Related Experiment Videos
Wnt-4 regulation by the Wilms' tumour suppressor gene, WT1
Edmund U-H Sim1, Aaron Smith, Elida Szilagi
1Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia.
Abstract:
The Wilms' tumour suppressor gene, WT1, encodes multiple nuclear protein isoforms, all containing four C-terminal zinc finger motifs. WT1 proteins can both activate and repress putative target genes in vitro, although the in vivo relevance of these putative target genes is often unverified. WT1 mutations can result in Wilms' tumour and the Denys-Drash Syndrome (DDS) of infantile nephropathy, XY pseudohermaphroditism and predisposition to Wilms' tumour. We have established stable transfectants of the mouse mesonephric cell line, M15, which express WT1 harbouring a common DDS point mutation (R394W). A comparison of the expression profiles of M15 and transfectant C2A was performed using Nylon-based arrays. Very few genes showed differential expression. However Wnt-4, a member of the Wnt gene family of secreted glycoproteins, was downregulated in C2A and other similar clones. Doxycycline induction of WT1-A or WT1-D expression in HEK293 stable transfectants also elicited an elevation in Wnt4 expression. Wnt4 is critical for the mesenchyme-to-epithelial transition during kidney development, making it an attractive putative WT1 target. We have mapped human Wnt-4 gene to chromosome 1p35-36, a region of frequent LOH in WT, have characterized the genomic structure of the human Wnt-4 gene and isolated 9 kb of immediate promoter. While several potential WT1 binding sites exist within this promoter, reporter analysis does not strongly support the direct regulation of Wnt4 by WT1. We propose that Wnt-4 regulation by WT1 occurs at a more distant promoter or enhancer site, or is indirect.
Insights
The Wilms' tumour suppressor gene (WT1) may indirectly regulate Wnt-4 expression, a gene crucial for kidney development. This finding offers new insights into Wilms' tumour pathogenesis and WT1 gene function.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- The Wilms' tumour suppressor gene (WT1) encodes proteins regulating gene expression, with mutations linked to Wilms' tumour and Denys-Drash Syndrome.
- WT1's in vivo target genes and regulatory mechanisms remain incompletely understood.
Purpose of the Study:
- To investigate the relationship between WT1 and Wnt-4, a gene vital for kidney development.
- To explore the potential of Wnt-4 as a direct or indirect WT1 target gene.
Main Methods:
- Established mouse mesonephric M15 cell lines stably expressing WT1 with a Denys-Drash Syndrome mutation.
- Utilized Nylon-based arrays to compare gene expression profiles.
- Performed reporter assays to assess Wnt-4 promoter activity.
- Mapped the human Wnt-4 gene and characterized its promoter region.
Main Results:
- Wnt-4 expression was downregulated in M15 cells expressing mutant WT1.
- WT1 induction in HEK293 cells elevated Wnt-4 expression.
- Reporter assays did not strongly support direct WT1 regulation of the Wnt-4 promoter.
- Human Wnt-4 gene mapped to chromosome 1p35-36, a region with frequent loss of heterozygosity in Wilms' tumour.
Conclusions:
- WT1 may indirectly regulate Wnt-4, potentially through distant regulatory elements or other factors.
- The findings suggest a complex regulatory relationship between WT1 and Wnt-4 in kidney development.
- This study provides a foundation for further research into WT1-mediated gene regulation in Wilms' tumour.