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Endocrinology Vol. 140, No. 10 4713-4724
Copyright © 1999 by The Endocrine Society

ARTICLES

Regulation of Insulin-Like Growth Factor I Receptor Promoter Activity by Wild-Type and Mutant Versions of the WT1 Tumor Suppressor1

Katsunori Tajinda2, Julie Carroll and Charles T. Roberts, Jr.

Department of Pediatrics, Oregon Health Sciences University, Portland, Oregon 97201

Address all correspondence and requests for reprints to: Dr. Charles T. Roberts, Jr., Department of Pediatrics, NRC-5, Oregon Health Sciences University, Portland, Oregon 97201. E-mail: robertsc{at}ohsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The insulin-like growth factor I (IGF-I) receptor is a transmembrane tyrosine kinase that mediates the growth-promoting effects of IGF-I and IGF-II. Changes in IGF-I receptor messenger RNA levels are reflected in cell surface receptor number, and modulation of IGF-I receptor levels affects tumorigenicity in numerous cellular models; thus, control of IGF-I receptor gene expression appears to be an important level at which cellular proliferation and tumorigenic potential may be regulated. We have previously shown that the product of the WT1 Wilms’ tumor suppressor gene represses IGF-I receptor gene expression both in vitro and in vivo, and that decreased WT1 levels are correlated with up-regulation of IGF-I receptor gene expression in Wilms’ tumor, benign prostatic hyperplasia, and breast cancer. Gene regulation by WT1 is complex, in that the WT1 gene encodes a variety of products as a result of alternative splicing and RNA editing, and a number of missense point mutations have been characterized in Wilms’ tumor-associated syndromes. Additionally, the WT1 protein has been demonstrated to self-associate through its N-terminal domain, although the role of this intermolecular interaction in transcriptional regulation by WT1 is unclear. In this report, we analyze the relative activity of wild-type and mutant versions of the WT1 protein with respect to IGF-I receptor promoter activity in transient transfection assays and assess the potential contribution of WT1 self-association to IGF-I receptor regulation using the yeast two-hybrid system. Of the naturally occurring variations in WT1 structure, only the presence of a three-amino acid KTS insert in the zinc finger domain introduced by alternative splicing of exon 9 had a significant effect on WT1 repression of IGF-I receptor promoter activity. The N- and C-terminal domains of WT1 also exhibited partial repression, as did the most common mutant version of the WT1 protein associated with Denys-Drash syndrome. Mutations in the WT1 N-terminus attenuated WT1 self-association in the yeast two-hybrid system, but did not impair transcriptional repression. Our results suggest that 1) the DNA-binding capacity of WT1 is critical for maximal repression of the IGF-I receptor promoter, but some effects may be mediated through protein-protein interactions involving the N-terminal domain; 2) WT1 self-association may not be required for repression of the IGF-I receptor promoter; and 3) the Denys-Drash syndrome version of the WT1 protein may exhibit residual or possible gain of function activity in some contexts rather than exerting dominant negative effects, as has been proposed previously.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INSULIN-LIKE growth factors, IGF-I and IGF-II, exert proliferative, antiapoptotic, and differentiative effects on many cell types, primarily through their activation of the IGF-I receptor, which is coupled to the mitogen-activated protein kinase and phosphoinositol 3-kinase cascades (1). The function of the IGF-I receptor in malignant transformation has been the subject of extensive study (reviewed in Ref. 2). Overexpression of the IGF-I receptor enhances ligand-dependent transformation in NIH-3T3 and Rat-1 fibroblasts, as shown by colony formation in soft agar and formation of tumors in nude mice (3). This transforming activity of the IGF-I receptor is blocked by truncated IGF-I receptors that lack a tyrosine kinase domain (4). Fibroblasts derived from IGF-I receptor knockout mice and cells expressing dominant negative mutant IGF-I receptors that contain amino acid substitutions in cytoplasmic tyrosine residues lose their ability to form tumors in nude mice (5, 6). A monoclonal antibody ({alpha}IR-3) directed against the {alpha}-subunit of the IGF-I receptor has been shown to inhibit the proliferation of various tumor cell types, including colorectal (7), osteosarcoma (8), and breast cancer (9). This evidence indicates that IGF-I receptor function is required for cellular transformation.

The relationship between reduced IGF-I receptor number and tumorigenicity has been demonstrated by various antisense strategies. Stable expression of an IGF-I receptor antisense RNA inhibited the growth of human T98G glioblastoma cells (10) and colony formation in soft agar in human FO-1 melanoma cells (11). Moreover, although both FO-1 cells and rat C6 glioblastoma cells develop tumors in nude mice, treatment of these cells with IGF-I receptor antisense oligonucleotides inhibited tumor development (11, 12). We have previously demonstrated that expression of a IGF-I receptor antisense RNA in MCF-7 cells resulted in a coordinate decrease in IGF-I receptor messenger RNA (mRNA) levels and cell surface receptor number, which reduced serum and IGF-I-stimulated cell proliferation (13). These studies suggest that changes in the level of IGF-I receptor gene expression can determine IGF-I receptor activity and thereby influence the possibility of malignant transformation.

In previous studies, we have demonstrated that the IGF-I receptor promoter is a target for the tumor suppressor protein encoded by the WT1 Wilms’ tumor suppressor gene. IGF-I receptor mRNA levels and WT1 mRNA levels are inversely related in Wilms’ tumor samples (14), and WT1 can repress IGF-I receptor promoter activity in transient transfection studies, binding to sites in both the 5'-flanking and 5'-untranslated regions (15). Constitutive expression of WT1 in stably transfected cells inhibits endogenous IGF-I receptor gene expression and inhibits IGF-I and serum-stimulated growth and colony formation in soft agar (16). We have previously proposed that loss of WT1 function may up-regulate IGF-I receptor gene expression in Wilms’ tumor itself (17), and that a similar loss of repression of the IGF-I receptor gene by WT1 may be involved in the pathogenensis of benign prostatic hyperplasia (18) and breast carcinoma (19). Thus, elucidation of the molecular mechanisms responsible for WT1 control of IGF-I receptor gene expression may be relevant to several types of proliferative disorders.

As shown in Fig. 1Go, the WT1 protein contains an N-terminal transcriptional regulatory and self-association domain, which includes two Pro-rich motifs, and a C-terminal zinc finger domain involved in DNA and RNA binding (reviewed in Ref. 20). The WT1 protein recognizes GC-rich and (TCC)n motifs (21), both of which are found in the proximal IGF-I receptor promoter (15, 22). Transcriptional regulation by WT1 is complicated by the existence of multiple versions of the protein due to alternative splicing of exon 5 and the use of an alternative splice site at the end of exon 9 (23). Additionally, WT1 mRNA is subject to RNA editing in exon 6 (24). As these multiple versions of the WT1 mRNA are usually coexpressed in varying proportions in tissues, an in-depth understanding of the control of any putative WT1 target gene requires analysis of the effects of each of the various versions of the WT1 protein. To extend our previous studies on WT1 regulation of the IGF-I receptor promoter that focused on a particular splice variant of the WT1 protein, we have characterized the activities of these versions of the WT1 protein on IGF-I receptor promoter activity. We have also examined the effect of deletion of the major Pro-rich domain and of WT1 point mutations described in the Wilms’ tumor-associated syndromes WAGR (a continuous gene syndrome characterized by Wilms’ tumor, Aniridia, Genito-urinary abnormalities, and mental Retardation (25) and Denys-Drash syndrome (DDS) (26). The latter class of mutation is of particular interest because DDS is associated with an increased incidence of Wilms’ tumor but is principally defined by early-onset nephropathy leading to end-stage renal disease. Finally, we have assessed the relationship between WT1 self-association and repression of IGF-I receptor promoter activity.



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  		Figure 1. WT1 protein structure. The WT1 N terminus includes two Pro-rich clusters, and the first 182 amino acids are involved in self-association. Amino acids 84–180 comprise a transcriptional repression domain in the context of PDGF A chain gene regulation. The WT1 C terminus contains four zinc finger DNA-binding motifs that exhibit significant sequence homology with the early growth response (Egr) gene family. Zinc fingers 2, 3, and 4 are involved in DNA binding, and zinc fingers 1, 2, and 3 have been identified as a RNA-binding domain (51 ). Although the domain(s) responsible for p53 association has not been determined yet, amino acids 297–381 are critical for stabilizing p53 protein. Zinc finger 1 and zinc finger 2 and 3 segments comprise the nuclear localization signal (46 ).

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid constructions: WT1 splice variants and domain constructs
Full-length human WT1 complementary DNAs (cDNAs) containing both alternatively spliced sequences (+/+) or lacking both (-/-) in pCB6+ vectors (27) were excised using ClaI and HincII, and these inserts, containing minimal 5'-untranslated region (5'-UTR) sequence and approximately 300 bp of 3'-UTR sequence were cloned into the ClaI and SmaI sites of the Gem7Zf(+) vector (Promega Corp., Madison, WI) for in vitro mutagenesis. WT1+/+ and WT1-/- inserts were excised from pGem7Zf(+) by cleaving with HindIII at the 5' end of the multiple cloning site, filling in with Klenow, and then cleaving with XbaI in the 3' end of the multiple cloning site. These inserts were then cloned into the pFastBac vector (Life Technologies, Inc., Gaithersburg, MD) that had been cut with NotI (and then filled in) and XbaI. These constructs were digested with KpnI and relegated to remove the EcoRI site present in the 3'-portion of the pGem7Zf(+) multiple cloning site. A FLAG epitope sequence was introduced by annealing oligonucleotides 1 and 2 (Table 1Go), phosphorylating the annealed oligonucleotides with ATP and polynucleotide kinase, and ligating this insert into WT1-pFastBac constructs that had been digested with EcoRI and ClaI. Sequencing verified that these constructs contained an EcoRI site followed by a FLAG epitope sequence that was in-frame with the WT1 open reading frame immediately following the ClaI site. EcoRI-HindIII (blunt ended) inserts from these constructs were then cloned into pcDNA3 vectors (Invitrogen, San Diego, CA) that had been digested with EcoRI and EcoRV. The resulting constructs were designated pcDNA3/WT1+/+ and pcDNA3/WT1-/-. Tripartite ligations using ClaI-AflIII and AflIII-XbaI fragments corresponding to exons 1–7 and exons 8–10 were performed to generate WT+/- and WT-/+ constructs. pcDNA3 constructs lacking the FLAG epitope were generated by cleavage with EcoRI and ClaI, filling in with Klenow, and relegating. The ClaI-AflIII and AflIII-XbaI fragments described above were cloned separately into pcDNA3 to generate constructs encoding the N-terminal 296 or 313 amino acids (with or without exon 5) or the C-terminal 133 or 136 amino acids (with or without the KTS insert). A simian virus 40-derived nuclear localization sequence was introduced into the N-terminal construct by annealing oligonucleotides 3 and 4 (Table 1Go) and ligating into the ClaI site of this construct. The full-length constructs used in this study did not contain the FLAG epitope, although this sequence had no significant effect in transient transfection experiments (data not shown). In some experiments, pCB6+ constructs containing full-length WT1 (-/-), the N-terminal 364 amino acids, or the C-terminal 135 amino acids (28) were used.


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  		Table 1. Oligonucleotides used in plasmid construction and in vitro mutagenesis

 
In vitro mutagenesis
The pGem7Zf(+) WT1+/+ and WT1-/- plasmids described above were used for in vitro mutagenesis (Stratagene, La Jolla, CA). In the major DDS-associated mutation, T1180 is replaced by C, changing amino acid Arg394 to Trp (26). In the case of a WAGR-associated point mutation, G602 is changed to adenine, which alters Gly201 to Asp (25). The substitution of nucleotide T842 by C (Leu280 to Pro) results from the posttranscriptional editing of WT1 mRNA (27). The larger of the two Pro-rich clusters located in the N-terminal region was also removed. This Pro-rich domain (amino acids 54–68) contains 13 prolines of 15 amino acids (N-PAPPPAPPPPPPPPP-C). The DDS, WAGR-associated, RNA editing, and Pro-deletion constructs were created in both +/+ and -/- backgrounds using oligonucleotides 5, 6, 7, and 8 (Table 1Go), respectively, as mutagenic primers in conjunction with a selection oligonucleotide according to the manufacturer’s instructions. After verification of in vitro mutagenesis by DNA sequencing, mutant WT1 inserts from pGem7Zf(+) were cloned into pcDNA3. Schematic diagrams of these mutants are shown in Fig. 2Go.



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  		Figure 2. Schematic representations of wild-type and variant forms of WT1 used in this study. Alternative splicing of exon 5 and exon 9 produces four variants of the WT1 protein. Top, The WT1 protein that includes both exon 5 and complete exon 9-encoded sequence, designated WT1+/+. The other naturally occurring variants are designated: WT1+/-, presence of exon 5 and absence of KTS insertion; WT1-/+, absence of exon 5 and presence of KTS insertion; and WT-/-, absence of both sites. The N-terminal construct includes the 313 amino acids encoded by exons 1–6 and the 5'-portion of exon 7; the C-terminal constructs encode the remaining amino acids that comprise the four zinc finger domains. Mutant forms of WT1 used in this study are as follows. The most common DDS mutation changes Arg394 to Trp in zinc finger 3, the WAGR-associated mutation is a Gly201 to Asp point mutation, RNA editing of WT1 mRNA changes Leu280 to Pro, and the Pro-rich deletion mutant lacks 15 amino acids constituting the N-terminal Pro-rich domain.

 
Reporter plasmids
A rat IGF-I receptor promoter sequence (-476 to +640) originally cloned by Werner et al. (29) into p0Luc was excised with HindIII, inserted into the pGL3 luciferase reporter plasmid (Promega Corp.), and designated pGL3/IGF-IR (-476/+640). A human IGF-II P3 promoter fragment (-574 to +136) cloned into the pGL2 luciferase promoter plasmid (Promega Corp.) was provided by L. Helman (NCI, Bethesda, MD) Transfection efficiencies were monitored by cotransfection with pCMVß-gal (CMV, cytomegalovirus; ß-gal, ß-galactosidase; CLONTECH Laboratories, Inc., Palo Alto, CA).

Two-hybrid plasmids
WT1 N-terminal domains (ClaI-AflIII fragments encompassing exons 1–7) corresponding to wild-type (with or without exon 5) or WAGR-associated RNA editing and Pro deletion mutants were cloned in-frame into the lexA DNA-binding domain plasmid pBTM116 that had been digested with EcoRI and filled in and into the pVP16 activation domain plasmid that had been digested with NotI and filled in (30). All constructs were verified by DNA sequencing.

Cell culture and transient transfections
Chinese hamster ovary (CHO) cells were grown in Ham’s F-12 nutrient medium with 10% heat-inactivated FBS at 37 C with 5% CO2. G401 cells were maintained in McCoy’s 5A medium supplemented with 10% heat-inactivated FBS. Transient transfections were carried out as follows. CHO or G401 cells were seeded in six-well plates at a density of 1 x 105 the day before transfection. The next day, variable amounts of expression plasmids (up to 5 µg) and 0.5 µg reporter constructs were cotransfected along with 0.5 µg pCMVß-gal for an internal control using lipofectin reagent (Life Technologies, Inc.) in OPTI-MEM reduced serum medium (Life Technologies, Inc.). The total DNA amount transfected was adjusted to 6.0 µg/well with empty pcDNA3 expression plasmid to prevent nonspecific squelching effects that could be ascribed to differences in the amount of CMV promoter between samples. Sixteen hours later, transfection medium was replaced with regular growing medium. Forty-eight hours posttransfection, cells were lysed with 150–200 µl luciferase assay buffer [1% (vol/vol) Triton X-100, 25 mM glycylglycine (pH 7.8), 15 mM MgSO4, and 4 mM EGTA]. Luciferase activity was measured by a luminometer (AutoLumat LB953, E.G.&G. Berthold) with 15–20 µl cell lysate, 300 µl luciferase assay buffer [25 mM glycylglycine (pH 7. 8), 15 mM MgSO4, 5 mM ATP, and 10 mM NaOH], and 100 µl luciferin solution (0.3%, wt/vol). For determination of ß-galactosidase activity, 30 µl of the lysate from the luciferase assay preparation was incubated with Z-buffer substrate [60 mM NA2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 4 mg/ml O-nitrophenyl-ß-D-galactopyranoside (ONPG), pH 7.0] for 1–6 h at 37 C. ONPG color conversion was determined on the basis of OD420. Luciferase assay data were normalized for ß-galactosidase, and luciferase levels in cells transfected with empty pGL3 vectors and insertless pcDNA3 were subtracted from experimental samples. Transfection of WT1 expression vectors did not have significant effects on the CMV promoter driving ß-galactosidase, as assessed by normalization of ß-galactosidase activities by the protein concentration of cell lysates. The luciferase data are shown as a percentage of the IGF-I receptor promoter activity in the control [i.e. cells transfected with pGL3/IGF-IR(-476/+640) and pcDNA3].

To assess the level of WT1 expression in these experiments, transiently transfected cells were lysed with Nonidet P-40 lysis buffer [1% (wt/vol) Nonidet P-40, 0.2% SDS, 150 mM NaCl, 20 mM Tris-Cl, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonylfluoride, 1 mM Na2VO4, 10 µg/ml leupeptin, and 10 µg/ml aprotinin], and 50–100 µg of the lysates were separated on SDS-PAGE, transferred to a nitro-cellulose membrane (Hybond ECL, Amersham Pharmacia Biotech, Arlington Heights, IL) using a wet transfer apparatus (Bio-Rad Laboratories, Inc., Richmond, CA). After blocking overnight at 4 C in 5% nonfat milk, membranes were incubated with an anti-WT1 antibody (C-19; 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at room temperature and then with horseradish peroxidase-conjugated donkey antirabbit IgG (1:5000; Amersham Pharmacia Biotech) for 1 h at room temperature. Between each step, membranes were washed three times in TBS-T [140 mM NaCl, 20 mM Tris-HCl (pH 7.6), and 0.1% (vol/vol) Tween-20] for 5 min each. WT1 protein was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).

Yeast two-hybrid assays
Transformations of yeast strains L40 (MATa trp1–901 leu2–3, 112His3{Delta}200 ade2 Lys::(lexAop)4-His3 URA3::(lexAop)8-lacZ) with pBTM116 plasmids and AMR70 (MAT{alpha}His3 Lys2 Trp1 Leu2 URA3::(lexAop)8-lacZ) with pVP16 plasmids (30) were carried out using lithium acetate. L40 and AMR70 transformants were selected by tryptophan and leucine prototrophy, respectively. Transformants were then mated, and the conjugates were plated on histidine-lacking plates with or without 1–40 mM 3-aminotriazole for 3-AT titration. Liquid ß-galactosidase assays were performed as described previously (31) on conjugants grown in tryptophan, leucine, and histidine drop-out medium.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential repression of IGF-IR promoter by splice variants of WT1
Although transcriptional repression by the form of WT1 that lacks alternatively spliced exon 5 and 9 sequences (-/-) has been demonstrated for many target genes, the relative specific repression activity of WT1 splice variants has not been examined in most cases. When increasing amounts of a pcDNA3 mammalian expression plasmid encoding WT1 (-/-) were transiently transfected into CHO cells along with a luciferase reporter construct containing nucleotides -476 to +640 of the rat IGF-I receptor promoter sequence [pGL3/IGF-IR (-476/+640)] and a pCMVß-gal plasmid as an internal control for transfection efficiency, WT1 protein driven by the pcDNA3 expression plasmid repressed IGF-IR promoter activity in a dose-dependent fashion (Fig. 3Go). These results replicate our previous data using pCB6+ constructs in transfected G401 cells (14, 15). Next, the effects of naturally occurring splice variants of WT1 were examined. Expression constructs encoding the four variants derived from the two alternative splice sites were transiently transfected into WT1-deficient G401 cells (Fig. 4Go, solid bars) and CHO cells (Fig. 3Go, dotted bars) in the same context as in Fig. 3Go. In both CHO and G401 cells, the +/- and -/- variants of WT1 repressed IGF-IR promoter activity by 80% compared with that in cells transfected with pcDNA3 vector alone (defined as 100%); however, transfection of the +/+ and -/+ variants resulted in approximately 40% repression of promoter activity. These results indicate that the presence or absence of the KTS insertion significantly affects the repression activity of WT1, presumably by disturbing DNA binding, as the KTS insertion is located between zinc fingers 3 and 4 that comprise the DNA-binding core. The relative repression activities of the +/+ and -/+ forms or the +/- and -/- forms were similar, implying that exon 5-encoded sequences do not influence IGF-IR promoter regulation by WT1, at least in the context of transient transfection assays.



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  		Figure 3. Dose-dependent IGF-I receptor promoter repression by WT1. Increasing amounts of a CMV promoter-driven WT1-/- expression plasmid [pcDNA3/WT1-/-] were transfected into CHO cells along with 0.5 µg of a luciferase reporter construct (pGL3) containing -476 to +640 nucleotides of the rat IGF-IR promoter sequence [pGL3/IGF-IR (-476/+640)] and 0.5 µg of a pCMVß-gal plasmid (CLONTECH Laboratories, Inc.) as an internal control for transfection efficiency. Cells were seeded at 1 x 105 in six-well plates 16 h before transfection. Transfection was carried out with lipofectin reagent (Life Technologies, Inc.) in OPTI-MEM reduced serum medium (Life Technologies, Inc.). Sixteen hours later, the medium was replaced with regular growing medium. At 44–48 h of posttransfection, the cells were processed for luciferase and ß-galactosidase assays. Luciferase data were normalized for ß-galactosidase activity. The experiment was performed in duplicate, and each experiment was repeated three times. Error bars represent the SDs (n = 2).

 


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  		Figure 4. Differential repression of IGF-I receptor promoter activity by WT1 splice variants. The WT1 gene encodes four naturally occurring splice variants: the presence or absence of exon 5 and the KTS insertion between zinc fingers 3 and 4. Five micrograms of pcDNA3/WT1 expression vectors were transiently transfected into the rhaboid tumor-derived cell line, G401 cells (solid bars) and CHO cells (dotted bars) along with 0.5 µg pGL3/IGF-IR (-476/+640) and 0.5 µg of pCMVß-gal plasmid. Luciferase data were normalized for ß-galactosidase activity. 100% represents the IGF-I receptor promoter-driven luciferase activity in CHO or G401 cells cotransfected with insertless pcDNA3 expression plasmid. The experiment was performed in duplicate, and each experiment was repeated three times. Error bars represent the SDs (n = 2).

 
Partial repression of IGF-I receptor promoter activity by WT1 domains
The WT1 protein is comprised of putative transcriptional regulatory domains in its N-terminus and zinc finger DNA-binding domains at the C-terminus. Although WT1 transcriptional repression is thought to be mediated by its binding to target DNA sequences, DNA-independent actions, presumably through protein-protein association, are also possible. To elucidate the role of individual WT1 domains in IGF-I receptor regulation, N- and C-terminal expression constructs were examined in reporter gene assays in CHO cells. The N-terminal portion of WT1 consists of 300 amino acids containing putative transcription regulatory domains, and the C-terminal WT1 fragment contains 130 amino acids, including four zinc-finger domains. As shown in Fig. 5Go, the C-terminal WT1 domains demonstrated different repression activities. Specifically, the +KTS C-terminus of WT1 (WT1C +KTS) exhibited only 20–25% repression, whereas the -KTS form (WT1C-KTS) exhibited nearly 60% repression, mimicking the data obtained with full-length constructs. Interestingly, the N-terminal domain itself (WT1N+exon 5) exhibited 50% repression activity. To verify that the observed activity of the WT1 domains was not a unique property of the pcDNA3 constructs, parallel studies were carried out using previously described pCB6+ vectors (28) encoding full-length WT1 (-/-), the WT1 N-terminus (without exon 5), or the WT1 C-terminus (without KTS). In multiple experiments, results similar to those described above were obtained, in that the C-terminal and N-terminal constructs repressed IGF-I receptor promoter activity by approximately 70% and 50%, respectively (data not shown). Additionally, cotransfection of CHO cells with N- and C-terminal pcDNA3 constructs and an IGF-II P3 luciferase reporter construct (in pGL2; provided by L. Helman, NCI) resulted in partial repression of promoter activity compared with that of full-length WT1 (data not shown). These results demonstrate that the residual activity seen with domain constructs was not unique to the IGF-I receptor promoter. The partial repression of both N- and C-terminal domains compared with full-length WT1 indicates that WT1 may exert its transcriptional suppression through both DNA binding and distinct protein-protein interactions involving the N-terminal (and, possibly, C-terminal) domains.



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  		Figure 5. Partial repression of IGF-I receptor promoter activity by WT1 N-terminal and C-terminal domains. Full-length WT1 lacking both exon 5 and the KTS insertion (WT1 (-/-)), an N-terminal domain containing exon 5 (WT1N +exon 5), and C-terminal domains with or without the KTS insertion (WT1C + KTS and WT1C -KTS) were examined for their transcriptional repression of the IGF-I receptor promoter. Five micrograms of expression plasmid, 0.5 µg pGL3/IGF-I receptor (-476/+640), and 0.5 µg pCMVß-gal were used for each transfection. Luciferase data were normalized for ß-galactosidase activity. The control represents the IGF-I receptor promoter-driven luciferase activity in CHO cells cotransfected with insertless pcDNA3 expression plasmid. The experiment was performed in duplicate, and each experiment was repeated three times. Error bars represent SDs (n = 2).

 
Effects of mutant and RNA-edited versions of WT1 on IGF-I receptor promoter repression
In addition to wild-type splice variants, a number of mutated forms of WT1 have been reported, and a posttranscriptionally modified form has been described. To determine the repression activity of these altered versions of WT1, transient transfection assays were performed with the WAGR-associated, Pro-rich domain deletion, and RNA-editing forms of WT1 (Fig. 6Go). The WAGR-associated mutant and RNA-editing forms of WT1 exhibited the same degree of repression as wild-type WT1-/-. The Pro-rich domain is one of the more distinct structural motifs found in WT1 and was originally speculated to play a role in gene regulation through protein-protein interaction. The Pro-rich deletion mutant exhibited repression activity at least as great as wild-type WT1-/-. In experiments using lower amounts of these expression vectors, the absolute degree of repression was less, but the activities of the mutant and RNA-edited forms were still equivalent to that of wild-type WT1.



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  		Figure 6. Effect of WAGR-associated mutant, RNA-editing, and Pro-rich domain deletion forms of WT1 in IGF-I receptor promoter repression. Five micrograms of wild-type or altered forms of WT1-/- expression vectors were transiently transfected into CHO cells along with 0.5 µg pGL3/IGF-I receptor (-476/+640) and 0.5 µg pCMVß-gal. Luciferase data were normalized for ß-galactosidase activity. The control is described in Fig. 5Go. The experiment was performed in duplicate, and each experiment was repeated three times. Error bars represent SDs (n = 2).

 
In DDS patients, mutations are commonly found in the zinc finger-coding region of one WT1 allele. In particular, a mutation of T1180 to C (amino acid Arg394 to Trp; R394W) in zinc finger 3 has been reported in 27 of 39 affected cases, making it the major missense mutation in DDS (26). DDS mutations function in an autosomal dominant fashion consistent with a dominant negative mode of action. The observations that WT1 could self-associate through its N-terminus and that the bacterially expressed DDS zinc finger domain lacks DNA-binding capability (32, 33) led to the idea that the DDS mutant behaves in a dominant negative fashion by interfering with the function of normal WT1 (34, 35). Unexpectedly, the R394W DDS mutant version of WT1 exhibited repression activity itself in a dose-dependent manner (Fig. 7Go). Both +/+ and -/- forms of the DDS mutant were moderately active (~40% repression at 5 µg) compared with wild-type +/+ and -/- versions of WT1 (30% and 70%, respectively). When wild-type and DDS forms of WT1 were cotransfected, increasing concentrations of the DDS mutant did not attenuate, but, rather, exhibited an additive effect on IGF-I receptor promoter repression. As shown in Fig. 8Go, transfection of 2 µg wild-type WT1+/+ and WT1-/- plasmids resulted in 20% and 60% repression, respectively, whereas cotransfection of increasing amounts of DDS mutant expression vectors (2–8 µg) with a fixed amount of wild-type WT1 plasmid (2 µg) resulted in additive effects on repression of IGF-I receptor promoter activity; this effect was more evident with the +/+ version of wild-type WT1, as the -/- version at 2 µg exhibited significant repression alone. Partial repression of the IGF-II P3 promoter construct described above was also seen with the DDS constructs (data not shown). Our findings that the DDS versions of the WT1 protein alone could repress the IGF-I receptor and IGF-II P3 promoters and did not attenuate the repression exerted by wild-type WT1 suggest that the DDS mutant form of WT1 may not function solely in a dominant negative manner, but may retain transcriptional regulatory activity different from that of the wild-type WT1 protein.



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  		Figure 7. Dose-dependent repression of IGF-I receptor promoter activity by the DDS-associated form of WT1. The DDS mutant contains a point mutation of T1180 to C, changing Arg349 to Trp in zinc finger 3. Increasing amounts (1–5 µg) of DDS plasmids (square, -/-; diamond, +/+) were transiently transfected into CHO cells along with 0.5 µg pGL3/IGF-I receptor (-476/+640) and 0.5 µg of the pCVMß-gal plasmid as an internal control for transfection efficiency. Wild-type WT1 variants are shown as a circle (-/-) and a triangle (+/+) at 5 µg for comparison. The control is described in Fig. 5Go. The experiment was performed in duplicate, and each experiment was repeated three times. Error bars represent SDs (n = 2).

 


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  		Figure 8. Lack of an inhibitory effect of DDS mutants in IGF-I receptor repression by wild-type WT1. Wild-type and DDS mutant forms of WT1 constructs were transiently cotransfected into CHO cells along with 0.5 µg pGL3/IGF-I receptor (-476/+640) and 0.5 µg pCMVßgal. A fixed amount (2 µg) of wild-type (+/+ or -/-) and increasing amounts (2–8 µg) of DDS expression plasmids (triangle, +/+; circle, -/-). Total DNA amount was adjusted to 10 µg with empty pcDNA3 plasmid. Wild-type variants are shown as a square (-/-) and a diamond (+/+) at 2 µg for comparison. The control is described in Fig. 5Go. The experiment was performed in duplicate, and each experiment was repeated three times. Error bars represent SDs (n = 2).

 
Expression of transfected WT1 proteins in CHO cells
To verify that the differential activities seen in the transient transfection data described above reflected intrinsic differences in the activities of the various versions of WT1 protein, the expression level of various forms of WT1 in CHO cells was examined using Western immunoblotting. Approximately 40–50 µg of all lysates were separated on a 10% SDS-PAGE gel. The expected size for full-length WT1 is 53–55 kDa, with a variation of 2–3 kDa due to the presence or absence of the exon 5 and KTS insertions. As shown in Fig. 9Go, all of the versions of WT1 protein analyzed were expressed at similar levels.



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  		Figure 9. Expression of exogenous WT1 proteins in CHO cells. Five micrograms of wild-type and mutant WT1 expression plasmids were transiently transfected into CHO cells along with 0.5 µg of the pCMVßgal construct as an internal control. Forty-four to 48 h posttransfection, cells were lysed. After normalization for ß-galactosidase activity, which did not significantly differ among samples, approximately 40–50 µg protein were separated by 10% SDS-PAGE. The proteins were transferred to nitro-cellulose membranes (Amersham Pharmacia Biotech). WT1 proteins were detected by an anti-WT1 antibody, C19, that recognizes the C-terminus of WT1. WT1 proteins are at 45–53 kDa. Bands at 60–65 kDa represent nonspecific binding.

 
Self-association of wild-type and mutant forms of WT1
Previous studies have demonstrated that WT1 can self-associate through its N-terminal region (35, 36, 37), and it has been suggested that this interaction may play a role in WT1 function. To address the potential differential self-association capability of wild-type and mutant forms of WT1, the yeast two-hybrid system was employed. N-terminal regions of wild-type, WAGR-associated, RNA editing, and Pro-rich deletion forms of WT1 were fused to LexA DNA-binding and VP16 transcriptional activation domains, and potential association was assessed by interaction mating analyses (38). First, yeast conjugants expressing various combinations of WT1 N-terminal domains were examined for ß-galactosidase activity (Fig. 10Go). Wild-type conjugants exhibited similar levels of ß-galactosidase activity regardless of the presence or absence of exon 5, indicating that exon 5 sequences do not appreciably influence protein-protein interaction. The conjugants expressing both wild-type and mutant forms of WT1, including WAGR-associated, RNA editing, and Pro-rich deletion forms exhibited ß-galactosidase activities similar to those of wild-type/wild-type conjugants. This result suggests that mutant WT1 N termini could still interact with wild-type N-termini. However, conjugants expressing only mutant forms of WT1 exhibited significantly reduced ß-galactosidase activity similar to that of the lamin C-negative control conjugants, indicating that self-association is disrupted by these alterations of the WT1 N-terminus.



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  		Figure 10. Quantitation of ß-galactosidase activity produced in yeast conjugates expressing fusion proteins containing wild-type and altered forms of WT1 N termini. N-terminal domains of wild-type and altered forms of WT1 containing the first 300 amino acids were fused to both the LexA DNA-binding domain and the VP16 transcriptional activation domain. Conjugants from various combinations were grown in histidine-lacking medium and subjected to ß-galactosidase assays. Mixed fusions used WT+ in pBTM116 and WT-, WAGR+, RNA-editing, and Pro-rich deletion mutants in pVP16. The activity was measured on the basis of color change of the ONPG substrate. The basal level of ß-galactosidase activity obtained in matings with lamin C is 1. A + or - indicates the presence or absence of exon 5. The experiment was repeated three times. Error bars represent SDs (n = 2).

 
Histidine synthase, the other reporter gene driven in the yeast two-hybrid system, was also used to assess WT1 self-association. 3-AT is a inhibitor of histidine synthase, and the strength of histidine prototrophy can be titrated by adding increasing concentrations of 3-AT. The conjugants used in the ß-galactosidase assays described above were examined using 3-AT titration (Fig. 11Go). Yeast conjugants containing wild-type WT1 were viable at a 3-AT concentration as high as 40 mM, indicating WT1 self-association. However, conjugants expressing both wild-type and altered forms of WT1 (WAGR-associated and RNA-edited forms) were only able to grow in significantly decreased concentrations of 3-AT (10 mM), implying disruption of self-association. Conjugants transformed with WAGR-associated and RNA-edited forms of WT1 were only viable in 10 or 20 mM 3-AT. The growth of Pro-rich deletion mutant-containing conjugants was abrogated at a low concentration of 3-AT (2 mM), indicating severely decreased self-association. No conjugates with the lamin C-negative control grew on plates supplemented with 3-AT. These results correlate well with the ß-galactosidase assay data described above and illustrate that mutations and deletions in the N-terminal domain of WT1 severely reduce self-association ability when both interacting fusion proteins carry the same alteration.



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  		Figure 11. Titration of growth of yeast transformed with wild-type and mutant versions of WT1 with 3-AT. N-Terminal domains of wild-type and altered forms of WT1 containing the first 300 amino acids were fused to both the LexA DNA-binding domain and the VP16 transcriptional activation domain. Conjugants from various combinations were plated out on histidine-lacking plates containing various concentrations of 3-AT, an inhibitor of histidine synthase. Mixed fusions used the WT1 N terminus (+exon 5) cloned into pBTM116 and WAGR, RNA-editing, and Pro-rich deletion N termini (+exon 5) cloned into pVP16. The values indicated are the highest concentration of 3-AT at which growth was detected after 2–3 days. A + or - indicates the presence or absence of exon 5. The experiment was repeated four times with identical results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Differential activity of naturally occurring WT1 variants and WT1 domains in the regulation of IGF-receptor gene expression
In this study, we examined the potential differential activity of WT1 variants in IGF-I receptor promoter repression. Transient transfection assays demonstrated that the +/- and -/- forms of WT1 repressed IGF-I receptor promoter activity more strongly than the +/+ and -/+ forms in CHO and G401 cells, suggesting that the presence or absence of the exon 5-encoded sequence has no effect, but that the presence of the KTS insertion between zinc fingers 3 and 4 influences transcriptional activity. This observation was expected, as differential repression activity of WT1 variants has been demonstrated with other gene promoters, including retinoic acid receptor-{alpha} (RAR{alpha}) (39), transforming growth factor-ß1 (40), platelet-derived growth factor (PDGF) A chain (41), Pax-2 (42), and WT1 (43). These reports illustrate that -KTS forms of WT1 have higher repression activity regardless of the presence or absence of the exon 5 sequence, presumably due to disruption of DNA-binding capability by the presence of the KTS insert. In support of this idea, DNA binding studies with these WT1 variants with the presence or absence of the KTS insertion using synthetic oligonucleotides (44) and native sequences found in the PDGF A chain gene promoter (21) and in the Pax-2 gene promoter (42) demonstrated that the +KTS form is less capable of binding DNA. Another type of naturally occurring WT1 variant results from RNA editing (24). The RNA-edited form of WT1 was shown to exhibit reduced repression of the Egr-1 gene promoter. However, the RNA-edited form of WT1 exhibited efficient repression of IGF-I receptor promoter activity.

In addition, individual WT1 domains were examined for IGF-I receptor promoter repression. Interestingly, the N- and C-terminal domains of WT1 both exhibited partial repression activity. The repression observed was 50% with the N-terminal domain and 30–60% with the C-terminal domain that contained or lacked the KTS insertion, respectively. This observation was unexpected, as N- and C-terminal domains of WT1 appeared to lack activity with the promoters of other putative WT1 target genes, including PDGF A chain (41), Egr-1 (28), RAR{alpha} (39), and novH (45). The lack of an effect of the N-terminal domain in some previous reports may have reflected the need for efficient nuclear targeting, as the nuclear localization sequence (NLS) of WT1 resides in the zinc finger domain (46). Our pcDNA3 constructs incorporated a simian virus 40-derived NLS, and the pCB6+ N-terminal construct encompassed the first zinc finger domain that comprises part of the WT1 NLS. The differences between our data and those obtained with the Egr-1 (28) and RAR{alpha} (39) promoters using constructs that contained partial WT1 NLS domains may reflect promoter- or cell type-specific effects. The effect of WT1 C-termini may also be to some extent promoter or cell type specific, as similar C-terminal constructs did not significantly affect the activity of the Egr-1 (28), PDGF A chain (47), and novH (45) promoters. However, the IGF-I receptor promoter is distinguished by the presence of multiple WT1-binding sites in both the 5'-flanking and 5'-UTR regions that contribute to repression by full-length WT1 (15), and the IGF-II P3 promoter that exhibited partial repression by the WT1 C-terminus also contains multiple WT1-binding sites that flank the transcription start site (48).

Our results suggest that there are several potential mechanisms through which WT1 may function in the context of IGF-I receptor promoter regulation. The effect of full-length WT1 is clearly influenced by the presence or absence of the KTS insert that modulates the affinity of WT1 for its cognate recognition sites on DNA. The partial repression seen with the C-terminus is also influenced by the KTS insert, suggesting that DNA binding per se may be one aspect of WT1 action, possibly by displacement of Egr-1-like factors that also recognize the GC-rich and (TCC)n motifs bound by WT1, or Sp1, which binds to multiple sites in the IGF-I receptor promoter, some of which overlap WT1-binding sites (15, 22). It is also conceivable that the WT1 C-terminus may exert some effects through protein-protein interaction with factors necessary for IGF-I receptor promoter activity, as zinc finger domains of the type found in WT1 are increasingly seen as responsible for protein-protein association as well as DNA binding (49). The partial repression seen with the WT1 N-terminus presumably involves an interaction with other transcriptional factors that are involved in IGF-I receptor promoter activity. These may include proteins previously demonstrated to interact with WT1, such as p53, Par-4, and Sumo-1 (20), or other, yet to be identified, proteins. This type of mechanism may also be relevant to the activity seen with the DDS version of the WT1 protein, as described below. It is probable that the relative contributions of these mechanisms to WT1 function may be target gene and cell type specific.

IGF-I receptor promoter repression by mutant forms of WT1
An examination of WT1 repression of IGF-I receptor gene promoter activity was extended to clinically derived and deletion forms of WT1. The WAGR mutation (substitution of Gly201 with Asp) exhibited 80% repression, which was similar to that seen with wild-type WT1. This is an intriguing observation, because Park et al. (25) demonstrated that this substitution converts the WT1 protein from a transcriptional repressor to an activator in the context of Egr-1 promoter regulation. In addition, a WT1 construct deleted in one of the Pro-rich clusters was examined in the same context. The Pro-rich deletion exhibited maximum repression activity among the WT1 constructs examined in this study. A similar Pro-rich deletion construct (deletion of amino acids 56–67) exhibited reduced repression of Egr-1 promoter activity (28). These results demonstrate that these alterations in the N-terminal region do not disrupt, but, rather, enhance, the WT1 transcriptional repression potential in the context of IGF-I receptor gene regulation and provide further evidence that the effects of various WT1 domains may vary with different target genes. The ability of these mutant versions of the WT1 protein to repress IGF-I receptor promoter activity is not inconsistent with the proposed role of IGF-I receptor gene up-regulation in Wilms’ tumorigenesis (17). Of these, only the WAGR-associated mutation has been seen in the context of Wilms’ tumor, and the patient from which the mutation was isolated carried a deletion of the other 11p13 locus, so that the WAGR-associated mutant version of the WT1 protein was functioning in a situation equivalent to WT1 haploinsufficiency. Thus, it would be difficult to ascribe the presence of Wilms’ tumor solely to the point mutation in the remaining allele.

Self-association is not required for IGF-I receptor repression by WT1
Previous reports of WT1 self-association defined regions responsible for the interaction to the first 182 amino acids (35, 37). To determine the effects of the WT1 mutations analyzed in this study on self-association activity, WT1 self-association assays were performed with N-terminal domains of wild-type and altered forms using the yeast two-hybrid system. Homologous combinations of WAGR-associated, RNA-editing, and Pro-rich deletion forms of WT1 N-terminal domains exhibited severely reduced self-association capability. This might be due to a redistribution of charge (WAGR-associated: Gly to Asp) or spatial hindrance (RNA-editing: Leu to Pro). Deletion of the Pro-rich region may have removed a critical domain required for self-association or may have induced a profound conformational change. As these mutations did not affect IGF-I receptor promoter repression, as shown above, these data strongly suggest that WT1 self-association is not absolutely required for IGF-I receptor promoter repression. A role for self-association in WT1 gene regulation has been suggested in another context by Reddy et al. (35), who concluded that self-association was important for WT1 function, because WT1 transcriptional activation using an artificial promoter construct, Egr3tkCAT, which contains three Egr-1-binding sites fused to the human tymidine kinase promoter and the chloramphenicol acetyltransferase gene, was disrupted by mutant WT1 proteins that lacked DNA-binding capability but could still self-associate. This study, however, used a synthetic promoter construct and involved transcriptional activation by WT1, an effect not seen with all native promoters of WT1 target genes. Moffett et al. (37) demonstrated that the WAGR-associated mutation had no effect on the self-association capability of WT1 by assessing wild-type and WAGR mutant WT1 proteins as a heterodimer using Far Western blotting. However, this WAGR mutation was found in a patient that has one allele of 11p13 deleted and the other containing the mutation. In this context, only mutant dimers or oligomers would be formed. In fact, differential behavior of heterodimers with wild-type/mutant forms of WT1 was observed. The self-association capability of heterodimers was drastically disrupted as determined by 3-AT titration, but not in ß-galactosidase assays. Physical interaction between wild-type and mutant forms of WT1 may occur, and this interaction may be strong enough to drive the ß-galactosidase reporter gene, but not the histidine synthase gene.

DDS mutant regulation of the IGF-I receptor gene
DDS mutations function in a autosomal dominant fashion (26). Moffett et al. (37) demonstrated that the most common DDS mutant (R394W) itself does not have repression activity and interfered with the function of wild-type WT1 in a cotransfection study with RAR{alpha} promoter constructs. More recently, Holmes et al. (34) reported that a short N-terminal fragment of WT1 inhibits transcriptional activity of the wild-type protein, but retains self-association capability. These findings suggested that the DNA binding-deficient DDS mutant probably functions in a dominant negative fashion. To evaluate that idea, the DDS mutant (R394W) was examined for regulation of IGF-I receptor promoter activity. Unexpectedly, the DDS mutant protein functions in this context. Both +/+ and -/- forms of DDS showed the same degree of repression (~50%). The DDS mutant in conjunction with wild-type WT1 exhibited an additive effect, further supporting the functional activity of the DDS mutant protein in the context of IGF-I receptor gene regulation. Therefore, we propose that the DDS mutant protein regulates the IGF-I receptor gene differently from wild-type WT1 and potentially recognizes novel target genes. Differential regulation of target genes by wild-type WT1 and the DDS mutant protein is consistent with the clinical differences between Wilms’ tumor and DDS.

This dominant function of the DDS mutant may require DNA binding, with the DDS mutant binding to novel target sequences (gain of function). Previously, the DDS mutant (R394W) protein produced in bacteria as a fusion protein or in an in vitro translation system using a rabbit reticulocyte lysate was unable to bind to the Egr-1 consensus binding sequence (32, 33). In addition, it was demonstrated that the bacterially produced zinc finger domain protein of the DDS mutant did not identify obvious DNA binding motifs using modified Egr-1 consensus recognition sequences, even though it might have slightly different binding properties from those of wild-type WT1. As these studies employed the bacterially produced protein, they may not reflect the biological activity of the full-length protein because of an aberrant conformation of the protein resulting from renaturing and refolding procedures in preparation or a lack of posttranslational modification. Our current findings are consistent with those of Vicanek et al. (50), who found that transfection of a similar DDS construct into HEK293 cells repressed the activity of an EGFR promoter reporter. Therefore, it is possible that the DDS mutant protein could bind to novel target sequences. Alternatively, if the DDS mutant protein does not retain DNA-binding capability, it might exert transcriptional regulation through its N-terminal domains. The similar degree of transcriptional repression seen with the +/+ and -/- forms of the DDS mutant protein is consistent with this idea.

In summary, we have demonstrated that alternative splicing of exon 5 does not influence WT1 repression of the IGF-I receptor promoter, whereas the presence of the KTS insert encoded by exon 9 reduces repression significantly. Although these data suggest that DNA binding is a major component of WT1 action on the IGF-I receptor promoter, the activity seen with +KTS versions of WT1 and with WT1 N- and C-termini are consistent with the idea that WT1 may function through additional mechanisms, such as RNA binding (51) or protein-protein associations involving N- and/or C-terminal domains. A comparison of the effects of several N-terminal mutations on transcriptional repression and on self-association in yeast suggests that WT1 self-association is not obligatory for repression of the IGF-I receptor promoter. Finally, our data suggest that the DDS version of the WT1 protein may represent a gain of function rather than operating solely through a dominant negative mechanism that would involve heterodimerization. It will be of interest to determine whether there are novel target genes that are regulated by the DDS protein that contribute to the nephropathy invariably associated with this syndrome.


   Acknowledgments
 
We thank Scott Kuhn for expert technical assistance, Dr. B. Druker for advice on two-hybrid assays, Dr. F. J. Rauscher III for the original WT1 expression vectors, and Dr. L. J. Helman for the IGF-II P3 promoter construct.


   Footnotes
 
1 This work was supported by grants from the Medical Research Foundation of Oregon and the NIH (DK-50810; to C.T.R.). Back

2 Present address: Yamanouchi Pharmaceuticals, Inc., Tokyo 103, Japan. Back

Received May 3, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Ataxia-Telangiectasia Mutated Gene Controls Insulin-Like Growth Factor I Receptor Gene Expression in a Deoxyribonucleic Acid Damage Response Pathway via Mechanisms Involving Zinc-Finger Transcription Factors Sp1 and WT1
Endocrinology, December 1, 2004; 145(12): 5679 - 5687.
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 J. Biol. Chem.Home page
G. Idelman, T. Glaser, C. T. Roberts Jr., and H. Werner
WT1-p53 Interactions in Insulin-like Growth Factor-I Receptor Gene Regulation
J. Biol. Chem., January 24, 2003; 278(5): 3474 - 3482.
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 EndocrinologyHome page
S. E. Damon, S. R. Plymate, J. M. Carroll, C. C. Sprenger, C. Dechsukhum, J. L. Ware, and C. T. Roberts Jr.
Transcriptional Regulation of Insulin-Like Growth Factor-I Receptor Gene Expression in Prostate Cancer Cells
Endocrinology, January 1, 2001; 142(1): 21 - 27.
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 JNCI J Natl Cancer InstHome page
H. Yu and T. Rohan
Role of the Insulin-Like Growth Factor Family in Cancer Development and Progression
J Natl Cancer Inst, September 20, 2000; 92(18): 1472 - 1489.
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A. Hossain and G. F. Saunders
The Human Sex-determining Gene SRY Is a Direct Target of WT1
J. Biol. Chem., May 11, 2001; 276(20): 16817 - 16823.
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