CHARACTERIZATION OF THE TRANSCRIPTIONAL REGULATORY REGION OF THE HUMAN WT1 GENE

Specific control of the expression of the Wilms tumor gene WT1 is impo rtant for normal development of the kidney. In order to characterise t he transcriptional control region of the WT1 gene we have isolated gen omic clones spanning the upstream region, the first WT1 exon and the 5 ' end of the Wit1 gene. DNA sequencing revealed that the WT1 promoter lacks a TATA box or CCAAT motif and has a GC content of 71%. Four tran scriptional start sites are clustered within a 32 bp region. GC-boxes are present at nucleotide positions - 413, - 160, + 84 and + 158. DNAa seI protection assays with purified Sp1 protein revealed the existence of 11 different binding sites in the WT1 promoter. WT1 and Wit1 promo ter activities were tested in COS-7 cells with luciferase reporter gen e constructs either containing or lacking an SV40 enhancer. WT1 promot er activity was found in a fragment extending from 449 bp upstream to 201 bp downstream of the WT1 start site. It was 26 fold lower in the a bsence of the SV40 enhancer than in the presence. Cotransfection with a Sp1 expression vector stimulated both constructs 3-4 fold. Wit1 prom oter activity was identified in a DNA fragment extending from 200 bp u pstream of the putative Wit1 TATA box to 130 bp downstream. Several po tential recognition sites for WT1/EGR, Pax-8, and GAGA-like transcript ion factors are present in the WT1 promoter.