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Differential Effects of Wilms Tumor WT1 Splice Variants on the Insulin Receptor Promoter
BIOCHEMICAL AND MOLECULAR MEDICINE ARTICLE NO.
62, 139–150 (1997)
MM972648
Differential Effects of Wilms Tumor WT1 Splice Variants on the Insulin Receptor Promoter Nicholas J. G. Webster,1 Yan Kong, Prem Sharma, Martin Haas, Saraswati Sukumar,* and B. Lynn Seely UCSD/Whittier Diabetes Program and the UCSD Cancer Center, University of California, San Diego, La Jolla, California 92093, and Medical Research Service, Department of Veterans Affairs Medical Center, San Diego, California 92161; and *Breast Cancer Program, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Received August 29, 1997, and in revised form September 25, 1997
locus implicated in the development of the pediatric nephroblastoma Wilms tumor on chromosome 11p13 (1). WT1 mutations have been found in the WAGR syndrome (Wilms tumor, aniridia, genitourinary abnormalities, and retardation) and the Denys – Drash syndrome (genitourinary abnormalities) in humans (2,3). During embryonic development, WT1 transcripts are present in a number of regions including the urogenital system, spinal cord, brain, and mesothelial lining of organs within the thoracic cavity (4). Mice deficient for WT1 die in utero from abnormal development of the kidney, gonads, heart, lung, and mesothelium, demonstrating an important role for WT1 in embryogenesis (5). In the kidney on embryonic day 11, the ureteric bud fails to grow out of the Wolffian duct toward the blastemal cells. The metanephric blastema shows many apoptotic cells in the null embryos, and no mesenchymal cells are detectable by day 12. In vitro organ culture experiments demonstrated that the blastemal cells are unable to respond to inducers of tubule formation (5). In the wild-type embryos, WT1 is detectable in the podocyte layer of the glomeruli but not the blastemal mesenchyme or tubules by immunohistochemistry (6). The WT1 gene encodes a transcription factor containing four putative zinc fingers that are closely related to the Egr1 family, but unlike Egr1, WT1 inhibits transcription (7). A number of cellular targets for WT1 have been identified in vitro, including the PDGF-A chain, IGF-II, the IGF-I receptor, CSFI,
The Wilms tumor gene WT1 has been implicated in the early development of the kidney. Mutations in WT1 are found in a small fraction of Wilms tumor, a pediatric nephroblastoma, and Denys–Drash syndrome, characterized by genitourinary abnormalities. The WT1 gene product functions as a transcriptional repressor of growth factor-related genes. The kidney is one of the major sites of insulin action in vivo and expresses high levels of insulin receptors (IR). IR expression has been detected during early embryogenesis, suggesting that it may play a role in development. We investigated whether two WT1 splice variants lacking or including a three-aminoacid (KTS) insertion between the third and fourth zinc finger in the DNA-binding domain could repress the IR promoter in vitro. We show that the /KTS variant effectively represses promoter activity under all conditions tested but the 0KTS variant was only able to repress in the presence of cotransfected C/EBPb or a dominant-negative p53 mutation. Deletional mapping indicated that distinct regions of the IR promoter mediated the effects of the two isoforms and DNaseI footprint analysis identified potential WT1 binding sites within these regions. q 1997 Academic Press
Insights into the development of the kidney have arisen from the cloning of the WT1 gene from a 1 To whom correspondence should be addressed at Department of Medicine (0673), University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0673. Fax: (619) 534-7181. E-mail: [email protected].
139 1077-3150/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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RARa, and TGF-b (8–13). However, few in vivo targets for WT1 have been identified. IGF-II and IGFIR are overexpressed in tumors containing mutations in WT1 (10,14) and, recently, Werner et al. demonstrated that IGF-IR expression and IGF-Istimulated mitogenesis could be inhibited in G401 cells engineered to express WT1 (15). This result underscores the importance of the insulin-like growth factors in kidney development and tumorigenesis. The EGFR is a second in vivo target for WT1, and WT1 induces apoptosis in cells by inhibiting EGFR expression (6). The gene also has two major sites of alternative splicing. The first is a 17amino-acid insertion at position 248 that is due to the inclusion of exon 5. The second is a 3-amino-acid (KTS) insertion at position 411 in the DNA-binding domain arising from the use of an alternative 3* splice site (16). The WT1 gene is also susceptible to RNA editing, resulting in a substitution of proline for leucine at position 280 (17). Due to the similarity between the DNA-binding domains of WT1 and Egr1, WT1 can bind to Egr1 sites (GCGGGGGCG) (18). The KTS insertion lies between the third and fourth zinc fingers in the DNA-binding domain and alters binding specificity. The 0KTS isoform of WT1 can recognize an Egr1 binding site, whereas the /KTS isoform does not (19). Recent studies have shown that both isoforms have a more relaxed binding specificity than the related Egr1 protein, presumably due to the presence of four zinc fingers rather than three (20,21). Other functional domains have been localized by deletional analysis. The domain involved in transcriptional repression has been mapped to residues 84–179, whereas a transcriptional activation domain has been mapped to residues 180–296 (22). Furthermore, the 17-amino-acid alternatively spliced exon 5 can function as an independent repressor module (23). Recent results have indicated that repression by WT1 is mediated by protein–protein interactions involving the repression domain and an accessory protein (24). The kidney, along with liver, muscle, and adipose tissue, is a major site of insulin action in vivo. One of insulin’s physiological roles in the kidney is to stimulate sodium readsorption. The kidney is also a major site of insulin metabolism, primarily in the proximal tubular epithelial cells, which express high levels of insulin receptors. In the developing mouse kidney on embryonic day 13, insulin receptor expression is detected in the plasmalemma cells of the ureteric bud and precursor cells of the rudimentary
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nephron including the condensing blastemal cells and S-shaped bodies (25). At later stages of development, IR expression is also detected in the tubular epithelial cells and after birth is restricted to only these tubular epithelial cells. The expression pattern of the IR is similar to that of the EGFR, which is known to be regulated by WT1 (15). Two pieces of evidence suggest a role for insulin receptors in kidney development (25). First, blockade of insulin receptors in vitro causes retardation of nephrogenesis and disruption of ureteric bud branching. Second, insulin treatment causes both a hypertrophy and hyperplasia of metanephrogenic cells. The expression of the IR in Wilms tumor, however, has not been investigated. These observations led us to investigate whether WT1 could modulate the IR promoter. The insulin receptor gene is unusual in that it has the characteristics of both a housekeeping and a tissue-specific gene. The IR gene promoter lacks a TATA box or initiator sequence, and the primary sequence is very GC rich (26–28). Basal promoter activity is mediated by the transcription factor Sp1 (29–31). The tissue-specific regulation of the IR gene is not well understood but transcription factors of the C/EBP family can stimulate the promoter in adipose and liver cells (32,33). We have also found that the promoter is under negative control and contains a tissue-specific silencer between 01 and 01.2 kb upstream of the translational start site (29). Computer analysis of the sequence of the GC-rich region of the promoter indicated the presence of multiple Egr1 sites, indicating that, potentially, the promoter could be regulated by WT1. In this paper, we demonstrate that the two KTS splice variants of WT1 have differential effects on the IR promoter. EXPERIMENTAL PROCEDURES Construction of Plasmids All plasmid constructions were performed using standard techniques. The construction of pIRC1 to 8 and the expression vector for C/EBPb have been described previously (29,32). Expression vectors encoding wild-type p53 and a mutant p53 (248Q) have been described previously (34). The parental expression vector pRcCMV was from Invitrogen (San Diego, CA). cDNAs encoding the two rat WT1 isoforms (/KTS and 0KTS) were subcloned into expression vector CB6/ (17). Both isoforms contained
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exon 5 and leucine at amino acid 280. Expression vectors for the DNA-binding domains of the two splice variants were constructed by amplifying the two C-terminal zinc finger domains (amino acids 312–448) by PCR and subcloning into expression vector CB6/. The 5* primer was designed to include a consensus Kozak sequence to ensure efficient translation. The Escherichia coli expression vectors pGEX / KTS and pGEX 0 KTS were constructed by amplifying the WT1 DNA-binding domains and subcloning into pGEX4T-1. This vector allows for the production of a glutathione-S-transferase (GST) fusion protein under control of the tac promoter (Pharmacia, Piscataway, NJ). Cultures were induced and the GST fusion protein purified following the manufacturer’s protocol. Cotransfections and CAT Assays HepG2 and HeLa cells were maintained in minimum essential medium plus Earle’s salts with 10% fetal calf serum (MEM-Earle’s). HepG2 cells were transfected using the calcium phosphate coprecipitation technique. The precipitate was removed after 8 h, and then the cells were subjected to a glycerol shock (10% glycerol in MEM-Earle’s) for 3 min at 377C. Saos-2 cells were maintained in McCoy 5a medium with 15% fetal calf serum. HeLa and Saos2 cells were transfected using Lipofectamine (BRL, Gaithersburg, MD) following the manufacturer’s protocol. In all cases the reference plasmid RSVlacZ, which contains the b-galactosidase gene under the control of the Rous sarcoma virus long terminal repeat, was cotransfected to correct for transfection efficiency. Cells were harvested 48 h after transfection, lysed in 250 ml of modified lysis buffer by three cycles of freeze–thaw, and centrifuged at 10,000g for 15 min at 47C, and the supernatant was stored at 0807C. The b-galactosidase activity and CAT activity were measured as described previously (32). Gel Shift and DNaseI Footprinting Assays GST fusion proteins containing the zinc finger DNA-binding domains of the WT1 isoforms were expressed in E. coli using the pGEX system. Cultures were induced with isopropyl-thio-galactopyranoside (IPTG) overnight. Fusion proteins were isolated from bacterial lysates by absorbing to glutathione– agarose beads followed by elution with glutathione following the manufacturer’s protocol. DNA probes were end-labeled with T4 polynucleotide kinase and
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50 mCi [g-32P]ATP and purified by gel electrophoresis. For the gel-shift assays, increasing amounts (0– 10 mg) of GST–WT1 fusion protein was preincubated with 5 mg poly(dI.C) and 0.1 mg/ml BSA in 20 ml 0.51 TGMEDK buffer (25 mM Tris–HCl, pH 7.9, 10% glycerol, 5 mM MgCl2 , 0.1 mM EDTA, 1 mM dithiothreitol, 100 mM KCl, 0.15 mM ZnCl2) for 30 min at 07C. The 32P-labeled probe (20,000 cpm) was added and the incubation continued for a further 15 min at room temperature. Free and bound probe were separated on a nondenaturing 5% polyacrylamide gel at 15 mA at room temperature in 0.251 TBE with recirculation of the buffer. Gels were dried and subjected to autoradiography. In competition experiments, unlabeled double-stranded oligonucleotides (10–100 pmol) were included in the incubation. DNaseI footprinting assays were performed as described previously (29). Immunoprecipitation of WT1 and p53 Chinese hamster ovary cells (CHO) were maintained in Ham’s F12 medium with 10% fetal calf serum. Cells were transfected with expression vectors (10 mg) for wild-type p53 and either the / or 0KTS isoform of WT1 using lipofectamine following the manufacturer’s protocol. Cells were harvested 48 h after transfection and lysed by three strokes of a Dounce homogenizer (tight pestle) in homogenization buffer (0.25 M sucrose, 15 mM Tris–HCl, pH 7.9, 15 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 60 mM KCl, 0.5% NP-40, 1 mM dithiothreitol) on ice in the presence of a cocktail of protease inhibitors (Complete, Boehringer Mannheim, Indianapolis, IN). The lysate was split into two fractions. A polyclonal antibody to WT1 (C-19) was added to one fraction and a monoclonal antibody to p53 (DO-1) was added to the other fraction to a concentration of 1 mg/ml; both antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA). Proteins were immunoprecipitated overnight at 47C on a rocking platform. A protein A–agarose conjugate was added and the incubation was continued for a further 2 h. Immune complexes were pelleted by centrifugation at 15,000g for 15 min at 47C. The supernatant was saved and the pellets washed three times in EBG (120 mM NaCl, 5 mM KCl, 1 mM MgSO4 , 1 mM CaCl2 , 25 mM Hepes, 10% glycerol, 0.05% Triton X-100). Finally the pellets were washed in 100 ml EBG containing 1% Triton X-100. The immunoprecipitated proteins were resuspended in
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sample buffer and boiled for 5 min. An aliquot (10%) of the initial supernatant and the 1% Triton X-100 wash were also boiled with sample buffer for 5 min. The proteins in the pellet, wash, and supernatant fractions were separated by 10% SDS–PAGE. Proteins were electrotransferred to Immobilon filters (Millipore, Bedford MA) overnight at 12 V in 25 mM Tris, 192 mM glycine, pH 8.3, at 47C. The filters were blocked with 5% nonfat dried milk in T-TBS (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) for 60 min at 257C. The polyclonal antibody to WT1 (C19) was added to the filter containing the p53 immunoprecipitates, and the monoclonal antibody to p53 (DO-1) was added to the filter containing the WT1 immunoprecipitates. Both antibodies were diluted 1:1000 in T-TBS. Filters were incubated for 2 h at 257C on a rocking platform. Filters were rinsed three times in T-TBS and then incubated for 60 min at 257C with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies, respectively. Proteins were detected by enhanced chemiluminescence (ECL, Amersham). RESULTS Inhibition of the hIR Promoter by WT1 To determine if the hIR promoter can be regulated by the Wilms’ tumor gene product WT1, we cotransfected the full-length insulin receptor promoter– CAT construction pIRC1 into HepG2 cells with increasing amounts of expression vectors for two splice variants of WT1. The HepG2 cell is derived from a human hepatoblastoma and has been useful as a model for studying insulin signaling. These cells express 25,000 insulin receptors per cell, do not express WT1, and have a wild-type p53 gene. The encoded cDNAs differ in the presence of three amino acids (KTS) in the third zinc finger of the WT1 DNAbinding domain (DBD). Both vectors contained fulllength WT1 including the 17-amino-acid exon 5 that encodes a repression domain (23). The parental expression vector CB6/ was included to normalize the amount of DNA in all transfections to minimize nonspecific effects. The RSV–lacZ plasmid was included to correct for transfection efficiency. A dose-dependent decrease in CAT activity was observed for both splice variants (Fig. 1A). The /KTS splice variant appeared to be a more potent inhibitor of the IR promoter, giving a 60% reduction in promoter activity at a reporter to expression vector ratio of 5:2 and
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increasing to 70% repression at a ratio of 1:1. This is similar to the reported 80% inhibition of IGF-IR promoter activity and 90% inhibition of the EGFR promoter (6,10). Both the IGF-IR and EGFR have been shown to be in vivo targets for WT1. It should be noted that a large excess of WT1 expression vector was required to obtain these inhibitions (20-fold and 5-fold excess for IGF-IR and EGFR, respectively). In the experiments reported here, the maximum amount of WT1 vector corresponded to a reporter:vector ratio of 1:1. This avoids nonspecific effects due to the vast overexpression of WT1 in the other studies. The 0KTS isoform only repressed promoter activity significantly (40%) at the highest amount of transfected vector (promoter:vector ratio of 1:1). As a control, the related transcription factor Egr1 had no effect on IR promoter activity (data not shown). To assess whether WT1 could repress the stimulated as well as the basal IR promoter, we studied the promoter in the presence of C/EBPb, a transcription factor known to stimulate the IR promoter (32,33). A titration was performed as above, but with the inclusion of an expression vector for C/EBPb. The absolute CAT activity in the absence of WT1 was increased 7- to 10-fold, as we have published previously (32). Both WT1 splice variants caused a similar dose-dependent decrease in CAT activity (Fig. 1B). Interestingly, the 0KTS variant was as effective in inhibiting C/EBP-stimulated IR promoter activity as the /KTS variant. Expression of the individual zinc finger DNA-binding domains of the two WT1 isoforms had no effect on IR promoter activity, suggesting that repression is not the result of competition for promoter binding sites (data not shown). Deletion Analysis of the IR Promoter To determine if the two isoforms recognize distinct binding sites, we attempted to localize the regions of the promoter required for repression by WT1. The effect of WT1 on a series of deletion mutants of the IR promoter was assessed to identify the approximate locations of the binding sites. Expression vectors for the / and 0KTS isoforms were transfected into HepG2 cells with a series of 5* deletions of the IR promoter at a promoter:vector ratio of 1:1 (Fig. 2). The deletions are numbered relative to the translational start site. The full-length promoter contains sequences from 01.8 to 0290 kb. Deletion of se-
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FIG. 1. Effect of WT1 on the IR promoter. HepG2 cells were transfected with the IR-CAT reporter plasmid pIRC1 (5 mg) and increasing amounts of expression vector (0–5 mg) for the two KTS isoforms of WT1.pCB6/ was used to normalize the amount of expression vector in each transfection. Transfection efficiency was monitored by cotransfecting the b-galactosidase plasmid RSVlacZ (1 mg). CAT and b-galactosidase activity were measured on cellular extracts prepared 48 h after transfection. CAT activity was normalized for b-galactosidase activity and then expressed as %max. (A) Repression of CAT activity by the two isoforms of WT1 in parental HepG2 cells. (B) Repression of CAT activity in the presence of an expression vector for C/EBPb. HepG2 cells were transfected as above except that an expression vector for C/EBPb (5 mg) was cotransfected. Basal CAT activity was elevated 7- to 10-fold. Results are the mean { SEM of two to three experiments performed in duplicate.
quences upstream of 0877 causes a tonic depression of promoter activity, as we have shown previously (29). The 0877 promoter deletion is very effectively repressed by both WT1 isoforms (84% by /KTS, 67% by 0KTS). The baseline promoter is decreased by
deletion to 0746 but is still repressed by both WT1 isoforms. Removal of a further 100 nucleotides to 0646 eliminates the inhibition by the 0KTS isoform, but not by the /KTS isoform. This suggests that there are binding sites for the 0KTS isoform be-
FIG. 2. Effect of WT1 on a series of 5* deletion mutants of the IR promoter. HepG2 cells were transfected with a series of 5* deletion mutants of pIRC1 (5 mg) with expression vectors for either WT1 isoform or the parental vector pCB6/ (5 mg). The parental reporter plasmid contained the IR promoter from 01.8 to 0290 kb relative to the translational start site. The 5* end of the promoter deletion is indicated on the x axis. RSV–lacZ was cotransfected to correct for transfection efficiency. CAT and b-galactosidase activity were measured on cellular extracts made 48 h after transfection. CAT activity is normalized for b-galactosidase activity and expressed as %max. Results are the mean { SEM of three experiments performed in duplicate.
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FIG. 3. Gel-shift analysis of region 0877 to 0487 of the IR promoter. Probes were generated by digestion of the IR promoter, followed by dephosphorylation and then labeling of the free 5*OH with [g-32P]ATP and T4 polynucleotide kinase. Probes were purified by electrophoresis on 5% polyacrylamide gels. Reactions contained increasing amounts of GST–DBD fusion proteins in the presence of poly(dI.C) (5 mg) and BSA (0.1 mg/ml). Free and bound probes were separated by electrophoresis of 5% polyacrylamide gels. (Left) A probe covering 0877 to 0646 containing the region implicated in repression by the 0KTS isoform. (Right) A probe covering 0645 to 0487 containing the region implicated in repression by the /KTS isoform.
tween 0646 and 0746 that are required for inhibition. It does not rule out the possibility of sites more distal or proximal in the promoter but such sites are not sufficient for repression. Removal of a further 79 nucleotides to 0577 eliminates most promoter activity, as we have shown previously, and also eliminates the inhibition by the /KTS isoform of WT1. The region between 0590 and 0640 is very GC-rich and has been shown to contain multiple functional sites for transcription factor Sp1 as well as predicted Egr1 sites (29–31). Thus, the deletional analysis indicates that sequences between 0746 and 0646 or 0646 and 0577 are important for repression by the 0KTS and /KTS WT1 isoforms, respectively. Identification of in Vitro WT1 Binding Sites on the IR Promoter The deletional analysis indicated that the promoter region between 0746 and 0577 appears to be important for repression by WT1 in vivo. The next experiments were designed to identify WT1 binding sites on fragments of the IR promoter in vitro. GST fusion proteins containing the DNA-binding domains from either WT1 isoform were expressed in E. coli and purified on glutathione–agarose beads. Promoter fragments end labeled with 32P were incubated with increasing amounts of fusion protein, then free and bound probe were separated on a nondenaturing polyacrylamide gel. Probes covering 0877 to 0646 and 0645 to 0486 gave multiple strong retarded complexes with both WT1 isoforms (Fig. 3).
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To analyze the actual sequences in the IR promoter that are recognized by the WT1 isoforms, we footprinted the region covering 0486 to 0744 of the promoter using the recombinant GST fusion proteins and DNaseI. A representative footprint is shown in Fig. 4A, and the results from a number of experiments using both strands are summarized in Fig. 4B. A GC-rich sequence between 0600 and 0620 was protected from digestion along with another at 0680. These sites resemble the GC-rich sequences that have been identified as WT1 binding sites in the PDGF-A promoter (11). A further binding site between 0590 and 0570 contains a direct repeat similar to the CTCCCTCCTCCTC motif in the A and B boxes defined as functional WT1 response elements in the EGFR promoter. The only differential footprint that was observed was at 0720. This region was protected by the 0KTS fusion protein only. Interestingly, this site does not conform to a GC-rich Egr1-like motif or TC box. Furthermore, this site lies between the 0746 and 0646 promoter deletions that are important for repression by the 0KTS isoform in vivo. Role of p53 in the Repression of the IR Promoter by WT1 Previous studies have suggested that p53 may be involved in the repression by WT1 (35). Furthermore, we have published that the IR promoter is repressed by p53 (34). To investigate whether p53 is required for repression of the IR promoter by WT1, we determined whether WT1 could repress the IR
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FIG. 4. DNaseI footprinting analysis of the IR promoter. Overlapping probes covering 0744 to 0575 were generated by digestion, dephosphorylation, and end labeling with [g-32P]ATP and T4 polynucleotide kinase. Probes were purified by electrophoresis on 5% polyacrylamide gels. DNaseI footprinting analysis was performed as published previously. Both strands were footprinted. A representative autoradiogram of the anti-sense strand between 0590 and 0744 is shown (A). Results from three experiments on both strands are summarized in the diagram (B). Protected regions are indicated by lines above or below the sequence for the sense and anti-sense strands, respectively. Dots indicate hypersensitive sites. The differential footprint at 0720 is indicated by the open boxes. The dashed lines represent potential WT1 binding sites that conform to the GC-rich Egr-like motif. The two arrows indicate direct repeats of the TCCCTCCC motif that have been identified as functional WT1 response elements in the EGFR promoter.
promoter in cells that do not express p53. The cells that we chose to use are HeLa cells, which contain very low levels of p53 due to destabilization by the HPV E6 protein, and Saos-2 cells, which have chromosomal deletions of the p53 gene. Reporter plasmid pIRC1 was transfected into these cells with increasing amounts of expression vectors for the two WT1 isoforms as above. As expected, the basal promoter activity was much lower in both HeLa and Saos-2 cells, which contain very few insulin receptors (õ3000/cell), than in HepG2 cells, which contain 25,000 receptors per cell (29). The WT (/KTS) isoform was able to repress promoter activity in all three cell lines in a similar manner (Fig. 5A). However, the 0KTS isoform was unable to repress in the HeLa and Saos-2 cells (Fig. 5B). These results demonstrate that (a) the WT1 (/KTS) isoform can inhibit transcription in a p53-independent manner and (b) WT1 (0KTS) does not repress in the absence of p53, confirming the results of Maheswaran et al. (35). It should be noted that the repressive effects of WT1 are weaker in these cell lines due to the lower activity of the IR promoter in fibroblast cells (29).
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FIG. 5. Effect of WT1 on the IR promoter in cells lacking p53. HepG2, HeLa, and Saos-2 cells were transfected with the IR-CAT reporter plasmid pIRC1 (5 mg) and increasing amounts of expression vector (0–5 mg) for the two KTS isoforms of WT1.pCB6/ was used to normalize the amount of expression vector in each transfection. Transfection efficiency was monitored by cotransfecting the bgalactosidase plasmid RSVlacZ (1 mg). CAT and b-galactosidase activity were measured on cellular extracts prepared 48 h after transfection. CAT activity is normalized for b-galactosidase activity and then expressed as %max. (A) Inhibition by the /KTS isoform of WT1. (B) Inhibition by the 0KTS isoform of WT1. Results are the mean { SEM of two to three experiments performed in duplicate.
Overexpression of p53 and WT1 in HepG2 Cells The next experiments were designed to test whether modulation of p53 function in the HepG2 cells, which contain wild-type p53, would affect the ability of the WT1 isoforms to inhibit promoter activity. We transfected pIRC1 into HepG2 cells with expression vectors for wild-type p53 to boost cellular p53 levels or with a vector for a dominant-negative p53 (R248Q) mutation to inhibit the endogenous p53. Expression of wild-type p53 inhibited basal IR promoter activity by 50% and the dominant-negative R248Q mutant elevated activity by 200%, as we have published previously (34). A titration with increasing amounts of the WT1 expression vectors was performed as above. The /KTS isoform was able to inhibit promoter function in the presence of either wild-type or mutant p53 (Fig. 6A). However, p53 does appear to modulate the response at low levels of /KTS expression, as repression of the IR promoter is more pronounced in the presence of mutant p53 (Fig. 6A; compare effects of p53 at 0.25 and 0.75 mg of WT1 vector). This confirms the results from the HeLa and Saos-2 cells that inhibition by the /KTS isoform does not absolutely require p53 and also suggests that p53 can modulate the magnitude of the response. The response to the 0KTS isoform, however, was markedly different. The 0KTS isoform inhibited the IR promoter in a dose-dependent manner only
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in the presence of the dominant-negative p53 (Fig. 6B). When wild-type p53 is overexpressed, the 0KTS isoform does not repress the IR promoter even at the highest concentration. These results indicate that p53 status has a major impact on the ability of WT1 0KTS to function as a transcriptional repressor and would be consistent with an interaction between this isoform and p53 (35). Coimmunoprecipitation of WT1 and p53 To test whether both isoforms of WT1 could interact with p53, we cotransfected expression vectors for wild-type p53 and either isoform of WT1 into CHO cells. CHO cells were used as they have a very high transfection efficiency. Whole-cell extracts were immunoprecipitated with antibodies to either p53 or WT1. The antibody to WT1 recognizes the C-terminus of the protein that is common to both isoforms. The immunoprecipitates were separated by SDS– PAGE and immunoblotted with antibodies to WT1 or p53, respectively. WT1 was detectable in the p53 immunoprecipitates (Fig. 7, lanes 1 and 4). There was no difference between the /KTS and 0KTS isoforms of WT1. No WT1 was detectable in the 1% Triton wash, indicating that the complexes were stable (Fig. 7, lanes 2 and 5). A small amount of WT1 was detected in the supernatants; however, only 10% of the supernatant was loaded on the gel. Therefore,
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FIG. 6. Effect of p53 on inhibition of the IR promoter by WT1. HepG2 cells were transfected with the IR-CAT reporter plasmid pIRC1 (5 mg) and increasing amounts of expression vector (0–5 mg) for the two KTS isoforms of WT1. Expression vectors for wild-type p53 or a dominant-negative p53 (248Q) (5 mg) were cotransfected along with the b-galactosidase plasmid RSVlacZ (1 mg). pCB6/ was used to normalize the amount of expression vector in each transfection. CAT and b-galactosidase activity were measured on cellular extracts prepared 48 h after transfection. CAT activity is normalized for b-galactosidase activity and then expressed as %max. (A) Inhibition by the /KTS isoform of WT1. (B) Inhibition by the 0KTS isoform of WT1. Results are the mean { SEM of two to three experiments performed in duplicate.
approximately 70% of the total WT1 was associated with p53. The identity of the higher molecular weight material that was also detected with the WT1 antibody was unknown. Immunoblotting WT1 immunoprecipitates with an antibody to p53 produced similar results; p53 was detectable in the WT1 immunoprecipitates (Fig. 7, lanes 7 and 10). The majority of p53, unlike WT1, was found in the supernatants (Fig. 7, lanes 9 and 12), indicating that only a small fraction of p53 was complexed with WT1. No difference was observed between the two KTS isoforms (Fig. 7, lanes 1 and 4 and 7 and 10). Similar experiments were performed using antibodies to C/ EBPb and Sp1; however, no association with p53 was observed (data not shown). The coprecipitation experiment showed that either WT1 isoform could be associated with p53 when both were overexpressed, but did not rule out the possibility that the two isoforms had different affinities for p53 in vivo. DISCUSSION In this paper, we show that the insulin receptor gene promoter is a target for WT1 in vitro. While WT1 has been shown to repress many GC-rich promoters in vitro, the interesting feature of repression of the IR promoter is the differential effect of the two KTS splice variants of WT1 and their sensitivity to p53. Many other promoters have been shown to
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be regulated differentially by the WT1 isoforms. In most of these cases, the promoter contains Egr1 binding sites and the differential effects can be explained by the failure of the /KTS isoform to bind to such sequences (36). However, in those cases
FIG. 7. Coimmunoprecipitation of WT1 and p53. CHO cells were cotransfected with expression vectors for p53 and either of the KTS isoforms of WT1. Whole-cell extracts were prepared 48 h after transfection. Proteins were immunoprecipitated with either a rabbit polyclonal antibody C-19 raised against WT1 or a mouse monoclonal antibody DO-1 against p53 and then immunoblotted with the converse antibody. Pellet, the immunoprecipitate; Wash, a 1% Triton X-100 wash of the pellet; Sup., the remaining supernatant. The wash and supernatant lanes represent 10% of the total volume. (A) Immunoprecipitation with antibody DO-1 against p53 followed by immunoblotting with antibody C19 against WT1. WT1 is indicated by the arrowhead. The origin of the higher molecular weight immunoreactive material is unknown. (B) Immunoprecipitation with antibody C-19 against WT1 followed by immunoblotting with antibody DO-1 against p53. p53 is indicated by the arrowhead.
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where both isoforms repress, the 0KTS isoform has a greater effect than the /KTS isoform. Both the isoforms tested in our experiments contain the exon 5 insertion that has been shown to disrupt the transcriptional activation domain; the two isoforms differ only by the presence of three amino acids (KTS) in the zinc finger domain (23). On the insulin receptor promoter, the WT1 variant containing the /KTS sequence inhibited promoter activity strongly under all conditions tested. The isoform lacking the KTS insert, on the other hand, could inhibit only in the presence of a dominant-negative p53 or when the IR promoter was stimulated by expression of C/EBPb. How can the differential effects of the KTS splice variants be explained? The DNA-binding domains from either isoform when expressed in E. coli can bind to the IR promoter in vitro as assessed by gel mobility shifts and DNaseI footprinting. Interestingly, one of the DNaseI footprints that we observed included direct repeats of a TCCCTCCC sequence. WT1 binding sites containing such repeats have been shown to be functional WT1 response elements in the EGFR promoter (15), an in vivo target for WT1, and similar sequences have been isolated in genomic binding site screens by Nakagama et al. (20) and Bickmore et al. (37). Other sites resembled GC-rich Egr1-like sites similar to those identified in other promoters, including the PDGF-A chain and IGF-II genes (11,12). We were only able to identify a single differential footprint at 0720 in the IR promoter in a region that was important for repression by the 0KTS isoform. This sequence was only recognized by the 0KTS WT1 isoform. However, all of the experiments used DNA-binding domains expressed in E. coli. The presence of threonine and serine residues in the KTS insert, potential protein kinase C phosphorylation sites, raises the intriguing possibility that the insert could be phosphorylated. This might be expected to have a dramatic effect on DNA binding. The isolated DNA-binding domains expressed in E. coli would not, of course, be phosphorylated. It will be interesting to determine whether phosphorylation modulates nuclear localization and DNA-binding activities of WT1 proteins. This may explain the observed differences in nuclear localization of the protein isoforms (38,39). What is the role of p53 in repression by WT1? An interaction of WT1 (0KTS) and p53 has been demonstrated by Maheswaran et al. (35). They were able to show complex formation in both transfected cell lines and primary Wilms tumors that expressed WT1. We have confirmed and extended their findings to show that both KTS isoforms are able to form
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stable complexes with p53 when overexpressed. What is the functional effect of this association? p53 was shown to switch the 0exon5/0KTS isoform of WT1 from a transcriptional activator to a repressor of a minimal promoter containing an Egr1 site (35). Recently it has been shown that WT1 stabilizes p53 and prevents its degradation (40). Both the 0KTS isoform of WT1 and an in-frame deletion mutant, WTAR, were able to stabilize p53. The WT1 domain involved in this stabilization maps to the first two zinc fingers of the DNA-binding domain (40). Thus, the /KTS isoform, which differs in three amino acids between the third and fourth zinc fingers, should also stabilize p53, although it has not been formally tested. Interestingly, only the 0KTS isoform was able to increase p53-mediated transactivation and inhibit p53-dependent transcriptional repression and apoptosis. Although the reason for this difference is not known, one can speculate that the effects that we observe with the two KTS isoforms of WT1 could be related to their differential effects on p53 signaling, particularly as we have shown that p53 can repress the IR promoter (34). ACKNOWLEDGMENTS This work was supported by grants from the NIH (DK44643), the Department of Veterans Affairs, and the American Diabetes Association. N.J.G.W. is a faculty member of the UCSD Biomedical Sciences Graduate Program.
REFERENCES 1. Call KM, Glaser T, Ito CY, Buckler AJ, Pelletier J, Haber DA, Rose EA, Kral A, Yeger H, Lewis WH. Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms’ tumor locus. Cell 60:509–520, 1990. 2. Glaser T, Lane J, Housman D. A mouse model of the aniridia-Wilms tumor deletion syndrome. Science 250:823–827, 1990. 3. Pelletier J, Bruening W, Kashtan CE, Mauer SM, Manivel JC, Striegel JE, Houghton DC, Junien C, Habib R, Fouser L. Germline mutations in the Wilms’ tumor suppressor gene are associated with abnormal urogenital development in Denys–Drash syndrome. Cell 67:437–447, 1991. 4. Sharma PM, Yang X, Bowman M, Roberts V, Sukumar S. Molecular cloning of rat Wilms’ tumor complementary DNA and a study of messenger RNA expression in the urogenital system and the brain. Cancer Res 52:6407–6412, 1992. 5. Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, Housman D, Jaenisch R. WT-1 is required for early kidney development. Cell 74:679–691, 1993. 6. Englert C, Hou X, Maheswaran S, Bennett P, Ngwu C, Re
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INHIBITION OF THE IR PROMOTER BY WT1 GG, Garvin AJ, Rosner MR, Haber DA. WT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis. EMBO J 14:4662–4675, 1995. 7. Madden SL, Cook DM, Morris JF, Gashler A, Sukhatme VP, Rauscher FJ, III. Transcriptional repression mediated by the WT1 Wilms tumor gene product. Science 253:1550– 1553, 1991. 8. Dey BR, Sukhatme VP, Roberts AB, Sporn MB, Rauscher FJ, Kim SJ. Repression of the transforming growth factorbeta 1 gene by the Wilms’ tumor suppressor WT1 gene product. Mol Endocrinol 8:595–602, 1994. 9. Harrington MA, Konicek B, Song A, Xia XL, Fredericks WJ, Rauscher FJ. Inhibition of colony-stimulating factor-1 promoter activity by the product of the Wilms’ tumor locus. J Biol Chem 268:21271–21275, 1993. 10. Werner H, Re GG, Drummond IA, Sukhatme VP, Rauscher FJ, Sens DA, Garvin AJ, LeRoith D, Roberts CTJ. Increased expression of the insulin-like growth factor I receptor gene, IGFIR, in Wilm’s tumor is correlated with modulation of IGFIR promoter activity by the WT1 Wilm’s tumor gene product. Proc Natl Acad Sci USA 90:5828–5832, 1993. 11. Gashler AL, Bonthron DT, Madden SL, Rauscher FJ, Collins T, Sukhatme VP. Human platelet-derived growth factor A chain is transcriptionally repressed by the Wilms tumor suppressor WT1. Proc Natl Acad Sci USA 89:10984–10988, 1992. 12. Drummond IA, Madden SL, Rohwer-Nutter P, Bell GI, Sukhatme VP, Rauscher FJ. Repression of the insulin-like growth factor II gene by the Wilms tumor suppressor WT1. Science 257:674–678, 1992. 13. Goodyer P, Dehbi M, Torban E, Bruening W, Pelletier J. Repression of the retinoic acid receptor-alpha gene by the Wilms’ tumor suppressor gene product, wt1. Oncogene 10:1125–1129, 1995. 14. Sharma PM, Bowman M, Yu BF, Sukumar S. A rodent model for Wilms tumors: Embryonal kidney neoplasms induced by N-nitroso-N*-methylurea. Proc Natl Acad Sci USA 91:9931– 9935, 1994. 15. Werner H, Shen-Orr Z, Rauscher FJ, Morris JF, Roberts CTJ, LeRoith D. Inhibition of cellular proliferation by the Wilms’ tumor suppressor WT1 is associated with suppression of insulin-like growth factor I receptor gene expression. Mol Cell Biol 15:3516–3522, 1995. 16. Haber DA, Sohn RL, Buckler AJ, Pelletier J, Call KM, Housman DE. Alternative splicing and genomic structure of the Wilms tumor gene WT1. Proc Natl Acad Sci USA 88:9618– 9622, 1991. 17. Sharma PM, Bowman M, Madden SL, Rauscher FJ, Sukumar S. RNA editing in the Wilms’ tumor susceptibility gene, WT1. Genes Dev 8:720–731, 1994. 18. Rauscher FJ, Morris JF, Tournay OE, Cook DM, Curran T. Binding of the Wilms’ tumor locus zinc finger protein to the EGR-1 consensus sequence. Science 250:1259–1262, 1990. 19. Drummond IA, Rupprecht HD, Rohwer-Nutter P, Lopez-Guisa JM, Madden SL, Rauscher FJ, Sukhatme VP. DNA recognition by splicing variants of the Wilms’ tumor suppressor, WT1. Mol Cell Biol 14:3800–3809, 1994. 20. Nakagama H, Heinrich G, Pelletier J, Housman DE. Se-
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quence and structural requirements for high-affinity DNA binding by the WT1 gene product. Mol Cell Biol 15:1489– 1498, 1995. 21. Hamilton TB, Barilla KC, Romaniuk PJ. High affinity binding sites for the Wilms’ tumour suppressor protein WT1. Nucleic Acids Res 23:277–284, 1995. 22. Madden SL, Cook DM, Rauscher FJ. A structure-function analysis of transcriptional repression mediated by the WT1, Wilms’ tumor suppressor protein. Oncogene 8:1713–1720, 1993. 23. Wang ZY, Qiu QQ, Huang J, Gurrieri M, Deuel TF. Products of alternatively spliced transcripts of the Wilms’ tumor suppressor gene, wt1, have altered DNA binding specificity and regulate transcription in different ways. Oncogene 10:415– 422, 1995. 24. Wang ZY, Qiu QQ, Gurrieri M, Huang J, Deuel TF. WT1, the Wilms’ tumor suppressor gene product, represses transcription through an interactive nuclear protein. Oncogene 10:1243–1247, 1995. 25. Liu ZZ, Kumar A, Ota K, Wallner EI, Kanwar YS. Developmental regulation and the role of insulin and insulin receptor in metanephrogenesis. Proc Natl Acad Sci USA 94:6758– 6763, 1997. 26. McKeon C, Moncada V, Pham T, Salvatore P, Kadowaki T, Accili D, Taylor SI. Structural and functional analysis of the insulin receptor promoter. Mol Endocrinol 4:647–656, 1990. 27. Mamula PW, Wong KY, Maddux BA, McDonald AR, Goldfine ID. Sequence and analysis of promoter region of human insulin-receptor gene. Diabetes 37:1241–1246, 1988. 28. Tewari DS, Cook DM, Taub R. Characterization of the promoter region and 3* end of the human insulin receptor gene. J Biol Chem 264:16238–16245, 1989. 29. Cameron KE, Resnik J, Webster NJ. Transcriptional regulation of the human insulin receptor promoter. J Biol Chem 267:17375–17383, 1992. 30. Levy JR, Hug V. Nuclear protein-binding analysis of a GCrich insulin-receptor promoter regulatory region. Diabetes 42:66–73, 1993. 31. Lee JK, Tam JW, Tsai MJ, Tsai SY. Identification of cis- and trans-acting factors regulating the expression of the human insulin receptor gene. J Biol Chem 267:4638–4645, 1992. 32. Webster NJG, Kong Y, Cameron KE, Resnik JL. An upstream element from the human insulin receptor gene promoter contains binding sites for C/EBPb and NF-1. Diabetes 43:305–312, 1994. 33. McKeon C, Pham T. Transactivation of the human insulin receptor gene by the CAAT/enhancer binding protein. Biochem Biophys Res Commun 174:721–728, 1991. 34. Webster NJG, Resnik JL, Reichart DB, Strauss B, Haas M, Seely BL. Repression of the insulin receptor promoter by the tumor suppressor gene product p53: A possible mechanism for receptor overexpression in breast cancer. Cancer Res 56:2781–2788, 1996. 35. Maheswaran S, Park S, Bernard A, Morris JF, Rauscher FJ, Hill DE, Haber DA. Physical and functional interaction between WT1 and p53 proteins. Proc Natl Acad Sci USA 90:5100–5104, 1993. 36. Drummond IA, Rupprecht HD, Rohwer-Nutter P, Lopez-Gu-
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37. Bickmore WA, Oghene K, Little MH, Seawright A, Van Heyningen V, Hastie ND. Modulation of DNA binding specificity by alternative splicing of the Wilms tumor wt1 gene transcript. Science 257:235–237, 1992. 38. Englert C, Vidal M, Maheswaran S, Ge Y, Ezzell RM, Isselbacher KJ, Haber DA. Truncated WT1 mutants alter the
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subnuclear localization of the wild-type protein. Proc Natl Acad Sci USA 92:11960–11964, 1995. 39. Larsson SH, Charlieu JP, Miyagawa K, Engelkamp D, Rassoulzadegan M, Ross A, Cuzin F, Van Heyningen V, Hastie ND. Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing. Cell 81:391–401, 1995. 40. Maheswaran S, Englert C, Bennett P, Heinrich G, Haber DA. The WT1 gene product stabilizes p53 and inhibits p53mediated apoptosis. Genes Dev 9:2143–2156, 1995.
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Differential Effects of Wilms Tumor WT1 Splice Variants on the Insulin Receptor Promoter Nicholas J. G. Webster,1 Yan Kong, Prem Sharma, Martin Haas, Saraswati Sukumar,* and B. Lynn Seely UCSD/Whittier Diabetes Program and the UCSD Cancer Center, University of California, San Diego, La Jolla, California 92093, and Medical Research Service, Department of Veterans Affairs Medical Center, San Diego, California 92161; and *Breast Cancer Program, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Received August 29, 1997, and in revised form September 25, 1997
locus implicated in the development of the pediatric nephroblastoma Wilms tumor on chromosome 11p13 (1). WT1 mutations have been found in the WAGR syndrome (Wilms tumor, aniridia, genitourinary abnormalities, and retardation) and the Denys – Drash syndrome (genitourinary abnormalities) in humans (2,3). During embryonic development, WT1 transcripts are present in a number of regions including the urogenital system, spinal cord, brain, and mesothelial lining of organs within the thoracic cavity (4). Mice deficient for WT1 die in utero from abnormal development of the kidney, gonads, heart, lung, and mesothelium, demonstrating an important role for WT1 in embryogenesis (5). In the kidney on embryonic day 11, the ureteric bud fails to grow out of the Wolffian duct toward the blastemal cells. The metanephric blastema shows many apoptotic cells in the null embryos, and no mesenchymal cells are detectable by day 12. In vitro organ culture experiments demonstrated that the blastemal cells are unable to respond to inducers of tubule formation (5). In the wild-type embryos, WT1 is detectable in the podocyte layer of the glomeruli but not the blastemal mesenchyme or tubules by immunohistochemistry (6). The WT1 gene encodes a transcription factor containing four putative zinc fingers that are closely related to the Egr1 family, but unlike Egr1, WT1 inhibits transcription (7). A number of cellular targets for WT1 have been identified in vitro, including the PDGF-A chain, IGF-II, the IGF-I receptor, CSFI,
The Wilms tumor gene WT1 has been implicated in the early development of the kidney. Mutations in WT1 are found in a small fraction of Wilms tumor, a pediatric nephroblastoma, and Denys–Drash syndrome, characterized by genitourinary abnormalities. The WT1 gene product functions as a transcriptional repressor of growth factor-related genes. The kidney is one of the major sites of insulin action in vivo and expresses high levels of insulin receptors (IR). IR expression has been detected during early embryogenesis, suggesting that it may play a role in development. We investigated whether two WT1 splice variants lacking or including a three-aminoacid (KTS) insertion between the third and fourth zinc finger in the DNA-binding domain could repress the IR promoter in vitro. We show that the /KTS variant effectively represses promoter activity under all conditions tested but the 0KTS variant was only able to repress in the presence of cotransfected C/EBPb or a dominant-negative p53 mutation. Deletional mapping indicated that distinct regions of the IR promoter mediated the effects of the two isoforms and DNaseI footprint analysis identified potential WT1 binding sites within these regions. q 1997 Academic Press
Insights into the development of the kidney have arisen from the cloning of the WT1 gene from a 1 To whom correspondence should be addressed at Department of Medicine (0673), University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0673. Fax: (619) 534-7181. E-mail: [email protected].
139 1077-3150/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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RARa, and TGF-b (8–13). However, few in vivo targets for WT1 have been identified. IGF-II and IGFIR are overexpressed in tumors containing mutations in WT1 (10,14) and, recently, Werner et al. demonstrated that IGF-IR expression and IGF-Istimulated mitogenesis could be inhibited in G401 cells engineered to express WT1 (15). This result underscores the importance of the insulin-like growth factors in kidney development and tumorigenesis. The EGFR is a second in vivo target for WT1, and WT1 induces apoptosis in cells by inhibiting EGFR expression (6). The gene also has two major sites of alternative splicing. The first is a 17amino-acid insertion at position 248 that is due to the inclusion of exon 5. The second is a 3-amino-acid (KTS) insertion at position 411 in the DNA-binding domain arising from the use of an alternative 3* splice site (16). The WT1 gene is also susceptible to RNA editing, resulting in a substitution of proline for leucine at position 280 (17). Due to the similarity between the DNA-binding domains of WT1 and Egr1, WT1 can bind to Egr1 sites (GCGGGGGCG) (18). The KTS insertion lies between the third and fourth zinc fingers in the DNA-binding domain and alters binding specificity. The 0KTS isoform of WT1 can recognize an Egr1 binding site, whereas the /KTS isoform does not (19). Recent studies have shown that both isoforms have a more relaxed binding specificity than the related Egr1 protein, presumably due to the presence of four zinc fingers rather than three (20,21). Other functional domains have been localized by deletional analysis. The domain involved in transcriptional repression has been mapped to residues 84–179, whereas a transcriptional activation domain has been mapped to residues 180–296 (22). Furthermore, the 17-amino-acid alternatively spliced exon 5 can function as an independent repressor module (23). Recent results have indicated that repression by WT1 is mediated by protein–protein interactions involving the repression domain and an accessory protein (24). The kidney, along with liver, muscle, and adipose tissue, is a major site of insulin action in vivo. One of insulin’s physiological roles in the kidney is to stimulate sodium readsorption. The kidney is also a major site of insulin metabolism, primarily in the proximal tubular epithelial cells, which express high levels of insulin receptors. In the developing mouse kidney on embryonic day 13, insulin receptor expression is detected in the plasmalemma cells of the ureteric bud and precursor cells of the rudimentary
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nephron including the condensing blastemal cells and S-shaped bodies (25). At later stages of development, IR expression is also detected in the tubular epithelial cells and after birth is restricted to only these tubular epithelial cells. The expression pattern of the IR is similar to that of the EGFR, which is known to be regulated by WT1 (15). Two pieces of evidence suggest a role for insulin receptors in kidney development (25). First, blockade of insulin receptors in vitro causes retardation of nephrogenesis and disruption of ureteric bud branching. Second, insulin treatment causes both a hypertrophy and hyperplasia of metanephrogenic cells. The expression of the IR in Wilms tumor, however, has not been investigated. These observations led us to investigate whether WT1 could modulate the IR promoter. The insulin receptor gene is unusual in that it has the characteristics of both a housekeeping and a tissue-specific gene. The IR gene promoter lacks a TATA box or initiator sequence, and the primary sequence is very GC rich (26–28). Basal promoter activity is mediated by the transcription factor Sp1 (29–31). The tissue-specific regulation of the IR gene is not well understood but transcription factors of the C/EBP family can stimulate the promoter in adipose and liver cells (32,33). We have also found that the promoter is under negative control and contains a tissue-specific silencer between 01 and 01.2 kb upstream of the translational start site (29). Computer analysis of the sequence of the GC-rich region of the promoter indicated the presence of multiple Egr1 sites, indicating that, potentially, the promoter could be regulated by WT1. In this paper, we demonstrate that the two KTS splice variants of WT1 have differential effects on the IR promoter. EXPERIMENTAL PROCEDURES Construction of Plasmids All plasmid constructions were performed using standard techniques. The construction of pIRC1 to 8 and the expression vector for C/EBPb have been described previously (29,32). Expression vectors encoding wild-type p53 and a mutant p53 (248Q) have been described previously (34). The parental expression vector pRcCMV was from Invitrogen (San Diego, CA). cDNAs encoding the two rat WT1 isoforms (/KTS and 0KTS) were subcloned into expression vector CB6/ (17). Both isoforms contained
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exon 5 and leucine at amino acid 280. Expression vectors for the DNA-binding domains of the two splice variants were constructed by amplifying the two C-terminal zinc finger domains (amino acids 312–448) by PCR and subcloning into expression vector CB6/. The 5* primer was designed to include a consensus Kozak sequence to ensure efficient translation. The Escherichia coli expression vectors pGEX / KTS and pGEX 0 KTS were constructed by amplifying the WT1 DNA-binding domains and subcloning into pGEX4T-1. This vector allows for the production of a glutathione-S-transferase (GST) fusion protein under control of the tac promoter (Pharmacia, Piscataway, NJ). Cultures were induced and the GST fusion protein purified following the manufacturer’s protocol. Cotransfections and CAT Assays HepG2 and HeLa cells were maintained in minimum essential medium plus Earle’s salts with 10% fetal calf serum (MEM-Earle’s). HepG2 cells were transfected using the calcium phosphate coprecipitation technique. The precipitate was removed after 8 h, and then the cells were subjected to a glycerol shock (10% glycerol in MEM-Earle’s) for 3 min at 377C. Saos-2 cells were maintained in McCoy 5a medium with 15% fetal calf serum. HeLa and Saos2 cells were transfected using Lipofectamine (BRL, Gaithersburg, MD) following the manufacturer’s protocol. In all cases the reference plasmid RSVlacZ, which contains the b-galactosidase gene under the control of the Rous sarcoma virus long terminal repeat, was cotransfected to correct for transfection efficiency. Cells were harvested 48 h after transfection, lysed in 250 ml of modified lysis buffer by three cycles of freeze–thaw, and centrifuged at 10,000g for 15 min at 47C, and the supernatant was stored at 0807C. The b-galactosidase activity and CAT activity were measured as described previously (32). Gel Shift and DNaseI Footprinting Assays GST fusion proteins containing the zinc finger DNA-binding domains of the WT1 isoforms were expressed in E. coli using the pGEX system. Cultures were induced with isopropyl-thio-galactopyranoside (IPTG) overnight. Fusion proteins were isolated from bacterial lysates by absorbing to glutathione– agarose beads followed by elution with glutathione following the manufacturer’s protocol. DNA probes were end-labeled with T4 polynucleotide kinase and
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50 mCi [g-32P]ATP and purified by gel electrophoresis. For the gel-shift assays, increasing amounts (0– 10 mg) of GST–WT1 fusion protein was preincubated with 5 mg poly(dI.C) and 0.1 mg/ml BSA in 20 ml 0.51 TGMEDK buffer (25 mM Tris–HCl, pH 7.9, 10% glycerol, 5 mM MgCl2 , 0.1 mM EDTA, 1 mM dithiothreitol, 100 mM KCl, 0.15 mM ZnCl2) for 30 min at 07C. The 32P-labeled probe (20,000 cpm) was added and the incubation continued for a further 15 min at room temperature. Free and bound probe were separated on a nondenaturing 5% polyacrylamide gel at 15 mA at room temperature in 0.251 TBE with recirculation of the buffer. Gels were dried and subjected to autoradiography. In competition experiments, unlabeled double-stranded oligonucleotides (10–100 pmol) were included in the incubation. DNaseI footprinting assays were performed as described previously (29). Immunoprecipitation of WT1 and p53 Chinese hamster ovary cells (CHO) were maintained in Ham’s F12 medium with 10% fetal calf serum. Cells were transfected with expression vectors (10 mg) for wild-type p53 and either the / or 0KTS isoform of WT1 using lipofectamine following the manufacturer’s protocol. Cells were harvested 48 h after transfection and lysed by three strokes of a Dounce homogenizer (tight pestle) in homogenization buffer (0.25 M sucrose, 15 mM Tris–HCl, pH 7.9, 15 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 60 mM KCl, 0.5% NP-40, 1 mM dithiothreitol) on ice in the presence of a cocktail of protease inhibitors (Complete, Boehringer Mannheim, Indianapolis, IN). The lysate was split into two fractions. A polyclonal antibody to WT1 (C-19) was added to one fraction and a monoclonal antibody to p53 (DO-1) was added to the other fraction to a concentration of 1 mg/ml; both antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA). Proteins were immunoprecipitated overnight at 47C on a rocking platform. A protein A–agarose conjugate was added and the incubation was continued for a further 2 h. Immune complexes were pelleted by centrifugation at 15,000g for 15 min at 47C. The supernatant was saved and the pellets washed three times in EBG (120 mM NaCl, 5 mM KCl, 1 mM MgSO4 , 1 mM CaCl2 , 25 mM Hepes, 10% glycerol, 0.05% Triton X-100). Finally the pellets were washed in 100 ml EBG containing 1% Triton X-100. The immunoprecipitated proteins were resuspended in
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sample buffer and boiled for 5 min. An aliquot (10%) of the initial supernatant and the 1% Triton X-100 wash were also boiled with sample buffer for 5 min. The proteins in the pellet, wash, and supernatant fractions were separated by 10% SDS–PAGE. Proteins were electrotransferred to Immobilon filters (Millipore, Bedford MA) overnight at 12 V in 25 mM Tris, 192 mM glycine, pH 8.3, at 47C. The filters were blocked with 5% nonfat dried milk in T-TBS (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) for 60 min at 257C. The polyclonal antibody to WT1 (C19) was added to the filter containing the p53 immunoprecipitates, and the monoclonal antibody to p53 (DO-1) was added to the filter containing the WT1 immunoprecipitates. Both antibodies were diluted 1:1000 in T-TBS. Filters were incubated for 2 h at 257C on a rocking platform. Filters were rinsed three times in T-TBS and then incubated for 60 min at 257C with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies, respectively. Proteins were detected by enhanced chemiluminescence (ECL, Amersham). RESULTS Inhibition of the hIR Promoter by WT1 To determine if the hIR promoter can be regulated by the Wilms’ tumor gene product WT1, we cotransfected the full-length insulin receptor promoter– CAT construction pIRC1 into HepG2 cells with increasing amounts of expression vectors for two splice variants of WT1. The HepG2 cell is derived from a human hepatoblastoma and has been useful as a model for studying insulin signaling. These cells express 25,000 insulin receptors per cell, do not express WT1, and have a wild-type p53 gene. The encoded cDNAs differ in the presence of three amino acids (KTS) in the third zinc finger of the WT1 DNAbinding domain (DBD). Both vectors contained fulllength WT1 including the 17-amino-acid exon 5 that encodes a repression domain (23). The parental expression vector CB6/ was included to normalize the amount of DNA in all transfections to minimize nonspecific effects. The RSV–lacZ plasmid was included to correct for transfection efficiency. A dose-dependent decrease in CAT activity was observed for both splice variants (Fig. 1A). The /KTS splice variant appeared to be a more potent inhibitor of the IR promoter, giving a 60% reduction in promoter activity at a reporter to expression vector ratio of 5:2 and
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increasing to 70% repression at a ratio of 1:1. This is similar to the reported 80% inhibition of IGF-IR promoter activity and 90% inhibition of the EGFR promoter (6,10). Both the IGF-IR and EGFR have been shown to be in vivo targets for WT1. It should be noted that a large excess of WT1 expression vector was required to obtain these inhibitions (20-fold and 5-fold excess for IGF-IR and EGFR, respectively). In the experiments reported here, the maximum amount of WT1 vector corresponded to a reporter:vector ratio of 1:1. This avoids nonspecific effects due to the vast overexpression of WT1 in the other studies. The 0KTS isoform only repressed promoter activity significantly (40%) at the highest amount of transfected vector (promoter:vector ratio of 1:1). As a control, the related transcription factor Egr1 had no effect on IR promoter activity (data not shown). To assess whether WT1 could repress the stimulated as well as the basal IR promoter, we studied the promoter in the presence of C/EBPb, a transcription factor known to stimulate the IR promoter (32,33). A titration was performed as above, but with the inclusion of an expression vector for C/EBPb. The absolute CAT activity in the absence of WT1 was increased 7- to 10-fold, as we have published previously (32). Both WT1 splice variants caused a similar dose-dependent decrease in CAT activity (Fig. 1B). Interestingly, the 0KTS variant was as effective in inhibiting C/EBP-stimulated IR promoter activity as the /KTS variant. Expression of the individual zinc finger DNA-binding domains of the two WT1 isoforms had no effect on IR promoter activity, suggesting that repression is not the result of competition for promoter binding sites (data not shown). Deletion Analysis of the IR Promoter To determine if the two isoforms recognize distinct binding sites, we attempted to localize the regions of the promoter required for repression by WT1. The effect of WT1 on a series of deletion mutants of the IR promoter was assessed to identify the approximate locations of the binding sites. Expression vectors for the / and 0KTS isoforms were transfected into HepG2 cells with a series of 5* deletions of the IR promoter at a promoter:vector ratio of 1:1 (Fig. 2). The deletions are numbered relative to the translational start site. The full-length promoter contains sequences from 01.8 to 0290 kb. Deletion of se-
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FIG. 1. Effect of WT1 on the IR promoter. HepG2 cells were transfected with the IR-CAT reporter plasmid pIRC1 (5 mg) and increasing amounts of expression vector (0–5 mg) for the two KTS isoforms of WT1.pCB6/ was used to normalize the amount of expression vector in each transfection. Transfection efficiency was monitored by cotransfecting the b-galactosidase plasmid RSVlacZ (1 mg). CAT and b-galactosidase activity were measured on cellular extracts prepared 48 h after transfection. CAT activity was normalized for b-galactosidase activity and then expressed as %max. (A) Repression of CAT activity by the two isoforms of WT1 in parental HepG2 cells. (B) Repression of CAT activity in the presence of an expression vector for C/EBPb. HepG2 cells were transfected as above except that an expression vector for C/EBPb (5 mg) was cotransfected. Basal CAT activity was elevated 7- to 10-fold. Results are the mean { SEM of two to three experiments performed in duplicate.
quences upstream of 0877 causes a tonic depression of promoter activity, as we have shown previously (29). The 0877 promoter deletion is very effectively repressed by both WT1 isoforms (84% by /KTS, 67% by 0KTS). The baseline promoter is decreased by
deletion to 0746 but is still repressed by both WT1 isoforms. Removal of a further 100 nucleotides to 0646 eliminates the inhibition by the 0KTS isoform, but not by the /KTS isoform. This suggests that there are binding sites for the 0KTS isoform be-
FIG. 2. Effect of WT1 on a series of 5* deletion mutants of the IR promoter. HepG2 cells were transfected with a series of 5* deletion mutants of pIRC1 (5 mg) with expression vectors for either WT1 isoform or the parental vector pCB6/ (5 mg). The parental reporter plasmid contained the IR promoter from 01.8 to 0290 kb relative to the translational start site. The 5* end of the promoter deletion is indicated on the x axis. RSV–lacZ was cotransfected to correct for transfection efficiency. CAT and b-galactosidase activity were measured on cellular extracts made 48 h after transfection. CAT activity is normalized for b-galactosidase activity and expressed as %max. Results are the mean { SEM of three experiments performed in duplicate.
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FIG. 3. Gel-shift analysis of region 0877 to 0487 of the IR promoter. Probes were generated by digestion of the IR promoter, followed by dephosphorylation and then labeling of the free 5*OH with [g-32P]ATP and T4 polynucleotide kinase. Probes were purified by electrophoresis on 5% polyacrylamide gels. Reactions contained increasing amounts of GST–DBD fusion proteins in the presence of poly(dI.C) (5 mg) and BSA (0.1 mg/ml). Free and bound probes were separated by electrophoresis of 5% polyacrylamide gels. (Left) A probe covering 0877 to 0646 containing the region implicated in repression by the 0KTS isoform. (Right) A probe covering 0645 to 0487 containing the region implicated in repression by the /KTS isoform.
tween 0646 and 0746 that are required for inhibition. It does not rule out the possibility of sites more distal or proximal in the promoter but such sites are not sufficient for repression. Removal of a further 79 nucleotides to 0577 eliminates most promoter activity, as we have shown previously, and also eliminates the inhibition by the /KTS isoform of WT1. The region between 0590 and 0640 is very GC-rich and has been shown to contain multiple functional sites for transcription factor Sp1 as well as predicted Egr1 sites (29–31). Thus, the deletional analysis indicates that sequences between 0746 and 0646 or 0646 and 0577 are important for repression by the 0KTS and /KTS WT1 isoforms, respectively. Identification of in Vitro WT1 Binding Sites on the IR Promoter The deletional analysis indicated that the promoter region between 0746 and 0577 appears to be important for repression by WT1 in vivo. The next experiments were designed to identify WT1 binding sites on fragments of the IR promoter in vitro. GST fusion proteins containing the DNA-binding domains from either WT1 isoform were expressed in E. coli and purified on glutathione–agarose beads. Promoter fragments end labeled with 32P were incubated with increasing amounts of fusion protein, then free and bound probe were separated on a nondenaturing polyacrylamide gel. Probes covering 0877 to 0646 and 0645 to 0486 gave multiple strong retarded complexes with both WT1 isoforms (Fig. 3).
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To analyze the actual sequences in the IR promoter that are recognized by the WT1 isoforms, we footprinted the region covering 0486 to 0744 of the promoter using the recombinant GST fusion proteins and DNaseI. A representative footprint is shown in Fig. 4A, and the results from a number of experiments using both strands are summarized in Fig. 4B. A GC-rich sequence between 0600 and 0620 was protected from digestion along with another at 0680. These sites resemble the GC-rich sequences that have been identified as WT1 binding sites in the PDGF-A promoter (11). A further binding site between 0590 and 0570 contains a direct repeat similar to the CTCCCTCCTCCTC motif in the A and B boxes defined as functional WT1 response elements in the EGFR promoter. The only differential footprint that was observed was at 0720. This region was protected by the 0KTS fusion protein only. Interestingly, this site does not conform to a GC-rich Egr1-like motif or TC box. Furthermore, this site lies between the 0746 and 0646 promoter deletions that are important for repression by the 0KTS isoform in vivo. Role of p53 in the Repression of the IR Promoter by WT1 Previous studies have suggested that p53 may be involved in the repression by WT1 (35). Furthermore, we have published that the IR promoter is repressed by p53 (34). To investigate whether p53 is required for repression of the IR promoter by WT1, we determined whether WT1 could repress the IR
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FIG. 4. DNaseI footprinting analysis of the IR promoter. Overlapping probes covering 0744 to 0575 were generated by digestion, dephosphorylation, and end labeling with [g-32P]ATP and T4 polynucleotide kinase. Probes were purified by electrophoresis on 5% polyacrylamide gels. DNaseI footprinting analysis was performed as published previously. Both strands were footprinted. A representative autoradiogram of the anti-sense strand between 0590 and 0744 is shown (A). Results from three experiments on both strands are summarized in the diagram (B). Protected regions are indicated by lines above or below the sequence for the sense and anti-sense strands, respectively. Dots indicate hypersensitive sites. The differential footprint at 0720 is indicated by the open boxes. The dashed lines represent potential WT1 binding sites that conform to the GC-rich Egr-like motif. The two arrows indicate direct repeats of the TCCCTCCC motif that have been identified as functional WT1 response elements in the EGFR promoter.
promoter in cells that do not express p53. The cells that we chose to use are HeLa cells, which contain very low levels of p53 due to destabilization by the HPV E6 protein, and Saos-2 cells, which have chromosomal deletions of the p53 gene. Reporter plasmid pIRC1 was transfected into these cells with increasing amounts of expression vectors for the two WT1 isoforms as above. As expected, the basal promoter activity was much lower in both HeLa and Saos-2 cells, which contain very few insulin receptors (õ3000/cell), than in HepG2 cells, which contain 25,000 receptors per cell (29). The WT (/KTS) isoform was able to repress promoter activity in all three cell lines in a similar manner (Fig. 5A). However, the 0KTS isoform was unable to repress in the HeLa and Saos-2 cells (Fig. 5B). These results demonstrate that (a) the WT1 (/KTS) isoform can inhibit transcription in a p53-independent manner and (b) WT1 (0KTS) does not repress in the absence of p53, confirming the results of Maheswaran et al. (35). It should be noted that the repressive effects of WT1 are weaker in these cell lines due to the lower activity of the IR promoter in fibroblast cells (29).
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FIG. 5. Effect of WT1 on the IR promoter in cells lacking p53. HepG2, HeLa, and Saos-2 cells were transfected with the IR-CAT reporter plasmid pIRC1 (5 mg) and increasing amounts of expression vector (0–5 mg) for the two KTS isoforms of WT1.pCB6/ was used to normalize the amount of expression vector in each transfection. Transfection efficiency was monitored by cotransfecting the bgalactosidase plasmid RSVlacZ (1 mg). CAT and b-galactosidase activity were measured on cellular extracts prepared 48 h after transfection. CAT activity is normalized for b-galactosidase activity and then expressed as %max. (A) Inhibition by the /KTS isoform of WT1. (B) Inhibition by the 0KTS isoform of WT1. Results are the mean { SEM of two to three experiments performed in duplicate.
Overexpression of p53 and WT1 in HepG2 Cells The next experiments were designed to test whether modulation of p53 function in the HepG2 cells, which contain wild-type p53, would affect the ability of the WT1 isoforms to inhibit promoter activity. We transfected pIRC1 into HepG2 cells with expression vectors for wild-type p53 to boost cellular p53 levels or with a vector for a dominant-negative p53 (R248Q) mutation to inhibit the endogenous p53. Expression of wild-type p53 inhibited basal IR promoter activity by 50% and the dominant-negative R248Q mutant elevated activity by 200%, as we have published previously (34). A titration with increasing amounts of the WT1 expression vectors was performed as above. The /KTS isoform was able to inhibit promoter function in the presence of either wild-type or mutant p53 (Fig. 6A). However, p53 does appear to modulate the response at low levels of /KTS expression, as repression of the IR promoter is more pronounced in the presence of mutant p53 (Fig. 6A; compare effects of p53 at 0.25 and 0.75 mg of WT1 vector). This confirms the results from the HeLa and Saos-2 cells that inhibition by the /KTS isoform does not absolutely require p53 and also suggests that p53 can modulate the magnitude of the response. The response to the 0KTS isoform, however, was markedly different. The 0KTS isoform inhibited the IR promoter in a dose-dependent manner only
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in the presence of the dominant-negative p53 (Fig. 6B). When wild-type p53 is overexpressed, the 0KTS isoform does not repress the IR promoter even at the highest concentration. These results indicate that p53 status has a major impact on the ability of WT1 0KTS to function as a transcriptional repressor and would be consistent with an interaction between this isoform and p53 (35). Coimmunoprecipitation of WT1 and p53 To test whether both isoforms of WT1 could interact with p53, we cotransfected expression vectors for wild-type p53 and either isoform of WT1 into CHO cells. CHO cells were used as they have a very high transfection efficiency. Whole-cell extracts were immunoprecipitated with antibodies to either p53 or WT1. The antibody to WT1 recognizes the C-terminus of the protein that is common to both isoforms. The immunoprecipitates were separated by SDS– PAGE and immunoblotted with antibodies to WT1 or p53, respectively. WT1 was detectable in the p53 immunoprecipitates (Fig. 7, lanes 1 and 4). There was no difference between the /KTS and 0KTS isoforms of WT1. No WT1 was detectable in the 1% Triton wash, indicating that the complexes were stable (Fig. 7, lanes 2 and 5). A small amount of WT1 was detected in the supernatants; however, only 10% of the supernatant was loaded on the gel. Therefore,
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FIG. 6. Effect of p53 on inhibition of the IR promoter by WT1. HepG2 cells were transfected with the IR-CAT reporter plasmid pIRC1 (5 mg) and increasing amounts of expression vector (0–5 mg) for the two KTS isoforms of WT1. Expression vectors for wild-type p53 or a dominant-negative p53 (248Q) (5 mg) were cotransfected along with the b-galactosidase plasmid RSVlacZ (1 mg). pCB6/ was used to normalize the amount of expression vector in each transfection. CAT and b-galactosidase activity were measured on cellular extracts prepared 48 h after transfection. CAT activity is normalized for b-galactosidase activity and then expressed as %max. (A) Inhibition by the /KTS isoform of WT1. (B) Inhibition by the 0KTS isoform of WT1. Results are the mean { SEM of two to three experiments performed in duplicate.
approximately 70% of the total WT1 was associated with p53. The identity of the higher molecular weight material that was also detected with the WT1 antibody was unknown. Immunoblotting WT1 immunoprecipitates with an antibody to p53 produced similar results; p53 was detectable in the WT1 immunoprecipitates (Fig. 7, lanes 7 and 10). The majority of p53, unlike WT1, was found in the supernatants (Fig. 7, lanes 9 and 12), indicating that only a small fraction of p53 was complexed with WT1. No difference was observed between the two KTS isoforms (Fig. 7, lanes 1 and 4 and 7 and 10). Similar experiments were performed using antibodies to C/ EBPb and Sp1; however, no association with p53 was observed (data not shown). The coprecipitation experiment showed that either WT1 isoform could be associated with p53 when both were overexpressed, but did not rule out the possibility that the two isoforms had different affinities for p53 in vivo. DISCUSSION In this paper, we show that the insulin receptor gene promoter is a target for WT1 in vitro. While WT1 has been shown to repress many GC-rich promoters in vitro, the interesting feature of repression of the IR promoter is the differential effect of the two KTS splice variants of WT1 and their sensitivity to p53. Many other promoters have been shown to
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be regulated differentially by the WT1 isoforms. In most of these cases, the promoter contains Egr1 binding sites and the differential effects can be explained by the failure of the /KTS isoform to bind to such sequences (36). However, in those cases
FIG. 7. Coimmunoprecipitation of WT1 and p53. CHO cells were cotransfected with expression vectors for p53 and either of the KTS isoforms of WT1. Whole-cell extracts were prepared 48 h after transfection. Proteins were immunoprecipitated with either a rabbit polyclonal antibody C-19 raised against WT1 or a mouse monoclonal antibody DO-1 against p53 and then immunoblotted with the converse antibody. Pellet, the immunoprecipitate; Wash, a 1% Triton X-100 wash of the pellet; Sup., the remaining supernatant. The wash and supernatant lanes represent 10% of the total volume. (A) Immunoprecipitation with antibody DO-1 against p53 followed by immunoblotting with antibody C19 against WT1. WT1 is indicated by the arrowhead. The origin of the higher molecular weight immunoreactive material is unknown. (B) Immunoprecipitation with antibody C-19 against WT1 followed by immunoblotting with antibody DO-1 against p53. p53 is indicated by the arrowhead.
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where both isoforms repress, the 0KTS isoform has a greater effect than the /KTS isoform. Both the isoforms tested in our experiments contain the exon 5 insertion that has been shown to disrupt the transcriptional activation domain; the two isoforms differ only by the presence of three amino acids (KTS) in the zinc finger domain (23). On the insulin receptor promoter, the WT1 variant containing the /KTS sequence inhibited promoter activity strongly under all conditions tested. The isoform lacking the KTS insert, on the other hand, could inhibit only in the presence of a dominant-negative p53 or when the IR promoter was stimulated by expression of C/EBPb. How can the differential effects of the KTS splice variants be explained? The DNA-binding domains from either isoform when expressed in E. coli can bind to the IR promoter in vitro as assessed by gel mobility shifts and DNaseI footprinting. Interestingly, one of the DNaseI footprints that we observed included direct repeats of a TCCCTCCC sequence. WT1 binding sites containing such repeats have been shown to be functional WT1 response elements in the EGFR promoter (15), an in vivo target for WT1, and similar sequences have been isolated in genomic binding site screens by Nakagama et al. (20) and Bickmore et al. (37). Other sites resembled GC-rich Egr1-like sites similar to those identified in other promoters, including the PDGF-A chain and IGF-II genes (11,12). We were only able to identify a single differential footprint at 0720 in the IR promoter in a region that was important for repression by the 0KTS isoform. This sequence was only recognized by the 0KTS WT1 isoform. However, all of the experiments used DNA-binding domains expressed in E. coli. The presence of threonine and serine residues in the KTS insert, potential protein kinase C phosphorylation sites, raises the intriguing possibility that the insert could be phosphorylated. This might be expected to have a dramatic effect on DNA binding. The isolated DNA-binding domains expressed in E. coli would not, of course, be phosphorylated. It will be interesting to determine whether phosphorylation modulates nuclear localization and DNA-binding activities of WT1 proteins. This may explain the observed differences in nuclear localization of the protein isoforms (38,39). What is the role of p53 in repression by WT1? An interaction of WT1 (0KTS) and p53 has been demonstrated by Maheswaran et al. (35). They were able to show complex formation in both transfected cell lines and primary Wilms tumors that expressed WT1. We have confirmed and extended their findings to show that both KTS isoforms are able to form
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stable complexes with p53 when overexpressed. What is the functional effect of this association? p53 was shown to switch the 0exon5/0KTS isoform of WT1 from a transcriptional activator to a repressor of a minimal promoter containing an Egr1 site (35). Recently it has been shown that WT1 stabilizes p53 and prevents its degradation (40). Both the 0KTS isoform of WT1 and an in-frame deletion mutant, WTAR, were able to stabilize p53. The WT1 domain involved in this stabilization maps to the first two zinc fingers of the DNA-binding domain (40). Thus, the /KTS isoform, which differs in three amino acids between the third and fourth zinc fingers, should also stabilize p53, although it has not been formally tested. Interestingly, only the 0KTS isoform was able to increase p53-mediated transactivation and inhibit p53-dependent transcriptional repression and apoptosis. Although the reason for this difference is not known, one can speculate that the effects that we observe with the two KTS isoforms of WT1 could be related to their differential effects on p53 signaling, particularly as we have shown that p53 can repress the IR promoter (34). ACKNOWLEDGMENTS This work was supported by grants from the NIH (DK44643), the Department of Veterans Affairs, and the American Diabetes Association. N.J.G.W. is a faculty member of the UCSD Biomedical Sciences Graduate Program.
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