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MEF-2 and Oct-1 Bind to Two Homologous Promoter Sequence Elements and Participate in the Expression of a Skeletal Muscle-specific Gene

1998; Elsevier BV; Volume: 273; Issue: 24 Linguagem: Inglês

10.1074/jbc.273.24.15217

1083-351X

Melissa M. Lakich, Thierry T. Diagana, Daniel North, Robert G. Whalen,

RNA modifications and cancer

The murine adult IIB myosin heavy chain (IIB MyHC) gene is expressed only in certain skeletal muscle fibers. Within the proximal promoter are two A + T-rich motifs, mAT1 and mAT2, which greatly enhance muscle-specific transcription; myogenic cells contain proteins that bind to these sequences. MEF-2 binds to both mAT1 and mAT2; a mutation abolishing its binding to mAT1 greatly diminishes the activity of the promoter. Both mAT motifs also form complexes with a protein requiring a target sequence typical of POU domain proteins, which migrate in electrophoretic mobility shift assays to the same position as a complex containing purified Oct-1 and which are supershifted by an antibody specific to Oct-1; this protein is therefore probably Oct-1. Footprinting experiments demonstrate that mAT1 is preferentially occupied by MEF-2 and mAT2 by Oct-1 and that these two proteins appear to bind cooperatively to their respective sites. Although the two mAT motifs have sequences that are very similar, they nonetheless exhibit distinct behaviors and perform differently in the activation of the promoter. The contribution of the IIB MyHC gene to specification of the myogenic phenotype is thus at least in part regulated by MEF-2 and Oct-1. The murine adult IIB myosin heavy chain (IIB MyHC) gene is expressed only in certain skeletal muscle fibers. Within the proximal promoter are two A + T-rich motifs, mAT1 and mAT2, which greatly enhance muscle-specific transcription; myogenic cells contain proteins that bind to these sequences. MEF-2 binds to both mAT1 and mAT2; a mutation abolishing its binding to mAT1 greatly diminishes the activity of the promoter. Both mAT motifs also form complexes with a protein requiring a target sequence typical of POU domain proteins, which migrate in electrophoretic mobility shift assays to the same position as a complex containing purified Oct-1 and which are supershifted by an antibody specific to Oct-1; this protein is therefore probably Oct-1. Footprinting experiments demonstrate that mAT1 is preferentially occupied by MEF-2 and mAT2 by Oct-1 and that these two proteins appear to bind cooperatively to their respective sites. Although the two mAT motifs have sequences that are very similar, they nonetheless exhibit distinct behaviors and perform differently in the activation of the promoter. The contribution of the IIB MyHC gene to specification of the myogenic phenotype is thus at least in part regulated by MEF-2 and Oct-1. Skeletal muscle represents an excellent model to examine tissue-specific controls on transcription, since most of the major muscle structural proteins have several isoforms that often have characteristic spatiotemporal patterns of expression (reviewed in Ref.1Bandman E. Dev. Biol. 1992; 154: 273-283Crossref PubMed Scopus (93) Google Scholar). The mouse adult IIB myosin heavy chain (IIB MyHC) 1The abbreviations used are: IIB MyHC, mouse adult IIB myosin heavy chain; EMSA, electrophoretic mobility shift assay; bHLH, basic helix-loop-helix; WT, wild type; mut., mutant; CAT, chloramphenicol acetyltransferase; MADS,MCM1-agamous-deficiens-serum response factor family of DNA-binding proteins. 1The abbreviations used are: IIB MyHC, mouse adult IIB myosin heavy chain; EMSA, electrophoretic mobility shift assay; bHLH, basic helix-loop-helix; WT, wild type; mut., mutant; CAT, chloramphenicol acetyltransferase; MADS,MCM1-agamous-deficiens-serum response factor family of DNA-binding proteins. gene, for example, is expressed only at a mature stage of development and only in the fast-twitch glycolytic fibers of differentiated skeletal muscle (2Butler-Browne G.S. Bugaisky L.B. Cuénod S. Schwartz K. Whalen R.G. Nature. 1982; 299: 830-833Crossref PubMed Scopus (113) Google Scholar, 3Russell S.D. Cambon N. Nadal-Ginard B. Whalen R.G. J. Biol. Chem. 1988; 263: 6370-6374Abstract Full Text PDF PubMed Google Scholar, 4Whalen R.G. Sell S.M. Butler-Browne G.S. Schwartz K. Bouveret P. Pinset-Härström I. Nature. 1981; 292: 805-809Crossref PubMed Scopus (412) Google Scholar). Transcriptional control is thought to be a primary level of regulation for many of the genes coding for these different isoforms (5Cox R.D. Buckingham M.E. Dev. Biol. 1992; 149: 228-234Crossref PubMed Scopus (59) Google Scholar, 6Cox R.D. Weydert A. Barlow D. Buckingham M.E. Dev. Biol. 1991; 143: 36-43Crossref PubMed Scopus (21) Google Scholar, 7Lompré A.-M. Nadal-Ginard B. Mahdavi V. J. Biol. Chem. 1984; 259: 6437-6446Abstract Full Text PDF PubMed Google Scholar). Among the transcription factors important for muscle gene expression are the myogenic basic helix-loop-helix (bHLH) regulatory factors (myf-5, MyoD, myogenin, and MRF4), which are able to induce muscle differentiation in non-myogenic cells (reviewed in Ref. 8Weintraub H. Cell. 1993; 75: 1241-1244Abstract Full Text PDF PubMed Scopus (924) Google Scholar). The MEF-2 proteins (summarized in Ref. 9Breitbart R.E. Liang C.-S. Smoot L.B. Laheru D.A. Mahdavi V. Nadal-Ginard B. Development. 1993; 118: 1095-1106Crossref PubMed Google Scholar) are also often required for the transcription of muscle genes. Many genes require interactions among several promoter-bound proteins, both tissue-specific and non-tissue-specific, to be expressed in a tissue-specific manner. We have previously characterized numerous potential binding sites for known transcriptional activators within the 5′-flanking region of the IIB MyHC gene (10Takeda S. North D.L. Diagana T. Miyagoe Y. Lakich M.M. Whalen R.G. J. Biol. Chem. 1995; 270: 15664-15670Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 11Takeda S. North D.L. Lakich M.M. Russell S.D. Kahng L.S. Whalen R.G. C. R. Acad. Sci. (Paris). 1992; 315: 467-472PubMed Google Scholar, 12Takeda S. North D.L. Lakich M.M. Russell S.D. Whalen R.G. J. Biol. Chem. 1992; 267: 16957-16967Abstract Full Text PDF PubMed Google Scholar). In particular, two regions rich in the nucleotides A and T, situated between −140 and −190 bp, enhance the transcriptional activity of those constructions that contain them. This activation is found only in differentiated myotubes and not in undifferentiated myoblasts, implying that these sites contribute to the restriction of the expression of the IIB MyHC gene to mature muscle tissue.There are numerous DNA-binding proteins that recognize and bind to sequences that are predominantly composed of A and T nucleotides. Among these are the ubiquitous SRF (13Treisman R. Ammerer G. Curr. Opin. Genet. Dev. 1992; 2: 221-226Crossref PubMed Scopus (99) Google Scholar) and the "related-to-SRF" proteins that share with SRF the existence of an amino-terminal MADS domain (13Treisman R. Ammerer G. Curr. Opin. Genet. Dev. 1992; 2: 221-226Crossref PubMed Scopus (99) Google Scholar,14Pollock R. Treisman R. Genes Dev. 1991; 5: 2327-2341Crossref PubMed Scopus (322) Google Scholar); the nuclear protein MEF-2 belongs to the related-to-SRF family. There are at least four different mef2 genes, each of which may be alternatively spliced to produce several isoforms of the MEF-2 protein, some of which are selectively expressed in muscle and brain (9Breitbart R.E. Liang C.-S. Smoot L.B. Laheru D.A. Mahdavi V. Nadal-Ginard B. Development. 1993; 118: 1095-1106Crossref PubMed Google Scholar, 15Martin J.F. Schwartz J.J. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5282-5286Crossref PubMed Scopus (218) Google Scholar, 16McDermott 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 (196) Google Scholar, 17Yu Y.-T. Breitbart R.E. Smoot L.B. Lee Y. Mahdavi V. Nadal-Ginard B. Genes Dev. 1992; 6: 1783-1798Crossref PubMed Scopus (381) Google Scholar). Proteins containing a homeodomain also generally have at least a short AT-rich motif (often TAAT) as part of their target sequence (reviewed in Ref. 18Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Würthrich K. Cell. 1994; 78: 211-233Abstract Full Text PDF PubMed Scopus (693) Google Scholar); among the homeodomain proteins are the POU domain proteins, such as Oct-1 and Pit-1 (19Rosenfeld M.G. Genes Dev. 1991; 5: 897-907Crossref PubMed Scopus (373) Google Scholar).The sequences of the two related AT-rich motifs found in the proximal region of the IIB MyHC are conserved among numerous distinct skeletal muscle MyHC genes of several vertebrate species (11Takeda S. North D.L. Lakich M.M. Russell S.D. Kahng L.S. Whalen R.G. C. R. Acad. Sci. (Paris). 1992; 315: 467-472PubMed Google Scholar), suggesting an important role for these "mAT" (myosinAT-rich) sites in the regulation of this family of genes. This observation led us to examine in detail the interactions among the two mAT sites and the DNA-binding proteins that presumably associate with them in vivo to effect transcriptional activation. In this study we demonstrate that Oct-1, bound to mAT2, and a MEF-2 protein, bound to the more downstream mAT1, are both involved in the regulation of the activity of the promoter. Muscle-specific transcription of this gene may thus be achieved by combinations of widely and narrowly expressed factors, and the interactions among them appear to be important for the activation of the IIB MyHC gene. Skeletal muscle represents an excellent model to examine tissue-specific controls on transcription, since most of the major muscle structural proteins have several isoforms that often have characteristic spatiotemporal patterns of expression (reviewed in Ref.1Bandman E. Dev. Biol. 1992; 154: 273-283Crossref PubMed Scopus (93) Google Scholar). The mouse adult IIB myosin heavy chain (IIB MyHC) 1The abbreviations used are: IIB MyHC, mouse adult IIB myosin heavy chain; EMSA, electrophoretic mobility shift assay; bHLH, basic helix-loop-helix; WT, wild type; mut., mutant; CAT, chloramphenicol acetyltransferase; MADS,MCM1-agamous-deficiens-serum response factor family of DNA-binding proteins. 1The abbreviations used are: IIB MyHC, mouse adult IIB myosin heavy chain; EMSA, electrophoretic mobility shift assay; bHLH, basic helix-loop-helix; WT, wild type; mut., mutant; CAT, chloramphenicol acetyltransferase; MADS,MCM1-agamous-deficiens-serum response factor family of DNA-binding proteins. gene, for example, is expressed only at a mature stage of development and only in the fast-twitch glycolytic fibers of differentiated skeletal muscle (2Butler-Browne G.S. Bugaisky L.B. Cuénod S. Schwartz K. Whalen R.G. Nature. 1982; 299: 830-833Crossref PubMed Scopus (113) Google Scholar, 3Russell S.D. Cambon N. Nadal-Ginard B. Whalen R.G. J. Biol. Chem. 1988; 263: 6370-6374Abstract Full Text PDF PubMed Google Scholar, 4Whalen R.G. Sell S.M. Butler-Browne G.S. Schwartz K. Bouveret P. Pinset-Härström I. Nature. 1981; 292: 805-809Crossref PubMed Scopus (412) Google Scholar). Transcriptional control is thought to be a primary level of regulation for many of the genes coding for these different isoforms (5Cox R.D. Buckingham M.E. Dev. Biol. 1992; 149: 228-234Crossref PubMed Scopus (59) Google Scholar, 6Cox R.D. Weydert A. Barlow D. Buckingham M.E. Dev. Biol. 1991; 143: 36-43Crossref PubMed Scopus (21) Google Scholar, 7Lompré A.-M. Nadal-Ginard B. Mahdavi V. J. Biol. Chem. 1984; 259: 6437-6446Abstract Full Text PDF PubMed Google Scholar). Among the transcription factors important for muscle gene expression are the myogenic basic helix-loop-helix (bHLH) regulatory factors (myf-5, MyoD, myogenin, and MRF4), which are able to induce muscle differentiation in non-myogenic cells (reviewed in Ref. 8Weintraub H. Cell. 1993; 75: 1241-1244Abstract Full Text PDF PubMed Scopus (924) Google Scholar). The MEF-2 proteins (summarized in Ref. 9Breitbart R.E. Liang C.-S. Smoot L.B. Laheru D.A. Mahdavi V. Nadal-Ginard B. Development. 1993; 118: 1095-1106Crossref PubMed Google Scholar) are also often required for the transcription of muscle genes. Many genes require interactions among several promoter-bound proteins, both tissue-specific and non-tissue-specific, to be expressed in a tissue-specific manner. We have previously characterized numerous potential binding sites for known transcriptional activators within the 5′-flanking region of the IIB MyHC gene (10Takeda S. North D.L. Diagana T. Miyagoe Y. Lakich M.M. Whalen R.G. J. Biol. Chem. 1995; 270: 15664-15670Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 11Takeda S. North D.L. Lakich M.M. Russell S.D. Kahng L.S. Whalen R.G. C. R. Acad. Sci. (Paris). 1992; 315: 467-472PubMed Google Scholar, 12Takeda S. North D.L. Lakich M.M. Russell S.D. Whalen R.G. J. Biol. Chem. 1992; 267: 16957-16967Abstract Full Text PDF PubMed Google Scholar). In particular, two regions rich in the nucleotides A and T, situated between −140 and −190 bp, enhance the transcriptional activity of those constructions that contain them. This activation is found only in differentiated myotubes and not in undifferentiated myoblasts, implying that these sites contribute to the restriction of the expression of the IIB MyHC gene to mature muscle tissue. There are numerous DNA-binding proteins that recognize and bind to sequences that are predominantly composed of A and T nucleotides. Among these are the ubiquitous SRF (13Treisman R. Ammerer G. Curr. Opin. Genet. Dev. 1992; 2: 221-226Crossref PubMed Scopus (99) Google Scholar) and the "related-to-SRF" proteins that share with SRF the existence of an amino-terminal MADS domain (13Treisman R. Ammerer G. Curr. Opin. Genet. Dev. 1992; 2: 221-226Crossref PubMed Scopus (99) Google Scholar,14Pollock R. Treisman R. Genes Dev. 1991; 5: 2327-2341Crossref PubMed Scopus (322) Google Scholar); the nuclear protein MEF-2 belongs to the related-to-SRF family. There are at least four different mef2 genes, each of which may be alternatively spliced to produce several isoforms of the MEF-2 protein, some of which are selectively expressed in muscle and brain (9Breitbart R.E. Liang C.-S. Smoot L.B. Laheru D.A. Mahdavi V. Nadal-Ginard B. Development. 1993; 118: 1095-1106Crossref PubMed Google Scholar, 15Martin J.F. Schwartz J.J. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5282-5286Crossref PubMed Scopus (218) Google Scholar, 16McDermott 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 (196) Google Scholar, 17Yu Y.-T. Breitbart R.E. Smoot L.B. Lee Y. Mahdavi V. Nadal-Ginard B. Genes Dev. 1992; 6: 1783-1798Crossref PubMed Scopus (381) Google Scholar). Proteins containing a homeodomain also generally have at least a short AT-rich motif (often TAAT) as part of their target sequence (reviewed in Ref. 18Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Würthrich K. Cell. 1994; 78: 211-233Abstract Full Text PDF PubMed Scopus (693) Google Scholar); among the homeodomain proteins are the POU domain proteins, such as Oct-1 and Pit-1 (19Rosenfeld M.G. Genes Dev. 1991; 5: 897-907Crossref PubMed Scopus (373) Google Scholar). The sequences of the two related AT-rich motifs found in the proximal region of the IIB MyHC are conserved among numerous distinct skeletal muscle MyHC genes of several vertebrate species (11Takeda S. North D.L. Lakich M.M. Russell S.D. Kahng L.S. Whalen R.G. C. R. Acad. Sci. (Paris). 1992; 315: 467-472PubMed Google Scholar), suggesting an important role for these "mAT" (myosinAT-rich) sites in the regulation of this family of genes. This observation led us to examine in detail the interactions among the two mAT sites and the DNA-binding proteins that presumably associate with them in vivo to effect transcriptional activation. In this study we demonstrate that Oct-1, bound to mAT2, and a MEF-2 protein, bound to the more downstream mAT1, are both involved in the regulation of the activity of the promoter. Muscle-specific transcription of this gene may thus be achieved by combinations of widely and narrowly expressed factors, and the interactions among them appear to be important for the activation of the IIB MyHC gene. We thank Dr. Shin'ichi Takeda for discussion and assistance. We also thank Dr. Barbara Demeneix for the dioctadecyl amidoglycyl spermine; Dr. R. G. Roeder for the Oct-1 antibody; Dr. P. C. van der Vliet for the Oct-1 protein; Dr. E. Olson for the MEF-2C expression vector; and Prof. François Gros for continuous support.