Figure 6

PAX3-FKHR-dependent transactivation of CCN3 required both the N-terminal repressor R- domain and the C-terminal FKHR AD. (a) Schematic representation of PAX3-FKHR mutants with modified R and AD domains. (b, c) The effect of wild type and AD replacement mutants of PAX3-FKHR on the CCN3 promoter (−143/+336)-CAT activity (b) and on the myogenin promoter (−214)-CAT (c) constructs. (d, e) The effect of R domain deletion on wild type and HD-mutant PAX3-FKHR transactivation of the CCN3 promoter (−143/+336)-CAT (d) and of the myogenin promoter (−214)-CAT (e) constructs. (b–e) C2 cells were transiently transfected with a total of 10 μg DNA containing 5 μg CAT DNA, 2 μg of CMV-lacZ and 1 μg pcDNA3 or plasmid pcDNA3-PAX3-FKHR using CaPO4-DNA. Lysates were prepared 48-h post-transfection for lacZ and CAT assays, as described in Materials and methods. Fold induction in CAT activity was defined as the level of CCN3 promoter-CAT activity over that of pKSCAT promoterless construct, which was assigned a value of 1. Results based on a minimum of three experiments.