TRANSCRIPTIONAL REPRESSION OF THE INSULIN-LIKE GROWTH-FACTOR-I RECEPTOR (IGF-I-R) GENE BY THE TUMOR-SUPPRESSOR WT1 INVOLVES BINDING TO SEQUENCES BOTH UPSTREAM AND DOWNSTREAM OF THE IGF-I-R GENE-TRANSCRIPTION START SITE

The insulin-like growth factor-I receptor (IGF-I-R) has been implicate d in the etiology and/or progression of Wilms' tumor, a pediatric mali gnancy of the kidney that is often associated with deletion or mutatio n of the WT1 tumor suppressor gene. The expression of the IGF-I-R gene is increased in Wilms' tumor as compared with normal kidney tissue. F urthermore, the levels of IGF-I-R mRNA in individual tumors have been shown to be inversely correlated to the levels of WT1 mRNA, suggesting that the expression of the IGF-I R gene is under the negative control of WT1. The activity of an IGF-I R promoter/luciferase construct in C hinese hamster ovary cells was reduced by cotransfection of a WT1 expr ession vector. An analysis of various reporter constructs containing d ifferent portions of the IGF-I-R 5'-flanking and 5'-untranslated regio ns suggested that the effect of WT1 depends on the number of WT1 bindi ng sites present, with sites located both upstream and downstream of t he IGF-I-R transcription start site involved in mediating this effect. Using the purified zinc finger domain of WT1 in gel retardation and D Nase I footprinting assays, we mapped five sites in the 5'-flanking an d six sites in the 5'-untranslated regions that were involved in WT1 b inding. In addition, the initiator element of the IGF-I-R gene contain s a sequence that binds WT1. Thus, the repression of IGF-I-R promoter activity by the WT1 tumor suppressor gene product involves multiple in teractions of its zinc finger domain with WT1 binding sites located bo th 5' and 3' of the transcription initiation site.