Figure 4

Ms1 promoter sensitivity to exogenous MyoD with targeted mutations of putative MyoD binding E-Box sequences. (A) Schematic representation of the reporter gene constructs used in luciferase assays. Wild type and mutant E-Box sequences are represented in white and black ovals respectively. (B) The E-Box sequences 1,2 and 3 (E1, E2 and E3) contained within the wild type P-1585/+60 construct were subjected to site directed mutagenesis. The subsequent single, double and triple E-Box mutant constructs were transiently co-transfected with MyoD into subconfluent C2C12 myoblasts for luciferase assays that were harvested 48 hours later, when the cells were 80% confluent, Luciferase activity, representing promoter sensitivity to ectopic MyoD expression (fold activation vs pcDNA), was inhibited by approximately 50% in the single E1 mutant with the double E1/E2 mutant resulting in a 67% reduction in activity with respect to wild type promoter sensitivity. Triple E1/E2/E3 mutant maintained the same level of MyoD sensitivity compared to the double E1/E2 mutant. (C) The putative TATA box sequence was subjected to site directed mutagenesis and transiently expressed in C2C12 myoblasts as described above and assayed for luciferase activity. TATA mutation resulted in a 95% reduction in luciferase activity with respect to wild type P-1585/+60 construct. The results are expressed as mean ± SE of at least three different experiments, in triplicate for each construct. Statistically significant differences compared to the appropriate WT construct are indicated by *P < 0.05 and **P < 0.05.