Abstract
The Wilms' tumor gene, WT1, encodes a zinc finger transcription factor that can repress transcription of a number of genes. WT1 mRNA undergoes alternative splicing at two locations, yielding four different mRNA species and protein products. One alternative splice alters the zinc finger region of WT1, resulting in the addition of three amino acids, Lys-Thr-Ser (KTS), between zinc fingers 3 and 4, altering the binding of WT1 to DNA. Here, we show that the WT1 protein with and without the KTS tripeptide can repress transcription from the human full-length WT1 promoter. Repression of transcription by WT1 has been shown to require two WT1 binding sites. We examined WT1 repression of the human minimal WT1 promoter, which contains two potential WT1 binding motifs. WT1 lacking the KTS tripeptide (WT1-KTS) was unable to repress transcription from a minimal WT1 promoter of 104 base pairs, whereas WT1 containing the KTS tripeptide (WT1+KTS) repressed transcription from the minimal promoter. The ability of WT1+KTS to repress transcription where WT1-KTS could not provided a functional assay to define differential WT1 binding motifs based on the presence or the absence of the KTS tripeptides. We present data defining the differential consensus DNA binding motifs for WT1-KTS and WT1+KTS. We demonstrate that WT1 zinc finger 1 plays a role in the differential DNA binding specificity of WT1-KTS and WT1+KTS.
Highlights
The human Wilms’ tumor gene WT1 is located at 11p13 and encodes a zinc finger transcription factor that functions as a tumor suppressor (Call et al, 1990; Gessler et al, 1990)
We demonstrate that WT1 zinc finger 1 plays a role in the differential DNA binding specificity of WT1 lacking the KTS tripeptide (WT1؊KTS) and WT1 containing the KTS tripeptide (WT1؉KTS)
We show that the WT1ϪKTS and WT1ϩKTS binding sites differed in the sequences recognized by zinc finger 1
Summary
Cell Culture and Chloramphenicol Acetyltransferase Assays—The WT1 promoter constructs containing the 652- and 104-bp promoter fragments have been previously described by Fraizer et al (1994), and the WT1 cDNA expression plasmids were described by Madden et al (1991). Transfection experiments were performed in HeLa cells (ATCC CCL2), a cell line derived from a human cervical carcinoma, as described previously (Hewitt et al, 1995a), using 3.75 g of WT1 promoter CAT reporter, plus 0 –10 g of expression plasmid containing the WT1 cDNA (Madden et al, 1991), and enough pCB6ϩ vector to bring the total amount of DNA to 10 g. The minimal promoter region used as a DNA probe in the EMSAs was amplified by polymerase chain reaction with primers previously described (Fraizer et al, 1994). The minimal promoter was labeled by incorporation of [␣-32P]dCTP into the amplified fragment during polymerase chain reaction amplification.
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