From the Division of Gastroenterology, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Cdx2 encodes for a homeodomain
protein that is expressed in intestinal epithelial cells. The Cdx2
protein triggers intestinal differentiation in cell lines and is
necessary for maintenance of the intestinal phenotype in mice. CBP
(cAMP response element-binding protein) is a transcriptional co-activator that interacts
with many transcription factors and components of the basal
transcriptional machinery. In this study, we demonstrate that CBP is
markedly induced upon differentiation of the Caco-2 intestinal cell
line and augments Cdx2-dependent transcriptional activity.
Cdx2 interacts with the amino-terminal domain of CBP, and the two
proteins coexist in vivo within the same nuclear protein
complex. Moreover, expression of the CBP domain that interacts with
Cdx2 acts as a dominant-negative inhibitor of transcriptional
activation by Cdx2. These findings demonstrate a direct interaction
between an intestinal homeodomain protein and CBP and suggest that CBP
participates in the network of transcriptional proteins that direct
intestinal differentiation.
In mammals, visceral endoderm gives rise to the gastrointestinal
epithelium. Several lines of evidence suggest that the
caudal-type homeobox gene Cdx2 is involved in the
regulation of intestinal epithelial cell development and maintenance of
the differentiated phenotype. Cdx2 encodes a transcription
factor that is expressed in differentiated enterocytes (1) and binds to
cis-elements present in the gene promoters of enterocyte-specific genes
including sucrase-isomaltase
(SI)1 (2) and intestinal
phospholipase A/lysophospholipase (3). Expression of Cdx2 in intestinal
cell lines and in an epithelial-mesenchymal co-culture system has shown
that it is involved in the control of intestinal cell proliferation and
differentiation (4-6). Finally, colon tumors develop upon loss of Cdx2
expression in colonocytes of mice that are heterozygous for the null
allele of Cdx2 (7).
The molecular mechanisms of how Cdx2 regulates transcriptional
initiation of enterocyte genes have not been well defined. We
hypothesized that co-factors that link DNA-binding proteins to the
basal transcriptional apparatus may be involved in regulation of
Cdx2-dependent transcription. CBP (cAMP
response element-binding protein) was first
characterized as a co-factor for the cAMP response element-binding
protein that potentiates transcriptional activity (8). Since then, CBP,
and its family member p300, have been shown to bind to interact with
many transcription factors in a number of different families in
addition to CREB, including helix-loop-helix proteins and nuclear
receptors (9-11). CBP functions as an adaptor protein for complex
transcriptional regulatory elements by enhancing the interaction
between transcription factors and components of the basal
transcriptional machinery. Additionally, inactivation of CBP is
critical for the adenoviral protein E1A to induce oncogenic transformation and to inhibit differentiation suggesting that CBP plays
a role in cell growth and differentiation (12, 13). Alterations of the
human CBP gene have been implicated in hematological malignancies as
well as in congenital malformation and mental retardation (14).
Recently, it has been shown that inhibition of CBP in
Caenorhabditis elegans leads to developmental arrest, with
most embryos showing a lack of endodermal cells (15).
In this study, we show that CBP interacts with Cdx2 and is functionally
important for Cdx2-dependent transactivation. These findings demonstrate that CBP interacts with intestinal homeodomain transcription factors and suggest that CBP may modulate the function of
Cdx2 in the regulation of enterocyte proliferation and differentiation.
Plasmids--
pRc/CMV-Cdx2 expression plasmid has been described
previously (2). The pFlag-Cdx2 was obtained by cloning the
HindIII fragment of pRc/CMV-Cdx2 into pFlag-CMV-2 (Eastman
Kodak Co.) HindIII restriction site.
The DNA sequence encoding for the amino acids 181 to 269 of Cdx2 was
generated by polymerase chain reaction. The oligonucleotides were
designed in manner to introduce a SalI and a XbaI
at the ends of the polymerase chain reaction products. The polymerase chain reaction product was cloned into SalI/XbaI
restriction sites of pSG424 vector encoding for the Gal4 DNA binding
domain (16). The resulting HindIII/XbaI fragment
was subcloned into pRc/CMV generating the pRc/CMV-Gal4-Cdx2(181-269)
plasmid. The pRc/CMV-Gal4 plasmid was obtained by subcloning the
HindIII/XbaI fragment of pSG424 into pRc/CMV
HindIII/XbaI restriction sites.
Mouse Flag-CBP encoding eukaryotic expression plasmid was a gift from
Dr. Rosenfeld (University of California, San Diego), expression vectors
coding for E1A, E1A( Cell Culture and Transfection--
Caco-2 and NIH-3T3 cells were
maintained in Dulbecco's modified Eagle's medium with high glucose,
10% fetal calf serum, and 1% penicillin/streptomycin. The cells were
placed in 6-well plates 24 h before transfection with Lipofectin
(Life Technologies, Inc.) following the manufacturer's
recommendations. Luciferase activity was determined 48 h after the
transfection using the luciferase assay kit (Promega Corp.). Each
transfection was performed in duplicate and repeated at least 3 times.
As a measure of transfection efficiency, 300 ng of
pCMV- In Vitro GST-Protein Interaction Assay--
GST-protein
interaction assays were performed as described elsewhere (19) with
slight modifications. Briefly, bacterial expression of GST fusion
proteins was induced in medium containing 0.1 µM
isopropyl-1-thio-
Proteins were labeled with [35S]methionine by coupled
in vitro transcription and translation using the TNT
reticulocyte lysate system (Promega) according to the manufacturer's
instructions. The GST beads with bound fusion proteins were incubated
with labeled proteins in 200 µl of HND buffer at 4 °C. The beads
were then washed with MTPBS (PBS, 0.1% Nonidet P-40) buffer and
proteins eluted from the beads in SDS sample buffer. The eluted
proteins were separated on a 10% acrylamide SDS-polyacrylamide gel
electrophoresis gel.
Co-immunoprecipitation and
Immunoblots--
Co-immunoprecipitations were performed using an
immunoprecipitation kit (Protein G) (Boehringer-Mannheim) following the
manufacturer's recommendations. Immunoprecipitation of Flag-tagged
proteins were performed using 3 µg of anti-Flag M2 antibodies
(Kodak). The immunoprecipitates were solubilized in SDS sample buffer
and loaded on a 4% acrylamide SDS-polyacrylamide gel electrophoresis
gel for detection of CBP and a 12% gel for Cdx2 and Flag-tagged
proteins. Proteins were transferred to polyvinylidene difluoride
membranes (Millipore, Bedford, MA). The membrane was blocked for 2 h in PBS (150 mM NaCl, 7 mM
Na2HPO4, 2 mM KCl, 800 µM KH2PO4, pH 7.4) containing 0.1% Tween 20 and 10% skim milk. Cdx2 was detected by using rabbit anti-Cdx2 polyclonal
antibody.2 CBP was detected
using rabbit polyclonal A-22 antibody (Santa Cruz Biotechnology, Santa
Cruz, CA). E-Cadherin was detected using mouse monoclonal 36 antibody
(Transduction Laboratories, Lexington, KY). Proteins were detected on
the membranes using ECL-plus (Amersham Pharmacia Biotech) in
combination with goat anti-rabbit antibodies for CBP and Cdx2 (Amersham
Pharmacia Biotech) and in combination with anti-mouse antibodies for
E-Cadherin (Santa Cruz Biotechnology).
Cdx2 is an important transcription factor for activating the SI
promoter in intestinal cell lines (2). Caco-2 cells, derived from a
human colonic adenocarcinoma, spontaneously differentiate following
confluence in culture, including a marked increase in the expression of
SI mRNA and protein (6, 20, 21). Therefore, we examined the
expression of Cdx2 protein during Caco-2 cell differentiation to
determine whether an increase in this transcription factor may be
involved in SI induction. The amount of Cdx2 protein was not different
in pre- and postconfluent cells in comparison to E-Cadherin, a protein
that is unchanged during Caco-2 differentiation (22) (Fig.
1). Because CBP is able to act as a
co-activator for many transcription factors, we also examined its
expression. CBP was not detectable in preconfluent Caco-2 cells and
only detectable at confluency after long exposure of the immunoblot
(Fig. 1). However, following confluency there was marked induction of
CBP protein (Fig. 1). This observation suggested that CBP may be an important co-factor for mediating the function of Cdx2 in the activation of SI gene transcription.
INTRODUCTION
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Abstract
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References
EXPERIMENTAL PROCEDURES
2-36) and E1ACXdl, and plasmids for
GST-CBP(1-450), GST-CBP(451-682), GST-CBP(1000-1500), HA-CBP(1-450), and HA-CBP(451-682) have been previously described (17). The reporter vectors pTK-luc, pTK-SIF1(+4)-luc vector, which
contains four SIF1 sites cloned upstream the TK promoter, and the
p(
183/+54)SI-luc have been described elsewhere (18).
-galactosidase were co-transfected in each experiment, and the
results were reported as light units per unit of
-galactosidase.
-D-galactopyranoside for 3 h. The
bacterial pellet was resuspended in 500 µl of PBS and sonicated to
disrupt the bacteria. The bacterial remnants were removed by
centrifugation, and the supernatant was incubated with 75 µl of GST
beads (Amersham Pharmacia Biotech) on a rotating wheel for 30 min at
4 °C. The beads were washed 4 times with PBS and once with HND
buffer (10 mg/ml bovine serum albumin, 20 mM Hepes, 50 mM NaCl, 0.1% Nonidet P-40, 5 mM dithiothreitol).
RESULTS AND DISCUSSION
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Fig. 1.
CBP and Cdx2 protein level during Caco-2 cell
differentiation. The expression of CBP and Cdx2 proteins during
Caco-2 cell differentiation were measured by immunoblots (see
"Experimental Procedures"). Total cellular protein was extracted 2 days before confluency (lane 1), at confluency (lane
2), 3 days after confluency (lane 3), 7 days after
confluency (lane 4), and 14 days after confluency
(lane 5). Protein integrity was confirmed by expression of
E-Cadherin.
Based on these findings in Caco-2 cells, we explored the functional
interaction of Cdx2 and CBP. The adenovirus E1A oncoprotein inhibits
the ability of CBP to co-activate transcription in a variety of cell
lines (23, 24). We examined the functional consequence of expression of
E1A on transactivation of the SI promoter by Cdx2 in Caco-2 cells,
which have been shown to express endogenous Cdx2 and support SI
promoter transcription via interaction of Cdx2 with the SIF1 promoter
element (2). Expression of wild-type E1A resulted in marked reduction
of SI promoter activity in Caco-2 cells and abolished superactivation
of the SI promoter when Cdx2 was over expressed (Fig.
2A). Moreover, an E1A mutant
that lacks the ability to interact with retinoblastonia protein but
does bind to CBP (E1ACXdl) also inhibited Cdx2 activation of the
promoter. In contrast, expression of a mutant form of E1A
(E1A(2-36)) that is unable to bind CBP, had no effect on SI
promoter activation by Cdx2 (Fig. 2A). We next examined the
ability of E1A to affect Cdx2-dependent activation of the
SI promoter in the nonintestinal fibroblast cell line NIH-3T3. Similar
to results in Caco-2 cells, E1A and the E1ACXdl mutant abolished Cdx2
activation of the SI promoter, whereas E1A(
2-36) had no effect
(Fig. 2B).
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These results suggested that CBP was involved in the control of SI
promoter activation. However, the region from nucleotides 183 to +54
that constitutes the SI promoter contains a number of DNA regulatory
binding sites (25), each of which bind proteins that might be affected
by E1A-CBP interactions. Because the presence of the Cdx binding site
is absolutely required for promoter activity (2), experiments with
mutations in this site could not alone answer the question of whether
other SI promoter elements are involved in mediating the effect of E1A
on Cdx2-dependent activation. Thus, to determine whether
transactivation by Cdx2 is specifically inhibited by E1A, the same
experiments were performed in Caco-2 cells using a reporter construct
that linked Cdx binding sites upstream of a minimal thymidine kinase
promoter (pTK-SIF1(+4)-luc) (18). As with the SI promoter, Cdx2-induced
transactivation of this construct was abolished by wild-type E1A, but
not by the E1A mutant that is unable to bind to CBP (Fig.
2C). These modifications in luciferase activity were
directly related to the presence of the SIF1 elements, because no
luciferase activity was unchanged when transfections were done using
pTK-luc (Fig. 2C). These results suggest that CBP may be
directly involved in the ability of Cdx2 to activate gene transcription.
The ability of CBP to directly augment transactivation by Cdx2 was
examined by co-transfection of expression vectors for Cdx2 and CBP with
the Cdx2-responsive reporter plasmid pTK-SIF1(+4) in Caco-2 (Fig.
3A). CBP overexpression in
Caco-2 cells resulted in a dose-dependent increase in
Cdx2-dependent transactivation with a maximum of a 3-fold
increase over that with Cdx2 alone (Fig. 3A). The induction
of transcription by CBP was greater in the presence of higher levels of
Cdx2, suggesting that Cdx2 may be limiting in Caco-2 cells. Addition of
the wild-type E1A expression vector resulted in loss of reporter gene
expression whereas E1A(2-36) had no effect on the ability of CBP to
augment Cdx2 transactivation (Fig. 3A). Transient
overexpression of CBP or Cdx2 in Caco-2 cells did not change the
endogenous expression of Cdx2 and CBP, respectively (Fig.
3B). Furthermore, transient overexpression of E1A or
E1A(
2-36) in Caco-2 cells did not interfere with Cdx2 and CBP
expression (Fig. 3B). These results demonstrate that in
Caco-2 cells, CBP is able to increase Cdx2 transactivation activity
when SIF1 elements are located immediately upstream of a heterologous
promoter.
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In previous studies, we have shown that Cdx2 is able to activate transcription when its DNA binding elements are placed in an enhancer position on heterologous promoters (18). These studies further showed that the cellular context is important for Cdx2 to activate transcription from an enhancer context, because Caco-2 cells supported Cdx2-dependent enhancer activation, whereas NIH-3T3 cells did not. These results led to the conclusion that there may be cell-specific adaptor proteins that are important for Cdx2 function on certain transcriptional elements (18). We found that CBP was not able to augment Cdx2 transactivation when Cdx binding elements were placed in an enhancer context (data not shown). Thus, consistent with the widespread expression of CBP, it does not appear to be responsible for the previously observed cell-specific function of Cdx2 on enhancer elements.
The mechanism by which CBP augments Cdx2 transcriptional activity could be via direct protein-protein interaction, by common interaction with a third protein, or by indirect effects on other components of the transcriptional machinery. To determine whether there is direct interaction between Cdx2 and CBP, in vitro protein interaction assays were performed. The bacterially expressed fusion protein GST-CBP(1-450) was able to interact with in vitro labeled Cdx2 (Fig. 4A). In contrast, there was very weak interaction with GST-CBP(451-682) and no interaction with GST-CBP(1000-1500). These data raised the question of whether the homeodomain of Cdx2 interacted with GST-CBP(1-450), similar to a recently described POU-homeodomain protein Pit-1 (26). A peptide containing the homeodomain of Cdx2 and excluding the majority of the amino-terminal and carboxyl-terminal domains was found to bind specifically to GST-CBP(1-450) (Fig. 4B).
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We next examined whether CBP and Cdx2 interact within the cell. Co-immunoprecipitation was performed to determine whether Cdx2 and CBP coexist within the same protein complex. Protein extracts of Caco-2 cells transfected with expression vectors for Flag-CBP and/or Flag-Cdx2 were subjected to immunoprecipitation followed by immunoblot analysis. Results of these experiments showed that Cdx2 protein was co-immunoprecipitated with Flag-CBP and that endogenous CBP was immunoprecipitated with Flag-Cdx2 (Fig. 4C). Nonimmune antibodies were unable to immunoprecipitate either Flag-CBP or Flag-Cdx2 and anti-Flag antibodies were unable to immunoprecipitate CBP or Cdx2 (Fig. 4C). Thus, CBP and Cdx2 are associated in an immunoprecipitable protein complex in Caco-2 cells.
The interaction of Cdx2 with a specific domain of CBP provided an avenue to test the functional specificity of the interaction. It was previously shown that short regions of the CBP protein have a dominant-negative effect on the functional interaction between CBP and nuclear hormone receptors (17). Therefore, we expressed HA-tagged domains of CBP with Cdx2 and CBP in Caco-2 cells to assess the ability of different domains to interfere with transcriptional activation. Expression of HA-CBP(1-450) inhibited CBP-dependent augmentation of Cdx2-induced transcription in a dose-dependent manner, whereas HA-CBP(451-682) had little effect (Fig. 5). This evidence substantiates the hypothesis that Cdx2 interacts with the amino-terminal domain of CBP in the cell nucleus.
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Taken together, these results demonstrate that the amino-terminal
domain of CBP is able to interact with Cdx2 and that this interaction
results in increased Cdx2-dependent transactivation. Recently, it has been shown that CBP interacts with the POU-homeodomain protein Pit-1 and that this interaction is functionally important for
transcriptional activation (26). Our findings represent the second
description of an interaction between homeodomain proteins and CBP and
extends the regulatory implications beyond the subfamily of
POU-homeodomain proteins. Cdx2 has been shown to be a critical component of the transcriptional machinery of intestinal epithelial cells and, as a result, plays a critical role in intestinal development and differentiation. Based on these studies, we hypothesize that CBP
may modulate the function of Cdx2 on complex promoters and thus may
influence intestinal differentiation and development.
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ACKNOWLEDGEMENTS |
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We thank Dr. Mitchell Lazar for his review of the manuscript and for many helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1-DK46704 and the Molecular Biology Core of the Center for Molecular Studies in Digestive Diseases at the University of Pennsylvania (P30-DK50306) (to P. G. T.).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.
Supported by a fellowship from the Fonds de la recherche en
Sauté du Québec.
§ To whom correspondence should be addressed: Hospital of the University of Pennsylvania, 100 Centrex, 34th and Spruce Sts., Philadelphia, PA 19104. Tel.: 215-662-2024; Fax: 215-349-5734; E-mail: traberp{at}mail.med.upenn.edu.
2 D. Silberg and P. G. Traber, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: SI, sucrase-isomaltase; CBP, cAMP response element-binding protein; GST, glutathione S-transferase; PBS, phosphate-buffered saline; HA, hemagglutinin.
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REFERENCES |
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