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Myospryn Is a Direct Transcriptional Target for MEF2A That Encodes a Striated Muscle, α-Actinin-interacting, Costamere-localized Protein

2006; Elsevier BV; Volume: 281; Issue: 10 Linguagem: Inglês

10.1074/jbc.m510499200

1083-351X

Jennifer T. Durham, Ondra M. Brand, Michael Arnold, Joseph G. Reynolds, Lavanya Muthukumar, Hartmut Weiler, James A. Richardson, Francisco J. Naya,

Cardiovascular Effects of Exercise

The full repertoire of proteins that comprise the striated muscle Z-disc and peripheral structures, such as the costamere, have yet to be discovered. Recent studies suggest that this elaborate protein network, which acts as a structural and signaling center for striated muscle, harbors factors that function as mechanosensors to ensure coordinated contractile activity. Mutations in genes whose products reside in this region often result in skeletal and cardio myopathies, demonstrating the importance of this macromolecular complex in muscle structure and function. Here, we describe the characterization of a direct, downstream target gene for the MEF2A transcription factor encoding a large, muscle-specific protein that localizes to the costamere in striated muscle. This gene, called myospryn, was identified by microarray analysis as a transcript down-regulated in MEF2A knock-out mice. MEF2A knock-out mice develop cardiac failure during the perinatal period with mutant hearts exhibiting several cardiac abnormalities including myofibrillar disarray. Myospryn is the mouse ortholog of a partial human cDNA of unknown function named cardiomyopathy-associated gene 5 (CMYA5). Myospryn is expressed as a single, large transcript of ∼12 kilobases in adult heart and skeletal muscle with an open reading frame of 3739 amino acids. This protein, belonging to the tripartite motif superfamily of proteins, contains a B-box coiled-coil (BBC), two fibronectin type III (FN3) repeats, and SPRY domains and interacts with the sarcomeric Z-disc protein, α-actinin-2. Our findings demonstrate that myospryn functions directly downstream of MEF2A at the costamere in striated muscle potentially playing a role in myofibrillogenesis. The full repertoire of proteins that comprise the striated muscle Z-disc and peripheral structures, such as the costamere, have yet to be discovered. Recent studies suggest that this elaborate protein network, which acts as a structural and signaling center for striated muscle, harbors factors that function as mechanosensors to ensure coordinated contractile activity. Mutations in genes whose products reside in this region often result in skeletal and cardio myopathies, demonstrating the importance of this macromolecular complex in muscle structure and function. Here, we describe the characterization of a direct, downstream target gene for the MEF2A transcription factor encoding a large, muscle-specific protein that localizes to the costamere in striated muscle. This gene, called myospryn, was identified by microarray analysis as a transcript down-regulated in MEF2A knock-out mice. MEF2A knock-out mice develop cardiac failure during the perinatal period with mutant hearts exhibiting several cardiac abnormalities including myofibrillar disarray. Myospryn is the mouse ortholog of a partial human cDNA of unknown function named cardiomyopathy-associated gene 5 (CMYA5). Myospryn is expressed as a single, large transcript of ∼12 kilobases in adult heart and skeletal muscle with an open reading frame of 3739 amino acids. This protein, belonging to the tripartite motif superfamily of proteins, contains a B-box coiled-coil (BBC), two fibronectin type III (FN3) repeats, and SPRY domains and interacts with the sarcomeric Z-disc protein, α-actinin-2. Our findings demonstrate that myospryn functions directly downstream of MEF2A at the costamere in striated muscle potentially playing a role in myofibrillogenesis. The cytoskeleton of striated muscle is comprised of a complex and highly organized assembly of proteins, which ensures that contraction is appropriately initiated and transmitted in a synchronized fashion while maintaining cellular integrity (1Clark K.A. McElhinny A.S. Beckerle M.C. Gregorio C.C. Annu. Rev. Cell Dev. Biol. 2002; 18: 637-706Crossref PubMed Scopus (462) Google Scholar, 2Chien K.R. Nature. 2000; 407: 227-232Crossref PubMed Scopus (165) Google Scholar). Alterations in the stoichiometry of cytoskeletal proteins or mutations in any of these components can destabilize this highly structured network often resulting in cardio and/or skeletal myopathies (3Arber S. Hunter J.J. Ross J. Hongo M. Sansig G. Borg J. Perriard J.C. Chien K.R. Caroni P. Cell. 1997; 88: 393-403Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, 4Pashmforoush M. Pomies P. Peterson K.L. Kubalak S. Ross J. Hefti A. Aebi U. Beckerle M.C. Chien K.R. Nat. Med. 2001; 7: 591-597Crossref PubMed Scopus (159) Google Scholar, 5Zhou Q. Chu P-H. Huang C. Cheng C-F. Martone M.E. Knoll G. Shelton G.D. Evans S. Chen J. J. Cell Biol. 2001; 155: 605-612Crossref PubMed Scopus (233) Google Scholar, 6Hauser M.A. Horrigan S.K. Salmikangas P. Torian U.M. Viles K.D. Dancel R. Tim R.W. Taivainen A. Bartoloni L. Gilchrist J.M. 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Whereas much progress has been made in elucidating the pathways of muscle dysfunction and in identifying essential structural and signaling proteins, a gap remains in our understanding of the true complexity of molecular interactions between known and novel proteins within this cytoarchitectural framework. The discovery of novel muscle-specific cytoskeletal proteins and their specific protein-protein interactions will undoubtedly reveal additional important pathways in the formation and maintenance of the cytoskeleton in muscle cells.The myocyte enhancer factor-2 (MEF2) 2The abbreviations used are: MEF2, myocyte enhancer factor-2; EST, expressed sequence tag; GST, glutathione S-transferase; ChIP, chromatin immunoprecipitation assay; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; BBC, B-box coiled-coil; CMYA5, cardiomyopathy-associated gene 5; TRIM, tripartite motif; FN, fibronectin; DM, double mutant; TM, triple mutant.2The abbreviations used are: MEF2, myocyte enhancer factor-2; EST, expressed sequence tag; GST, glutathione S-transferase; ChIP, chromatin immunoprecipitation assay; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; BBC, B-box coiled-coil; CMYA5, cardiomyopathy-associated gene 5; TRIM, tripartite motif; FN, fibronectin; DM, double mutant; TM, triple mutant. family of transcription factors regulates the expression of numerous muscle-specific genes that include components of the elaborate cytoskeletal network in striated muscle cells (10Black B.L. Olson E.N. Annu. Rev. Cell Dev. Biol. 1998; 14: 167-196Crossref PubMed Scopus (843) Google Scholar). Consistent with its role in the regulation of muscle-specific gene expression, MEF2 loss-of-function mutations exhibit defects in striated muscle development and function with associated alterations in the expression of genes encoding proteins of the contractile apparatus (11Lilly B. Zhao B. Ranganayakulu G. Paterson B.M. Schulz R.A. Olson E.N. Science. 1995; 267: 688-693Crossref PubMed Scopus (419) Google Scholar, 12Lin Q. Schwarz J. Bucana C. Olson E.N. Science. 1997; 276: 1404-1407Crossref PubMed Scopus (769) Google Scholar, 13Lin Q. Lu J. Yanagisawa H. Webb R. Lyons G.E. Richardson J.A. Olson E.N. Development. 1998; 125: 4565-4574Crossref PubMed Google Scholar). One of the more complex MEF2 knock-out phenotypes is that of MEF2A. MEF2A is one of the four MEF2 genes in vertebrates, MEF2A, -2B, -2C, and -2D expressed in muscle and non-muscle cell lineages and activates genes involved in cell proliferation and differentiation (14McKinsey T.A. Zhang C.L. Olson E.N. Trends Biochem. Sci. 2002; 27: 40-47Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). A null allele for MEF2A in mice results in cardiac sudden death with cytoarchitectural defects, mitochondrial deficiency, and conduction disturbances (15Naya F.J. Black B.L. Wu H. Bassel-Duby R. Richardson J.A. Hill J.A. Olson E.N. Nat. Med. 2002; 8: 1303-1309Crossref PubMed Scopus (255) Google Scholar). In humans, a dominant negative mutation of the mef2a gene has been linked to cardiovascular disease arising from defects in the coronary arteries (16Wang L. Fan C. Topol S.E. Topol E.J. Wang Q. Science. 2003; 302: 1578-1581Crossref PubMed Scopus (299) Google Scholar). Subsequent reports have failed to demonstrate linkage of this mutation to coronary artery disease (17Weng L. Kavaslar N. Ustaszewska A. Doelle H. Shackwitz W. Hebert S. Cohen J.C. McPherson R. Pennacchio L.A. J. Clin. Investig. 2005; 115: 1016-1020Crossref PubMed Scopus (97) Google Scholar, 18Kajimoto K. Shioji K. Tago N. Tomoike H. Nonogi H. Goto Y. Iwai N. Circ. J. 2005; 69: 1192-1195Crossref PubMed Scopus (27) Google Scholar) though a different polymorphism in MEF2A is now implicated in myocardial infarctions (19Gonzalez P. Garcia-Castro M. Reguero J.R. Batalla A. Ordonez A.G. Palop R.L. Lozano I. Montes M. Alvarez V. Coto E. J. Med. Genet. 2005; (in press)Google Scholar). Nevertheless, whereas a considerable amount is known regarding the mechanisms by which MEF2 transcription factors are modified to regulate gene expression (14McKinsey T.A. Zhang C.L. Olson E.N. Trends Biochem. Sci. 2002; 27: 40-47Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar) the downstream pathways that they regulate are not completely understood. Thus, the identification of novel downstream target genes for MEF2A will provide further insight into the cellular pathways regulated by this transcription factor in the cardiovascular system.To investigate the molecular mechanisms of cardiac abnormalities in mef2a mutant mice and identify novel MEF2A target genes we compared gene expression levels between wild type and mef2a knock-out hearts using DNA microarray (15Naya F.J. Black B.L. Wu H. Bassel-Duby R. Richardson J.A. Hill J.A. Olson E.N. Nat. Med. 2002; 8: 1303-1309Crossref PubMed Scopus (255) Google Scholar). This gene expression profiling approach identified numerous dysregulated expressed sequence tags (ESTs) down-regulated in mef2a mutant hearts. One of these mouse ESTs is down-regulated ∼2-fold and is homologous to a partial human cDNA in the NCBI data base named cardiomyopathy-associated gene 5 (CMYA5). CMYA5 was discovered by expression profiling of a human cardiac muscle library but its function has not been reported. Because the expression of this EST is dependent on MEF2A transcriptional activity we focused our efforts on the cloning and characterization of the mouse ortholog of the CMYA5 gene. While this work was in progress, the mouse ortholog of CMYA5, named Myospryn, was identified as a dysbindin-interacting protein (20Benson M.A. Tinsley C.L. Blake D.J. J. Biol. Chem. 2004; 279: 10450-10458Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Dysbindin is a ubiquitously expressed, cytoplasmic protein that interacts with α-dystrobrevin, which itself associates with the dystrophin glycoprotein complex (21Benson M.A. Newey S.E. Martin-Rendon E. Hawkes R. Blake D.J. J. Biol. Chem. 2001; 276: 24232-24241Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Myospryn encodes a large, novel member of the tripartite motif (TRIM) superfamily of proteins (22Reymond A. Meroni G. Fantozzi A. Merla G. Cairo S. Luzi L. Riganelli D. Zanaria E. Messali S. Cainarca S. Guffanti A. Minucci S. Pelicci P.G. Ballabio A. EMBO J. 2001; 20: 2140-2151Crossref PubMed Scopus (1033) Google Scholar). We demonstrate that Myospryn is a direct target for MEF2A and is localized specifically to the costamere of striated muscle cells where it interacts with the sarcomeric Z-disc protein, α-actinin-2. The characterization of this MEF2A-dependent gene will contribute to our understanding of the extensive protein interactions within the costamere and Z-disc complex and its potential role in muscle function and disease.MATERIALS AND METHODSMicroarray Analysis and Bioinformatics—Total RNA was prepared using TRIzol reagent (Invitrogen) from wild type and mef2a mutant hearts and used with Mouse GEM1 array (Incyte Genomics) as previously described (15Naya F.J. Black B.L. Wu H. Bassel-Duby R. Richardson J.A. Hill J.A. Olson E.N. Nat. Med. 2002; 8: 1303-1309Crossref PubMed Scopus (255) Google Scholar). The down-regulated EST AA413670 was used to search the NCBI and Celera data bases to assemble the full-length mouse cDNA by PCR from a Marathon skeletal muscle cDNA library (Clontech) and is identical to Myospryn (GenBank™ accession number AJ575748).Creation of Mef2A Transgenic Mice—The mouse MEF2A cDNA was amplified from a Marathon cardiac muscle cDNA library (Clontech) and cloned into the αMHC-hGH poly(A) vector. The promoter-cDNA cassette was released from the vector backbone by digestion with NotI, gel-purified using QiaQuick columns (Qiagen) and injected into the male pronuclei of fertilized mouse oocytes. Two transgenic positive founders were obtained and both transmitted the transgene to their offspring.RT-PCR Analysis—Total RNA was isolated from cardiac muscle using the TRIzol method. First strand cDNA was prepared from total RNA with random hexamers using reverse transcriptase. cDNAs were subjected to PCR, fractionated on a 5% native polyacrylamide gel, and visualized with a PhosphorImager using ImageQuant Program (Molecular Dynamics). RT-PCR was performed in the linear range (26 cycles) with primers specific for Myospryn within the 3′-end of the gene. Primers for detecting Myospryn were as follows: 5′-GTTAACCTCCCTCCCAACGACAATTACTT-3′;5′-TCGTCACGTACATGGTACTCCAGAG-3′.Northern Blot Analysis and in Situ Hybridization—A mouse multiple tissue poly(A)+ RNA blot (Clontech) was hybridized with a 32P-labeled HindIII fragment (nucleotides 5,391-6,455) of the Myospryn cDNA. An antisense cDNA fragment corresponding to nucleotides 5,391-5,915 was used for radioactive in situ hybridization. Embryos at various developmental time points were fixed according to standard procedures and sagittal mouse sections were hybridized with antisense Myospryn cDNA labeled with 35S-UTP.Antibodies and Immunocytochemistry—Rabbit polyclonal anti-Myospryn antibodies were raised against GST fusion proteins (Cocalico Biologicals) generated from three different regions of the Myospryn cDNA. Antibodies UT266 (GST fusion-containing nucleotides 4,764-5,391), UT264 (nucleotides 5,391-6,455), and UT262 (nucleotides 9,583-9,868) were IgG-purified using MabTrap II (Amersham Biosciences). All three antibodies exhibited specificity for Myospryn in transfected cells. However, the UT266 antibody exhibited the highest specificity with heart cryosections. For immunostaining of heart cryosections, adult mice were perfused with 4% paraformaldehyde, hearts were dissected and cryoprotected by immersion in 30% sucrose/phosphate-buffered saline at 4 °C for 24-48 h. Cryoprotected hearts were then placed in embedding compound (OCT) and frozen at -80 °C. Hearts were cryosectioned at 15 μm and air-dried on Superfrost Plus Slides (Fisher). Primary antibodies for Myospryn (UT266, IgG fraction, dilution 1:100), mouse monoclonal sarcomeric α-actinin (Sigma, 1:100) and secondary antibodies (1:200) for anti-Myospryn (anti-rabbit Texas Red) and anti-actinin (anti-mouse fluorescein isothiocyanate) were used on heart cryosections. After immunostaining procedure, Vectashield (Vector Labs) was applied to heart cryosections and protected with cover slips.Transient Transfections, Coimmunoprecipitations, and Western Blots—COS-7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mm glutamine, and penicillin/streptomycin. For reporter assays, COS cells in 6-well plates, each well containing ∼500,000 cells, were transfected with 0.2 μg of the wild-type or mutant forms of the myospryn promoter in pGL3-Basic (Promega) and 0.8 μg of human MEF2A in pcDNA 1 (Invitrogen). Cells were transfected using FuGENE 6 transfection reagent (Roche Applied Sciences), and were harvested after 36-48 h. Luciferase assays were performed using the Dual Luciferase Reporter Assay system (Promega), and protein concentrations were normalized via Bradford assay. For co-immunoprecipitations, COS cells in 100-mm dishes (at 50% confluency) were transfected with 20 μg of expression plasmids for full-length Myospryn and 5 μg for truncated forms of Myospryn and α-actinin, using FuGENE 6 reagent. Myospryn proteins were fused with an N-terminal Myc epitope, and α-actinin was fused with an N-terminal FLAG epitope. Forty eight hours after transfection, cells were harvested in ELB buffer (50 mm HEPES, pH 7.0, 250 mm NaCl, 5 mm EDTA, 0.1% IGE-PAL, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Complete, Roche Applied Sciences). Extracts were immunoprecipitated for 2 h at 4°C using protein A/G-agarose and 1 μg of monoclonal anti-Myc antibody (Santa Cruz Biotechnology). Subsequently, the pellet was washed with ELB buffer and subjected to SDS-PAGE followed by transfer to Immobilon polyvinylidene difluoride membrane (Bio-Rad) and immunoblotting using anti-FLAG antibodies (Sigma).Gel Shift and Chromatin Immunoprecipitation Assays—For gel shift assays, whole cell extracts were isolated from adult mouse hearts by homogenization and incubated in the presence of radiolabeled, double-stranded oligonucleotides corresponding to each of the four MEF2 sites in the myospryn promoter as described previously (15Naya F.J. Black B.L. Wu H. Bassel-Duby R. Richardson J.A. Hill J.A. Olson E.N. Nat. Med. 2002; 8: 1303-1309Crossref PubMed Scopus (255) Google Scholar). For ChIP analysis, primary neonatal rat cardiomyoyctes were isolated and seeded at a density of 5 × 106 cells in 10-cm dishes. C2C12 myoblasts seeded at a density of 1 × 106 were transfected with 15 μg of FLAG-MEF2A expression vector. Cardiomyocytes and C2C12 myoblasts were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mm glutamine, and penicillin/streptomycin for 48-72 h. Cells were cross-linked with 37% formaldehyde, incubated for 10 min at room temperature, and subsequently harvested. ChIP assays were performed using the ChIP assay kit (Upstate Cell Signaling Solutions), and procedures were carried out according to the Upstate protocol, with the following modifications. Harvested cells were sonicated in 400 μl of SDS lysis buffer containing protease inhibitors. Each 400-μl sample was subsequently split into 200 μl, and then diluted ∼6-fold in ChIP dilution buffer containing protease inhibitors. Immunoprecipitations were performed using 2 μg of anti-acetyl-histone H3 antibody (Upstate Cell Signaling Solutions) or 4 μg of anti-FLAG. PCR primers flanking the -1985 site in the myospryn promoter were used for the PCR analysis. The rat, mouse, and human myospryn promoter sequences were aligned using the MatInspector program, and primers were designed using sequences that were found to be identical in both the rat and mouse promoters. 5′ primer sequence: 5′-CCT GGT TTT ATT TTC CTC ATG ATA GG-3′.3′ primer sequence: 5′-CTG TAG GCC TAC AAG AAG TTT G-3′.RESULTSMicroarray Analysis and Identification of Myospryn—In our ongoing effort to gain further insight into the cellular pathways regulated by MEF2A in the postnatal heart we have continued to analyze microarray data previously reported for the characterization of the mef2a knock-out phenotype (15Naya F.J. Black B.L. Wu H. Bassel-Duby R. Richardson J.A. Hill J.A. Olson E.N. Nat. Med. 2002; 8: 1303-1309Crossref PubMed Scopus (255) Google Scholar). mef2a mutant mice die in the perinatal period and display severe cytoarchitectural and mitochondrial abnormalities in cardiac muscle cells. Aside from the known MEF2 target genes dysregulated in mef2a mutant hearts, numerous novel ESTs also exhibited alterations in gene expression levels in these abnormal hearts. One of these ESTs (accession number AA413760) is down-regulated 1.9-fold. While this work was in preparation, the full-length gene product of mouse EST AA413760, named Myospryn, was identified as a dysbindin-interacting protein (20Benson M.A. Tinsley C.L. Blake D.J. J. Biol. Chem. 2004; 279: 10450-10458Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Dysbindin is a ubiquitously expressed cytosolic protein that interacts with α-dystrobrevin, which itself is a component of the dystrophin glycoprotein complex (21Benson M.A. Newey S.E. Martin-Rendon E. Hawkes R. Blake D.J. J. Biol. Chem. 2001; 276: 24232-24241Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The down-regulation of Myospryn was subsequently confirmed by semi-quantitative RT-PCR with cDNA from postnatal day 5 (P5) hearts of mef2a mutant mice. Transcripts for Myospryn were expressed at 0.41 ± 0.09 (n = 3) of wild-type levels in P5 hearts of mef2a mutant mice (data not shown).MEF2A-dependent Activation of Endogenous Myospryn Gene—To further demonstrate that endogenous myospryn gene expression is sensitive to MEF2A dosage, we generated mice that overexpressed the fulllength mouse MEF2A cDNA in the heart under the control of the α-myosin heavy chain (αMHC) promoter (Fig. 1A). An observed induction of the myospryn gene would strengthen the notion that it is a relevant in vivo target downstream of MEF2A. In combination with the mef2a knock-out microarray data, these transgenic mice will also provide us with an important reagent to identify additional genes most likely to be directly activated by MEF2A. This complementary approach will be useful in dissecting the MEF2A-dependent transcriptional network in the postnatal heart. 3J. Reynolds and F. Naya, manuscript in preparation.Two independent lines of αMHC-MEF2A transgenic mice, MHC2A19 and MHC2A20, were obtained, and expression of the transgene was confirmed by Northern analysis (data not shown). Western analysis using a MEF2A antibody (C-21, Santa Cruz Biotechnology) demonstrated an approximately 2-fold increase in MEF2A protein over basal levels in these transgenic hearts (Fig. 1B). We noted that this increase in MEF2A expression resulted in a modest enlargement of the heart by three months of age (Fig. 1C) and the induction of atrial natriuretic factor (ANF) a known MEF2 target and hypertrophic marker gene (data not shown). These results are consistent with the observed phenotype in transgenic mice overexpressing human MEF2A in the heart 4J. D. Molkentin, personal communication.. Additional molecular characterization of this phenotype will be described elsewhere.To examine transcript levels for endogenous myospryn, RNA was isolated from 2-month-old transgenic hearts, prior to any visible cardiac enlargement and subjected to quantitative real time (qRT)-PCR (Applied Biosystems). Measurement of transcripts for myospryn using qRT-PCR revealed a 2.9 ± 1.0 (n = 3) enhancement in basal expression levels (Fig. 1D) demonstrating that forced expression of MEF2A alone induces this gene in the heart. Other well characterized MEF2 target genes and novel ESTs, previously demonstrated to be down-regulated in mef2a knock-out hearts, were induced in transgenic mice overexpressing MEF2A validating that these genes are downstream of MEF2A and are likely to be direct targets for this transcription factor in the heart. These results demonstrate that MEF2A is sufficient to activate the endogenous myospryn gene and supports the notion that it is a bona fide in vivo target for MEF2A in the heart.Regulation of Myospryn Transcription by MEF2A—To test whether myospryn represents a direct transcriptional target for MEF2A we identified the 5′-end of the gene using a combination of the EST data base, 5′-RACE PCR, primer extension, and RNase protection (data not shown). Based on this information, we isolated 3.5 kb of mouse genomic sequence upstream of the first exon and cloned this fragment into the pGL3BASIC-luciferase reporter. MEF2A-responsiveness of this 5′ region was tested in transfected COS cells. As demonstrated in Fig. 2A, this 3.5-kb fragment alone exhibited background levels of activity in COS cells. However, when co-transfected with a MEF2A expression vector, the 3.5-kb fragment was induced 5.2-fold (Fig. 2A) indicating that the myospryn promoter responds to MEF2A activity.FIGURE 2Direct activation of myospryn promoter by MEF2A. A, MEF2A expression vector or pcDNA3 was co-transfected into COS cells with the wild-type myospryn promoter or a series of mutant promoter constructs. The fold activation is relative to the luciferase activity (normalized to 1) of the wild-type or mutant reporter transfected with pcDNA3 vector backbone. DM, -1452/-1985; TM1, +33/-1452/-1985; TM2, -1452/-1985/-2834. Error bars represent S.D. for three experiments. Values obtained for the -1452, -1985, DM, and TM mutants are statistically significant at p < 0.05. B, sequence of the myospryn proximal promoter and additional upstream sequences. The four MEF2 sites and a putative SRF binding site (CArG-like) are underlined. C, MEF2A binds four MEF2 sites present in myospryn promoter. In vitro translated MEF2A or whole cell lysates from adult mouse hearts were subjected to gel shift assays. Protein-DNA complexes were formed with each of the MEF2 sites within the mysospryn promoter. MEF2 DNA binding activity was confirmed by incubation with a MEF2A-specific antibody resulting in a slower migrating supershifted complex. D, sequence alignment of rat, mouse, and human myospryn promoter regions. The conserved -1985 MEF2 site is noted in the boxed region. Also shown are the -1452 and -2834 MEF2 sites aligned with their rat and human counterparts. No significant alignment was obtained with the +33 MEF2 site. Upper right panel, the conserved -1985 MEF2 site of the myospryn promoter is located in a region of acetylated chromatin. Lower right panel, FLAG-MEF2A binds the mouse -1985 site in C2C12 myoblasts. ChIP analysis was performed on cross-linked chromatin isolated from neonatal rat primary cardiomyocytes and C2C12 myoblasts (transfected with a FLAG-MEF2A expression vector), and immunoprecipitations were performed using the anti-acetyl-histone H3 antibody and anti-FLAG respectively. A no-antibody immunoprecipitation serves as the control. Immunoprecipitated DNA from each sample, as well as aliquots of non-immunoprecipitated DNA used as input controls for each sample, were subjected to PCR using primers flanking the -1985 MEF2 site.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Given the ability of MEF2A to transactivate this upstream fragment we examined this region for MEF2 binding sites and identified four putative MEF2 sequences exhibiting similarity to the consensus MEF2 site, YTA(A/T)4TAR (Fig. 2B). To determine whether MEF2A could bind these sequences we performed gel mobility shift assays. The MEF2 sequences located at +33 (CTATTTAAAG), -1452 (TTATAATTAG), -1985 (TTATAAATAA), and -2834 (TTATTTTTAA) relative to the putative transcription start site bound to in vitro translated MEF2A (Fig. 2C, left panel), and MEF2A-specific binding was confirmed by supershifting the protein-DNA complex with an anti-MEF2A antibody. To validate that endogenous MEF2A from cardiomyocytes could also bind these sequences protein extracts from adult mouse hearts were subjected to gel shift assays. MEF2 DNA binding activity was readily apparent for each of the four MEF2 binding sites and incubation of these extracts with MEF2A antibodies resulted in a slower migrating supershifted complex (Fig. 2C, right panel). These results confirm that MEF2A protein directly binds to all four MEF2 sites within the myospryn promoter.To determine whether the myospryn promoter was being activated through one or more of the MEF2 sites, mutations that disrupt MEF2 binding were introduced within each of the core sequences in the context of the 3.5-kb proximal promoter. As shown in Fig. 2A, mutations within each of the individual MEF2 binding sites had varying effects on transactivation potential. Mutations of the +33 and the -2834 MEF2 sites had no significant effect and a 20% reduction, respectively, in transcriptional activity directed by MEF2A. In contrast, mutations within the -1452 and -1985 MEF2 sites affected transcriptional activity by 30 and 45%, respectively. Given the greater inhibitory effects of the -1452 and -1985 mutations we generated a double mutation (DM) within the 3.5-kb promoter and determined its ability to be transactivated by MEF2A. This double mutation resulted in an additional inhibitory effect (60% reduction relative to wild type) in transcriptional activity. Because the double mutation did not completely eliminate transcriptional response of this promoter to MEF2A, we asked whether mutations in either the +33 or the -2834 MEF2 sites in the context of the DM construct would result in a more pronounced inhibitory effect. Mutations in the +33 and -2834 MEF2 sites were introduced into the DM construct resulting in two different triple mutations, TM1 (+33/-1452/-1985) and TM2 (-1452/-1985/-2834). Each of these triple mutant promoter constructs were tested for their ability to respond to MEF2A. Both TM1 and TM2 mutations did not result in a further reduction in MEF2A responsiveness.