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DNA binding capacity of the WT1 protein is abolished by Denys-Drash syndrome WT1 point mutations

M Little1, G Holmes, W Bickmore

  • 1Centre for Molecular and Cellular Biology, University of Queensland, St Lucia, Brisbane, Australia.

Human Molecular Genetics
|March 1, 1995
PubMed

Insights

Denys-Drash syndrome (DDS) results from Wilms' tumour suppressor gene 1 (WT1) mutations. These mutations disrupt WT1's DNA binding, impacting gene regulation and potentially causing disease through dominant-negative effects or altered isoform balance.

Area of Science:

  • Molecular Biology
  • Genetics
  • Developmental Biology

Background:

  • Denys-Drash syndrome (DDS) is a rare genetic disorder characterized by Wilms' tumour, renal failure, and pseudohermaphroditism.
  • Constitutional point mutations in the zinc finger (ZF) region of the Wilms' tumour suppressor gene 1 (WT1) are the underlying cause of DDS.
  • WT1 mutations are categorized into three main types based on their effect on DNA interaction, zinc complexing, or ZF removal.

Purpose of the Study:

  • To investigate the DNA-binding affinity of wild-type and mutant Wilms' tumour suppressor gene 1 (WT1) fusion proteins.
  • To determine how different classes of Denys-Drash syndrome (DDS) mutations affect WT1's interaction with its DNA targets.
  • To elucidate the molecular mechanisms by which DDS mutations lead to disease, considering dominant-negative effects or altered isoform dosage.

Main Methods:

  • Expression of WT1 zinc finger domains as glutathione-S-transferase (GST) fusion proteins, with and without the KTS splice.
  • Creation of WT1 fusion constructs incorporating three classes of DDS mutations.
  • Assay of DNA-binding affinity for wild-type and mutant fusion proteins using four known WT1 DNA targets (EGR1, IGF2P2, PDGFA, +P5).

Main Results:

  • Wild-type WT1-KTS bound to all four DNA targets, while WT1 + KTS specifically bound to the +P5 target.
  • All three classes of investigated DDS mutations, regardless of the KTS splice presence, completely abolished DNA-binding affinity for all tested targets.
  • This loss of DNA binding indicates a significant disruption of WT1's transcriptional regulatory function.

Conclusions:

  • Denys-Drash syndrome (DDS) mutations in the Wilms' tumour suppressor gene 1 (WT1) zinc finger region abolish DNA binding.
  • These mutations likely exert their pathogenic effects through a dominant-negative antimorph mechanism or by disrupting WT1 isoform dosage balance.
  • Understanding these mechanisms is crucial for comprehending DDS pathogenesis and developing potential therapeutic strategies.

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