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Differential effects of Wilms tumor WT1 splice variants on the insulin receptor promoter
N J Webster1, Y Kong, P Sharma
1UCSD/Whittier Diabetes Program, University of California, San Diego, La Jolla 92093, USA. nwebster@ucsd.edu
Abstract:
The Wilms tumor gene WT1 has been implicated in the early development of the kidney. Mutations in WT1 are found in a small fraction of Wilms tumor, a pediatric nephroblastoma, and Denys-Drash syndrome, characterized by genitourinary abnormalities. The WT1 gene product functions as a transcriptional repressor of growth factor-related genes. The kidney is one of the major sites of insulin action in vivo and expresses high levels of insulin receptors (IR). IR expression has been detected during early embryogenesis, suggesting that it may play a role in development. We investigated whether two WT1 splice variants lacking or including a three-amino-acid (KTS) insertion between the third and fourth zinc finger in the DNA-binding domain could repress the IR promoter in vitro. We show that the +KTS variant effectively represses promoter activity under all conditions tested but the -KTS variant was only able to repress in the presence of cotransfected C/EBP beta or a dominant-negative p53 mutation. Deletional mapping indicated that distinct regions of the IR promoter mediated the effects of the two isoforms and DNaseI footprint analysis identified potential WT1 binding sites within these regions.
Insights
The Wilms tumor (WT1) gene regulates kidney development. This study shows WT1 splice variants differentially repress the insulin receptor (IR) promoter, impacting kidney development and function.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- The Wilms tumor (WT1) gene is crucial for kidney development and acts as a transcriptional repressor.
- Insulin receptors (IR) are vital for kidney function and are expressed during embryonic development.
- WT1 has splice variants that differ by a three-amino-acid (KTS) insertion, potentially affecting gene regulation.
Purpose of the Study:
- To investigate the role of WT1 splice variants in regulating the insulin receptor (IR) promoter.
- To determine if WT1 variants differentially repress IR gene expression.
- To identify potential WT1 binding sites on the IR promoter.
Main Methods:
- In vitro assays using WT1 splice variants (+KTS and -KTS) and the IR promoter.
- Transfection experiments with cofactors C/EBP beta and dominant-negative p53.
- Deletional mapping of the IR promoter.
- DNaseI footprint analysis to identify WT1 binding sites.
Main Results:
- The +KTS WT1 variant repressed the IR promoter under all tested conditions.
- The -KTS WT1 variant showed repression only when co-transfected with C/EBP beta or dominant-negative p53.
- Distinct promoter regions were responsible for the differential repression by the WT1 isoforms.
- Potential WT1 binding sites were identified on the IR promoter.
Conclusions:
- WT1 splice variants exhibit distinct regulatory activities on the insulin receptor promoter.
- These findings suggest a role for WT1 in modulating insulin signaling during kidney development.
- Differential regulation of the IR promoter by WT1 isoforms may contribute to kidney development and disease.