Control of Cardiac-specific Transcription by p300 through Myocyte Enhancer Factor-2D*

Tatiana I. SlepakDagger , Keith A. WebsterDagger , Jie ZangDagger , Howard Prentice§, Ann O'Dowd§, Martin N. Hicks, and Nanette H. BishopricDagger ||

From the Dagger  Department of Molecular and Cellular Pharmacology, University of Miami, Miami, Florida 33101 and the Departments of § Molecular Genetics and  Medical Cardiology, Glasgow Royal Infirmary, University of Glasgow, Glasgow G11 6 NU, United Kingdom

Received for publication, May 29, 2000, and in revised form, October 16, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The transcriptional integrator p300 regulates gene expression by interaction with sequence-specific DNA-binding proteins and local remodeling of chromatin. p300 is required for cardiac-specific gene transcription, but the molecular basis of this requirement is unknown. Here we report that the MADS (MCM-1, agamous, deficiens, serum response factor) box transcription factor myocyte enhancer factor-2D (MEF-2D) acts as the principal conduit for cardiac transcriptional activation by p300. p300 activation of the native 2130-base pair human skeletal alpha -actin promoter required a single hybrid MEF-2/GATA-4 DNA motif centered at -1256 base pairs. Maximal expression of the promoter in cultured myocytes and in vivo correlated with binding of both MEF-2 and p300, but not GATA-4, to this AT-rich motif. p300 and MEF-2 were coprecipitated from cardiac nuclear extracts by an oligomer containing this element. p300 was found exclusively in a complex with MEF-2D at this and related sites in other cardiac-restricted promoters. MEF-2D, but not other MEFs, significantly potentiated cardiac-specific transcription by p300. No physical or functional interaction was observed between p300 and other factors implicated in skeletal actin transcription, including GATA-4, TEF-1, or SRF. These results show that, in the intact cell, p300 interactions with its protein targets are highly selective and that MEF-2D is the preferred channel for p300-mediated transcriptional control in the heart.



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ABSTRACT
INTRODUCTION
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Transcriptional coactivators, or integrators, are members of a class of transcription factors that bring about tissue- and stimulus-specific changes in gene expression by coordinating groups of signal responsive proteins in the cell. Coactivators do not bind independently to DNA, but are thought to stabilize the formation of specific transcription factor-DNA complexes and to cause the local unwinding of chromatin directly or by recruitment of histone-remodeling enzymes (1, 2). Specific coactivator families have been identified for regulation of steroid hormone-responsive genes (SRC-1, SRC-3) (3-5) and fatty acid regulatory proteins (PGC-1) (6, 7). Gene targeting experiments have confirmed that the activities of these coactivators in vivo are highly tissue-restricted, although the molecular basis of this specificity is not well understood (4, 6, 8-10).

An important subgroup of coactivators is represented by the closely related transcriptional integrators p300 and cAMP-responsive element binding protein-binding protein (CBP).1 These large proteins share extensive sequence homology and many structural features and appear to have arisen as part of an ancestral gene duplication (11-13). The cellular levels and activities of p300 and CBP are tightly regulated through steroid, adrenergic, and growth factor signals as well as during the cell cycle (14). Critical roles for p300 and CBP have been identified in growth, differentiation, apoptosis, and tissue-specific gene expression, reflecting their interaction with multiple cellular regulatory proteins (13). A particular requirement for p300 is observed in the heart. Mice deficient in p300 have an embryonic lethal phenotype characterized by failure of cardiac myocyte proliferation and muscle-specific gene expression (10). In the postnatal heart, adenovirus E1A selectively inhibits cardiac muscle-specific gene expression by binding to p300 and/or related proteins (15-17). Although p300 is also required for skeletal muscle gene transcription by the tissue-specific basic helix-loop helix protein MyoD, the heart lacks any equivalent to this transcription factor (18). The molecular partners and pathways of p300-mediated transcriptional regulation in the heart are not known.

Expression of the human skeletal alpha -actin gene is tightly restricted to striated muscle. In the myocardium, skeletal actin is one of a group of "fetal" genes up-regulated in response to hypertrophic stresses such as pressure overload in vivo, and during the response to adrenergic stimulation, growth factors, and other neurohormonal effectors in cell culture models (19-22). Although skeletal actin is the predominant sarcomeric actin isoform in the human heart, it is further up-regulated during hypertrophy (23-25) and is considered an important marker of hypertrophy in the rat (20, 26-29). Regulation of skeletal actin expression in the heart involves both extracellular signal-responsive and cardiac-specific transcription factors; the latter have not yet been identified. The -2130 bp human skeletal actin promoter has two major transcriptional activation domains, proximal (-153 to -87) and distal (-2130 to -710), which are required for maximal tissue-specific expression in both skeletal and cardiac myocytes (19). The proximal promoter contains functional binding sites for Sp-1, SRF, and TEF-1 (30). Here, we report the sequence of the distal human skeletal actin (hSA) promoter, and demonstrate that only one DNA motif within the entire 2130-bp transcriptional unit is capable of transmitting the p300 activation signal. This motif, centered at -1256, binds the MADS (MCM-1, agamous, deficiens, serum response factor) box transcription factor MEF-2 as well as GATA-4, and is required for maximal expression in both neonatal and adult rat cardiac myocytes. The motif also binds endogenous cardiac nuclear p300, and coordinates synergistic activation of the hSA promoter by p300 and MEF-2D. Remarkably, p300-dependent activation did not involve interaction with GATA-4, SRF, or TEF-1, indicating that p300 selectively targets MEF-2 in the context of a native promoter. Based on these findings and on recent data showing interactions between MEF-2 and HDAC-4 (31), we propose that MEF-2 governs expression of the cardiac phenotype by acting as a primary channel for chromatin remodeling on cardiac-specific promoters.


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Materials-- Expression vectors encoding MEF-2A-D were generously provided by Dr. Eric Olson. A p300 expression vector (pCMVp300beta ) was the kind gift of Dr. R. Eckner. Polyclonal antibodies against GATA-4, MEF-2, and p300 were obtained from Santa Cruz Biotechnology. The monoclonal antibody against p300 (NM-11) was supplied by PharMingen, and restriction enzymes were from New England Biolabs. All other molecular biology reagents were purchased from Sigma except as indicated, and were of the highest grade available.

Plasmid Construction-- The distal hSA promoter sequence was determined by automated DNA sequencing using an ABI 377 DNA sequencer in the Biochemistry Core Facility, University of California, San Francisco, CA. The resulting information was used to construct skeletal actin promoter-luciferase chimeras. A 2335-bp HindIII genomic fragment containing the hSA promoter sequence, comprising 2130 bp 5' and 203 bp 3' to the start of transcription, including all of exon 1, was cloned in sense orientation into the HindIII site of pGL-2Basic (Promega). Truncations and point mutations were generated by cloning and/or PCR-mediated mutagenesis as described (32). Fig. 1 shows the structure of each construct used in this paper and the specific point mutations introduced. Primers used for introduction of point mutations are shown in Table I. Mutated bases are shown in bold letters. Numbering reflects the position of hSA gene sequences relative to the transcription start site. Our numbering reflects a systematic difference of +16 bp with the sequence previously published by Muscat et al. (33) as nucleotides -1282 through -1177; the corresponding numbers in our sequence are -1298 through -1193.


                              
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Table I
Oligonucleotides used for point mutagenesis

Internal and 5' deletions of the distal promoter were generated by digestion of the hSA at unique restriction sites at -1787, -1656, -1298, and -1243, and internal religation or ligation to compatible sites in the polylinker (Fig. 1B). The p1787luc1 plasmid was created by digestion at the hSA SpeI site and the polylinker NheI site, and re-ligating. p1656 was generated by digestion at the hSA NdeI site and religation of the proximal end to the polylinker BglII site. Digestion with NdeI and partial digestion with XbaI removed a small fragment of 358 bp, resulting in the dNX plasmid. The dNP plasmid was created by double digestion with NdeI and PstI, gel purification of the vector fragment, and religation.

Introduction of point mutations to phiGATA and ATr sites was performed as described (34) with slight modifications. Briefly, two adjacent primers were designed on opposite DNA strands with the mutation encoded at the 5' end of one primer. Both primers were phosphorylated before PCR. PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) was used to increase fidelity of DNA replication and create blunt-ended PCR products. The amplified plasmid with the introduced mutation was isolated from agarose, religated and transformed to bacteria. Mutations in the proximal promoter were made by directional cloning of double-stranded 52-bp oligomers containing mutations in CArG I (m8, m9), a TEF-1 site (mTEF), or sequences 5' to the CArG box (m6), flanked by XbaIII and XhoI sites at the 5' and 3' ends, respectively. The parental pluc1 vector was linearized with XhoI and subjected to partial digestion with XbaIII; each oligonucleotide was then ligated into this vector to generate m6, m8, m9, and mTEF constructs. All clones were screened by restriction analysis and sequenced to confirm the presence of each mutation.

In Vivo Gene Transfer to Rat Myocardium-- Male Harlan Sprague-Dawley rats (250-400 g) were premedicated with a mixture of 10-20 mg/kg fluanisone, 0.315-0.630 mg/kg fentanyl citrate (Hypnorm, Jansen Pharmaceuticals), and 0.5-1.0 mg/kg midazolam (Hypnovel, Roche Pharmaceuticals) given intraperitoneally. Animals were ventilated (0.04-0.06 liters/min/kg) on a small animal respirator with 0.5-1.0 cmH2O of positive end-expiratory pressure and maintained under anesthesia with a mixture of nitrous oxide and oxygen in a 1:1 ratio plus 0.5-1% halothane. The chest was opened by a left thoracotomy and the pericardium removed for DNA injection. DNA was directly injected into the apex of the left ventricle, using 100 µl/injection in a Hamilton syringe (25 µg of internal control plasmid and 50 µg of hSA-luciferase plasmid suspended in phosphate-buffered saline). Postoperative analgesia was administered for at least the next 24 h with 0.2 mg/kg intramuscular buprenorphine (Vetergesic, Reckitt & Coleman). Seven days after surgery, animals were sacrificed with a lethal dose of pentobarbital sodium and the heart excised for assays of reporter gene expression. Enzyme determinations in tissue extracts were performed as described previously (35). pRSV-luciferase and pCAT3PV (SV40 promoter linked to CAT) were used as internal controls and were obtained from Promega Biotech (Madison, WI). All procedures were performed under license in accordance with National Institutes of Health guidelines or in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986.

Cell Culture and Transfection-- HeLa cells were grown in MEM with Eagle's salts, penicillin, streptomycin, and 10% fetal bovine serum. Neonatal rat myocardial cells were isolated as described previously (19) by gentle trypsinization and mechanical dissociation over a period of 4-5 h, and plated at a density of 3.5-4 × 106 cells/60-mm culture dish. Cultures were enriched for myocardial cells by preplating for 30-60 min to deplete the population of nonmyocardial cells. Prior to and during transfection, cells were maintained in MEM with Eagle's salts, penicillin, streptomycin, and 5% fetal bovine serum (MEM-FBS). Following transfection, cells were incubated in a serum-free medium consisting of MEM supplemented with insulin, transferrin, vitamin B12, penicillin, and streptomycin (MEM-TIB). All cells were maintained in 5% CO2 atmosphere at 37 °C and transfected as described below.

Cardiac myocytes were transfected with reporter plasmids and other constructs on the day following plating, using an adaptation of the calcium phosphate method (36). Equal numbers of myocytes were cotransfected with 10 µg of reporter plasmid and either 5 µg of p300 expression vector, 5 µg of MEF-2 expression plasmid, or an equal amount of blank CMV expression vector. 24 h after transfection, cells were washed twice with MEM-TIB and maintained in that medium for an additional 48 h. Cells were then washed twice with phosphate-buffered saline, pH 7.4, and collected in 1× reporter lysis buffer (Promega).

HeLa and C2C12 cells were transfected at a confluence of 50-70%, also using the calcium phosphate method. Cells were maintained prior and during transfection in MEM (HeLa) or DMEM (C2C12) supplemented with 10% fetal bovine serum, penicillin, and streptomycin. On the day after transfection, cells were rinsed twice with fresh media and incubated for 24-48 h before harvesting as described above. A commercially available kit was used to measure luciferase activity in cell lysates (Promega).

Gel Mobility Shift Assays-- For preparation of nuclear extracts, cardiac myocytes were plated in 10-cm dishes and cultured in MEM-FBS for 2 days, then switched to MEM-TIB for 3-5 days. Nuclear extracts were prepared essentially as described previously (37), except that the final extract was desalted on a 1-2-ml Sephadex G-25 chromatography mini-column instead of by dialysis. Protein concentrations were determined using the Bio-Rad protein assay kit. Aliquots of nuclear extract were frozen and stored at -80 °C.

Double-stranded oligonucleotide gel shift probes containing the wild-type and mutant phiGATA and ATr motifs were synthesized as shown in Table II. Gel-purified oligonucleotide pairs were annealed and end-labeled with [32P]ATP using T4 polynucleotide kinase (New England Biolabs) and [gamma -32P]ATP (PerkinElmer Life Sciences). Gel mobility shift assays were performed as described previously (38) (69). In brief, equal amounts of radioactive probe (1.5-2.5 × 104 cpm) were added to binding reactions that contained 6 µg of nuclear extract protein in 20 µl of a buffer containing 4 mM Tris (pH 7.8), 12 mM HEPES (pH 7.9), 60 mM KCl, 30 mM NaCl, 0.1 mM EDTA, 1 µg/ml poly(dI-dC) (Amersham Pharmacia Biotech). Reactions were incubated for 20 min at 22 °C and then separated on a nondenaturing 5% polyacrylamide gel at 4 °C. Where indicated, antibodies (2-4 µg/reaction) were incubated with the binding reactions for 30 min at 22 °C before addition of the probe. For determination of sequence-specific binding, a 100-fold molar excess of unlabeled oligonucleotides was added immediately before the probe. No DNA-antibody interaction was observed in the absence of nuclear protein (data not shown).

Immunoprecipitation-- 50 µg of nuclear extract from cardiac myocytes (prepared as described above) was mixed in equal amounts with 2× immunoprecipitation buffer (2% Triton X-100, 300 mM NaCl, 20 mM Tris, pH 7.4, 2 mM EDTA, 2 mM EGTA, pH 8.0, 0.4 mM sodium orthovanadate, 0.4 mM phenylmethylsulfonyl fluoride, 1.0% Nonidet P-40). Subsequently, 1-5 µg of either MEF-2 or p300 antibody was added. Mixtures were incubated for 1 h at 4 °C. with constant agitation. Protein A-agarose (20 µl; Santa Cruz Biotechnology) was then added to the protein-antibody mixture, and tubes were incubated for an additional 30 min at 4 °C. At the end of the incubation, agarose beads were spun down, and the supernatant was reserved to quantitate unbound proteins. Beads were washed three times with 1× immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM sodium orthovanadate, 0.2 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40), resuspended in 30 ml of 2× electrophoresis sample buffer (250 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, 2% beta -mercaptoethanol) and then boiled for 5 min. Aliquots of the original nuclear extract, unbound proteins, and wash fractions were also mixed with 2× electrophoresis sample buffer and boiled. All protein samples were resolved on 6% SDS-polyacrylamide gels (for p300) or 8% SDS-polyacrylamide (for detection of MEF-2 proteins). Gels were transferred overnight to nitrocellulose membranes using a Transblot electrophoresis transfer cell (Bio-Rad). Membranes were probed with polyclonal MEF-2 antibody (SC-313, Santa Cruz Biotechnology), polyclonal p300 (N-15) (sc-584, Santa Cruz Biotechnology), and monoclonal p300 NM11 (14991A, PharMingen). Antigen-antibody complexes were visualized by enhanced chemiluminescence (Pierce).

Coprecipitation of MEF-2/p300 Complexes Using a Biotinylated Probe-- A biotinylated oligonucleotide containing the antisense ATr motif (5'-biotin-TTACCAGAGCCTGCTGCAGGTTCTATTTATATCA-3') was annealed to the complementary ATr (R) sense oligonucleotide (see Fig. 7A) at a final concentration of 10 µM and linked to Dynabeads M-280 streptavidin (Dynal Inc., Lake Success, NY) as described previously (39). For coprecipitation of MEF-2 and p300, the beads were initially washed three times with phosphate-buffered saline, pH 7.4, containing 0.1% bovine serum albumin, and twice with a buffer containing 1 M NaCl, 10 mM Tris, 1 mM EDTA (TE-NaCl, pH 7.5). The beads were then mixed with 10 pmol of double-stranded biotinylated probe and incubated 20 min at room temperature in TE-NaCl. Sequential washes with TE-NaCl and 1× binding buffer (10 mM Tris, pH 7.5, 50 mM KCl, 1 mM MgCl2, 1 mM EDTA, 5.5 mM dithiothreitol, 5% glycerol, 0.3% Nonidet P-40) were used to remove excess unbound oligonucleotide and to equilibrate the beads with the binding buffer. Cardiac myocyte nuclear extract (50 µg) was mixed with 1× binding buffer for 5 min at room temperature and incubated with the probe-linked beads for 20 min. Unbound proteins were washed from the beads three times with 1× buffer containing 0.5 µg/ml poly(dI-dC), and aliquots of each wash were retained for analysis. Finally, proteins specifically bound to the beads were eluted with 1× binding buffer containing 1 M NaCl.

All fractions were resolved by SDS-PAGE (6% for p300 and 12% for MEF-2) and analyzed by Western blot using antibodies to p300 and MEF-2 as described above.


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ABSTRACT
INTRODUCTION
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MEF-2, SRF, and TEF-1 Binding Sites in the hSA Promoter Are Required for Maximal Cardiac Expression in Vivo-- p300/CBP does not bind DNA directly, but is recruited to specific gene control regions by transcription factors that bind to these sites. Thus, we looked for binding sites for known DNA-binding transcription factors in the hSA promoter that could represent partners or mediators of p300-dependent transactivation. The proximal human skeletal actin promoter contains consensus binding sites for SRF, TEF-1, and Sp-1, similar to the chick, mouse, and rat skeletal actin promoters (Refs. 41, 35, and 56, respectively; see Fig. 1A), and additional regulatory elements have been localized to the distal promoter (5' to -710) (19). To characterize these elements, we sequenced the distal human skeletal actin promoter from -710 to -2130 (data not shown). Within this sequence are two DNA elements resembling AP-1 binding sites (centered at -1494 and -1300, Fig. 1A), as well as six variant E-boxes (CANNTG; data not shown). A previously described AT-rich motif was found centered at -1256 (33) (Fig. 1, A and C). This AT-rich site differs by one nucleotide from the consensus binding site for MEF-2 (CTA(A/T)4TAG; Ref. 40). We also searched for GATA binding sites by BLAST screening of the hSA promoter with the GATA binding site from the brain natriuretic peptide promoter (CTGATAAATCAGAGATAACC). This search revealed two potential GATA binding sites, one of which overlapped with the MEF-2 consensus sequence. For convenience, the compound MEF-2/GATA binding motif was called "ATr." An additional putative GATA site was centered at - 1798 (Fig. 1, A and C) and designated phiGATA.



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Fig. 1.   Diagram of the hSA promoter and mutants. A, schematic diagram of the hSA promoter, showing location of major restriction sites and DNA motifs resembling binding sites for AP-1 (white boxes), GATA-4 (phiGATA), GATA-4/MEF-2 (ATr), serum response factor (CArG), and TEF-1 as indicated. The complete sequence has been published in GenBankTM (accession no. AF288779). B, deletion and point mutants of hSA promoter constructs. Arrow indicates transcriptional start site. Shaded box, exon 1; open box, start of luciferase sequence in pGL2Basic. X indicates site of point mutations illustrated in C. C, sequence of point mutations introduced in designated hSA promoter constructs. The targeted DNA binding motifs are boxed. Specific mutated bases are shown below the corresponding wild type nucleotides (in bold). Details of plasmid construction are included under "Experimental Procedures."

To test the functional relevance of these elements, the hSA promoter was subjected to serial 5' truncations and point mutagenesis (Fig. 1, B and C). The resulting constructs were assayed for activity in adult myocardium by direct injection as described under "Experimental Procedures" (Fig. 2). These studies showed that the ATr site at -1256 (mATr; Fig. 2), as well as the TEF-1 and SRF binding sites in the proximal promoter (mutants mTEF and m9, respectively; Fig. 2), were required for maximal expression of the human skeletal actin promoter in the adult rat heart. Deletion of sequences upstream of -1298 did not reduce, and in fact enhanced expression, indicating that elements in this region (including phiGATA) do not confer tissue-specific activation in the heart. In contrast, point mutagenesis of ATr reduced hSA promoter activity by about 50% (Fig. 2), suggesting that this element forms the core of the previously reported distal tissue-specific element (19).



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Fig. 2.   Expression of hSA promoter in adult rat myocardium requires proximal and distal enhancer motifs. DNA was delivered by injection into rat myocardium as described under "Experimental Procedures," and luciferase activity was measured and normalized after 1 week. p2130, p2130luc1; p1656, p1656luc1; p1298, p1298luc1; p87, p87luc1 (please refer to Fig. 1B). This graph summarizes data from a minimum of 4 different animals and at least two different plasmid preparations per construct.

A Distal AT-rich Site Is the Major p300-responsive Element-- We next looked for specific DNA sequences in the hSA promoter that mediate its activation by p300. Although the full-length hSA promoter (p2130luc1) has constitutively high basal activity in cardiac myocytes, comparable to that of beta  actin (19), coexpression of p300 further activated it by more than 3-fold (Fig. 3). Similar to our findings in vivo, two different point mutations of the proximal SRE (CArG I) and mutagenesis of the TEF-1 binding site each reduced basal hSA promoter activity in cardiac myocytes by > 60% (mutants m8, m9, and mTEF; Fig. 3). However, these sites were not required for transactivation by p300 (Fig. 3). Mutation of the Sp-1 site in the proximal promoter also reduced basal activity, but did not affect p300 transactivation (data not shown). Minimal expression of the basal promoter (truncated at -87) was increased in the presence of p300, possibly reflecting interaction of p300 with TATAA binding factors (41). In contrast, point mutation of the AT-rich motif centered at -1256 (ATr) not only reduced basal hSA promoter activity, but also abrogated transactivation by p300 (Fig. 3, mutants mATr and mATrphiGA). Although in some experiments mutation of the phiGATA site at -1798 appeared to further reduce p300 transactivation, this effect was not reproducible and did not achieve statistical significance (p > 0.08). Deletion or mutation of other distal sites had no significant on p300 transactivation. These findings indicated that transcriptional activation by p300 required a single tissue-specific AT-rich enhancer element in the hSA promoter.



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Fig. 3.   Identification of a single p300-responsive element in the hSA promoter. In vitro activity of the 2130-bp hSA promoter construct and mutants in the presence and absence of cotransfected p300. Transfections were performed in neonatal rat cardiac myocyte cultures as detailed under "Experimental Procedures," and luciferase activity was measured 40-48 h later. Light bars, hSA-luciferase construct alone; dark bars, hSA luciferase construct + pCMVp300beta . For all constructs, p <=  0.05 for comparison between -p300 and +p300, except mATr (p = 0.3705) and mATrphiGA (p = 1.0) (asterisks). These data summarize a minimum of eight different transfection experiments and three separate plasmid preparations per construct.

The ATr Element Is a MEF-2 Binding Site-- The ATr site is assumed to be a MEF-2 binding site based on its homology to similar sites in other muscle-specific promoters (42). To identify cardiac nuclear proteins binding to the p300-responsive ATr element, we synthesized oligonucleotides containing both the wild type and mutant sequences used for functional assays above (Table II). Electrophoretic mobility shift assays (EMSAs) were performed with nuclear extracts from cardiac myocytes and from endothelial cells. Cardiac nuclear proteins interacting with the ATr element formed at least three sequence-specific bands (labeled 1-3 in Fig. 4A). The upper two bands required the core ATr sequence, and did not form on a mutant ATr site (Fig. 4A, lane 6), or on the GATA-like sequence at -1798, phiGATA (Fig. 4A, lane 2). The third nucleoprotein complex formed specifically on both ATr and mATr, suggesting that it interacts with sequences flanking the core ATATA sequence (Fig. 4A, lanes 6 and 12). All three major nucleoprotein complexes were muscle-restricted, as they were absent in endothelial cell nuclear extracts (Fig. 4A, lanes 13 and 14).


                              
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Table II
Oligonucleotides used in EMSA assays



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Fig. 4.   Muscle-specific proteins bind to the p300-responsive element. A, cardiomyocyte and endothelial cell nuclear protein interactions with hSA promoter. Oligonucleotides corresponding to phiGATA and ATr motifs (see Fig. 1C and Table I) were synthesized and used to define nucleoprotein interactions with these sites. A mutant ATr motif was also used as a probe (mATr). The ATr motif forms three specific protein complexes with cardiac myocyte nuclear proteins (Cardiomyocytes; arrows labeled 1, 2, and 3). None of these bands are seen when endothelial cell nuclear extract is used. -, labeled probe only; NE, probe + nuclear extract; S, 100× unlabeled self probe competitor; mphiGA, unlabeled mutant phiGATA oligonucleotide competitor; ATr, unlabeled ATr probe competitor; mATr, unlabeled mutant ATr competitor. B, MEF-2 binds to the hSA p300-responsive element. The p300-responsive element (ATr) was labeled and used as a probe in EMSAs as in Fig. 4A. For comparison, a commercially available synthetic MEF-2 oligonucleotide (MEF-2) was also used as a probe. Cardiac nuclear proteins formed three sequence-specific bands with both of these probes (labeled 1, 2, and 3 as in Fig. 4A). A MEF-2 antibody supershifted bands 1 and 2 on both oligonucleotides; three novel supershifted bands are seen (black arrowheads). -, probe alone; NE, probe + nuclear extract; S, 100× cold self competitor; MEF, unlabeled synthetic MEF-2 oligonucleotide; ATr, unlabeled ATr oligonucleotide; Ab, antibody against MEF-2.

We next confirmed that MEF-2 proteins bind to the ATr site. The same three nucleoprotein complexes identified in Fig. 4A were again seen forming on the ATr site, as well as on a synthetic MEF-2 site (Fig. 4B). The synthetic MEF-2 oligonucleotide also competed effectively for the three complexes binding to ATr. In both cases, bands 1 and 2 were specifically and quantitatively supershifted by a MEF-2 antibody that recognizes MEF-2 subtypes A, C, and D (Fig. 4B, lanes 5 and 10), whereas a control MyoD antibody did not (data not shown). Thus, MEF-2 is present in two of three tissue-specific complexes bound to the ATr site.

The ATr site has significant homology to a GATA-4 binding site from the B-type natriuretic peptide promoter that is required for its cardiac-specific activation (43). To determine whether GATA-4 also bound to the ATr site, we synthesized shorter oligomers representing sequences centered at -1260 ("left"), -1252 ("right"), and -1256 ("center") (Fig. 5A). All three previously detected complexes formed on each of these shorter oligonucleotides (Fig. 5B), but with varying efficiency. Complexes 1 and 2 preferentially interacted with the right side of ATr (Fig. 5B, lane 9), whereas complex 3 preferentially bound to the left (Fig. 5B, lane 5). For all three oligonucleotides, only complexes 1 and 2 were supershifted by the MEF-2 antibody (Fig. 5B, lanes 3, 7, and 11). Thus, complex 3 binds a site adjacent to and overlapping that of complexes 1 and 2, and does not contain MEF-2. In contrast, a polyclonal anti-GATA-4 antibody selectively depleted ATr complex 3, suggesting that it contained GATA-4 (Fig. 5C, lane 4). This depletion was accompanied by appearance or enhancement of a band comigrating with complex 1 (lane 4). When a synthetic GATA binding site was used as the probe (Fig. 5C, lanes 5-9), we observed a single cardiac nucleoprotein complex that comigrated with ATr complex 3 and was effectively competed by an ATr oligomer (Fig. 5C, lane 8). Incubation with the GATA-4 antibody generated a supershifted complex that migrated roughly in tandem with complex 1 on the ATr site (Fig. 5C, compare lanes 9 and 4). Thus, the enhanced binding in lane 4 appears to be identical with a supershifted GATA-DNA complex, although we cannot formally exclude the possibility that this represents enhanced binding of MEF-2. In either case, it is clear that the most rapidly migrating nucleoprotein complex on the ATr sequence contains GATA-4.



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Fig. 5.   Overlapping binding sites for MEF-2 and GATA-4 on the p300-responsive element. A, sequences of original and truncated ATr oligonucleotides. Sequences are aligned with a GATA consensus binding site to show the relative position of the GATA (bold) and MEF-2 consensus sequences (boxed). The position of nucleotide -1256 in the hSA promoter sequence is shown. B, bands 1, 2, and 3 exhibit differential affinity for truncated ATr sites. The oligonucleotides shown in A were labeled and used as probes in EMSAs with cardiac myocyte nuclear extracts as in Fig. 4. A MEF-2-specific antibody was used to confirm the presence of MEF-2 in complexes 1 and 2 (bracket), forming at least two supershifted bands (white arrowheads). Note that complex 3 (arrow) is enhanced on the "left" truncated oligomer (Left) and absent or diminished from the "right" oligomer (Right). As in Fig. 4, complex 3 is not supershifted by the MEF-2 antibody. -, probe alone; NE, probe + nuclear extract; S, 100× cold self competitor; ab, probe, nuclear extract, and MEF-2-specific antibody. C, GATA-4 binds to the hSA p300-responsive element. EMSA of cardiac nuclear extracts was performed as above using the ATr (Left) oligonucleotide and a commercially available oligonucleotide containing two GATA binding sites (Santa Cruz Biotechnology). A band similar to complex 3 appears on the GATA consensus probe and is effectively competed by ATr (black arrowheads; bar labeled 3). Both complexes 3 are supershifted by a GATA-4-specific antibody (white arrowheads). -, probe alone; NE, probe + nuclear extract; S, 100× cold self competitor; ATr, 100× cold ATr oligonucleotide competitor; ab, GATA-4-specific antibody.

Taken together, these data show that the distal hSA cardiac-specific element is occupied by at least three muscle-specific protein complexes: two containing MEF-2, and one with GATA-4. Moreover, MEF-2 and GATA-4 complexes form on distinct but overlapping sites within the ATr motif.

p300 Binds Specifically to the Skeletal Actin MEF-2 Binding Site-- We next looked for evidence that p300 interacts directly with the MEF-2 DNA-binding complexes. The ATr and Atr-Left oligonucleotides shown in Fig. 5A were labeled and allowed to interact with cardiac nuclear proteins in the presence or absence of specific antibodies. As in Figs. 4B and 5B, the polyspecific MEF-2 antibody completely supershifted bands 1 and 2 (Fig. 6, lane 8). A second, MEF-2D-specific antibody reacted only with band 1 (Fig. 6, lane 7.) Band 3 on both oligonucleotides was supershifted by a GATA-4 antibody (Fig. 6, lanes 3 and 10). These results show that the more rapidly migrating complex is likely to contain MEF-2A, MEF-2C, or both, whereas the slower moving complex (band 1) probably contains only the MEF-2D isoform.



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Fig. 6.   Both MEF-2 and p300 contact the p300-responsive element. EMSAs were performed as above, using the original ATr (ATr (Full)) and ATr (ATr (Left)) motifs as probes (please refer to Fig. 5A). A polyspecific antibody against MEF-2 isoforms (MEF2) and antibodies specific for MEF-2D (MEF2D), p300 (p300), and GATA-4 (GATA) were used to identify these proteins in DNA-protein complexes, as indicated. The three major protein-DNA complexes are numbered (1, 2, and 3). ATr (Full) panels, as in Figs. 4B and 5B, the MEF-2 antibody generated three supershifted bands (black arrows) and depleted bands 1 and 2 (lane 8). In contrast, the MEF-2D-specific antibody supershifted only band 1 (lane 7, black arrowhead). The p300 antibody also selectively depleted band 1 and generated at least 1 supershifted band (white arrowhead). Band 3 (white arrow) is supershifted exclusively by a GATA-4 antibody. ATr (Left) panels, the left Atr motif was examined for interaction with p300 since this oligomer maximizes GATA-4 binding. As with the full-length ATr, band 3 is supershifted by a GATA-4 antibody, but not by a p300 antibody. None of the complexes formed on the ATr (left) motif appear to interact with p300. NE, nuclear extract.

Band 1, containing MEF-2D, was also the only complex that could be shown to contain p300. A polyclonal antibody directed against the NH2 terminus of p300 reacted very specifically with the MEF-2D-ATr complex (Fig. 6, lane 9); bands 2 and 3 were not affected. None of the protein-DNA complexes on the Atr-Left oligomer reacted with the p300 antibody. These data show that p300 is specifically present in a complex with MEF-2D on the ATr, and not with GATA-4 or other MEF-2 species. Morever, 3'-flanking sequences of the ATr are required for formation of this complex.

A MEF-2 site in the cardiac alpha -myosin heavy chain (alpha -MHC) promoter is required for maximal activity in cardiac myocytes (44-46). We were interested in determining whether p300 could be identified at these sites, or at a binding site for the related MADS protein, SRF, in the proximal hSA promoter (Fig. 1A). Oligonucleotide sequences used for these studies are given in Table II. Fig. 7 shows that the AT-rich motifs from the alpha -MHC and M-creatine kinase (M-CK) form complexes similar to hSA complexes 1, 2, and 3 (Fig. 7, lanes 2, 7, and 12). In each case, complexes 1 and 2 were supershifted by a MEF-2 antibody (lanes 4, 9, and 14). Furthermore, each complex 1 was supershifted by the p300 antibody (lanes 5, 10, and 15). No other bands were visibly affected. Neither of two distinct sequence-specific complexes formed on the hSA SRE was supershifted by the MEF-2 or p300 antibodies. These results suggest that p300 interacts differentially with these two MADS proteins and is selective for MEF-2 over SRF (Fig. 5, lanes 19 and 20).



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Fig. 7.   p300 is present at other cardiac promoter MEF-2 sites, but not at the hSA SRF site. Homologous AT-rich sites in the cardiac alpha -myosin heavy chain (alpha -MHC, Ref. 48) and muscle creatine kinase (M-CK) promoters were synthesized as shown in Table II and used as probes in EMSAs. The hSA p300 responsive element (hSA ATr) and a proximal serum response element centered at -93, CArG I (hSA-SRE), were used for comparison. Complexes 1, 2, and 3 can be identified on all three MEF-2 sites but not on the SRE. Black arrowheads, MEF-2 antibody-supershifted bands. White arrowheads, p300 antibody-supershifted bands. Lanes are labeled as in Fig. 5 except as follows: Ab, antibody used for supershift; M, MEF-2 antibody; P, p300 antibody.

Endogenous Cardiac MEF-2 and p300 Interact on the ATr Element-- We next asked whether MEF-2 and p300 bound to each other directly or via contact with DNA. Initially, we attempted to coimmunoprecipitate MEF-2 and p300 from cardiac nuclear extracts, using antibodies against both p300 and MEF-2. The polyclonal MEF-2 antibody was able to quantitatively immunoprecipitate at least two MEF-2 species (Fig. 8A, lane 4). However, we detected no p300 protein in the MEF-2 immunoprecipitates using either monoclonal or polyclonal p300 antibodies (Fig. 8A, lane 9). Conversely, the anti-p300 monoclonal antibody (NM-11) successfully immunoprecipitated p300 from cardiac nuclear extracts (Fig. 8A, lane 10), but did not coprecipitate detectable MEF-2 species (data not shown). This may be attributable to inaccessibility of the interaction domains in the presence of antibody or to the lack of high affinity interaction between the two proteins in the absence of DNA.



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Fig. 8.   Cardiac MEF-2 and p300 interact in a DNA-dependent manner. A, independent immunoprecipitation of MEF-2 and p300. A MEF-2 antibody recognizing MEF-2A, -C, and -D was used to immunoprecipitate cardiac nuclear extracts. Immunoprecipitates were subjected to Western analysis using the same MEF-2 antibody (lanes 1-4) and a polyclonal p300 antibody (lanes 5-9). Lane 10 shows a p300 Western analysis of cardiac nuclear proteins immunoprecipitated with a p300 antibody for comparison. MEF-2 is nearly quantitatively precipitated from extracts by this method and appears as a doublet at around 70 kDa (black arrowhead). p300 is not detectable in the MEF-2 immunoprecipitates, but is clearly seen in the p300 immunoprecipitates (black arrow, right). T, total nuclear extract; U, unbound proteins; W1 and W2, proteins in wash fractions 1 and 2, respectively. Positions of size markers are shown to left of autoradiogram. E, proteins specifically eluted from beads. B, MEF-2 and p300 associate in the presence of the ATr site. A "pull-down" assay was performed as described under "Experimental Procedures," using a biotinylated hSA ATr oligonucleotide linked to streptavidin-coated beads for affinity purification of cardiac nuclear proteins. Nuclear extracts were incubated with the DNA-linked beads for defined intervals and samples of the total input protein (T), unbound protein (U), wash fractions (W3), and specific eluates (E) were subjected to Western analysis with MEF-2 and p300 antibodies as in panel A. Nuclear extracts from C2C12 myoblasts were used as positive controls (T (C2)). Upper panel is a MEF-2 Western blot. Cardiac MEF-2 is eluted as a narrow doublet from the beads (arrows). Note the presence of additional MEF-2 species in the C2C12 extract. Lower panel is a p300 Western blot. A band corresponding to p300 is visible in the total cardiac nuclear extract (T) and in the specific eluate (E). Note a second band of smaller size also recognized by the p300 antibody and specifically eluted from the affinity beads. The identity of this protein is not known, but it may represent a degradation product of p300.

To determine whether MEF-2 and p300 interacted through DNA binding, we performed DNA "pull-down" assays using Dynal beads linked to a double-stranded oligonucleotide containing the hSA MEF-2 site (ATr) as described previously (39). Cardiac nuclear proteins binding specifically to this oligonucleotide were eluted from the beads and characterized by Western analysis with MEF-2 and p300 antibodies. Two MEF-2 proteins eluted from the hSA ATr site (Fig. 8B, upper panel, lane 4). Western analysis of the same blot using a MEF-2D-specific antibody confirmed that the upper band contains MEF-2D (data not shown). Importantly, a band corresponding to p300 was present in the same eluate (Fig. 8B, lower panel, lane 4). A second protein of smaller size (about 140 kDa) was also detected by the NM-11 antibody and may represent a degradation product. A biotinylated mutant ATr did not bind to either MEF-2 or p300 (data not shown). These data show that endogenous cardiac MEF-2 and p300 form a specific complex with the ATr element, and they support the results of the gel mobility retardation assays shown above.

MEF-2, but Not GATA-4, Is Synergistic with p300-- To address the functional significance of the MEF-2-p300 interaction, we asked whether the two proteins could activate cardiac transcription synergistically. We measured the activity of the hSA wild-type promoter, and the same promoter containing a point mutation in the ATr motif (p2130luc1 and mATr, Fig. 1, B and C) in the presence and absence of p300 and one of the four MEF-2 species. Parallel experiments were performed in cardiac myocytes and HeLa cells (Fig. 9A). Alone, none of the individual MEF-2 subtypes significantly activated either the wild type or mutant hSA promoters in cardiac myocytes, although a small amount of activation was seen in HeLa cells (Fig. 9A, white bars). In cardiac myocytes, activation of the wild-type promoter by MEF-2A, -B, or -C did not significantly increase in the presence of p300 (Fig. 9A, blue bars). However, the combination of MEF-2D and p300 significantly activated hSA expression, compared with either blank vector or p300 alone (p < 0.01, Fig. 9A). Because it is not possible to establish the relative expression of the different MEF-2 species from these vectors, it may be that the divergent behavior of MEF-2C and MEF-2D is due to quantitative differences in MEF-2 delivery or expression. However, all vectors contained the same CMV promoter and were expressed at measurable levels in a HeLa cell background, suggesting that transfer and expression were not qualitatively defective for the MEF-2C vector (data not shown).



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Fig. 9.   MEF-2 but not GATA-4 is synergistic with p300. A, functional interaction between MEF-2D and p300. Transcriptional activities of the intact hSA promoter (p2130luc1) and a mutant lacking the p300-responsive site (mATr) were compared in cardiac myocytes and HeLa cells as indicated, in the presence and absence of p300, and with or without one of the four MEF-2 isoforms (A-D). 35-mm dishes of cardiac myocytes were transfected with the indicated constructs at a ratio of reporter:p300:MEF = 2:1:1. Total transfected DNA was kept constant by addition of appropriate amounts of blank CMV expression vector. This graph summarizes three independent experiments in which substantially similar data were obtained. B, GATA-4 and p300 are not synergistic for skeletal actin promoter transcription. Cardiac myocytes were transfected with the intact hSA promoter (p2130luc1, black bars) or the p300 responsive site mutant (mATr, light bars), alone or in the presence of p300, GATA-4, or both. Transfections were performed as in panel A and represent three independent determinations. *, p < 0.05; **, p < 0.01, both for comparison with control.

In HeLa cells, the combination of p300 and MEF-2D failed to activate hSA transcription above 10% of its activity in cardiac myocytes, and this activation was largely independent of the ATr site, suggesting that additional cell type-specific factors are required for maximal expression of this promoter. As expected, neither p300 nor MEF-2 species activated an hSA mutant lacking the ATr site (mATr) in cardiac myocytes. These results show that p300 and MEF-2D are synergistic for cardiac transcription of the hSA promoter and that this synergy is exerted through the ATr site at -1256.

The skeletal actin gene has not previously been shown to be a target for activation by GATA-4. However, as shown above, GATA-4 binds to the hSA ATr site, flanking the MEF-2 binding sequences. Thus, GATA-4 might also transactivate the hSA promoter, or participate in activation of hSA by p300 through the ATr site. To investigate these possibilities, a GATA-4 expression vector was cotransfected with the hSA wild type and ATr site mutants. The ATr mutant was expressed at ~50% of wild type promoter levels, as reported above. Coexpression of GATA-4 did not activate basal expression of the wild type promoter at any concentration (Fig. 9 and data not shown), and significantly reduced its activation by p300 in the presence of an intact ATr (Fig. 9, light bars). Together with the observed lack of physical interaction in vivo, these observations suggest that GATA-4 is not directly involved in p300-mediated activation of skeletal actin transcription.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Studies described here identify the myogenic transcription factor MEF-2D as a specific physical and functional target of p300 in the heart. Activation of the native 2180-bp human skeletal actin promoter by p300 required a single short DNA sequence in the distal promoter, a hybrid binding site for MEF-2 and GATA-4. Full myocardial expression of the skeletal actin promoter required this same element, both in vitro and in vivo. p300 bound exclusively to this element, as part of a complex with MEF-2D, and could not be identified in DNA complexes with GATA-4 or in a structurally related DNA-SRF complex. Furthermore, p300 and MEF-2D displayed cooperative transcriptional activation of the hSA promoter. This functional synergy was not observed in HeLa cells, suggesting a requirement for additional cell type-specific factors. Taken together, these findings identify MEF-2D as a dominant partner for p300 in the myocardium and place p300 in the regulatory hierarchy of the cardiac phenotype (10, 15-17).

Our data show that p300 targeting of MEF-2D takes priority over its potential interactions with several other proteins that bind to the 2130-bp hSA promoter, including GATA-4 and SRF. This observation is remarkable, since other studies have shown that p300 can bind to both SRF and members of the GATA family and can activate transcription through their cognate binding sites (47, 48, 49). In contrast, we did not observe physical or functional interaction between p300 and GATA-4 on the hSA promoter, nor did we detect p300 at GATA-4 binding sites in the B-type natriuretic peptide, beta -myosin heavy chain (50), or angiotensin type I receptor promoters (51) under equivalent conditions (data not shown). Our results do not exclude the possibility that GATA-4 and p300 interact independently of DNA or under defined conditions in vitro. Further work will be required to establish the relationship between GATA-4- and MEF-2-dependent transcriptional activation in the heart.

The MEF-2 family of transcription factors belongs to the MADS group of DNA-binding proteins, characterized by their homology within an amino-terminal domain ("MADS box"). Although MEF-2 is widely expressed, its role in cardiac-specific transcription been well documented. MEF-2C is required for normal cardiac morphogenesis, and most if not all cardiac genes possess functionally important MEF-2 binding sites (42). MEF-2 species form DNA-binding homo- and heterodimers and also bind to myogenic helix-loop-helix proteins as coregulators. In skeletal muscle, MEF-2 proteins collaborate with MyoD and p300 to promote myogenesis and muscle-specific transcription and are thought to act in a positive autoregulatory loop that maintains myogenic differentiation (18, 40, 52, 53). Other partners for MEF-2 proteins in cardiac transcriptional activation remain to be identified.

Our data suggest that the MEF-2D isoform may be targeted by p300 in preference to other MEF species. We were able to demonstrate cooperative transcriptional activation between p300 and MEF-2D, but not with the other MEF-2 isoforms, and p300 was localized to the DNA complex containing MEF-2D. Our data do not exclude limited functional interaction between p300 and other MEF-2 species at this promoter, but it is also possible that the small positive interaction between the other MEF species and p300 is mediated by heterodimerization with endogenous MEF-2D. This finding is important because, to date, there are few examples of functional differences between the four known MEF-2 species. All four mammalian MEF-2 species are expressed in the heart (reviewed in Ref. 42), and all subtypes recognize the same AT-rich consensus sequence (YTA(A/T)4TAR). Differential recruitment of essential coactivator proteins may confer distinct transcriptional activation properties on MEF-2D.

The observed lack of coactivation between MEF2C and p300 at this promoter was unexpected. Several previous findings suggest that p300 and MEF-2C may act cooperatively in the cardiovascular system; p300-deficient and MEF-2C-deficient mice have overlapping defects in vascularization, and both have defects in cardiac development, although these are dissimilar (54). MEF2C and D have closely parallel expression patterns throughout cardiac development, and MEF2C expression actually precedes that of MEF2D in the cardiac mesoderm (42). Furthermore, cotranslated p300 and MEF2C have been shown to interact in vitro (18). However, the physical and functional data presented here suggest that MEF-2-p300 interactions may be subject to modification by cell type-specific or promoter-specific factors. One possibility is that MEF2C-p300 interactions play critical roles in mesodermal patterning and in vasculogenesis, whereas MEF2D-p300 may be more important for tissue-specific gene expression in the differentiated cardiac myocyte. Further studies of MEF2D in gene-targeted mice will help to clarify its specific roles.

The muscle-specific actions of MEF-2 cannot be explained by tissue-restricted expression, or by differences between MEF-2 binding sites in muscle-specific and ubiquitously expressed genes (55). Instead, MEF-2 activity appears to be subject to significant post-transcriptional control by phosphorylation, and by the recruitment of additional factors (40, 56, 59). In support of this hypothesis, it has been suggested that MEF-2-dependent transcription is silenced in nonmuscle cell types by recruitment of HDAC-4, a histone deacetylase (31). We propose that the converse is also true; activation of MEF-2-dependent genes in cardiac muscle requires recruitment of histone acetyltransferase via p300. These findings point to a unique role for MEF-2D in channeling both the activation and silencing signals of chromatin-remodeling enzymes to cardiac-specific promoters.

Our studies provide the first direct evidence that MEF-2 and p300 interact to regulate cardiac-specific transcription. Although MEF-2 proteins and p300 have been shown to interact in vitro (52, 18), and at artificial MEF-2-dependent promoters in nonmuscle cells (18), the significance of these findings in tissue-specific transcription has been unclear. In skeletal muscle, the MEF-2-p300 interaction may serve to stabilize critical MyoD-p300 complexes on adjacent E boxes (52). However, our data indicate that the MEF-2-p300 complex can activate the tissue-specific transcription of promoters that lack essential E-boxes, and can do so in the apparent absence of basic helix-loop-helix proteins analogous to MyoD or NeuroD/Beta2 (60, 61). It seems likely that additional, unidentified cell type-specific factors cooperate with MEF-2 and p300 in the cardiac myocyte.


    ACKNOWLEDGEMENTS

We thank Drs. R. Eckner, D. Livingston, and E. Olson for the generous gift of cDNA clones used in this paper. Dr. G. Q. Zeng assisted in construction of several of the promoter mutants. We especially appreciate the excellent technical support of Daryl Discher and Mary Gardiner, and the helpful comments of Dr. Gary Grotendorst.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL49891 (to N. H. B.) and HL44578 (to K. A. W.), by an Established Investigator grant from the American Heart Association (to N.H.B.), by a grant from the Miami Heart Research Institute (to N. H. B.), by a grant from the British Heart Foundation (to H. P.), and by a Wellcome Trust Foundation collaborative travel grant (to H. P., K. A. W., and N. H. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF288779.

|| To whom correspondence should be addressed: Dept. of Molecular and Cellular Pharmacology, University of Miami, P.O. Box 106189 (R-189), Miami, FL 33101. Tel.: 305-243-6775; Fax: 305-243-6082; E-mail: nhb@chroma.med.miami.edu.

Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M004625200


    ABBREVIATIONS

The abbreviations used are: CBP, cAMP-responsive element binding protein-binding protein; MEF, myocyte enhancer factor; bp, base pair(s); MEM, minimal essential medium; MEM-TIB, minimal essential medium supplemented with insulin, transferrin, vitamin B12, penicillin, and streptomycin; MEM-FBS, minimal essential medium with Eagle's salts, penicillin, streptomycin, and 5% fetal bovine serum; alpha -MHC, alpha -myosin heavy chain; hSA, human skeletal actin; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay; SRE, serum response element; MADS, MCM-1, agamous, deficiens, serum response factor.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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