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 Wilms' tumour suppressor gene 1 (WT1) lead to Denys-Drash syndrome (DDS). Patients with this-syndrome display renal failure, Wilms' tumour (WT) acid pseudohermaphroditism. DDS WT1 mutations fall into three major categories: (a) missense mutations altering amino acids which directly interact with the DNA target; (b) substitution of amino acids involved in zinc complexing; and (c) nonsense mutations leading to the removal of at least two zinc fingers. We have expressed the WT1 zinc fingers as glutathione-S-transferase fusion proteins, with the lysine-threonine-serine (KTS) alternate splice between ZF3 and ZF4 either present or 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 DNA targets: an EGR1 consensus site; murine insulin-like growth factor 2 promoter 2 (IGF2P2); a (TCC)n motif from the PDGFA-chain promoter; and +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 binding to all four targets. This provides evidence that DDS mutations act either as dominant-negative antimorphs, or elicit their effect through disturbed isoform dosage balance.