(Received for publication, September 21, 1995)
From the
The WT1 Wilms' tumor suppressor gene encodes a
zinc finger transcription factor which plays a critical role in renal
and genitourinary development. The WT1 protein was reported to both
activate and repress transcription. We found that the transcriptional
effect of WT1 on the Egr1 promoter could be modulated by the
use of expression vectors containing different promoters. WT1 activated
the Egr1 promoter when expression of WT1 was driven by the
Rous sarcoma virus promoter. In contrast, a cytomegalovirus (CMV)
promoter-containing WT1 expression vector repressed the Egr1 promoter. However, WT1 activated transcription of a simple test
promoter, EGRtkCAT, regardless of the expression
vector used. Co-transfection of the parental CMV-based vector strongly
depressed the basal activity of the Egr1-CAT reporter, suggesting that
the CMV promoter competes with the Egr1 promoter for
transcription factors or co-factors which may be required for
activation by WT1. In support of this hypothesis, WT1 was converted
from an activator to a repressor by co-transfection of an excess of the
parental CMV-based vector. These results provide an important caveat to
the interpretation of co-transfection studies and confirm the
bi-functional nature of the WT1 transcription factor.
The Wilms' tumor suppressor 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 zinc finger transcription factors, termed WT1(A-D)(3, 4, 5) . These isoforms arise form the inclusion of a 17-amino acid segment in the amino terminus of the protein and a 3-amino acid sequence (KTS) in the zinc finger region of the protein. The A and B isoforms, also known as WT1(-KTS), bind to the same DNA sequence as the Egr1 transcription factor (5`-GCGGGGGCG-3`)(6, 7) . Isoforms C and D bind poorly to the Egr1 binding site due to the insertion of three amino acids (KTS) between zinc fingers 3 and 4(7) .
WT1 can both activate and repress transcription, and several factors have been shown to influence the transcriptional activity of WT1. Studies on the platelet-derived growth factor A-chain (PDGF-A) and other promoters suggest that WT1 represses transcription when it is bound both upstream and downstream of the start site of a promoter(8, 9, 10, 11, 12) . Conversely, WT1 activates transcription when WT1 binding sites are present only 5` or 3` to the transcription start site of the PDGF-A promoter or when WT1 binding sites are inserted upstream of the HSV-tk or MMTV promoters(10, 13, 14, 15) . However, WT1 was also reported to repress transcription from upstream binding sites in its native state(16, 17, 18) and when expressed as a GAL4-WT1 fusion protein(19) . Finally, interaction between WT1 and the p53 tumor suppressor protein can determine whether WT1 acts as a repressor or an activator of transcription(20) .
In this study, we identified a new
factor influencing the transcriptional effects of WT1 which may help to
resolve some of the apparent inconsistencies in this field. We show
that WT1 can either activate or repress transcription from the Egr1-CAT
reporter, depending on which promoter is used to drive the expression
of WT1. In contrast, WT1 activates transcription from the simple
EGRtkCAT reporter gene regardless of the
expression vector used, confirming the role of promoter architecture in
determining the transcriptional function of WT1. WT1 repressed
transcription of the Egr1 promoter only when expressed by the
pCB6+ vector, which contains the strong cytomegalovirus (CMV) (
)promoter. Repression mediated by the pCB6+ vector was
not related to the higher levels of protein expressed by this
construct. Rather, repression was related to the ability of the
pCB6+ vector itself to profoundly depress the basal activity of
the Egr1 promoter. We confirmed the importance of this
promoter competition effect by demonstrating that WT1 expressed from
the RSV vector was converted from an activator to a repressor of the Egr1 promoter by co-transfection of an excess of pCB6+
vector lacking a WT1 cDNA. Taken together, these results
suggest that competition between the CMV and Egr1 promoters
for transcription factors and/or co-factors results in changes in the
basal activity of the Egr1 promoter and that only under
conditions of low basal activity is the Egr1 promoter able to
be repressed by WT1. These data suggest that WT1 may require one or
more co-factors to activate transcription. In the absence of these
co-factors the protein acts as a repressor. Our results also provide an
important caveat regarding choice of expression vector in transfection
experiments.
Figure 1:
Schematic
representation of the EGRtkCAT and Egr1-CAT
reporter genes and the RSV-WT1, rat
-actin-WT1, and pCB6-WT1
expression vectors used for co-transfection experiments. The locations
of transcription factor binding sites in the target promoters are
indicated. RSV LTR, promoter and enhancer sequences derived
from the long terminal repeats of the Rous sarcoma virus. MCS,
multiple cloning site. SV40 Ori/E, SV40 virus promoter and
enhancer sequences, including the viral replication origin. SV40T
IVS, pA, intervening sequences and 3` polyadenylation site of the
T antigen gene of the SV40 virus. NeoR-neomycin resistance
gene. hGH Term, transcriptional termination sequences from the
human growth hormone gene.
Our previous work (14) showed that WT1 was a
transcriptional activator of a simple test promoter containing three
Egr1/WT1 binding sites upstream of the HSV-tk promoter
(EGRtkCAT) (Fig. 1). In contrast, Madden et al.(16) found that WT1 repressed transcription of
the Egr1 promoter. The complex Egr1 promoter contains
three potential Egr1/WT1 binding sites as well as binding sites for
AP-1 and SRF upstream of the start site of transcription (23, 27) (Fig. 1). To investigate the
conditions that might affect the ability of WT1 to activate or repress
transcription, we co-transfected both NIH 3T3 cells and CV1 cells with
an Egr1 promoter-containing reporter gene and the RSV-WT1
expression vector. To our surprise, we found that expression of murine
WT1 activated rather than repressed transcription of this promoter (Fig. 2, A and B). The same RSV-based
expression vector also activated the simple
EGR
tkCAT reporter gene ( Fig. 3and (14) ). To further explore this phenomenon, we constructed an
expression vector for WT1 containing the
-actin promoter and
obtained a CMV-based expression vector for WT1 used by many other
groups for co-transfection studies (pCB6+(16) ; gift of V.
Sukhatme). The Egr1 promoter was activated by WT1 expressed
from the RSV or
-actin promoters. Repression of the Egr1 promoter only occurred when WT1 was expressed from the pCB6+
vector (Fig. 2, A and B). While only modest
repression was observed at 10 µg of input effector plasmid,
stronger repression was seen at a dose of 20 µg (Fig. 2, A and B). In contrast, WT1 always activated
transcription of the EGR
tkCAT reporter, regardless
of the expression vector used (Fig. 3).
Figure 2:
WT1 can activate or repress transcription
in the same cell type, depending upon the expression vector utilized. A, NIH 3T3 cells were transfected with 0.5 µg of Egr1-CAT
reporter and 10 or 20 µg of RSV, -actin, or pCB6+
expression vectors, either lacking or containing the WT1 cDNA,
as indicated. The intrinsic activity of the Egr1-CAT reporter by was
determined by co-transfection with pBluescript alone. Transcriptional
activity is expressed as percent conversion of chloramphenicol to
acetylated chloramphenicol, normalized for the protein concentration of
the extracts and the incubation time of the CAT assay. The results are
presented as the average of four independent determinations. The fold
activation or repression is indicated. B, the same experiment
as in A was performed in CV-1 cells. The results are presented
as the average of two independent determinations.
, empty
expression vector;
, WT1 expression
vector.
Figure 3:
The
EGRtkCAT reporter gene cannot be repressed by
CMV-WT1. A, the EGR
tkCAT reporter plasmid
(2 µg) was co-transfected with 10 µg of the indicated
expression vector or control vector lacking the WT1 cDNA
insert. The intrinsic activity of the EGR
tkCAT
reporter by was determined by co-transfection with pBluescript alone.
Transcriptional activity is expressed as percent conversion of
chloramphenicol to acetylated chloramphenicol, normalized for the
protein concentration of the extracts and the incubation time of the
CAT assay. The fold activation or repression is indicated. The results
are presented as the average of four independent determinations.
, empty expression vector;
, WT1 expression
vector.
We then examined the
reasons why the choice of expression vector might influence the
transcriptional activity of WT1. The effect was not cell
type-dependent, since activation and repression were effected by
RSV-WT1 and pCB6-WT1, respectively, both in NIH 3T3 cells and CV-1
cells (Fig. 2, A and B). Second, since
pCB6-WT1 contains the human WT1 cDNA, whereas RSV-WT1 and
-actin-WT1 contain the murine WT1 cDNA, we wanted to rule out the
possibility of a species-specfic effect. Therefore, we cloned the
murine WT1 cDNA into the pCB6+ vector and found that it repressed
the Egr1 promoter in an identical manner to the human cDNA
(data not shown).
Next, we determined whether the transcriptional
effect of WT1 might depend on the level of protein expressed from the
various expression vectors. We performed immunoblotting analysis on the
same transfected cell extracts used in the CAT assays. Transfection of
pCB6-WT1 yielded a much higher level of WT1 protein expression than
that achieved by either the RSV or -actin-based expression vectors
in both NIH 3T3 and CV-1 cells (Fig. 4A). Transfection
of NIH 3T3 cells with 5 or 0.5 µg of each expression vector showed
that the pCB6+ expression vector yields approximately 10-fold more
WT1 protein than the RSV vector (Fig. 4B).
Dose-dependent transcriptional effects have been reported for the Drosophila Krüppel protein, which activates
transcription at low doses but represses transcription at high
doses(28) . It was therefore possible that WT1 had a similar
dose-dependent transcriptional effect. However, we could not confirm
this hypothesis, as transfection of even up to 40 µg of RSV-WT1
still activated the Egr1 promoter (data not shown).
Conversely, we could not observe activation of the Egr1 promoter by low doses of pCB6-WT1 (data not shown).
Figure 4:
The
pCB6+ expression vector yields a 10-fold higher level of WT1
protein expression than the RSV or -actin expression vectors.
Extracts from transfected NIH 3T3 or CV-1 cells were subjected to
immunoblot analysis with affinity-purified polyclonal anti-WT1 C19
antibody (Santa Cruz Biotechnology). A, immunoblot of extracts
from NIH 3T3 cells or CV-1 cells transfected with 10 µg of RSV-WT1,
-actin-WT1, or pCB6-WT1. B, immunoblot of extracts from
NIH 3T3 cells transfected with the indicated amounts of the RSV-WT1 or
pCB6-WT1 expression vectors.
Finally, we
determined whether the actions of the strong CMV promoter itself might
be affecting the results of the transient co-transfection assay. Almost
all previously reported studies which showed transcriptional repression
by WT1 have utilized the pCB6+ expression vector. In these
experiments, a total of 20 µg of pCB6+ vector, with or without
the WT1 cDNA insert, has been used per 100-mm dish of cells.
We noted that transfection of the empty pCB6+ vector significantly
inhibited transcription of the Egr1 promoter as compared with
the level activity directed by the promoter co-transfected with
pBluescript (Fig. 2, A and B). The RSV and
-actin expression vectors affected Egr1-CAT expression to a
significantly lesser extent than the pCB6+ vector (
8-fold, Fig. 2A). A dose-response experiment confirmed that
addition of increasing amounts of pCB6+ to the transfection
progressively and severely depressed the basal transcriptional activity
of the Egr1 promoter. At a dose of 10 µg of pCB6+, Egr1 promoter activity was 44-fold depressed compared with the
level obtained from cells co-transfected with pBluescript (Fig. 5). This indicates that the pCB6+ vector itself was
inducing a profound alteration in the Egr1 promoter activity,
most likely by sequestering transcription factors and/or co-factors
away from the Egr1 promoter. Therefore, WT1 might regulate
transcription differently on this ``depressed'' Egr1 promoter compared with its effects on high level transcription
observed in the absence of pCB6+.
Figure 5: The basal activity of the Egr1 promoter is repressed by co-transfection of the pCB6+ expression vector. The Egr1-CAT reporter (0.5 µg) was transfected into NIH 3T3 cells along with the indicated amount of pCB6+ vector lacking the WT1 cDNA insert. The total amount of DNA in the transfection was kept constant by the addition of pBluescript. Transcriptional activity is expressed as percent conversion of chloramphenicol to acetylated chloramphenicol, normalized for the protein concentration of the extracts and the incubation time of the CAT assay. The results are presented as the average of three independent determinations.
To dissect apart the effect of WT1 protein levels from the effect of the pCB6+ expression vector, we co-transfected the Egr1 promoter with the RSV-WT1 expression vector and determined the effect of additional co-transfection of pCB6+ lacking a WT1 cDNA insert. As before, RSV-WT1 activated the Egr1 promoter (Fig. 6A). Co-transfection of pCB6+ vector in combination with RSV-WT1 both decreased the basal activity of the Egr1 promoter and converted WT1 from an activator to a repressor of the Egr1 promoter (Fig. 6A). In the absence of pCB6+, WT1 activated transcription approximately 2-fold, whereas in the presence of 20 µg of co-transfected pCB6+, WT1 repressed transcription approximately 4-fold. Immunoblotting analysis of extracts from these transfected cells showed that there was a lower level of WT1 expressed in the presence of pCB6+ than in the absence of pCB6+, ruling out the possibility that repression by WT1 depended on high levels of protein expression (Fig. 6B). We also performed the converse experiment. In this experiment, the activity of the Egr1-CAT reporter was repressed 60% (1.7-fold) by co-transfection with 20 µg of pCB6-WT1. Further co-transfection with the RSV expression vector lacking an insert was still associated with 20% inhibition of the Egr1 promoter. It is possible that some of the WT1 protein expressed from the CMV promoter bound to the RSV promoter instead of the Egr1 target reporter hence diluting the repressive effect of WT1 protein. In addition less WT1 protein may be produced in the presence of both the RSV and pCB6+ expression vectors (Fig. 6B). Nevertheless, WT1 expressed from the CMV promoter was not converted into an activator by the presence of RSV vector. Together from these data we conclude that WT1 is a default activator of the Egr1 promoter and suggest that repression of this promoter by transfection of pCB6-WT1 is facilitated by competition between the CMV and Egr1 promoters.
Figure 6:
WT1 is converted from an activator to a
repressor of the Egr1 promoter by co-transfection with the
pCB6+ expression vector. A, the Egr1-CAT reporter (0.5
µg) was transfected into NIH 3T3 cells along with 10 µg of the
RSV expression vector, either lacking or containing the WT1 cDNA, in the absence or presence of pCB6+ lacking the WT1 cDNA as indicated. The total amount of DNA in each transfection
was kept constant by the addition of pBluescript. Transcriptional
activity is expressed as percent conversion of chloramphenicol to
acetylated chloramphenicol, normalized for the protein concentration of
the extracts and the incubation time of the CAT assay. The fold
activation or repression is indicated. The experiment was repeated
several times in duplicate, and the results of a representative
experiment are presented as the average of two independent
determinations. , empty expression vector;
, WT1
expression vector. B, protein extracts from the transfected
cells were subjected to immunoblot analysis to determine the level of
WT1 protein expressed in the presence or absence of pCB6+. A
representative experiment is presented.
While the co-transfection assay is a valuable tool which can
provide much information about the regulation of a promoter by a
particular protein, there are significant limitations of this method.
Removal of a promoter from its endogenous chromosomal context may
result in changes in its transcriptional activity and its ability to be
regulated by specific transcription factors. For example, the Egr1 promoter fragment used in these experiments is highly active in
the absence of co-transfected CMV-based expression vector, yielding
50-fold more CAT activity than the HSV-tk promoter (data not
shown). In contrast, the endogenous cellular Egr1 gene is
expressed only transiently in response to various types of
growth-promoting signals(29) . This implies that sequences or
chromatin structures required for proper regulation of the Egr1 gene are not present in the promoter fragment being studied. As
another example, it was found that the MYB protein potently activated a
co-transfected mim-1 reporter gene but failed to activate the
endogenous mim-1 gene. Only when the MYB protein was
co-expressed with C/EBP- was the endogenous gene
activated(30) . This indicates that the requirements of
reporter genes and endogenous genes for transcriptional regulation may
differ.
The transcriptional effects of WT1 depend on the experimental context. Many co-transfection assays using promoters containing GC-rich Egr1/WT1 binding sites showed WT1 to be a repressor of transcription (8, 9, 12, 16, 18, 19, 31-34). WT1 activated transcription of a truncated form of the PDGF-A chain promoter(9) , while it repressed the full-length PDGF-A promoter in the same cell type(9, 33) . Subsequent reports confirmed the ability of WT1 to activate promoter targets(13, 14) . By both deletion analysis and fusion of portions of the WT1 protein to the GAL4 DNA binding domain, the repression domain was mapped to amino acids 85-124 of the protein(10, 15, 19) . A potent transactivation domain was mapped between amino acids 181 and 250, adjacent to the repression region(10, 15) . These studies indicate that the WT1 transcription factor is intrinsically bi-functional in nature. Similarly, adjacent activation and repression domains were noted in the Drosophila Krüppel and even-skipped proteins as well as the Egr1 protein(35, 36) . In particular, the full-length Krüppel protein was shown to both activate and repress transcription(28, 37) .
Given that WT1 contains both an activation and repression domain, several experimental factors may determine what transcriptional effect the protein exhibits, including the nature of the target promoter. In particular, WT1 tends to repress promoters containing binding sites both 5` and 3` to the start site of transcription, but not truncated promoters containing either 5` or 3` sites(8, 9, 12) . It is interesting to note that the repression domain of WT1 maps to the same region as a recently identified self-association domain(14) . This suggests that WT1 self-association might play a role in repression, perhaps by causing looping of DNA between WT1 molecules bound both 5` and 3` to the start site of transcription.
A second factor that may affect the function of WT1 is the cell type in which the protein is expressed. A recent report showed that WT1 activated the insulin-like growth factor-I receptor (IGFI-R) promoter in human embryonic kidney 293 cells and repressed the same promoter in the G401 cell line(38) , originally thought to represent a Wilms' tumor cell line(39) , but now thought to be derived from a rhabdoid tumor of the kidney(40) . Similarly, Madden et al.(19) found that a GAL4-WT1 fusion protein containing amino acids 84-180 of WT1 repressed transcription in NIH 3T3 cells but not in 293 cells. These findings suggest that there may be cell type-specific co-factors required for activation or repression.
A third potential factor which may influence transcriptional regulation is the level of protein expression. In Drosophila Schneider cells, low doses of Krüppel protein activate transcription, while high doses repress transcription(28) . However, this result could not be reproduced in mammalian cells(41) , again pointing to cell type-specific functions of transcriptional repressors. We could not find a clear relationship between WT1 expression level and transcriptional function, as both high and low levels of WT1 protein were associated with both transcriptional activation and repression, depending on the target promoter and presence or absence of the pCB6+ expression vector.
We propose a novel factor which influences the transcriptional effect of WT1, namely that the promoter driving protein expression can have a profound effect on the observed action of the WT1 protein. In reviewing the literature, virtually all prior studies which demonstrated repression by WT1 used the pCB6-WT1 expression vector (8, 9, 12, 16, 18, 19, 31-33), with the exception of the work of Malik et al.(34) who showed 2-fold repression of the WT1 promoter by WT1 expressed from an inducible metallothionein promoter. In contrast we found that WT1, as expressed from the RSV promoter, activated the Egr1 promoter. RSV-WT1-mediated activation was converted to repression by co-transfection of a CMV promoter-containing expression vector lacking the WT1 cDNA insert. The converse, however, was not true, as CMV-WT1-mediated repression was not converted to activation by co-transfection with an empty RSV promoter-containing vector. We believe that the CMV promoter, which has approximately 10-fold higher activity than the RSV promoter (Fig. 4B), could draw cooperating factors or co-factors away from the Egr1 promoter, depressing its basal state and defeating the ability of WT1 to activate transcription. Under these circumstances the repression domain of WT1 predominates (Fig. 7). Alternatively, a co-factor might mask the repression domain of WT1. The CMV promoter could draw this factor away from WT1, again allowing repression to occur (Fig. 7). Although other instances of promoter competition have been documented in the literature(42, 43, 44, 45) , to our knowledge this is the first report of promoter competition playing a role to alter the effect of a specific transcription factor.
Figure 7: A model for the bi-functional transcriptional potential of WT1. A, in cooperation with either DNA binding transcription factors or transcriptional co-factors, WT1 activates the Egr1 promoter. B, in the presence of the strong CMV promoter, co-factors are stripped from the Egr1 promoter and/or from WT1 protein itself. Under these conditions, WT1 represses the Egr1 promoter.
Our
data could be interpreted in an alternative manner. The WT1 protein
could be posited to always be a repressor but its availability to
affect a reporter gene is modulated by the choice of expression vector.
According to this view, WT1 expressed from the RSV promoter is
completely sequestered by the RSV promoter, releasing general
transcription factors for use by the Egr1 promoter. The result
would be activation not directly by binding of WT1 to the Egr1 promoter but indirectly by relief of promoter competition between
the RSV expression vector and the Egr1 reporter. Continuing
along this line of reasoning, if WT1 expressed from the CMV promoter
could not bind to the CMV promoter to release basal factors for use by
the Egr1 promoter, then WT1 protein would not relieve promoter
competition. In addition WT1 protein would bind to the Egr1 reporter gene and directly repress its transcription. Several
factors argue against this model and indicate that WT1 is a direct
activator of transcription. First, WT1 activates the Egr1-CAT reporter
gene in CV-1 cells above the level of the promoter in the presence of
empty RSV vector, indicating that WT1 directly activates the promoter (Fig. 2B). Second, recent data indicate that the
induction of WT1 protein in the tetracycline-repressible system (46) WT1 activates rather than represses the Egr1 reporter gene(47) . Third, we found that RSV-expressed WT1
can activate a RSV--galactosidase reporter (data not shown),
suggesting that WT1 can bind to the RSV promoter, probably through
GC-rich sites and activate transcription directly rather than by a
promoter competition effect. Last, it was demonstrated (48) that WT1 protein binds to pCB6+ through the GC-rich
sites of the SV40 promoter in this expression vector (Fig. 1B). Together this information supports the more
simple model that WT1 directly binds the target gene and may be a
default activator. The transcriptional effect of WT1 is then modulated
by the presence or absence of co-factors which can be sequestered by
the strong CMV promoter.
In further support of the hypothesis that
WT1 needs to cooperate with factors in addition to the basal
transcriptional machinery to activate transcription, our preliminary
data show that WT1 cannot activate transcription through binding sites
upstream of a minimal TATA box-containing promoter, ()whereas WT1 is a potent activator when these same sites
are placed upstream of the HSV-tk promoter ( Fig. 3and (14) ). It is not yet known which transcription factors and/or
co-factors act to augment the activation or repression functions of
WT1. These proteins could be other sequence-specific DNA-binding
proteins and/or transcriptional co-factors such as the transcription
factor IID-associated proteins (reviewed in (49) ). One
candidate factor might be the p53 protein, which was shown to affect
the transcriptional activity of WT1, changing it from an activator to a
repressor of transcription(20) . Wang et al.(15) recently presented data for the the existence of an
as yet unidentified nuclear protein that interacts with WT1 to effect
transcriptional repression. In vitro transcription studies
using fractionated basal factors and transcriptional co-factors will be
required to define the biochemical requirements for activation and
repression by WT1.
Although all expression vectors co-transfected
with the Egr1 reporter gene down-regulated the reporter gene
to some extent, the pCB6+ vector was the most potent competitor
and the only vector among the ones tested that cooperated with WT1 to
facilitate transcriptional repression. The pCB6+ vector contains
two promoters: the CMV major immediate-early promoter, which drives the
expression of the insert cDNA, and the SV40 late promoter, present in
the SV40 origin of replication, which drives expression of a neomycin
resistance gene (described in (48) ) (Fig. 1). The
Egr1-CAT reporter contains nucleotides -957 to +248 of the
murine Egr1 promoter and includes binding sites for Egr1,
AP-1, CREB, and SRF (27, 50) (Fig. 1). The
serum response element is likely responsible for the induction of the Egr1 gene in the G-G
transition(29, 50) . Since the rat
-actin
and RSV promoters contain serum response elements, whereas the CMV
promoter does not(25, 51, 52, 53) ,
it is unlikely that SRF or other proteins that bind to the serum
response element are involved in determining the activation or
repression activity of WT1. However, the CMV promoter contains multiple
binding sites for Sp1, AP1, AP2, and CREB and might exert its strong
negative effect on the Egr1 promoter by competing for binding
of these or other sequence-specific DNA binding transcription factors
or their respective co-factors(25) . pCB6+ also differs
from the RSV and
-actin expression vectors as it contains a pUC18
rather than a pBR322 backbone. However, the pUC18 backbone is unlikely
to play a role in the unique ability of pCB6-WT1 to convert WT1 into a
repressor, as co-transfection of RSV-WT1 along with pBluescript,
another pUC18-based plasmid, still leads to activation of the Egr1 reporter gene ( Fig. 6and data not shown).
Our data
indicate a relationship between the ability of the target reporter gene
to be competed by the pCB6+ promoter and the ability of WT1 to
repress the reporter. The pCB6+ plasmid has a significantly
smaller effect on the basal activity of EGRtkCAT
(8-fold) than on Egr1-CAT (>40-fold) ( Fig. 2and Fig. 5). In turn, WT1 expressed from the pCB6+ vector can
still activate transcription from EGR
tkCAT (Fig. 3). However, it is important to note that pCB6-WT1
activated the EGR
tkCAT promoter less efficiently
than either RSV-WT1 or
-actin-WT1, despite the fact that
approximately 10 times more WT1 protein is produced by pCB6-WT1 than by
the other vectors (Fig. 4B). This latter result
suggests that WT1 expressed at these high levels leads to ineffective trans-activation due to transcriptional
self-squelching(41, 54) . Alternatively, promoter
competition by pCB6+ might partially alter the transcriptional
state of EGR
tkCAT, making it less responsive to trans-activation by WT1.
Our results highlight a major
pitfall in the use of co-transfection experiments to assay
transcriptional effects. The use of a potent expression vector may have
a profound effect on the target gene. While the -actin and RSV
promoters had relatively modest effects on the Egr1 target
gene, the CMV promoter led to as much as a 40-100-fold decrease
in Egr1 promoter activity (Fig. 2A and data
not shown). Therefore the action of the expression vector may overwhelm
the effect of the expressed protein, as in this report, where the CMV
expression vector changed WT1 from an activator to a repressor. A clear
implication of this work is the need to devise more refined procedures
for the identification of authentic WT1 target genes. One such method
would be to stably express WT1 in a suitable target cell line in an
inducible manner and examine the expression pattern of the putative
target genes. This approach was recently taken, using the
tetracycline-repressor system (46) to tightly induce expression
of WT1 in osteosarcoma cells(47) . Many of the genes previously
identified as WT1 targets by co-transfection assay failed to respond
WT1 induction in this endogenous gene assay. A notable exception was
the epidermal growth a factor receptor, which was down-regulated by the
expression of WT1. Of note, transfection of the Egr1 reporter
gene into SAOS cells followed by induction of WT1 expression by
tetracycline withdrawal was associated with activation of the Egr1 promoter, in accordance with our data. Another group forced the
constitutive expression of WT1 in G401 cells leading to the
down-regulation of both an IGFI-R-luciferase reporter gene and the
endogenous IGFI-R gene(38) . This correlated with a
reduction of the growth rate and IGFI responsiveness of the G401-WT1
cells. More studies of this type, focusing on the response of
endogenous genes in correlation with a phenotype relevant to tumor
suppression or kidney development, will be required to fully understand
the direction and magnitude of the transcriptional actions of WT1.