©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
WT1-mediated Transcriptional Activation Is Inhibited by Dominant Negative Mutant Proteins (*)

Josina C. Reddy (1)(§), John C. Morris (2), Jing Wang (3) (4), Milton A. English (1), Daniel A. Haber (5), Yang Shi (4), Jonathan D. Licht (1)(¶)

From the (1) Brookdale Center for Molecular Biology and Molecular Medicine Division, Department of Medicine and (2) The Derald H. Ruttenberg Cancer Center, Division of Neoplastic Diseases, Mount Sinai School of Medicine, New York, New York 10029, the (3) Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute and the (4) Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, and the (5) Massachussetts General Hospital Cancer Center, Charlestown, Massachusetts 02129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The WT1 tumor suppressor gene encodes four isoforms of a zinc finger transcription factor with both activation and repression functions which are dependent upon promoter architecture. Using a simple HSV- tk promoter containing 5`-Egr-1/WT1-binding sites, we found that WT1 isoforms (A) and (B) strongly activated transcription. WT1(A) and (B) bound equally well to the Egr-1/WT1-binding site, but WT1(B), which contains a 17 amino acid insertion compared to WT1(A), was a consistently stronger activator of transcription than WT1(A). Transcriptional activation by wild-type WT1 was inhibited by coexpression of WT(PM) or WT(AR), genetically defined dominant negative alleles of WT1. In vitro, as well as in the yeast two-hybrid system, WT1 protein associated with itself and with dominant negative mutant proteins. The major domain required for self-association and inhibition of transcriptional activation mapped to the first 182 amino acids of WT1. Dominant negative WT1 alleles may play a role in tumorigenesis by associating with wild-type WT1 proteins and decreasing their transcriptional activity.


INTRODUCTION

The Wilms' tumor-associated gene WT1 was isolated by positional cloning based on the presence of a constitutional deletion of chromosome 11p13 in patients with the WAGR syndrome (Wilms' tumor, aniridia, genitourinary malformations, and mental retardation) (1, 2) . The WT1 gene yields four alternatively spliced mRNAs of approximately 3 kilobases which encode polypeptides of apparent molecular mass 52-54 kDa (3, 4, 5) . All four isoforms have four cysteine and histidine-containing (CH) zinc fingers near the carboxyl terminus of the protein and a glutamine and proline-rich domain in the amino-terminal portion of the protein. The (A) isoform contains neither alternative splice; the (B) isoform includes an NH-terminal splice which codes for 17 additional amino acids; the (C) isoform includes a COOH-terminal splice which results in the insertion of 3 amino acids (KTS) between zinc fingers 3 and 4; and the (D) isoform contains both the 17 amino acid and 3 amino acid insertions. The ratio of these four spliced mRNAs in human fetal kidney is 1 A:2.5 B:3.8 C:8.3 D; this ratio does not appear to be regulated during development (4) .

The (A) and (B) isoforms of WT1 were shown to bind to the same DNA sequence as the Egr-1 transcription factor (5`-GCGGGGGCG-3`) (6, 7) . Isoforms (C) and (D) bind poorly to the Egr-1 sequence due to the 3 amino acid insertion between zinc fingers 3 and 4 (7) . WT1(A) can repress transcription from a variety of cellular genes involved in growth and proliferation (8, 9, 10, 11, 12, 13) . Like many transcription factors, WT1 can also regulate transcription of its own promoter (14) . WT1 can also activate transcription. WT1 isoform (A) stimulates promoters containing Egr-1 sites either upstream or downstream of the transcription start site, but suppresses transcription when Egr-1 sites are present both upstream and downstream of the start site of transcription (15, 16, 17) .

Up to 5-10% of Wilms' tumors (18, 19) are associated with homozygous null WT1 mutations.This figure may be an underestimate, because in most studies, only the zinc finger domains were examined, and when searched for, mutations can be found which alter other portions of the WT1 protein (19) . Heterozygous WT1 mutations are also associated with disease. Such WT1 mutations were isolated both from patients with Wilms' tumor and from patients with the Denys-Drash syndrome (DDS),() a syndrome which includes nephropathy, intersex disorders, and predisposition to the development of Wilms' tumor. These mutants were proposed to act in a dominant negative manner by affecting the function of the wild-type allele. Consonant with this, we found that two genetically defined dominant negative alleles, which yield proteins unable to bind to the Egr-1/WT1 consensus site, inhibited the transcriptional function of wild-type WT1. The domain of the WT1 mutant proteins required for inhibition mapped to the NH-terminal 182 amino acids. This domain was found both by in vitro biochemical assays and by the in vivo yeast two-hybrid system to mediate self-association of WT1 protein.


EXPERIMENTAL PROCEDURES

Plasmid Construction

The expression vectors for the murine WT1(A) and (B) isoforms and the two WT(AR) mutant proteins were described previously (20) . The expression vector for WT(PM) was constructed by PCR amplification of sequences encoding amino acids 100-256 using the primers: 5`-GGCCAGTTCACCGGTACA-3` and 5`-GTCGACGTCGACTTATCCACTCTCGTACCCTATAC-3`. The PCR product was then digested with AgeI and SalI and inserted into AgeI/ SalI digested RSV-WT1(A). A RSV vector lacking the WT1 coding region was constructed by restriction of the RSV-WT1(A) plasmid with BglII to release the WT1 coding region, followed by recircularization. The EGR tkCAT reporter plasmid (Fig. 1 B) was constructed by the insertion of three tandem oligonucleotides containing the Egr-1-binding site (underlined) from the zif268 promoter: 5`-TCGACCCTCGCCCCCGCGCCGGGC-3`; 3`-GGGAGCGGGGGCGCGGCCCGAGCT-5` (21) into the SalI site of pBLCAT2 (22) . The thymidine kinase growth hormone ( tk-GH) (Fig. 1 B) internal control plasmid was described previously (23) .


Figure 1: A, expression vectors for production of WT1 proteins in transfected cells. Expression of WT1 was directed by the RSV promoter. 17 refers to the 17 amino acids inserted in WT1 as the result of alternative splicing of the WT1 mRNA. The zinc finger domains are indicated by black boxes and are numbered. B, reporter genes utilized. EGR tkCAT contains three Egr-1/WT1 binding sites inserted upstream of the HSV thymidine kinase ( tk) promoter and the CAT gene. In tk-hGH, the HSV- tk promoter is linked 5` to the human growth hormone gene. Secretion of human growth hormone from transfected cells is used as an internal control for transfection efficiency.



To construct a vector for in vitro translation of WT1 protein, a 1.5-kilobase Sau3AI fragment derived from the RSV-WT1(A), (B), or WT(AR) expression vectors was cloned into the BamHI site of pSP64 (Promega, Madison, WI). The pSP64-WT(PM) vector was constructed by cloning a HindIII/ SalI fragment of RSV-WT(PM) into HindIII/ SalI digested pSP64. The pSP64-WT(1-182) vector was made by digestion of pSP64-WT1(A) with NcoI followed by ligation to the duplex oligonucleotide: 5`-CATGTGAGTCGACTCA-3`, 3`-ACTCAGCTGAGTGTAC-5` containing a stop codon. To express amino acids 1-182 of WT1 in transfected cells, a Sau3AI- SalI fragment of pSP64-WT(1-182) was cloned into BglII/ SalI digested RSV vector. Vectors for translation of the zinc finger moeities of WT1(A) and WT(AR) were prepared by PCR amplification of the WT1 cDNA using an NH-terminal primer 5`-GGATCCGGATCCACCATGTGTGCATACCCAGGC-3` and a COOH-terminal primer 5`-GAATTCGAATTCTCAAAGCGCCACGTGGAGTTT-3`. The resultant fragments were digested with BamHI and EcoRI and inserted into pSP64.

To construct a vector for expression of a glutathione S-transferase (GST)-WT1 fusion protein in Escherichia coli, the pSP64-WT1A plasmid was digested with HincII and EcoRI with the resulting fragment cloned into SmaI/ EcoRI digested pGEX-2tk (Pharmacia, Uppsala, Sweden) yielding the plasmid pGEX-WT1(A). The resulting fusion protein contains an additional 20 amino acids (GSPTLEDRRSGEPSASEPHL) after the GST moiety. To construct pGEX-WT1(1-183), pGEX-WT1(A) was restricted with NcoI, blunted with the Klenow fragment of DNA polymerase I, and ligated to a duplex oligonucleotide containing stop codons in all three reading frames (24) .

Transfection Assays

CV-1 African green monkey kidney cells, were grown in Dulbecco's modified Eagle's medium containing 10% calf serum, while NIH 3T3 mouse fibroblast cells (gift of N. Landau), and 293 human embryonic kidney cells were cultured in 10% fetal calf serum. Cells were transfected with 2 µg of chloramphenicol acetyltransferase (CAT) reporter plasmid, up to 20 µg of WT1 expression vector or RSV vector lacking the WT1 insert as indicated, and 1 µg of tkGH plasmid as an internal control for transfection efficiency as described (25) . Carrier DNA (pBluescript, Stratagene, La Jolla, CA) was added to a total of 20-40 µg of DNA. Normalized CAT activity was determined as percent conversion of chloramphenicol divided by the growth hormone value in nanograms/milliliter (25) .

Immunoblotting

Transfected cells were boiled in 1 SDS sample buffer without -mercaptoethanol, and extract concentrations were determined with the Bio-Rad DC Protein Assay Kit (Bio-Rad) using bovine -globulin as a standard. Following this, -mercaptoethanol was added to a final concentration of 5%, and equal quantities of cellular proteins were separated by electrophoresis through a 12% SDS-polyacrylamide gel. The gel was then electrophoretically transferred to Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA) in a buffer containing 192 mM glycine and 25 mM Tris base. The membranes were probed with 0.1 µg/ml rabbit polyclonal anti-WT1 C19 Antibody (Santa Cruz Biotechnology, Santa Cruz, CA) as directed by the manufacturer. Immunoreactive proteins were visualized by chemiluminescence and autoradiography (ECL kit, Amersham, Buckinghamshire, United Kingdom).

Electrophoretic Mobility Shift Assays

Proteins were translated from pSP64-based vectors using the TNT rabbit reticulocyte lysate system (Promega, Madison, WI). In each case, a reaction containing [S]methionine was performed in parallel with each non-labeled reaction. Equal volumes of lysate from the labeled reaction were subjected to SDS-polyacrylamide gel electrophoresis and quantified on a PhosphorImager using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Binding reactions were performed with equimolar quantities of translated WT1 proteins as described previously (26) , except that the total volume of the reaction was 20 µl, the amount of probe used was 0.2 ng (approximately 10 counts/min/ng), and the electrophoresis was performed for 1.5 h at 300 V at room temperature.

In Vitro Protein Binding Assays

GST and GST fusion proteins were purified as described (27) , and the yield of each protein was determined by SDS-polyacrylamide gel electrophoresis analysis and Coomassie Blue staining. Volumes of bacterial lysate containing approximately 1 µg of GST or GST fusion protein were incubated with 25 µl of a 50% slurry of glutathione-agarose beads (Sigma) in NET-50 (20 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl) for 15 min at room temperature with rocking. GST proteins bound to the beads were then collected by brief centrifugation and washed once with 400 µl of NET-50. The beads were rocked for 10 min at room temperature in 200 µl of binding buffer (25 mM HEPES, pH 7.5, 12.5 mM MgCl, 20% glycerol, 0.1% Nonidet P-40, 150 mM KCl, 1 mM dithiothreitol, 150 µg/ml bovine serum albumin, 200 µg/ml ethidium bromide (EtBr) as indicated). Coupled in vitro transcription-translation reactions containing [S]methionine (1175 Ci/mmol) were programmed with the indicated pSP64-WT or control SP6-luciferase plasmid and were diluted 1:10 in binding buffer (without dithiothreitol, bovine serum albumin, or EtBr). Diluted programmed lysate (5 µl) was incubated with immobilized GST or GST-WT proteins for 1 h at room temperature. The beads were collected by centrifugation and washed three times with 1 ml of NETN (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) in the presence or absence of 200 µg/ml EtBr as indicated. Bound proteins were eluted from the beads by boiling in 1 loading buffer (62.5 mM Tris, pH 6.9, 10% glycerol, 2% SDS, 5% -mercaptoethanol) and were separated by electrophoresis through a 10 or 15% SDS-polyacrylamide gel. The gel was fixed in 30% methanol, 10% acetic acid for 1 h, washed in water for 0.5 h, soaked in 1 M sodium salicylate for 0.5 h, dried, and exposed to XAR film at -80 °C. The percentage of input S-labeled protein bound to GST protein was quantified by exposure of the dried gel on a PhosphorImager and analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Mapping of WT1 Self-association Domains Using the Two-hybrid System in Yeast

A LexA-WT1 expression vector was constructed by excision of the WT1(A) cDNA from pGEM7zf(+)WT1 (gift of Frank J. Rauscher, III) using EcoRI and ClaI, blunting these ends with the Klenow fragment of DNA polymerase I, and cloning the resultant fragment into pEG202 (28) . To create LexA-WT1(1-180), LexA-WT1 was restricted with BamHI to release the COOH-terminal two-thirds of WT1, and the plasmid was religated. LexA-WT1(175-301) and LexA-WT1(275-429) were created by PCR amplification of the indicated portion of the WT1 coding sequence and ligation of these PCR fragments into pEG202. An expression vector for the B42 acidic activation domain-WT1 fusion protein was constructed by digestion of pGEM7zf(+)WT1 with EcoRI and XhoI and insertion of the resulting fragment into EcoRI/ XhoI cut pJG4-5 (29) . LexA-WT1 and B42-WT1 expression vectors were transformed in pairs into the yeast strain EGY48 containing the reporter LexAop-LacZ (28) . After growth in 5-bromo-4-chloro-3-indolyl--D-galactopyranoside, the yeast colonies were scored for blue color. -Galactosidase activity of individual colonies was determined as described (30) .

Graphics

Autoradiographs were imaged using a Silverscan flatbed digital scanner (La Cie, Ltd., Beaverton, OR). Image processing was performed on a Power PC (Apple, Cupertino, CA) using Adobe Photoshop (Adobe, Mountain, CA) and Aldus Persuasion (Aldus, Seattle, WA). Densitometry was performed using the NIH Image 1.56 program for Power PC. Figures were printed using a XL7700 Digital Continuous Tone Printer (Kodak).


RESULTS

Transcriptional Activation by WT1 Proteins

To characterize the transcriptional effects of WT1 proteins, the EGR tkCAT reporter containing three Egr-1 consensus sequences, capable of binding WT1(A) and (B) (7) (Fig. 1 B) was transfected into CV-1 cells along with increasing amounts of WT1(A) or (B) expression vectors (Fig. 1 A). Both WT1(A) and (B) acted as dose-dependent transcriptional activators of this simple test promoter. Transfection of 10 µg of the WT1(A) or (B) expression vector produced a 10-15-fold increase in reporter gene transcription. Transcriptional activation was binding site dependent, as neither WT1(A) or (B) affected transcription from the tk-hGH plasmid or from a tk-CAT reporter gene (data not shown). Similar results were obtained in NIH 3T3 cells and in human embryonic kidney 293 cells (data not shown) indicating that activation was not cell type-specific. In addition, transcriptional activation by WT1(A) was linear and non-saturable at the doses used in these experiments (Fig. 2 C), implying that any cofactors required for WT1(A) activity are not limiting in CV-1 cells. After six independent determinations using three different preparations of expression vector plasmid, transcriptional activation by WT1(B) was consistently 20-50% stronger than activation by WT1(A) (Fig. 2 A). However, immunoblotting and densitometric analysis indicated that after transfection, WT1(B) was expressed at somewhat lower levels than WT1(A) (Fig. 2 B). In addition, in vitro translated WT1(A) and (B) bound to the Egr-1/WT1 site to a similar extent (Fig. 2 D). Therefore, increased DNA binding is unlikely to account for the increased transactivational activity of WT1(B).


Figure 2: A, transcriptional activation of EGR tkCAT by WT1 in CV-1 cells. The average fold induction of transcription (normalized CAT activity) and standard error from six independent experiments is presented. B, immunoblotting analysis of extracts from CV-1 cells transfected with 5 µg of RSV-WT1(A) or RSV-WT1(B), probed with anti-WT1 C19 antibody (Santa Cruz Biotechnology). The autoradiogram was digitally scanned and densitometrically quantified. C, transcriptional activation by WT1 increases linearly with the dose of WT1 expression vector. The EGR tkCAT reporter was cotransfected into CV-1 cells with increasing amounts of WT1 expression vector. The fold induction of transcription for a representative experiment is indicated below the chromatogram. D, WT1(A) and (B) bind to the Egr-1 site to the same extent as determined by electrophoretic mobility shift assay. 1 = probe only; 2 = unprogrammed reticulocyte lysate; 3 = WT1(A); 4 = WT1(B). The specific WT1DNA complex is indicated by an arrow.



Two Dominant Negative WT1 Alleles Inhibit Transcriptional Activation by WT1(A)

The WT(AR) allele of WT1 was isolated from a patient with a sporadic, unilateral Wilms' tumor. The mutation alters a splice donor site, leading to a deletion of zinc finger 3 of the WT1 protein (31) and yields a protein which binds poorly to the Egr-1 consensus binding site (7) . Since the WT(AR) mutation was present in the heterozygous state, it was proposed to be a dominant negative allele, which suppresses the function of the normal allele (31) . The protein products of this mutant allele, referred to as WT(AR-) and WT(AR+), differ due to the absence or presence, respectively, of the 17 amino acids coded for by the alternatively spliced exon 5 of WT1. The COOH-terminal splice region is eliminated by the deletion of the third zinc finger (Fig. 1).

The WT(PM) mutation was isolated in the heterozygous state from germline DNA from a patient with DDS (32) . Patients with this syndrome exhibit nephropathy and intersex disorders and occasionally develop Wilms' tumor. Insertion of a single guanine residue in this allele results in the truncation of the WT1 protein at amino acid 256, with an extra amino acid (glycine) added at the COOH terminus (32) (Fig. 1). This patient presented with nephropathy, ambiguous genitalia, and bilateral Wilms' tumors. While the tumors were most likely homozygous for the mutant WT1 allele (32) , its presence in the heterozygous state in germline DNA suggests that it may act as a dominant negative allele resulting in genitourinary malformations and nephropathy.

We first determined whether, consonant with their proposed dominant negative genetic function, the WT(AR) proteins could interfere with transcriptional activation by WT1(A). The WT(AR) isoforms alone had no effect on transcription from the EGR tkCAT reporter in CV-1 cells at doses of 4 and 8 µg (data not shown). At doses of 10 µg and above, the WT(AR) proteins produced an approximately 50% increase in transcription from the EGR tkCAT reporter (data not shown), compared with an over 10-fold increase in transcription by WT1(A) (Fig. 2 A). Although full-length WT(AR) does not bind to the Egr-1 consensus sequence (7, and see below), it can bind to sequences other than the Egr-1 consensus (33) . At high concentrations, WT(AR) could possibly bind to sites in the backbone of the reporter plasmid, resulting in modest activation of transcription.

Transfection of a fixed amount (4 µg) of WT1(A) expression vector with increasing amounts (4, 8, or 16 µg) of WT1(B) further stimulated transcription (Fig. 3 A). In contrast, transfection of increasing amounts of either WT(AR) expression vector inhibited transcriptional activation by WT1(A) (Fig. 3 B). At the highest dose (16 µg) of transfected WT(AR) plasmid, inhibition was less marked, perhaps due to the modest stimulatory activity of WT(AR) observed at high inputs.


Figure 3: Two dominant negative WT1 alleles interfere with transcriptional activation by WT1 ( A). The EGR tkCAT reporter was cotransfected into CV-1 cells with 4 µg of RSV-WT1(A) and the indicated amounts of RSV-WT1(B) ( A), RSV-WT(AR-), or RSV-WT(AR+) ( B), and RSV-WT(PM) or RSV-WT1(1-182) ( C). The amount of transcription obtained is presented as the percent of activation directed by WT1(A), where the data are averaged from two independent experiments for WT1(B), WT(AR-) and WT(AR+), from five independent experiments for WT(PM), and from three independent experiments for WT1(1-182).



To determine which domain of the mutant WT1 protein was required for inhibition of transcriptional activation, we utilized the WT(PM) mutant allele. This protein contains only the first 256 amino acids of WT1 and cannot bind to the Egr-1 site since it lacks zinc finger motifs (see below). Expression of either WT(PM) or of a protein containing the first 182 amino acids of WT1 inhibited transactivation by full-length WT1 in a dose-dependent manner (Fig. 3 C). Unlike WT(AR), WT(PM) and WT1(1-182) continued to inhibit transactivation at the highest dose of input plasmid. Therefore, we conclude that the NH-terminal 182 amino acids of WT1 are sufficient to mediate down-modulation of transcriptional activation by WT1(A).

WT(AR) and WT(PM) Do Not Interfere with DNA Binding by WT1

To determine whether expression of either dominant negative mutant protein might interfere with DNA binding by WT1(A), we performed electrophoretic mobility shift assays with in vitro translated WT1(A), WT(AR-), and WT(PM) proteins. As predicted, WT(AR-) and WT(PM) did not bind to the Egr-1 consensus site (Fig. 4, lanes 3 and 7) while WT1(A) bound strongly to this site (Fig. 4, lanes 2 and 6). This complex was specific as addition of unlabeled Egr-1 consensus site oligonucleotide eliminated the WT1(A)DNA complex, and addition of an antiserum against WT1, but not preimmune serum, slowed the mobility of the WT1(A)DNA complex (data not shown). Preincubation of WT1(A) with either an equimolar amount or a 2-fold molar excess of WT(AR) or WT(PM) did not interfere with DNA binding by WT1 (Fig. 4, lanes 4, 5, 8, and 9).


Figure 4: Dominant negative WT1 proteins do not interfere with DNA binding by wild-type WT1. A, lane 1: unprogrammed reticulocyte lysate. Lane 2, WT1(A). Lane 3, WT(AR-). Lane 4, WT1(A) preincubated with an equimolar amount of WT(AR-). Lane 5, WT1(A) preincubated with a 2-fold excess of WT(AR-). Lane 6, WT1(A). Lane 7, WT(PM). Lane 8, WT1(A) preincubated with an equimolar amount of WT(PM). Lane 9, WT1(A) preincubated with a 2-fold excess of WT(PM). The WT1DNA complex is indicated by an arrow.



Mutant and Wild-type WT1 Proteins Interact in Vitro

To further investigate the mechanism of action of the dominant negative WT1 proteins, we determined whether WT1(A) could associate with wild-type WT1 proteins or with proteins encoded by the WT(AR) or WT(PM) alleles. GST-WT1(A) fusion protein immobilized on glutathione-agarose beads was incubated with [S]methionine-labeled WT1(A), WT1(B), or mutant WT(AR-), WT(AR+), or WT(PM) proteins, produced in reticulocyte lysates. Bound S-labeled proteins were eluted, electrophoretically separated, and visualized by fluorography. The two WT(AR) mutant isoforms, as well as wild-type WT1(A) and (B), bound to GST-WT1(A) (Fig. 5 A, rows 1-4), while a control luciferase protein did not (Fig. 5 A, row 5). None of the proteins bound to naked glutathione-agarose beads or to GST protein-coated beads (Fig. 5 A). The interaction between WT1 proteins was unlikely to be due to coprecipitation of these DNA-binding proteins through interaction with DNA fragments in the reaction mixture, since such DNA-protein interactions would be expected to be destabilized by the addition of 200 µg/ml EtBr (34) . As seen in Fig. 5 A, EtBr had no effect on interaction between these full-length WT1 proteins.


Figure 5: Mutant and wild-type WT1 proteins interact in vitro. S-Labeled proteins (indicated at left) were incubated with either naked glutathione-agarose beads or with GST or GST-WT1 A proteins bound to the beads (indicated at top), in the presence or absence of 200 µg/ml EtBr. After washing and elution, bound proteins were electrophoretically separated and visualized by fluorography. For comparison, the input amount of S-labeled protein for each reaction was loaded in the far left lane. A, full-length wild-type and mutant S-labeled WT1 proteins and S-labeled luciferase protein were incubated with the indicated GST fusion proteins. B, S-labeled full-length WT1(A) or truncated WT1 proteins containing the indicated amino acids were incubated with the indicated GST fusion proteins.



We then mapped the domain required for WT1 self-association. Full-length WT1(A) and WT(AR) bound both to GST-WT1(A)(1-429) and to a GST-WT1 fusion protein containing only amino acids 1-183 (Fig. 5 B, row 1). Interaction between full-length proteins was consistently more efficient than interaction between full-length WT1 and the NH terminus of WT1. Similarly, a greater extent of interaction was observed between S-labeled WT1 containing amino acids 1-182 with GST-WT1(1-183) (35% of input labeled protein retained on the beads) than with the full-length GST-WT1(A)(1-429) fusion protein (6% of input labeled protein retained on the beads) (Fig. 5 B, row 5). This suggests that the full-length protein is folded in such a way that the NH-terminal interaction surface is partially inaccessible, and when two full-length molecules interact, this blockage is relieved.

The WT(PM) protein, containing amino acids 1-256 of WT1, bound strongly to both GST-WT1(A)(1-429) and GST-WT1(1-183) (Fig. 5 B, row 4). In addition, a truncated protein containing only wild-type WT1 or WT(AR) zinc finger domains (amino acids 307-429) did not bind to GST-WT1(1-183) but bound weakly to the full-length GST-WT1 protein (Fig. 5 B, rows 2 and 3). Intriguingly, the binding of the wild-type WT1 or WT(AR) zinc finger domain (amino acids 307-429) to the full-length protein was intensified upon addition of EtBr (Fig. 5 B, rows 2 and 3). This suggests that the zinc fingers of WT1 can also mediate a protein-protein interaction in vitro, which becomes stronger if DNA binding by the zinc fingers is precluded by EtBr. The presence of two self-association domains within the WT1 protein may also help explain why full-length WT1 molecules interact better with themselves than with NH-terminal truncated proteins.

Mapping of WT1 Self-association Domains in Vivo Using the Two-hybrid System in Yeast

To determine the ability of WT1 protein to self-associate in vivo, we used a yeast two-hybrid system (28, 29) . The full-length WT1 coding region, or smaller fragments of the coding region, was fused in-frame to the DNA-binding domain of the bacterial LexA protein. The second hybrid fused WT1 to the B42 acidic activation domain. Plasmids encoding these hybrids were introduced into yeast strain EGY48, containing the 2-µm-based reporter plasmid LexAop-LacZ. This reporter contains two Lex operators and the GAL1 promoter linked to the E. coli -galactosidase reporter gene. Interaction between WT1 fused to LexA and the WT1/B42 acidic activation domain hybrid will stimulate transcription from the reporter gene. As seen in , WT1 interacted with itself in this assay as scored by qualitative (blue/white) and quantitative -galactosidase assays. The domain of WT1 responsible for self-association mapped to the NH-terminal 180 amino acids of the protein. These results are consistent with our finding that in transfection studies, WT1(1-182) was a potent inhibitor of transactivation by full-length WT1 (Fig. 3 B) and that the NH-terminal 182 amino acids mediated self-association of WT1 molecules in vitro (Fig. 5 B). In addition, these results suggest that association between the zinc finger domains of WT1 may not contribute to self-association of WT1 in vivo.


DISCUSSION

Transcriptional Activation by WT1

Consistent with previous work (15, 16) , we found that the WT1 protein activated the HSV- tk promoter through upstream binding sites. WT1(B) was a stronger transcriptional activator than WT1(A); however, WT1(A) and (B) bound DNA to similar extents in vitro and WT1(B) was expressed at a somewhat lower level than WT1(A) in transfected cells. Together this information suggests that the 17 additional amino acids in WT1(B) augment the transcriptional activation domain of WT1. This insertion (MAAGSSSSVKWTEGQSN) contains a potential protein kinase C phosphorylation site (SVK). Other factors such as the cyclic AMP response element-binding protein (CREB) (35) and c- jun(36) can be dynamically regulated by phosphorylation. While a prior study did not detect phosphorylation of WT1(A) (5) , the phosphorylation state of WT1(B) has not been examined. Alternatively, the inserted amino acids in WT1(B) could interact directly with a component of the transcription machinery to stimulate transcription, or could effect a conformational change in the entire WT1 protein, altering its interaction with the transcriptional machinery.

Mechanism of Action of Dominant Negative WT1 Alleles

The ability of the dominant negative alleles WT(AR) and WT(PM) to inhibit transcriptional activation by WT1 could be explained by one of three mechanisms: competition for binding sites (37) , competition for co-factors (``squelching'') (38) , or physical interaction between wild-type and mutant proteins (39) . Competition between wild-type and mutant proteins for binding to the Egr-1-binding site is unlikely to play a role in this effect, since the WT(AR) and WT(PM) mutant proteins do not bind to this site (7, and Fig. 4 ). In addition, neither dominant negative protein interfered with DNA binding by WT1(A) in vitro (Fig. 4). Since cofactors for transcriptional activation by WT1 do not appear to be limiting in our cell lines (Fig. 2 C), the mutant proteins are also not likely to inhibit transcription by squelching.

By an in vitro biochemical assay and by the in vivo yeast two-hybrid assay, WT1 was found to self-associate. We therefore propose that the dominant negative WT(AR) and WT(PM) proteins, which cannot bind to the Egr-1 site, interact with WT1 bound to DNA and inhibit the function of wild-type protein, perhaps by partially shielding its activation domain. WT(PM) was a more potent inhibitor of transactivation by WT1(A) than was WT(AR). This may be due to the fact that WT(PM) is incapable of binding to DNA while WT(AR) can bind to sites other than the Egr-1 site used in this study (33) . Some of the WT(AR) protein in the cell may be bound to DNA sequences other than the Egr-1 consensus and would therefore be unavailable for protein-protein association with wild-type WT1 required for inhibition of transactivation. In contrast, WT(PM) cannot bind to any DNA sequence and thus can only function through protein-protein interaction with wild-type WT1. Based on analysis of WT1 mutations isolated from DDS patients, Bardeesy et al.(40) predicted that a domain encoded by exons 1, 2, and 3 of WT1 (amino acids 1-222) would mediate a dominant negative effect. We confirm this prediction by mapping the domain responsible for self-association and for the dominant negative effect to amino acids 1-182 of WT1.

WT1 also represses transcription of a variety of growth-related genes (8, 9, 10, 11, 12, 13) , with this effect dependent on promoter architecture (15, 16, 17) and on interaction of WT1 with the p53 protein (41) . Studies are now underway to determine whether dominant negative WT1 proteins also inhibit transcriptional repression by WT1. Even if only activation is affected, the resultant imbalance between the repression and activation activities of WT1 might be sufficient to cause neoplasia, developmental abnormalities, or nephropathy.

Heterozygous null mutations of WT1, as found in the WAGR syndrome, result in genitourinary malformations and predisposition to Wilms' tumor, but not nephropathy. Patients with such mutations would have a 50% reduction in their effective WT1 activity. Our results suggest that heterozygous dominant negative mutations of WT1, such as WT(AR) and WT(PM), can result in an inhibition of function of the remaining wild-type WT1 allele. The maximal inhibition we observed was approximately 3-fold for WT(AR) and 5-fold for WT(PM). In patient AR, this could result in a level of WT1 activity that is less than 50% of normal in the small subset of kidney cells containing the acquired mutation. This might be sufficient to produce a disregulation of growth control of these cells, resulting in tumor formation. Since WT(AR) can bind to specific DNA sequences other than the Egr-1 consensus (33) , it could also deregulate growth by binding to novel target genes not normally regulated by WT1.

According to our hypothesis, DDS patients, who have a heterozygous germline WT1 mutation, would also have a level of effective WT1 activity substantially lower than hemizygous WAGR patients. The remaining level of WT1 activity in DDS patients must still be sufficient to allow kidney development, since mice with a homozygous disruption of the WT1 locus fail to form kidneys (42) . WT1 continues to be expressed in the podocytes of the adult kidney, which are responsible for the maintenance of the glomerular basement membrane (43) . Therefore, inhibition of WT1 activity by a dominant negative mutant protein in the podocytes of DDS patients might contribute to the novel phenotype of progressive glomerulonephropathy.

  
Table: WT1 self-association assayed using the two-hybrid system in yeast

Plasmids encoding the indicated chimeric fusion proteins were transformed in pairs into the yeast strain EGY48 containing the reporter LexAop-LacZ. Interaction between fusion proteins was scored qualitatively by blue (positive) or white (negative) color of yeast colonies grown on plates containing X-gal. Transformed yeast containing selected pairs of plasmids were then colorimetrically assayed for -galactosidase activity as described (30). Results are expressed as the mean ± standard deviation for three independent isolates.



FOOTNOTES

*
This work was supported by a Basil O'Connor Starter Grant from the March of Dimes, the Council for Tobacco Research, and National Institutes of Health Grant CA59998 (to J. D. L.), by NIH Grant CA58997 (to Y. S.), and NIH Grant CA58596 (to D. A. H.). This is publication 216 from the Brookdale Center for Molecular Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by NIH Medical Scientist Training Program Grant T32GM07280-17 and by the Wendy Will Case Cancer Fund.

To whom correspondence should be addressed: Box 1126, Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Tel.: 212-241-9427; Fax: 212-860-9279; Internet: jlicht@smtplink.mssm.edu.

The abbreviations used are: DDS, Denys-Drash syndrome; PCR, polymerase chain reaction; RSV, Rous sarcoma virus; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; EtBr, ethidium bromide; HSV, herpes simplex virus.


ACKNOWLEDGEMENTS

We thank Dr. Ned Landau for the NIH 3T3 cell line, Dr. Frank J. Rauscher, III, for plasmid constructions, and Dr. George F. Atweh for careful review of the manuscript.


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