Muscle regulatory factor gene: zebrafish (Danio rerio) myogenin cDNA

Myogenin is one of the basic helix-loop-helix proteins that regulate muscle-specific gene expression. Using reverse transciption-polymerase chain reaction (RT-PCR), 5'- and 3'-rapid amplification of cDNA ends (RACE), zebrafish myogenin cDNA was cloned from mRNA of embryos at 10-96 h post-fertilization. The cDNA, at 1384 base pairs (bp), contained a 771-bp open reading frame with 113- and 500-bp flanking regions at the 5'- and 3'-ends, respectively. The deduced amino acid sequences of zebrafish myogenin encoded a 256-amino-acid polypeptide. In a comparison with myogenin of carp, trout, Xenopus, chicken and human, zebrafish myogenin shared 90.9, 77.6, 70.3, 62.9 and 51.5% amino acid identity, respectively. The basic helix-loop-helix domains in myogenin are all conserved. The molecular phylogenic tree demonstrated that myogenin of zebrafish is more closely related to that of fish than to the myogenin of other vertebrates.     

Myogenin induces the myocyte-specific enhancer binding factor MEF-2 independently of other muscle-specific gene products.

The myocyte-specific enhancer-binding factor MEF-2 is a nuclear factor that interacts with a conserved element in the muscle creatine kinase and myosin light-chain 1/3 enhancers (L. A. Gossett, D. J. Kelvin, E. A. Sternberg, and E. N. Olson, Mol. Cell. Biol. 9:5022-5033, 1989). We show in this study that MEF-2 is regulated by the myogenic regulatory factor myogenin and that mitogenic signals block this regulatory interaction. Induction of MEF-2 by myogenin occurs in transfected 10T1/2 cells that have been converted to myoblasts by myogenin, as well as in CV-1 kidney cells that do not activate the myogenic program in response to myogenin. Through mutagenesis of the MEF-2 site, we further defined the binding site requirements for MEF-2 and identified potential MEF-2 sites within numerous muscle-specific regulatory regions. The MEF-2 site was also found to bind a ubiquitous nuclear factor whose binding specificity was similar to but distinct from that of MEF-2. Our results reveal that MEF-2 is controlled, either directly or indirectly, by a myogenin-dependent regulatory pathway and suggest that growth factor signals suppress MEF-2 expression through repression of myogenin expression or activity. The ability of myogenin to induce MEF-2 activity in CV-1 cells, which do not activate downstream genes associated with terminal differentiation, also demonstrates that myogenin retains limited function within cell types that are nonpermissive for myogenesis and suggests that MEF-2 is regulated independently of other muscle-specific genes.     

Transcriptional analysis of the titin cap gene

Mutations in titin cap (Tcap), also known as telethonin, cause limb-girdle muscular dystrophy type 2G (LGMD2G). Tcap is one of the titin interacting Z-disc proteins involved in the regulation and development of normal sarcomeric structure. Given the essential role of Tcap in establishing and maintaining normal skeletal muscle architecture, we were interested in determining the regulatory elements required for expression of this gene in myoblasts. We have defined a highly conserved 421 bp promoter proximal promoter fragment that contains two E boxes and multiple putative Mef2 binding sequences. This promoter can be activated by MyoD and myogenin in NIH3T3 fibroblast cells, and maintains the differentiated cell-specific expression pattern of the endogenous Tcap in C2C12 cells. We find that while both E boxes are required for full activation by MyoD or myogenin in NIH3T3 cells, the promoter proximal E box has a greater contribution to activation of this promoter in C2C12 cells and to activation by MyoD in NIH3T3 cells. Together, the data suggest an important role for MyoD in activating Tcap expression through the promoter proximal E box. We also show that myogenin is required for normal expression in vivo and physically binds to the Tcap promoter during embryogenesis.