DNA-BINDING CAPACITY OF THE WT1 PROTEIN IS ABOLISHED BY DENYS-DRASH SYNDROME WT1 POINT MUTATIONS

Constitutional point mutations in the zinc finger (ZF) region of the W ilms' tumour suppressor gene 1 (WT1) lead to Denys-Drash syndrome (DDS ). Patients with this-syndrome display renal failure, Wilms' tumour (W T) acid pseudohermaphroditism. DDS WT1 mutations fall into three major categories: (a) missense mutations altering amino acids which directl y interact with the DNA target; (b) substitution of amino acids involv ed in zinc complexing; and (c) nonsense mutations leading to the remov al of at least two zinc fingers. We have expressed the WT1 zinc finger s as glutathione-S-transferase fusion proteins, with the lysine-threon ine-serine (KTS) alternate splice between ZF3 and ZF4 either present o r absent. WT1 fusion constructs with all three classes of DDS mutation were also created. Wild-type and mutant fusion proteins were assayed for their DNA-binding affinity using four previously identified WT1 DN A targets: an EGR1 consensus site; murine insulin-like growth factor 2 promoter 2 (IGF2P2); a (TCC)n motif from the PDGFA-chain promoter; an d +P5, a genomic fragment isolated by its affinity for WT1 + KTS. WT1 - KTS bound all four targets, but WT1 + KTS only bound +P5. All three classes of DDS mutation investigated, with or without KTS, abolished b inding to all four targets. This provides evidence that DDS mutations act either as dominant-negative antimorphs, or elicit their effect thr ough disturbed isoform dosage balance.