Abstract
The p53 protein has been proposed as a modulator of the Wilms' tumour-suppressor protein (WT1) transcriptional regulation activity. To investigate this putative p53 role, the promoter P3 of the mouse insulin-like growth factor II gene (Igf2) was used as a target for WT1 regulation in primary cell cultures derived from p53 wild-type (p53+/+) and knock-out (p53-/-) mouse embryos. In these cells, the WT1 transcriptional activity was observed to be independent of p53 genotype. Furthermore, the two WT1 zinc finger (ZF) isoforms were for the first time found to have opposite effects on gene expression from a single promoter in the same cell type, WT1[-KTS] activating Igf2 P3, whereas WT1[+KTS] repressed its activity. In addition, we have mapped the WT1 binding sites and investigated the effect on WT1 binding activity of individual ZF deletions and Denys-Drash syndrome point mutations to this target.
Highlights
The two WT1 zinc finger (ZF) isoforms were for the first time found to have opposite effects on gene expression from a single promoter in the same cell type, WTI [-KTS] activating Ig2 promoter P3, whereas WT1 [+KTS] repressed its activity
Identification of WT1 and EGR1 binding sites in the IGF2 gene (Igf2) P3 promoter Binding of WT1[+KTS], WT1[-KTS] and EGR1 to the 1gJ2 P3 promoter was analysed by DNAase I footprinting
Both EGRI and WT1[-KTS] ZF fusion proteins bound to several sites in the fragment spanning nucleotides -162 to +70 of P3 (Figure 1)
Summary
The transcriptional fusion between the Igf P3 promoter (isolated from cosIGF4; Rotwein and Hall, 1990) and the firefly luciferase reporter gene, pP3MM, was previously described (Caricasole and Ward, 1993). To construct WT1 (pCMV-KTS, pCMV+KTS, pCMV-Drash) and EGRI (pCMV-EGR) mammalian expression vectors, the corresponding murine cDNA sequences (Lemaire et al, 1990; Buckler et al, 1991) were subcloned into pcDNA I/Amp (Invitrogen), downstream of the cytomegalovirus enhancer/ promoter. The bacterial expression constructs used to prepare glutathione S-transferase (GST) fusion proteins were formed using the pGEX-3X vector (Pharmacia) and cDNA sequences encoding either WT1 or EGRI ZF domains. Details of EGRI (Caricasole et al, 1996) and both wild-type (Bickmore et al, 1992) and mutant WT1 constructs (Little et al, 1995) were published elsewhere
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