Dual tandem promoter elements containing CCAC-Like motifs from the tetrodotoxin-resistant voltage-sensitive Na+ channel (rSkM2) gene can independently drive muscle-specific transcription in L6 cells

cis-Elements in the -129/+124 promoter segment of the rat tetrodotoxin-resistant voltage-gated sodium channel (rSkM2) gene that are responsible for reporter gene expression in cultured muscle cells were identified by deletion and scanning mutations. Nested 5' deletion constructs, assayed in L6 myotubes and NIH3T3 cells, revealed that the minimum promoter allowing muscle-specific expression is contained within the -57 to +1 segment relative to the major transcription initiation site. In the context of the -129/+1 construct, however, scanning mutations in the -69/+1 segment failed to identify any critical promoter elements. In contrast, identical mutations in a minimal promoter (-57/+124) showed that all regions except -29/-20 are essential for expression, especially the -57/-40 segment, consistent with the 5' deletion analysis. Further experiments showed that the distal (-129/-58) and proximal promoter (-57/+1) elements can independently drive reporter expression in L6 myotubes, but not in NIH3T3 fibroblasts. This pair of elements is similar in sequence and contains Spl sites (CCGCCC), CCAC-like motifs, but no E-boxes or MEF-2 sites. The two segments form similarly migrating complexes with L6 myotube nuclear extracts in gel-shift assays. Critical elements within the distal promoter element were defined by 10 base pair scanning mutations in the -119 to -60 region in the context of the -129/+1 segment containing a mutated -59/-50 segment that inactivates the proximal promoter. Nucleotides in the -119/-90 region, especially -109/-100, were the most important regions for distal promoter function. We conclude that the -129/+1 segment contains two tandem promoter elements, each of which can independently drive muscle-specific transcription. Supershifts with antibodies to Spl and myocyte nuclear factor (MNF) implicate the involvement of Spl, MNF, and other novel factors in the transcriptional regulation of rSkM2 gene expression.