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New insights into DNA-binding behavior of Wilms tumor protein (WT1)--a dual study
Elmar Nurmemmedov1, Raymond K Yengo, Hüseyin Uysal
1Center for Molecular Protein Science, Lund University, Getingevägen 60, 221 00, Lund, Sweden.
Abstract:
Wilms Tumor suppressor protein (WT1) is a transcription factor that is involved in a variety of developmental functions during organ development. It is also implicated in the pathology of several different cancer forms. The protein contains four C(2)H(2)-type zinc fingers and it specifically binds GC-rich sequences in the promoter regions of its target genes, which are either up or down regulated. Two properties make WT1 a more unusual transcription factor - an unconventional amino acid composition for zinc finger 1, and the insertion of a tri-peptide KTS in some of the splice isoforms of WT1. Using six WT1 constructs in which zinc fingers are systematically deleted, a dual study based on a bacterial 1-hybrid system and surface plasmon resonance measurements is performed. The experiments show that the effect of zinc finger 1 is not significant in terms of overall DNA-binding kinetics, however it influences both the specificity of target recognition and stability of interaction in presence of KTS. The KTS insertion, however, only mildly retards binding affinity, mainly by affecting the on-rate. We suggest that the insertion disturbs zinc finger 4 from its binding frame, thus weakening the rate of target recognition. Finally, for the construct in which both zinc fingers 1 and 4 were deleted, the two middle fingers 2-3 still could function as a 'minimal DNA-recognition domain' for WT1, however the formation of a stable protein-DNA complex is impaired since the overall affinity was dramatically reduced mainly since the off-rate was severely affected.
Insights
The Wilms Tumor suppressor protein (WT1) uses its zinc fingers to bind DNA. The KTS insertion and specific zinc fingers influence DNA binding stability and target recognition, impacting gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Wilms Tumor suppressor protein (WT1) is a crucial transcription factor in organ development.
- WT1 dysregulation is linked to various cancers.
- WT1 features C(2)H(2) zinc fingers that bind GC-rich DNA sequences.
Purpose of the Study:
- To investigate the role of individual WT1 zinc fingers and the KTS insertion in DNA binding kinetics and specificity.
- To elucidate the contribution of different WT1 domains to protein-DNA complex formation and stability.
Main Methods:
- Utilized a bacterial one-hybrid system to study DNA-binding interactions.
- Employed surface plasmon resonance (SPR) measurements to quantify binding kinetics and affinity.
- Systematically deleted zinc fingers from WT1 constructs for functional analysis.
Main Results:
- Zinc finger 1 has minimal impact on overall DNA binding kinetics but affects specificity and stability with KTS.
- The KTS insertion mildly reduces binding affinity by slowing the on-rate, potentially by displacing zinc finger 4.
- Zinc fingers 2-3 can act as a minimal DNA-binding domain, but deleting fingers 1 and 4 severely impairs stable complex formation due to reduced affinity and increased off-rate.
Conclusions:
- WT1's DNA-binding properties are modulated by specific zinc fingers and the KTS insertion.
- The interplay between zinc fingers and the KTS peptide is critical for precise target recognition and stable complex formation.
- Understanding these interactions provides insights into WT1's role in development and disease.
