From the Microbiology and Molecular Genetics
Department, School of Medicine, University of California, Irvine,
California 92697-4025 and the ¶ Department of Human Genetics,
David Geffen School of Medicine, UCLA, Los
Angeles, California 90095
Received for publication, December 27, 2002, and in revised form, February 6, 2003
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ABSTRACT |
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Transcription of the lymphoid enhancer factor-1
(LEF1) gene is aberrantly activated in sporadic colon
cancer, whereas this gene is not expressed in the normal adult colon.
We have shown previously that promoter 1 of the LEF1 gene
is activated by T cell factor (TCF)- Wnt ligand-Frizzled receptor interactions at the plasma membrane
trigger a cytoplasmic signal transduction cascade that propagates to
the nucleus via the stabilization and release of cytoplasmic Some of the Wnt target genes encode components of the Wnt signal
transduction pathway, and their increased expression may have
inhibitory or facilitatory effects on Wnt signaling (13-19). For
example, although it is not expressed in the normal adult colon, the
LEF1 gene is aberrantly expressed in the majority of sporadic colon cancers, in which Wnt signaling is constitutive (20).
Although the underlying mechanism of aberrant LEF1
expression is not fully understood, only the promoter for full-length
forms of LEF-1 is activated. A second intronic promoter that
produces dominant negative forms of LEF-1 remains silent. We have shown that the promoter for full-length LEF-1 is likely to be a Wnt target
gene, because it is activated by TCF-1- E-tail isoforms of TCF-1, TCF-3, and TCF-4 are generated from
alternative splicing at the 3'-end of the pre-mRNA to encode a
longer alternative C-terminal region (21-23). The LEF1
locus does not produce an E-tail isoform, because it is missing an
E-tail-specific exon. Instead, the most abundant isoform of LEF-1
carries an "N"-tail. Neither the expression pattern nor the
function of TCF E-tail isoforms is very well characterized except that
they are detected as one of the most abundant isoforms in cell lines,
and the TCF-4E and TCF-3 E-tails are able to bind CtBP, a transcription
repressor. We show here for the first time that the E-tail of TCF-1
carries a novel transcription-activating function with respect to the LEF1 promoter. This activity is not present in TCF-3E or
LEF-1N but remains dependent on Transient Transfection Assay--
COS cells were transiently
transfected with Effectene reagent according to the manufacturer's
protocol (Qiagen). COS-7 cells were plated at a density of 150,000 cells/well in six-well plates 20 h before transfection. Luciferase
reporter constructs (LEF1 promoter, Western Blot Analysis--
COS cells were transiently
transfected with either 0.5 µg of the indicated TCF constructs using
the Effectene reagent (Fig. 2) or 10 µg using a BTX 600 electroporator (Fig. 1, Ref. 20). 3% (Fig. 2C) or 20%
(Fig. 1C) of whole cell lysates from transfected cells were
separated by electrophoresis on SDS-10% polyacrylamide gels. Blots of
these gels were probed with either TCF-1 monoclonal antibody
(anti-TCF-1, Upstate Biotechnology) or LEF-1N polyclonal rabbit
antiserum at a 1:1000 dilution (20).
Immunofluorescence--
Immunofluorescence detection of TCF-1E
proteins in transiently transfected COS cells was performed as
described previously with the following modifications (24). Cells were
transfected by electroporation and plated on glass coverslips. After
24 h, cells were fixed with 3.7% formaldehyde, washed three times
in 1× phosphate-buffered saline, and permeabilized with 0.5% Nonidet P-40. Cells were blocked with horse serum (1:100) and hybridized with
rabbit polyclonal LEF-1 antiserum (1:1000), which detects all
LEF/TCFs, for 1 h at room temperature. Cells were washed and hybridized to goat anti-rabbit-conjugated fluorescein isothiocyanate serum (1:1000, Amersham Biosciences) for 1 h at room
temperature. Cells were dipped in 4',6-diamidino-2-phenylindole
solution for 10 s, washed, and mounted on slides for viewing.
DNase I Footprinting--
Partially purified recombinant LEF-1N
and TCF-1E were used in standard DNase I footprinting assays as
described previously (20). Equivalent amounts of protein were judged by
SDS-PAGE and Coomassie staining.
Plasmid Construction--
The LEF1 promoter
luciferase reporter plasmid has been described previously (20). The EVR
eukaryotic expression plasmid was used to express LEF and TCF coding
sequences (34). An overlap extension PCR method was used to generate
nucleotide substitutions within the two LEF/TCF response elements
(+190, +283) (35, 36). Complete details of plasmid construction and
mutational analysis are described in the Supplemental Material.
Isoform-specific Activation of the LEF1 Promoter--
In a
previous study we showed that a fragment of the LEF1
promoter was activated 7-fold by TCF-1-
That LEF/TCFs should differ so dramatically in their ability to
activate transcription is surprising, because although they are encoded
by separate genes, they are highly homologous. They share a highly
similar DNA-binding and The E-tail Can Convert LEF-1 into an Autoregulatory Factor--
To
determine whether this unique activity is solely the result of
sequences in the E-tail or whether other regions unique to TCF-1 are
also necessary, a chimeric LEF-1N protein was created in which coding
sequences for the E-tail from TCF-1E were cloned in-frame to the end of
the full-length LEF1 open reading frame. This LEF-1E chimera
was tested in a transient transfection assay with the LEF1
promoter. Both TCF-1E and LEF-1E were able to activate expression
~8-fold, whereas wild type LEF-1N was again inactive in the assay
(Fig. 1C). Western blot analysis of the expression of these
three proteins showed that protein levels are equivalent. Thus the
dramatic differences in activity are not caused by a change in
expression level or stability; rather the results show that sequences
in the E-tail and not other unique sequences within TCF-1 are
sufficient to convert LEF-1 into an activator of its own promoter.
Mutational Analysis of the E-tail Activation Domain--
To
identify the sequences within the long E-tail that are necessary for
LEF1 promoter activation, a set of C-terminal TCF-1E truncations were created (Fig. 2A). Four C-terminal
truncations were designed with end points at amino acid 376 (
The low level of expression of the TCF-1E and LEF-1N Bind to the Same Wnt Response Elements in the
LEF1 Promoter--
The CR peptide motifs are highly conserved among
the TCF homologs, are located near the high mobility group DNA-binding
domain, and are highly enriched in basic residues. This result suggests that these CR motifs might function as independent or auxiliary DNA-binding domains and thus modify the ability of TCF-1E to recognize alternative DNA sequences. Alternative sequence recognition could explain why TCF-1E and TCF-4E and not LEF-1N, TCF-1B, or TCF-3E are
able to activate the promoter. To test for alternative DNA sequence
recognition, we produced recombinant LEF-1N and TCF-1E protein in
bacteria. Partially purified preparations of both proteins were used in
a DNase I footprint assay with a fragment from the LEF1
promoter ( Transfer of the E-tail-sensitive Wnt Response Elements to a
Heterologous Promoter--
To test whether this downstream region of
the LEF1 promoter contains the TCF-1E-specific response
element(s), we transferred a fragment containing the footprints
(nucleotides +104 to +308) to a position ( We have described a new activity of the E-tail isoform of TCF-1
that enables Several possible mechanisms may enable activation of the
LEF1 promoter by the E-tail. Such possibilities include the
location of response elements relative to the start site of
transcription, regulation of different types of basal promoters,
differences in DNA sequence recognition, or interactions with other
regulatory proteins. The first two possible mechanisms, the
downstream location of the Wnt response elements or the type of basal
promoter, are unlikely to affect activation because movement of the
response elements to a position 5' of the start site of transcription
of the heterologous TATA-containing Herpesvirus TK
promoter does not change the patterns of activity that we observed
(Fig. 5). We conclude that the E-tail activity is not likely to
subserve promoter-specific or location-specific regulation. The third
mechanism, conformation of DNA sequence recognition or affinity,
remains a possibility because the DNase I footprinting experiments with recombinant LEF-1N and TCF-1E protein show minor differences in DNaseI
hypersensitivities (Fig. 4). Difficulties with the purification of full-length TCF-1E have prevented us from directly comparing DNA-binding affinities with LEF-1N protein. However, to address whether
different DNA binding affinities could account for the disparate
activities of TCF-1E and LEF-1N, we created a mutant LEF1 promoter reporter plasmid in which the sequences of the
two Wnt response elements at +190 and +283 were changed to the high affinity LEF/TCF response sequences present in the TOPFLASH reporter plasmid. Again only TCF-1E was able to work with If the E-tail modifies target gene recognition through nucleic acid
interactions or protein interactions, it does so via a novel
independent mechanism with the CR1 and CR2 motifs playing an important
role. The TCF-1 E-tail is a 137-amino acid domain, and for most of this
domain, there is little sequence conservation with other TCF E-tails.
The exception is the high level of sequence conservation in the most
N-terminal part centered over the CR motifs (Fig. 2B). The
CR1 and CR2 peptide motifs of 10 and 15 amino acids, respectively, show
the highest conservation between TCF-1 and TCF-4, with TCF-3 having the
greatest sequence variance and missing the 9 amino acid CR1 motif
completely (Fig. 2B). Drosophila TCF and the
Caenorhabditis elegans ortholog, Pop-1, have nearly identical CR1 and CR2 motifs. We have introduced amino acid
substitution mutations in CR1 and CR2, and E-tail activity is abolished
in our assays (Fig. 2B). Interestingly, van de Wetering
et al. (4) identified a mutation in the
Drosophila TCF/pangolin locus that causes lethality. This
mutant allele, dTCF1, carries a single
alanine-to-valine substitution in the CR1 motif, underscoring the
functional importance of this region.
Although a detailed understanding of the function of the CR
region (also referred to as the CRAR region (3)) is lacking, its
activities are likely to be independent and not intrinsically associated with LEF/TCF proteins only. A search of
GenBankTM proteins containing similar CR1 and CR2 motifs
identified two additional DNA-binding proteins (Fig. 2B).
The first, a 513-amino acid protein called papillomavirus regulatory
factor (Prf-1) and papillomavirus binding factor (PBF) was identified
in a yeast one-hybrid assay for cellular proteins binding to the E2
binding site, P2, of human papillomavirus type 8 (29). The second
factor, GLUT4-enhancer factor (G4EF), is highly similar to Prf-1/PBF
and was identified as a 387-amino acid DNA-binding protein that
recognizes the enhancer for the human GLUT4 transporter gene (30). On
the basis of a
CX4C-X12H-X4H
sequence in the middle portion of each protein, both Prf-1/PBF and G4EF are proposed to contain a TFIIIA-like zinc finger DNA-binding domain.
Indeed Prf-1/PBF has been shown to bind specifically to DNA sequences
with CCGG at the core (29). Unlike the closely juxtaposed high mobility
group DNA-binding domain and CR1/2motifs of the TCFs, the homologous
CR1/2 motifs in Prf-1/PBF lie at the extreme C-terminal end of the
protein, 139 residues away from the putative zinc finger DNA-binding
domain. Thus, if CR1/2 motifs influence DNA binding for all these
different proteins, they must do so as independent and auxiliary motifs
able to cope with variable placement relative to different types of
DNA-binding domains.
The results that we report in this study support the intriguing
possibility that not all LEF/TCFs are functionally equivalent and that
different isoforms may preferentially regulate different subsets of Wnt
target genes. Such a possibility is consistent with genetic data from
knock-out and transgenic mice experiments in which the removal of the
LEF1 gene or TCF1 gene generated partially non-redundant phenotypes in tissues where their expression patterns overlapped or where overexpression of dominant negative forms of
LEF/TCFs produced non-identical phenotypes (31-33). Whether a portion
of these non-redundant activities is a result of the expression of
E-tail isoforms and whether such specialized functions play a role in
tumor development are unknowns that are important to address if we are
to understand the role that Wnt signals play in promoting cancer.
-catenin complexes in
transient transfection assays, suggesting that LEF1 is a
target of the Wnt pathway in colon cancer. To further explore the link
between LEF1 expression and the Wnt pathway, we studied two
response elements in the promoter. Surprisingly we found that the
LEF1 promoter is selectively activated by specific isoforms
of the LEF/TCF transcription factor family that contain an alternative
C-terminal "E" tail. These isoforms, TCF-1E and TCF-4E, activate
the promoter in a
-catenin-dependent manner. We show
that a complete E-tail domain is necessary for full activity and
delimits residues within two highly conserved peptide motifs within the
tail that are required (KKCRARFG; WCXXCRRKKKC). These peptide motifs are not only conserved among the TCF family members but are also found in two newly identified DNA-binding proteins
named papillomavirus binding factor and GLUT4 enhancer factor.
This study thus identifies a new and unique set of motifs used by the
Wnt pathway for target gene regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin, an armadillo repeat protein with nuclear import/export capabilities (reviewed most recently in Refs. 1 and
2).1 Nuclear-localized
-catenin forms a transcription regulatory complex with any member of
the lymphoid enhancer factor/T cell factor family
(LEF/TCF)2 (LEF-1, TCF-1,
TCF-3, and TCF-4) (reviewed recently in Ref. 3). All four LEF/TCFs bind
to Wnt target genes through their high mobility group
DNA-binding domains and position a potent regulatory domain within
-catenin to activate transcription (4-6). When Wnt signals operate
properly, the changes in gene expression direct cells to adopt specific
cell fates and differentiate or to grow and divide (reviewed in Refs.
3, 7, and 8). Unregulated constitutive Wnt signaling, which is enabled
by mutations in cytoplasmic Wnt signaling components, is thought to be
an initiating event for many types of cancer, including colon cancer
(5, 9, 10). The most common mutations in colon cancer, truncation
mutations of adenomatous polyposis coli, disallow
-catenin
degradation and increase the concentration of
-catenin protein
in cells (9, 11, 12). High levels of
-catenin cause an increase in
the formation of LEF/TCF-
-catenin complexes in the nucleus,
which leads to inappropriate activation of Wnt target genes. This is proposed to be a key component of Wnt-linked carcinogenesis.
-catenin and
TCF-4-
-catenin complexes and is sensitive to cellular levels of
-catenin. Inappropriate LEF1 expression may contribute to
increased Wnt signal strength by increasing the concentration of
LEF/TCF-
-catenin complexes in the nucleus, and such increases appear
to be Wnt pathway-directed. We show in this study that the promoter for
full-length LEF-1 is activated by
-catenin through two Wnt response
elements, but surprisingly this occurs only when
-catenin is
recruited by a specific TCF isoform that contains an alternative
C-terminal tail referred to as the E-tail.
-catenin recruitment.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
672,+314, or thymidine
kinase (TK) promoter,
64,+46) (0.5 µg)) were co-transfected with
-catenin (0.4 µg), LEF/TCFs (0.2 µg), and
-galactosidase (0.1 µg) expression constructs. The TOPFLASH luciferase reporter plasmid
(0.4 µg) was co-transfected with 0.02 µg of LEF/TCF expression
vector. Cells were harvested 18-20 h after transfection, and
-galactosidase activity was determined using the Galacton-Plus
substrate (Applied Biosystems) to normalize luciferase activity for
each point (each point was performed in duplicate). The -fold induction
was calculated relative to the luciferase reporter plasmid alone.
Results were repeated from 4 to 11 times independently.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin and
TCF-4-
-catenin complexes in a transient transfection assay (20).
Activation was dependent on
-catenin, because no activation was seen
when a mutant
-catenin that could not bind to LEF/TCFs was
co-expressed. Two putative response elements were localized by DNaseI
footprinting with recombinant LEF-1 protein at +190 and +283, and
deletion of this downstream region abrogated activation by
TCF-
-catenin. Furthermore the depletion of
-catenin protein in a
colon cancer cell line by the reintroduction of wild type adenomatous
polyposis coli lowered both the activity of the LEF1
promoter in a transient co-transfection assay and also the levels of
endogenous LEF-1 protein (20). These data suggested that
LEF1 is a target of the Wnt pathway and that the response
elements for LEF/TCF-
-catenin complexes are located in a position
~200 nucleotides downstream of the start site of transcription. These
data also suggested that LEF1 is subject to autoregulation
by LEF-1-
-catenin complexes, because all LEF/TCF family members
recognize the same DNA consensus sequence. To test for this
possibility, we co-transfected LEF-1 and
-catenin expression vectors
with the LEF1 promoter reporter plasmid (Fig.
1A). Surprisingly, we were
unable to observe activation of the promoter (1.5-fold). In contrast,
co-expression of full-length TCF-1 with
-catenin-enabled gene
activation similar to that we had observed previously with TCF-1 and
TCF-4 (20-fold; Fig. 1A) (20). TCF-3 was also unable to
activate the LEF1 promoter.
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Fig. 1.
The LEF1 promoter is
activated by specific LEF/TCF isoforms that contain a C-terminal
E-tail. A, COS cells were transiently transfected with
a LEF1 promoter luciferase reporter plasmid and expression
vectors for the indicated proteins. B, cells were
transiently transfected with the TOPFLASH reporter construct and
indicated expression vectors. C, transfections were
performed in COS cells as described in A. Whole cell
extracts from transfected COS cells were analyzed by Western blotting
with LEF-1N polyclonal antiserum (inset;
asterisk indicates expressed protein), which detects all
LEF/TCF proteins (20).
-catenin-binding domain, and thus they both
bind
-catenin and recognize the identical DNA consensus sequence.
Nevertheless, regions of LEF/TCFs exist that are not highly conserved
among the four family members. One of the most notable differences
occurs between the alternative C-terminal tails for each protein.
Previously we reported that both TCF-1 and TCF-4 were able to activate
the LEF1 promoter (20). The forms of the two proteins
expressed in those assays contained full-length E-tails (TCF-1E and
TCF-4E). In this study, full-length TCF-1E was used because it is the
most active of the two (13). The LEF1 gene does not encode
an E-tail isoform because it does not have the E-tail-specific exon
(22). Instead the most common isoform of LEF-1 that is expressed
contains an N-tail, and this isoform was used in our assays. To
determine whether the difference in activation of the LEF1
promoter was caused by the alternative C-terminal tail, we compared the
activities of TCF-1E with TCF-1B and TCF-3E in our assay (Fig.
1A). TCF-1E and TCF-1B are identical except for the
C-terminal tail, and although TCF-3E carries an E-tail, the amino acid
sequence is significantly divergent from TCF-1 and TCF-4 (see Fig.
2B). We found that, like
LEF-1N, TCF-1B was completely inactive in the assay. TCF-3E was not
very active either, because only a 3.5-fold activation of luciferase
expression was observed (Fig. 1A). The dramatic difference
in activity of these LEF/TCF isoforms was not caused by varying
expression levels or the inability to interact with
-catenin,
because each of these isoforms was equivalent in its ability to
activate the TOPFLASH reporter plasmid (Fig. 1B). The
TOPFLASH reporter plasmid contains three consensus binding sites for
LEF/TCF factors upstream of the c-fos minimal promoter and
is often used as a standard Wnt-responsive reporter plasmid (4). Each
isoform was able to recruit
-catenin for activation of TOPFLASH
~40-fold. Thus even though all LEF/TCFs are highly active with
respect to a standard Wnt responsive reporter plasmid, only two
isoforms, TCF-1E and TCF-4E, are able to activate the LEF1
promoter in a
-catenin-dependent manner (Fig.
1A and Ref. 20).
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Fig. 2.
Conserved sequences in the E-tail of
TCF-1E. A, TCF-1E is a 524-amino acid protein; the last
amino acid of each C-terminal truncation is shown. CR1mt and CR2mt are
full-length TCF-1E proteins with a 5-amino acid substitution mutation
in a CR motif (indicated by an X); the sequence of the CR
mutations is shown in B. B, E-tail homologous
sequences from the human LEF/TCF family are aligned with the TCF
orthologs. (Amino acid sequences for TCF orthologs were derived
from the following GenBankTM entries: Drosophila
melanogaster (dTCF, accession numbers AAC47464 and CAA70343); and
C. elegans (Pop-1, accession number AAC05308); and
Prf-1/PBF (accession number AAF73463), and G4EF (accession number
AAF97516)). A consensus CR1 and CR2 is shown above the alignment. The
sequence of the CR1 and CR2 mutations are shown
(CR1mt, CR2mt). The asterisk after the
PBF and G4EF proteins indicates the end of the protein.
C376),
amino acid 388 (
C388), amino acid 440 (
C440), and amino acid 457 (
C457). At the beginning of the E-tail are three tandemly conserved
regions (CRs) found in TCF orthologs and homologs (CR1, CR2, and CR3, Fig. 2B) (22). The first two of these peptide motifs are
enriched in cysteines, basic residues, and aromatic amino acids. These motifs also share similarities with nuclear localization signals. The
end points of the largest C-terminal truncations were designed to end
near these conserved motifs, with the largest deletion,
C376,
eliminating CR2 but not CR1. Each of these truncation mutants was
tested in the transient transfection assay with the LEF1
promoter reporter plasmid. Fig.
3A shows that only full-length
TCF-1E was capable of robust LEF1 promoter activation
(14-fold); all C-terminal truncations were compromised in their ability
to activate the reporter (4-fold). Expression analysis of the mutant
TCF-1E proteins showed that although most were expressed to levels
similar to wild type TCF-1E,
C376 and
C388 were either not
expressed very well or were not stable. An intact E-tail may be
important for overall structure and activity.
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Fig. 3.
Sequences in the E-tail are necessary for
LEF1 promoter activation. A, COS cells were
transiently transfected as described in Fig. 1A. Whole cell
extracts from transfected COS cells were analyzed by Western blotting
with monoclonal TCF-1 antibody (Upstate Biotech, see inset).
B, immunofluorescence detection of full-length wild type
(WT) TCF-1E and CR1mt and CR2mt proteins (transiently
transfected as in A) shows that all are localized to the
nucleus of COS cells. C, COS cells transfected with the
TOPFLASH reporter and the indicated expression plasmids show that both
E-tail mutants are able to work with -catenin to activate the
reporter.
C376 and
C388 truncations made
it difficult to conclude whether the CR1 and CR2 peptide motifs are
important for E-tail activity. Therefore, to more directly test whether
the CR motifs are necessary for activation, a more targeted set of
mutations was designed. These mutations consisted of a 5-amino acid
substitution of CR1 (CR1mt) or CR2 (CR2mt) within the context of the
full-length TCF-1E protein (Fig. 2, A and B). To
minimize general disruption of protein folding, the pentapeptide VALAL
was used in the substitution. This sequence is similar to VAHAL, which
adopts
-sheet or
-helical structures depending on the surrounding
context of the sequence (25). These full-length mutant proteins were
compared with wild type full-length TCF-1E in the transient
transfection assay, and their subcellular localization was examined by
immunofluorescence (Fig. 3, A and B). Western analysis showed that CR1mt and CR2mt were expressed as well as wild
type TCF-1E protein (inset, Fig. 3A). Amino acid
substitutions in either of the conserved CR motifs abrogated
TCF-1E activity; CR1mt expression exhibited a 1.8-fold activation of
the reporter plasmid, and CR2mt exhibited a 2.5-fold activation.
Immunofluorescence detection of CR1mt and CR2mt protein showed
that both mutants were localized to the nucleus as is true of the wild
type TCF-1E protein (Fig. 3B). Therefore, mislocalization of
the protein cannot account for the dramatic loss of activity. The
ability of CR1mt and CR2mt to activate the TOPFLASH reporter plasmid
was also assessed (Fig. 3C). Each mutant was able to elicit
between 20- and 30-fold activation of the luciferase
reporter plasmid. This is in dramatic contrast to the pattern observed
with the LEF1 promoter reporter in which CR1mt and CR2mt are
inactive (Fig. 3A). The deletions
C440 and
C457 were
also tested for their ability to activate TOPFLASH. Each deletion
mutant was able to activate TOPFLASH in a
-catenin-dependent manner, however, only to half the
level of those observed with wild type TCF-1E (data not shown). We
conclude that transcription activation of the LEF1 promoter
requires an intact E-tail, whereas a standard Wnt-responsive reporter
plasmid such as TOPFLASH is not heavily dependent on those sequences. Indeed, LEF/TCF family members that do not contain an E-tail such as
LEF-1N and TCF-1B can activate TOPFLASH as well as TCF-1E (Fig. 1B). We conclude that the CR1 and CR2 motifs are not
involved in nuclear localization or basic activities associated with
-catenin recruitment and transcription activation. Rather these
unique C-terminal peptide motifs as well as an intact E-tail appear to be required for activation of certain promoters or Wnt response elements.
64 to +317) spanning the region that contains both
previously identified LEF-1N binding sites (Fig.
4). The results show that both LEF-1N and
TCF-1E proteins generate footprints over the same sequence in the
downstream region including the +190 site (not shown). No additional
footprints exist over any other portion of the fragment. The borders of
the LEF-1N and TCF-1E footprints are the same, showing that the E-tail
region does not make extended contacts with the DNA such that an
additional region is protected from DNaseI digestion. Slight
differences exist in the DNaseI hypersensitivities surrounding the
LEF-1N and TCF-1E footprints (Fig. 4, arrows). Because
LEF/TCF proteins induce a strong bend in the DNA on binding, such small
differences in the DNase I digestion pattern may indicate differences
in bending. We conclude that LEF-1N and TCF-1E recognize the same DNA
response elements, but minor differences in the DNaseI footprint
pattern suggest that the conformation of each protein bound to these
sequences may differ.
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Fig. 4.
Comparative DNase I footprint analysis of
LEF-1N and TCF-1E. Partially purified recombinant LEF-1N extracts
(0.6, 3.0, and 6.0 µg of protein) and TCF-1E (0.1, 0.3, 0.6, 1.5, 3.0, and 4.5 µg of protein) were used in DNaseI footprint
analysis with a 32P-labeled LEF1 promoter
fragment ( 64, +317). Maxim-Gilbert reactions were used to map the
protected regions indicated by the solid bars (not shown).
Arrows denote differences in DNase I hypersensitivities
induced by the two proteins. The footprint that protects +190 is not
visible in this figure, but the protection by LEF-1N and TCF-1E is the
same. The relative position, orientation, and core sequences of the
+190 and +283 response elements are shown in the bottom section of
the figure.
46) relative to the start
site of transcription of the minimal Herpesvirus TK promoter
in a luciferase reporter plasmid (TK2FP/WT, Fig.
5, Ref. 26). This promoter differs
significantly from the LEF1 promoter in that it has a well
defined TATA box, a single start site of transcription, and is not
GC-rich. In a parallel construct, we introduced nucleotide
substitution mutations in each of the two TCF-1E footprints to destroy
binding (Fig. 5, TK2FP/MT). DNaseI footprint analysis with
LEF-1N and TCF-1E recombinant protein was used to confirm that the
LEF/TCF binding sites were completely
destroyed.3 The results from
the transient transfection analysis are shown in Fig. 5A.
Both TCF-1E-
-catenin and LEF-1E-
-catenin complexes were able to
activate transcription from the TK2FP/WT reporter, whereas LEF-1N was
inactive in the assay. Mutation of the two LEF/TCF footprints in
TK2FP/MT abrogated activation by TCF-1E-
-catenin, and control
transfections with the parent TK promoter reporter plasmid showed that
none of these factors activates the minimal promoter alone (data not
shown). Thus, E-tail-dependent activation of the
LEF1 promoter occurs via TCF-1E binding to the two
previously identified Wnt response elements. We also tested the E-tail
deletion and substitution mutations for their effect on the TK2FP/WT
reporter to determine whether the same residues of the E-tail were
involved in activation of the TATA-containing promoter. The results are identical to that of the LEF1 promoter (Fig. 5B).
C-terminal deletions of the E-tail compromise activity, and mutation of
either CR motif abrogates activity. Thus, the same residues are
involved in activation of the LEF1 and TK promoters through
the Wnt response elements identified with DNase I footprinting.
Activation is the same whether from a downstream or an upstream
location.
View larger version (26K):
[in a new window]
Fig. 5.
E-tail-dependent activation is
independent of position of the response element or promoter type.
A, a DNA fragment encompassing nucleotides +104 to +308 of
the LEF-1 promoter was subcloned at position 46 of the
herpesvirus TK promoter. Site-directed mutations in both of the core
Wnt response elements were introduced (TK2FP/WT and TK2FP/MT reporter
constructs are TK promoter with wild type and mutant TCF-1E response
elements from the LEF1 promoter, respectively). These
reporter constructs and the indicated expression vectors were
transiently transfected into COS cells for analysis. Selective
activation by TCF-1E is transferred, and mutation of the core binding
sites destroys E-tail-dependent regulation. B,
description is the same as for A except that TCF-1E mutant
expression plasmids were co-transfected with TK2FP/WT only. The
activities of each mutant are the same as those observed with the
LEF1 promoter reporter plasmid (Fig. 3A).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin to activate transcription of the
LEF1 promoter in transient transfection assays. The E-tail
isoform of TCF-4 is also able to activate the LEF1 promoter
(shown previously in Ref. 20). Other isoforms of TCF-1 as well as
LEF-1N and the disparate E-tail isoform of TCF-3 do not function with
-catenin at this promoter. This is true despite the fact that all
LEF/TCF isoforms are equally active with the standard TOPFLASH reporter plasmid. These data suggest that expression of the LEF1
locus may be particularly sensitive to regulation by
-catenin
complexes containing TCF-E-tail isoforms. Such forms are detected in
colon cancer cell lines that aberrantly express LEF1 (23,
27).
-catenin to activate this reporter.4 Thus
if the E-tail modifies DNA sequence affinity, such differences may only
be revealed with reporters containing one or two separated response
elements and not the closely apposed multiple elements that are found
in the TOPFLASH reporter. Current efforts are under way to more
carefully analyze DNA binding affinity, DNA bending, and protein-DNA
conformations of the LEF1 promoter response sites by LEF-1N
and TCF-1E. A fourth possible mechanism involves the engagement of
E-tail motifs in protein interactions as opposed to direct DNA binding.
The absolute conservation of the cysteine residues in each motif among
TCF orthologs suggests a cysteine-based tertiary structure but not one
that matches any known metal-chelating domain (Fig. 2B).
Co-activators of transcription are obvious candidates if protein
interaction is involved, but one direct possibility could be that the
E-tail enables a better and more stable interaction with
-catenin.
This possibility is unlikely, given that CR1mt and CR2mt are as active
as wild type TCF-1E for
-catenin-dependent activation of
TOPFLASH and that LEF/TCFs without an E-tail efficiently co-immunoprecipitate with
-catenin in COS-7 cells (28).
However, multimerized response elements may mask differences between
family members in their ability to recruit
-catenin to target
promoters, and only careful assessment of
-catenin-binding
affinities to DNA-bound LEF/TCFs can resolve this issue. If
co-regulators are recruited through interactions with the E-tail
domain, then such regulators are most likely to be ubiquitously
expressed, because we have observed E-tail-specific activation of the
LEF1 promoter in different cell types including mature and
immature human T lymphocyte lines (Jurkat and 2017) and COS-1 cells, a
monkey kidney cell line.5
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ACKNOWLEDGEMENTS |
---|
We acknowledge the generosity of Dr. Hans Clevers and Dr. Elaine Fuchs for sharing TCF cDNAs. We thank Dr. Klemens Hertel and members of the Waterman laboratory for ideas and critiques of the project and the manuscript.
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Addendum |
---|
While this manuscript was under review, similar observations for TCF-4E regulation of the Cdx1 promoter and the importance of CR1 and CR2 motifs (referred to as CRARF) were published by Drs. Hecht and Stemmler (37).
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant CA83982.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.
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Material.
§ Supported by NCI, National Institutes of Health Training Grant CA09054 from the University of California Irvine Cancer Research Institute.
To whom correspondence should be addressed. Tel.:
949-824-2885; Fax: 949-824-8598; E-mail: mlwaterm@uci.edu.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M213218200
1 The following websites provide updated referenced reviews of Wnt signaling: http://www.stanford.edu/~rnusse/wntwindow.html and http://stke.sciencemag.org.
3 T. Li and F. Atcha, data not shown.
4 F. A. Atcha, data not shown.
5 F. A. Atcha, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: LEF/TCF, lymphoid enhancer factor/T cell factor; TK, thymidine kinase; GLUT, glucose transporter; G4EF, GLUT4 enhancer factor; Prf, papillomavirus regulatory factor; PBF, papillomavirus binding factor; CR, conserved region.
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