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Functional characterization of WT1 binding sites within the human vitamin D receptor gene promoter
1Department of Biochemistry, McGill Cancer Center, McGill University, Montreal, Quebec, Canada H3G 1Y6.
Abstract:
The Wilms' tumor suppressor gene, wt1, encodes a zinc finger transcription factor that can regulate gene expression. It plays an essential role in tumorigenesis, kidney differentiation, and urogenital development. To identify WT1 downstream targets, gene expression profiling was conducted using a cDNA array hybridization approach. We confirm herein that the human vitamin D receptor (VDR), a ligand-activated transcription factor, is a WT1 downstream target. Nuclear run on experiments demonstrated that the effect of WT1 on VDR expression is at the transcriptional level. Transient transfection assays, deletion mutagenesis, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays suggest that, although WT1 is presented with a possibility of three binding sites within the VDR promoter, activation of the human VDR gene appears to occur through a single site. This site differs from a previously identified WT1-responsive site in the murine VDR promoter (Maurer U, Jehan F, Englert C, Hübinger G, Weidmann E, DeLucas HF, and Bergmann L. J Biol Chem 276: 3727-3732, 2001). We also show that the products of a Denys-Drash syndrome allele of wt1 inhibit WT1-mediated transactivation of the human VDR promoter. Our results indicate that the human VDR gene is a downstream target of WT1 and may be regulated differently than its murine counterpart.
Insights
The Wilms' tumor suppressor gene (WT1) regulates gene expression and is crucial for kidney development. This study identifies the vitamin D receptor (VDR) as a direct WT1 target, revealing a new mechanism in gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- The Wilms' tumor suppressor gene (WT1) encodes a transcription factor vital for kidney and urogenital development.
- WT1's role in tumorigenesis highlights the importance of understanding its downstream targets.
- Identifying WT1 targets is key to elucidating its regulatory functions in development and disease.
Purpose of the Study:
- To identify downstream targets of the WT1 transcription factor.
- To confirm the human vitamin D receptor (VDR) gene as a WT1 target.
- To investigate the mechanism of WT1 regulation on VDR expression.
Main Methods:
- Gene expression profiling using cDNA array hybridization.
- Nuclear run-on assays to assess transcriptional regulation.
- Transient transfection, deletion mutagenesis, EMSA, and ChIP assays to map WT1 binding sites on the VDR promoter.
Main Results:
- The human vitamin D receptor (VDR) is confirmed as a direct transcriptional target of WT1.
- WT1 regulates human VDR gene expression through a single binding site in its promoter, distinct from the murine VDR promoter site.
- A Denys-Drash syndrome allele of WT1 inhibits transactivation of the human VDR promoter.
Conclusions:
- The human VDR gene is a downstream target of WT1.
- WT1 regulation of the human VDR promoter differs from that of the murine VDR promoter.
- Understanding WT1-VDR interaction provides insights into urogenital development and WT1-associated disorders.