Ornithine decarboxylase is a transcriptional target of tumor suppressor WT1.
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.
Department of Biochemistry, University of Washington, Seattle, Washington, 98195, USA.
R S
RS
Li
G L
GL
Law
R A
RA
Seifert
P J
PJ
Romaniuk
D R
DR
Morris
D016475:3T3 Cells; D000818:Animals; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016162:Genes, Wilms Tumor; D007668:Kidney; D051379:Mice; D009955:Ornithine Decarboxylase / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D025721:WT1 Proteins
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D025721:WT1 Proteins; D009955:Ornithine Decarboxylase