Sonic Hedgehog-induced Activation of the Gli1
Promoter Is Mediated by GLI3*
Ping
Dai
,
Hiroshi
Akimaru
§,
Yasunori
Tanaka
,
Toshio
Maekawa
§,
Masato
Nakafuku¶, and
Shunsuke
Ishii
§
From the
Laboratory of Molecular Genetics, Tsukuba
Life Science Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki
305-0074, Japan, the ¶ Division of Neurobiology, Department of
Neuroscience, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and § CREST,
Japan Science and Technology
 |
ABSTRACT |
Drosophila transcription factor
cubitus interruptus (Ci) and its co-activator CRE (cAMP
response element)-binding protein (CBP) activate a group of target
genes on the anterior-posterior border in response to hedgehog protein
(Hh) signaling. In the anterior region, in contrast, the
carboxyl-truncated form of Ci generated by protein processing represses
Hh expression. In vertebrates, three Ci-related transcription factors
(glioblastoma gene products (GLIs) 1, 2, and 3) were identified, but
their functional difference in Hh signal transduction is unknown. Here,
we report distinct roles for GLI1 and GLI3 in Sonic hedgehog (Shh)
signaling. GLI3 containing both repression and activation domains acts
both as an activator and a repressor, as does Ci, whereas GLI1 contains only the activation domain. Consistent with this, GLI3, but not GLI1,
is processed to generate the repressor form. Transcriptional co-activator CBP binds to GLI3, but not to GLI1. The
trans-activating capacity of GLI3 is positively and
negatively regulated by Shh and cAMP-dependent protein
kinase, respectively, through a specific region of GLI3, which contains
the CBP-binding domain and the phosphorylation sites of
cAMP-dependent protein kinase. GLI3 directly binds to the
Gli1 promoter and induces Gli1 transcription in
response to Shh. Thus, GLI3 may act as a mediator of Shh signaling in
the activation of the target gene Gli1.
 |
INTRODUCTION |
Hedgehog protein (Hh)1
plays an important role in the pattern formation of many species
including insects and vertebrates (for review, see Refs. 1 and 2). The
hh gene was originally identified as a segment polarity gene
in Drosophila. Hh is a secreted and diffusible protein and
is a critical signaling molecule for the pattern formation of the
anterior-posterior (A-P) axis. Hh is expressed only in the posterior
compartment and acts locally at the compartment border to induce the
expression of border-specific genes in anterior cells, such as
decapentaplegic (dpp), wingless (wg), and gooseberry (gsb) (3-8). The
zinc finger-containing protein (Ci) encoded by the segment polarity
gene cubitus interruptus (ci), which has a
homology with the vertebrate family of glioblastoma (Gli) transcription
factors (9), mediates Hh-induced transcriptional activation (10). On
the other hand, in the anterior region, located far from the A-P
border, Ci is processed into a C-terminal truncated form that functions
as a repressor of hh expression (11, 12). Hh signaling
suppresses this processing on the A-P border. Also on the A-P border,
extracellular Hh binds to the transmembrane protein Patched (Ptc),
preventing its normal inhibition of Smoothened (Smo), another
transmembrane protein of the receptor complex (13-16). This allows Smo
to transduce a signal to Ci through the positive regulator Fused (Fu),
which is a serine-threonine kinase (17). Hh signaling also negates the
inhibitory effects of Costal2 (Cos2), a kinesin-related protein (18,
19), cAMP-dependent protein kinase (PKA) (20-24), and
Suppressor of fused (Su(fu)) (25). Ci, Fu, Su(fu), and Cos2 are
normally found in a complex that docks at microtubules, and tight
binding of this complex to microtubules is prevented by Hh signaling
(18, 19, 25) These facts suggest that Hh signaling prevents association
of the complex with the cytoskeleton and cleavage of Ci into its repressor form.
The most studied member of the hh multigene family in
vertebrates is Sonic hedgehog (Shh). Targeted
disruption of mouse Shh leads to multiple defects in
embryonic tissues, including the notochord, floor plate, and limb
structures (26). The mechanism of Shh signaling is thought
to resemble to that of Drosophila hh. For instance, the
negative regulation of Shh signaling by PKA was also
observed in vertebrate embryos (27). It was further demonstrated that
suppression of PKA activity is sufficient to activate targets of the
Shh signaling pathway in the vertebrate central nervous
system (28). The three vertebrate genes Gli1, Gli2, and Gli3 have a homology with ci
(29, 30). All three GLI proteins bind to the consensus sequence
5'-TGGGTGGTC-3' through their metal finger regions (31, 32).
Gli1 is downstream of Shh, and ectopic
Shh signaling induces its expression, whereas expression of
Gli3 is down-regulated by ectopic Shh signaling (33-35). In fact, Gli1 and Ptc, both of which
are downstream of Shh, are expressed in similar domains in
diverse regions of developing mouse embryo (36), and ectopic expression
of Gli1 in Xenopus and mouse embryos induced the
expression of midline neural plate markers, such as Hnf-3
(34, 35), indicating that GLI1 is a positive regulator of
Shh signaling. As in the case of Ci, which acts as a
repressor in the anterior compartment, GLI3 also represses Shh expression in the anterior region of the limb bud (37). Moreover, GLI1 is an activator of transcription, whereas GLI3 represses
transcription of an Hnf-3
enhancer element (38, 39). Although these facts suggest functional differences between GLI1 and
GLI3 proteins, their precise roles in Shh signaling remain unclear.
Translocations and mutations of the human Gli3 gene cause
abnormal pattern formation through haploinsufficiency in humans and
mice. The associated human syndromes are known as Greig
cephalopolysyndactyly syndrome (GCPS) (40) and Pallister-Hall syndrome
(PHS) (41), and the mouse mutants such as extra-toe
(Xt) have the mutations in the Gli3 gene (42).
Mutations or deletions of the human Cbp gene at chromosome
16p13.3 also cause the Rubinstein-Taybi syndrome (RTS) through
haploinsufficiency (43). RTS consists of a wide variety of
developmental defects, including craniofacial malformations, broad
thumbs, broad big toes, and mental retardation (44). Disruption of one
copy of the mouse Cbp gene by gene targeting causes skeletal abnormality partially resembling that of RTS (45). CBP was originally identified as a transcriptional co-activator of CREB (46, 47) and has
recently been found to be required for many other transcription factors
(for review, see Ref. 48). RTS and GCPS are distinct syndromes, but
both include preaxial limb anomalies and craniofacial features. A
recent genetic study of Drosophila CBP mutants indicated that CBP is used as a co-activator of Ci and plays an important role in
hh signaling (49). The structural homology of Ci and GLI3
supports the idea that CBP acts as a co-activator of GLI3 in
vertebrates. As described above, however, GLI3 was reported to be a
repressor of Shh transcription, not an activator of Shh targets.
To investigate the role of CBP in vertebrate pattern formation, we have
examined whether CBP directly interacts with human GLI3 and GLI1. Our
results indicate that GLI3 and GLI1 have multiple differences in their
domain structures including the CBP-binding domain. CBP binds only to
GLI3. Furthermore, a specific region in the GLI3 protein that includes
the CBP-binding domain can mediate Shh-induced
trans-activation. We have also demonstrated that GLI3 directly binds to the mouse Gli1 promoter and mediates
Shh-induced activation of the Gli1 promoter in
Shh-responsive multipotential neural stem (MNS)-70 cells. Thus, our
results demonstrate distinct roles for GLI3 and GLI1 in Shh signaling.
 |
EXPERIMENTAL PROCEDURES |
Plasmid Construction--
The human Gli1 and
Gli3 cDNA was kindly provided by Drs. Kenneth W. Kinzler
and Bert Vogelstein. The plasmids used for in vitro
transcription/translation of the various forms of human GLI3 were
generated by inserting various fragments of GLI3 cDNA, which were
prepared by the polymerase chain reaction (PCR)-based method, into the
NcoI-XbaI site of pSPUTK (Stratagene). For all constructs generated by PCR, it was confirmed by sequencing that they
do not contain mutations. The pA10CAT6GBS reporter plasmid containing
the GLI-binding sites was constructed by inserting six tandem copies of
the GLI-binding site (5'-GCGTGGACCACCCAAGACGAAATT-3') (32) into the
BglII site of pA10CAT2, in which the SV40 early promoter was
linked to the CAT gene (50). The plasmids pSR
-GLI3 and pSR
-GLI1
to express human GLI3 and GLI1 in cultured cells were made by placing
the Gli3 and Gli1 cDNA downstream of the SR
promoter, respectively (51). To generate the plasmids
pact-Flag-GLI3 and pact-Flag-GLI1 encoding the Flag-linked GLI3 and
GLI1, the synthetic oligonucleotides containing one copy of the Flag
sequence were inserted downstream of the chicken cytoplasmic
-actin
promoter and fused in frame to the N terminus of GLI3 and GLI1,
respectively. The plasmids to express Gal4-GLI3 and Gal4-GLI1, in which
various portions of GLI3 and GLI1 were fused to the DNA-binding domain of Gal4 (amino acids 1-147), were constructed by the PCR-based method
using the CMV promoter-containing vector. The two Gal4-GLI3 fusions
containing the N-terminal region or the CBP-binding domain contained
amino acids 1-397 and 827-1132 of GLI3, respectively. The luciferase
reporter plasmid containing the mouse Gli1 promoter was
generated by inserting the 3.5-kilobase
HindIII-EcoRI fragment containing the
Gli1 promoter upstream of the luciferase gene in the plasmid
pGL3-basic vector (Promega). The Gli1 promoter
fragment2 was kindly provided
by Drs. Heidi Park and Alexandra L. Joyner. The mutant Gli1
promoter, in which the eight GLI3-binding sites were disrupted, was
constructed by the PCR-based method. The plasmids to express CBP and
E1A were described previously (52). The Shh expression plasmid pJT4/Shh
and the Gal4 site-containing luciferase reporter, in which three tandem
repeats of Gal4 sites were linked to the thymidine kinase promoter, was
kindly provided by Drs. Sumihare Noji, Kazuhiko Umesono, and Ronald M. Evans, respectively.
GST Pull-down Assay and Co-immunoprecipitation--
The GST
pull-down assay using the GST-CBP and the in vitro
translated GLI3 was performed as described (52).
For co-immunoprecipitation, a mixture of 5 µg of the CBP expression
plasmid pSR
-CBP and 5 µg of the Flag-linked GLI3 expression plasmid pact-Flag-GLI3 or the Flag-linked GLI1 expression plasmid pact-Flag-GLI1 were transfected into 293T cells. Forty hours after transfection, cells were lysed in the lysis buffer (50 mM
Hepes, pH 7.5, 250 mM NaCl, 0.2 mM EDTA, 10 mM NaF, 0.5% Nonidet P-40), and whole cell lysates were
prepared. Lysates were immunoprecipitated using anti-CBP antibodies CT
(Upstate Biotechnology) and A-22 (Santa Cruz), and the immune complexes
were separated on 7% SDS-polyacrylamide gels and analyzed by Western
blotting using anti-Flag antibodies (Upstate Biotechnology) and ECL
detection reagents (Amersham Pharmacia Biotech).
Transient Co-transfection Assays--
The amount of each plasmid
DNA used for transfection is described in the legends to the figures.
The co-transfection experiments and CAT assays were performed as
described (52). Dual luciferase assays were done as described by the supplier.
Examination of Proteolysis of GLI1 and GLI3
Proteins--
Extracts were prepared from mouse embryos at 10.5 days
post-coitus as described (45), and separated on 7 and 10% gels to detect GLI3 and GLI1, respectively. Western blotting was performed using anti-GLI3 and anti-GLI1 antibodies (Santa Cruz). The anti-GLI3 antibody that recognizes the N-terminal region of GLI3 was further purified by using the GST-GLI3 affinity resin.
To examine the processing of GLI3 in cultured cells, 4 µg of the
N-terminal Flag-linked GLI3 expression plasmid, pact-Flag-GLI3, or the
GLI1 expression plasmid, pSR
-GLI1, was transfected into 293T cells
with or without 1 µg of the Shh expression plasmid and 1 µg of the
plasmid to express the catalytic subunit of PKA. Forty hours after
transfection, cells were lysed in the lysis buffer (50 mM
Hepes, pH 7.5, 250 mM NaCl, 0.2 mM EDTA, 10 mM NaF, 0.5% Nonidet P-40), and whole cell lysates were
prepared. Lysates were separated on 7% SDS-polyacrylamide gels, and
analyzed by Western blotting using anti-Flag antibodies (Upstate
Biotechnology), anti-GLI1 antibodies (Santa Cruz), and ECL detection
reagents (Amersham Pharmacia Biotech).
Induction of Endogenous Gli1 Gene Expression by Shh in MNS-70
Cells--
MNS-70 cells were transfected with a mixture of 4 µg of
the Shh expression plasmid pJT4/Shh and 4 µg of the plasmid to
express the GLI3-binding domain of CBP (amino acids 454-718) or GLI3, or the control plasmid lacking the cDNA to be expressed. Forty hours after transfection, total RNA was prepared using TRIZOL (Life
Technologies, Inc.), and reverse transcription-PCR was performed as
described by Sasaki et al. (38) to measure Gli1
mRNA and cytoplasmic
-actin mRNA as a control.
 |
RESULTS |
Binding of CBP to GLI3 but not to GLI1--
We first examined
whether human GLI3 and GLI1 could interact directly with mouse CBP
in vitro (Fig. 1A).
The full-length form of human GLI3 was in vitro translated
and mixed with the GST-CBP resin containing the 265-amino acids region
of CBP, which is responsible for binding to multiple transcription
factors, including phospho-CREB, c-Jun, and c-Myb (46, 52, 53). GLI3
efficiently bound to GST-CBP but not to the GST resin alone (Fig.
1A, left panel). PKA treatment of GLI3 did not increase the
efficiency of binding of GLI3 to CBP. To examine the possibility that
the in vitro translated GLI3 is already phosphorylated by
some kinase(s), we treated the in vitro translated GLI3 with
potato acid phosphatase. The phosphatase treatment did not affect the
binding of GLI3 to CBP. In contrast to GLI3, in vitro
translated GLI1 protein did not bind to CBP in the GST pull-down assay
using the same GST-CBP resin (Fig. 1A, right panel). GLI1
also did not bind to the GST-CBP fusions containing various other
regions of CBP, which covered the entire CBP molecule (data not shown).
These results indicate that CBP binds to GLI3 in a
phosphorylation-independent manner, but not to GLI1.

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Fig. 1.
Binding of CBP to GLI3 but not to GLI1.
A, GST pull-down assay. At the top, GLI3 and
GLI1, which share 88% identical amino acids in their metal finger
regions, are schematically shown. The GST-CBP fusion containing the
N-proximal region of CBP (amino acids 454-718) or the GST resin alone
was mixed with the in vitro translated
[35S]GLI3 or [35S]GLI1, and the bound
proteins were analyzed by 10% SDS-PAGE. To examine the effect of PKA,
[35S]GLI3 or [35S]GLI1 was treated with PKA
(+PKA) or control buffer ( PKA) and used for the
binding assay. To investigate the effect of phosphatase, the binding
of GLI3 to CBP was similarly analyzed with (+APase) or
without ( APase) treatment of [35S]GLI3 by
potato acid phosphatase. The amount of GLI3 protein bound to GST-CBP
was about 18% of the input protein, whereas the GST control resin
bound to less than 1% of the input protein (data not shown).
B, co-immunoprecipitation. Whole cell lysates were prepared
from 293T cells transfected with the two plasmids to express CBP or the
Flag-linked GLI3, and used for immunoprecipitation with the anti-CBP
antibody CT, A-22, or the control antibody anti- -galactosidase. The
immune complexes were analyzed by Western blotting using the anti-Flag
antibody. In the left lane (Western), an aliquot
of whole cell lysate was directly used for Western blotting.
C, identification of the CBP-binding domain in GLI3. The
structures of the various forms of GLI3 used are shown below. The GST
pull-down assays were performed as described above, and the results of
binding assays shown below are indicated on the right. The
binding activities of the mutants are designated + and , which
indicate the binding of 15-20% and 1% of the input proteins,
respectively. In the input lanes, various forms of GLI3 indicated above
each lane were synthesized in vitro and analyzed by 10%
SDS-PAGE. Various forms of GLI3 synthesized were mixed with the GST-CBP
resin, and the bound proteins are shown on the right half
(Bound to CBP) of the bottom panel. The amount of protein in
the input lanes was 10% of that used for the binding assay. The lanes
for the 827/1132 protein were exposed for a longer time than the other
lanes, because the efficiency of in vitro translation of
this protein was relatively low. D, identification of the
GLI3-binding domain in CBP. The domains of CBP are indicated at the
top: C/H, cysteine- and histidine-rich domain;
KIX, CBP-binding domain; Bromo, bromo domain. The
structures of the various forms of CBP that were reported previously
(52) are shown below. The GST pull-down assays were performed as
described above, and the results of the binding assays shown below are
indicated on the right. The binding activities of the
mutants are designated + and , which indicate the binding of 15-20%
and 1% of the input proteins, respectively. The results of binding
assays using CREB and c-Myb to each GST-CBP resin, which were
previously reported (52), are also indicated.
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A possible in vivo interaction between GLI3 and CBP was
investigated by co-immunoprecipitation (Fig. 1B). Whole cell
lysates were prepared from 293T cells transfected with the two plasmids to express CBP and Flag-linked GLI3, and incubated with anti-CBP antibodies or a control antibody, anti-
-galactosidase. The
immunoprecipitates were separated on an SDS-polyacrylamide gel and the
N-terminal Flag-linked GLI3 was detected by Western blotting using the
anti-Flag antibody. A significant amount of Flag-GLI3 was
co-precipitated with the CBP C-terminal region-specific antibody (CT),
whereas the CBP N-terminal region-specific antibody (A-22)
co-precipitated only a small amount of Flag-GLI3. The low efficiency of
co-immunoprecipitation with the A-22 antibody could be due to the
proximity of the epitope for this antibody with the GLI3-binding domain
in the CBP molecule. The control antibody, anti-
-galactosidase, did
not co-immunoprecipitate Flag-GLI3.
To investigate which domain of GLI3 interacts with CBP, we used a
series of deletion mutants of GLI3 in the in vitro binding assay (Fig. 1C). Various forms of GLI3 were synthesized
using an in vitro translation system, mixed with the GST-CBP
affinity resin, and the bound proteins were analyzed. The results
showed that the 306-amino acid region between amino acids 827 and 1132 in the C-terminal half of GLI3 bound to CBP with almost the same efficiency as full-length GLI3.
By using a series of GST-CBP proteins containing various portions of
the CBP molecule, we confirmed that regions other than the N-terminal
region containing the CREB-binding domain (KIX: the region in CBP
molecule that binds to the kinase-inducible activation domain (KID) of
CREB) did not bind to GLI3 (data not shown). This region of CBP is
known to directly interact with multiple transcription factors, such as
CREB and c-Myb (52). To map the GLI3-binding domain more precisely, we
used three additional GST-CBP resins for the GST-pull down assay (Fig.
1D). This revealed that the region containing KIX and the
N-terminal adjacent region (amino acids 461-661) was required for
binding to GLI3.
GLI3 Has the Activation and Repression Domains--
To examine
whether CBP acts as a co-activator of GLI3, we then performed CAT
co-transfection experiments using mouse fibroblast NIH3T3 cells (Fig.
2, A-C). The plasmid
pA10CAT6GBS, in which six tandem repeats of the GLI-binding site were
linked to the SV40 early promoter, was used as a reporter.
Co-transfection of this reporter with a GLI3 expression vector into
NIH3T3 cells increased the level of CAT activity in a
dose-dependent manner (Fig. 2A). Increasing
amounts of the CBP expression plasmid were then co-transfected with the
reporter in the presence or absence of 6 µg of the GLI3 expression
plasmid (Fig. 2B). In the absence of CBP expression plasmid,
6 µg of the GLI3 expression plasmid activated promoter activity
2.5-fold. Co-transfection of increasing amounts of the CBP expression
plasmid increased the degree of trans-activation up to a
maximum of 8.2-fold (obtained with 16 µg of the CBP expression
plasmid in the presence of the GLI3 expression plasmid). In the control
experiment without GLI3 expression plasmid, addition of the CBP
expression plasmid did not affect the level of CAT activity at all.
Thus, CBP potentiates GLI3-induced trans-activation. Consistent with this, adenovirus wild type 12SE1A and its mutant form
121-150, which is unable to interact with any retinoblastoma-family protein but retains the capacity to bind to CBP, inhibited GLI3-induced trans-activation, but another mutant
30-85, which is
unable to interact with CBP/p300, did not (Fig. 2C).

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Fig. 2.
Presence of the activation and repression
domains in GLI3. A, transcriptional activation by GLI3.
trans-Activation by GLI3 was examined by co-transfection
assays in NIH3T3 cells using the CAT reporter containing the
GLI-binding sites. A mixture of 0, 3, 6, 9, 12, or 15 µg of the GLI3
expression plasmid pSR -GLI3, 6 µg of the CAT reporter plasmid
pA10CAT6GBS, and 1 µg of the internal control plasmid
pact- -galactosidase was transfected into NIH3T3 cells. The total
amount of plasmid DNA was adjusted to 22 µg by adding the control
plasmid DNA pSR 0 lacking the GLI3-coding region. CAT assays were
done as described under "Experimental Procedures." The experiments
were repeated three times, and the differences between the experiments
were no more than 20%. Typical results are indicated by a bar
graph. The shaded bar shows the data obtained in the
presence of the GLI3 expression plasmid. B, potentiation of
GLI3-induced trans-activation by CBP. The effect of CBP on
GLI3-induced trans-activation was investigated by further
addition of increasing amounts of the CBP expression plasmid. A mixture
of 0, 4, 8, 12, or 16 µg of the CBP expression plasmid pSR -CBP, 5 µg of the CAT-reporter plasmid pA10CAT6GBS, 0 or 6 µg of the GLI3
expression plasmid pSR -GLI3, and 1 µg of the internal control
plasmid pact- -galactosidase was transfected into NIH3T3 cells, and
CAT assays were done. The total amount of plasmid DNA was adjusted to
27 µg by adding the control plasmid DNA pSR 0. The experiments were
repeated three times, and the average is shown as the relative CAT
activity, with S.D. as indicated. C, repression of
GLI3-dependent transcriptional activation by E1A. A mixture
of 6 µg of the CAT-reporter plasmid pA10CAT6GBS, 9 µg of the GLI3
expression plasmid pSR -GLI3, 1 µg of the plasmid to express wild
type 12SE1A, 121-150, or 30-85 mutant, and 1 µg of the
internal control plasmid pact- -galactosidase was transfected into
NIH3T3 cells, and CAT assays were done. The total amount of plasmid DNA
was adjusted to 20 µg by adding the control plasmid DNA pSR 0.
D, activation and repression domains in GLI3. The two Gal4
fusion proteins containing the N-terminal region (Gal4-N) and the
CBP-binding domain (Gal4-CBD) of GLI3 are shown below. Co-transfection
assays using the plasmid to express these Gal4-GLI3 fusions were done
using the luciferase reporter containing Gal4-binding sites. A mixture
of 4 µg of the Gal4 site-containing reporter plasmid pGal-TK-luc, 0.2 µg of the plasmid to express various forms of Gal4-GLI3 fusion or
Gal4 DNA-binding domain alone as a control, 3 µg of the CBP
expression plasmid pSR -CBP, 1 µg of the E1A expression plasmid,
and 1 µg of the internal control plasmid pRL-CMV, in which the sea
pansy luciferase gene is linked to the CMV promoter, was transfected
into NIH3T3 cells, and luciferase assays were done. The total amount of
plasmid DNA was adjusted to 10 µg by adding the control plasmid DNA.
The shaded bar shows the data obtained with the Gal4-GLI3
expression plasmid. E, Shh-responsiveness of GLI3. A mixture
of 0, 1, 2, 3, or 4 µg of the Shh expression plasmid pJT4/Shh, 4 µg
of the Gal4 site-containing reporter plasmid pGal4-TK-luc, 0.2 µg of
the plasmid to express Ga4-GLI3 fusion, which contains the full-length
GLI3 or the CBP-binding domain, or Gal4 DNA-binding domain alone as a
control, 3 µg of the PKA catalytic subunit expression plasmid
pSR -PKA, and 1 µg of the internal control plasmid pRL-CMV was
transfected into MNS-70 cells, and the luciferase assay was performed.
The total amount of plasmid DNA was adjusted to 12.2 µg by adding the
control plasmid DNA. The experiments were repeated three times, and the
average is shown with S.E. The data indicating the positive regulation
by Shh is shown by a shaded bar.
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The results of co-transfection experiments using various GLI3 deletion
mutants with the GLI site-containing reporter suggested that the
N-terminal region of GLI3 contains the transcriptional repression
domain, whereas the CBP-binding domain mediates transcriptional activation (data not shown). To confirm this, we made the two Gal4-GLI3
fusions, which consisted of the Gal4 DNA-binding domain and the
N-terminal region or CBP-binding domain of GLI3, and examined their
capacity to modulate luciferase expression from the Gal4 site-containing luciferase reporter (Fig. 2D). The Gal4
fusion containing the N-terminal 397 amino acids (Gal4-N) repressed
luciferase expression to 35% of the level of the control Gal4
DNA-binding domain alone (Fig. 2D, cf. lanes 1 and 2), whereas the Gal4 fusions containing the CBP-binding
domain (Gal4-CBD) stimulated luciferase expression 5-fold. The
expression levels of these two fusion proteins were similar (data not
shown). Co-expression of CBP enhanced trans-activation by
the CBP-binding domain (Fig. 2D, cf. lanes 5 and
6) but did not affect the function of the N-terminal
repression domain (cf. lanes 2 and 3). E1A
inhibited CBD-dependent trans-activation
(cf. lanes 5 and 7). These results indicated that
GLI3 has both the repression and activation domains.
GLI3 Mediates the Shh-induced Transcriptional Activation--
The
identification of the CBP-binding domain of GLI3 allowed us to examine
the effect of Shh and PKA on the capacity of this domain to regulate
transcription. For this purpose, we used a neural stem cell line,
MNS-70, that is able to express different sets of ventral-specific
genes including Isl-1, Nkx-2.1, and Nkx-2.2 in response to
Shh (54). The Gal4 site-containing reporter and the plasmid to express
the Gal4 fusion protein containing GLI3 were co-transfected into MNS-70
cells with increasing amounts of the Shh expression plasmid in the
presence or absence of the PKA catalytic subunit expression plasmid
(Fig. 2E). We first examined the effect of Shh and PKA on
trans-activation by the Gal4 fusion containing full-length
GLI3 (Fig. 2E, left panel). The Gal4-fusion containing
full-length GLI3 repressed luciferase expression to 65% of the level
of the control Gal4 DNA-binding domain. Co-expression of Shh increased
luciferase expression in a dose-dependent manner, whereas
PKA suppressed trans-activating capacity. We then performed a similar experiment using a Gal4 fusion with the CBP-binding domain
(Fig. 2E, right panel). In the absence of PKA, Shh only slightly enhanced trans-activation by the CBP-binding
domain. PKA negatively regulated this trans-activation, and
co-expression of Shh restored this trans-activation in a
dose-dependent manner in the presence of PKA. Thus, the
CBP-binding domain mediates the antagonistic actions of PKA and Shh.
The Shh responsiveness of full-length GLI3 in the absence of PKA may
suggest that Shh may enhance GLI3 activity through not only the
CBP-binding domain but also other region. On the other hand, the
trans-repression mediated by Gal4 fusion containing the
N-terminal repression domain was affected neither by Shh nor by PKA
(data not shown).
Negative Regulation of GLI1 Activity by Shh--
To compare the
functional domains of GLI3 and GLI1, we next examined the functional
domains of GLI1. In the CAT co-transfection experiments using the GLI
site-containing reporter and NIH3T3 cells, GLI1 increased the level of
CAT activity (Fig. 3A).
Consistent with the results of the GST pull-down assays, co-expression
of CBP did not potentiate GLI1-induced trans-activation
(Fig. 3B). To identify the transcriptional activation domain
in GLI1, three different portions of GLI1 were fused to the Gal4
DNA-binding domain, and their capacity to regulate transcription was
investigated by co-transfection assays using the Gal4 site-containing
reporter in MNS-70 cells (Fig. 3C). The expression levels of
these three fusion proteins were similar (data not shown). Full-length
GLI1 fusion and the Gal4 fusion containing the C-terminal 394 amino acids enhanced luciferase expression (Fig. 3C, left panel).
These results indicated that GLI1 has the activation domain in its
C-terminal region and does not have a repression domain. The Shh and
PKA responsiveness of the Gal4 fusion containing full-length GLI1 was
investigated (Fig. 3C, right panel). The full-length GLI1 activity was suppressed by Shh, whereas PKA did not affect GLI1 activity.

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Fig. 3.
Domain structure and Shh
responsiveness of GLI1. A, transcriptional activation
by GLI1. trans-Activation by GLI1 was examined by
co-transfection assays in NIH3T3 cells using the CAT reporter
containing the GLI-binding sites. A mixture of 0, 4, or 8 µg of the
GLI1 expression plasmid pSR -GLI1, 4 µg of the CAT-reporter plasmid
pA10CAT6GBS, and 1 µg of the internal control plasmid
pact- -galactosidase was transfected into MNS-70 cells, and CAT
assays were performed. The total amount of plasmid DNA was adjusted to
13 µg by adding the control plasmid DNA pSR 0. The experiments were
repeated three times, and the differences between the experiments were
no more than 20%. Typical results are indicated by a bar
graph. The shaded bars show the data obtained in the
presence of the GLI expression vector. B, there was no
effect of CBP on GLI1-induced trans-activation. The effect
of CBP on GLI1-induced trans-activation was investigated by
the addition of the increasing amounts of the CBP expression plasmid. A
mixture of 0, 4, or 8 µg of the CBP expression plasmid pSR -CBP, 5 µg of the CAT-reporter plasmid pA10CAT6GBS, 0 or 5 µg of the GLI1
expression plasmid pSR -GLI1, and 1 µg of the internal control
plasmid pact- -galactosidase was transfected into MNS-70 cells, and
CAT assays were done. The total amount of plasmid DNA was adjusted to
21 µg by adding the control plasmid DNA. The experiments were
repeated three times, and the average is shown as the relative CAT
activity, with S.D. indicated. C, regulation of GLI1
activity by Shh and PKA. The structure of Gal4-GLI1 fusions used is
indicated, and the trans-activating capacity of each
construct is shown to the right. Left panel,
identification of the activation domain. A mixture of 2 µg of the
Gal4 site-containing reporter plasmid pGal-TK-luc, 0.3 µg of the
plasmid to express various forms of Gal4-GLI1 fusion or Gal4
DNA-binding domain alone, and 0.5 µg of the internal control plasmid
pRL-CMV was transfected into MNS-70 cells, and luciferase assays were
done. The total amount of plasmid DNA was adjusted to 5 µg by adding
the control plasmid DNA. Right panel, inhibition of
full-length GLI1 activity by Shh. A mixture of 0, 0.5, 1, or 2 µg of
the Shh expression plasmid pJT4/Shh, 2 µg of the Gal4 site-containing
reporter plasmid pGal4-TK-luc, 0.5 µg of the plasmid to express
Ga4-full-length GLI1 fusion or Gal4 DNA-binding domain alone as a
control, 1 µg of the PKA catalytic subunit expression plasmid
pSR -PKA, and 0.5 µg of the internal control plasmid pRL-CMV was
transfected into MNS-70 cells, and the luciferase assay was performed.
The total amount of plasmid DNA was adjusted to 6 µg by adding the
control plasmid DNA.
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Processing of GLI3 but not GLI1--
Our results indicated that
GLI3 has both activation and repression domains, as does the
Drosophila homolog Ci, whereas GLI1 has only the activation
domain. Because Ci is processed into a repressor form lacking the
activation domain in the anterior compartment (11), we examined GLI3
for similar processing (Fig. 4). Extracts were prepared at 11.5 days post-coitus from mouse embryos, which express both GLI3 and GLI1, and were used for Western blotting (Fig.
4A). An antibody that can recognize the C-terminal region (amino acids 1577-1596) of mouse GLI3 detected 190- and 110-kDa proteins. Analysis of the short, 110-kDa form of GLI3 by SDS-PAGE using
two in vitro translated N-truncated mutants as markers
suggested that this short form was generated probably by processing at
a site between amino acids 650 and 750, corresponding to the region between the zinc finger region and the CBP-binding domain. Another fragment generated by this processing should have the N-terminal repressor domain and the zinc finger. In fact, an antibody recognizing the N-terminal region (amino acids 2-20) of GLI3 detected a 100-kDa protein in addition to the full-length 190-, 60-, and 50-kDa proteins. In contrast, the GLI1-specific antibody recognizing the N-terminal region (amino acids 2-17) of GLI1 detected only a 150-kDa protein, which corresponds to the predicted size of full-length GLI1.

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Fig. 4.
Processing of GLI3 but not GLI1.
A, analysis of GLI3 in mouse embryo. Extracts were prepared
from the 11.5-day post-coitus mouse embryos and separated on 7 and 10%
SDS-polyacrylamide gels. Western blotting was performed using the
anti-GLI1, which recognizes amino acids 2-17 of GLI1, or anti-GLI3,
which recognizes amino acids 1577-1596 (anti-GLI3C) or 1-19
(anti-GLI3N) of GLI3. The short form of GLI3, which may be generated by
protein processing, is indicated by an arrow. In the
lower right panel, the two N-truncated mutants of GLI3 were
in vitro translated and analyzed on a 7% SDS-polyacrylamide
gel. By using these two in vitro translated proteins as
markers, the approximate position of GLI3 protein processing was
estimated and is shown at the top. B, Shh
inhibits the processing of GLI3. The plasmid to express Flag-GLI3
(left panel) or GLI1 (right panel) was
transfected with (+) or without ( ) the PKA or Shh expression plasmid
into 293T cells. Whole cell lysates were prepared and subjected to
SDS-PAGE followed by Western blotting using anti-Flag (left
panel) or anti-GLI1 antibody (right panel).
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To further confirm the processing of GLI3 and also to examine whether
Shh signaling prevents the processing of GLI3 as reported in the case
of Ci of Drosophila, we tested the processing of GLI3 in
cultured cells. The plasmid to express the N-terminal Flag-linked GLI3
protein was transfected into the highly transfectable 293T cells, and
the GLI3 proteins expressed from the transfected DNA was analyzed by
Western blotting using anti-Flag antibody (Fig. 4B, left
panel). When the Flag-GLI3 expression plasmid alone was transfected, only the full-length form of GLI3 was detected, indicating that the processing did not occur. However, when the plasmid to express
the catalytic subunit of PKA was co-expressed with Flag-GLI3, the
95-kDa GLI3 protein was generated. The size of this protein is close to
but slightly smaller than that (100 kDa) of one of the GLI3 proteins
detected in mouse embryonic lysates. This small difference in the
molecular mass could be due to retardation of mobility because of high
amount of proteins loaded in Fig. 4A, and appears to be
within error. The 60- and 50-kDa GLI3 proteins detected in mouse
embryonic lysates may be artifactual degradation products, because the
similar size of proteins were not detected in the transfected cells.
These results suggest that PKA stimulates the processing of GLI3, as in
the case of Ci. When the Shh is co-expressed with Flag-GLI3 and PKA,
this processing was inhibited. In the similar experiment with the GLI1
expression plasmid and the PKA expression plasmid, only the full-length
form of GLI1 was detected, and the processing of GLI1 was not observed
(Fig. 4B, right panel). Thus, consistent with their domain
structures, GLI3, but not GLI1, is processed to generate a repressor form.
Shh-induced Activation of the Gli1 Promoter Is Mediated by
GLI3--
It was recently reported that the transcription of the
Gli1 gene, but not the Gli3 gene, is induced by
ectopic Shh signaling (15, 34, 35). These reports and our finding that
GLI3, but not GLI1, has an Shh-induced activation domain suggested that GLI3 could mediate Shh-induced activation of the Gli1
promoter. To investigate this possibility, we examined whether GLI3
directly binds to the mouse Gli1 promoter region by gel
mobility shift assays using the GST-GLI3 fusion protein containing the
metal finger region of GLI3 (Fig.
5A). Among six DNA fragments
covering the mouse Gli1 promoter region (55),2
only two DNA fragments, the BamHI-XhoI 210-base
pair fragment (fragment A) and the adjacent
XhoI-EcoRI 800-base pair fragment (fragment B)
containing the two exons that encode the 5'-untranslated region of
Gli1 mRNA, bound to the GST-GLI3 recombinant protein and
generated the specifically retarded bands (Fig. 5A, left
panel). Increasing the amount of GST-GLI3 protein generated
multiple retarded bands (Fig. 5A, middle and
right panels), suggesting the presence of multiple
GLI3-binding sites in these two fragments. In fact, the DNA sequences
of both fragments indicated the presence of three and five putative
GLI-binding sites in fragments A and B, respectively (Fig. 5B,
left panel). A comparison of these DNA sequences with the
previously reported consensus sequence (5'-TGGGTGGTC) for the
GLI-binding site (32) indicated that these eight DNA sequences have
fewer than three mismatches with the reported consensus sequence. To
confirm that these sites are really responsible for GL13 binding, the
sixth G residue of all of these sites was mutated to an A residue, and
the mutated fragments were used for the gel retardation assays. The
introduction of this mutation into these putative GLI3-binding sites
abolished GST-GLI3 binding (Fig. 5B, right panel).

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Fig. 5.
GLI3 directly activates the Gli1
promoter in response to Shh. A, direct binding of
GLI3 protein to the Gli1 promoter. The structure of the
genomic clone containing the two exons corresponding to the 5'-untranslated region of the mouse Gli1 gene
is indicated at the top. B, BamHI;
E, EcoRI; H, HindIII;
Hi, HincII; K, KpnI;
X, XhoI. The direction of transcription is from
left to right. The eight putative GLI3-binding
sites are indicated by vertical arrows. The gel mobility
shift assays were performed using the GST fusion protein containing the
metal finger region of GLI3 and the six DNA fragments prepared from the
indicated genomic clone as probes. Of six fragments, only two, the
210-base pair BamHI-XhoI fragment (fragment
A) and the 800-base pair XhoI-EcoRI fragment
(fragment B), bound to GST-GLI3. The results using these two
fragments are shown below. In the left panel, the
32P-labeled fragment A or fragment B was incubated with the
GST-GLI3 fusion (200 ng), GST (200 ng), or control buffer and analyzed
on a 4% nondenaturing gel. In the right two panels,
increasing amounts of GST-GLI3 fusion proteins (0, 40, 80, 160, or 320 ng) were incubated with the fragment A or fragment B probe.
B, multiple GLI3-binding sites in the Gli1
promoter region. The DNA sequence of eight putative GLI3-binding sites
in fragments A and B are indicated on the left. The
previously reported consensus sequence for the GLI-binding site (32) is
shown below. On the right, the gel mobility shift assays
were performed using 25 ng of GST-GLI3 protein with the wild type and
mutant fragments, in which the sixth G residue in the eight putative
GLI3-binding sites was changed to A. C, transcriptional
activation of the Gli1 promoter by GLI3 in NIH3T3 cells. A
mixture of 0, 0.2, 0.5, 1, 2, 3, 4, or 5 µg of the GLI3 expression
plasmid pSR -GLI3, 3 µg of the luciferase reporter plasmid pHR-luc,
which contains the 3.5-kilobase HindIII-EcoRI
fragment of Gli1 promoter, and 0.5 µg of the internal
control plasmid pRL-SV, in which the SV40 early promoter was linked to
the sea pansy luciferase gene, was transfected into NIH3T3 cells, and
luciferase assays were performed. The total amount of plasmid DNA was
adjusted to 8.5 µg by adding the control plasmid DNA pSR 0. The
experiments were repeated three times, and the differences between the
experiments were no more than 20%. Typical results are indicated by a
bar graph. The shaded and open bars
indicate the data with and without the GLI3 expression plasmid,
respectively. D, Shh-induced activation of the
Gli1 promoter by GLI3 in MNS-70 cells. A mixture of 0, 0.5, 1, or 2 µg of the Shh expression plasmid pJT4/Shh, 3 µg of the
Gli1 wild type promoter-containing luciferase reporter
plasmid pHR-luc, 3 µg of the GLI3 expression plasmid pSR -GLI3, 1 µg of the PKA catalytic subunit expression plasmid pSR -PKA, and
0.5 µg of the internal control plasmid pRL-SV was transfected into
MNS-70 cells, and luciferase assays were performed. The total amount of
plasmid DNA was adjusted to 8.5 µg by adding the control plasmid DNA.
The experiments were repeated three times, and the average is shown as
the relative luciferase activity with S.D. E, requirement of
GLI3-binding sites for Shh-induced activation of the Gli1
promoter. Co-transfection experiments were done as described in
D using the reporter containing the mutant Gli1
promoter in which the eight GLI3-binding sites were mutated.
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When either of fragment A or fragment B was inserted upstream of the
luciferase gene, the resulting constructs expressed a significant level
of luciferase in transfected NIH3T3 and MNS-70 cells, indicating that
both of these fragments have promoter activity (data not shown). We
also constructed a luciferase reporter construct that has the
3.5-kilobase HindIII-EcoRI fragment containing
both fragments A and B and used it in the following experiments.
Co-transfection of the GLI3 expression plasmid with this reporter into
NIH3T3 cells enhanced the luciferase activity in a
dose-dependent manner (Fig. 5C). To investigate
the role of GLI3 in Shh-induced activation of the Gli1
promoter, increasing amounts of the Shh expression plasmid were
co-transfected into MNS-70 cells with the Gli1
promoter-containing luciferase reporter in the presence or absence of
GLI3 and the PKA expression plasmid (Fig. 5D). Neither Shh
nor PKA affected Gli1 promoter activity in the absence of
exogenous GLI3. In the presence of exogenous GLI3, PKA suppressed
Gli1 promoter activity, whereas Shh enhanced Gli1
promoter activity in a dose-dependent manner. This
antagonistic regulation by Shh and PKA was not observed with the mutant
promoter in which the eight GLI3-binding sites of the Gli1
promoter were disrupted (Fig. 5E).
Inhibition of Shh-induced Gli1 Expression by Overexpression of the
GLI3-binding Domain of CBP--
Ectopic expression of Shh in MNS-70
cells induces expression of the endogenous Gli1 gene (38),
and MNS-70 cells express endogenous GLI3 at a level that may be
sufficient for the induction of endogenous target genes by Shh. To
confirm that GLI3 functions as a mediator of Shh induction of
Gli1 mRNA, we examined the effect of overexpression of
the GLI3-binding domain of CBP on the induction of endogenous
Gli1 mRNA by Shh (Fig. 6).
If the CBP-binding domain of GLI3 really mediates Shh-induced
activation of the Gli1 promoter, the GLI3-binding domain of
CBP should mask the CBP-binding domain of GLI3 and inhibit the
induction of the endogenous Gli1 gene by Shh. As reported by
Sasaki et al. (38), transfection of the Shh expression
plasmid increased the level of Gli1 mRNA by about 6.3-fold. Co-transfection of the Shh expression plasmid with the GLI3
expression plasmid strongly enhanced the Gli1 mRNA level by 10.5-fold. In contrast, co-expression of the GLI3-binding domain of
CBP with Shh significantly lowered the level of induction of Gli1 mRNA by about half. These results further confirm
that GLI3 is a mediator of Shh-dependent transcriptional
activation of Gli1.

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Fig. 6.
Inhibition of Shh-induced Gli1
expression by overexpression of the GLI3-binding domain of
CBP. MNS-70 cells were transfected with a mixture of 4 µg of the
Shh expression plasmid pJT4/Shh and 4 µg of the plasmid to express
GLI3 or the GLI3-binding domain of CBP or the control plasmid lacking
the cDNA to be expressed. Total RNA was prepared from the
transfected cells, and Gli1 expression was analyzed by
reverse transcription-PCR. Cytoplasmic -actin was used as a control.
On the right, the degree of Gli1 expression is
indicated by a bar graph.
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DISCUSSION |
Distinct Roles for GLI3 and GLI1 in Shh Signaling--
In
Drosophila, Hh signaling inhibits association of the
Ci-Cos2-Fu-Su(fu) complex with the cytoskeleton and cleavage of Ci into
a repressor form on the A-P border (18, 19, 25, 11). Although the
pathway of Shh signaling has not yet defined completely, there is
evidence to suggest that it is well conserved between Drosophila and vertebrates (Fig.
7). First, GLI3, like Ci, is primarily
localized in the cytosol (34). Second, GLI3 has a domain structure
similar to that of Ci, with both proteins having transcriptional
activation and repression domains. Third, GLI3, like Ci, appears to be
cleaved into a repressor form. These facts suggest that Shh signaling
blocks association of the GLI3-Cos2-Fu-Su(fu) complex to the
cytoskeleton and processing of GLI3 into a repressor form.

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Fig. 7.
Schematic representation of the role of GLI3
and GLI1 in Shh signaling. Shh signaling enhances the
trans-activating capacity of GLI3 through the region
containing the CBP-binding domain and the PKA phosphorylation sites and
induces Gli1 transcription through the direct action of GLI3
on the Gli1 promoter. The expressed GLI1 then activates
specific target genes including Hnf-3 and the
Gli1 gene itself in an Shh-independent manner.
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Recently, PKA was demonstrated to directly phosphorylate the multiple
sites adjacent to the dCBP-binding domain in Ci protein and to enhance
the proteolysis of Ci (56). The multiple phosphorylation sites for PKA
are well conserved in GLI3, and our results indicate that the small
segment of GLI3 containing the CBP-binding domain and the putative PKA
phosphorylation sites is sufficient for positive regulation by Shh and
negative regulation by PKA (Fig. 2). In addition, PKA enhances the
proteolysis of GLI3 to generate a repressor form, and Shh signaling
inhibits this (Fig. 4). Phosphorylation of GLI3 by PKA does not affect
binding to CBP. One possibility is that direct phosphorylation of
GLI3 by PKA retains GLI3 as part of the cytoskeleton complex in the
cytosol and enhances the proteolysis of GLI3. Interestingly, in the
absence of PKA, Shh does not enhance trans-activation
by Gal4-CBD (Fig. 2E, right panel), suggesting that the role
of Shh is to negate the action of PKA. This is consistent with the
previous report that suppression of PKA activity is sufficient to
activate targets of the Shh signaling pathway in the mouse central
nervous system (28). However, the Shh responsiveness of full-length
GLI3 in the absence of PKA (Fig. 2E) may suggest that Shh
may enhance GLI3 activity through not only the CBP-binding domain but
also other region. In addition, Shh and PKA enhanced the
Gli1 promoter activity to the higher level than that with
Shh alone (Fig. 5D). This may be consistent with the recent
report by Ohlmeyer and Kalderon (57) that increased PKA activity can
induce ectopic Hh target gene expression without changes in Ci protein
concentration. At present, it remains unknown whether CBP is merely act
as a co-activator of full-length GLI3 or is important for Shh and PKA
responsiveness of GLI3. Because Ci lacks an obvious nuclear
localization signal (9, 58), its movement into the nucleus could be
mediated by its ability to bind to dCBP, which would carry it there.
Although GLI3 has one putative nuclear localization signal in the
N-terminal region, vertebrate CBP may also act as a carrier of GLI3
into the nuclei.
Unlike GLI3, GLI1 is a simple transcriptional activator encoded by a
target gene of Shh signaling. It appears to contain only the
transcriptional activation domain and is not cleaved. These facts
suggest the following cascade between Shh and the induction of its
target genes (Fig. 7). Shh first enhances GLI3 activity via the region
containing the CBP-binding domain, and then the activated GLI3 directly
binds to the Gli1 promoter and induces Gli1
transcription. This leads to accumulation of GLI1, which then induces a
second wave of transcription involving Shh target genes such as
Hnf-3
and possibly the Gli1 gene itself,
because GLI1 can activates the Gli1 promoter in our
co-transfection assays (data not shown).
We observed that the level of Gli1 mRNA in various
regions of Gli3 heterozygous and homozygous mouse embryos
(Xt/+ and Xt/Xt) was significantly lower than
that of the wild type (data not shown). However, Gli1
mRNA did not completely disappear, even in the Gli3 homozygous mutant. This may be due to a redundant function of GLI3 and
GLI2. Redundant functions of GLI3 and GLI2 was suggested by a study of
Gli2 and Gli3 double mutant mice (59). In MNS-70 cells, Gli1 mRNA is expressed at very low level,
although a significant level of Gli3 and Gli2
mRNA expression is still observed (data not shown). Because
overexpression of the CBP fragment containing the GLI3-binding domain
significantly lowered the Shh-dependent induction of
Gli1 mRNA, CBP may also act as a co-activator of GLI2.
Gli3 is not thought to be expressed in the ventral midline of the central nervous system, where the floor plate is induced by Shh.
In this region, GLI2 may play the same role as GLI3 to induce
Gli1 expression. This is consistent with the recent study of
Gli2 mutant mice, in which Gli1 is not detected
ventrally at E9.5 and the floor plate does not form (60). Recently, it
was reported that Gli2 and Gli3 repress the
ectopic induction of frog floor plate cells by Gli1 in
co-injection assays and inhibit endogenous floor plate differentiation
(61). However, a large amount of repressor form of GLI3 and only a
small amount of full-length activator form could be generated in this
system, because Shh signaling may not act on large amount of GLI3
protein generated from injected mRNA. If this is a case, a
repressor form of GLI3 may inhibit the expression of Shh target genes
and antagonize Gli1 function. The antagonizing activities of
Gli2 and Gli3 on Gli1 function are
also not consistent with the recent loss-of function studies with
Gli1 and Gli2 mutants in mouse (60).
Relationship between Gli3 Mutations and Genetic
Diseases--
Mutations and translocations of the human
Gli3 gene are responsible for two human disorders, GCPS and
PHS (40, 41). The characteristics of each of these disorders overlap
with one another but still remain sufficiently distinct to be
classified as separate disorders. For instance, although both GCPS and
PHS have polysyndactyly and abnormal craniofacial features, GCPS has
commonly postaxial polydactyly of the hands and preaxial polydactyly of
the feet, whereas PHS has typically central or postaxial polydactyly.
GCPS does not cause the hypothalamic hamartoma observed in PHS. In addition, PHS is not associated with hypertelorism or broadening of the
nasal root or forehead seen in GCPS. The different anomalies in these
two disorders may be partly due to the generation of truncated proteins
having different characteristics. In GCPS, GLI3 was found to be
truncated upstream or within the zinc finger domain (40, 62), whereas
mutations found in PHS truncate GLI3 after the zinc finger (41). Our
domain analysis indicated that the N-terminal region upstream from the
zinc finger region is a repressor domain. Therefore, the truncated
protein found in PHS could still retain repressor activity, unlike the
protein truncated upstream or within the zinc finger domain, which was found in GCPS.
Direct Binding of CBP to GLI3: a Molecular Link between RTS and
Genetic Diseases caused by Gli3 Mutations--
Our results indicate
that GLI3 utilizes CBP as a co-activator, as in Drosophila,
in which Ci uses dCBP. Thus, the interaction of GLI3/Ci with CBP/dCBP
is conserved between mammals and insects. Some of the characteristics
of GCPS and PHS are similar to RTS. GCPS and RTS are caused by
haploinsufficiency, and GCPS, PHS, and RTS are all associated with
craniofacial, hand, and foot defects. Thus, direct interaction between
GLI3 and CBP could explain the similar characteristics of these
disorders. However, some characteristics of these syndromes are
distinct. For instance, broad thumbs and broad halluces are associated
with both GCPS and RTS, but polydactyly and syndactyly are commonly
observed only in GCPS. These differences between GCPS and RTS could be
explained by multiple mechanisms. In the embryos of multiple
polydactylous mouse mutants, such as Xt and Hemimelic
extra toes (Hx), Shh is ectopically expressed at the
anterior margin of the limb buds (63). By analogy with the
Ci-dependent repression of hh expression in the
anterior compartment of Drosophila, the repressor form of
GLI3 lacking the C-terminal activation domains may repress Shh
expression. Therefore, the reduction or loss of repressor activity of
GLI3 may lead to polydactyly. Although the truncated forms found in PHS
still retain the N-terminal repressor domain and the zinc finger
domain, these truncations may lead to increased instability of these
proteins or to low repressor activity. In contrast, the deficiency of
CBP should not affect the repressor activity of GLI3, and this may
explain the lack of polydactyly in RTS. In addition, CBP affects the
activity of many transcription factors, and some of the features seen
in RTS could be explained by decreased activity of transcription factors other than GLI3. dCBP is a co-activator of Dorsal, a
Drosophila homolog of NF-
B, and dCBP mutations cause the
loss of Dorsal-dependent expression of the twist
gene (64). Because mutations of the human Twist gene are
associated with the autosomal dominant Saethre-Chotzen syndrome, which
is clinically analogous to RTS (65, 66), some defects may be caused by
decreased expression of the Twist gene.
 |
ACKNOWLEDGEMENTS |
We thank Alexandra L. Joyner and Heidi Park
for the mouse Gli1 promoter clone and helpful comments,
Hiroshi Sasaki for advice on Gli1 mRNA detection by PCR
and discussion, Kenneth W. Kinzler and Bert Vogelstein for the human
Gli1 and Gli3 cDNAs, Richard H. Goodman for
the murine CBP expression plasmid and the protocol for
co-immunoprecipitation, Sumihare Noji for the Shh expression plasmid,
and Ken-ichi Arai for the PKA expression plasmid.
 |
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. Tel.:
81-298-36-9031; Fax: 81-298-36-9030; E-mail:
sishii{at}rtc.riken.go.jp.
2
H. Park and A. L. Joyner, manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
CBP, CRE (cAMP
response element)-binding protein;
Ci, cubitus interruptus
gene product;
GLI, glioblastoma gene product;
Hh, hedgehog protein;
PKA, cAMP-dependent protein kinase;
Shh, Sonic
hedgehog gene product;
A-P, anterior-posterior;
RTS, Rubinstein-Taybi syndrome;
GCPS, Greig cephalopolysyndactyly syndrome;
PHS, Pallister-Hall syndrome;
PCR, polymerase chain reaction;
MNS, multipotential neural stem;
CAT, chloramphenicol acetyltransferase;
CMV, cytomegalovirus;
Shh, Sonic hedgehog.
 |
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