 |
INTRODUCTION |
The Wilms' tumor gene WT1 is involved in tumorigenesis
in humans, and its mouse homolog, Wt1, has a distinct role
in the development of several organs during embryogenesis (1, 2). The
gene was originally identified by positional cloning and sequencing on
the basis of its association with the rare childhood kidney tumor,
Wilms' tumor (3-5). Specific mutations in the Wilms' tumor gene
cause two different types of genitourinary abnormalities called
Denys-Drash syndrome (DDS)1
(6) and Frasier syndrome (7). Patients often showed male to female sex
reversal, male pseudohermaphroditism, and cryptorchidism.
WT1 is a zinc finger containing DNA-binding protein and acts as a
transcriptional activator or repressor depending on the cellular or
chromosomal context (8, 9). It has four major isoforms, due to 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 (Fig. 1A) (10). These
four isoforms are conserved among mammals. WT1 binds to the highly
GC-rich canonical early growth response gene-1 (EGR-1) DNA-binding
motif GCGGGGGCG (8). Many genes are proposed to be regulated by WT1.
Most of them are involved in growth regulation and cellular
differentiation as a result of the tumor suppressor action of WT1
(11-13). A second group is involved in sex development and includes
Müllerian inhibiting substance (Mis) (14). Nachtigal et
al. (14) reported that SF1 and WT1 synergistically activate the
Mis promoter. DNA binding of WT1 is not essential for this
synergistic activation. Mutations in MIS or its receptor
cause male pseudohermaphroditism in humans, and deletion of Mis or the
Mis receptor also causes pseudohermaphroditism in mice (15, 16). Thus
the regulation of MIS by WT1 has physiological effects and suggests
that pseudohermaphroditism appears in DDS patients as a result
of deregulation of MIS. Another common trait of DDS is sex reversal
(17), and our objective is to identify the cause of sex reversal in DDS patients.
One possible target gene of WT1 is the sex-determining gene
SRY. SRY is an HMG box containing transcription
factor. It is expressed during the critical period of male sex
determination and is able to direct male sex determination in a
genetically XX mouse (18). Little is known about the regulation of SRY
during sex development (19). The level of expression of Sry in the mouse is important for sex determination (20). Characterization of the
regulation of SRY has been hampered by the poor conservation of the
upstream sequences of SRY across species. It is known that Sry is
expressed only for a short period during male sex determination in the
Sertoli's cell precursor in mouse (21-23). However, SRY expression in
humans is less tissue- and stage-specific (24, 25). Gonadal expression
of both WT1 (25, 26) and SRY (25) overlaps in humans. WT1 first appears
at a low level at 31 days post-ovulation in the gonadal ridges of both
male and female embryos. Expression levels significantly increase at 41 days post-ovulation, when SRY expression begins in the gonadal ridge.
SRY is most strongly expressed at 44 days post-ovulation, and both WT1
and SRY expression persist later in fetal development (25, 26). Our
hypothesis is that because mutation of both genes causes sex reversal
in humans and WT1 is expressed before SRY in the gonadal ridge, WT1 may
regulate the expression of SRY. Our preliminary data also showed that
the SRY promoter is positively regulated by WT1 (27). In
this report, we provide clear evidence that WT1 activated the SRY gene and initiated a regulatory gene cascade in the male
sex determination and differentiation pathway.
 |
EXPERIMENTAL PROCEDURES |
Plasmids--
The expression vectors with the four isoforms of
WT1 (pCBWT1
/
, +/
,
/+, and +/+) were described previously (28).
pcDNA3WT1 constructs were made by PCR amplifications of wild-type
WT1 from CMVWT1
/
expression vector. The resulting PCR products were
subsequently subcloned into a modified pcDNA3 vector (29) harboring
the 5'-untranslated region of the herpes simplex virus-thymidine kinase
gene. All WT1 mutants were created from this pcDNAWT1
/
background by site-directed mutagenesis, and mutations were verified by sequencing.
The plasmids in which the SRY upstream sequence drove transcription of
the luciferase gene, a 1979- and 313-base pair fragment from SRY-CAT1
and SRY-CAT2 (30), were isolated and cloned in front of the luciferase
gene pGL3 basic vector (Promega) and were designated
1938SRYP and
272SRYP, respectively. The SRY promoter deletion constructs were
generated by PCR amplification using
272SRYP as template. Resulting
PCR fragments were subcloned into the pGL3 basic vector. All constructs
were verified by sequencing from both directions.
All site-directed mutagenesis of the SRY promoter constructs
was performed with the Quick Change site-directed mutagenesis kit
(Stratagene). Details of PCR primers used for mutagenesis can be
obtained from the authors. All mutations were confirmed by
sequencing from both directions.
Cell Cultures and Transfection--
TM4, HeLa, and NT2D1 cells
were grown at 37 °C in Dulbecco's modified Eagle's medium/Ham's
F-12 supplemented with 10% fetal calf serum in 5%
CO2.
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.
CMV-
-galactosidase (10 ng) was cotransfected as an internal control
to normalize the transfection efficiency. The transfections were
carried out using LipofectAMINE-Plus reagent according to manufacturer's recommendations (Life Technologies, Inc.), 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).
-Galactosidase was measured with the Galacto-Light
plus kit (Tropix).
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 with wild-type or
mutant oligonucleotides, a 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 anti-WT1 antibody (C-19, Santa Cruz
Biotechnology) for 15 min at room temperature, and labeled probe was
added with further incubation on ice for 30 min.
Generation of Cells with Inducible WT1 Expression--
A
full-length WT1 construct was assembled from pcDNA3WT1,
cloned downstream from the tetracycline-regulated promoter in pEC1214A (31), and confirmed as wild-type by sequencing the entire coding region. NT2D1 cells were used to generate stably transfected clones with tightly regulated inducible expression of wild-type
WT1.
NT2D1 cells stably transfected with a tetracycline-repressible WT1
expression vector and control vector were grown to about 70%
confluency. Tetracycline analog doxycycline was removed from the cells.
The cells were harvested after 12 h, and total RNA was isolated
with the RNeasy mini Kit (Qiagen). RT was performed with random
hexamers and the Superscript II kit (Life Technologies, Inc.). The
products from the RT were aliquoted and directly subjected to PCR
amplification. The following forward (F) and reverse (R) primers were
used for PCR amplification: SRY-F GAAGATCAGGGGCTGGCAGA), SRY-R
(CAGAGGCGCAAGATGGCTCT), GAPDH-F(CCAGCCGAGCCACATCGCTC), and GAPDH-R
(ATGAGCCCCAGCCTTCTCCAT). PCR was performed with the high fidelity PCR kit (Roche Molecular Biochemicals), and the following conditions were used: 1 cycle at 94 °C for 2 min and 22 cycles of
denaturation at 94 °C, annealing at 60 °C, and extension at 72 °C. PCR products were separated on a 1.5% agarose gel.
Western Blotting--
Whole cell extracts were prepared with
cell lysis buffer consisting of 50 mM Tris-HCl (pH 8.0),
150 mM NaCl, 1.0% Nonidet P-40, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml each aprotinin, leupeptin, and benzamidine. Western blots were
developed by enhanced chemiluminescence (Amersham Pharmacia Biotech).
The primary antibody and secondary antibodies used were as rabbit
anti-WT1 at a dilution of 1:100 (C19, Santa Cruz Biotechnology), goat
anti-human SRY at a dilution of 1:200 (C180, Santa Cruz Biotechnology), anti-rabbit IgG conjugated with peroxidase (Amersham Pharmacia Biotech), and anti-goat IgG conjugated with peroxidase (Santa Cruz Biotechnology).
 |
RESULTS |
Ectopic Expression of WT1 Activates the SRY Promoter in Different
Cell Lines--
To test whether the SRY promoter is
regulated by WT1, we used two luciferase reporter gene constructs. One
of these,
1938SRYP, contains a large region upstream of the
SRY promoter, and the other,
272SRYP, contains the minimal
promoter and 5' sequence. Both constructs have similar promoter
activities in TM4 and Ltk cells (30). Fig.
1B shows that both
1938SRYP
and
272SRYP were activated severalfold in response to WT1
/
(Fig.
1B) in the human teratocarcinoma cell line NT2D1 derived
from testicular tumor and the mouse Sertoli cell line TM4. The NT2D1
and TM4 cell lines have been shown to express most of the genes known
to be involved in mammalian sex determination (32). For further
studies, we chose the cervical carcinoma cell line HeLa, because most
(if not all) male sex-specific genes and WT1 are not expressed in HeLa
cells, and HeLa is highly transfectable. We found that the basal level
of reporter gene activity was very low in HeLa cells compared with
NT2D1 and TM4 cells. Both
1938SRYP (data not shown) and
272SRYP
were robustly activated (>50-fold over the control) by WT1
/
in
HeLa cells in a dose-dependent manner (Fig. 1C). We used
272SRYP for most of our subsequent studies, because both
1938SRYP and
272SRYP showed similar transactivational activities. The transactivation of the SRY promoter was dependent on the
KTS isoforms, WT1
/
and WT1+/
. Both +KTS isoforms, WT1+/+ and
WT1
/+, were unable to transactivate the SRY promoter (Fig.
1D). 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 post-transcriptional level (33).
These data also clearly indicate that WT1 is able to transactivate the human SRY promoter.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1.
WT-KTS isoforms specifically activated
the SRY promoter in different cell lines.
A, isoforms of WT1. Alternative splicing of exon 5 and the
alternative usage of two different splice-donor sites at the 3' end of
exon 9 produce four different splice forms. + and indicate
inclusion and exclusion, respectively, of the sites. B,
NT2D1 and TM4 cells were transfected with 0.2 µg of the promoter from
human SRY driving the luciferase reporter constructs and 0.2 µg of
pCB6WT1 / or the empty expression vector, pCB6+, as a control. Two
forms of the SRY promoter were used, one containing nt
1938 to +41 ( 1938SRYP) and the other containing nt 272 to +41
( 272SRYP). Ten nanograms of CMV promoter-driven -galactosidase
gene, CMV- gal (Promega), was cotransfected
with each sample to control the transfection efficiency. The assay was
performed 40 h after transfection. All results are expressed as
mean ± S.D. of at least three experiments. C, HeLa
cells were transfected with different amounts of pCB6.WT1 / with
272SRYP and 10 ng of CMV- gal. Empty pCB6+
was added to keep the plasmid concentration to 0.4 µg. The assay was
performed 40 h after transfection. All results are expressed as
mean ± S.D. of at least three experiments. D, HeLa
cells were transfected with different isoforms of WT1 in pCB6 with
272SRYP and 10 ng of CMV- gal. The assay was
performed 40 h after transfection. All results are expressed as
mean ± S.D. of at least three experiments.
|
|
Activation of the SRY Promoter Required nt
32 to +41--
To
determine the location of the WT1-responsive element in the
SRY promoter, we made a series of deletion constructs as
follows:
272SRYP,
221SRYP,
147SRYP,
71SRYP, and
32SRYP (Fig.
2A). All of these constructs
were similarly activated by WT1 (Fig. 2B). However, empty
pGL3 basic vector was not activated over the basal level by WT1 (Fig.
2B). The deletion constructs mapped the WT1-responsive
element (WTE) to the region between nt
32 and +41, within the
SRY promoter.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
WTE within the SRY
promoter. A, schematic representation of the
1938SRYP.pGL3 basic luciferase reporter constructs and deletion
mutants. WTE sequences are shown on top of the 1938SRYP
reporter construct. Restriction enzyme sites are indicated, and all
mutants were confirmed by sequencing. B, reporter gene
assays with 1938SRYP and its derivatives in HeLa cells. The cells
were cotransfected with 0.2 µg of deletion mutant or empty pGL3 basic
vector and 0.2 µg of pCB6WT1 / or empty vector. Reporter gene
activity was normalized to that of -galactosidase. All results are
expressed as mean ± S.D. of at least three experiments.
|
|
WT1-responsive Element Is Essential for the Activation of the SRY
Promoter--
Analysis of this 73-base pair minimal WT1-responsive
region revealed only one EGR-1-like potential WT1-responsive element (GAGGGGGGTG) (Fig. 2A). To test the functional significance
of this site, single mutations were introduced at several positions (Fig. 3A). These constructs
were assayed for their responsiveness to WT1 in HeLa cells and compared
with wild-type
272SRYP. Substitution of any G to T did not
significantly reduce the reporter gene activity (data not shown).
Single nucleotide substitution of the A to a G (M2) had little or no
effect on reporter gene activity. On the other hand, substitution of
the T with a G (M1) was reduced in reporter gene activity by
50%. Moreover, substitution of the both the A and T to Gs (M3)
completely blocked transactivation by WT1.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
A, sequences of the WTE and its mutant
derivatives (underlined). B, reporter gene assay
with 272SRYP and its potential WT1-binding site mutants in HeLa
cells. The cells were transfected with 0.2 µg of reporter gene with
pCB6WT1 / or 0.2 µg of pCB6. CMV- gal (10 ng) was
cotransfected to control for the transfection efficiency. All results
are expressed as mean ± S.D. of at least three experiments.
|
|
The WT1-responsive sequence was further characterized by gel shift
assays. The gel shift assay in Fig. 4
shows that WT1 synthesized in vitro binds to a
32P-labeled WTE oligonucleotide probe. Binding was competed
completely by unlabeled wild-type probe and also by unlabeled EGR-1
probe (Fig. 4, lanes 3 and 5). The mutants M1 and
M2, which showed partial or no reduction in reporter gene activity,
also competed with the wild-type probe (Fig. 4, lanes 8 and
9). The mutant M3 in which both the A and T were changed to
Gs did not compete with the wild-type probe and has no transactivation
abilities in reporter gene assays. This indicates that both A and T
residues at positions 2 and 8 were very important for WT1-mediated
transactivation of the human SRY promoter. As a positive
control, a 32P-labeled EGR-1 consensus probe was used in a
WT1-mediated DNA binding assay (Fig. 4B, lane 6). Antibody
against WT1 supershifted WT1-DNA complexes, indicating that WT1 was
present in that complex. These data suggest that the WTE sequence in
the human SRY promoter is essential for the binding of WT1
to this promoter as well as for WT1-mediated transactivation of the
SRY promoter.

View larger version (89K):
[in this window]
[in a new window]
|
Fig. 4.
Gel mobility shift assays were performed with
radiolabeled WT1-binding elements in SRY promoter
(GTGGGGGAG) and incubated with in vitro translated
WT1. The amount of IVT WT1 protein used was 5 µl. The
EGR-1-binding sequence served as a positive control. WT1-DNA complexes
were competed with a 100-fold excess of wild-type unlabeled probe or an
unlabeled 100-fold excess of EGR-1 probe (GCGGGGGCG). WT1-DNA complexes
were also partially competed with a 100-fold excess of M1 and M2, but a
100-fold excess of M3 could not compete the DNA-protein complexes.
Anti-WT1 antibody (1.0 µg) was used to supershift the specific
WT1-DNA complex. The arrow indicates the specific WT1-DNA
complexes, and the arrowhead indicates the supershifted
band.
|
|
Different WT1 Mutants Had Different Effects on the SRY
Promoter--
The DDS phenotype often arises as a result of alteration
of one allele by a missense point mutation, usually in the zinc finger DNA binding domain of WT1. One mutational hot spot is codon 394, in
which arginine 394 is changed to tryptophan. To determine how these
mutations affect the SRY promoter, we created two types of
mutations. One group of mutants contained changes in the zinc finger
region (C330Y, R366C, R366H, H377Y, and R394W), and the other group
contained (F154S, P180S, and S273A) changes outside that region. These
mutants were tested for their ability to transactivate the
SRY promoter in HeLa and NT2D1 cells. The most common DDS mutants R366C, R366H, H377Y, and R394W failed to activate the SRY promoter in reporter gene assays. In contrast, the
mutants with mutations outside the zinc fingers region (F154S, P180S, and S273A) had transactivational potentials similar to that of wild-type WT1. Another mutant (C330Y) retained some transactivational ability even though its mutation was in the zinc finger region. Western
blot data from HeLa cell extracts showed that all the mutant proteins
were expressed at similar levels (Fig.
5C).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Activation of the human SRY
promoter in HeLa cells (A) and NT2D1 cells
(B) expressing the mutants of WT1. Cells were
transfected with 272SRYP (0.2 µg) with different point mutants of
the WT1 in modified pcDNA3. Empty pcDNA3 was added to keep the
plasmid amounts equal in each well. The assays were performed 40 h
after transfection. All results are expressed as mean ± S.D. of
at least three experiments. C, Western blot analysis using
wild-type and mutant WT1 expression vector transfected HeLa cell
extracts using anti-WT1 antibody. Expression level of wild-type WT1 and
mutants are almost identical in all cell extracts. Fifty micrograms of
the whole cell extracts were loaded in each lane.
|
|
The DNA binding abilities of these mutants were assessed by gel shift
assays. Mutations in the DNA binding region (the zinc finger regions)
caused loss of DNA binding ability (Fig.
6). However, the mutant C330Y, which has
a mutation in the DNA binding region, retained some transactivational
ability as well as DNA binding ability (Fig. 6). This implies that the
cysteine in codon 330 in WT1 is not 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 SRY promoter (Fig. 6).

View larger version (123K):
[in this window]
[in a new window]
|
Fig. 6.
Mutations in the zinc finger region
abolished the DNA binding of WT1. Gel mobility shift assays were
performed with radiolabeled WT1-binding elements in the SRY
promoter (GTGGGGGAG) and incubated with IVT WT1 and its point mutants.
The amount of IVT WT1 proteins used was 5 µl. Specific WT1-DNA
complexes are indicated by the arrow. WT1-DNA complexes were
competed with a 100-fold excess of wild-type unlabeled probe. The
arrow indicates the WT1-DNA complexes.
|
|
DDS Mutants Failed to Act in a Dominant Negative Manner on the SRY
Promoter--
To determine the molecular mechanisms underlying the
pathogenesis of DDS, we determined whether DDS mutants act in a
dominant negative manner by sequestering wild-type WT1. There are
conflicting reports about this issue (14, 34). We tested this
hypothesis in the context of SRY promoter activation. HeLa
cells were cotransfected with the wild-type WT1
/
with or without a
5-fold excess of the different mutants in the presence of
272SRYP.
The mutants (F154S, P180S, S273A, and C330Y) that had transactivation
ability had additive effects both in HeLa and NT2D1 cells (Fig.
7). However, the DDS mutants (R366C,
R366H, H377Y, and R394W) did not have additive or dominant negative
effects, i.e. there was no significant reduction of promoter
activation by wild-type WT1 (Fig. 7). This experiment was repeated at
least three times with different plasmid preparations, and similar
results were obtained. These data clearly indicated that the DDS
mutants did not act in a dominant negative manner, at least in the
activation of the SRY promoter.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 7.
DDS mutants failed to act in a dominant
negative manner. Cells were transfected with 272SRYP (0.2 µg)
and different point mutants of WT1 in modified pcDNA3 (0.5 µg)
with or without wild-type pcDNA3WT1 / (0.1 µg). Empty
pcDNA3 was added to keep the plasmid amounts equal in each well.
The assays were performed 40 h after transfection. Reporter gene
activity was normalized to -galactosidase activity. All results
are expressed as mean ± S.D. of at least three experiments.
|
|
Increase in Endogenous SRY Expression by the Inducible Expression
of WT1 in NT2D1 Cells--
To investigate the effect of WT1 on
endogenous SRY expression, we constructed a system for
tetracycline-regulated inducible expression of WT1
/
in the human
teratocarcinoma cell line NT2D1. We chose NT2D1 because it expresses
SRY at very low levels but does not express WT1 (Fig.
8A). Also, we assumed that
other components for the expression of SRY are present in this cell
line. It has been reported that some clones of NT2D1 constitutively
express high amounts of SRY (35). However, our lines constitutively expressed low amounts. WT1
/
was placed under the control of the
tetracycline transactivator. In the presence of the tetracycline analog
doxycycline, WT1 expression was turned off. Withdrawal of doxycycline
from the medium caused strong expression of WT1, as determined by
Western blotting (Fig. 8A). Fig. 8B shows that NT2D1 cell expressing WT1 did up-regulate SRY mRNA, as determined by RT-PCR. To confirm these results, we performed Western blottings using the whole cell extracts of the induced and uninduced NT2D1 cells
and anti-SRY antibody. As expected, WT1-expressing NT2D1 cells
expressed severalfold more SRY protein (Fig. 8C). These results showed clearly that WT1 was capable of up-regulating the endogenous SRY gene in its native chromosomal context.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 8.
Up-regulation of SRY by inducible expression
of WT1 in NT2D1 cells. A, Western blot analysis of WT1
expression in NT2D1 cells harboring WT1 / under the control of the
inducible tetracycline promoter or vector alone, after growth for
6 h with or without the tetracycline analog doxycycline
(DOX). WT1 expression was up-regulated in the absence of
doxycycline. Fifty micrograms of the whole cell extracts was loaded in
each lane. B, RT-PCR analysis of NT2D1 cells with
doxycycline regulated expression of WT1 / or vector, following
growth in the presence or absence of the doxycycline for 12 h. SRY
expression is up-regulated in association with inducible expression of
WT1 / . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
was used as an internal RT-PCR control. C, Western blot
analysis of NT2D1 cells with tetracycline-regulated expression of
WT1 / or vector, after growth for 12 h in the presence or
absence of doxycycline. SRY expression is up-regulated by induced
expression of WT1 / . Fifty micrograms of the whole cell extracts
were loaded in each lane. -Actin antibody was used for the protein
control.
|
|
 |
DISCUSSION |
The molecular mechanisms underlying the pathogenesis of DDS and
how WT1 participates in the sex determination pathway have not been
well understood. That heterozygous germ line mutations of
WT1 cause DDS in humans has been known for a long time.
Numerous genes have been proposed as target genes of WT1, but the
physiological relevance of those genes remains unclear. By a candidate
gene approach, we identified SRY as one of the direct
target genes of WT1. SRY fulfills the criteria of a
direct target of WT1 in the sex determination pathway for the following
reasons. First, mutations in both SRY and WT1
cause male to female sex reversal in genetically XY individuals.
Second, the expression patterns of SRY and WT1 overlap in the human
gonads. Third, WT1 can positively regulate the SRY promoter
and can directly bind to a cis-acting element in this promoter.
Finally, WT1 can directly transactivate endogenous SRY expression in
its native chromosomal context. Thus, we conclude that SRY is a
physiologically relevant target of WT1 in the sex determination pathway.
WT1 was identified as a tumor suppressor and was predicted to be a
transcriptional repressor on the basis of its tumor suppressor action.
However, some of its biological roles cannot be explained by its
transcriptional repressor activity. Later it was found that WT1 has at
least two independent transactivation domains in its N-terminal region.
Transcriptional activation of the SRY promoter is not an
unusual case. Recently, many genes have been shown to be up-regulated
by WT1 (11, 12, 14). Amphiregulin has been
identified as an in vivo target of WT1 by DNA
microarray screening (12). In the same study, they found other genes
were also up-regulated to different extents by WT1; however,
no genes were down-regulated by WT1. It seems that
transcriptional activation of the down stream genes is a major function
of WT1 in vivo.
The WTE described in this study consists of high affinity WT1-binding
sequences located near the transcription initiation site of the
SRY promoter. WT1 can bind to DNA sequences showing variations in the EGR-1 consensus-binding site,
GXGXGGGXG (13). This WTE (GAGGGGGTG)
is similar to the consensus EGR-1-binding site. However, WTE displays
higher affinity than the EGR-1 consensus site (Fig. 4). There is
another WT1-binding site far upstream from the WTE in the
SRY promoter. Mutational analysis revealed that the second
binding site does not have a significant role in the transactivation of
the SRY promoter. Full transcriptional activation of the
Mis promoter requires at least two factors (14, 32). It is
likely that additional factors may also be important for the regulation
of the SRY promoter. Several TCF-binding sequences are present in the SRY promoter. The contribution of these
sites to the activation of the SRY promoter is being
investigated. The WT1-binding sequence in the SRY promoter
is conserved among closely related primates (data not shown). There are
virtually no similarities in the 5'-untranslated and promoter regions
between the mouse and human genes. The WT1-binding site in the human
SRY promoter is not conserved in the mouse, but there are
similar sequences in the mouse Sry promoter. It will
be interesting to see whether Wt1 also regulates mouse Sry during sex
determination in the mouse.
SRY has been known for its pivotal role in sex determination for a
decade. Transcriptional regulation of SRY is relatively uncharacterized. Partial sex reversal has been found resulting from
mutations in the 5' and 3' region of the SRY gene (36). It
indicates that regulation of the SRY gene is important in
male sex determination. De-regulated expression of the SRY
may have pleiotropic effects in the critical period of sex
determination. In addition to SRY and WT1, other
genes also play an important role in male sex development (19). SRY
initiates the differentiation of the indifferent gonads to the male
pathway and creates an environment for other critical factors to carry
on this process. Thus the regulation of SRY by
WT1 is an important step in this pathway. Since DDS mutant
proteins were unable to activate the SRY promoter, it is
likely that SRY expression is affected in DDS patients.
DDS usually results from point mutations in the WT1 zinc
finger region that abrogate DNA binding. The resulting mutant proteins are predicted to act in a dominant negative manner to inhibit the
action of the wild-type protein by sequestering it from the transcriptional machinery (6). However, the validity of this hypothesis
is in question for several reasons. If DDS mutants act in a dominant
negative fashion, then that would inactivate the protein product of the
wild-type allele, creating a WT1-null situation during embryogenesis.
Wt1-nulls are embryonic lethals in mice, suggesting that WT1-null
situations in human embryos would likewise not be viable. However,
heterozygous WT1 lethality does not occur and has not been seen in DDS
patients. Moreover, there is a mouse model of DDS (37). Clear
elucidation of the molecular mechanism of DDS was compromised by many
complexities; however, it was clear from the mouse model of DDS that
simple squelching of the wild-type protein by the mutant did not occur. In our study we did not see dominant negative effects of DDS mutations in the SRY promoter. Our observations are in good agreement
with others (14, 34). Discrepancies with others are most likely due to
the amount of the DNA used in the transfection experiments; we used
nanogram quantities, whereas they used microgram quantities. Thus, the
dominant negative effects were probably an artifact of the in
vitro system and varied under different experimental conditions.
Our model for DDS is that heterozygous point mutations cause functional
loss of one allele of WT1 and thus haploinsufficiency. This
reduces the amount of transcriptionally active
KTS isoform of WT1. As
a result, expression of downstream target genes such as SRY
and MIS is affected during the critical period of sex
determination and differentiation in male gonads.