From the
The WT1 tumor suppressor gene encodes four isoforms of
a zinc finger transcription factor with both activation and repression
functions which are dependent upon promoter architecture. Using a
simple HSV- tk promoter containing 5`-Egr-1/WT1-binding sites,
we found that WT1 isoforms (A) and (B) strongly activated
transcription. WT1(A) and (B) bound equally well to the
Egr-1/WT1-binding site, but WT1(B), which contains a 17 amino acid
insertion compared to WT1(A), was a consistently stronger activator of
transcription than WT1(A). Transcriptional activation by wild-type WT1
was inhibited by coexpression of WT(PM) or WT(AR),
genetically defined dominant negative alleles of WT1. In
vitro, as well as in the yeast two-hybrid system, WT1 protein
associated with itself and with dominant negative mutant proteins. The
major domain required for self-association and inhibition of
transcriptional activation mapped to the first 182 amino acids of WT1.
Dominant negative WT1 alleles may play a role in tumorigenesis by
associating with wild-type WT1 proteins and decreasing their
transcriptional activity.
The Wilms' tumor-associated gene WT1 was isolated
by positional cloning based on the presence of a constitutional
deletion of chromosome 11p13 in patients with the WAGR syndrome
(Wilms' tumor, aniridia, genitourinary malformations, and mental
retardation)
(1, 2) . The WT1 gene yields four
alternatively spliced mRNAs of approximately 3 kilobases which encode
polypeptides of apparent molecular mass 52-54 kDa
(3, 4, 5) . All four isoforms have four cysteine
and histidine-containing (C
The (A) and (B) isoforms of WT1 were shown to bind to the same DNA sequence as the Egr-1
transcription factor (5`-GCGGGGGCG-3`)
(6, 7) . Isoforms
(C) and (D) bind poorly to the Egr-1 sequence due to the 3 amino acid
insertion between zinc fingers 3 and 4
(7) . WT1(A) can repress
transcription from a variety of cellular genes involved in growth and
proliferation
(8, 9, 10, 11, 12, 13) .
Like many transcription factors, WT1 can also regulate transcription of
its own promoter
(14) . WT1 can also activate transcription. WT1
isoform (A) stimulates promoters containing Egr-1 sites either upstream
or downstream of the transcription start site, but suppresses
transcription when Egr-1 sites are present both upstream and downstream
of the start site of transcription
(15, 16, 17) .
Up to 5-10% of
Wilms' tumors
(18, 19) are associated with
homozygous null WT1 mutations.This figure may be an
underestimate, because in most studies, only the zinc finger domains
were examined, and when searched for, mutations can be found which
alter other portions of the WT1 protein
(19) . Heterozygous
WT1 mutations are also associated with disease. Such WT1
mutations were isolated both from patients with Wilms' tumor and
from patients with the Denys-Drash syndrome (DDS),
To construct a vector for
expression of a glutathione S-transferase (GST)-WT1 fusion
protein in Escherichia coli, the pSP64-WT1A plasmid was
digested with HincII and EcoRI with the resulting
fragment cloned into SmaI/ EcoRI digested pGEX-2tk
(Pharmacia, Uppsala, Sweden) yielding the plasmid pGEX-WT1(A). The
resulting fusion protein contains an additional 20 amino acids
(GSPTLEDRRSGEPSASEPHL) after the GST moiety. To construct
pGEX-WT1(1-183), pGEX-WT1(A) was restricted with NcoI,
blunted with the Klenow fragment of DNA polymerase I, and ligated to a
duplex oligonucleotide containing stop codons in all three reading
frames
(24) .
The WT(PM) mutation was isolated in the
heterozygous state from germline DNA from a patient with DDS
(32) . Patients with this syndrome exhibit nephropathy and
intersex disorders and occasionally develop Wilms' tumor.
Insertion of a single guanine residue in this allele results in the
truncation of the WT1 protein at amino acid 256, with an extra amino
acid (glycine) added at the COOH terminus
(32) (Fig. 1).
This patient presented with nephropathy, ambiguous genitalia, and
bilateral Wilms' tumors. While the tumors were most likely
homozygous for the mutant WT1 allele
(32) , its presence in the
heterozygous state in germline DNA suggests that it may act as a
dominant negative allele resulting in genitourinary malformations and
nephropathy.
We first determined whether, consonant with their
proposed dominant negative genetic function, the WT(AR) proteins could interfere with transcriptional activation by
WT1(A). The WT(AR) isoforms alone had no effect on
transcription from the EGR
Transfection of a fixed amount (4
µg) of WT1(A) expression vector with increasing amounts (4, 8, or
16 µg) of WT1(B) further stimulated transcription
(Fig. 3 A). In contrast, transfection of increasing
amounts of either WT(AR) expression vector inhibited transcriptional
activation by WT1(A) (Fig. 3 B). At the highest dose (16
µg) of transfected WT(AR) plasmid, inhibition was less marked,
perhaps due to the modest stimulatory activity of WT(AR) observed at
high inputs.
The WT(PM) protein, containing amino acids 1-256 of
WT1, bound strongly to both GST-WT1(A)(1-429) and
GST-WT1(1-183) (Fig. 5 B, row 4). In
addition, a truncated protein containing only wild-type WT1 or WT(AR)
zinc finger domains (amino acids 307-429) did not bind to
GST-WT1(1-183) but bound weakly to the full-length GST-WT1
protein (Fig. 5 B, rows 2 and 3).
Intriguingly, the binding of the wild-type WT1 or WT(AR) zinc finger
domain (amino acids 307-429) to the full-length protein was
intensified upon addition of EtBr (Fig. 5 B, rows 2 and 3). This suggests that the zinc fingers of WT1 can
also mediate a protein-protein interaction in vitro, which
becomes stronger if DNA binding by the zinc fingers is precluded by
EtBr. The presence of two self-association domains within the WT1 protein may also help explain why full-length WT1 molecules
interact better with themselves than with NH
By an in vitro biochemical assay and by the in vivo yeast two-hybrid assay, WT1 was found to self-associate. We
therefore propose that the dominant negative WT(AR) and
WT(PM) proteins, which cannot bind to the Egr-1 site, interact
with WT1 bound to DNA and inhibit the function of wild-type protein,
perhaps by partially shielding its activation domain. WT(PM) was a more
potent inhibitor of transactivation by WT1(A) than was WT(AR). This may
be due to the fact that WT(PM) is incapable of binding to DNA while
WT(AR) can bind to sites other than the Egr-1 site used in this study
(33) . Some of the WT(AR) protein in the cell may be bound to
DNA sequences other than the Egr-1 consensus and would therefore be
unavailable for protein-protein association with wild-type WT1 required
for inhibition of transactivation. In contrast, WT(PM) cannot bind to
any DNA sequence and thus can only function through protein-protein
interaction with wild-type WT1. Based on analysis of WT1 mutations
isolated from DDS patients, Bardeesy et al.(40) predicted that a domain encoded by exons 1, 2, and 3 of WT1
(amino acids 1-222) would mediate a dominant negative effect. We
confirm this prediction by mapping the domain responsible for
self-association and for the dominant negative effect to amino acids
1-182 of WT1.
WT1 also represses transcription of a variety of
growth-related genes
(8, 9, 10, 11, 12, 13) ,
with this effect dependent on promoter architecture
(15, 16, 17) and on interaction of WT1 with the
p53 protein
(41) . Studies are now underway to determine whether
dominant negative WT1 proteins also inhibit transcriptional repression
by WT1. Even if only activation is affected, the resultant imbalance
between the repression and activation activities of WT1 might be
sufficient to cause neoplasia, developmental abnormalities, or
nephropathy.
Heterozygous null mutations of WT1, as found in the
WAGR syndrome, result in genitourinary malformations and predisposition
to Wilms' tumor, but not nephropathy. Patients with such
mutations would have a 50% reduction in their effective WT1 activity.
Our results suggest that heterozygous dominant negative mutations of
WT1, such as WT(AR) and WT(PM), can result
in an inhibition of function of the remaining wild-type WT1 allele. The maximal inhibition we observed was approximately
3-fold for WT(AR) and 5-fold for WT(PM). In patient AR, this could
result in a level of WT1 activity that is less than 50% of normal in
the small subset of kidney cells containing the acquired mutation. This
might be sufficient to produce a disregulation of growth control of
these cells, resulting in tumor formation. Since WT(AR) can bind to
specific DNA sequences other than the Egr-1 consensus
(33) , it
could also deregulate growth by binding to novel target genes not
normally regulated by WT1.
According to our hypothesis, DDS
patients, who have a heterozygous germline WT1 mutation, would
also have a level of effective WT1 activity substantially lower than
hemizygous WAGR patients. The remaining level of WT1 activity in DDS
patients must still be sufficient to allow kidney development, since
mice with a homozygous disruption of the WT1 locus fail to form kidneys
(42) . WT1 continues to be expressed in the podocytes of the
adult kidney, which are responsible for the maintenance of the
glomerular basement membrane
(43) . Therefore, inhibition of WT1
activity by a dominant negative mutant protein in the podocytes of DDS
patients might contribute to the novel phenotype of progressive
glomerulonephropathy.
Plasmids encoding the indicated chimeric fusion proteins were
transformed in pairs into the yeast strain EGY48 containing the
reporter LexAop-LacZ. Interaction between fusion proteins was scored
qualitatively by blue (positive) or white (negative) color of yeast
colonies grown on plates containing X-gal. Transformed yeast containing
selected pairs of plasmids were then colorimetrically assayed for
We thank Dr. Ned Landau for the NIH 3T3 cell line, Dr.
Frank J. Rauscher, III, for plasmid constructions, and Dr. George F.
Atweh for careful review of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H
) zinc fingers near
the carboxyl terminus of the protein and a glutamine and proline-rich
domain in the amino-terminal portion of the protein. The (A) isoform
contains neither alternative splice; the (B) isoform includes an
NH
-terminal splice which codes for 17 additional amino
acids; the (C) isoform includes a COOH-terminal splice which results in
the insertion of 3 amino acids (KTS) between zinc fingers 3 and 4; and
the (D) isoform contains both the 17 amino acid and 3 amino acid
insertions. The ratio of these four spliced mRNAs in human fetal kidney
is 1 A:2.5 B:3.8 C:8.3 D; this ratio does not appear to be regulated
during development
(4) .
(
)
a syndrome which includes nephropathy, intersex disorders,
and predisposition to the development of Wilms' tumor. These
mutants were proposed to act in a dominant negative manner by affecting
the function of the wild-type allele. Consonant with this, we found
that two genetically defined dominant negative alleles, which yield
proteins unable to bind to the Egr-1/WT1 consensus site, inhibited the
transcriptional function of wild-type WT1. The domain of the WT1 mutant
proteins required for inhibition mapped to the NH
-terminal
182 amino acids. This domain was found both by in vitro biochemical assays and by the in vivo yeast two-hybrid
system to mediate self-association of WT1 protein.
Plasmid Construction
The expression vectors for
the murine WT1(A) and (B) isoforms and the two WT(AR) mutant proteins were described previously
(20) . The
expression vector for WT(PM) was constructed by PCR amplification of
sequences encoding amino acids 100-256 using the primers:
5`-GGCCAGTTCACCGGTACA-3` and 5`-GTCGACGTCGACTTATCCACTCTCGTACCCTATAC-3`.
The PCR product was then digested with AgeI and SalI
and inserted into AgeI/ SalI digested RSV-WT1(A). A
RSV vector lacking the WT1 coding region was constructed by
restriction of the RSV-WT1(A) plasmid with BglII to release
the WT1 coding region, followed by recircularization. The
EGR tkCAT reporter plasmid (Fig. 1 B)
was constructed by the insertion of three tandem oligonucleotides
containing the Egr-1-binding site (underlined) from the zif268 promoter: 5`-TCGACCCTCGCCCCCGCGCCGGGC-3`;
3`-GGGAGCGGGGGCGCGGCCCGAGCT-5`
(21) into the SalI site
of pBLCAT2
(22) . The thymidine kinase growth hormone
( tk-GH) (Fig. 1 B) internal control plasmid was
described previously
(23) .
Figure 1:
A, expression vectors for production of
WT1 proteins in transfected cells. Expression of WT1 was
directed by the RSV promoter. 17 refers to the 17 amino acids
inserted in WT1 as the result of alternative splicing of the WT1 mRNA. The zinc finger domains are indicated by black boxes and are numbered. B, reporter genes utilized.
EGR tkCAT contains three Egr-1/WT1 binding sites
inserted upstream of the HSV thymidine kinase ( tk) promoter
and the CAT gene. In tk-hGH, the HSV- tk promoter is
linked 5` to the human growth hormone gene. Secretion of human growth
hormone from transfected cells is used as an internal control for
transfection efficiency.
To construct a vector for in
vitro translation of WT1 protein, a 1.5-kilobase
Sau3AI fragment derived from the RSV-WT1(A), (B), or WT(AR)
expression vectors was cloned into the BamHI site of pSP64
(Promega, Madison, WI). The pSP64-WT(PM) vector was constructed by
cloning a HindIII/ SalI fragment of RSV-WT(PM) into
HindIII/ SalI digested pSP64. The
pSP64-WT(1-182) vector was made by digestion of pSP64-WT1(A) with
NcoI followed by ligation to the duplex oligonucleotide:
5`-CATGTGAGTCGACTCA-3`, 3`-ACTCAGCTGAGTGTAC-5` containing a stop codon.
To express amino acids 1-182 of WT1 in transfected cells, a
Sau3AI- SalI fragment of pSP64-WT(1-182) was
cloned into BglII/ SalI digested RSV vector. Vectors
for translation of the zinc finger moeities of WT1(A) and WT(AR) were
prepared by PCR amplification of the WT1 cDNA using an
NH-terminal primer 5`-GGATCCGGATCCACCATGTGTGCATACCCAGGC-3`
and a COOH-terminal primer 5`-GAATTCGAATTCTCAAAGCGCCACGTGGAGTTT-3`. The
resultant fragments were digested with BamHI and
EcoRI and inserted into pSP64.
Transfection Assays
CV-1 African green monkey
kidney cells, were grown in Dulbecco's modified Eagle's
medium containing 10% calf serum, while NIH 3T3 mouse fibroblast cells
(gift of N. Landau), and 293 human embryonic kidney cells were cultured
in 10% fetal calf serum. Cells were transfected with 2 µg of
chloramphenicol acetyltransferase (CAT) reporter plasmid, up to 20
µg of WT1 expression vector or RSV vector lacking the WT1 insert as indicated, and 1 µg of tkGH plasmid as an internal
control for transfection efficiency as described
(25) . Carrier
DNA (pBluescript, Stratagene, La Jolla, CA) was added to a total of
20-40 µg of DNA. Normalized CAT activity was determined as
percent conversion of chloramphenicol divided by the growth hormone
value in nanograms/milliliter
(25) .
Immunoblotting
Transfected cells were boiled in 1
SDS sample buffer without
-mercaptoethanol, and extract
concentrations were determined with the Bio-Rad DC Protein Assay Kit
(Bio-Rad) using bovine
-globulin as a standard. Following this,
-mercaptoethanol was added to a final concentration of 5%, and
equal quantities of cellular proteins were separated by electrophoresis
through a 12% SDS-polyacrylamide gel. The gel was then
electrophoretically transferred to Immobilon polyvinylidene difluoride
membrane (Millipore, Bedford, MA) in a buffer containing 192
mM glycine and 25 mM Tris base. The membranes were
probed with 0.1 µg/ml rabbit polyclonal anti-WT1 C19 Antibody
(Santa Cruz Biotechnology, Santa Cruz, CA) as directed by the
manufacturer. Immunoreactive proteins were visualized by
chemiluminescence and autoradiography (ECL kit, Amersham,
Buckinghamshire, United Kingdom).
Electrophoretic Mobility Shift Assays
Proteins
were translated from pSP64-based vectors using the TNT rabbit
reticulocyte lysate system (Promega, Madison, WI). In each case, a
reaction containing [S]methionine was performed
in parallel with each non-labeled reaction. Equal volumes of lysate
from the labeled reaction were subjected to SDS-polyacrylamide gel
electrophoresis and quantified on a PhosphorImager using ImageQuant
software (Molecular Dynamics, Sunnyvale, CA). Binding reactions were
performed with equimolar quantities of translated WT1 proteins as
described previously
(26) , except that the total volume of the
reaction was 20 µl, the amount of probe used was 0.2 ng
(approximately 10
counts/min/ng), and the electrophoresis
was performed for 1.5 h at 300 V at room temperature.
In Vitro Protein Binding Assays
GST and GST fusion
proteins were purified as described
(27) , and the yield of each
protein was determined by SDS-polyacrylamide gel electrophoresis
analysis and Coomassie Blue staining. Volumes of bacterial lysate
containing approximately 1 µg of GST or GST fusion protein were
incubated with 25 µl of a 50% slurry of glutathione-agarose beads
(Sigma) in NET-50 (20 mM Tris, pH 8.0, 1 mM EDTA, 50
mM NaCl) for 15 min at room temperature with rocking. GST
proteins bound to the beads were then collected by brief centrifugation
and washed once with 400 µl of NET-50. The beads were rocked for 10
min at room temperature in 200 µl of binding buffer (25 mM
HEPES, pH 7.5, 12.5 mM MgCl, 20% glycerol, 0.1%
Nonidet P-40, 150 mM KCl, 1 mM dithiothreitol, 150
µg/ml bovine serum albumin, 200 µg/ml ethidium bromide (EtBr)
as indicated). Coupled in vitro transcription-translation
reactions containing [
S]methionine (1175
Ci/mmol) were programmed with the indicated pSP64-WT or control
SP6-luciferase plasmid and were diluted 1:10 in binding buffer (without
dithiothreitol, bovine serum albumin, or EtBr). Diluted programmed
lysate (5 µl) was incubated with immobilized GST or GST-WT proteins
for 1 h at room temperature. The beads were collected by centrifugation
and washed three times with 1 ml of NETN (20 mM Tris, pH 8.0,
100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) in the
presence or absence of 200 µg/ml EtBr as indicated. Bound proteins
were eluted from the beads by boiling in 1
loading buffer (62.5
mM Tris, pH 6.9, 10% glycerol, 2% SDS, 5%
-mercaptoethanol) and were separated by electrophoresis through a
10 or 15% SDS-polyacrylamide gel. The gel was fixed in 30% methanol,
10% acetic acid for 1 h, washed in water for 0.5 h, soaked in 1
M sodium salicylate for 0.5 h, dried, and exposed to XAR film
at -80 °C. The percentage of input
S-labeled
protein bound to GST protein was quantified by exposure of the dried
gel on a PhosphorImager and analyzed using ImageQuant software
(Molecular Dynamics, Sunnyvale, CA).
Mapping of WT1 Self-association Domains Using the
Two-hybrid System in Yeast
A LexA-WT1 expression vector was
constructed by excision of the WT1(A) cDNA from pGEM7zf(+)WT1
(gift of Frank J. Rauscher, III) using EcoRI and
ClaI, blunting these ends with the Klenow fragment of DNA
polymerase I, and cloning the resultant fragment into pEG202
(28) . To create LexA-WT1(1-180), LexA-WT1 was restricted
with BamHI to release the COOH-terminal two-thirds of WT1, and
the plasmid was religated. LexA-WT1(175-301) and
LexA-WT1(275-429) were created by PCR amplification of the
indicated portion of the WT1 coding sequence and ligation of these PCR
fragments into pEG202. An expression vector for the B42 acidic
activation domain-WT1 fusion protein was constructed by digestion of
pGEM7zf(+)WT1 with EcoRI and XhoI and insertion
of the resulting fragment into EcoRI/ XhoI cut
pJG4-5
(29) . LexA-WT1 and B42-WT1 expression vectors were
transformed in pairs into the yeast strain EGY48 containing the
reporter LexAop-LacZ
(28) . After growth in
5-bromo-4-chloro-3-indolyl--D-galactopyranoside, the
yeast colonies were scored for blue color.
-Galactosidase activity
of individual colonies was determined as described
(30) .
Graphics
Autoradiographs were imaged using a
Silverscan flatbed digital scanner (La Cie, Ltd., Beaverton, OR). Image
processing was performed on a Power PC (Apple, Cupertino, CA) using
Adobe Photoshop (Adobe, Mountain, CA) and Aldus Persuasion (Aldus,
Seattle, WA). Densitometry was performed using the NIH Image 1.56
program for Power PC. Figures were printed using a XL7700 Digital
Continuous Tone Printer (Kodak).
Transcriptional Activation by WT1 Proteins
To
characterize the transcriptional effects of WT1 proteins, the
EGR tkCAT reporter containing three Egr-1 consensus
sequences, capable of binding WT1(A) and (B)
(7) (Fig. 1 B) was transfected into CV-1 cells
along with increasing amounts of WT1(A) or (B) expression vectors
(Fig. 1 A). Both WT1(A) and (B) acted as dose-dependent
transcriptional activators of this simple test promoter. Transfection
of 10 µg of the WT1(A) or (B) expression vector produced a
10-15-fold increase in reporter gene transcription.
Transcriptional activation was binding site dependent, as neither
WT1(A) or (B) affected transcription from the tk-hGH plasmid
or from a tk-CAT reporter gene (data not shown). Similar
results were obtained in NIH 3T3 cells and in human embryonic kidney
293 cells (data not shown) indicating that activation was not cell
type-specific. In addition, transcriptional activation by WT1(A) was
linear and non-saturable at the doses used in these experiments
(Fig. 2 C), implying that any cofactors required for
WT1(A) activity are not limiting in CV-1 cells. After six independent
determinations using three different preparations of expression vector
plasmid, transcriptional activation by WT1(B) was consistently
20-50% stronger than activation by WT1(A)
(Fig. 2 A). However, immunoblotting and densitometric
analysis indicated that after transfection, WT1(B) was expressed at
somewhat lower levels than WT1(A) (Fig. 2 B). In
addition, in vitro translated WT1(A) and (B) bound to the
Egr-1/WT1 site to a similar extent (Fig. 2 D). Therefore,
increased DNA binding is unlikely to account for the increased
transactivational activity of WT1(B).
Figure 2:
A, transcriptional activation of
EGR tkCAT by WT1 in CV-1 cells. The average fold
induction of transcription (normalized CAT activity) and standard error
from six independent experiments is presented. B,
immunoblotting analysis of extracts from CV-1 cells transfected with 5
µg of RSV-WT1(A) or RSV-WT1(B), probed with anti-WT1 C19 antibody
(Santa Cruz Biotechnology). The autoradiogram was digitally scanned and
densitometrically quantified. C, transcriptional activation by
WT1 increases linearly with the dose of WT1 expression vector. The
EGR
tkCAT reporter was cotransfected into CV-1
cells with increasing amounts of WT1 expression vector. The fold
induction of transcription for a representative experiment is indicated
below the chromatogram. D, WT1(A) and (B) bind to the Egr-1
site to the same extent as determined by electrophoretic mobility shift
assay. 1 = probe only; 2 = unprogrammed
reticulocyte lysate; 3 = WT1(A); 4 =
WT1(B). The specific WT1
DNA complex is indicated by an
arrow.
Two Dominant Negative WT1 Alleles Inhibit Transcriptional
Activation by WT1(A)
The WT(AR) allele of WT1 was isolated from a patient with a sporadic, unilateral
Wilms' tumor. The mutation alters a splice donor site, leading to
a deletion of zinc finger 3 of the WT1 protein
(31) and yields a protein which binds poorly to the Egr-1
consensus binding site
(7) . Since the WT(AR) mutation
was present in the heterozygous state, it was proposed to be a dominant
negative allele, which suppresses the function of the normal allele
(31) . The protein products of this mutant allele, referred to
as WT(AR-) and WT(AR+), differ due to the absence or
presence, respectively, of the 17 amino acids coded for by the
alternatively spliced exon 5 of WT1. The COOH-terminal splice
region is eliminated by the deletion of the third zinc finger
(Fig. 1).
tkCAT reporter in CV-1
cells at doses of 4 and 8 µg (data not shown). At doses of 10
µg and above, the WT(AR) proteins produced an approximately 50%
increase in transcription from the EGR
tkCAT
reporter (data not shown), compared with an over 10-fold increase in
transcription by WT1(A) (Fig. 2 A). Although full-length
WT(AR) does not bind to the Egr-1 consensus sequence (7, and see
below), it can bind to sequences other than the Egr-1 consensus
(33) . At high concentrations, WT(AR) could possibly bind to
sites in the backbone of the reporter plasmid, resulting in modest
activation of transcription.
Figure 3:
Two dominant negative WT1 alleles
interfere with transcriptional activation by WT1 ( A). The
EGR tkCAT reporter was cotransfected into CV-1
cells with 4 µg of RSV-WT1(A) and the indicated amounts of
RSV-WT1(B) ( A), RSV-WT(AR-), or RSV-WT(AR+) ( B),
and RSV-WT(PM) or RSV-WT1(1-182) ( C). The amount of
transcription obtained is presented as the percent of activation
directed by WT1(A), where the data are averaged from two independent
experiments for WT1(B), WT(AR-) and WT(AR+), from five
independent experiments for WT(PM), and from three independent
experiments for WT1(1-182).
To determine which domain of the mutant WT1 protein was required for inhibition of transcriptional activation,
we utilized the WT(PM) mutant allele. This protein contains
only the first 256 amino acids of WT1 and cannot bind to the Egr-1 site
since it lacks zinc finger motifs (see below). Expression of either
WT(PM) or of a protein containing the first 182 amino acids of WT1
inhibited transactivation by full-length WT1 in a dose-dependent manner
(Fig. 3 C). Unlike WT(AR), WT(PM) and WT1(1-182)
continued to inhibit transactivation at the highest dose of input
plasmid. Therefore, we conclude that the NH-terminal 182
amino acids of WT1 are sufficient to mediate down-modulation of
transcriptional activation by WT1(A).
WT(AR) and WT(PM) Do Not Interfere with DNA Binding by
WT1
To determine whether expression of either dominant negative
mutant protein might interfere with DNA binding by WT1(A), we performed
electrophoretic mobility shift assays with in vitro translated
WT1(A), WT(AR-), and WT(PM) proteins. As predicted, WT(AR-)
and WT(PM) did not bind to the Egr-1 consensus site (Fig. 4,
lanes 3 and 7) while WT1(A) bound strongly to this
site (Fig. 4, lanes 2 and 6). This complex was
specific as addition of unlabeled Egr-1 consensus site oligonucleotide
eliminated the WT1(A)DNA complex, and addition of an antiserum
against WT1, but not preimmune serum, slowed the mobility of the
WT1(A)
DNA complex (data not shown). Preincubation of WT1(A) with
either an equimolar amount or a 2-fold molar excess of WT(AR) or WT(PM)
did not interfere with DNA binding by WT1 (Fig. 4, lanes
4, 5, 8, and 9).
Figure 4:
Dominant negative WT1 proteins do not
interfere with DNA binding by wild-type WT1. A, lane
1: unprogrammed reticulocyte lysate. Lane 2, WT1(A).
Lane 3, WT(AR-). Lane 4, WT1(A) preincubated with an
equimolar amount of WT(AR-). Lane 5, WT1(A) preincubated
with a 2-fold excess of WT(AR-). Lane 6, WT1(A).
Lane 7, WT(PM). Lane 8, WT1(A) preincubated with an
equimolar amount of WT(PM). Lane 9, WT1(A) preincubated with a
2-fold excess of WT(PM). The WT1DNA complex is indicated by an
arrow.
Mutant and Wild-type WT1 Proteins Interact in
Vitro
To further investigate the mechanism of action of the
dominant negative WT1 proteins, we determined whether WT1(A) could
associate with wild-type WT1 proteins or with proteins encoded
by the WT(AR) or WT(PM) alleles. GST-WT1(A) fusion
protein immobilized on glutathione-agarose beads was incubated with
[S]methionine-labeled WT1(A), WT1(B), or mutant
WT(AR-), WT(AR+), or WT(PM) proteins, produced in reticulocyte lysates. Bound
S-labeled proteins were eluted, electrophoretically
separated, and visualized by fluorography. The two WT(AR) mutant isoforms, as well as wild-type WT1(A) and (B), bound to
GST-WT1(A) (Fig. 5 A, rows 1-4), while a
control luciferase protein did not (Fig. 5 A, row
5). None of the proteins bound to naked glutathione-agarose beads
or to GST protein-coated beads (Fig. 5 A). The
interaction between WT1 proteins was unlikely to be due to
coprecipitation of these DNA-binding proteins through interaction with
DNA fragments in the reaction mixture, since such DNA-protein
interactions would be expected to be destabilized by the addition of
200 µg/ml EtBr
(34) . As seen in Fig. 5 A,
EtBr had no effect on interaction between these full-length WT1 proteins.
Figure 5:
Mutant and wild-type WT1 proteins
interact in vitro. S-Labeled proteins (indicated
at left) were incubated with either naked glutathione-agarose beads or
with GST or GST-WT1 A proteins bound to the beads (indicated at
top), in the presence or absence of 200 µg/ml EtBr. After
washing and elution, bound proteins were electrophoretically separated
and visualized by fluorography. For comparison, the input amount of
S-labeled protein for each reaction was loaded in the
far left lane. A, full-length wild-type and mutant
S-labeled WT1 proteins and
S-labeled
luciferase protein were incubated with the indicated GST fusion
proteins. B,
S-labeled full-length WT1(A) or
truncated WT1 proteins containing the indicated amino acids
were incubated with the indicated GST fusion
proteins.
We then mapped the domain required for WT1
self-association. Full-length WT1(A) and WT(AR) bound both to
GST-WT1(A)(1-429) and to a GST-WT1 fusion protein containing only
amino acids 1-183 (Fig. 5 B, row 1).
Interaction between full-length proteins was consistently more
efficient than interaction between full-length WT1 and the NH terminus of WT1. Similarly, a greater extent of interaction was
observed between
S-labeled WT1 containing amino acids
1-182 with GST-WT1(1-183) (35% of input labeled protein
retained on the beads) than with the full-length
GST-WT1(A)(1-429) fusion protein (6% of input labeled protein
retained on the beads) (Fig. 5 B, row 5). This
suggests that the full-length protein is folded in such a way that the
NH
-terminal interaction surface is partially inaccessible,
and when two full-length molecules interact, this blockage is relieved.
-terminal
truncated proteins.
Mapping of WT1 Self-association Domains in Vivo Using the
Two-hybrid System in Yeast
To determine the ability of WT1
protein to self-associate in vivo, we used a yeast two-hybrid
system
(28, 29) . The full-length WT1 coding region, or
smaller fragments of the coding region, was fused in-frame to the
DNA-binding domain of the bacterial LexA protein. The second hybrid
fused WT1 to the B42 acidic activation domain. Plasmids encoding these
hybrids were introduced into yeast strain EGY48, containing the
2-µm-based reporter plasmid LexAop-LacZ. This reporter contains two
Lex operators and the GAL1 promoter linked to the E. coli -galactosidase reporter gene. Interaction between WT1 fused
to LexA and the WT1/B42 acidic activation domain hybrid will stimulate
transcription from the reporter gene. As seen in , WT1
interacted with itself in this assay as scored by qualitative
(blue/white) and quantitative
-galactosidase assays. The domain of
WT1 responsible for self-association mapped to the
NH
-terminal 180 amino acids of the protein. These results
are consistent with our finding that in transfection studies,
WT1(1-182) was a potent inhibitor of transactivation by
full-length WT1 (Fig. 3 B) and that the
NH
-terminal 182 amino acids mediated self-association of
WT1 molecules in vitro (Fig. 5 B). In addition,
these results suggest that association between the zinc finger domains
of WT1 may not contribute to self-association of WT1 in vivo.
Transcriptional Activation by WT1
Consistent
with previous work
(15, 16) , we found that the WT1
protein activated the HSV- tk promoter through upstream binding
sites. WT1(B) was a stronger transcriptional activator than WT1(A);
however, WT1(A) and (B) bound DNA to similar extents in vitro and WT1(B) was expressed at a somewhat lower level than WT1(A) in
transfected cells. Together this information suggests that the 17
additional amino acids in WT1(B) augment the transcriptional activation
domain of WT1. This insertion (MAAGSSSSVKWTEGQSN) contains a potential
protein kinase C phosphorylation site (SVK). Other factors such as the
cyclic AMP response element-binding protein (CREB)
(35) and
c- jun(36) can be dynamically regulated by
phosphorylation. While a prior study did not detect phosphorylation of
WT1(A)
(5) , the phosphorylation state of WT1(B) has not been
examined. Alternatively, the inserted amino acids in WT1(B) could
interact directly with a component of the transcription machinery to
stimulate transcription, or could effect a conformational change in the
entire WT1 protein, altering its interaction with the
transcriptional machinery.
Mechanism of Action of Dominant Negative WT1
Alleles
The ability of the dominant negative alleles WT(AR) and WT(PM) to inhibit transcriptional activation by
WT1 could be explained by one of three mechanisms: competition
for binding sites
(37) , competition for co-factors
(``squelching'')
(38) , or physical interaction
between wild-type and mutant proteins
(39) . Competition between
wild-type and mutant proteins for binding to the Egr-1-binding site is
unlikely to play a role in this effect, since the WT(AR) and
WT(PM) mutant proteins do not bind to this site (7, and
Fig. 4
). In addition, neither dominant negative protein
interfered with DNA binding by WT1(A) in vitro (Fig. 4).
Since cofactors for transcriptional activation by WT1 do not appear to
be limiting in our cell lines (Fig. 2 C), the mutant
proteins are also not likely to inhibit transcription by squelching.
Table:
WT1
self-association assayed using the two-hybrid system in yeast
-galactosidase activity as described (30). Results are expressed
as the mean ± standard deviation for three independent isolates.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.