From the Max-Planck-Institut für Immunbiologie, Stübeweg 51, D-79108 Freiburg, Germany
Received for publication, October 2, 2002, and in revised form, November 22, 2002
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ABSTRACT |
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Wnt growth factors control numerous cell fate
decisions in development by altering specific gene expression patterns
through the activity of heterodimeric transcriptional activators. These consist of Multicellular organisms characteristically employ a
limited number of signaling systems in order to generate the panoply of different cell types found in the body. The repeated use of the same
growth factor families and signaling pathways poses the question of how
the same effector proteins can generate distinct, tissue-specific responses (1). The canonical Wnt/ In mammals, four genes encode the TCF family members TCF1, LEF1, TCF3,
and TCF4, which have some structural features in common (5). The
extreme N terminus harbors the binding site for The participation of Wnt/ To better understand the mechanisms whereby Wnt signals selectively
activate target genes, we began to compare TCF family members with
respect to their ability to support Plasmids--
Expression vectors for murine Electrophoretic Mobility Shift Assays (EMSA)--
For EMSA, LEF1
and TCF4 proteins were transcribed and translated in vitro
using the SP6 TNT system (Promega). Translation efficiency was analyzed
by Western blotting with anti-HA antibodies (3F10; Roche Applied
Science). DNA probes used were complementary pairs of synthetic
oligonucleotides derived from the TCR Cell Culture, Transient Transfections, and Reporter Gene
Assays--
Human 293 embryonic kidney cells (ATCC number CRL-1658)
and U-2 OS osteosarcoma cells (ATCC number HTB-96) were cultured as described (21). To monitor TCF protein expression, 2.5 × 105 293 cells, seeded into 35-mm dishes, were transfected
with 2.5 µg of expression vector using FuGENE6 reagent (Roche Applied
Science). For immunoprecipitations, 293 cells were transfected as
before (21). For reporter gene assays, cells were plated into 24-well plates (5 × 104 cells/well) and transfected 4 h
later with a FuGENE6/DNA mixture containing 10 ng of pRL-CMV or pCMV Western Blotting and Immunoprecipitation--
Protein extracts
from cells transfected in 35-mm dishes were made by lysing cells in 200 µl of SDS-PAGE sample buffer containing "Complete" protease
inhibitor mix (Roche Applied Science). After boiling for 5 min and
sonication, one-tenth of each lysate was loaded onto an 8%
SDS-polyacrylamide gel. Co-immunoprecipitations were performed as
described (21) except that TCF4/p300 immunoprecipitates were washed for
10 min at 4 °C once with 1 ml of immunoprecipitation buffer (50 mM Tris/HCl, pH 7.6, 5 mM MgCl2,
0.1% Nonidet P-40) containing 120 mM NaCl and twice with 1 ml of immunoprecipitation buffer with 75 mM NaCl. For
Western blot analyses, immunoprecipitates were eluted from protein
G-Sepharose beads in SDS-gel loading buffer and run on 6%
polyacrylamide gels. After electrophoresis, proteins were transferred
onto nitrocellulose membranes and probed with anti-HA (3F10; Roche
Applied Science), anti-p300 (catalog no. 05-267; Upstate Biotechnology,
Inc.) or anti- GST Pull-down Assays--
For GST pull-down assays, 2 µg of
GST or the various GST-TCF4 fusion proteins were immobilized on
glutathione-Sepharose beads as described (21). After preincubation in
binding buffer containing 0.5% bovine serum albumin for 30 min at
4 °C, [35S]methionine-labeled p300 was added, after
which binding proceeded for 2 h at 4 °C. Following extensive
washing with binding buffer without bovine serum albumin, material
retained on the glutathione-Sepharose matrix was eluted in SDS-PAGE
loading buffer, separated by SDS-PAGE on 6% gels, and visualized by
fluorography. Radiolabeled p300 for these experiments was transcribed
and translated in vitro using the SP6-based TNT system
(Promega). For each binding reaction, we used a 4-µl aliquot of a
50-µl TNT reaction.
Phosphatase Assays--
Transfected 293 cells were lysed for 30 min on ice in 1 ml of IPK buffer (50 mM Tris/HCl, pH 7.6, 75 mM KCl, 1 mM dithiothreitol, 10 mM NaF, 0.1 mM sodium orthovanadate, and
"Complete" protease inhibitor mix). After clearance by
centrifugation at 20,000 × g for 15 min, 1-2 µl of
cell lysate were treated with LEF1 and TCF4E Exhibit Promoter-specific Transactivation
Properties--
To test whether downstream components of the
Wnt/ The C Terminus of TCF4E Harbors a Promoter-specific Activation
Domain--
To gain insight into the mechanisms underlying its
promoter-specific activity, we determined which domain in TCF4E is
required for activation of the Cdx1 promoter. A panel of
deletion mutants was constructed (Fig.
2A), and Western blotting
experiments were performed to confirm that all mutants were expressed
at similar levels (Fig. 2B). Functional testing revealed
that all mutants that lack the C1.1 domain (amino acids 436-482 of the
TCF4E C terminus) are unable to synergize with LEF1 Forms a Nonproductive Transcription Factor Complex at the Cdx1
Promoter--
To rule out the possibility that LEF1 and the inactive
TCF4 mutants were unable to activate the Cdx1 promoter
because they could not access it, we performed competition experiments.
For this, activation of the Cdx1 promoter by
Since LEF1 can occupy the Cdx1 promoter construct, we
wondered whether the addition of the C-terminal activation domain of TCF4E would endow LEF1 with the ability to stimulate Cdx1
promoter activity. A LEF1-TCF4E chimera was generated (Fig. 3,
C and D), and when coexpressed together with
either LEF1 and TCF4 Possess Different DNA Binding Properties in
Vitro--
Wnt target gene activation depends on sequence-specific
promoter recognition by TCFs and on TCF-
Whereas TCF4E possesses a markedly reduced affinity for a single TBE
when compared with LEF1, this difference was not seen in EMSAs with a
Cdx1 promoter fragment containing all three TBEs together.
TCF4E, TCF4 The Promoter-specific Activation Domain of TCF4E Interacts with the
Transcriptional Coactivator p300--
High level induction of the Cdx1
reporter constructs required simultaneous expression of
Complex formation between TCF4E and p300 was further characterized by
GST pull-down experiments with bacterially expressed GST-TCF4 fusions
and p300, which was transcribed and translated in vitro. All
GST-TCF4C fusions containing the C1.1 domain interacted with p300 (Fig.
5, B and C, constructs A, D, and F), whereas all mutants lacking the C1.1 domain did not (Fig. 5, B and
C, constructs B, C, and E). Thus, the C1.1 domain in TCF4E,
which is required for promoter-specific transactivation, also mediates
an interaction with p300.
TCFs are multifunctional transcription factors that control the
expression status of Wnt target genes by forming multiprotein promoter
complexes that incorporate either repressing or activating cofactors (5). Due to a common overall structure and seemingly identical DNA binding specificities, TCF proteins were long considered to be largely interchangeable components of the Wnt signaling cascade.
However, recent reports revealed functional differences between TCF
family members and between isoforms derived from the same TCF gene. For
example, LEF1 appears to mainly act as a
The domain of TCF4E responsible for its promoter-specific activity is
located at the C terminus and contains the "CRARF" amino acid motif
and a second block of mostly positively charged amino acids. The
"CRARF" domain is evolutionarily conserved and specifically present
in the "E" variants of TCF1 and TCF4 gene products (5, 34).
However, to date, no specific function has been assigned to this
domain. Based on our results, it may be involved in at least two
different processes, namely DNA binding and selective activation of
certain promoters. Although LEF1 and TCF4E recognized the same DNA
sequences, their affinities for single or multimerized recognition
motifs varied considerably. Pukrop et al. (31) also observed
3-7-fold differences in affinity for single TBEs between LEF1 and
other TCF family members. These differences are likely to be of
importance, because TCF proteins are not only mediators of Wnt
signaling but also contribute to Wnt/ Although differences in DNA binding may contribute to the differential
activation of the Cdx1 promoter by LEF1 and TCF4E, we
believe that the C-terminal activation domain in TCF4 performs additional functions. LEF1 and TCF4 mutants lacking the C1.1 domain displayed differences in promoter recognition, but there was no clear
correlation between the ability to activate the Cdx1
promoter and the mode of promoter binding in vitro. TCF4 Expression of the murine Cdx1 gene begins in ectodermal and
mesodermal cells of gastrulating embryos around day 7.5 of gestation. It occurs in a graded fashion along the body axis with highest levels
of expression in posterior parts. From day 14 of embryonic development
onward, Cdx1 is also expressed in the endoderm of the
developing intestine (46-48). The latter tissue expresses both TCF4
and LEF1 (49, 50). Based on our results, TCF4E may be responsible for
Cdx1 regulation in the intestine. However, TCF4 is not
expressed in the early embryonic Cdx1 expression domain (51). We suggest that there activation of the Cdx1 promoter is mediated by an isoform derived from the TCF1 gene. Preliminary results indicate that TCF1E, but not TCF1B, which resembles LEF1, can
cooperate with Although p300 and CBP have already been linked to differential gene
regulation by -catenin and one of the four members of the T-cell factor
(TCF) family of DNA-binding proteins. How can the
Wnt/
-catenin pathway control various sets of target genes in
distinct cellular settings with such a limited number of nuclear
effectors? Here we asked whether different TCF proteins could perform
specific, nonredundant functions at natural
-catenin/TCF-regulated
promoters. We found that TCF4E but not LEF1 supported
-catenin-dependent activation of the Cdx1
promoter, whereas LEF1 specifically activated the Siamois
promoter. Deletion of a C-terminal domain of TCF4E prevented
Cdx1 promoter induction. A chimeric protein consisting of
LEF1 and the C terminus of TCF4E was fully functional. Therefore, the
TCF4E C terminus harbors a promoter-specific transactivation domain.
This domain influences the DNA binding properties of TCF4 and
additionally mediates an interaction with the transcriptional coactivator p300. Apparently, the C terminus of TCF4E cooperates with
-catenin and p300 to form a specialized transcription factor complex
that specifically supports the activation of the Cdx1 promoter.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin signal transduction pathway provides an interesting model system to address this problem. Wnt growth factors constitute a large family of secreted glycoproteins that control numerous developmental processes in a wide range of
organisms (2, 3). In order to evoke transcriptional responses, the
Wnt/
-catenin signaling cascade utilizes a small number of bipartite
transcription factor complexes. These complexes are formed by
-catenin, which provides a transcriptional activation function, and
by a member of the T-cell factor
(TCF)1 family of DNA-binding
proteins, which guides
-catenin to the promoter regions of specific
target genes (4, 5).
-Catenin belongs to an evolutionarily conserved
family of proteins, which, as a signature motif, carry multiple copies
of a 42-amino acid module, the Armadillo repeat (6). In
-catenin, 12 copies of this repeat form the large, central domain of the protein and provide the interaction surface for most of the known binding partners
of
-catenin (7-10). The Armadillo repeat domain is flanked by short
N- and C-terminal extensions, which harbor the transcriptional activation domains of
-catenin (11-13). In addition to its role in
Wnt signaling,
-catenin also performs a function in cell-cell adhesion, where it is crucial for the formation of cadherin-catenin complexes (2, 3).
-catenin. The
recognition and occupation of specific DNA sequence motifs is mediated
by nearly identical HMG-box domains, which are located near the C
terminus or in the middle of TCFs. Interspersed between the
-catenin-binding domain and the HMG-box are sequences that interact with Groucho/TLE transcriptional corepressors (14-17). Groucho/TLE factors are histone-binding proteins and additionally interact with a histone deacetylase (18, 19). This suggests that their
function is to set up a specialized repressive chromatin structure that
prevents inappropriate activation of
-catenin/TCF target genes in
the absence of a Wnt signal. Since TCF proteins are functionally
neutral on their own, their role in gene regulation is likely to serve
as chromosomal docking sites for various interaction partners, thus
promoting the formation of different transcription factor complexes
with distinct properties.
-catenin signaling in multiple
developmental programs necessitates that
-catenin-TCF
complexes induce only subsets of all potential Wnt/
-catenin target
genes at any particular time and that different sets of genes are
addressed depending on the particular cellular background. How this is
achieved is not understood. Several different mechanisms have been
described whereby the formation and activity of
-catenin-TCF
complexes is controlled by covalent modification of TCFs or competitive binding to
-catenin (3-5). These mechanisms, however, appear to
affect the expression of Wnt target genes indiscriminately. Alternatively,
-catenin could employ transcriptional coactivators in
a promoter-specific manner. Among the cofactors of
-catenin are
BCL9/Legless, Brg-1, p300, and the closely related CBP (20-25). CBP
and p300 are widely used transcriptional coactivators that can provide
a link to the basal transcription machinery or can target chromatin
structure through their intrinsic acetylase activity (26). Both p300
and CBP have been implicated in differential promoter activation by
-catenin (21, 27). In addition, several observations closely link
TCF family members to mechanisms that generate promoter-specific
transcriptional responses. TCFs can interact with Smad proteins, and
this interaction appears to be critically involved in the combinatorial
regulation of the Xenopus laevis Twin gene
promoter by the transforming growth factor
and Wnt signaling
pathways (28, 29). Similarly, an interaction between LEF1 and the basic
helix-loop-helix/leucine zipper protein microphtalmia-associated
transcription factor has been implicated in the melanocyte-specific
expression of Wnt target genes (30). In addition, multiple isoforms
with different functionalities arise from TCF genes by way of
alternative splicing and the use of dual promoters, and different TCF
family members perform distinct tasks in developmental processes
(31-36). It thus appears that TCF family members and their isoforms
are intrinsically different and can support the execution of different
developmental programs.
-catenin-mediated activation of
different
-catenin/TCF target gene promoters. Here we report that
LEF1 and the TCF4E isoform have opposing capabilities to synergize with
-catenin at the Siamois and the Cdx1
promoters. A promoter-specific transcriptional activation domain was
identified at the C terminus of TCF4E, which can interact with the p300
transcriptional adaptor. The interaction between TCF4E and p300 differs
from the
-catenin/p300 interaction, because
-catenin induces a
posttranslational modification of p300, whereas TCF4E does not. We
propose that TCF4E supports the promoter-specific assembly of a dormant
transcription factor complex, the transcriptional activity of which is
triggered by
-catenin-induced phosphorylation of p300.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin, human
p300, human TCF4E, and mouse LEF1 were the same as in Hecht et
al. (21). All other plasmids were generated by standard cloning
techniques or polymerase chain reaction-based strategies (37). Details of the constructs are available upon request.
gene enhancer (TCR
25,
5'-TCGACCTGCAGGTAGGGCACCCTTTGAAGCTCTCCC-3' and 5'-TCGAGGGAGAGCTTCAAAGGGTGCCCTACCTGCAGG-3'; TCF
consensus motifs underlined) or a BamHI/XbaI
fragment with Cdx1 promoter sequences from positions
224
to
52 (38, 39). DNA fragments were labeled by fill-in with Klenow
enzyme and [
-32P]dCTP (3000 Ci/mmol). For DNA binding,
0.5, 1.0, and 2.0 µl of the TNT samples containing similar amounts of
LEF1 and TCF4 proteins and 5 fmol of radiolabeled probe were combined
in 20 mM Hepes/NaOH, pH 7.9, 75 mM NaCl, 1 mM dithiothreitol, 100 µg of bovine serum albumin and
0.25 µg of poly(dI:dC). Total volume was 15 µl. Control reactions
received 2 µl of unprogrammed reticulocyte lysate diluted 1:1 with
phosphate-buffered saline. Binding reactions were incubated for 30 min
on ice, supplemented with 2 µl of a solution containing 0.25% (w/v)
bromphenol blue in 10 mM Tris/HCl, pH 7.8, and loaded onto
5 or 6% polyacrylamide gels with 0.5× Tris-borate-EDTA running buffer
(37). Electrophoresis was carried out at 150 V constant voltage. Gels
were transferred onto Whatman paper, dried, and exposed to Biomax MR
film (Eastman Kodak Co.) at
70 °C.
(Promega) as internal standard, 100 ng of luciferase reporter plasmid,
and expression vectors for
-catenin (50 ng), TCF (10 ng), and p300
(250 ng) together with increasing amounts of competitor as indicated.
Total amounts of DNA were kept constant by adding empty expression
vector where needed. Firefly luciferase reporters were p01234, pCdx1-4
Luc, and pCdx1 (
350/+72) Luc (39, 40). Firefly luciferase and
-galactosidase activities in cell lysates were determined as before
(21) using 96-well plates and a Labsystems Luminoskan Ascent
luminometer. To measure Renilla luciferase activity, 100 µl of a solution with 0.5 µM coelenterazine
(Calbiochem) in 25 mM Tris/HCl, pH 7.5, 100 mM
NaCl, 1 mM EDTA was injected per well of a 96-well plate,
and after a delay of 0.5 s, light emission was recorded for 5 s. Reporter gene activities shown are average values and their S.D.
values, obtained from at least three independent experiments after
normalization against
-galactosidase or Renilla luciferase activities.
-catenin (Transduction Laboratories) primary
antibodies and the appropriate horseradish peroxidase-labeled secondary
antibodies (Jackson ImmunoResearch Laboratories). Antibody-antigen
complexes were visualized by chemiluminescence (ECL system; Amersham Biosciences).
-phosphatase (New England Biolabs) for
20 min at 37 °C in a total volume of 10 µl in the buffer supplied
with the enzyme. Untreated or phosphatase-treated samples were loaded
onto 6% SDS-polyacrylamide gels and separated by electrophoresis at 75 V. Run times were extended for up to 2 h after the bromphenol blue
tracking dye had left the gel. After transfer onto nitrocellulose
membranes, p300 was visualized by Western blotting as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin signal transduction pathway contribute to the
differential regulation of Wnt target genes, we asked whether LEF1 and
TCF4E were equally capable of supporting activation of Wnt-regulated
promoters by
-catenin and p300. LEF1 and the TCF4E splice variant
are representatives of the short and long isoforms of TCF family
members (5). Their main differences are the extended C terminus in
TCF4E with binding motifs for the transcriptional corepressor CtBP and
a context-dependent transactivation domain at the N
terminus of LEF1 (Fig. 1A).
Transactivation properties of LEF1 and TCF4E were compared at the
promoters of the mouse Cdx1 gene and the Siamois
gene of X. laevis (39, 40). Both of these genes
are Wnt/
-catenin targets, which contain multiple TCF-binding
elements (TBEs) in their promoter regions, and in their natural
contexts they are regulated in a highly cell type-specific manner.
Combinations of expression vectors for epitope-tagged TCF4E and LEF1,
an activated form of
-catenin with Ala substitutions in the
N-terminal destruction box (
-catS33A) (3, 41), and p300 were
transfected into the human embryonic kidney cell line 293 and the human
osteosarcoma cell line U-2 OS together with the pCdx1-4 Luc and p01234
luciferase reporter constructs (Fig. 1, C and D).
When
-catenin, TCF4E, LEF1, and p300 were expressed individually,
they had little or no effect on promoter activity. Pairwise
combinations of
-catenin and LEF1 or
-catenin and TCF4E only
weakly activated the luciferase reporters. Low levels of reporter gene
expression were also induced by
-catenin and p300. In the absence of
transfected TCFs,
-catenin presumably employs a limited pool of
endogenous TCFs (14). In contrast, high levels of reporter gene
activation were obtained when
-catenin, p300, and one of the TCFs
were expressed simultaneously. Importantly, however, TCF4E and LEF1
clearly differed in their ability to synergize with
-catenin and
p300, although they are expressed at similar levels (Fig. 1,
B and D). In both 293 and U-2 OS cells, TCF4E supported
-catenin/p300-dependent activation of the
Cdx1 promoter but not of the Siamois promoter.
Conversely, LEF1 mediated activation of the Siamois promoter
but not of the Cdx1 promoter (Fig. 1D). Even when
we raised the amount of transfected plasmid, LEF1 did not support
activation of the Cdx1 reporter (not shown). Thus, the two
TCF family members are functionally different and act in a
promoter-specific manner.
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Fig. 1.
LEF1 and TCF4E differ in their
ability to support -catenin
(
-cat)/p300-mediated
activation of the Siamois and Cdx1
promoters. A, scheme of human TCF4E and mouse
LEF1 constructs. Binding sites for
-catenin (
BD), TLE
proteins, CtBP, the context-dependent transactivation
domain in LEF1 (CTA), the DNA-binding domains
(HMG), the adjacent nuclear localization signals
(black boxes), and the C-terminal HA epitope tags
are indicated. B, Western blot analyses of LEF1 and TCF4E
expression in transfected 293 cells with a monoclonal antibody against
the HA epitope tag. Note that the anti-HA antibodies detect TCF4E
proteins at positions according to their theoretical molecular weight
and an additional slower migrating form (marked with an
asterisk in lane 3), which arises as
the result of an unknown posttranslational modification.
Mw, molecular weight standard. C,
schematic representation of luciferase reporter gene constructs. In
pCdx1-4 Luc, a mouse Cdx1 promoter fragment from position
3600 to +72 drives firefly luciferase expression. In p01234, an
800-bp promoter fragment from the X. laevis
Siamois gene controls luciferase expression. Positions of
functional TBEs are indicated by black boxes, and
nonfunctional TBEs are depicted as light gray
boxes. D, 293 (top panels)
or U-2 OS cells (bottom panels) were transfected
with expression vectors for
-catenin, p300, LEF1, TCF4E, the
Cdx1 or Siamois luciferase reporters, and the
pCMV
vector for internal standardization as indicated. A
stabilized
-catenin mutant (
-catS33A) was used. Luciferase
reporter gene activities are shown as relative values compared with the
activity measured in lysates from cells transfected with only the
luciferase reporter (relative activity of 1).
-catenin and p300 in
Cdx1 promoter activation (TCF4
C, TCF4
C1, and
TCF4
C1.1) (Fig. 2D). Constructs that retain this domain
but have deletions in other parts of the TCF4E C terminus (TCF4
C2
and TCF4
C1.2) activate the Cdx1 promoter. From these
results, we conclude that the C1.1 domain contains at least essential
parts, if not all, of a promoter-specific transactivation domain. In
addition, because activation of the Cdx1 promoter by TCF4E
was seen with the short reporter construct used in these experiments
(Fig. 2C), it appears that the proximal promoter region with
its TBEs contains all of the determinants that make the Cdx1
promoter specifically responsive to TCF4E.
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Fig. 2.
The C terminus of TCF4E harbors a
promoter-specific activation domain. A, scheme of TCF4E
deletion constructs. Amino acid positions of the deletion end points
are given. Other features of TCF4E are depicted as in Fig. 1. All
constructs contain the C-terminal HA epitope tag. B,
expression of the TCF4E deletion mutants was analyzed by Western
blotting with an anti-HA antibody as before (Fig. 1B).
Mw, molecular weight standard. Asterisks
mark posttranslationally modified forms of TCF4E and its derivatives.
C, schematic representation of the pCdx1( 350/+72)Luc
reporter. The promoter fragment in this reporter harbors only TBE3a,
-3, and -4. D, 293 cells were transfected with combinations
of expression vectors for the TCF4E deletion mutants,
-catS33A,
p300, the Cdx1 reporter, and pCMV
as indicated.
Luciferase reporter gene activities were determined as in Fig. 1.
Reporter gene activity without
-catenin (
-cat), TCF4E,
and p300 was arbitrarily assigned the value of 1. WT, wild
type.
-catenin,
p300, and TCF4E was challenged by coexpressing increasing amounts of
LEF1 and TCF4
C. As shown in Fig.
3A, higher levels of both
factors progressively inhibited Cdx1 reporter activation.
Deletion of the DNA-binding domain of LEF1 (Fig. 3A,
LEF1
HMG) and, to a lesser extent, removal of the
-catenin-binding
domain (Fig. 3A, LEF1
N) impaired this effect.
Accordingly, the dominant negative activity of LEF1, and presumably of
the TCF4
C mutant, most likely results from a combination of promoter
blockade and sequestration of
-catenin. Therefore, LEF1 is capable
of binding to the Cdx1 promoter in living cells and of
competing with TCF4E. However, LEF1 and TCF4 isoforms lacking the C1.1
domain do not form productive transcription factor complexes at the
Cdx1 promoter.
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Fig. 3.
A, LEF1 and TCF4 C can occupy the
Cdx1 promoter in vivo and act as dominant
negative factors. 293 cells were transfected with combinations of
expression vectors for
-catS33A, p300, the pCdx1-4 Luc and pRL-CMV
reporter plasmids, and increasing amounts of expression vectors for
wild-type or mutant TCF4E and LEF1 (10, 50, and 250 ng) as indicated.
TCF4
C is schematically shown in C. LEF1
N and
LEF1
HMG are defective for binding of
-catenin or DNA because they
lack amino acids 7-89 (
N) and 320-339
(
HMG), respectively. B, a LEF1-TCF4 chimera
can functionally replace TCF4E at the Cdx1 promoter. 293 cells were transfected with combinations of expression vectors for
TCF4E, the TCF4
C mutant, LEF1, the LEF1-TCF4 chimera (LEF1-T4C), the
LEF1
-T4C mutant,
-catS33A, p300, the pCdx1-4 Luc and
pRL-CMV reporter plasmids as indicated. A and B,
reporter gene activities were determined as in Fig. 1 except that
Renilla luciferase activity was used for normalization.
Reporter gene activity without
-catenin, TCF4E, and p300 was
arbitrarily assigned the value of 1. C, schematic
representation of TCF4E, TCF4
C, LEF1, and the LEF1-TCF4 chimeric
constructs with a wild-type (LEF1-T4C) or mutant DNA-binding domain
(LEF1
-T4C). Functionally relevant domains and interaction sites for
TCF4E/LEF1 binding factors are marked as in Fig. 1A. Amino
acid end points of the deletion mutants are given. D,
expression of LEF1 and TCF4E constructs in whole cell lysates from
transfected 293 cells was analyzed by Western blotting with an anti-HA
antibody. Posttranslationally modified forms of the TCF4E constructs
are marked with asterisks. Mw, molecular
weight standard.
-catenin or p300 alone, the LEF1-T4C fusion mediated even
higher levels of Cdx1 reporter activity than TCF4E (Fig.
3B). In the presence of both p300 and
-catenin, TCF4E and
LEF1-T4C activated the Cdx1 promoter equally well. For its
function, the chimeric protein required an intact DNA-binding domain.
This shows that the LEF1-T4C chimera must be able to bind to the
Cdx1 promoter in order to activate and rules out the
possibility that LEF1-T4C stimulates the reporter only indirectly.
Thus, the presence of the TCF4E C terminus turns LEF1 into an activator
at the Cdx1 promoter. Together with the previous results,
this demonstrates that the transactivation domain present at the C
terminus of TCF4E is necessary and sufficient to confer promoter
specificity upon TCF proteins.
-catenin complex formation. However, no differences were seen with respect to the ability of LEF1,
TCF4E, or TCF4
C to interact with
-catenin (not shown). Therefore,
we compared the DNA binding properties of LEF1, wild-type and mutant
TCF4E, and the LEF1-T4C chimera by EMSA with two different DNA probes
(Fig. 4). The TCR
25 probe harbors a
single consensus TBE derived from the T-cell receptor
-chain
enhancer (38). The second probe was a Cdx1 promoter
fragment, which contains three TCF binding motifs: TBE3a, TBE3, and
TBE4 (39). The TBE3a element is a newly discovered TBE that was not
reported previously. The TCF proteins were transcribed and translated
in vitro, and similar amounts of the various factors, as
determined by Western blotting (Fig. 4A), were used.
Although both probes were bound by LEF1 and TCF4E, LEF1 exhibited an
affinity for the single binding site of the TCR
25 probe that was at
least 5 times higher than that of TCF4E (Fig. 4C, compare
lanes 3-5 with lanes
6-8). Interestingly, this difference was also seen when
oligonucleotides with a single TBE (either TBE3a, -3, or -4) derived
from the Cdx1 promoter were used as probes (not shown). The
C terminus of TCF4E appears to be at least partly responsible for the
reduced DNA binding capacity of TCF4E, as indicated by the
complementary increases and decreases in the DNA-binding capabilities
of TCF4
C1.1 and the LEF1-T4C chimera (Fig. 4C, compare
lanes 6-8 with lanes 9-11
and lanes 3-5 with lanes
12-14).
View larger version (45K):
[in a new window]
Fig. 4.
Comparison of the DNA binding properties of
TCF4E and LEF1. A, Western blot analyses of LEF1 and
TCF4 proteins used for EMSA. Wild-type (WT) and mutant
TCF4E, LEF1, and the LEF1-T4C chimera, which had been transcribed and
translated in vitro, were detected with an anti-HA antibody.
Mw, molecular weight standard. B,
schematic representation of the TCR 25 and Cdx1 DNA
fragments used as probes in EMSAs. End points of the Cdx1
promoter fragment relative to the transcriptional start site are given.
TCF consensus motifs are shown as black boxes.
C, comparison of the DNA binding properties of different
forms of LEF1 and TCF4E with probes containing a single or multiple
TCF-binding sites. LEF1 and TCF4E were transcribed and translated
in vitro and used for DNA-binding experiments with the
radiolabeled TCR
25 oligonucleotide or the Cdx1 promoter
fragment with three TBEs. Positions of the free probe and the
DNA-protein complexes are marked. The faster migrating complex labeled
with an asterisk in lanes 12-14 is
caused by incompletely translated forms of LEF1-T4C that do not contain
the TCF4 C1.1 domain and therefore possess a higher affinity for the
probe compared with the full-length protein. Lanes
1 and 15, binding reaction without reticulocyte
lysate or TCF proteins. Lanes 2 and
16, binding reactions with unprogrammed reticulocyte
lysate.
C1.1, and the chimeric LEF1-T4C protein readily generated
protein-DNA complexes representing single, double, and triple occupancy
of the Cdx1 probe (Fig. 4C, lanes
20-28). In contrast, under the conditions used, LEF1
generated primarily a single species of protein-DNA complexes (Fig.
4C, lanes 17-19). Still, LEF1 can
recognize all three Cdx1 TBEs also in the context of the entire
promoter fragment as shown by DNase I footprinting experiments.2 Because the
same protein preparations were used for EMSA with the Cdx1
promoter fragment and the single TBEs, the observed differences in DNA
binding are inherent properties of the TCF proteins and not due to
variable efficiencies of protein folding. In addition, these
experiments also confirm the potential regulatory functions of the
TCF4E C terminus. As seen in EMSAs with a single TBE, we observed that
deletion of the C1.1 domain increased the ability of TCF4E to bind to
the Cdx1 promoter fragment. This is evident from the greater
abundance of DNA complexes with all three TBEs being simultaneously
occupied by the TCF4
C1.1 mutant (Fig. 4C, compare
lanes 20-22 with lanes
23-25). In addition to this inhibitory activity, which
seems to be linked to the C1.1 domain, the TCF4E C terminus also
appears to harbor an opposing activity, which promotes the binding of
TCFs to multiple TBEs. We conclude this from the observation that the
LEF1-T4C chimera interacts with the Cdx1 promoter more
efficiently than the parental LEF1 protein (Fig. 4C, compare
lanes 17-19 with lanes
26-28). Aside from this, the pattern of Cdx1
promoter complexes formed by the LEF1-T4C chimera differed from all
other patterns seen. The molecular weights of TCF4E and LEF1-T4C are
very similar, and with the TCR
25 probe, TCF4E and LEF1-T4C complexes
migrated at nearly identical positions (Fig. 4C,
lanes 6-8 and 12-14). However, the
LEF1-T4C-Cdx1 complexes are much more retarded than
expected (Fig. 4C, compare lanes
20-22 and 26-28). Since TCF proteins are
architectural transcription factors that are known to change DNA
topology (5), it is possible that these migratory differences between
TCF4E and LEF1-T4C arise from different conformations of the DNA
complexes containing either TCF4E or LEF1-T4C. Taken together, the
results of our comparative DNA-binding experiments show that the DNA
binding properties of TCF4E differ significantly from those of LEF1 and
that the affinity of TCF4E for DNA-binding elements changes depending
on the number of motifs present and in response to regulatory functions
present at its C terminus.
-catenin,
p300, and TCF4E. However, pairwise combinations of p300 and TCF4E and
especially the LEF1-T4C chimera also elicited a significant response of
the Cdx1 promoter (e.g. Fig. 3B).
Therefore, we speculated that the C terminus of TCF4E might contribute
to the formation of an active transcription factor complex at the
Cdx1 promoter through protein-protein interactions with
p300. Indeed, p300 co-immunoprecipitated with epitope-tagged TCF4E from
lysates of transfected 293 cells (Fig.
5A, lane
3). To clarify whether the presence of p300 was simply due
to coprecipitation with
-catenin, which was also detected in the
immunoprecipitate and which can interact with p300 on its own (21), we
performed control experiments with the TCF4
N mutant lacking the
-catenin-binding domain of TCF4E. As expected,
-catenin no longer
coprecipitated with TCF4
N, whereas p300 was still present in the
immunoprecipitate (Fig. 5A, lane 4).
In contrast, deletion of the C1.1 domain significantly reduced the
amount of p300 coprecipitating with TCF4
C1.1 even in the presence
of
-catenin (Fig. 5A, lane 5).
Apparently, the C terminus of TCF4E contributes to an interaction
between TCF4E and p300.
View larger version (30K):
[in a new window]
Fig. 5.
The C terminus of TCF4E mediates
an interaction with p300. A, TCF4E and p300
co-immunoprecipitate from cell lysates. 293 cells were transfected with
expression plasmids for Glu-Glu-tagged p300 (p300-EE; 15 µg) and HA-tagged wild-type (WT) or mutant TCF4E (5 µg)
as indicated. Cell extracts were prepared 40 h after transfection
and used for immunoprecipitation (IP) with anti-HA
antibodies. Immunoprecipitated material and a fraction of each cell
lysate were resolved by SDS-PAGE and analyzed by Western blotting
(IB) with antibodies as shown. B, the
promoter-specific activation domain at the TCF4E C terminus interacts
with p300 in vitro. GST or GST-TCF4 fusion proteins shown
schematically in C were bound to glutathione-Sepharose beads
and incubated with radiolabeled p300. Proteins retained by GST or the
GST-TCF4 fusions and 10% of the input material were analyzed by
SDS-PAGE and fluorography. C, structure of the GST-TCF4
fusion proteins and, for comparison, of TCF4E. Amino acid end points of
the TCF4 fragments are given. Functionally relevant domains and
interaction sites for TCF4E-binding factors are indicated as before.
Hatched bar, GST.
-Catenin, but Not TCF4E, Induces Phosphorylation of
p300--
The observation that the C1.1 domain can interact with p300
raises the possibility that p300 is recruited to the Cdx1
promoter by TCF4E, which would make
-catenin seemingly dispensable
for promoter activation. However, when we analyzed p300 in total cell lysates using extended electrophoresis on denaturing gels and Western
blotting, we noticed that p300 from 293 cells expressing both p300 and
-catenin migrated more slowly than p300 from control cells or cells
expressing p300 and TCF4E (Fig.
6A). This indicates that
-catenin induces a covalent, posttranslational modification of p300,
whereas TCF4E does not. Since p300 is a phosphoprotein and regulated
phosphorylation contributes to its functionality as a transcriptional
coactivator (26), we tested whether p300 becomes phosphorylated in the
presence of
-catenin. Upon treating protein samples containing p300
with
-phosphatase, the p300 bands sharpened, and material from
control cells and
-catenin-expressing cells migrated at the same
relative positions (Fig. 6B). Control experiments performed
in the presence of the phosphatase inhibitors NaF and EDTA or in the
absence of Mn2+ ions (essential phosphatase cofactors)
prove that the changes in electrophoretic mobility of p300 are due to
dephosphorylation rather than protein degradation (Fig. 6C).
Based on this, we propose that TCF4E recruits a dormant form of p300 to
the Cdx1 promoter, whereas
-catenin might trigger
promoter activation by inducing phosphorylation of p300.
View larger version (31K):
[in a new window]
Fig. 6.
-Catenin
(
-cat), but not TCF4E,
induces phosphorylation of p300 in vivo.
A, altered electrophoretic mobility of p300 in the presence
of
-catenin. 293 cells were transfected with expression plasmids for
p300-EE (15 µg),
-catS33A, or HA-tagged TCF4E (5 µg) as
indicated. Cell extracts were prepared 40 h after transfection. A
fraction of each cell lysate was resolved by SDS-PAGE and analyzed by
Western blotting with antibodies against p300. The position of
anti-p300 immunoreactive material in the presence or absence of
-catS33A or TCF4E is indicated at the right
side of the blot. B, reduced electrophoretic
mobility of p300 in the presence of
-catenin is due to
phosphorylation. Cell extracts prepared as in A were treated
with increasing amounts of
-phosphatase (
PPase) for
20 min at 37 °C. Samples were then resolved by SDS-PAGE on 6% gels
and analyzed by Western blotting with anti-p300 antibodies.
C, specificity of the phosphatase effect. Fractions of the
same cell extracts as in B were subjected to phosphatase
treatment either in the absence of the essential
-phosphatase
cofactor MnCl2 or in the presence of the phosphatase
inhibitors EDTA and sodium fluoride (NaF). Samples were analyzed by
SDS-PAGE and Western blotting as in B.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin-dependent transcriptional activator, whereas
the repressor activities of TCF3 prevail over its activating function
(42-44). The molecular basis for these differences is unknown, but in
the case of TCF proteins from X. laevis it was
shown that activating properties require the presence of an
alternatively spliced exon at their N termini (31, 35). Here we report
that also LEF1 and TCF4 have different transactivation capacities,
although both factors contain the activating N-terminal exon.
Additionally, whereas both LEF1 and TCF4 can act as activators, they
appear to have distinct spectra of target genes, and a novel,
promoter-specific transcriptional activation domain was identified in
the TCF4E variant.
-catenin-independent gene
regulation, for example at the TCR
enhancer or at the HIV-1 promoter
(38, 45). How can inappropriate activation of these regulatory elements
by
-catenin-TCF complexes be prevented? Perhaps significantly, both
the TCR
enhancer and the HIV-1 promoter contain single TCF binding
motifs (38, 45), whereas the known Wnt target genes typically contain
multiple TCF recognition elements. Under competitive conditions, when
TCF levels are low, or when only a particular TCF family member is
expressed in a cell, the differential recognition and occupancy of
single versus multiple binding sites could be one way to
distinguish Wnt target genes from nontarget genes.
C
and TCF4
C1.1 did not activate the Cdx1 promoter and
blocked Cdx1 induction by wild-type TCF4E in our competition
experiments, yet they bound to the Cdx1 promoter in
vitro even more efficiently than TCF4E or LEF1 (Figs. 3 and 4).
Moreover, the pattern of DNA-protein complexes produced by the LEF1-T4C
chimera differed from those of all other factors analyzed, although the
chimera is functionally equivalent to TCF4E. Thus, an influence on
promoter occupation or topology is unlikely to be the only mechanism by
which the Cdx1-specific activation domain at the C terminus
of TCF4E functions. An additional mode of action could be that the C1.1
domain mediates protein-protein interactions. Indeed, it interacts with
p300 in vitro, and p300 co-immunoprecipitated together with
TCF4 from cellular lysates. Although p300 may not be the only factor
that interacts with the C1.1 domain, the match between the ability to
form a complex with p300 and the ability to support activation of the
Cdx1 promoter strongly suggests that the interaction between TCF4E and p300 is of physiological relevance.
-catenin and p300 to activate the Cdx1
promoter.2 Temporal and tissue-specific inducibility of the
Cdx1 gene by Wnt signaling thus may in part be restricted
through the availability of an appropriate TCF isoform. The presence of
TCF4E or TCF1E may hence be a prerequisite, but it is not sufficient
for Cdx1 activation, because the expression domains of TCF1
and TCF4 exceed the expression domains of the Cdx1 gene
(51). Many factors have been identified that interfere with the gene
regulatory activity of
-catenin and TCFs and could help to shape
specific gene expression patterns. However, inhibitors like ICAT act
upstream of target gene promoter activation and cause a global
inhibition of Wnt signaling rather than a selective and differential
regulation of Wnt target genes (3, 5, 52). More likely, mechanisms that
govern tissue-specific Wnt target gene regulation act at the level of
individual regulatory elements and their associated transcription
factors. For example, a hierarchy of regulatory events permits Wnt
effectors to maintain expression of the murine Brachyury gene but only
after the initiation of transcription by a different regulatory system
(53). Alternatively, the activation of Wnt target genes may depend on
the simultaneous input from multiple signaling pathways such as a
combination of Wnt and transforming growth factor
signals (54, 55).
At the molecular level, the two pathways are integrated through
physical interactions of the Smad proteins and members of the TCF
family, necessitating the presence of DNA recognition motifs for both
families of DNA-binding proteins (28, 29). Thereby, only those genes
with the proper combination of cis-active DNA elements in their
promoter regions will respond to the combinatorial input from Wnt and
transforming growth factor
-signaling pathways. This underscores
that the DNA sequences of promoter regions and the interaction partners of TCF proteins are critical determinants for the differential regulation of Wnt target genes.
-catenin (21, 27), it was unexpected to find an
interaction between TCF4E and a transcriptional coactivator like p300.
Intuitively, one would surmise that recruitment of p300 by TCF4E
catalyzed transcription even in the absence of a Wnt signal. However,
even the low level Cdx1 promoter activation upon expression
of p300 and TCF4E was seen only if TCF4 contained the N-terminal
-catenin binding domain. Thus, Cdx1 transcriptional activation is strictly dependent on
-catenin and simple recruitment of p300 by TCF4 is insufficient for promoter activation. A similar separation between recruitment of p300 or the highly related CBP and
transcriptional activation has been described by Soutoglou et
al. (56). An additional step that could be required for
transcriptional activation is the phosphorylation of p300, which we
found can be induced by
-catenin. Phosphorylation of p300 by several
different kinases has been observed, but both activating and inhibitory effects have been described (26, 57, 58). A possible explanation for
these conflicting observations could be that the particular settings at
a given promoter ultimately determine the molecular mechanisms by which
p300 is used as a transcriptional coactivator and whether its activity
needs to be additionally regulated. Such a context dependence would
explain how p300 can be utilized by
-catenin at both the
Siamois and the Cdx1 promoters but apparently in
two different manners. At the Siamois promoter and in
conjunction with LEF1, recruitment and activation of p300 by
-catenin appears to be sufficient, whereas at the Cdx1
promoter an additional contribution by TCF4E is required. What could be
the promoter-specific determinants for these distinctions? The
interaction of LEF1 and TCF4E with Cdx1 promoter fragment
resulted in protein-DNA complexes with distinct topologies. Different
three-dimensional structures of the resulting transcription factor
complexes may preclude or even require the incorporation of specific
coactivators in order to facilitate the formation of a productive
transcription complex (59) (Fig.
7A). Indeed, the promoters of
the Siamois, Twin, and cyclin D1 genes and
an artificial reporter construct displayed a differential
responsiveness to coexpression of
-catenin and p300 (21). An
alternative but not necessarily mutually exclusive explanation is based
on the ability of p300 to interact with many different transcription
factors. Based on this property of p300, it is possible that its
recruitment to the Cdx1 promoter depends on multiple
interactions not only with
-catenin and TCF4E but in addition with
an as yet unknown factor that specifically recognizes Cdx1
promoter sequences (Fig. 7B). According to this model, LEF1 fails to activate the Cdx1 promoter because it cannot
provide a vital contact for p300. Such a mechanism represents a new
variation of the combinatorial input and multiple contact model that is already used for the regulation of Wnt target gene expression. Whether
formation of a transcriptionally active complex occurs in one step or
whether
-catenin joins a preformed complex and triggers its activity
by inducing phosphorylation of p300 remains to be determined. Other
issues that need to be addressed are the mechanisms that prevent
activation of the Siamois promoter by TCF4E and make this
promoter dependent on LEF1 or TCF3 (40). In addition, the repeated
cooperation in differential gene regulation and the varying functional
implications of their joint activities warrants further analysis of the
interactions between
-catenin, TCFs, and p300/CBP. This will provide
further insight into the mechanisms of tissue-specific and inducible
gene regulation by Wnt growth factors during embryonic development.
View larger version (31K):
[in a new window]
Fig. 7.
Models for activation of the Cdx1
promoter by transcription factor complexes containing TCF4E,
-catenin
(
-cat), and p300.
A, in this model, promoter specificity is generated, because
the assembly of a transcriptionally active complex at the
Cdx1 promoter requires a particular promoter topology that
is induced specifically by TCF4E (I) but not LEF1
(II). B, the combinatorial input model is an
alternative, but not mutually exclusive, possibility. Here, TCF4E and
an additional promoter-specific DNA-binding protein (X) synergize to
recruit p300 to the Cdx1 promoter by providing multiple
contact points (I). In this case, LEF1 fails to support
-catenin and p300-dependent activation of the
Cdx1 promoter, because, unlike TCF4E, it does not provide an
essential interaction site for p300 (II). XRE,
factor X-responsive promoter element. Transcriptionally active
complexes may assemble at the promoter de novo, or, as
indicated here,
-catenin may enter preformed complexes containing
p300 and TCF4E. By inducing phosphorylation of p300,
-catenin would
thereby turn the resident but dormant protein into an active cofactor
(symbolized by the appearance of a star-shaped outline
around p300). Note that the exact composition of the protein complexes
and the stoichiometric relationships are unknown.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Katja Bruser for excellent technical assistance, to Rolf Kemler for continuous and generous support, to Karl-Heinz Klempnauer for helpful discussions, and to Louise M. Campano for critically reading the manuscript.
![]() |
FOOTNOTES |
---|
* 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: Institut für
Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany. Tel.: 49-761-2707182; Fax: 49-761-2707177; E-mail:
hecht@mm11.ukl.uni-freiburg.de.
§ This work was done in partial fulfillment of Ph.D. requirements at the University of Freiburg.
Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M210081200
2 A. Hecht, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TCF, T-cell factor; TBE, TCF-binding element; EMSA, electrophoretic mobility shift assay; CBP, CREB-binding protein; HA, hemagglutinin; GST, glutathione S-transferase.
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