Figure 3. Pax7 Dominantly Binds Hoemobox Motif.

(A–B) Electromobility shift assay (EMSA) showing Pax3 and Pax7 binding to hbox and prd motif taken from Myf5 ECR111 locus. Pax7 shows higher affinity to hbox motif than Pax3 (Panel A). On the other hand both Pax3 and Pax7 show equal affinity for prd motif. Also see Figure S3 and Supplementary Materials and Methods. (C) Three copies of ECR111 were directionally cloned into a pGL4.10 reporter vector driven by a Myf5 minimal promoter (Summerbell et al., 2000). 10T1/2 fibroblasts were co-transfected with Pax3- and Pax7-VP16 fusion constructs consisting of Pax3 or Pax7 fused in-frame with a VP16 transcriptional activation domain (Lin et al., 1991) at the C-terminus. Expression of luciferase was measured and normalized to renilla expression. Relative luciferase activity was normalized against a control (VP16) reporter construct. Both Pax3- and Pax7-VP16 drove luciferase expression to high levels, demonstrating that ECR111 has enhancer activity and suggests that a combination of paired and homeodomain motifs produce synergistic effects on enhancer activity. (D) Luciferase reporter assay showing transcriptional output of Pax3- and Pax7-VP16 on prd and hbox motifs. Three copies of the prd or hbox of ECR111 were concatamerized and cloned into pGL4.10 luciferase construct as described. Pax7-VP16 produced a significant increase in luciferase expression relative to controls and Pax3-VP16. Error bars represent standard deviation.