Cloning and expression analysis of myogenin from flounder (Paralichthys olivaceus) and promoter analysis of muscle-specific expression.

Myogenin is a bHLH transcription factor of the MyoD family. It plays a crucial role in myoblast differentiation and maturation. We report here the isolation of flounder myogenin gene and the characterization of its expression patterns. Sequence analysis indicated that flounder myogenin shared a similar structure and the conserved bHLH domain with other vertebrate myogenin genes. Flounder myogenin gene contains 3 exons and 2 introns. Sequence alignment and phylogenetic showed that flounder myogenin was more homologous with halibut (Hippoglossus hippoglossus) myogenin and striped bass (Morone saxatilis) myogenin. Whole-mount embryo in situ hybridization revealed that flounder myogenin was first detected in the medial region of somites that give rise to slow muscles, and expanded later to the lateral region of the somite that become fast muscles. The levels of myogenin transcripts dropped significantly in matured somites at the trunk region. Its expression could only be detected in the caudal somites, which was consistent with the timing of somite maturation. Transient expression analysis showed that the 546 bp flounder myogenin promoter was sufficient to direct muscle-specific GFP expression in zebrafish embryos.

Purkinje fibers of the avian heart express a myogenic transcription factor program distinct from cardiac and skeletal muscle.

A rhythmic heart beat is coordinated by conduction of pacemaking impulses through the cardiac conduction system. Cells of the conduction system, including Purkinje fibers, terminally differentiate from a subset of cardiac muscle cells that respond to signals from endocardial and coronary arterial cells. A vessel-associated paracrine factor, endothelin, can induce embryonic heart muscle cells to differentiate into Purkinje fibers both in vivo and in vitro. During this phenotypic conversion, the conduction cells down-regulate genes characteristic of cardiac muscle and up-regulate subsets of genes typical of both skeletal muscle and neuronal cells. In the present study, we examined the expression of myogenic transcription factors associated with the switch of the gene expression program during terminal differentiation of heart muscle cells into Purkinje fibers. In situ hybridization analyses and immunohistochemistry of embryonic and adult hearts revealed that Purkinje fibers up-regulate skeletal and atrial muscle myosin heavy chains, connexin-42, and neurofilament protein. Concurrently, a cardiac muscle-specific myofibrillar protein, myosin-binding protein-C (cMyBP-C), is down-regulated. During this change in transcription, however, Purkinje fibers continue to express cardiac muscle transcription factors, such as Nkx2.5, GATA4, and MEF2C. Importantly, significantly higher levels of Nkx2.5 and GATA4 mRNAs were detected in Purkinje fibers as compared to ordinary heart muscle cells. No detectable difference was observed in MEF2C expression. In culture, endothelin-induced Purkinje fibers from embryonic cardiac muscle cells dramatically down-regulated cMyBP-C transcription, whereas expression of Nkx2.5 and GATA4 persisted. In addition, myoD, a skeletal muscle transcription factor, was up-regulated in endothelin-induced Purkinje cells, while Myf5 and MRF4 transcripts were undetectable in these cells. These results show that during and after conversion from heart muscle cells, Purkinje fibers express a unique myogenic transcription factor program. The mechanism underlying down-regulation of cardiac muscle genes and up-regulation of skeletal muscle genes during conduction cell differentiation may be independent from the transcriptional control seen in ordinary cardiac and skeletal muscle cells.

High-level expression and purification of MyoD, myogenin, and E12.

The myogenic regulatory factors (MRFs), MyoD, myogenin, Myf-5, and MRF4, function as transcriptional activators of muscle-specific gene expression by forming heterodimers with the ubiquitously expressed products of the E2A gene, E12 and E47. To enable quantitative biochemical and biophysical analyses of the wild-type proteins, as well as mutants designed to reveal structure-function relationships, we developed protocols for the high-level expression and rapid purification of milligram quantities of MyoD, myogenin, and E12 using conventional biochemical techniques. T7 expression systems were used to direct expression of cDNA encoded proteins in Escherichia coli. Whereas MyoD and E12 were expressed well without alteration, high-level expression of myogenin required changing several rare arginine codons by in vitro mutagenesis to a commonly used E. coli arginine codon. Presumably, inefficient translation of the rare arginine codons inhibited high-level expression of myogenin in the original expression plasmid. Purification protocols are described which involve a simple strategy of cell lysis, ammonium sulfate precipitation, and ion-exchange chromatography. Using this approach, 20 to 50 mg of MyoD, myogenin, or E12 can be purified to 90-95% homogeneity from induced cell pellets in 1 day's time.

Physical interaction between the mitogen-responsive serum response factor and myogenic basic-helix-loop-helix proteins.

Terminal differentiation of muscle cells results in opposite effects on gene promoters: muscle-specific promoters, which are repressed during active proliferation of myoblasts, are turned on, whereas at least some proliferation-associated promoters, such as c-fos, which are active during cell division, are turned off. MyoD and myogenin, transcription factors from the basic-helix-loop-helix (bHLH) family, are involved in both processes, up-regulating muscle genes and down-regulating c-fos. On the other hand, the serum response factor (SRF) is involved in the activation of muscle-specific genes, such as c-fos, as well as in the up-regulation of a subset of genes that are responsive to mitogens. Upon terminal differentiation, the activity of these various transcription factors could be modulated by the formation of distinct protein-protein complexes. Here, we have investigated the hypothesis that the function of SRF and/or MyoD and myogenin could be modulated by a physical association between these transcription factors. We show that myogenin from differentiating myoblasts specifically binds to SRF. In vitro analysis, using the glutathione S-transferase pull-down assay, indicates that SRF-myogenin interactions occur only with myogenin-E12 heterodimers and not with isolated myogenin. A physical interaction between myogenin, E12, and SRF could also be demonstrated in vivo using a triple-hybrid approach in yeast. Glutathione S-transferase pull-down analysis of various mutants of the proteins demonstrated that the bHLH domain of myogenin and that of E12 were necessary and sufficient for the interaction to be observed. Specific binding to SRF was also seen with MyoD. In contrast, Id, a natural inhibitor of myogenic bHLH proteins, did not bind SRF in any of the situations tested. These data suggest that SRF, on one hand, and myogenic bHLH, on the other, could modulate each other's activity through the formation of a heterotrimeric complex.