Myocyte - specific enhancer factor 2 acts cooperatively with a muscle activator region to regulate Drosophila tropomyosin

MEF2 (myocyte-specific enhancer factor 2) is a MADS box transcription factor that is thought to be a key regulator of myogenesis in vertebrates. Mutations in the Drosophila homologue of the mef2 gene indicate that it plays a kty role in regulating myogenesis in Drosophila. We show here that the Drosophila tropomyosin I (TmI) gene is a target gene for mef2 regulation. The TmI gene contains a proximal and a distal muscle enhancer within the first intron of the gene. We show that both enhancers contain a MEF2 binding site and that a mutatign in the MEF2 binding site of either enhancer significantly reduces reporter gene expression in embryonic, larval, and adult somatic body wall muscles of transgenic flies. We also show that a high level of proximal enhancer-directed repjbrter gene expression in somatic muscles requires the cooperative activity ofMEF2 and a cis-acting muscle activator region located within the enhancer. Thus, mef2 null iiutant embryos show a significant reduction but not an elimination of TmI expression in the body wall myoblasts and muscle fibers that are present. Surprisingly, there is little effect in these mutants on TmI expression in developing visceral muscles and dorsal vessel (heart), despite the fact that MEF2 is expressed in these muscles in wild-type embryos, indicating that TmI expression is regulated differently in these muscles. Taken together, our results show that mef2 is a positive regulator of tropomyosin gene transcription that is necessary but not sufficient for high level expression in somatic muscle of the embryo, larva, and adult. Much is now known about axis formation and segmentation of the Drosophila embryo; however, relatively little is known about morphogenesis of the internal organs such as muscle. In vertebrates, myogenesis is controlled in part by the MyoD family of basic helix-loop-helix transcriptidn factors and a second factor, the myocyte-specific enhancer factor 2 (MEF2) (reviewed in refs. 1-3). In Drosophila the only MyoD muscle homologue identified thus far is the nau/Dmyd gene; however, the timing and restricted pattern of expression of the naul Dmyd gene suggests that it probably has a more limited role in muscle differentiation than its vertebrate homologues (4, 5). A single mef2 gene has been identified in DrosQphila (6, 7). The mef2 gene is expressed in the mesoderm of early embryos shortly after gastrulation, and continues to be expressed jn cardioblasts and in visceral and somatic muscle lineages throughout embryogenesis. This pattern of expression suggests that mef2 may be important in regulating the earlier stages of myogenesis that establish mesoderm and muscle lineages, as well as the later stages of myoblast fusion and differentiation. Mutations in the mef2 gene, however, suggest that the role of mef2 in regulating Drosophila myogenesis may be limited to later aspects of myogenesis because muscle patterning in mef2 mutant embryos appears to be normal up to the stage of myoblast fusion and differentiation to form myotubes (8, 9). Thus far no target genes for MEF2 regulation have been identified in Drosophila. The MEF2 factors belong to the MADS box family (named for MCM1, agamous, deficiens, and serum response factor) of transcription factors (reviewed in refs. 3, 10, and 11), and bind DNA through a 56-amino acid MADS box domain that recognizes A+T rich sequences found in the enhancers of many vertebrate muscle genes (3). MEF2 can also form dimers through the MADS box domain and the adjacent 29-amino acid MEF2 domain that is highly homologous among the MEF2 family members. In the experiments presented here, we have examined the role of the mef2 gene in regulating transcription of the Drosophila tropomyosin I (TmI) gene. We show that each of two previously identified TmI muscle enhancers (12, 13) contains a consensus MEF2 binding site that is a direct target for mef2-regulated transcription in somatic body wall muscles during all stages of development. Interestingly, regulation of TmI expression in visceral muscle and dorsal vessel (heart) does not appear to require MEF2 despite the fact that it is also expressed in these two muscle types. MATERIALS AND METHODS Nuclear Extract Preparation and Gel Mobility Shift Assay. Nuclear extracts were prepared from isolated nuclei as described (14) except that KCl was used instead of (NH4)2SO4 to extract the nuclei. Gel mobility shift assays were performed according to Parmacek et al. (15) with slight modifications. Gel mobility shift assays of the 1B(c) fragment (see Fig. 2A), and those of the double-stranded oligonucleotides (see Fig. 2B) were in 5 and 8% polyacrylamide gels, respectively. The rabbit polyclonal anti-MEF2 antibody has been described (8). DNA Cloning and Plasmid Preparation. The reporter gene P-element vector construct containing the Drosophila hsp7O gene promoter and the Escherichia coli lacZ gene (pWhsp7Olac) and the reporter constructs 1B (proximal enhancer), 1B(c), and muscle activator (MA) DNA fragment [referred to previously as 1B(a)] have been described (12, 13). The 1BAMEF2(a and b) constructs were made by mutating the 1B plasmid using the Altered Site mutagenesis system (Promega) and the single-stranded oligonucleotides indicated below. The mutated fragments were amplified by PCR and cloned into the KpnI-NotI site of the pWhsp7Olac transformation vector. The MA+MEF2, MA+AMEF2, MEF2, and AMEF2 constructs were made using the double-stranded oligonucleotides listed below which contain 5' BamHI and 3' Notl protruding ends. Abbreviations: TmI, Drosophila tropomyosin I; MA, muscle activator; MEF2, myocyte-specific enhancer factor 2; IFM, indirect flight muscles; TDT, tergal depressor of the trochanter (jump) muscles. tTo whom reprint requests should be addressed at: Department of Biochemistry M/C536, University of Illinois College of Medicine, 1853 West Polk Street, Chicago, IL 60612. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.