Two E-boxes and a MEF2 site are critical for activity of the MCK -SIE. (A) Deletions and mutations tested in MCK-SIE. The currently accepted consensus motifs for the E-box and MEF2 motifs are shown. Proven MAF half-site and AP-1 control element sequences are also indicated. Stars indicate sequences that were experimentally proven to recruit the labeled factors and do not represent consensus binding motifs. The wild-type mouse sequences of these elements within the MCK-SIE (Wt), the deletion sequences (Del) and two mutation sequences (M1 and M2) used in this study are shown on successive lines. Base pair deletions are indicated as hyphens, point mutations are shown as changed bases and asterisks indicate unchanged bases. (B) Mutational analysis of control elements within the MCK-SIE. The E-box, MAF/AP-1 and MEF2 motifs in the MCK-proximal promoter-CAT (MCK-SIE-PP-CAT) (diagrammed with elements in their relative positions) were deleted (gray bars) or subjected to two mutations (white bars) within core bases (Figure 2A) and were tested for transcriptional activity in differentiated MM14 skeletal myocyte cultures. The relative activities of these constructs were compared to the MCK-SIE-PP-CAT construct (scaled to equal 1.0) and PP-CAT alone (black bars). Each construct was tested in twelve plates in three separate experiments, and activities shown are averages of those experiments. Error bars represent ±1 standard deviation.