Related Experiment Videos
Differential function of Wilms' tumor gene WT1 splice isoforms in transcriptional regulation
S M Hewitt1, G C Fraizer, Y J Wu
1Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston, 77030, USA.
Abstract:
The Wilms' tumor gene, WT1, encodes a zinc finger transcription factor that can repress transcription of a number of genes. WT1 mRNA undergoes alternative splicing at two locations, yielding four different mRNA species and protein products. One alternative splice alters the zinc finger region of WT1, resulting in the addition of three amino acids, Lys-Thr-Ser (KTS), between zinc fingers 3 and 4, altering the binding of WT1 to DNA. Here, we show that the WT1 protein with and without the KTS tripeptide can repress transcription from the human full-length WT1 promoter. Repression of transcription by WT1 has been shown to require two WT1 binding sites. We examined WT1 repression of the human minimal WT1 promoter, which contains two potential WT1 binding motifs. WT1 lacking the KTS tripeptide (WT1-KTS) was unable to repress transcription from a minimal WT1 promoter of 104 base pairs, whereas WT1 containing the KTS tripeptide (WT1+KTS) repressed transcription from the minimal promoter. The ability of WT1+KTS to repress transcription where WT1-KTS could not provided a functional assay to define differential WT1 binding motifs based on the presence or the absence of the KTS tripeptides. We present data defining the differential consensus DNA binding motifs for WT1-KTS and WT1+KTS. We demonstrate that WT1 zinc finger 1 plays a role in the differential DNA binding specificity of WT1-KTS and WT1+KTS.
Insights
The Wilms' tumor 1 (WT1) protein
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- The Wilms' tumor 1 (WT1) gene encodes a transcription factor involved in gene repression.
- WT1 mRNA exhibits alternative splicing, producing four protein variants.
- A key splice variant includes the Lys-Thr-Ser (KTS) tripeptide, affecting DNA binding.
Purpose of the Study:
- To investigate the differential transcriptional repression activity of WT1 protein variants.
- To define the distinct DNA binding motifs for WT1 with and without the KTS tripeptide.
- To elucidate the role of WT1 zinc finger 1 in differential DNA binding specificity.
Main Methods:
- Analysis of WT1 protein variants' ability to repress transcription from full-length and minimal WT1 promoters.
- Functional assay to differentiate WT1 binding motifs based on KTS presence.
- Examination of WT1 zinc finger 1's role in DNA binding specificity.
Main Results:
- Both WT1+KTS and WT1-KTS proteins repressed the full-length WT1 promoter.
- WT1-KTS failed to repress transcription from a minimal 104 bp WT1 promoter, while WT1+KTS did.
- Differential consensus DNA binding motifs for WT1-KTS and WT1+KTS were defined.
- WT1 zinc finger 1 was shown to be critical for differential DNA binding specificity.
Conclusions:
- The presence or absence of the KTS tripeptide in WT1 protein significantly alters its transcriptional repression activity on specific DNA elements.
- WT1+KTS and WT1-KTS exhibit distinct DNA binding specificities, enabling functional differentiation.
- WT1 zinc finger 1 is a key determinant of this differential binding specificity, providing insights into WT1 gene regulation.