PAX-8 REGULATES HUMAN WT1 TRANSCRIPTION THROUGH A NOVEL DNA-BINDING SITE

The Wilms' tumor gene (WT1) is an essential gene for kidney and gonada l development, although how WT1 expression is induced in these tissues is not known, One kidney transcription factor likely to play a role i n this regulation is PAX 8, The co-expression of WT1 and PAX 8 during kidney development and in Wilms' tumors with an epithelium predominant histology suggested a possible interaction, and indeed, we identified potential core PAX-binding sites in the WT1 promoter, Endogenous PAX 8 plays an important role in the activation of the WT1 promoter, since promoter activity is much stronger in cells with PAX 8 than without, Using binding assays, we searched for evidence of PAX 8-DNA interactio ns throughout the 652-base pair human WT1 promoter and found only one functional PAX 8 site with DNA binding activity, located 250 base pair s 5' of the minimal promoter, The responsiveness of the PAX 8 site was con firmed by assessing its ability to function as an enhancer signif icantly activating the minimal promoter in a position-and orientation- independent manner. Using transfection assays, we demonstrated that ei ther endogenous or exogenously added PAX 8 activated the WT1 promoter and that this promoter up-regulation depended upon the presence of an intact PAX 8-binding site, In contrast, the previously reported core P AX 8-binding sites identified by computer analysis of the WT1 promoter failed to specifically bind in vitro translated PAX 8 protein or acti vate the minimal promoter, Thus, we identified a novel functional bind ing site for the transcription factor PAX 8, suggesting that part of i ts role in kidney development may be as a modulator of WT1 expression in the kidney.