Myocyte enhancer factor 2 and chorion factor 2 collaborate in activation of the myogenic program in Drosophila.

A major function of the mesodermal germ layer during embryogenesis is the generation of myogenic tissue and the subdivision of cells into skeletal, cardiac, and visceral muscle precursors. This process has been broadly conserved during evolution. Specification of the three major muscle lineages is conserved from Drosophila to mammals, and conserved structural genes are activated in each lineage to provide power for locomotion, circulation, and digestion (reviewed in reference 10). The transcription factors that mediate muscle cell differentiation are also broadly conserved. Foremost among these proteins are members of the myocyte enhancer factor 2 (MEF2) family of transcriptional regulators (reviewed in reference 6). MEF2 proteins dimerize and bind to DNA via conserved MADS and MEF2 protein domains, and MEF2 proteins recognize an AT-rich sequence (2), which is found in the promoters and enhancers of numerous muscle-specific genes. MEF2-encoding genes are found in all animal genomes, from single-gene copies in Caenorhabditis elegans (named CeMef2) and Drosophila melanogaster (named Mef2), to four genes in mammals (named mef2a to -d). Consistent with the pervasiveness of MEF2 sites in muscle-specific genes and the conservation of this gene family during evolution, mutational studies in several animal models have established important roles for MEF2 factors in muscle differentiation. In Drosophila, inactivation of the single Mef2 gene resulted in a complete failure of differentiation for all muscle lineages (7, 27). In mice, inactivation of mef2c resulted in a failure of normal cardiac and visceral muscle development (5, 31, 30). Furthermore, inactivation of murine mef2a caused cardiac failure shortly after birth (38). Also, expression of dominant-negative isoforms of MEF2 predicted to inhibit all MEF2 function resulted in the inhibition of skeletal myogenesis in mammalian cells in vitro (40) and in interference in cardiac development in vivo (23). Finally, a point mutation in the human gene encoding MEF2A is associated with susceptibility to cardiac disease (19). These studies present a strong argument for the importance of MEF2 proteins in muscle development and disease. Despite the critical requirement of MEF2 for muscle differentiation, MEF2 proteins are not themselves capable of activating the myogenic program in naive cells in tissue culture (36). Similarly in Drosophila, ectopic expression of Mef2 in the ectoderm caused the activation of some muscle-specific markers, and yet failed to induce myogenesis at high levels (31). These findings suggested that MEF2 must act with specific cofactors in order to control myogenesis. Much progress has subsequently been made in defining both positive and negative cofactors for MEF2, in a variety of muscle tissues. MEF2 collaborates positively with skeletal muscle-specific members of the basic helix-loop-helix family of transcriptional regulators, including MyoD and myogenin, to control skeletal myogenesis (36, 37). In addition, the SAP domain proteins Myocardin and MASTR also stimulate the transcriptional activity of MEF2 in cardiac and skeletal muscle tissue, respectively (11). MEF2 proteins also interact with ubiquitous factors, such as the p300 coactivator (46) and members of the histone deacetylase family of transcriptional repressors (32). In Drosophila, there is relatively little direct data concerning the identification of cofactors that might collaborate with MEF2 to control myogenesis. Nevertheless, a number of enhancers for muscle-specific genes have been described, many of which contain MEF2 binding sites that are critical for full gene activation (3, 13, 29, 34, 42). Among these, we recently described the promoter for the Act57B actin gene in Drosophila (24). Act57B is the predominant embryonic muscle actin (15, 48), being expressed at high levels in all of the major muscle lineages and also being one of the earliest markers of muscle differentiation in the embryo (24). We found that activation of Act57B expression in all embryonic muscle lineages was controlled by a proximal 595-bp promoter element. Within this region, full transcriptional activation required the integrity of a single MEF2 binding site at position −209 relative to the transcriptional start site, validating the importance of MEF2 as a direct regulator of myogenesis in this system. This finding was also supported by the study of Sandmann et al. (44), who showed, using microarray analysis, that Act57B expression was strongly downregulated in Mef2 mutants. Furthermore, these authors utilized chromatin immunoprecipitation-microarray analyses to identify the MEF2 site at position −209 as an in vivo target of MEF2 (44). Our studies also demonstrated that an ∼300-bp region distal to the MEF2 site was critical for full Act57B activation in embryonic muscle tissue, suggesting the existence of a MEF2 cofactor that interacted with this distal sequence. This analysis permitted us to carry out a directed approach to the identification and functional characterization of a MEF2 collaborating factor in Drosophila. We delineate here sequences within the 300-bp distal region that are required for full gene activation in vivo, and we show that a nuclear factor binds to this region with the characteristics identical to that of the C2H2 zinc finger transcriptional regulator chorion factor 2 (CF2) (22). We further show that MEF2 and CF2 can each activate Act57B expression in vitro and in vivo and that these factors function synergistically to maintain high levels of actin expression in the Drosophila embryo. More globally, MEF2 and CF2 synergistically activate other muscle structural genes, and loss of CF2 function results in reductions in the expression levels of several muscle structural genes, including Act57B. Our findings describe the first collaborating factor for MEF2 in the Drosophila system and further delineate the network of transcriptional events required for normal muscle development.