Department of Genetics, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania
19104-6145
Received 24 May 2001/Returned for modification 13 July
2001/Accepted 8 August 2001
Notch signaling commences with two ligand-mediated proteolysis
events that release the Notch intracellular domain, NICD, from the
plasma membrane. NICD then translocates into the nucleus and interacts
with the DNA binding protein CSL to activate transcription. We found
that NICD expression also potentiates activity of the transcription
factor LEF-1. NICD stimulation of LEF-1 activity was context dependent
and occurred on a subset of promoters distinct from those activated by
-catenin. Importantly, the effect of NICD does not appear to be
mediated through canonical components of the Wnt signaling pathway or
downstream components of the Notch pathway. In vitro assays show a weak
association between the C-terminal transactivation domain of NICD and
the high-mobility group domain of LEF-1, suggesting that the two
proteins interact in vivo. Our data therefore describe a new nuclear
target of Notch signaling and a new coactivator for LEF-1.
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INTRODUCTION |
Notch signaling involves a series of
precisely regulated events. Notch resides at the plasma membrane as a
heterodimer due to proteolysis by a furin-like convertase at a site
designated S1 (25). In response to ligand, Notch is
cleaved at two additional sites, S2 and S3, by TACE and a
-secretase-like activity, respectively (6; for a
review, see references 20 and 27).
Cleavage at S3 releases the Notch intracellular domain (NICD) from the
membrane. NICD has two nuclear localization signals that target it to
the nucleus where it interacts with the DNA binding factor CSL (CBF-1, Suppressor of Hairless, LAG-1; also known as RBP-J) (1, 18, 37,
38). In the absence of NICD, CSL acts as a transcriptional repressor. CSL can mediate repression in vitro by interacting with
TFIID and TFIIA (29) and in vivo by interacting with
corepressors and histone deacetylases (15, 19, 45). NICD
and the corepressors bind to the same region of CSL; thus, entry of
NICD into the nucleus leads to the displacement of the CSL-associated
corepressors (15, 19, 45). Binding of NICD to CSL is
mediated by the Notch RAM and Ankyrin domains (14), and
transcriptional activation occurs as a consequence of the NICD
transcription activation domain recruiting coactivators, such as
PCAF and GCN-5 (21, 22).
The LEF-1 transcription factor was originally identified as a
T-cell-specific factor that regulates the T-cell receptor 
enhancer
(40). While LEF-1 was once thought to play an
architectural role in transcriptional activation, it is now clear that
LEF-1 can act as a conventional transcription factor (10,
13). LEF-1 acts in conjunction with several other DNA binding
proteins to activate the TCR- enhancer (12) using a
context-dependent activation domain (8, 11). LEF-1 can
also interact with the coactivators -catenin and ALY to induce gene
expression (5, 7). The activities of -catenin and ALY
toward LEF-1 are highly context dependent and have not been found to
overlap (16). While -catenin can activate certain
promoters containing multiple LEF-1 binding sites, ALY cannot.
Conversely, ALY is able to stimulate activity of the TCR- enhancer,
while -catenin has no effect.
Results presented here identify NICD as a coactivator for the LEF-1
transcription factor. The effects of Notch on LEF-1 activity are direct
and not due to modulation of components of the Wnt signaling cascade or
due to effects of Notch-mediated activation of CSL. Potentiation of
LEF-1 activity by NICD defines new roles for both Notch and LEF-1 in
the regulation of gene expression.
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MATERIALS AND METHODS |
Plasmids and transfections.
The Notch expression plasmids
NICD, NICD
R, NICDRA, and NICDTAD were generated by PCR from a
full-length human Notch 1 cDNA. Amino acid positions included in
each Notch fragment are 1760 to 2556 for NICD, 1859 to 2556 for
NICDR, 2094 to 2556 for NICDRA, and 1760 to 2094 for NICDTAD.
Each PCR product was cloned into pcDNA3.1(
) Myc-HisC (Invitrogen), in
frame with the Myc-His tags, and sequenced to ensure correct cloning
and sequence. NICD (or NICD fragments) was subcloned using traditional
methods to create GAL4-NICDRA, GST-NICDTAD, GST-NICDRA,
MIGR-NICD, and NICD-ER (details available upon request). The parental
MIGR retroviral construct was provided by W. Pear (University of
Pennsylvania), and the parental estrogen receptor (ER) fusion
retroviral construct was given by M. McMahon (University of California,
San Francisco). The LEF-1, 56LEF, -catenin, and GST-ALY
expression vectors, as well as the 7xLEF-luc and fos-luc reporter
plasmids, were the generous gifts of R. Grosschedl (University of
Munich). The LEF-OT and LEF-OF reporter constructs were gifts of B. Vogelstein (Johns Hopkins University). The Notch3 NICD, TCF-1, and
CSL-VP16 (previously known as pCMX-VP16-RBP-J) expression vectors were
gifts from U. Lendahl (Karolinska Institute), H. Clevers (University
Hospital Utrecht Medical School), and T. Honjo (Kyoto University),
respectively. Reporter constructs containing promoters from the Xtwn,
Cyclin D1, and WISP-1 genes were gifts of L. Attisano (University of Toronto), A. Rustgi (University of Pennsylvania), and A. Levine (Rockefeller University), respectively.
All cell lines were maintained in Dulbecco's minimal Eagle's medium
(Gibco-BRL) supplemented with 10% fetal bovine serum, Pen/Strep, and
glutamine. To generate the MIGR, MIGR-NICD, ER, and NICD-ER transduced
cells, NIH 3T3 cells were infected with ecotropic retrovirus and
selected with 2 µg of puromycin per ml (for ER and NICD-ER) or by
green fluorescent protein (GFP)-positive fluorescence-activated cell
sorting (for MIGR and MIGR-NICD). Transfections of NIH 3T3 and Neuro-2A
were carried out using CaPO4 DNA coprecipitation
(Clontech) or Fugene (Boehringer-Mannheim), as per manufacturers'
instructions. Jurkat cells were transfected by electroporation with the
Gene Pulser II electroporator (Bio-Rad). Jurkat cells were transfected
during logarithmic growth phase, using 2 × 106 cells, in 4 mM Gap cuvettes with settings of
0.250 kV and 975 µF. Transfections typically contained 100 ng of
reporter and 1 ng of pRL-CMV (Promega) to assess relative transfection
efficiencies. Unless otherwise noted, transfections also contained 500 ng of expression vector(s). All cells were harvested 42 to 48 h
after transfection. Firefly and Renilla luciferases were
assayed following the instructions provided with the Dual Luciferase
Assay kit (Promega). All transfections are shown as the means ± standard errors of the means of at least three separate transfections.
GST interaction assays.
Glutathione-S-transferase
(GST), GST-NICDTAD, GST-NICDRA, and GST-ALY were expressed in the
BL21 strain of Escherichia coli (Stratagene). GST proteins
were induced with 0.2 mM
isopropyl--D-thiogalactopyranoside (IPTG)
(Promega), and the bacteria were allowed to grow an additional 4 to
5 h. Following induction, cells were lysed by freeze-thawing in
phosphate-buffered saline and protease inhibitors. GST proteins were
bound to glutathione resin (Pharmacia) and washed five times with
phosphate-buffered saline-0.2% NP-40 (Sigma).
The LEF-1 deletion fragment proteins were generated in a manner similar
to that described in reference 4. Briefly, two rounds of
PCR amplification were used: the first, to generate the deletion
fragments with a common 5' end containing a consensus Kozak sequence
and a 3' stop codon; the second, to generate deletion fragments
containing a 5' T7 promoter. After the first amplification reaction the
PCR products were gel purified, and after the second amplification
reaction the samples were purified using the QIAquick PCR purification
kit (Qiagen). The PCR products purified from the second round of PCR
were then in vitro transcribed and translated with the TNT T7 coupled
reticulocyte lysate system (Promega) in the presence of
[S35]methionine.
GST interaction assays were performed with whole-cell extracts from
transfected 293T cells or in vitro-synthesized proteins that were
prebound with glutathione resin. GST fusion proteins were equilibrated
in bead binding buffer (25 mM HEPES [pH 7.5], 150 mM KCl, 5 mM
MgCl2, 1 mM dithiothreitol, 0.1% NP-40, 1%
glycerol) and then incubated with the whole-cell extracts or in
vitro-synthesized proteins at 4°C for 1 h. After incubation,
glutathione bead-GST fusion protein complexes were collected by
centrifugation and washed five times with bead binding buffer. The
washed beads were then resuspended in sodium dodecyl sulfate loading
buffer and boiled, and Western blotting was performed.
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RESULTS AND DISCUSSION |
NICD potentiates LEF-1 activity.
During experiments designed
to investigate interactions between components of the Notch and Wnt
signaling pathways, we noted the ability of Notch1 NICD to augment the
activity of LEF-1 on certain promoters. The reporter 7xLEF-luc (Fig.
1A) harbors a promoter consisting of
seven LEF-1 binding sites upstream of a minimal Fos promoter and is
responsive to -catenin acting through LEF-1 (16). When
assayed in Neuro-2A cells, -catenin alone did not activate 7xLEF-luc
and LEF-1 activated the reporter only weakly. However, LEF-1 plus
-catenin had a marked effect, stimulating the reporter greater than
ninefold (Fig. 1B, left panel). NICD had very little effect on the
reporter either in the presence or in the absence of LEF-1. Very
different results were obtained with a second LEF-1 responsive
reporter, LEF-OT (Fig. 1B, right panel), whose promoter carries three
LEF-1 binding sites upstream of the E1b TATA box. First, activity of
LEF-OT was stimulated approximately 15-fold by LEF-1 alone and the
addition of -catenin gave rise to only a modest further increase in
LEF-1 activity (less than twofold). Second, the addition of NICD
stimulated reporter activity approximately 5-fold over that seen with
LEF-1 alone (Fig. 1B) and up to 10-fold in other cell lines (data not
shown). NICD had no effect if LEF-1 was not included in the
transfection or if the reporter harbored mutant LEF-1 binding sites
(LEF-OF). We conclude that NICD stimulation of LEF-OT is mediated
through LEF-1.
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FIG. 1.
Notch potentiates LEF-1 activity. (A) Schematic
representations of reporters used to assay LEF-1 activity. Binding
sites for LEF-1 are shown as gray (consensus) or black (mutant) ovals.
(B) Effects of LEF-1, -catenin, and NICD on 7xLEF-luc and LEF-OT in
Neuro-2A cells. Relative luciferase values for reporters containing
seven LEF-1 binding sites (7xLEF-luc; gray bars) or no LEF-1 binding
sites (fos-luc; black bars) are shown in the left panel. Values for
reporters having three consensus LEF-1 binding sites (LEF-OT; gray
bars) or three mutant LEF-1 binding sites (LEF-OF; black bars) are
shown in the right panel. Values for fold induction were determined
relative to those obtained for each reporter in the absence of any
expression plasmids. (C) Effects of LEF-1 and NICD on naturally
occurring promoters. Transfections of Neuro-2A cells were carried out
with reporters containing promoters from the WISP-1 (white bars),
Cyclin D1 (striped bars), or Xtwn (black bars) genes. Expression
plasmids that were cotransfected with each set of reporters are
indicated. Values are given as fold induction relative to the reporter
alone.
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These findings are reminiscent of reports demonstrating promoter
specificity for the LEF coactivators -catenin and ALY (7, 16). We attempted to determine the nature of the NICD-LEF
promoter specificity by generating hybrids of LEF-OF and 7xLEF-luc. In one instance we fused the LEF sites of LEF-OF to the core promoter in
7xLEF-luc (c-Fos), and in another, we fused the LEF sites of 7xLEF-luc
to the core promoter of LEF-OF (a TATA box). Surprisingly, both hybrid
reporters were activated by NICD in the presence of LEF-1 (data not
shown). Hence, the NICD-LEF promoter specificity cannot be easily explained.
We also examined the responses of three naturally occurring promoters
that carry LEF-1 binding sites (Fig. 1C). The WISP-1 promoter is an
example of a promoter with LEF-1 binding sites that are not necessary
for stimulation by -catenin or Wnt signaling (43). Both
LEF-1 alone and NICD alone activated the WISP-1 promoter to some
degree, and LEF-1 plus Notch exerted a small additive effect. The LEF-1
sites in the Xenopus Twin (Xtwn) promoter are responsive to -catenin and Wnt signaling, but are also active independently of -catenin (23, 28). LEF-1 alone and
NICD alone induced the Xtwn promoter fourfold and twofold,
respectively. However, LEF-1 plus NICD gave a strong synergistic
activation of the Xtwn promoter (43-fold). The Cyclin D1 promoter has
also been identified as having LEF-1 binding sites; but unlike the WISP-1 promoter, these sites are highly responsive to Wnt
signaling through LEF-1 and -catenin (35, 39). As with
the WISP-1 promoter, NICD did not potentiate LEF-1 activity on the
Cyclin D1 promoter. We conclude that naturally occurring promoters,
like the artificial promoters, fall into two groups: those that are
stimulated by NICD through LEF-1 and those that are not.
Activation is limited to subsets of each protein family.
In
vertebrates, multiple Notch genes exist; therefore, we sought to
identify whether the potentiation of LEF-1 is specific to Notch1.
Notch1 NICD stimulated LEF-1 activity approximately fivefold, while
a full-length Notch1 receptor had no effect on LEF-1 activity (Fig.
2A). Notch2 and Notch3 were also tested
for the ability to activate LEF-1. Notch2 NICD potentiated LEF-1
activity, although not as strongly as Notch1 NICD; however, Notch3
NICD was unable to stimulate LEF-1. The ability of Notch1 and Notch2, but not Notch3, to enhance LEF-1 activity is consistent with the idea
that both Notch1 and Notch2 are transcriptional activators, while
Notch3 is not (3, 22).
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FIG. 2.
Specific Notch proteins activate a subset of HMG domain
transcription factors. (A) Ability of Notch 1, 2, and 3 to potentiate
LEF-1. LEF-1 activity was assayed in NIH 3T3 cells using the LEF-OT
reporter in the presence of Notch1 (N1) NICD, full-length Notch1 (FL
N1), Notch2 (N2) NICD, or Notch3 (N3) NICD. Values are given as fold
induction relative to LEF-OT alone. (B) Effects of NICD on other HMG
box transcription factors. Neuro-2A cells were transfected with LEF-OT
and expression vectors for LEF-1, TCF-1, HAF-2, or HAF-1 in the
presence (striped bars) or absence (black bars) of NICD. Values are
give as fold induction as for panel A.
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LEF-1 is a member of the high-mobility group (HMG) box family of DNA
binding proteins. To determine if NICD potentiates the activity of
other members of this family, we tested the response of additional HMG
box transcription factors, including TCF-1, HAF-1, and HAF-2 (the last
two are also referred to as Sox 17 and Sox 18, respectively
[36]). Like LEF-1, TCF-1 was also stimulated by NICD,
although not as strongly (Fig. 2B). Reasons for the variance in
potentiation by NICD are not clear, as Western analysis shows no
apparent difference in protein levels (data not shown). By contrast,
HAF-1 and HAF-2 stimulated the activity of LEF-OT, but NICD had no
additional effect. The effect of NICD is therefore restricted to a
subset of HMG box proteins.
The effect of NICD on LEF-1 does not involve other components of
the canonical Wnt or Notch signaling pathways.
We considered the
possibility that NICD may stimulate LEF-1 by modulating the components
of the Wnt signaling pathway that lead to an increase in nuclear
-catenin. We felt that this was unlikely since NICD and -catenin
were most effective on distinct reporters (Fig 1A). However, to test
directly if Notch potentiates LEF-1 through -catenin, NICD was
assayed in the presence of a LEF-1 deletion mutant, 56LEF, that
lacks the -catenin interaction domain. As expected, -catenin was
unable to stimulate 7xLEF-luc in the presence of 56LEF (Fig.
3, left panel). By contrast, 56LEF induced LEF-OT eightfold and NICD resulted in a further twofold stimulation (Fig. 3, right panel). While Western blot analysis comparing wild-type LEF-1 and 56LEF expressions indicated that 56LEF was present at lower levels, increasing the amount of
transfected 56LEF was unable to match the degree of potentiation
observed with wild-type LEF-1 and NICD (data not shown). Although this overall level of stimulation was below that obtained with wild-type LEF-1, we conclude that NICD does not augment LEF-1 activity through -catenin.
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FIG. 3.
NICD activation of LEF-1 is independent of -catenin.
NICD augments activity of 56LEF. Neuro-2A cells were transfected
with 7xLEF-luc and expression plasmids for 56LEF (LEF-1 lacking the
-catenin interaction domain) and -catenin as indicated (left
panel, black bars). Cells were also transfected with LEF-OT and
expression plasmids for 56LEF and NICD as indicated (right panel,
gray bars). Results are given as fold induction relative to the
reporters alone.
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The effect of NICD is also not mediated by prototypical Notch target
genes. CSL-VP16 is a fusion protein that activates Notch target genes
in the absence of Notch signaling. Although CSL-VP16 was able to
activate a CSL-dependent reporter, it had no effect on LEF-1 (data not
shown). Additionally, potentiation of LEF-1 was not observed with a
hybrid protein that carries the VP16 activation domain in place of the
NICD activation domain (Fig. 4B). The
latter result suggests that the effect of NICD specifically requires the Notch activation domain. NICD comprises three functional domains: the RAM domain (R), which mediates interaction with CSL; the Ankyrin repeats (A), which bind a number of proteins including CSL; and the
C-terminal transcriptional activation domain (TAD). We generated a
series of proteins that contain one or more of these domains and
assessed their abilities to activate LEF-1 on the LEF-OT reporter (Fig.
4C). Both NICD and NICDR (lacking the RAM domain) potentiated LEF-1.
The C terminus of NICD that encompasses the TAD but lacks both
CSL-interaction domains (NICDRA) also activated LEF-1, while a
fragment that contains the RAM and ankyrin domains (NICDTAD) did
not. NICDRA and LEF-1 also gave synergistic activation of the Xtwn
promoter, while NICDTAD-VP16 did not (data not shown). Although
induction of LEF-1 by NICDRA was slightly less than that observed
for NICD, this is likely due to lower protein levels (data not shown).
These data show that the Notch TAD is necessary and sufficient for the
observed effects on LEF-1 and argue further that the effects are not
mediated indirectly through the induction of CSL-responsive genes.
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FIG. 4.
NICD activates LEF-1 independently of CSL. (A) Schematic
diagrams of the NICD deletion fragments are shown. R, RAM domain; A,
Ankyrin repeats; TAD, C-terminal transcription activation domain; VP16
A.D., VP16 transcription activation domain. (B) NICDTAD-VP16 does
not augment activity of LEF-1. Neuro-2A cells were transfected with a
CSL-dependent reporter, CSL-luc (black bars), or LEF-OT (gray bars) and
the expression vectors as indicated. NICDTAD-VP16 carries the VP16
TAD in place of the Notch TAD. Results are given as fold induction
relative to the reporters alone. (C) The Notch TAD is sufficient for
LEF-1 activation. NIH 3T3 cells were transfected with LEF-OT (gray
bars) or LEF-OF (black bars) and the NICD fragments indicated. Results
are presented as fold induction relative to the reporter alone.
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NICD and LEF-1 interact physically.
Next, we carried out
experiments to investigate whether NICD and LEF-1 interact physically.
First we used a modified mammalian two-hybrid assay to assess in vivo
interactions. Specifically, a Gal4-NICDRA fusion protein was tested
for transcriptional activity in the absence and presence of LEF-1 (Fig.
5A). Gal4-NICDRA alone was able to
stimulate the Gal4 responsive reporter, confirming the presence of an
activation domain within the C terminus of NICD (22).
While LEF-1 had little effect on the Gal4 DNA binding domain alone or a
Gal4-VP16 fusion, it enhanced the activity of Gal4-NICDRA
approximately threefold to fourfold (Fig. 5A and data not shown). As
anticipated by earlier results, Gal4-NICDRA was also activated by
56LEF (data not shown). The increase in transcriptional response is
most likely due to the consequences of physically linking two
activation domains, one provided by NICD and the other by LEF-1. This
would occur when NICD interacts with DNA-bound LEF-1 (Fig. 1) or when
LEF-1 interacts with DNA-bound NICD (Fig. 5A).
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FIG. 5.
The Notch TAD physically interacts with the LEF-1 HMG
domain. (A) LEF-1 activates Gal4-NICDRA. Neuro-2A cells were
transfected with a Gal4 responsive reporter and either the DNA binding
domain of Gal4 (Gal4) or a Gal4 fused to the TAD of Notch
(Gal4-NICDRA) in the presence (gray bars) or absence (black bars) of
LEF-1 as indicated. Results are shown as fold induction relative to the
reporter plus the Gal4 DNA binding domain. (B) LEF-1 interacts with the
Notch TAD in vitro. 293T cells were transfected with plasmids
expressing HA-LEF-1 and FLAG-CSL, extracts were incubated with GST,
GST-NICDTAD, or GST-NICDRA, and bound proteins were analyzed by
Western analysis using anti-HA antibodies (top panel) or anti-FLAG
antibodies (lower panel). Samples of untreated cell extracts were
loaded in each of the far left lanes, corresponding to 1/350 of the
input analyzed for HA-LEF-1 (top) and 1/5 of the input analyzed for
FLAG-CSL (bottom). (C) LEF interacts with the Notch TAD and ALY with
comparable affinities. 293T cells were transfected with HA-LEF,
extracts were incubated with GST, GST-NICDRA or GST-ALY as
indicated, and bound proteins were analyzed by Western analysis with an
anti-HA antibody. Lane 1 contains untreated extract corresponding to
1/300 of the input. (D) Schematic diagram of the LEF-1 fragments used
to map the interaction with NICD. Amino acid positions and several
functionally defined domains are indicated. CID, -catenin
interaction domain; CAD, context-dependent activation domain; HMG, HMG
domain. (E) The LEF-1 HMG box mediates interactions with the Notch TAD.
The indicated radiolabeled LEF-1 fragments were generated by in vitro
transcription and translation (lanes 1, 4, 7, 10, and 13) and analyzed
for binding to GST (lanes 2, 5, 8, 11, and 14) and GST-NICDRA (lanes
3, 6. 9, 12, and 15). Untreated samples represent 1/100 of the input
used for each binding analysis. Positions of molecular mass standards
(in kilodaltons) are shown at the left.
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To measure interactions in vitro, GST-NICDRA (containing the Notch
TAD) and GST-NICDTAD (lacking the TAD) fusion proteins were
generated and tested for their abilities to interact with LEF-1.
Initially, whole-cell extracts were used to investigate whether NICD
and LEF-1 could interact in the context of a large array of cellular
proteins. Cells transfected with hemagglutinin (HA)-tagged LEF-1
or FLAG-tagged CSL (as a positive control) were used in these assays
(Fig. 5B). LEF-1 was retained by GST-NICDRA, but not by GST alone or
GST-NICDTAD (Fig. 5B, upper panel). As expected, CSL was retained by
GST-NICDTAD, but not by GST or GST-NICDRA (Fig. 5B, lower panel).
Given the strong potentiation of LEF-1 by NICD, it was somewhat
surprising that the NICD-LEF-1 interaction was so weak relative to that
of NICD and CSL (see amounts retained versus input). Addition of
ethidium bromide did not affect interactions between GST-NICDRA and
LEF-1, indicating that nucleic acid is not mediating their association
(data not shown). We therefore compared the interaction of LEF-1 with
NICD to that of LEF-1 with ALY, a coactivator known to functionally interact with LEF-1 (7, 16). GST fusions of NICDRA or
ALY were tested for their abilities to interact with LEF-1 from
transfected whole-cell extracts (Fig. 5C). GST-NICDRA and GST-ALY
retained similar amounts of LEF-1, suggesting comparable binding
affinities. We could not compare these interactions with those
involving -catenin since we were unable to identify
-catenin-LEF-1 or NICD-LEF-1 complexes by immunoprecipitation (data
not shown). Stable interactions between NICD and LEF-1 were also not
seen using mobility shift assays of transfected-cell extracts or
recombinant proteins (data not shown). While robust with respect to
NICD's effect on LEF-1 activity in vivo, the interaction between NICD
and LEF-1 is physically weak in vitro.
The region of LEF-1 that interacts with NICD was determined using
radiolabeled in vitro-synthesized LEF-1 deletions (Fig. 5D). All
deletions from the N terminus of LEF-1 retained the ability to interact
with GST-NICDRA (Fig. 5E). While the HMG domain of LEF-1 was clearly
sufficient to mediate an interaction with the Notch TAD, the observed
interaction was weaker than that seen with the other deletion
fragments. The diminished interaction between NICD and the HMG domain
alone might suggest that sequences immediately N terminal to the DNA
binding domain are involved in NICD-LEF interactions. This could
explain the lower potentiation ability of NICD for TCF-1 (Fig. 2B),
which differs from LEF-1 outside the HMG domain. Removal of the LEF-1
HMG domain eliminated the interaction with NICDRA. Thus,
interactions are localized to the Notch TAD domain and the LEF-1 HMG
domain. Since crude extracts were used to demonstrate NICD-LEF-1
interactions and these are physically weak, we cannot rule out the
possibility that the interaction is indirect and mediated by an unknown
bridging protein.
Stimulation of LEF-1 requires high levels of NICD.
The
apparent low in vitro affinity of NICD for LEF-1 prompted us to assess
the relative in vivo responses of LEF-dependent and CSL-dependent
promoters (Fig. 6A). The activity of NICD
towards the CSL-dependent reporter was linear and apparent at low input concentrations (0.1 µg) of the NICD expression vector. By contrast, activity of the LEF-dependent reporter (LEF-OT) required higher amounts
of NICD (0.25 µg) before the response was seen and became linear. We
also carried out an experiment in which we transfected Jurkat cells
with full-length Notch1 and mimicked ligand-mediated activation by
treating cells with EDTA (32). Under these conditions, the
CSL-dependent reporter was activated roughly sixfold, while the
LEF-dependent reporter was unaffected (data not shown). One possible
explanation for these results is that low cellular concentrations of
NICD bind exclusively to CSL and binding to LEF occurs only when NICD
concentrations functionally exceed those of CSL. Consistent with this,
the ability of NICD to stimulate the LEF-responsive reporter was
completely inhibited in the presence of overexpressed CSL (Fig. 6B).
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FIG. 6.
High-level expression of NICD is required for LEF-1
activation. (A) Low levels of NICD stimulate a CSL reporter but do not
potentiate LEF-1. Fold induction of CSL-luc, with increasing amounts of
NICD expression plasmid, is shown relative to the reporter alone (left
graph). Stimulation of LEF-1 in the presence of increasing amounts of
NICD compared to the LEF-OT reporter and LEF-1 alone is shown (right
graph). (B) Excess CSL inhibits NICD's ability to stimulate LEF-1
activity. Neuro-2A cells were transfected with the LEF-OT reporter and
the indicated expression vectors. Results are given as fold induction
relative to the reporter alone. (C) Activity of NICD in the presence of
endogenous LEF-1. Jurkat cells were transfected with reporters
containing wild-type (LEF-OT) or mutant (LEF-OF) LEF-1 binding sites,
plus or minus an expression vector for NICD as indicated. Data are
represented as fold induction relative to LEF-OF alone.
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To examine the effect of NICD on endogenous LEF-1, we used Jurkat
cells, a T-cell line that normally expresses LEF-1. In the absence of
NICD the transfected LEF-OT reporter was approximately 20-fold more
active than the promoter containing mutant LEF-1 sites (LEF-OF) (Fig.
6C). This is presumably due to endogenous LEF-1/TCF-1 activity. Upon
transfection of an NICD expression plasmid, activity was increased to
approximately 50-fold over that of LEF-OF. Similarly to what is
depicted in Fig. 1A, the 7xLEF-luc reporter had no activity in
Jurkat cells in the absence of -catenin and was not induced
with NICD (data not shown).
Retroviral expression of NICD has been used in a mouse model for
Notch-induced leukemogenesis in humans (30, 31). The relatively high level of Notch expression obtained with retroviruses is
necessary for the development of T-cell tumors and has been proposed to
mimic the level obtained in human T-ALL carrying the t(7;9)
translocation (17). We generated NIH 3T3 cells that harbor either a retrovirus that carries NICD linked to an IRES-GFP (MIGR-NICD) or a retrovirus that contains only the IRES-GFP (MIGR). When the two
cell populations were transfected with LEF-1 and either LEF-OT or
LEF-OF (to establish a baseline), we observed a 30% increase in
activity in the cells stably expressing NICD (Fig.
7A). These data argue that there is a
significant enhancement of LEF-1 activity in cells harboring
NICD-expressing retroviruses. To better establish that the observed
effect is direct, we generated cells that contain a retrovirus that
expresses an NICD-ER fusion protein. In control experiments we showed
that activity of a CSL-dependent reporter in those cells was stimulated
roughly 10-fold by tamoxifen (data not shown). Consistent with the
previous result using NICD-expressing retroviruses, LEF-OT activity was
also increased approximately 30% by tamoxifen (Fig. 7B). Induction
required the LEF binding sites in the promoter, did not occur in the
absence of the fusion protein (Fig. 7B), and occurred within 12 h
(data not shown). These data argue that the effect of tamoxifen is due
to the activation of NICD and its binding to LEF-1.
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FIG. 7.
Retrovirally expressed NICD stimulates LEF-1 activity.
(A) NIH 3T3 cells transduced with either the MIGR (black bars) or
MIGR-NICD (gray bars) retroviruses and then transfected with LEF-OF or
LEF-OT reporters and a LEF-1 expression vector. Results for each
transduced cell population are shown as fold induction relative to the
LEF-OF plus LEF-1. (B) Tamoxifen-responsive NICD stimulates LEF-1
activity. NIH 3T3 cells were transduced with retroviruses expressing
either the parental ER element (ER cells) or NICD fused to the estrogen
receptor element (NICD-ER cells). LEF-1 activity was assayed in the
absence (black bars) or presence (gray bars) of 25 nM tamoxifen,
following the transfection of ER and NICD-ER cells with the LEF-OF or
LEF-OT reporters and a LEF-1 expression vector. Results are shown as
fold induction relative to LEF-OF and LEF-1 with no tamoxifen.
|
|
Our results describe a new activity for NICD and a new coactivator for
LEF-1. To date, the only nuclear target for NICD has been the DNA
binding protein CSL and the only coactivators for LEF-1 have been
-catenin and ALY. Our data therefore expand the repertoire of genes
that may be influenced by Notch signaling and increase the known
variety of ways in which genes can be activated through LEF-1. Numerous
reports have described both negative and positive genetic interactions
between the Notch and Wingless pathways in Drosophila
melanogaster (reviewed in reference 26). It
has been proposed, for example, that Wingless signaling can inhibit Notch through direct binding of Disheveled to the Notch C terminus (2). (We have been unable to demonstrate any effects of
mouse Disheveled on NICD activity towards a CSL-dependent promoter
[data not shown].) Wingless itself has also been reported to be a
Notch ligand, thereby serving as a direct stimulator of Notch signaling (reviewed in references 26, 41, and 42). Our
results with the various reporters imply that the promoters activated
by NICD do not necessarily overlap with those activated by -catenin; thus, our data do not explain how the two pathways may or may not
interact. Although the Xtwn promoter can be activated by -catenin (23, 28) and by NICD (Fig. 1), Xtwn gene activation during embryonic development occurs prior to the induction of Notch signaling. Thus, it remains to be determined if there are genes whose activity is
influenced directly by both pathways. Interestingly, and perhaps directly relevant to our results, it has been shown that Notch can
modulate the activity of the Drosophila
UbxVMB enhancer through dTCF (24).
Modulation of dTCF activity was shown to be independent of the
components of the canonical Wingless pathway and of Su(H). Although
this report showed that Notch can inhibit dTCF activity, deletion of
the Notch RAM and Ankyrin domains resulted in stimulation of the
UbxVMB enhancer and expression in regions where
it was previously undetected. The latter set of results may reflect the
type of activity we have described here.
Our experiments show that LEF-1 is likely to be activated only in those
cells where NICD levels are high (i.e., with transfection experiments
or transduced cell lines). Low levels of nuclear Notch are sufficient
to activate CSL-dependent reporters (33) and may support
the majority of Notch's signaling tasks during embryonic development.
However, high levels of NICD are found in the nuclei of various cell
types, including cortical neurons (34) and certain cancers
(9, 44), and these have a higher likelihood of supporting the signaling pathway described here.
This work was supported by a grant from the National Institutes of
Health to T.K. (RO1 GM58228); D.R. was supported by a National Institutes of Health National Cancer Institute training grant (T32 CA09140).
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Abstract
Introduction
