FIG. 4.

Mutational analysis of the Dax-1 promoter and inhibition of WT1-mediated transactivation by DDS alleles of wt1 and par-4 genes. (A) Mutational analysis of WT1 binding sites within the Dax-1 promoter. Mutations were introduced by PCR primer-directed mutagenesis to alter site WB1 (5′ GTCCCCCTC 3′) to WB1(m) (5′ GTCACCCTC 3′) and site WB2 (5′ AGGAGGAGGCGG 3′) to WB2(m) (5′ A---GG 3′) (changes are indicated in boldface, and a dash represents a deletion). Open box, wild-type WT1 binding site; filled box, mutated WT1 binding site. Transfections were performed with 1 μg of reporter plasmid, 10 μg of CMV/WT1(−/−) expression vector, and 2 μg of CMV/β-gal. CAT and β-galactosidase assays were performed with whole-cell extracts from transiently transfected COS-7 and 293T cells. The values represent the average fold activations of at least two experiments performed in duplicate. (B) DDS alleles inhibit WT1-mediated transactivation. One microgram of the pmDAX-1/CAT reporter plasmid was cotransfected into COS-7 cells with 5 μg of CMV/WT1(−/−) and the indicated amounts of WT1(−/−, R→W). The total transfected DNA concentration was kept constant by the addition of empty expression vector, pcDNA3, to make up differences in amounts of expression vector between tubes. CAT activity was determined from whole-cell extracts prepared 48 h after transfection. The standard deviation for each experiment is shown by an error bar. (C) Par-4 inhibits transcriptional activation of Dax-1 by WT1(−/−) in a dose-dependent fashion. For these experiments, 5 μg of WT1(−/−) expression vector was cotransfected with 1 μg of pmDAX-1/CAT and the indicated amounts of Par-4 expression vector. The total transfected DNA concentration was kept constant by the addition of empty expression vector, pcDNA3, to make up differences in amounts between tubes. CAT activity was determined from whole-cell extracts prepared 48 h after transfection. The standard deviation for each experiment is shown by an error bar.