Regulation of Expression of the Ornithine Decarboxylase Gene by Tumor Suppressor
WT1
Run-Shang Li, G. Lynn Law, Ronald A. Seifert, Paul J. Romaniuk and David
R. Morris
The product of the Wilms' 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 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 5-fold 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.