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Ornithine decarboxylase is a transcriptional target of tumor suppressor WT1
1Department of Biochemistry, University of Washington, Seattle, Washington, 98195, USA.
Abstract:
The product of the Wilm's tumor suppressor gene, WT1, is a zinc-finger DNA-binding protein, which is thought to be a transcription factor. Two genes, those encoding epidermal growth factor receptor and syndecan-1, are known to be endogenous targets of WT1. Previous studies had identified binding sites for WT1 in the promoter of the ornithine decarboxylase (ODC) gene. In this paper, we tested whether the endogenous ODC gene might be a target of WT1 by establishing lines of baby hamster kidney (BHK) cells that expressed WT1 isoform A under control of a tetracycline-regulated expression system. When expression of WT1 was activated in BHK cells, the cellular level of ODC mRNA declined, with kinetics that correlated with the increase in WT1 level, demonstrating that the endogenous ODC gene was indeed responsive to cellular level of WT1. WT1 isoforms A and B inhibited the activity of the ODC promoter by approximately fivefold in transiently transfected BHK cells, while isoforms C and D, which have altered DNA binding domains, had no significant effect. The sequence CTCCCCCGC, located at nucleotides -106 to -98 relative to the site of transcriptional initiation in the ODC gene, interacted with the zinc-finger domain of isoforms A and B of WT1 with high affinity and specificity. A mutation in the binding site that disrupted this interaction partially removed the inhibition of ODC promoter activity by WT1, as did mutation of the two E-box sequences in intron I of the ODC gene. Simultaneous mutation of the WT1-binding motif and the two E-boxes completely abolished inhibition by WT1 of ODC promoter activity. These results, taken together, implicate the ODC gene as a downstream target of the tumor suppressor WT1.
Insights
The Wilm's tumor suppressor gene (WT1) directly regulates ornithine decarboxylase (ODC) gene expression. This study confirms ODC as a downstream target of WT1, impacting cellular ODC mRNA levels and promoter activity.
Area of Science:
- Molecular Biology
- Cancer Genetics
- Gene Regulation
Background:
- The Wilm's tumor suppressor gene (WT1) encodes a zinc-finger DNA-binding protein, functioning as a transcription factor.
- Known WT1 targets include genes for epidermal growth factor receptor and syndecan-1.
- Previous research indicated potential WT1 binding sites within the ornithine decarboxylase (ODC) gene promoter.
Purpose of the Study:
- To investigate whether the endogenous ornithine decarboxylase (ODC) gene is a direct target of the Wilm's tumor suppressor gene (WT1).
- To elucidate the mechanism by which WT1 influences ODC gene expression and promoter activity.
Main Methods:
- Established tetracycline-regulated baby hamster kidney (BHK) cell lines expressing WT1 isoform A.
- Measured ODC mRNA levels and ODC promoter activity in response to WT1 expression.
- Utilized site-directed mutagenesis to assess the role of specific WT1 binding sites and E-boxes in ODC gene regulation.
Main Results:
- Inducible WT1 expression in BHK cells led to a decrease in cellular ODC mRNA levels.
- WT1 isoforms A and B significantly inhibited ODC promoter activity, while isoforms C and D did not.
- A specific DNA sequence (CTCCCCCGC) in the ODC promoter bound WT1 isoforms A and B with high affinity.
- Mutating the WT1 binding site or E-boxes partially reduced WT1's inhibitory effect; simultaneous mutation abolished it.
Conclusions:
- The ornithine decarboxylase (ODC) gene is confirmed as a downstream target of the Wilm's tumor suppressor gene (WT1).
- WT1 directly represses ODC gene expression through specific DNA binding interactions.
- These findings highlight a novel regulatory pathway involving WT1 and ODC in cellular processes.