Identification and characterization of the Mustang promoter: Regulation by AP-1 during myogenic differentiation

We previously identified Mustang (musculoskeletal temporally activated novel gene) with expression exclusively in the musculoskeletal system. Although its expression is almost undetectable in intact bone, it is robustly upregulated during bone regeneration. It is also abundantly expressed in adult skeletal muscle and tendon. As such, Mustang represents a marker for these cells and thus identifying its promoter would enable us to characterize its transcriptional regulation. To this end, we have isolated and characterized a 1512-bp mouse genomic clone representing the Mustang 5′-flanking region and identified a transcription start site, a TATA box, and multiple putative transcription factor binding sites (including AP-1 and AP-2). The activity of this promoter was detected in musculoskeletal cells and embryonic fibroblasts, even exceeding levels (145%) of the control SV40 promoter (in C2C12 cells). Further, the contribution of specific AP-1 and AP-2 sites was determined with serially deleted and mutated promoter constructs. Results indicate that one of the four AP-1 sites is required for substantial transcriptional activation, as its specific deletion or mutation decreases promoter activity by 32% and 40%, respectively. In contrast, deletion of both identified AP-2 sites results in only a 12% decrease in promoter activity. We further characterized the key AP-1 site by EMSA and determined that in both proliferating and differentiating C2C12 cells, only c-Fos, Fra-2 and JunD were required for transcriptional activation. Mustangʹs restricted tissue specificity and strong promoter makes this gene an ideal candidate for utilization in cell lineage studies that could unveil cellular/molecular mechanisms responsible for musculoskeletal development and regeneration.