), which together synergistically regulate expression of the ANF and α-actin genes in cardiomyocytes (23Belaguli N.S. Sepulveda J.L. Nigam V. Charron F. Nemer M. Schwartz R.J. Mol. Cell. Biol. 2000; 20: 7550-7558Crossref PubMed Scopus (155) Google Scholar, 24Morin S. Paradis P. Aries A. Nemer M. Mol. Cell. Biol. 2001; 21: 1036-1044Crossref PubMed Scopus (89) Google Scholar). The studies discussed above suggest that cardiac-expressed GATA factors interact with an array of heterotypic transcription factors in the heart. In addition to transcription factor interactions, GATA4 interacts with discrete transcriptional co-activators or general repressors. For example, GATA4 was recently shown to directly interact with p300/CBP (cAMP-response element-binding protein-binding protein) resulting in synergistic gene activation (25Dai Y.S. Markham B.E. J. Biol. Chem. 2001; 276: 37178-37185Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The N- and C-terminal zinc finger domains of GATA4 directly interacted with the cysteine/histidine-rich (CH3) region of p300 (25Dai Y.S. Markham B.E. J. Biol. Chem. 2001; 276: 37178-37185Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Given the ability of p300/CBP to interact with a heterogeneous array of transcription factors (reviewed in Ref. 26Vo N. Goodman R.H. J. Biol. Chem. 2001; 276: 13505-13508Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar), the observed GATA4-p300 interaction suggested a mechanism whereby a diverse array of cardiac-expressed transcription factors could simultaneously interact through a p300 scaffold. GATA4 also interacts with the transcriptional modifying protein friend of GATA-2 (FOG-2) through a physical interaction involving the N-terminal zinc finger of GATA-4 (27Lu J.R. McKinsey T.A., Xu, H. Wang D.Z. Richardson J.A. Olson E.N. Mol. Cell. Biol. 1999; 19: 4495-4502Crossref PubMed Scopus (177) Google Scholar, 28Svensson E.C. Tufts R.L. Polk C.E. Leiden J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 956-961Crossref PubMed Scopus (235) Google Scholar, 29Tevosian S.G. Deconinck A.E. Cantor A.B. Rieff H.I. Fujiwara Y. Corfas G. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 950-955Crossref PubMed Scopus (173) Google Scholar). This interaction is conserved in Drosophila where the friend of GATA-2 homologue, U-shaped (Ush), interacts withpannier, a GATA homologue (30Haenlin M. Cubadda Y. Blondeau F. Heitzler P. Lutz Y. Simpson P. Ramain P. Genes Dev. 1997; 11: 3096-3108Crossref PubMed Scopus (165) Google Scholar). Interestingly,GATA4, Fog-2, and p300 gene-targeted mice each die during embryogenesis with significant cardiac abnormalities (31Tevosian S.G. Deconinck A.E. Tanaka M. Schinke M. Litovsky S.H. Izumo S. Fujiwara Y. Orkin S.H. Cell. 2000; 101: 729-739Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 32Svensson E.C. Huggins G.S. Lin H. Clendenin C. Jiang F. Tufts R. Dardik F.B. Leiden J.M. Nat. Genet. 2000; 25: 353-356Crossref PubMed Scopus (149) Google Scholar, 33Yao 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, 34Molkentin J.D. Lin Q. Duncan S.A. Olson E.N. Genes Dev. 1997; 11: 1061-1072Crossref PubMed Scopus (936) Google Scholar, 35Kuo C.T. Morrisey E.E. Anandappa R. Sigrist K., Lu, M.M. Parmacek M.S. Soudais C. Leiden J.M. Genes Dev. 1997; 11: 1048-1060Crossref PubMed Scopus (853) Google Scholar). Like GATA4 gene-targeted mice, disruption of the gene encoding the transcription factor dHAND results in embryonic lethality because of cardiac abnormalities, suggesting non-redundant roles for each of these factors in specifying developmental gene expression in vivo (36Srivastava D. Thomas T. Lin Q. Kirby M.L. Brown D. Olson E.N. Nat. Genet. 1997; 16: 154-160Crossref PubMed Scopus (551) Google Scholar). Whereas less is understood of the manner in which dHAND regulates target genes in the heart, PCR-mediated site selection identified a series of specific E-box consensus elements verifying the ability of dHAND to bind DNA (37Dai Y.S. Cserjesi P. J. Biol. Chem. 2002; 277: 12604-12612Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Here we demonstrate that the transcription factors GATA4 and dHAND physically interact with one another to synergistically regulate expression of cardiac gene promoters. The identified functional interaction is mediated through GATA, but not E-box DNA-binding sites, suggesting a dHAND-binding site-independent mechanism of regulation. Finally, dHAND was shown to physically interact with the transcriptional co-activator p300, which was necessary for functional synergy with GATA4. These data suggest a paradigm whereby cardiac-expressed transcription factors form large multisubunit complexes in conjunction with p300/CBP. GATA4-ΔN (N-terminal zinc finger) in the pMT2 expression vector was generated using PCR to delete amino acids 216–240, whereas the ΔC construct deleted amino acids 270–294 of GATA4. Expression vectors encoding GATA4 amino acids 253–441, wild type and site-specific mutants, were generated by PCR and subsequently subcloned into the pcDNA3.1-His vector (25Dai Y.S. Markham B.E. J. Biol. Chem. 2001; 276: 37178-37185Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). pcDNA3.1-pFlag-GATA4 was described previously (25Dai Y.S. Markham B.E. J. Biol. Chem. 2001; 276: 37178-37185Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). pFLAG-CMV-2-BAP encoding a FLAG-tagged bacterial alkaline phosphatase (Sigma) was used as a control plasmid in mammalian transfection experiments. The GATA4 C-terminal zinc finger site-specific mutant expression plasmids were described previously (25Dai Y.S. Markham B.E. J. Biol. Chem. 2001; 276: 37178-37185Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), as was pcDNA-His-dHAND (37Dai Y.S. Cserjesi P. J. Biol. Chem. 2002; 277: 12604-12612Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). pCMV-bHLH-Nuc-Myc is a mammalian expression vector encoding the bHLH region of dHAND (amino acids 98–157), which was cloned as a NcoI-XhoI fragment in-frame with three nuclear localization signals contained within the pCMV/Nu/Myc vector (Invitrogen). For construction of pcDNA3.1-His-dHAND ΔC, full-length dHAND was digested with StuI at amino acids 166 and the resultant fragment (encoding amino acids 1–166) was cloned into pcDNA3.1-His vector in-frame. pcDNA3.1-His-dHANDΔN was generated by deleting the first 85 amino acids using a NarI restriction site. pcDNA3.1-His-bHLH was generated by deleting amino acids 86–166 of dHAND using NarI and StuI restriction enzyme sites. pcDNA3.1-His-dHAND ΔGTA was generated by deleting amino acids 102–104 of dHAND using the QuikChange mutagenesis kit (Stratagene), which was further modified by substituting amino acid residues 107–111 of dHAND (i.e.KERRR) with 5 alanine residues using a combined PCR and ligase chain reaction technique as described previously (38Bi W. Stambrook P.J. Anal. Biochem. 1998; 256: 137-140Crossref PubMed Scopus (45) Google Scholar). The resultant PCR product was cloned into the NcoI and XhoI sites of the pCMV/Nuc/Myc vector for mammalian expression. For in vitro translation of the basic mutation of dHAND, anNcoI-NotI fragment from pCMV-dHAND basic mutant-Nuc-NLS vector was cloned into pGBKT7 vector (CLONTECH). PRSET 2b-dHAND was generated by cloning the NcoI fragment of dHAND into pRSET 2b vector. GST-dHAND was constructed by cloning the NcoI fragment of dHAND into the SmaI site in the pGEX-2T vector (AmershamBiosciences). GST GATA4 fragments were described previously (25Dai Y.S. Markham B.E. J. Biol. Chem. 2001; 276: 37178-37185Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The mouse CMV-E12 expression vector was a gift of Dr. Andrew Lassar (Harvard, Boston MA). The mammalian expression vector CMV-p300 CH3Δ (missing amino acids 1737–1836) was purchased from Upstate Biotech. The CMV-p300 expression vector was a gift from Dr. David Livingston (Harvard). The E1A mammalian expression vector and the GST-ElA bacterial vector were previously described (25Dai Y.S. Markham B.E. J. Biol. Chem. 2001; 276: 37178-37185Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 39Chakravarti D. Ogryzko V. Kao H.Y. Nash A. Chen H. Nakatani Y. Evans R.M. Cell. 1999; 96: 393-403Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). An Id1 cDNA was derived from a human heart yeast two-hybrid library and was subcloned into the pCI-neo mammalian expression vector (Promega) at an EcoRI site. cDNA fragments encoding p300 N1, N2, N3, and C fragments were generated by PCR and cloned into theXhoI and HindIII sites in pRSET 2C vector to permit in vitro translation (Invitrogen) (25Dai Y.S. Markham B.E. J. Biol. Chem. 2001; 276: 37178-37185Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). PRSET2C-p300 N3 (amino acids 1186–1860) was digested with NcoI at amino acid 1587 and PvuII at amino acid 1817 to generate the p300 CH3 construct (amino acids 1517–1817). The p300 CH3 (amino acids 1587–1817) was then cloned into pGEX4T-1 at a SmaI site in-frame to obtain the GST-CH3 fusion protein construct, which was previously described as GST-N3 (25Dai Y.S. Markham B.E. J. Biol. Chem. 2001; 276: 37178-37185Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The ANF-luciferase promoter construct (−638 base pairs upstream from the transcriptional start site) was described previously (40Knowlton K.U. Baracchini E. Ross R.S. Harris A.N. Henderson S.A. Evans S.M. Glembotski C.C. Chien K.R. J. Biol. Chem. 1991; 266: 7759-7768Abstract Full Text PDF PubMed Google Scholar). The GATA site-ANF-luciferase reporter was derived by cloning 6 copies of the GATA site from the ANF proximal promoter (starting at position −124 base pairs upstream from the transcriptional start site) into the pGL2-basic vector as described before (25Dai Y.S. Markham B.E. J. Biol. Chem. 2001; 276: 37178-37185Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The α-MHC-luciferase (−330 base pairs upstream from the transcriptional start site) and the GATA site-MHC-luciferase reporter were described previously (2Molkentin J.D. Kalvakolanu D.V. Markham B.E. Mol. Cell. Biol. 1994; 14: 4947-4957Crossref PubMed Google Scholar). The BNP-luciferase reporter (−116 base pairs upstream from the transcriptional start site) was described previously (1Thuerauf D.J. Hanford D.S. Glembotski C.C. J. Biol. Chem. 1994; 269: 17772-17775Abstract Full Text PDF PubMed Google Scholar). The dHAND E box artificial reporter 4xE-box-TATA-Luc was generated by cloning 4 copies of the optimized dHAND-binding site (CATCTG) into pTATA-Luc at a XhoI site (37Dai Y.S. Cserjesi P. J. Biol. Chem. 2002; 277: 12604-12612Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The pTATA-Luc contains the TATA box derived from the α-MHC minimal promoter in pGL2 basic vector (Promega) (25Dai Y.S. Markham B.E. J. Biol. Chem. 2001; 276: 37178-37185Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). HeLa cells were transfected with pFlag-GATA4 and CMV-dH-bHLH-nuc-Myc. The cells were lysed at 4 °C in lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5% Triton-100) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 μg/ml aprotinin). Lysates were cleared by centrifugation at 18,000 × g for 10 min. Lysate proteins were immunoprecipitated overnight at 4 °C with FLAG antibody-agarose (Sigma). The agarose was washed and the bound proteins were resolved in SDS-PAGE and Western blotted. The blot was incubated with mouse anti-Myc (Sigma). A T7 mouse monoclonal antibody was purchased from Novagen, whereas GATA4 antiserum was purchased from Santa Cruz. All GST fusion proteins were overexpressed in Escherichia coli BL21 cells. Binding assays were performed with labeled proteins synthesized in vitrousing the TnT coupled reticulocyte lysate system (Promega) in the presence of 35S-labeled methionine (AmershamBiosciences) as described previously (25Dai Y.S. Markham B.E. J. Biol. Chem. 2001; 276: 37178-37185Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Equal amounts of immobilized GST fusion proteins were incubated for 2 h at 4 °C with 10 μl of 35S-labeled proteins in GST binding buffer containing 40 mm Hepes, pH 7.2, 50 mm Na acetate, pH 7.0, 200 mm NaCl, 2 mm EDTA, 5 mmdithiothreitol, 0.5% Nonidet P-40, protease inhibitors, and 2 μg of bovine serum albumin/ml. After four washes in GST binding buffer, beads were boiled in SDS sample buffer to elute bound protein, which was subsequently resolved by SDS-PAGE and analyzed by autoradiography. HeLa and HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mm glutamine, streptomycin (10 g/liter), and penicillin (10 g/liter). All transfections were performed in 6-well plates with Lipofectin and LipofectAMINE Plus reagent as suggested by the manufacturer (Invitrogen) or with Tfx-20 reagent (Promega). Cells were transfected with 0.3 μg of DNA containing the various reporter plasmids; α-MHC-GATAx4-Luc, α-MHC-LUC, ANF-LUC, ANF-GATAx6-LUC, and 0.3 μg of expression vectors for pFlag-GATA4, pFlag-BAP, pcDNA3-p300HAT, pMT2-GATA4, pMT2GATA4 mutants, pcDNA3-dHAND, and pcDNA3-dHAND mutants, whereas 0.6 μg of CMV-p300 and 0.1 μg of CMV-Id1, CMV-E1A, CMV-E12, and pcDNA3-p300 CH3 were used. CMV-β-galactosidase (20 ng in each well) was used as internal control. Luciferase activity was measured in a luminometer, which was normalized to β-galactosidase activity using Tropix's Galacto-Star reporter assay system (Tropix). Each value presented is the average of triplicate samples and is representative of multiple independent experiments. The data were statistically analyzed with a Student's t test. Cardiomyocyte cultures were prepared as described previously (41De Windt L.J. Lim H.W. Haq S. Force T. Molkentin J.D. J. Biol. Chem. 2000; 275: 13571-13579Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Cultures were transfected in serum-free M199 medium and plated in triplicate 6-cm plates with 1 μg of ANF-luc reporter and 0.2 μg of Flag-GATA4 and 0.8 μg of pcDNA-His-dHAND in 6 μl of Tfx-20 reagent (Promega). The cardiomyocytes were washed with phosphate-buffered saline 14 h post-transfection. The cardiomyocytes were lysed and luciferase activity was measured 24 h post-transfection. Conditions for EMSA were described previously (37Dai Y.S. Cserjesi P. J. Biol. Chem. 2002; 277: 12604-12612Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The GATA DNA-binding site (from the ANF promoter) was 5′-CTGATAACTCTGATAACTCTGATAACTGGTAC, whereas the dHAND-binding site consisted of the sequence 5′-TCGACAGGGCCATCTGGCATTG. To gain greater insight into the transcriptional mechanisms whereby GATA transcription factors regulate cardiac-specific gene expression, we surveyed the ability of GATA4 to function in cooperation with the cardiac-enriched bHLH protein dHAND. Promoters from the ANF , BNP, and α-MHC genes were employed for analysis because each was previously shown to require GATA DNA binding activity for cardiac-specific expression (reviewed in Ref. 63Molkentin J.D. J. Biol. Chem. 2000; 275: 38949-38952Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar). In the presence of both GATA4 and dHAND expression vectors, the ANF promoter showed ∼40- and 60-fold induction in transiently transfected neonatal cardiomyocytes and HeLa cells, respectively (Fig.1 A). Whereas a similar degree of transcriptional synergy was observed in each cell type, HeLa cells lack some cardiac-expressed transcription factors that might otherwise dominantly regulate promoter activity. Indeed, transient transfection of the BNP and α-MHC luciferase fusion constructs into HeLa cells demonstrated a similar degree of synergy in the presence of co-transfected GATA4 and dHAND (Fig. 1, B and C). Importantly, co-transfection of GATA4 and dHAND expression constructs did not result in squelching of either factor compared with individually transfected cells (Fig. 1 D). These results indicate that the transcription factors GATA4 and dHAND functionally synergize to enhance expression of the assayed cardiac-expressed gene promoters. Finally, we also observed that the closely related transcription factors GATA5 and GATA6 synergized with dHAND on the ANF promoter, whereas eHAND (HAND1) did not synergize with GATA4 (data not shown). To analyze the mechanism of this observed transcriptional synergy between GATA4 and dHAND in more detail, multiple deletion constructs were generated and assayed. Deletion of the C terminus of dHAND did not significantly reduce GATA4 synergy on the ANF promoter, suggesting that this domain was dispensable for interaction (Fig.2 A). In contrast, deletion of the N-terminal transactivation domain in dHAND, or both N- and C-terminal domains together, reduced or eliminated transcriptional synergy (Fig. 2 A). However, it should be noted that dHAND contains a strong transcriptional activation domain only within its N terminus, suggesting that deletion of this domain could simply reduce the transcriptional potency of any underlying interaction (37Dai Y.S. Cserjesi P. J. Biol. Chem. 2002; 277: 12604-12612Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Last, the basic domain of dHAND was directly mutated within the context of the full-length protein, which eliminated all transcriptional synergy (Fig. 2 A). Collectively, these results are consistent with the interpretation that GATA4 interacts with dHAND through the bHLH domain to augment transcriptional activation. To more carefully elucidate the critical interacting domains within GATA4, a similar series of deletion mutants was generated and assayed for functional synergy with dHAND. Deletion of the N-terminal zinc finger domain of GATA4 did not alter transcriptional synergy with dHAND, although deletion of the C-terminal zinc finger domain severely compromised the functional interaction (Fig. 2 B). These data indicate that the C-terminal zinc finger of GATA4 is most critical for mediating transcriptional synergy with dHAND. The transcriptional synergy observed between GATA4 and dHAND could result from either independent binding of each factor to its cognate site, or from a direct physical interaction. To test this later possibility, glutathione S-transferase (GST) fusion constructs were generated and used for in vitro precipitation experiments. cDNA fragments encoding multiple domains of GATA4 were fused to the GST coding sequence to permit generation of each recombinant protein in bacteria. Each purified fusion protein was loaded onto a glutathione column and in vitro translated dHAND protein (35S-labeled methionine) was subsequently added to assay for interaction (Fig. 3). The data demonstrate that the C-terminal zinc finger domain of GATA4 strongly interacted with the in vitro translated dHAND, whereas the N-terminal zinc finger domain only showed a minimal interaction (Fig.3). Comparable quantity of each GST-GATA4 fusion construct was loaded onto GST beads (data not shown), suggesting that the zinc finger domains of GATA4 are capable of physically interacting with dHANDin vitro. To more carefully elucidate the interactive surface within the C-terminal zinc finger of GATA4, a series of sequential site-directed mutants was generated for in vitro translation and subsequent interaction with GST-dHAND. All site-directed GATA4 mutant proteins were produced at roughly similar levels by in vitrotranslation (Fig. 4, top panel). The data demonstrate that each GATA4 mutant was capable of physically interacting with dHAND, except the WRR-SSS mutant, which alters residues at the tip of the C-terminal zinc finger (Fig. 4). GST alone did not interact with any of the mutant or deletion constructs (Figs. 3 and 4). As a further control, deletion of the entire C-terminal zinc finger domain in GATA4 severely attenuated the interaction with dHAND (Fig. 4). Collectively, these results not only confirm the requirement of the C-terminal zinc finger domain for mediating the dHAND interaction, but they also suggest that residues within the tip of the GATA4 C-terminal zinc finger are most critical. To examine the domain of dHAND that facilitates GATA4 interaction, a series of dHAND deletion constructs were generated for production ofin vitro translated protein. Analysis of protein levels showed equivalent amounts of each dHAND deletion protein, except for the bHLH domain, which was only detected at ∼10% of the signal of the full-length protein (Fig 5 A). This decreased signal likely reflects the presence of far fewer methionine residues available for in vitrotranslation-dependent radioactive labeling compared with the larger fragments. In any event, the data demonstrate that the bHLH domain in dHAND is sufficient to mediate a physical interaction with a GST construct fused to the C-terminal zinc finger domain of GATA4, but not with GST alone (Fig. 5 A). Last, mutatio" @default.
• W2083136237 created "2016-06-24" @default.
• W2083136237 creator A5007435444 @default.
• W2083136237 creator A5009169258 @default.
• W2083136237 creator A5059467245 @default.
• W2083136237 creator A5075550603 @default.
• W2083136237 date "2002-07-01" @default.
• W2083136237 modified "2023-10-10" @default.
• W2083136237 title "The Transcription Factors GATA4 and dHAND Physically Interact to Synergistically Activate Cardiac Gene Expression through a p300-dependent Mechanism" @default.
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