FIG. 3.

Transactivation of the human and murine Dax-1 promoters by WT1(−KTS) isoforms. (A) Schematic representation of reporter plasmids and expression vectors. The pmDax-1/CAT and phDAX-1/CAT reporter plasmids contain genomic DNA sequences from nucleotides −218 to +3 (for mouse) and −234 to +3 (for human), respectively (Fig. 1B). The murine promoter is indicated by solid boxes, the human Dax-1 gene promoter is indicated by shaded boxes, and the CAT gene is indicated by open boxes. Expression vectors driving the production of murine WT1 isoforms are also represented. The first alternative splice site (exon V) consists of 17 amino acids (VAAGSSSSVKWTEGDSN), and the second alternative splice site consists of three amino acids (KTS). The exon boundaries of wt1 are denoted above WT1(+/+), and the positions of the first and last amino acids are indicated below each construct. (B) Transcriptional activation of the murine and human Dax-1 promoters by WT1(−/−). Cotransfections of COS-7 cells were performed with 1 μg of reporter plasmid and 0, 5, or 10 μg of CMV/WT1(−/−) expression plasmid. Individual DNA precipitates were adjusted to contain equal amounts of total DNA by the addition of the empty expression vector, pcDNA3. To normalize for transfection efficiency, the cells were cotransfected with 2 μg of pRSV/β-gal. At 48 h after transfection, the cells were harvested and assayed for β-galactosidase and CAT activity. The average fold activation and standard error for CAT determinations are indicated below the chromatogram and represent the value obtained from three independent experiments. (C) Effect of WT1 isoforms on the transcriptional regulation of the murine Dax-1 promoter. The average relative transactivation (normalized CAT activity) and standard error is presented, with the relative transactivation values obtained with pcDNA3 and CMV/WT1(−/−) taken as 0 and 100%, respectively. The values shown are taken from three independent experiments. A representative autoradiogram is shown on the right. R→W indicates a missense mutation in WT1 zinc finger III converting an Arg residue to a Trp. This is the commonest DDS allele.