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• W2000884265 abstract "Myocyte enhancer factor 2 (MEF2) transcription factors play pivotal roles in striated muscle, neuron, and lymphocyte gene expression and are targets of stress- and calcium-mediated signaling. All MEF2 gene products have a common DNA binding and dimerization domain, but MEF2 transcripts are alternatively spliced among coding exons to produce splicing isoforms. In vertebrate MEF2A, -C, and -D, a splice versus no-splice option gives forms that include or exclude a short domain that we designate β. We show that mRNAs containing β are expressed predominantly in striated muscle and brain and that splicing to include β is induced during myocyte differentiation. MEF2 β+ isoforms are more robust than β- forms in activating MEF2-responsive reporters despite similar expression levels. One-hybrid transcription assays using Gal4-MEF2 fusions show similar distinctions in the transactivation produced by β+ versus β- isoforms in all cell types tested, including myocytes. β function is position-independent and exists in all MEF2 splicing variant contexts. The activity is not due to cis effects on MEF2 DNA binding or dimerization nor are established transcription factor or coactivator interactions involved. Each MEF2 β domain contains multiple acidic residues, mutation of which abolishes function. Despite a location between the p38 MAPK docking domain and Thr phosphoacceptors of MEF2A and MEF2C, inclusion of β does not influence responses of these factors to this signaling pathway. Thus, a conserved pattern of alternative splicing in vertebrate MEF2 genes generates an acidic activation domain in MEF2 proteins selectively in tissues where MEF2 target genes are highly expressed. Myocyte enhancer factor 2 (MEF2) transcription factors play pivotal roles in striated muscle, neuron, and lymphocyte gene expression and are targets of stress- and calcium-mediated signaling. All MEF2 gene products have a common DNA binding and dimerization domain, but MEF2 transcripts are alternatively spliced among coding exons to produce splicing isoforms. In vertebrate MEF2A, -C, and -D, a splice versus no-splice option gives forms that include or exclude a short domain that we designate β. We show that mRNAs containing β are expressed predominantly in striated muscle and brain and that splicing to include β is induced during myocyte differentiation. MEF2 β+ isoforms are more robust than β- forms in activating MEF2-responsive reporters despite similar expression levels. One-hybrid transcription assays using Gal4-MEF2 fusions show similar distinctions in the transactivation produced by β+ versus β- isoforms in all cell types tested, including myocytes. β function is position-independent and exists in all MEF2 splicing variant contexts. The activity is not due to cis effects on MEF2 DNA binding or dimerization nor are established transcription factor or coactivator interactions involved. Each MEF2 β domain contains multiple acidic residues, mutation of which abolishes function. Despite a location between the p38 MAPK docking domain and Thr phosphoacceptors of MEF2A and MEF2C, inclusion of β does not influence responses of these factors to this signaling pathway. Thus, a conserved pattern of alternative splicing in vertebrate MEF2 genes generates an acidic activation domain in MEF2 proteins selectively in tissues where MEF2 target genes are highly expressed. Myocyte enhancer factor 2 (MEF2) 1The abbreviations used are: MEF2, myocyte enhancer factor 2; MEF2S, MEF2 signature domain; Cabin1, calcineurin binding protein 1; cdk, cyclin-dependent kinase; CPT-IB, carnitine palmitoyltransferase I; DME, Dulbecco's modified Eagle's medium; DMEF2, Drosophila myocyte enhancer factor 2; Erk, extracellular signal responsive protein kinase; Gal4DBD, DNA binding domain of yeast Gal4p; MADS, MCM1, agamous, deficiens, serum response factor; MAPK, mitogen-activated protein kinase; RPA, ribonuclease protection assay. proteins are members of the MADS (MCM1, agamous, deficiens, serum response factor)-box family of transcriptional regulators (1.Black B.L. Olson E.N. Annu. Rev. Cell Dev. Biol. 1998; 14: 167-196Crossref PubMed Scopus (856) Google Scholar, 2.McKinsey T.A. Zhang C.L. Olson E.N. Trends Biochem. Sci. 2002; 27: 40-47Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 3.Naya F.J. Olson E. Curr. Opin. Cell Biol. 1999; 11: 683-688Crossref PubMed Scopus (259) Google Scholar). MEF2 was originally recognized as a sequence-specific DNA-binding activity at conserved elements in the promoters of various genes encoding muscle structural proteins and as products of cDNAs encoding proteins related to serum response factor (4.Yu Y.T. Breitbart R.E. Smoot L.B. Lee Y. Mahdavi V. Nadal-Ginard B. Genes Dev. 1992; 6: 1783-1798Crossref PubMed Scopus (385) Google Scholar, 5.Pollock R. Treisman R. Genes Dev. 1991; 5: 2327-2341Crossref PubMed Scopus (323) Google Scholar). Four distinct vertebrate genes encoding MEF2 forms were subsequently recognized, MEF2A, MEF2B, MEF2C, and MEF2D (6.Martin J.F. Miano J.M. Hustad C.M. Copeland N.G. Jenkins N.A. Olson E.N. Mol. Cell. Biol. 1994; 14: 1647-1656Crossref PubMed Scopus (188) Google Scholar, 7.Martin J.F. Schwarz J.J. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5282-5286Crossref PubMed Scopus (220) Google Scholar, 8.Breitbart R.E. Liang C.S. Smoot L.B. Laheru D.A. Mahdavi V. Nadal-Ginard B. Development. 1993; 118: 1095-1106Crossref PubMed Google Scholar, 9.McDermott J.C. Cardoso M.C. Yu Y.T. Andres V. Leifer D. Krainc D. Lipton S.A. Nadal-Ginard B. Mol. Cell. Biol. 1993; 13: 2564-2577Crossref PubMed Scopus (199) Google Scholar). Initial studies of MEF2 largely considered a role in myogenesis and muscle structural protein expression, but a wider province is now appreciated. Thus, MEF2 target genes include those encoding transporters and metabolic enzymes of striated muscle (10.Mora S. Pessin J.E. J. Biol. Chem. 2000; 275: 16323-16328Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 11.Knight J.B. Eyster C.A. Griesel B.A. Olson A.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14725-14730Crossref PubMed Scopus (82) Google Scholar, 12.Scarpulla R.C. Biochim. Biophys. Acta. 2002; 1576: 1-14Crossref PubMed Scopus (516) Google Scholar, 13.Czubryt M.P. Olson E.N. Recent. Prog. Horm. Res. 2004; 59: 105-124Crossref PubMed Scopus (59) Google Scholar) and effectors of stress signaling in various cell types (14.Kato Y. Zhao M. Morikawa A. Sugiyama T. Chakravortty D. Koide N. Yoshida T. Tapping R.I. Yang Y. Yokochi T. Lee J.D. J. Biol. Chem. 2000; 275: 18534-18540Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 15.Zhao M. New L. Kravchenko V.V. Kato Y. Gram H. di Padova F. Olson E.N. Ulevitch R.J. Han J. Mol. Cell. Biol. 1999; 19: 21-30Crossref PubMed Scopus (379) Google Scholar). In addition, MEF2 proteins interact directly with neuron-specific transcription factors (16.Black B.L. Ligon K.L. Zhang Y. Olson E.N. J. Biol. Chem. 1996; 271: 26659-26663Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and play a critical role in differentiation and programmed cell death in this cell type (17.Gillardon F. Steinlein P. Burger E. Hildebrandt T. Gerner C. 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Sherman K. Lipton S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7561-7566Crossref PubMed Scopus (173) Google Scholar). Finally, critical roles for MEF2 factors in leukocyte functions have been established, including T lymphocyte apoptosis (25.Youn H.D. Liu J.O. Immunity. 2000; 13: 85-94Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 26.Youn H.D. Chatila T.A. Liu J.O. EMBO J. 2000; 19: 4323-4331Crossref PubMed Scopus (182) Google Scholar) and activation (27.Pan F. Ye Z. Cheng L. Liu J.O. J. Biol. Chem. 2004; 279: 14477-14480Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), maintenance of Epstein-Barr virus latency in B cells (28.Gruffat H. Manet E. Sergeant A. EMBO Rep. 2002; 3: 141-146Crossref PubMed Scopus (101) Google Scholar), and macrophage activation (29.Han J. Jiang Y. Li Z. Kravchenko V.V. Ulevitch R.J. Nature. 1997; 386: 296-299Crossref PubMed Scopus (688) Google Scholar). The four MEF2 genes are differentially expressed spatially and temporally during development and in mature tissues (30.Edmondson D.G. Lyons G.E. Martin J.F. Olson E.N. Development. 1994; 120: 1251-1263Crossref PubMed Google Scholar, 31.Subramanian S.V. Nadal-Ginard B. Mech. Dev. 1996; 57: 103-112Crossref PubMed Scopus (48) Google Scholar). MEF2 isotype functions partially overlap, but distinct roles for the different genes remain to be fully elucidated. Murine gene disruption studies provide genetic evidence in support of discrete MEF2 isotype-specific functions (32.Lin Q. Schwarz J. Bucana C. Olson E.N. Science. 1997; 276: 1404-1407Crossref PubMed Scopus (786) Google Scholar, 33.Naya 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 (265) Google Scholar). Thus, mef2-c null mice die at embryonic day 9 due to failure of cardiac development, and the animals also exhibit vascular defects (34.Lin Q. Lu J. Yanagisawa H. Webb R. Lyons G.E. Richardson J.A. Olson E.N. Development. 1998; 125: 4565-4574Crossref PubMed Google Scholar). Because the other mef2 isotype genes are expressed at normal or supraphysiological levels in the mef2-c null animals, lack of compensation by these forms indicates a unique role for mef2-c in cardiac and vascular development. mef2-a null mice survive to the neonatal period, or in some cases to adulthood, but severe myocardial mitochondrial defects are present that predispose to sudden death (33.Naya 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 (265) Google Scholar). Expression of the other mef2 genes is also up-regulated in these animals, again indicating lack of compensation in vivo for selective MEF2 gene loss. At present it is not clear whether MEF2 isotype-selective functions relate solely to distinctions in temporospatial expression, or to unique features of the MEF2 protein forms encoded by the different genes, although we have provided recent evidence to suggest that the latter is likely (35.Zhu B. Gulick T. Mol. Cell. Biol. 2004; 24: 8264-8275Crossref PubMed Scopus (68) Google Scholar). Regulation of MEF2 function is complex and occurs at many levels. Abundance of MEF2 proteins is controlled at transcriptional (36.Wang D.Z. Valdez M.R. McAnally J. Richardson J. Olson E.N. Development. 2001; 128: 4623-4633Crossref PubMed Google Scholar, 37.De Val S. Anderson J.P. Heidt A.B. Khiem D. Xu S.M. Black B.L. Dev. Biol. 2004; 275: 424-434Crossref PubMed Scopus (66) Google Scholar, 38.Dodou E. Xu S.M. Black B.L. Mech. Dev. 2003; 120: 1021-1032Crossref PubMed Scopus (112) Google Scholar, 39.Dodou E. Verzi M.P. Anderson J.P. Xu S.M. Black B.L. Development. 2004; 131: 3931-3942Crossref PubMed Scopus (315) Google Scholar), 2T. Gulick and G.-S. Yu, submitted for publication. translational (41.Black B.L. Lu J. Olson E.N. Mol. Cell. Biol. 1997; 17: 2756-2763Crossref PubMed Scopus (47) Google Scholar), and degradation (23.Okamoto S. Li Z. Ju C. Scholzke M.N. Mathews E. Cui J. Salvesen G.S. Bossy-Wetzel E. Lipton S.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3974-3979Crossref PubMed Scopus (119) Google Scholar, 42.Li M. Linseman D.A. Allen M.P. Meintzer M.K. Wang X. Laessig T. Wierman M.E. Heidenreich K.A. J. Neurosci. 2001; 21: 6544-6552Crossref PubMed Google Scholar) steps. The transactivation function of MEF2 proteins is regulated by various means, including through protein-protein interactions with other transcription factors (16.Black B.L. Ligon K.L. Zhang Y. Olson E.N. J. Biol. Chem. 1996; 271: 26659-26663Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 43.Grayson J. Bassel-Duby R. Williams R.S. J. Cell. 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Chem. 2000; 275: 18534-18540Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 15.Zhao M. New L. Kravchenko V.V. Kato Y. Gram H. di Padova F. Olson E.N. Ulevitch R.J. Han J. Mol. Cell. Biol. 1999; 19: 21-30Crossref PubMed Scopus (379) Google Scholar, 21.Gong X. Tang X. Wiedmann M. Wang X. Peng J. Zheng D. Blair L.A. Marshall J. Mao Z. Neuron. 2003; 38: 33-46Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar); and by determinants of MEF2 and MEF2 co-regulator subcellular localization, including calcium-dependent effects on MEF2 interactors and interactions (2.McKinsey T.A. Zhang C.L. Olson E.N. Trends Biochem. Sci. 2002; 27: 40-47Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 51.Lazaro J.B. Bailey P.J. Lassar A.B. Genes Dev. 2002; 16: 1792-1805Crossref PubMed Scopus (84) Google Scholar, 61.De Angelis L. Borghi S. Melchionna R. Berghella L. Baccarani-Contri M. Parise F. Ferrari S. Cossu G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12358-12363Crossref PubMed Scopus (64) Google Scholar). MEF2 mRNAs, proteins, and sequence-specific DNA-binding activities are widely expressed, but target gene activation is highly restricted among tissues and cell types (20.Heidenreich K.A. Linseman D.A. Mol. Neurobiol. 2004; 29: 155-166Crossref PubMed Scopus (69) Google Scholar, 62.Dodou E. Sparrow D.B. Mohun T. Treisman R. Nucleic Acids Res. 1995; 23: 4267-4274Crossref PubMed Scopus (35) Google Scholar, 63.Ornatsky O.I. McDermott J.C. J. Biol. Chem. 1996; 271: 24927-24933Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 64.Blaeser F. Ho N. Prywes R. Chatila T.A. J. Biol. Chem. 2000; 275: 197-209Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 65.Swanson B.J. Jack H.M. Lyons G.E. Mol. Immunol. 1998; 35: 445-458Crossref PubMed Scopus (69) Google Scholar). Some of this discordance clearly involves one or more of these regulatory mechanisms, but additional conditions may also be pertinent, including regulated expression of splicing isoforms with distinct functions (35.Zhu B. Gulick T. Mol. Cell. Biol. 2004; 24: 8264-8275Crossref PubMed Scopus (68) Google Scholar). Each vertebrate MEF2 gene gives rise to multiple isoforms through alternative splicing patterns that are conserved among vertebrates (35.Zhu B. Gulick T. Mol. Cell. Biol. 2004; 24: 8264-8275Crossref PubMed Scopus (68) Google Scholar). 3B. Ramachandran and T. Gulick, submitted for publication. These splicing patterns include use of bona fide alternative exons, a splice versus no-splice option, and use of alternative splice acceptors within one exon. In contrast to extensive work on MEF2 gene and MEF2 protein regulation at the various levels noted above, the differential expression and unique roles and responses of MEF2 splicing variants has only recently been explored (35.Zhu B. Gulick T. Mol. Cell. Biol. 2004; 24: 8264-8275Crossref PubMed Scopus (68) Google Scholar, 67.Janson C.G. Chen Y. Li Y. Leifer D. Brain Res. Mol. Brain Res. 2001; 97: 70-82Crossref PubMed Scopus (25) Google Scholar). 3B. Ramachandran and T. Gulick, submitted for publication. No MEF2 protein-protein interactions or MEF2 protein modifications have been identified that involve domains unique to the splicing variants. One genetic study of splicing variants of the sole Drosophila MEF2 gene, DMef2, has been reported (68.Gunthorpe D. Beatty K.E. Taylor M.V. Dev. Biol. 1999; 215: 130-145Crossref PubMed Scopus (41) Google Scholar). No significant differences were noted in the ability of the various DMEF2 splicing forms to rescue muscle differentiation defects in a DMef2 mutant. However, distinctions in splicing variant function may have been obscured by the particular conditions in this study. Furthermore, DMef2 gene structure and alternative splicing patterns are not analogous to those of the vertebrate MEF2 genes, such that these findings do not specifically inform function of the vertebrate MEF2 splicing forms. Previously, we have shown that the MEF2C gene is unique in having alternative splice acceptors at the last exon that generate MEF2C variants that either have or lack a phosphoserine-dependent transrepressor domain called γ (35.Zhu B. Gulick T. Mol. Cell. Biol. 2004; 24: 8264-8275Crossref PubMed Scopus (68) Google Scholar). 3B. Ramachandran and T. Gulick, submitted for publication. In the present report, we show a second distinction among MEF2 splicing isoforms that involves an autonomous acidic transactivation domain that is selectively included in MEF2A, MEF2C, and MEF2D proteins expressed in muscle and nerve. Expression of the β domain is governed by tissue-selective inclusion of a short exon in MEF2 mRNAs. Plasmid Construction—Plasmid sequences are available from the corresponding author. All construct sequences were verified by dideoxy sequencing (69.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Chanda V.B. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1998: 4.1.1-4.1.5Google Scholar). [MEF2CPT-IB]3-tk-Luc, [-150/+75]-jun-Luc, and G5Luc were described (35.Zhu B. Gulick T. Mol. Cell. Biol. 2004; 24: 8264-8275Crossref PubMed Scopus (68) Google Scholar). MEF2 coding regions were obtained using reverse transcription-PCR with human or murine heart, brain or skeletal muscle RNA as templates. pET-MEF2A α1 containing the human MEF2A α1 isoform coding region was described (35.Zhu B. Gulick T. Mol. Cell. Biol. 2004; 24: 8264-8275Crossref PubMed Scopus (68) Google Scholar). Combination PCR on a pET-MEF2A α1 template was used with vector- and gene-specific mutagenic complementary primers for introduction of the β domain with an XhoI restriction site signature. Primers used were 5′-CGGAGGAAGAGGAAcTcGAGTTGAACACCCAAAGGATCAGTAG-3′ and 5′-GTTCAACTCgAgTTCCTCTTCCTCCGATAGTGGAGGCATCATGCC-3′. The BglII- and HindIII-restricted amplicons were substituted into similarly restricted pET-MEF2A α1 to give pET-MEF2A α1.β. Substitution of a NheI to SphI fragment from expressed sequence tag IMAGE clone 3896491 into these plasmids gave pET-MEF2A α2 and α2.β. pET-MEF2C α1 and pET-MEF2C α1.γ, containing the human MEF2C α1 and α1.γ isoform coding regions, were described (35.Zhu B. Gulick T. Mol. Cell. Biol. 2004; 24: 8264-8275Crossref PubMed Scopus (68) Google Scholar). Additional MEF2C isoform coding regions were obtained by swapping a PstI to Acc65I fragment of an reverse transcription-PCR amplicon, obtained using a human skeletal muscle RNA template, to give α1.β and α1.β.γ forms. The MEF2C β SalI site served as a signature. pM-MEF2D α1 was described (35.Zhu B. Gulick T. Mol. Cell. Biol. 2004; 24: 8264-8275Crossref PubMed Scopus (68) Google Scholar). The MEF2D α1.β coding region was obtained by swapping a PstI to XmaI fragment of an reverse transcription-PCR amplicon, obtained using a human skeletal muscle RNA template. The MEF2D β BglII site served as a signature. MEF2 isoform coding regions from pET28 were introduced into pCDNA3.hygro (Invitrogen) (pCDNA-MEF2C) and pM (Clontech) (pM-MEF2C) for expression of native factor isoforms and Gal4DBD fusions, respectively. pM-ΔN86 MEF2C α1 and α1.γ were described (35.Zhu B. Gulick T. Mol. Cell. Biol. 2004; 24: 8264-8275Crossref PubMed Scopus (68) Google Scholar). PstI to NotI fragments from the full-length MEF2C constructs were substituted to give pM-ΔN86 MEF2C α1.β and α1.β.γ. Acc65I to EcoRI segment swapping from a previously described MEF2C γ mutant gave α1.γS388A and α1.β.γS396A inserts in this ΔN86 MEF2C context. The initial MEF2C β domain mutant was created in the pM-ΔN86 MEF2C α1.β background using PCR with a forward vector primer and reverse primer 5′-gggcgcGTCGACATCCTCAGcCACTGATGGCATCGTATTCTTGgatCCTGGTG-3′, installing a BamHI site 5′ to β by silent mutation. The PstI- and SalI-digested amplicon was substituted into the vector to give pM-ΔN86 MEF2C α1.βS271A. BamHI- and NotI-restricted PCR amplicons were substituted into this construct to make additional MEF2C β domain mutants. Amplicons were created using a reverse vector primer with forward primers 5′-gggcgcGGatcCAAGAATACGATGCCATCAGTGTCTcAGGATG-3′ (α1.βE272Q), 5′-gggcgcGGatcCAAGAATACGATGCCATCAGTGTCTGAGaATGTtGACCTG-3′ (α1.βD273N), 5′-gggcgcGGatcCAAGAATACGATGCCATCAGTGTCTGAGGATGTtaACC TGCTTTTG-3′ (α1.βD275N), and 5′-gggcgcGGatcCAAGAATACGATGCCATCAGTGTCTcAGaATGTtaACCTGCTTTTG-3′ (α1.βDEβNQ), with AlwNI, SalI, and HpaI serving as MEF2C β mutant signatures. Human MEF2D sequence 3′ of the MADS box/MEF2S domain was amplified by PCR using forward primer 5′-cccgcgccATGgtgACCCTGAGGAAGAAGGGCTTC-3′ and reverse vector primer on a pM-MEF2D α1 template. The NcoI- and PstI-digested amplicon was reintroduced into similarly restricted pM-MEF2D α1 and α1.β to give pM-ΔN86 MEF2D α1 and α1.β, respectively. A forward vector primer was used with reverse primers in PCR to generate MEF2D β domain mutants. Primers were 5′-cccgcgAGATCTAAATGGTCCTCAGcCAAGTGATGCATTAggCCtTTTCCTGC-3′ (α1.βT286A) and 5′-cccgcgAGATCTAAAgcGTCCTCAGTCAAGTGgTGCATTAACC-3′ (α1.βH289A), and the PstI- and BglII-restricted amplicons were swapped into pM-ΔN86 MEF2D α1. Combination PCR with vector primers and complementary gene-specific mutagenic primers was used to generate an amplicon for PstI to XmaI fragment swapping to give pM-ΔN86 MEF2D α1.βDEβNQ. Primers were 5′-TCACTTGACTcAGaACCATTTAaATCTGAAC-3′ and 5′-TTGTTCAGATtTAAATGGTtCTgAGTCAAG-3′. BglII, NsiI, StuI, and DraI served as MEF2D β mutant signatures. pM-βMEF2D was constructed using PCR with primer 5′-gccgtggcggccgctttaCAGATCTAAATGGTCCTCAGTcatgaggaattccggcgatacagtc-3′ in combination with a forward SV40 promoter primer on a pM template, followed by introduction of an XhoI- and NotI-restricted amplicon into pM. Complimentary oligonucleotides (5′-aattcACTGAGGACCATTTAGATCTGggcggaACTGAGGACCATTTAGATCTGggcggaACTGAGGACCATTTAGATCTGtaagc-3′ and 5′-ggccgcttaCAGATCTAAATGGTCCT CAGTtccgccCAGATCTAAATGGTCCTCAGTtccgccCAGATCTAAATGGTCCTCAGTg-3′) were annealed and inserted into EcoRI- and NotI-restricted pM to give pM-(βMEF2D)3. The reverse primer 5′-gccgtggcggccgctttaCAGATCTAAATGGTCCTCAGTtccacctgcaggCTTTAATGTCCAGGTATCAAGC-3′ was used with a forward vector primer on a pET-MEF2D α1 template, followed by introduction of the BamHI- to NotI-restricted amplicon into pM-MEF2D α1 to give pM-ΔN86 MEF2D α1.βCOOH. The reverse primer 5′-gtggaggcggccgctttaCAAAAGCAGGTCGACATCCTCAGAtcctgcaggTGTTGCCCATCCTTCAG-3′ was used with a forward vector primer on pET-MEF2C α1 template, followed by introduction of the EcoRI- to NotI-restricted amplicon into pM-MEF2C α1 to give pM-ΔN86 MEF2C α1/βCOOH. Cultured Cell Transfection—C2C12 cells were maintained and differentiated as described (35.Zhu B. Gulick T. Mol. Cell. Biol. 2004; 24: 8264-8275Crossref PubMed Scopus (68) Google Scholar). HeLa, COS7, and 293 cells were maintained in DME with 10% fetal bovine serum. Cells were split into 12-well plates 1 day prior to transfection with Superfect (Invitrogen). Triplicate wells received 1.0 μg of reporter plasmid, 0.1 or 0.3 μg of control reporter, and 1.0 μg of expression vector(s) except where otherwise indicated, and cells were harvested for reporter activity determinations 24-48 h after transfection. Luciferase readings were corrected for transfection efficiency using β-galactosidase activity from pSV40βGal or Renilla luciferase activity from pRL-tk (Promega). Protein Analyses—Western blotting of PAGE-separated proteins was performed with horseradish peroxidase chemiluminescence assays (ECL, Amersham Biosciences). Primary antibodies recognized skeletal muscle myosin heavy chain (70.Bader D. Masaki T. Fischman D.A. J. Cell Biol. 1982; 95: 763-770Crossref PubMed Scopus (796) Google Scholar) (MF20, Development Studies Hybridoma Bank, University of Iowa), α-actin (MAB1501R, Chemicon), the Gal4DBD (SC510, RK5C1, Santa Cruz Biotechnology), or MEF2A, MEF2C, and MEF2D. 2T. Gulick and G.-S. Yu, submitted for publication. Secondary structures of MEF2 isoforms were predicted using various applications (71.Combet C. Blanchet C. Geourjon C. Deleage G. Trends Biochem. Sci. 2000; 25: 147-150Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar, 72.Geourjon C. Deléage G. Comput. Appl. Biosci. 1995; 11: 681-684PubMed Google Scholar, 73.Garnier J. Gibrat J.-F. Robson B. Methods Enzymol. 1996; 266: 540-553Crossref PubMed Google Scholar). RNA Analyses—Murine tissue and C2C12 cell RNA was isolated by conventional procedures (69.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Chanda V.B. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1998: 4.1.1-4.1.5Google Scholar). RNA was harvested from C2C12 myoblasts daily during growth to confluence in DME with 20% fetal calf serum, and daily during differentiation after cell confluence in DME with 2% horse serum. Ribonuclease protection assays (RPA) and radio-labeled cRNA probe syntheses were carried out as described (35.Zhu B. Gulick T. Mol. Cell. Biol. 2004; 24: 8264-8275Crossref PubMed Scopus (68) Google Scholar, 74.Yu G.S. Lu Y.C. Gulick T. J. Biol. Chem. 1998; 273: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The template for the mef2-a cRNA probe was a 169-bp SpeI to BglII fragment from a mouse mef2-a β+ cDNA subcloned into XbaI- and BamHI-restricted pBS-KS (Stratagene). The template for mef2-d cRNA probe was a 198-bp NsiI to PstI β- cDNA subcloned into PstI-restricted pBS-SK. The mef2-c template was a 387-bp BglII to HcII β-/γ- cDNA subcloned into BamHI- and HcII-restricted pBS-SK. Alternative Splicing Patterns Are Conserved among Vertebrate MEF2 Genes—The structures of the four vertebrate MEF2 genes, MEF2A, MEF2B, MEF2C, and MEF2D, have either been described (35.Zhu B. Gulick T. Mol. Cell. Biol. 2004; 24: 8264-8275Crossref PubMed Scopus (68) Google Scholar, 36.Wang D.Z. Valdez M.R. McAnally J. Richardson J. Olson E.N. Development. 2001; 128: 4623-4633Crossref PubMed Google Scholar, 75.Suzuki E. Lowry J. Sonoda G. Testa J.R. Walsh K. Cytogenet. Cell Genet. 1996; 73: 244-249Crossref PubMed Scopus (13) Google Scholar) or can be deduced from genomic and cDNA sequences in GenBank™. All but MEF2B have highly similar gene str" @default.
• W2000884265 created "2016-06-24" @default.
• W2000884265 creator A5042214986 @default.
• W2000884265 creator A5085954687 @default.
• W2000884265 creator A5086372610 @default.
• W2000884265 date "2005-08-01" @default.
• W2000884265 modified "2023-10-09" @default.
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