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Male Reproductive Tract |
Department of Biochemistry and Molecular Biology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030-4009
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene regulation, male reproductive tract, mullerian ducts
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A zinc finger containing DNA-binding protein, WT1 acts as a transcriptional activator or repressor depending on the cellular or chromosomal context [13, 14]. It has four major isoforms because of the insertion of three amino acids (KTS) between zinc fingers 3 and 4 and the insertion of an alternatively spliced, 17-amino acid segment encoded by exon 5 in the middle of the protein [15]. These four isoforms are conserved among mammals. Recent reports have described specific in vivo function of the different isoforms of WT1 in embryonic development. Severe defects in kidney and gonads are found in mice lacking the WT1(-KTS) isoforms, and mice lacking the WT1(+KTS) isoform show defects of kidney and male-to-female sex reversal [16]. Natoli et al. [17] reported that mice carrying a deletion of exon 5 have no developmental defects and are fertile.
WT1 binds to the highly GC-rich canonical early growth response gene-1 DNA-binding motif [13]. Many genes are proposed to be regulated by WT1. Specific mutations in WT1 cause two different types of genitourinary abnormalities, Denys-Drash syndrome (DDS) [18] and Frasier syndrome [19]. Besides kidney abnormalities, patients often showed male-to-female sex reversal, male pseudohermaphroditism, and cryptorchidism. Most of the missense mutations in the WT1 gene found in patients with DDS are in DNA-binding zinc finger domains of WT1 [20, 21]. If pseudohermaphroditism in patients with DDS is caused by the deregulation of MIS gene activation by WT1, then most likely WT1 needs to bind DNA to regulate the MIS promoter. To understand the pathogenesis of DDS, it is important to know whether WT1 regulates the Mis promoter. The role of SOX9 with WT1 in this process is not clear. In the present report, we provide evidence that WT1 can activate the murine Mis gene in the presence of SOX9 as well as SF1 and that WT1 must bind DNA to do that.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression vectors, pcDNA3WT1 (-KTS) and pcDNA3WT1 (+KTS), were made by polymerase chain reaction (PCR) amplification of wild-type WT1 from pCB6WT1-/- and pCB6WT1+/+, respectively. The resulting PCR products were subsequently subcloned into a modified pcDNA3 vector [22] harboring the 5' untranslated region of the herpes simplex virus-thymidine kinase gene. All WT1 mutants were created from this vector, pcDNAWT1 (-KTS), by site-directed mutagenesis, and the mutations were verified by sequencing.
The 203-base pair (bp) Mis upstream sequence that drove transcription of the luciferase gene was amplified by PCR from mouse Mis (nucleotides -180 to +23) and cloned in front of the luciferase gene in the vector pGL3 basic (Promega, Madison, WI). The construct was designated -180MISP. All site-directed mutagenesis of the promoter constructs was performed with the Quick-change site-directed mutagenesis kit (Stratagene, La Jolla, CA). All constructs were confirmed by sequencing from both directions.
Cell Culture and Transfection
HeLa and TM4 cells were grown at 37°C in Dulbecco modified Eagle medium-Ham F-12 supplemented with 10% fetal calf serum in 5% CO2. The cells were seeded at a density of 50 000–70 000 cells/well in 12-well plates 16–18 h before transfection. The cells were cotransfected with expression and reporter plasmids as indicated in the figure legends. The plasmidCMV-ß-galactosidase (10 ng) was cotransfected as an internal control to normalize for differences in transfection efficiency. The transfections were performed with Lipofectamine-Plus reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations, and the cells were harvested after 40–48 h. Luciferase activity was measured with a luciferase assay kit (Tropix) and a Lumat LB9507 luminometer (EG&G Berthold). ß-Galactosidase was measured with the Galacto-Light plus kit (Tropix).
Statistical analyses performed on transfection data included a two-way, paired analysis of variance followed by Student-Newman-Keuls analysis with a 95% confidence score. These analyses were performed using STATISTICA 6 program (Stat Soft, Tulsa, OK).
Gel Shift Assay
Gel shift reactions were performed in a total volume of 20 µl on ice. Radiolabeled probes were prepared by end labeling with [
-32P]ATP, and 100 pmol of each labeled probe and 2.5 µl of in vitro-translated (IVT) protein were used for each reaction. For competition of wild-type and mutant oligonucleotides, a 50- or 100-fold excess of unlabeled oligonucleotides was added to the reaction mixture before addition of the labeled probe. Thirty minutes later, the reaction mixture was loaded onto a 5% polyacrylamide gel in Tris-glycine buffer, and electrophoresis was performed at 150 V for 3 h. In the supershift with antibody assay, reaction mixture without labeled probe was incubated with 2.0 µg of rabbit anti-WT1 antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) for 15 min at room temperature, and labeled probe was added with further incubation on ice for 30 min.
Western Blot Analysis
Whole-cell extracts were prepared with cell lysis buffer consisting of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1.0% NP-40, 1 mM dithiothreitol, 1 mM PMSF, and 5 µg/ml each of aprotinin, leupeptin, and benzamidine. Western blots were developed by enhanced chemiluminescence (Amersham, Piscataway, NJ). The primary and secondary antibodies used were N-terminal-specific rabbit anti-WT1 at a 1:100 dilution (N180; Santa Cruz Biotechnology), anti-rabbit immunoglobulin (Ig) G conjugated with peroxidase (Amersham).
Chromatin Immunoprecipitation Assay
Formaldehyde was added to TM4 cells (1 x 107) at a final concentration of 1%. Fixation was allowed to proceed at room temperature for 15 min and was stopped by addition of glycine to a final concentration of 0.125 M. The cells were then washed with PBS and collected by centrifugation. Next, the cells were incubated with buffer A (10 mM potassium acetate, 15 mM magnesium acetate, and 0.1 mM Tris [pH 7.4] with Roche protease inhibitor cocktail; Roche Applied Science, Indianapolis, IN) on ice for 20 min and homogenized with a dounce homogenizer. The nuclei were collected by centrifugation, resuspended in sonication buffer, and incubated on ice for 15 min. The samples were sonicated on ice with an sonicator (Fisher model 100 Sonic Dismembrator; Fisher Scientific, Inc., Pittsburgh, PA) at a setting of 10 for six 20-sec pulses to an average length of approximately 1000 bp (confirmed by electrophoresis) and then microcentrifuged. The chromatin solution was precleared with protein A sepharose for 15 min at 4°C. Immunoprecipitations (IP) were performed overnight at 4°C using 1 µg of anti-WT1 antibody (C-19). After the final ethanol precipitation, each IP sample was resuspended in 30 µl of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (TE). Total input chromatin samples were resuspended in 30 µl of TE and further diluted to 1:100. Each 50 µl of PCR reaction mixture contained 5 µl of IP sample; 1.5 mM MgCl2; 50 ng of each primer; 300 µM each of ATP, dGTP, dCTP, and dTTP; 1x PCR buffer (PerkinElmer, Wellesley, MA); and 1.25 U of Taq DNA polymerase (PerkinElmer). After 35 cycles of amplification, 5 µl of the PCR products were electrophoresed on a 1.5% agarose gel, and the DNA was stained with ethidium bromide and visualized under ultraviolet light. The sequences and positions (parentheses) of primers used for PCR were as follows: MisPF, (-182) CAG GCC TCT GCA GTT ATG; MisPR, CAT GGT GGT ACA GCA AGG (+10).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We next examined the contribution of SF1 to WT1 and SOX9 activation of the Mis promoter. The SF1 was cotransfected with different combinations of WT1 and SOX9 and the presence of a reporter gene into HeLa cells. Alone, SF1 also could not activate the reporter gene in HeLa cells; however, SF1 could activate the reporter gene in the presence of either WT1 or SOX9. The WT1-SOX9 combination was the most potent activator, followed by WT1-SF1 and SOX9-SF1. Maximum luciferase reporter gene expression was achieved by all three of these factors together (Fig. 3A). These results indicate that neither factor alone is sufficient to activate the Mis promoter. At least two factors are needed, and maximal activation is achieved with all three factors. The synergistic activation of the Mis promoter by WT1 in combination with either SOX9 or SF1 was found to be statistically significant, with a confidence value of 0.05. The activation of the Mis promoter by WT1, SF1, and SOX9 when transfected alone was variable in many independent experiments, and the differences were not always statistically significant (Figs. 1–3 and data not shown). From these results, we concluded that WT1 can act as a cofactor with SOX9 as well as with SF1, at least in activation of the Mis promoter. However, WT1(+KTS) isoforms, either alone or in the presence of SOX9 or SF1, could not activate the Mis promoter (Fig. 3B). These results are consistent with the hypothesis that -KTS isoforms are involved in transcriptional activity, whereas +KTS isoforms are associated with the spliceosome and are involved in the regulation of certain genes at the posttranscriptional level (i.e., Sry [23]).
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The DDS phenotype often arises from the alteration of one allele by a missense point mutation, usually in the zinc finger DNA-binding domain of WT1 [20]. Most WT1 mutations found in patients with DDS cause loss of the DNA-binding ability of WT1 [20]. We cotransfected several mutant WT1s with either SOX9 or SF1 and reporter constructs containing the Mis promoter. One group of mutants (C330Y, R366H, D396N, H377Y, and R394W) contained changes in the zinc finger region, and other mutants (S273A, F154S, and P180S) had changes outside that region. The most common DDS mutants (D396N, R366H, H377Y, and R394W) failed to synergistically activate the Mis promoter with SOX9 or SF1 (Fig. 4, A and B). In contrast, WT1 with a mutation outside the zinc finger region (S273A, P180S, and F154S) had a cotransactivational potential similar to that of wild-type WT1. Another mutant (C330Y) retained some transactivation ability even though its mutation was in the zinc finger region. This mutant behaves similarly in the activation of the SRY promoter [21]. Western blot data showed that all the mutant proteins were expressed at similar levels (Fig. 4C).
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The SOX9-binding site is required for Mis gene activation in vivo [8]. Although the SF1-binding site is essential for the activation of the Mis promoter in vitro [12], mutation in this binding site decreased expression of the MIS, but mullerian ducts regressed normally [8]. Nachtigal et al. [12] have shown that WT1 can synergistically activate the Mis promoter with SF1. They also reported that WT1 DNA binding is not required for the activation of this promoter [12]. However, most patients with DDS are pseudohermaphrodites because of a mutation in the DNA-binding domain of WT1 [20], and mutations within the zinc finger DNA-binding region abrogate transactivation of the Mis promoter by WT1, indicating that at least some WT1 DNA binding is required to activate the Mis promoter (Fig. 4, A and B).
To resolve this apparent contradiction, we re-examined whether DNA binding of WT1 is essential. We identified the region responsible for WT1-mediated activation of this promoter. Because the Mis promoter is quite small, we used a site-directed mutagenesis approach to identify the WT1 DNA-binding site in the Mis promoter. No consensus WT1 DNA-binding site was found in the 180-bp Mis promoter (Fig. 5A). However, one GC-rich region (caggcccagggccac) was similar to the WT1 consensus site near the transcription start site of the Mis gene. To test the functional significance of this site, we introduced mutations at several positions (Fig. 5B), and these constructs were assayed for their responsiveness to WT1 in HeLa cells and compared with the wild-type construct (-180MISP). A single A-to-G substitution in the second or 14th position did not significantly reduce reporter gene activity. An A-to-G substitution at both those positions reduced reporter gene activity by 50%, and replacement of the A at the eighth position with a G reduced reporter gene activity by 40%. Substitution of all three As with Gs almost completely blocked transactivation by WT1. Because this putative WT1-binding site was very close to the transcription initiation site, it is possible that it interfered with the basal promoter activity. However, that was not the case, because these base substitutions did not affect the activation by SOX9 and SF1 (Fig. 6).
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The WT1-responsive sequence was further characterized by gel shift assays. The gel shift assay in Figure 6 shows that WT1 synthesized in vitro bound to a 32P-labeled, 16-bp, WT1-binding site oligonucleotide. Binding was competed by unlabeled wild-type probe (Fig. 7, lanes 4 and 5). The mutants, which showed complete (M5), partial (M2 and M4), or no reduction (M1 and M3) in reporter gene activity, also competed accordingly with the wild-type probe (Fig. 7, lanes 6–10). A WT1 N-terminal-specific antibody (Fig. 7, lane 11) substantially reduced binding of the specific WT1-DNA complex. This binding site was further characterized by gel shift assays (Fig. 8). Two overlapping binding sites are found in the Mis promoter. The WT1 can bind to the half-binding site (cagggccac) in the Mis promoter (Fig. 8, lane 2) but can compete with wild-type probe (Fig. 8, lane 4). The IVT WT1 did not bind to mutant binding sites (M4 and M5) and also showed loss of cotransactivational properties in reporter gene assays (Fig. 6). However, the mutants M1, M2, and M3 showed some degree of to full reporter gene activity by WT1 in the presence of SF1 or SOX9, and WT1 was also able to bind in these DNA sequences (Fig. 8). These data suggest that the WT1-binding sequence we identified in the Mis promoter is required for binding of WT1 to the Mis promoter and for WT1-mediated transactivation of the Mis promoter.
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To explore the mechanism of DDS mutants, the DNA-binding abilities of DDS mutants were assessed by gel shift assays. Mutations in the DNA-binding region of WT1 caused loss of DNA-binding ability (Fig. 9, lanes 5–9 and 11) and concomitant loss of transactivational properties. Although mutant D396N retained some of its DNA-binding ability (Fig. 9, lane 11), it showed loss of transactivational activity (Fig. 4, A and B). However, the mutant C330Y, which has a mutation in the zinc finger DNA-binding region, retained some cotransactivational ability as well as DNA-binding ability (Fig. 9, lane 5). This implies that the cysteine in codon 330 in WT1 is not so crucial for DNA binding, although the mutant did lose some of its activity. On the other hand, mutations outside the DNA-binding region did not have major effects on DNA binding to the Mis promoter (Fig. 9, lanes 4, 9, and 10). Interestingly, mutations outside the zinc finger region of WT1 are rarely seen in patients with DDS. These data clearly indicate that some of the DDS phenotype arises from the loss of DNA-binding ability of WT1 because of the mutations in the zinc finger region.
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To address more fully the possibility of WT1-mediated regulation of Mis gene expression, we used the chromatin immunoprecipitation assay to determine whether WT1 bound the endogenous Mis promoter. We choose the TM4 cell line, because it expresses a significant amount of endogenous WT1 (Fig. 10A) and Mis (data not shown). Analysis by PCR of formaldehyde cross-linked chromatin from TM4 cells immunoprecipitated with antibodies specific to the C-terminal region of WT1 revealed that WT1 bound to the Mis promoter sequences (220-bp band) (Fig. 10B), whereas rabbit IgG antibody controls did not. As a negative control, PCR was done using the Pax6-specific primers to show that WT1 antibody specifically immunoprecipitated only the Mis promoter-bound Wt1-DNA complex and not the nonspecific genomic DNA (Fig. 10B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We were unable to detect an interaction between WT1 and SOX9 by coimmunoprecipitation and the yeast two-hybrid system. It is possible that WT1 and SOX9 and SF1 do not interact directly, although they bind in very close proximity on the Mis promoter. Alternatively, WT1 may bind to another, yet-to-be-identified cofactor, and only that complex may associate with SOX9 and regulate the Mis promoter. The WT1-binding site in the Mis promoter is between the TATA box and the transcription start site. The WT1 may help to form the transcription initiation complex in the Mis promoter, and SOX9 may just allow WT1 to bind to its cognate binding site. Further investigation is needed to determine which of these hypotheses is correct.
The role of WT1 in sex determination and differentiation is multiple in both mice and humans. The WT1 plays many roles in each step of this process. Different isoforms of WT1 also have very different roles in the process. It has been shown recently that WT1-KTS isoforms also activate the SF1 promoter [25]. Different isoforms of WT1 are also involved in regulation of SRY, DAX1, and MIS [12, 16, 21, 26]. These observations indicate the importance of isoforms of WT1 in different stages of mammalian sex determination and differentiation. Involvement of the WT1(-KTS) isoforms in up-regulation of the SRY promoter [21] and of the WT1(+KTS) isoform in stabilization of the SRY transcript is linked to sex reversal in patients with DDS [16]. This syndrome has variable phenotypes, occurs only in men, and is most likely caused by the haploinsufficiency of WT1. The MIS as well as androgen receptors may be deregulated for this phenotype in patients with DDS. The MIS gene is a good candidate gene for this phenotype in patients with this syndrome, both because WT1 is expressed in Sertoli cells during the production of the MIS and because its promoter is regulated by WT1 [12, 27]. We propose here that Mis meets all the criteria of a true WT1 target gene. Expression of the Mis in Sertoli cells during mullerian duct regression is closely correlated with WT1 expression [27]. The Mis promoter is directly regulated by WT1 with SOX9 and SF1 [12; present study]. The WT1 must bind to DNA to regulate the Mis promoter. Our chromatin immunoprecipitation studies showed that WT1 directly bound to the Mis promoter in vivo. Therefore, we conclude that deregulated MIS expression because of the mutation in WT1 may cause the pseudohermaphroditism in patients with DDS.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Grady F. Saunders, Department of Biochemistry and Molecular Biology, Box-117, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030. FAX: 713 791 9478; gsaunders{at}odin.mdacc.tmc.edu
Received: 24 January 2003.
First decision: 22 February 2003.
Accepted: 26 June 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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