MEF2-dependent Recruitment of the HAND1 Transcription Factor Results in Synergistic Activation of Target Promoters
2005; Elsevier BV; Volume: 280; Issue: 37 Linguagem: Inglês
10.1074/jbc.m507640200
ISSN1083-351X
AutoresSteves Morin, Gina Pozzulo, Lynda Robitaille, James C. Cross, Mona Nemer,
Tópico(s)Congenital Heart Disease Studies
ResumoHAND proteins are tissue-restricted members of the basic helix-loop-helix transcription factor family that play critical roles in cell differentiation and organogenesis including placental, cardiovascular, and craniofacial development. Nevertheless, the molecular basis underlying the developmental action of HAND proteins remains undefined. Within the embryo, HAND1 is first detected in the developing heart where it becomes restricted to the atrial and left ventricular compartments, a pattern identical to that of the Nppa gene, which encodes atrial natriuretic factor, the major secretory product of the heart. We hereby report that the cardiac atrial natriuretic factor promoter is directly activated by HAND1, making it the first known HAND1 transcriptional target. The action of HAND1 does not require heterodimerization with class I basic helix-loop-helix factors or DNA binding through E-box elements. Instead, HAND1 is recruited to the promoter via physical interaction with MEF2 proteins. MEF2/HAND1 interaction results in synergistic activation of MEF2-dependent promoters, and MEF2 binding sites are sufficient to mediate this synergy. MEF2 binding to DNA is not enhanced in the presence of HAND1. Instead, cooperativity likely results from corecruitment of co-activators such as CREB-binding protein. The related HAND2 protein can also synergize with MEF2. Thus, HAND proteins act as cell-specific developmental co-activators of the MEF2 family of transcription factors. These findings identify a novel mechanism for HAND action in the heart and provide a general paradigm to understand the mechanism of HAND action in organogenesis. HAND proteins are tissue-restricted members of the basic helix-loop-helix transcription factor family that play critical roles in cell differentiation and organogenesis including placental, cardiovascular, and craniofacial development. Nevertheless, the molecular basis underlying the developmental action of HAND proteins remains undefined. Within the embryo, HAND1 is first detected in the developing heart where it becomes restricted to the atrial and left ventricular compartments, a pattern identical to that of the Nppa gene, which encodes atrial natriuretic factor, the major secretory product of the heart. We hereby report that the cardiac atrial natriuretic factor promoter is directly activated by HAND1, making it the first known HAND1 transcriptional target. The action of HAND1 does not require heterodimerization with class I basic helix-loop-helix factors or DNA binding through E-box elements. Instead, HAND1 is recruited to the promoter via physical interaction with MEF2 proteins. MEF2/HAND1 interaction results in synergistic activation of MEF2-dependent promoters, and MEF2 binding sites are sufficient to mediate this synergy. MEF2 binding to DNA is not enhanced in the presence of HAND1. Instead, cooperativity likely results from corecruitment of co-activators such as CREB-binding protein. The related HAND2 protein can also synergize with MEF2. Thus, HAND proteins act as cell-specific developmental co-activators of the MEF2 family of transcription factors. These findings identify a novel mechanism for HAND action in the heart and provide a general paradigm to understand the mechanism of HAND action in organogenesis. The HAND1 and HAND2 proteins form an evolutionarily conserved subgroup of the tissue-restricted bHLH 3The abbreviations used are: bHLH, basic helix-loop-helix; ANF, atrial natriuretic factor; CREB, cAMP-response element-binding protein; GST, glutathione S-transferase; HA, hemagglutinin; HLH, helix-loop-helix; PBS, phosphate-buffered saline; BSA, bovine serum albumin; CBP, CREB-binding protein; MCK, muscle creatine kinase.3The abbreviations used are: bHLH, basic helix-loop-helix; ANF, atrial natriuretic factor; CREB, cAMP-response element-binding protein; GST, glutathione S-transferase; HA, hemagglutinin; HLH, helix-loop-helix; PBS, phosphate-buffered saline; BSA, bovine serum albumin; CBP, CREB-binding protein; MCK, muscle creatine kinase. factors that are expressed in several neural crest and mesodermal derivatives, most notably in the heart and limbs (1Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (582) Google Scholar, 2Srivastava D. Cserjesi P. Olson E.N. Science. 1995; 270: 1995-1999Crossref PubMed Scopus (449) Google Scholar, 3Sparrow D.B. Kotecha S. Towers N. Mohun T.J. Mech. Dev. 1998; 71: 151-163Crossref PubMed Scopus (45) Google Scholar, 4Cserjesi P. Brown D. Lyons G.E. Olson E.N. Dev. Biol. 1995; 170: 664-678Crossref PubMed Scopus (209) Google Scholar, 5Firulli A.B. Gene (Amst.). 2003; 312: 27-40Crossref PubMed Scopus (88) Google Scholar). During embryogenesis, the two HAND genes are initially co-expressed throughout the heart tube, but their expression pattern becomes complementary as the heart develops, with HAND1 marking the atria and left ventricle while HAND2 marks the right ventricle. Genetic studies have demonstrated clearly that both proteins are required for proper cardiac cell differentiation and heart morphogenesis (6Riley P. Anson-Cartwright L. Cross J.C. Nat. Genet. 1998; 18: 271-275Crossref PubMed Scopus (424) Google Scholar, 7Firulli A.B. McFadden D.G. Lin Q. Srivastava D. Olson E.N. Nat. Genet. 1998; 18: 266-270Crossref PubMed Scopus (296) Google Scholar, 8Srivastava D. Thomas T. Lin Q. Kirby M.L. Brown D. Olson E.N. Nat. Genet. 1997; 16: 154-160Crossref PubMed Scopus (556) Google Scholar, 9Yelon D. Ticho B. Halpern M.E. Ruvinsky I. Ho R.K. Silver L.M. Stainier D.Y. Development (Camb.). 2000; 127: 2573-2582PubMed Google Scholar, 10McFadden D.G. Barbosa A.C. Richardson J.A. Schneider M.D. Srivastava D. Olson E.N. Development (Camb.). 2005; 132: 189-201Crossref PubMed Scopus (251) Google Scholar). Targeted inactivation of hand2 leads to embryonic lethality around embryonic day 10.5 because of cardiovascular defects that include right ventricular hypoplasia and vascular abnormalities (8Srivastava D. Thomas T. Lin Q. Kirby M.L. Brown D. Olson E.N. Nat. Genet. 1997; 16: 154-160Crossref PubMed Scopus (556) Google Scholar, 11Yamagishi H. Olson E.N. Srivastava D. J. Clin. Investig. 2000; 105: 261-270Crossref PubMed Scopus (120) Google Scholar). In the case of HAND1, analysis of its role in heart development was initially complicated by its requirement for proper placental development; mice lacking hand1 died at embryonic day 8–8.5 of placental and extraembryonic defects, tissues where HAND1 is abundantly expressed (6Riley P. Anson-Cartwright L. Cross J.C. Nat. Genet. 1998; 18: 271-275Crossref PubMed Scopus (424) Google Scholar, 7Firulli A.B. McFadden D.G. Lin Q. Srivastava D. Olson E.N. Nat. Genet. 1998; 18: 266-270Crossref PubMed Scopus (296) Google Scholar). Nevertheless, tetraploid aggregation experiments showed that hand1 null embryonic stem cells could not contribute to the left ventricle, suggesting a cell autonomous requirement for HAND1 in this cardiac lineage (6Riley P. Anson-Cartwright L. Cross J.C. Nat. Genet. 1998; 18: 271-275Crossref PubMed Scopus (424) Google Scholar). Gain-of-function studies in transgenic mice showed that misexpression of HAND1 in the whole ventricles altered expression of several genes including Nppa, the gene encoding atrial natriuretic factor (ANF), and Hand2 (12Togi K. Kawamoto T. Yamauchi R. Yoshida Y. Kita T. Tanaka M. Mol. Cell. Biol. 2004; 24: 4627-4635Crossref PubMed Scopus (37) Google Scholar). Interestingly, this study concluded that HAND1 is not a master regulator of the left ventricular myocyte lineage but rather that it acts cooperatively with other transcription factors to control dorso-ventral patterning. More recently, conditional ablation of hand1 in the heart provided support for these previous observations and revealed that absence of HAND1 in cardiac myocytes leads to structural cardiac defects, consistent with an essential role in cardiomyocyte differentiation (10McFadden D.G. Barbosa A.C. Richardson J.A. Schneider M.D. Srivastava D. Olson E.N. Development (Camb.). 2005; 132: 189-201Crossref PubMed Scopus (251) Google Scholar). Targeted gain- and loss-of-function studies have also pointed to important roles for HAND proteins in placental (6Riley P. Anson-Cartwright L. Cross J.C. Nat. Genet. 1998; 18: 271-275Crossref PubMed Scopus (424) Google Scholar), vascular (11Yamagishi H. Olson E.N. Srivastava D. J. Clin. Investig. 2000; 105: 261-270Crossref PubMed Scopus (120) Google Scholar, 13Morikawa Y. Cserjesi P. Development (Camb.). 2004; 131: 2195-2204Crossref PubMed Scopus (61) Google Scholar), craniofacial (14Yanagisawa H. Clouthier D.E. Richardson J.A. Charite J. Olson E.N. Development (Camb.). 2003; 130: 1069-1078Crossref PubMed Scopus (110) Google Scholar), and limb (15McFadden D.G. McAnally J. Richardson J.A. Charite J. Olson E.N. Development (Camb.). 2002; 129: 3077-3088PubMed Google Scholar, 16Fernandez-Teran M. Piedra M.E. Rodriguez-Rey J.C. Talamillo A. Ros M.A. Dev. Dyn. 2003; 226: 690-701Crossref PubMed Scopus (23) Google Scholar) development. However, the molecular mechanisms underlying the essential role of HAND proteins in organogenesis remain largely undefined.HAND1 was initially isolated by virtue of its ability to heterodimerize with the ubiquitous class I bHLH factors, E12/E47 (1Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (582) Google Scholar, 4Cserjesi P. Brown D. Lyons G.E. Olson E.N. Dev. Biol. 1995; 170: 664-678Crossref PubMed Scopus (209) Google Scholar). However, several independent studies showed that, unlike the MyoD/NeuroD subfamily (17Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Crossref PubMed Scopus (1365) Google Scholar), HAND1-containing heterodimers do not bind canonical E-boxes (1Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (582) Google Scholar, 4Cserjesi P. Brown D. Lyons G.E. Olson E.N. Dev. Biol. 1995; 170: 664-678Crossref PubMed Scopus (209) Google Scholar, 18Scott I.C. Anson-Cartwright L. Riley P. Reda D. Cross J.C. Mol. Cell. Biol. 2000; 20: 530-541Crossref PubMed Scopus (184) Google Scholar). In vitro site selection revealed binding of HAND1/E47 to distinct DNA elements containing degenerate E-box motifs (1Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (582) Google Scholar), but the relevance of these elements has yet to be confirmed in the natural context of HAND1 target genes. Interestingly, the ability of HAND1 to bind as a heterodimer and/or activate transcription from these sites may itself be regulated by other cofactors (18Scott I.C. Anson-Cartwright L. Riley P. Reda D. Cross J.C. Mol. Cell. Biol. 2000; 20: 530-541Crossref PubMed Scopus (184) Google Scholar). One such cofactor may be the LIM domain-containing FHL2 transcription factor, which was recently shown to interact with the bHLH domain of HAND1 and repress HAND1/E12dependent transcription (19Hill A.A. Riley P.R. Mol. Cell. Biol. 2004; 24: 9835-9847Crossref PubMed Scopus (38) Google Scholar). Finally, the ability of HAND1 to form homodimers versus heterodimers with E12 can also be regulated at the level of phosphorylation; two residues within helix 1 of the bHLH domain have been identified the phosphorylation status of which alters E-protein heterodimer formation and biologic activity in the limbs (20Firulli B.A. Howard M.J. McDaid J.R. McIlreavey L. Dionne K.M. Centonze V.E. Cserjesi P. Virshup D.M. Firulli A.B. Mol. Cell. 2003; 12: 1225-1237Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Other studies suggest that HAND1 may act essentially as a repressor by titrating class I bHLH factors in a manner reminiscent of that of the Id proteins (20Firulli B.A. Howard M.J. McDaid J.R. McIlreavey L. Dionne K.M. Centonze V.E. Cserjesi P. Virshup D.M. Firulli A.B. Mol. Cell. 2003; 12: 1225-1237Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar); additionally, HAND1 was shown to contain transcriptional repressor domains capable of down-regulating GAL-E47-dependent transcription (1Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (582) Google Scholar, 21Knofler M. Meinhardt G. Bauer S. Loregger T. Vasicek R. Bloor D.J. Kimber S.J. Husslein P. Biochem. J. 2002; 361: 641-651Crossref PubMed Scopus (54) Google Scholar). At present, the question as to whether and how HAND1 acts as a transcriptional activator is not yet settled. Evidently, analysis of HAND1 action on target promoters would provide invaluable insight into its transcriptional pathways.In the case of the related HAND2 factor, two modes of action have been identified so far. One of them is E-box-dependent and involves DNA binding as a heterodimer with ubiquitous class 1 proteins (22Dai Y.S. Cserjesi P. J. Biol. Chem. 2002; 277: 12604-12612Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The other pathway does not depend on HAND2 binding to DNA; instead, HAND2 is recruited to the promoter through protein/protein interaction with GATA-4 (23Dai Y.S. Cserjesi P. Markham B.E. Molkentin J.D. J. Biol. Chem. 2002; 277: 24390-24398Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Interestingly, interaction with GATA proteins may be a general property of class II bHLH factors as shown previously for SCL and GATA-1 in hematopoietic cells (24Wadman I.A. Osada H. Grutz G.G. Agulnick A.D. Westphal H. Forster A. Rabbitts T.H. EMBO J. 1997; 16: 3145-3157Crossref PubMed Scopus (723) Google Scholar, 25Cohen-Kaminsky S. Maouche-Chretien L. Vitelli L. Vinit M.A. Blanchard I. Yamamoto M. Peschle C. Romeo P.H. EMBO J. 1998; 17: 5151-5160Crossref PubMed Scopus (51) Google Scholar). Finally, HAND2 has been shown to interact with the homeodomain-containing Nkx2.5 protein and, in combination with E12, enhance its activation of the ANF promoter (26Thattaliyath B.D. Firulli B.A. Firulli A.B. J. Mol. Cell. Cardiol. 2002; 34: 1335-1344Abstract Full Text PDF PubMed Scopus (55) Google Scholar). In this study, the modest ability of HAND2 to activate the ANF promoter could be enhanced by the addition of E12, although no interaction between HAND2/E12 and any of the ANF E-boxes could be detected. In fact, all E-boxes were dispensable for HAND2/E12 action, which apparently required the Nkx2.5 binding site. This was suggested to be relevant for ANF regulation in the right ventricular compartment (26Thattaliyath B.D. Firulli B.A. Firulli A.B. J. Mol. Cell. Cardiol. 2002; 34: 1335-1344Abstract Full Text PDF PubMed Scopus (55) Google Scholar).In addition to their overlapping expression pattern, two recent studies suggest that ANF may be a transcriptional target for HAND1. For example, the restricted pattern of HAND1 expression in heart atria and left ventricles is highly reminiscent of that of the Nppa gene, which encodes ANF, the major secretory product of the heart. Moreover, mice with cardiac-specific inactivated hand1 alleles have decreased ANF transcripts in the left ventricles, whereas in hand1/2 double mutant mice, ANF expression is completely abrogated (10McFadden D.G. Barbosa A.C. Richardson J.A. Schneider M.D. Srivastava D. Olson E.N. Development (Camb.). 2005; 132: 189-201Crossref PubMed Scopus (251) Google Scholar). On the other hand, ectopic expression of HAND1 in the right ventricle was sufficient to induce transcription of the endogenous ANF gene (12Togi K. Kawamoto T. Yamauchi R. Yoshida Y. Kita T. Tanaka M. Mol. Cell. Biol. 2004; 24: 4627-4635Crossref PubMed Scopus (37) Google Scholar). We therefore tested whether the cardiac ANF promoter may be a transcriptional target for HAND1. Our results show that HAND1 is a transcriptional activator of ANF and identify for the first time a direct transcriptional target for HAND1. The studies also unravel a novel mechanism for HAND action, which involves direct physical association and cooperative interaction with MEF2 proteins. These findings have broad implications for understanding the molecular mechanisms underlying HAND functions in cardiac and non-cardiac cells.MATERIALS AND METHODSCell Cultures and Transfections—Neonatal cardiomyocytes were prepared from 4-day-old Sprague-Dawley rats and plated at a density of 250,000 cells/9.5-cm2 culture dish in six-well Primaria plates as described previously (27Charron F. Paradis P. Bronchain O. Nemer G. Nemer M. Mol. Cell. Biol. 1999; 19: 4355-4365Crossref PubMed Scopus (192) Google Scholar). HeLa and NIH3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfections were carried out using calcium phosphate as described previously (28Morin S. Charron F. Robitaille L. Nemer M. EMBO J. 2000; 19: 2046-2055Crossref PubMed Scopus (277) Google Scholar). In general, the results reported are the mean of three to four experiments carried out in duplicate with two different DNA preparations.Plasmids—ANF-luciferase reporters containing various mutations and deletions of the rat ANF promoter as well as MEF2 and GATA-4 expression vectors have been described previously or were prepared using similar mutagenesis (28Morin S. Charron F. Robitaille L. Nemer M. EMBO J. 2000; 19: 2046-2055Crossref PubMed Scopus (277) Google Scholar, 29Morin S. Paradis P. Aries A. Nemer M. Mol. Cell. Biol. 2001; 21: 1036-1044Crossref PubMed Scopus (90) Google Scholar). The endothelin-1 (ppET-1Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (582) Google Scholar) luciferase reporter was described in Nemer and Nemer (30Nemer G. Nemer M. Development (Camb.). 2002; 129: 4045-4055PubMed Google Scholar). The HAND1 (eHAND) expression vectors were described previously (18Scott I.C. Anson-Cartwright L. Riley P. Reda D. Cross J.C. Mol. Cell. Biol. 2000; 20: 530-541Crossref PubMed Scopus (184) Google Scholar). The myc-Hand2 expression vector was a kind gift of Dr. Robert Schwartz.Recombinant Protein Production and Pull-down Assays—HAND1 was bacterially produced as described previously (28Morin S. Charron F. Robitaille L. Nemer M. EMBO J. 2000; 19: 2046-2055Crossref PubMed Scopus (277) Google Scholar) with few modifications. After transformation of Escherichia coli (BL21, DE3) with GST fusion vectors pGST-HAND1 (pGEX-3X, Amersham Biosciences), individual colonies were picked and grown in 500 ml of LB to an optical density of 0.6. Isopropyl thiogalactopyranoside was then added at a final concentration of 0.1 mm, and bacterial cultures were grown at 37 °C for 2 h. The cultures were centrifuged, and the bacteria were resuspended in 20 ml of 1% Triton X-100 cold PBS and lysed by sonication. Purification was performed using 1 ml of 50% glutathione-agarose beads (Sigma) in PBS, mixed at 4 °C with agitation for 2 h, and then centrifuged at 1500 rpm at 4 °C for 5 min to pellet the resin. The beads were washed three times in 50 ml of 1% Triton X-100 PBS, and the GST fusion proteins were analyzed on SDS-PAGE. Recombinant E47 and MEF2 proteins were produced in vitro using the TnT-coupled in vitro transcription translation system (Promega Corp., Madison, WI). In vitro binding studies were performed as described previously (28Morin S. Charron F. Robitaille L. Nemer M. EMBO J. 2000; 19: 2046-2055Crossref PubMed Scopus (277) Google Scholar) with minor modifications. Briefly, 3–5 μl of 35S-labeled MEF2 and E47 proteins were incubated with 300 ng of immobilized HAND1 fusion proteins in 500 μl of IpH buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 5 mm EDTA, 0.1% Nonidet P-40, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 0.25% bovine serum albumin (BSA)) for 2 h at 4°C with agitation and then centrifuged for 2 min at 1500 rpm at 4 °C. The amount of total 35S-labeled proteins was kept constant (8 μl) in each binding reaction by adding 35S-labeled luciferase to complete. Beads were washed three times by vortexing in 500 μl of IpH buffer without BSA. The protein complexes were released after boiling in Laemmli buffer and resolved by SDS-PAGE. Labeled proteins were visualized and quantified using a phosphorimaging screen and a STORM system (Amersham Biosciences).Immunoprecipitations and Immunoblots—Co-immunoprecipitation of FLAG-HAND1 and hemagglutinin (HA)-MEF2A (pCGN-MEF2A) was carried out using nuclear extracts of 293T cells overexpressing the relevant proteins as described previously (28Morin S. Charron F. Robitaille L. Nemer M. EMBO J. 2000; 19: 2046-2055Crossref PubMed Scopus (277) Google Scholar). Co-immunoprecipitation reactions were carried out on 50 μg of nuclear extracts using 1 μl of anti-HA (12CA5, Roche Diagnostics) antibody in 500 μl of binding buffer without BSA, and bound immunocomplexes were washed and subjected to SDS-PAGE followed by transfer to a Hybond polyvinylidene difluoride membrane and immunoblotting.Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift assays were carried out essentially as described previously (28Morin S. Charron F. Robitaille L. Nemer M. EMBO J. 2000; 19: 2046-2055Crossref PubMed Scopus (277) Google Scholar) using nuclear extracts from cells transfected with the indicated expression vectors. The probes for the MCK and ANF Mef2 sites were described previously (28Morin S. Charron F. Robitaille L. Nemer M. EMBO J. 2000; 19: 2046-2055Crossref PubMed Scopus (277) Google Scholar).RESULTSHAND1 Is a Transcriptional Activator of ANF—To test whether ANF was a potential downstream target for HAND1, we carried out cotransfection studies using a luciferase reporter driven by the –700 ANF promoter. Previous studies have established that these promoter sequences are sufficient to recapitulate spatiotemporal regulation of the endogenous ANF gene in the heart. As shown in Fig. 1A, HAND1 activated transcription from the ANF promoter in HeLa cells in a dose-dependent manner. This effect was also observed in cardiomyocytes and in other non-cardiac cells (Fig. 1B). Interestingly, the class I bHLH factors E47 and E12 also activated the ANF promoter (Fig. 1, A and B and data not shown). However, other bHLH proteins including MyoD, myogenin, Hey1, and Hey2 did not result in transcriptional activation of the promoter (Fig. 1A and data not shown). In fact, Hey 1 and 2 resulted in a dose-dependent repression of the ANF promoter especially in cardiomyocytes. Interestingly, the related HAND2 protein did not significantly activate the ANF promoter, a finding consistent with previous studies (23Dai Y.S. Cserjesi P. Markham B.E. Molkentin J.D. J. Biol. Chem. 2002; 277: 24390-24398Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 26Thattaliyath B.D. Firulli B.A. Firulli A.B. J. Mol. Cell. Cardiol. 2002; 34: 1335-1344Abstract Full Text PDF PubMed Scopus (55) Google Scholar) (Fig. 1A). These results indicate that the ANF promoter is responsive to HAND1 and provides a tool to elucidate the mechanisms of action of HAND1. Next, we carried out a limited structure-function study to determine the domains of HAND1 required for its transcriptional effect. Activation required the HLH domain of HAND1 but not its N terminus (Fig. 1C), a region associated previously with transcriptional repression (21Knofler M. Meinhardt G. Bauer S. Loregger T. Vasicek R. Bloor D.J. Kimber S.J. Husslein P. Biochem. J. 2002; 361: 641-651Crossref PubMed Scopus (54) Google Scholar). In fact, removal of the N terminus produced a superactivator (Fig. 1C). Because E12/E47 also activated the promoter, it was important to determine whether the action of HAND1 on the ANF promoter involved interaction with class I bHLH proteins or heterodimerization with other factors. A tethered HAND1-HAND1 homodimer and a tethered HAND1-ITF2 heterodimer were tested in cotransfection studies. Both failed to activate the ANF promoter (Fig. 1C), suggesting that HAND1 activation of ANF may involve interaction with other transcription factors but likely not class I bHLH.HAND1 and E47 Target Distinct DNA Elements on ANF—To gain further insight into the mechanism of HAND1 action, we used mutational analysis of the ANF promoter to localize the HAND1 and E47-responsive regions. Removal of sequences between –640 and –500 bp reduced HAND1 activation by 5-fold (Fig. 2A) but had no effect on the E47 activation that was lost when sequences between –500 and –480 were deleted (Fig. 2B). This region harbors a conserved E-box, which is evolutionarily conserved in ANF genes from different species. Gel shift analysis confirmed that the ANF E-box at position –490 bp is a bona fide binding site for E47 as shown in direct binding and competition experiments (Fig. 2D). These results suggested that HAND1 and class I bHLH activate the ANF promoter via distinct pathways. Next, we undertook to map the HAND1 regulatory element(s). Previously, we showed that sequences between –640 and –500 contain a Mef2 element (28Morin S. Charron F. Robitaille L. Nemer M. EMBO J. 2000; 19: 2046-2055Crossref PubMed Scopus (277) Google Scholar). Unexpectedly, mutations of this element drastically reduced HAND1 activation (Fig. 2C), raising the possibility that HAND1 action involves functional cooperation with (endogenous) MEF2 proteins. We tested whether HAND1 affected MEF2 binding to its site using either the ANF (Fig. 2E) or MCK (data not shown) Mef2 probes. In repeated experiments using nuclear extracts expressing MEF2A alone or together with HAND1, we could not detect any increase in MEF2 binding in the presence of HAND1 (Fig. 2E). HAND1 alone did not bind to the A/T-rich Mef2 site or to an adjacent E-box (data not shown) suggesting that transcriptional activation of the ANF promoter by HAND1 does not involve direct binding of HAND1 to DNA. We also tested whether HAND1 activation could be due to interaction with endogenous GATA factors (GATA2 is abundantly expressed in HeLa cells (28Morin S. Charron F. Robitaille L. Nemer M. EMBO J. 2000; 19: 2046-2055Crossref PubMed Scopus (277) Google Scholar)) because the related HAND2 protein was shown to interact with GATA-4 (23Dai Y.S. Cserjesi P. Markham B.E. Molkentin J.D. J. Biol. Chem. 2002; 277: 24390-24398Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Mutation of the two high affinity GATA sites in the context of the native –700 bp promoter (27Charron F. Paradis P. Bronchain O. Nemer G. Nemer M. Mol. Cell. Biol. 1999; 19: 4355-4365Crossref PubMed Scopus (192) Google Scholar) significantly reduced but did not abrogate HAND1 activation (Fig. 2C). In contrast, mutation of other regulatory elements, including the proximal SRE or the proximal NKE, had no effect on HAND1 activation (Fig. 2C and data not shown). Together, these results suggest that HAND1 is recruited to the ANF promoter via its interaction with GATA or MEF2 proteins.FIGURE 2Mapping HAND1 and E47 response elements. The cotransfections with various deletions and point mutations (mut) of the ANF promoter were carried out in NIH3T3 cells (A and B) or HeLa cells (C). The exact positions of the different binding sites are indicated in the text. M1, M2, and M3 are three different scanning mutants of the A/T-rich mef2 site. The experiments were carried out as in Fig. 1 using 1 μg of expression vector and 1 μg of reporter. The data are the means of two different experiments, each carried out in duplicate with different DNA preparations (S.D. <10%). Note how the response to HAND1 and E47 map to distinct elements. Mutations are in the following sites: NKE (mut-N), SRE (mut-S), GATA (mut-G), and MEF2 (mut-M). D, gel shift analysis confirms that the –490 ANF E-box is a binding site for E47. Nuclear extracts were prepared from L cells mock-transfected (L) or transfected with E47 expression vector (L+E47). ANF1 and ANF2 correspond to double-stranded oligonucleotides of 25 or 27 bp spanning the E-box site. ANF2 mut contains a mutation in the E-box consensus site. The TRE oligonucleotide corresponds to a neighboring E-box region that contains an AP-1 binding site. E, gel shift analysis on the ANF-Mef2 probe using nuclear extracts from 293T cells transfected with empty vector (Ctl) or with HA-MEF2A (MEF) in the presence or not of HAND1. Competitor DNA are used at 100×. Mef2 (MCK) and E-box probes were derived from the MCK promoter. The identity of the MEF2 complex is also confirmed using anti-HA antibody (αHA) blocking. All probes are described in Ref. 28Morin S. Charron F. Robitaille L. Nemer M. EMBO J. 2000; 19: 2046-2055Crossref PubMed Scopus (277) Google Scholar.View Large Image Figure ViewerDownload Hi-res image Download (PPT)HAND1 Is a MEF2 Cofactor—Next, we directly tested whether HAND1 and MEF2 interact functionally. This was done by cotransfecting the ANF-luc reporter with either MEF2A or HAND1 or both in NIH3T3 cells that have no detectable GATA activity. As shown in Fig. 3A, the presence of both HAND1 and MEF2A resulted in synergistic transcriptional activation. This synergy was also observed with MEF2C (Fig. 3E). Addition of the related HAND2 protein resulted in similar transcriptional cooperativity with MEF2 (Fig. 3A). The DNA binding domain of MEF2 (MEF2A DIVE) was sufficient to observe synergy (albeit to a lower extent) suggesting that MEF2 proteins acted to recruit HAND1 to target promoters. Consistent with that, HAND1/MEF2 synergy was also observed on a minimal ANF promoter driven by a multimerized Mef2 element (Fig. 3B). Given that we showed previously that MEF2 proteins could also be recruited to target promoters via GATA proteins (28Morin S. Charron F. Robitaille L. Nemer M. EMBO J. 2000; 19: 2046-2055Crossref PubMed Scopus (277) Google Scholar), we tested whether GATA-4 could also interact with a MEF2
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