From the Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, Massachusetts 02129
Received for publication, October 23, 2000, and in revised form, January 5, 2001
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ABSTRACT |
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The Wilms tumor gene WT1 encodes a
zinc finger transcription factor that is required for normal kidney
development. WT1 was identified as a transcriptional repressor, based
on its suppression of promoter reporters, but analysis of native
transcripts using high density microarrays has uncovered
transcriptional activation, rather than repression, of potential target
genes. We report here that WT1 binds to the transcriptional coactivator
CBP, leading to synergistic activation of a physiologically
relevant promoter. The physical interaction between WT1 and CBP is
evident in vitro and in vivo, and the two
proteins are co-immunoprecipitated from embryonic rat kidney cells. The
WT1-CBP association requires the first two zinc fingers of WT1 and the
adenovirus 5 E1A-binding domain of CBP. Overexpression of this domain
of CBP is sufficient to inhibit WT1-mediated transcriptional activation
of a promoter reporter, as is co-transfection of E1A. Retrovirally
driven expression of either the CBP fragment or of E1A in human
hematopoietic cells suppresses the induction by WT1 of its endogenous
target gene, p21Cip1. These observations support a model of
WT1 as a transcriptional activator of genes required for cellular differentiation.
The tumor suppressor gene WT1 provides a paradigm for
the link between normal organ development and cancer. WT1
was originally identified by its inactivation in a subset of Wilms
tumors and in the germline of children with genetic predisposition to
this pediatric kidney cancer (1-4). Expression of WT1 is
restricted to specific cell types in the fetal kidney, gonads,
mesothelium, and hematopoietic lineages (5-8). In the developing
kidney, WT1 is present at low levels in blastemal stem cells, and very
high levels of expression are observed as these cells differentiate to
form glomerular podocytes. The critical role of WT1 in
normal renal development is demonstrated by the apoptosis of renal
blastemal cells in WT1-null mice, leading to complete
failure of kidney differentiation (9).
The DNA-binding domain of WT1 is encoded by four C-terminal
Cys-His zinc fingers, which mediate recognition of both GC- and TC-rich
sequences (10, 11). The affinity of WT1 for these motifs is greatly
reduced by the product of an alternatively spliced transcript, in which
the three amino acids KTS are inserted between zinc fingers three and
four (10, 12). The WT1( As a strategy to identify physiologically regulated WT1
target genes, we recently used high density oligonucleotide arrays representing 6,800 genes and expressed sequence tags to compare the
expression profile of cells before and shortly after inducible expression of WT1 (27). Ectopic expression of
WT1( Cell Culture and Expression Constructs--
NIH3T3 cells were
grown in Dulbecco's modified Eagle's medium with 10% fetal calf
serum. RSTEM cells with inducible WT1 expression (27) were
grown at 32 °C in the same media, supplemented with 1 µg/ml
tetracycline. For WT1 induction, cultures at 70% confluence were extensively washed with phosphate-buffered saline and allowed to
grow for an additional 30 h in medium without tetracycline before
protein extraction. CMV-driven constructs encoding
WT1( Transient Transfections and Luciferase Reporter Assays--
NIH
3T3 cells were transfected using the calcium phosphate DNA
precipitation method. For protein extraction following transfection with WT1 deletion constructs, 5 µg of each plasmid was
used. For luciferase reporter assays, 0.5 µg of
CMV-WT1( Immunoprecipitation and Western Blotting--
For
immunoprecipitation experiments, RSTEM or NIH 3T3 cells were lysed on
ice for 20 min in hypotonic buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 5 mM EDTA)
supplemented with protease inhibitors. Cellular extracts were incubated
with either anti-WT1 antibody (C19, Santa Cruz) or anti-CBP antibodies (SC-369, SC-583, Santa Cruz; 06-294, Upstate Technology) at 4 °C
for 1.5 h. Immunoprecipitates were resolved by 7.5%
SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting
(ECL) using monoclonal antibodies against WT1 (13) or CBP. For GST
pull-down assays, DNA fragments encoding the four relevant domains of
CBP were generated by polymerase chain reaction, containing a
primer-derived T7 polymerase-binding site and Kozak consensus sequence
(GCCGCCATGGCT) at their 5' end. Proteins were in vitro
translated in the presence of [35S]methionine using
TNT-coupled transcription/translation kit (Promega), and incubated with
GST-WT1 affinity matrix (27) in phosphate-buffered saline plus 1%
Nonidet P-40 for 1 h at room temperature. After extensive washing,
the precipitates were resolved by SDS-polyacrylamide gel
electrophoresis and analyzed by autoradiography.
Retroviral Infection--
For retroviral expression, DNA
sequences encoding E1A, E1A Enhancement of WT1-mediated Transactivation by CBP--
We made
use of AR, as a potentially physiological
WT1-target gene, to test the effect of CBP
expression on WT1-mediated transcriptional activation. The
WT1-responsive element (WRE) in the AR promoter, 5'-CCGTGGGTGG-3', is located at position In Vivo Physical Association between WT1 and CBP--
In the fetal
kidney, physiological expression of WT1 is restricted to
blastemal stem cells and differentiating glomerular precursors (8). To
identify WT1-interacting proteins in an appropriate cellular context,
we used RSTEM cells, derived from day 12.5 embryonic rat kidney.
Expression of endogenous WT1 in these cells is low but clearly
detectable by immunoblotting, and the presence of a stably transfected
tetracycline-regulated WT1 construct allows a 5-fold
increase in WT1 protein levels without inducing any discernible
alterations in cellular properties (27). Extraction of RSTEM cellular
lysates using nonionic detergents, followed by immunoprecipitation with
antibody against the C terminus of WT1 (C19), and immunoblotting using
anti-CBP antibody demonstrated co-immunoprecipitation of these two
proteins (Fig. 2). Similarly, immunoprecipitation of lysates with anti-CBP antibody, followed by
Western blotting with anti-WT1 antibody showed co-precipitation of WT1
with endogenous CBP. Comparison of the amounts of co-precipitated WT1
or CBP with the total cellular proteins directly immunoprecipitated from cellular lysates (Fig. 2) indicated that ~10% of cellular CBP
is co-precipitated with WT1, while a similar fraction of cellular WT1
is associated with CBP. The two major isoforms of WT1, resulting from
the KTS alternative splice between zinc fingers 3 and 4, demonstrated
comparable binding to CBP. In many cell types, CBP is thought to be
limiting in its cellular concentration, resulting in possible
competition for binding with different transcription factors. The
levels of WT1 expression achieved in RSTEM cells are
comparable to those seen in glomerular precursor cells, in which
WT1 is presumed to play an important role in mediating
cellular differentiation. The stoichiometry of the interaction between WT1 and CBP observed in RSTEM cells may therefore be physiologically relevant in the developing kidney.
Domains Involved in the Interaction between WT1 and CBP--
We
used five deletion constructs spanning the entire length of the
WT1 coding region to map the site required for its
interaction with CBP. The HA epitope-tagged deletion constructs were
transfected into NIH 3T3 cells along with a plasmid expressing
CBP, and whole cell extracts were subjected to
immunoprecipitation Western analysis (Fig.
3). Equal expression of the transfected
constructs was determined, and anti-CBP immunoprecipitates were
analyzed for the presence of WT1 using antibody against the HA epitope.
A WT1 product lacking the first two zinc fingers failed to
co-immunoprecipiate with CBP, indicating that this portion of the WT1
DNA-binding domain is required for this protein interaction.
To identify the domain of CBP required for its interaction with WT1, we
incubated a bacterially synthesized protein containing the four WT1
zinc fingers with in vitro translated domains of CBP. This
in vitro approach was used to confirm the direct interaction between these two proteins. CBP encodes a protein of 300 kDa, but four domains mediate its known interactions with transcription factors (34-39). Polymerase chain reaction-generated fragments encoding amino acids 1-436 (CBP1: binding site for nuclear hormone receptors), amino acids 437-786 (CBP2: binding site for CRE-binding factors), amino acids 1625-1991 (CBP3: binding site for E1A), and
amino acids 1992-2442 (CBP4: binding site for p53) were translated in vitro, and incubated with GST-WT1 affinity matrix. Only
CBP3 bound to WT1 (Fig. 4), indicating
that the E1A-binding domain of CBP is also responsible for its
interaction with WT1. Consistent with the stoichiometric protein
interaction observed in vivo, the in vitro
association between WT1 and CBP was quantitative, with ~1/3 of input
CBP protein associated with WT1. Of note, the WT1(+KTS) isoform also
showed high affinity binding to CBP (Fig. 4). The possibility that this
alternative splice product of WT1, which has been previously linked to
a role in pre-mRNA processing (16), also plays a role in
transcriptional regulation warrants further investigation.
Disruption of the WT1-CBP Interaction Abrogates WT1 Target Gene
Expression--
Having defined the domains required for the
interaction of WT1 with CBP, we sought to determine the effect of
disrupting this protein association on WT1-mediated transcriptional
activation. We first tested whether CBP3, the domain that mediates the
interaction of CBP with WT1 but lacks functional histone
acetyltransferase activity, could function as a dominant negative
construct in reporter assays. Co-transfection of NIH 3T3 cells with the
WRE-containing reporter (pGL2-B-
Transcriptional coactivation by both CBP and P300 is also known to be
disrupted by their physical association with the adenovirus 5 early
gene product E1A (31, 40), and the binding of both WT1 and E1A to the
CBP3 domain suggests that these proteins may compete for this binding
site. We therefore examined the effect of E1A expression on
WRE-dependent transcription. Co-transfection of
CMV-driven E1A demonstrated a
dose-dependent suppression of CBP-mediated coactivation of
the WT1-responsive promoter (Fig. 6). To
extend these studies to an endogenous WT1-target gene, we examined the
effect of E1A expression on induction by WT1 of the
cyclin-dependent kinase inhibitor p21Cip1. WT1
directly activates the p21Cip1 promoter, and induction of
the endogenous p21Cip1 gene is linked to WT1-mediated cell
cycle arrest (14, 21, 41). More recently, we have found that high titer
retroviral infection of U937 human leukemia cells with a mouse stem
cell virus promoter-driven WT1 construct, leads to
p21Cip1 induction and triggers growth arrest and
differentiation.2 We
therefore used this in vivo assay to examine the effect of E1A, as well as the dominant negative fragment CBP3, on expression on a
physiological WT1 target.
U937 cells were first infected with virus expressing E1A, CBP3, or
vector control along with the neomycin resistance gene. Uncloned pools
of infected cells were selected by treatment with G418, and then
subjected to a second round of infection with virus encoding
WT1( Concluding Remarks--
We have described a functional interaction
between the tumor suppressor gene product WT1 and the transcriptional
coactivator CBP. Recruitment of CBP to a target promoter is thought to
enhance transcription by providing a platform that facilitates the
assembly of additional transcription factors and components of the
basic transcriptional machinery, and by mediating histone acetylation (for review, see Refs. 42 and 43). The observation that WT1 encodes a bona fide transcriptional activator that
associates with CBP in vivo was unexpected, since WT1 has
long been thought to act as a transcriptional repressor, whose function
as a tumor suppressor is linked to the repression of
proliferation-inducing genes (19). However, the identification of
physiologically induced WT1 target genes (27, 44, 45), and
the analysis of alterations and naturally occurring mutations that
specifically disrupt transcriptional activation but not repression by
WT1 (41, 46), have suggested an alternative role for WT1, as an
inducer of genes involved in cellular differentiation.
According to this model, inactivation of WT1 during Wilms
tumorigenesis may result in the failure of renal cells to
differentiate, leading to the persistence of pluripotent stem cells
susceptible to malignant transformation.
The interaction between WT1 and CBP also has implications for
understanding functional properties of WT1 that have been reported to
date. The consequences of WT1 expression in baby rat kidney cells, a well established primary cell transformation model in which
cellular immortalization is achieved by expression of adenovirus E1A,
should be interpreted with caution, given the likely disruption of
WT1-mediated transcriptional activation by E1A (47, 48). These observations also provide insight into the functional and physical association between WT1 and p53. These two proteins do not
interact directly in vitro, but are co-immunoprecipitated from cellular lysates, and expression of wild-type p53 suppresses transcriptional activation by WT1, while expression of WT1 inhibits p53-mediated apoptosis (29, 25). CBP is likely to mediate this indirect
interaction, since it binds to both WT1 and p53 through adjacent
domains, and WT1 zinc fingers 1-2 are required for its interaction
with both CBP and p53. Overexpression of either WT1 or p53 may thus
modulate the transactivational properties of the other (29, 25).
A similar indirect association may underlie the synergistic effect of
WT1 and steroidogenic factor 1, an orphan nuclear receptor implicated
in gonadal differentiation. WT1 and steroidogenic factor 1 act
synergistically to activate the promoter of Mullerian inhibitory substance, but WT1 does not bind directly to the Mullerian inhibitory substance promoter, nor do WT1 and steroidogenic factor 1 co-immunoprecipitate from cellular lysates (49). Given the known
interaction between nuclear receptors and CBP, the effect of WT1 may
therefore also result from an indirect association mediated by CBP.
Likewise, the activation by WT1 of the CRE site in the
amphiregulin promoter may result from its enhancement of
CBP-dependent transactivation mediated by CRE-binding
factors (27). Finally, these observations raise the possibility that
the WT1(+KTS) isoform, which does not appear to bind to a known DNA
sequence with high affinity, may in fact play an indirect role in
transcriptional activation by virtue of its association with CBP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
KTS) isoform modulates transcriptional
activity of reporter constructs, and its ectopic expression leads to
either cell cycle arrest or apoptosis (13-15). In contrast, the
WT1(+KTS) isoform is inactive in such assays, and its discrete
subnuclear localization has been linked to a potential role in
pre-mRNA splicing (16). The N terminus of WT1 encodes a
proline/glutamine-rich domain similar to the transactivation domain of
other transcription factors, such as Sp1 (17). Fusion of this
transactivation domain to a GAL4 DNA-binding domain results in
transcriptional repression of a reporter construct (18), and WT1 itself
has been shown to repress GC- and TC-rich promoters in transient
transfection assays (for reviews, see Refs. 19 and 20). The observation
that the transcriptional activity of promoters from many
growth-promoting genes, including early growth response 1 (18),
insulin-like growth factor 2 (21), insulin-like growth factor receptor
(22), platelet-derived growth factor-A (23, 24), and epidermal growth
factor receptor (13), is suppressed by WT1 has led to the model that
WT1 functions as a tumor suppressor by transcriptional repression of
genes required for cellular proliferation (19). However, variations in
experimental conditions, including promoter context (11), presence or
absence of p53 (25), and even choice of expression vector (26), appear to modulate the properties of WT1 in transient transfection assays, leading to transcriptional activation as well as repression.
Furthermore, analysis of cells with inducible expression of
WT1 demonstrated that few endogenous genes with putative
WT1-repressible promoters are in fact regulated by WT1 in
vivo (13).
KTS) did not result in reduced expression
of any transcripts represented on the microarrays. However, a small
number of genes were strongly induced following WT1(
KTS)
expression, notably amphiregulin
(AR),1 encoding a
secreted growth factor of the epidermal growth factor family capable of
stimulating cellular differentiation in organ culture systems, and the
cyclin-dependent kinase inhibitor p21Cip1. A
physiologically relevant interaction between WT1 and AR was suggested
by their precise co-localization within differentiating glomeruli of
the developing kidney, and by the ability of recombinant AR to induce
epithelial differentiation in cultured kidney rudiments (27).
WT1(
KTS) binds to a high affinity site (WRE) in the AR promoter, adjacent to a CRE site. The synergistic transactivation mediated through the WRE and CRE sites raised the possibility that WT1
itself might interact with CBP/P300, a coactivator known to enhance
CRE-dependent transcriptional activation by CRE-binding factors (28). We show here that WT1 binds to CBP stoichiometrically both in vitro and in vivo, and that this protein
interaction contributes to WT1-dependent
transcriptional activation. These observations support the role of WT1
as a transcriptional activator, which may participate in the induction
of target genes directly, as well as through potential interactions
with other transcriptional regulators.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
KTS) and WT1(+KTS)
have been previously described (13), as have WT1-deletion constructs (29) and constructs encoding CBP (30),
P300 (31), and E1A (32). Constructs encoding
different CBP domains were generated by inserting the appropriate
polymerase chain reaction fragments into pCMV-myc vector (Invitrogen).
The AR promoter reporter (luciferase) PGL2-B-
CRE contains
the WT1-responsive site and lacks an adjacent CRE site (27).
KTS) and/or CBP expression
plasmids were transfected along with 1 µg of the PGL2-B-
CRE luciferase reporter construct. Transfections with CBP domains were done
using 2 µg of PGL2-B-
CRE, 4 µg of CMV-WT1(
KTS), and increasing
amounts (2-8 µg) of each of the four CBP expression constructs
(CMV-CBP1-4). Cells were collected 40 h following transfection and equal transfection efficiency was confirmed by co-transfecting a
human growth hormone expression plasmid and quantitation of human
growth hormone levels in the medium (Nichols Institute). Equal amounts
of CMV promoter sequences were transfected in all cases by addition of
vector plasmid, and experiments were performed in triplicate. In
transfection experiments with E1A, cell viability was ascertained by
vital dye staining, to ensure that differences in transcriptional
activity were not the result of selective cell killing.
CR2, CBP3, and WT1 were cloned into
murine stem cell virus vectors as previously described (33). Virus
encoding these constructs or vector control was prepared from
supernatants of 293T cells transfected with the appropriate plasmids as
well as plasmids expressing viral envelope proteins. For the first
infection, 1 ml of viral supernatant was added onto 1 ml of U937 cells
(105 cells/ml) grown in 24-well plate. Cells were
centrifuged for 1 h and returned to the incubator. Viral
supernatant was replaced with fresh media after 12 h and cells
were allowed to recover for another 12 h. The procedure was
repeated once, and cells were then selected with G418 for 4-7 days to
remove non-infected populations. For the second infection,
G418-resistant cells were treated with viral supernatants according to
the same procedure. 48 h after the second infection, cell lysates
were prepared and the expression level of WT1 and p21Cip1
was examined by Western blotting.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
292 to
283, adjacent to a
CRE element at position
274 to
267 (27). To restrict our analysis
to WRE-dependent transactivation, we deleted the CRE site
from a minimal reporter (pGL2-B-
CRE), which was co-transfected into
NIH 3T3 cells together with cytomegalovirus (CMV)-driven constructs
encoding WT1(
KTS) and CBP. 2-Fold
transcriptional activation of the reporter was observed following
transfection of small amounts (0.5 µg) of
WT1(
KTS) plasmid alone. Coexpression of
CBP (0.5 µg) resulted in 10-fold activation of the
reporter, while CBP alone had no effect (Fig.
1). Transfection of CMV-driven P300, a transcriptional coactivator that is closely related
to CBP, also enhanced WT1-dependent
transactivation, but to a lesser extent.
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Fig. 1.
Enhancement of WT1-dependent
activation by CBP and P300. Relative luciferase activity following
transient transfection of the amphiregulin promoter-reporter
PGL-2B- CRE (1 µg) into NIH 3T3 cells, together with CMV
promoter-driven constructs encoding WT1(
KTS)
(0.5 µg), CBP (0.5 µg), and P300 (0.5 µg). Equal transfection
efficiency was confirmed by co-transfection of a human growth hormone
construct, and the total amount of CMV promoter sequence transfected
was equalized by addition of empty vector. Experiments were performed
in triplicate.
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Fig. 2.
In vivo association between WT1
and CBP. Immunoprecipitation Western analysis of cellular lysates
extracted from RSTEM cells, 30 h following withdrawal of
tetracycline and induction of WT1 expression. Equal amount of lysates
from cells expressing either WT1( KTS) or WT1(+KTS) were
immunoprecipitated with antibodies against WT1, CBP, or FasL
(nonspecific), followed by immunoblotting using the complementary
antibody. Total lysate (1%) was analyzed directly to show the
migration position of the endogenous protein; for the WT1 immunoblot, a
longer exposure of the total lysate lane from the same gel is shown.
The stoichiometry of the protein interaction is estimated by comparing
the amount of CBP co-precipitated using
-WT1 antibody with that
directly immunoprecipitated using
-CBP antibody (upper
panel), and the amount of WT1 co-immunoprecipitated using
-CBP
antibody with that directly immunoprecipitated using
-WT1 antibody
(lower panel).
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Fig. 3.
Interaction between CBP and zinc fingers 1-2
of WT1. A, immunoprecipitation Western analysis of NIH
3T3 cells, transfected with plasmids encoding HA-epitope tagged
WT1 deletion constructs along with full-length
CBP. Equal amounts of cellular lysates were
immunoprecipitated using anti-CBP antibody, followed by immunoblotting
analysis using antibody to the HA epitope. B, comparable
expression of the WT1 deletion constructs is shown by direct
Western analysis of the cellular lysates. A background band is present
at 50 kDa. C, a schematic representation of the deletions
within WT1 is shown, demonstrating that the 282-364 construct lacks
zinc fingers 1-2.
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Fig. 4.
In vitro interaction between
WT1 and the E1A-binding domain of CBP. A,
co-immunoprecipitation of radiolabeled in vitro translated
domains of CBP, following incubation with GST-WT1 affinity matrix.
Comparable expression of the four known protein-association domains of
CBP was confirmed by direct analysis of 4% of input lysate. Incubation
of these reticulocyte lysates with bacterially synthesized
GST-WT1( KTS) or GST-WT1(+KTS) was followed by GST pull-down and
autoradiography. B, a schematic representation of the
protein-interaction domains of CBP is shown, including the domains
involved in its association with nuclear hormone receptors
(NR), CRE-binding factors (CREB), and Jun, E1A,
and p53. The E1A-binding domain of CBP (amino acids (aa)
1625-1991) also mediates its interaction with WT1.
CRE) along with
CMV-WT1(
KTS) alone (4 µg) resulted in 8-fold
transcriptional activation (Fig. 5,
upper panel). Coexpression of increasing amounts of
CMV-CBP3 (2-8 µg), fused to an SV40-derived nuclear
localization signal, resulted in a dose-dependent reduction in activation. In contrast, co-transfection of CMV-driven
constructs encoding other domains of CBP that fail to interact with WT1
had no effect (Fig. 5, upper panel), despite
equal expression levels (Fig. 5, lower panel).
Transcriptional activation by WT1 may therefore be specifically
inhibited by disruption of its interaction with endogenous CBP.
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Fig. 5.
Disruption of WT1-dependent
transactivation by dominant negative CBP mutant. Relative
luciferase activity following transient transfection of the
amphiregulin promoter-reporter PGL-2B- CRE (2 µg) into
NIH 3T3 cells, together with CMV promoter-driven constructs encoding
WT1(
KTS) (4 µg), and increasing amounts (2-8
µg) of plasmids expressing each of the four CBP domains described in
the legend to Fig. 4. Nuclear localization of the CBP fragments was
ensured by insertion of a SV40-derived nuclear localization signal and
comparable expression was demonstrated by Western blotting (data not
shown). The total amount of CMV promoter sequence transfected was
equalized by addition of empty vector. Data presented are derived from
two sets of duplicate experiments and presented as fold induction
relative to that of vector alone, with standard deviation shown. The
expression level of each CBP deletion construct (5 µg) is shown by
Western blotting (lower panel).
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Fig. 6.
Inhibition of WT1-mediated transactivation by
E1A. E1A suppresses CBP-mediated enhancement of transcriptional
activation by WT1. NIH 3T3 cells were transfected with the
PGL-2B- CRE reporter (2 µg), along with CMV-driven WT1(
KTS) and
increasing amounts (0.5 to 2.5 µg) of CMV-driven E1A. Equal amount of
DNA was transfected in each experiment and luciferase activity was
compared with that of vector-transfected controls in each experiment.
No difference in total cell numbers and viability were observed between
samples. Experiments were performed in triplicate.
KTS) or vector, linked to the green
fluorescent protein (GFP) gene within a bicistronic
construct. The expression level of each protein after selection was
confirmed by Western blotting (Fig.
7A, lower panel). Following
the second retroviral infection, ~35% of both vector- and
WT1-infected cell populations expressed the GFP marker. No
differences in cell viability were observed within the time studied (48 h after the second infection). The expression of p21Cip1 in
infected cells was quantitated by Western blotting and corrected for
that of WT1 itself. As expected, U937 cells expressing WT1(
KTS) demonstrated dramatic induction of p21Cip1 (Fig.
7B). In contrast, E1A-infected cells reproducibly
demonstrated a 2-fold reduction in p21Cip1 induction by
WT1. Ectopic expression of E1A is associated with cellular toxicity,
which limits the levels of protein expressed following retroviral
infection. A variant E1A construct, E1A
CR2, lacking the CR2 domain
between amino acids 121 and 139, has a preserved CBP interaction and
reduced cellular toxicity, allowing for higher levels of protein
expression (Ref. 33 and Fig. 7A, lower panel). In multiple
experiments, retroviral infection of U937 cells with constructs
encoding E1A
CR2 reproducibly demonstrated a 4-fold reduction in
endogenous p21Cip1 induction by WT1 (Fig. 7B).
Infection of U937 cells with a construct expressing the dominant
negative fragment CBP3 also resulted in a 2.5-fold reduction in
p21Cip1 induction by WT1. These observations suggest that
disruption of the WT1-CBP interaction by either E1A or dominant
negative CBP fragment, suppresses the transcriptional activation of an endogenous target gene by WT1.
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Fig. 7.
Suppression of endogenous p21Cip1
induction by WT1 in cells expressing E1A or CBP3. A,
upper panel: flow chart describing the sequential retroviral
infection strategy. U937 cells were first infected with retroviral
constructs encoding vector, CBP3, E1A, or the E1A CR2 construct with
reduced cell toxicity, all linked to the neomycin resistance gene.
Following G418 selection (4-7 days), uncloned pools of resistant cells
were infected with retroviral constructs encoding either vector or
WT1(
KTS) linked to GFP. Equal infection efficiency (~35%) and cell
viability was confirmed for these constructs, and expression of
p21Cip1 and WT1 were monitored by Western blotting (ECL).
Lower panel, expression level of each retrovirally encoded
construct following G418 selection of uncloned cell populations is
shown by Western blotting. B, inhibition by E1A and CBP3
of endogenous p21Cip1 induction by WT1. The relative
intensity of p21Cip1 expression was normalized to that of
WT1 itself (p21/WT1 ratio) in U937 cell populations stably infected
with vector, E1A, E1A
CR2, or CBP3, 48 h following infection
with vector or WT1(
KTS). The p21/WT1 ratio in E1A-expressing cells
is expressed as a fraction of that in vector-infected cells. A
representative experiment is shown.
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ACKNOWLEDGEMENTS |
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Expression constructs for P300, CBP, and E1A were kindly provided by Drs. J. DeCaprio, M. Montminy and N. Dyson. We thank members of the Haber lab for helpful discussions and Dr. J. Settleman for critical review of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA58596 (to D. A. H.) and the National Foundation for Cancer Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: MGH Cancer Center, CNY
7, Bldg. 149, 13 Street, Charlestown, MA 02129. Tel.: 617-726-7805; Fax: 617-724-6919; E-mail: Haber@helix.mgh.harvard.edu.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M009687200
2 L. W. Ellisen and D. A. Haber, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: AR, amphiregulin; CBP, CREB binding protein; CRE, cyclic AMP response elements; CMV, cytomegalovirus; GST, glutathione S-transferase; WRE, WT1-responsive element.
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REFERENCES |
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