title: DNA binding capacity of the WT1 protein is abolished by Denys-Drash syndrome WT1 point mutations.
abstract: 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) and 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.
authors.affiliation: Centre for Molecular and Cellular Biology, University of Queensland, St Lucia, Brisbane, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Little
authors.forename: G
authors.initials: G
authors.lastname: Holmes
authors.forename: W
authors.initials: W
authors.lastname: Bickmore
authors.forename: V
authors.initials: V
authors.lastname: van Heyningen
authors.forename: N
authors.initials: N
authors.lastname: Hastie
authors.forename: B
authors.initials: B
authors.lastname: Wainwright
mesh_terms: D000015:Abnormalities, Multiple / Q000235:genetics*; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012734:Disorders of Sex Development / Q000235:genetics*; D005835:Genitalia / Q000002:abnormalities; D005982:Glutathione Transferase / Q000378:metabolism; D018238:Gonadoblastoma / Q000235:genetics; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051437:Renal Insufficiency / Q000235:genetics; D013577:Syndrome; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D025721:WT1 Proteins; D016335:Zinc Fingers / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025721:WT1 Proteins; D005982:Glutathione Transferase