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INTRODUCTION |
The Zic genes encode zinc finger proteins that mediate
diverse events in vertebrate development (1, 2). The genes are expressed in the developing or mature central nervous system, somites,
and limb buds in a spatially restricted manner (3-8). A
Zic1-deficient mouse shows cerebellar abnormalities and
marked ataxia (9). Mutations in human ZIC2 or mouse
Zic2 lead to a congenital brain anomaly known as
holoprosencephaly (10-12). The human ZIC3 mutations result
in a disturbance of the left-right body axis (13). In
Xenopus, Zic genes play a role in the initial phase of neural and neural crest development (5, 7). The zinc finger
domain was highly conserved not only in the vertebrate Zic
family but also in Drosophila odd-paired, which regulates segmentation and midgut development (14, 15). All of these findings indicate that Zic genes exert an essential
role in multiple aspects of development.
In addition to Zic orthologues in other species, the zinc finger
domains of the Zic proteins, which consist of five tandemly repeated
C2H2 motifs, show a notable homology to those of Gli family
proteins (1). At present, Gli1, Gli2, and
Gli3 have been reported in human, mouse, and frog (16-21)
in addition to their nonvertebrate homologues Drosophila Cubitus
interruptus (Ci; Ref. 22) and Caenorhabditis
elegans Tra1 (23). Gli proteins are also known to regulate events
in vertebrate development. In particular, it has been reported that Gli
and Cubitus interruptus proteins function downstream of the Sonic
hedgehog-Patched signaling pathway as transcriptional regulators
(24-26). For example, in response to the Sonic hedgehog signal,
activated Gli proteins bind directly to the enhancer element of a
winged helix transcription factor, hepatocyte nuclear factor-3
, the
expression of which is critical for floor plate development (27,
28).
The sequence-specific DNA binding of the GLI proteins has been studied
in detail. A consensus nonamer target site, 5'-TGGGTGGTC-3' (GLI-BS),1 has been
identified for human GLI1 (29). A crystallographic study showed that
GLI1 bound DNA through the last four fingers via extensive base
contacts, which are highly conserved between GLI and Zic families (2,
30). Tra1 protein can also bind the GLI-BS possibly through the last
three zinc fingers, because elimination of the first two fingers had
little effect on its binding to DNA (23). Furthermore, the GLI3 zinc
finger domain (GLI3-ZF) can also bind the GLI-BS (31). The target has
been optimized to an extended 16-nucleotide sequence including the GLI-BS (GLI3-BS).
The fact that Zic proteins are nuclear proteins and have zinc finger
domains highly similar to those of Gli proteins led us to infer that
the Zic proteins might act as transcription factors in a manner similar
to that of Gli proteins. However, little is known about the regulation
of gene expression by the Zic proteins. The only finding in support of
this to date is that the zinc finger domain of Zic1 (Zic1-ZF) can bind
the GLI-BS (1). Therefore, the Zic proteins may regulate the expression
of target genes by interacting with a GLI-BS-like sequence.
Nevertheless, no methodical analysis of the DNA recognition sequence
for Zic family proteins has been performed to date. We therefore
undertook the task of empirically determining the Zic binding sequence
and characterizing its binding properties. Furthermore, we examined
transcriptional regulation by Zic proteins and tested whether the Zic
proteins affected the GLI-DNA interaction and GLI-mediated
transcriptional activation. This study should pave the way for a better
understanding of the molecular mechanism of Zic-mediated
transcriptional regulation.
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EXPERIMENTAL PROCEDURES |
Expression and Purification of the Zinc Finger Domain of Zic1,
Zic2, Zic3, and GLI3--
cDNAs corresponding to the zinc finger
domain of Zic1 (Zic1-ZF, amino acids 213-403),
Zic2 (Zic2-ZF, amino acids 243-434), Zic3
(Zic3-ZF, amino acids 238-425), and GLI3 (GLI3-ZF, amino acids 460-641) were prepared by polymerase chain reaction (PCR) using
the following primers and the mouse Zic1, Zic2, and
Zic3 (2) and human GLI3 cDNAs (17) as
templates: Zic1, 5'-CCGGATCCTTCTTCCGCTATATG-3' and
5'-CCGTCGACGACTCATACCCCGA-3'; Zic2, 5'-CCGGATCCTTTTTCCGCTACATG-3' and
5'-CCGTCGACGACTCGTAGCCAGA-3'; Zic3,
5'-CCGGATCCTTCTTCCGTTACATG-3' and
5'-CCGTCGACGATTCATAGCCTGAAC-3'; and GLI3, 5'-CCGGATCCCTTGTCAAGGAGGAA-3' and 5'-CCGTCGACTTCTTGGTGACATGAG-3'.
The resultant cDNA fragments were cut with BamHI
and SalI, and inserted into pGEX4T2 (Amersham Pharmacia
Biotech) or pQE-30 (Qiagen). The constructs were checked by DNA
sequencing. Bacterially expressed proteins were prepared and purified
according to the manufacturer's instructions. Glutathione
S-transferase (GST) fusion proteins were purified by
glutathione-Sepharose 4B (Amersham Pharmacia Biotech) without
denaturation. The histidine-tagged proteins were affinity-purified by
nickel-chelate affinity (Qiagen), denatured by urea, and subsequently
renatured in the purification process. The purified proteins were
finally dialyzed against a solution consisting of 25 mM
HEPES, pH 7.5, 50 mM KCl, 5 mM
MgCl2, 10 µM ZnSO4, 1 mM
dithiothreitol, 0.1% Nonidet P-40, and 12% glycerol and stored at
80 °C in small aliquots.
Electrophoretic Mobility Shift Assay (EMSA)--
EMSA was
performed in a binding buffer consisting of 25 mM HEPES, pH
7.5, 50 mM KCl, 5 mM MgCl2, 10 µM ZnSO4, 1 mM dithiothreitol, 0.1% Nonidet
P-40, 12% glycerol, 32P-end-labeled, double-stranded
oligonucleotides, 150 ng of poly(dI-dC), and 480 ng of the fusion
protein. The binding reactions (20 µl) were incubated at room
temperature for 30 min, resolved by 4% native polyacrylamide gel
electrophoresis for 60 min, and visualized by autoradiography.
Binding Site Selection and Mutational Analysis--
Initially,
DNA fragments consisting of random oligonucleotides flanked on each
side by a known sequence were generated by PCR amplification of R62AB
(5'-AGACGGATCCATTCAG(N)30TGTCAGGAATTCGGAC-3') using primers A1
(5'-AGACGGATCCATTCAG-3') and B1 (5'-GTCCGAATTCCTGACA-3') by 25 cycles
of PCR, in which each cycle consisted of denaturation at 94 °C (30 s), annealing at 52 °C (30 s), and extension at 72 °C (1 min).
The PCR products were purified on a CHROMA SPIN TE 30 column
(CLONTECH). A fraction of the PCR product was
32P-labeled with T4 polynucleotide kinase. Unincorporated
nucleotides were removed by column chromatography over a TE Micro
Select-D G-25 microcentrifuge spin column (5 Prime
3 Prime Inc.,
Boulder, CO).
After the first EMSA reaction, the bands corresponding to the
protein-DNA complexes, which were identified by autoradiography, were
excised from the dried gel and eluted into water for 1 h at room
temperature. The eluted DNA was amplified by PCR with the A1 and B1
primers and purified by the same procedure as described above. Four
additional cycles of EMSA, elution, and PCR amplification were
performed. After the fifth cycle, the PCR products were digested with
BamHI and EcoRI and subcloned into pBluescript
(Stratagene). Insert-containing clones were sequenced, and sequences
were analyzed by DNASIS (Hitachi Software).
For mutational analysis, double-stranded oligonucleotides were prepared
by annealing of complementary oligonucleotides, and an EMSA was
performed using 80 ng of GST-Zics-ZF or 8 ng of GST-GLI3-ZF protein.
Calculation of the Equilibrium Binding Constant--
The GLI3-BS
probe, which contains the entire optimized target sequence of GLI3
(31), was used to determine the Kd. The nucleotide
sequence of the probe was
5'-AGACGGATCCATTGCATCTGTGATTTTCGTCTTGGGTGGTCTCCCTCCTGTAGGAATTCGGAC-3', where the underline indicates the optimized target sequence flanked by
PCR primer sequences. The procedures for the PCR amplification and
radiolabeling of the probe were the same as those described above. The
following equilibrium equation was used to calculate Kd2 from the midpoint of the curves:
B = Bmax * F/(Kd + F), where B
and F are the concentration of the protein-DNA complex and
unbound probe, respectively. Bmax is the total
concentration of the protein.
Plasmid Construction--
A luciferase reporter plasmid driven
by a thymidine kinase (TK) promoter, TK-0GBS-Luc, was constructed by
ligating a herpes simplex virus TK promoter derived from plasmid pRL-TK
(Promega) into the HindIII and BglII sites of the
pGL2-Basic vector (Promega). Six tandem copies of the GLI-BS (32) were
inserted upstream of the TK promoter in TK-0GBS-Luc, giving rise to
TK-6GBS-Luc. A luciferase reporter plasmid driven by a major late
promoter of adenovirus, MLP-Luc, was constructed by removing the GAL4
binding sites from pG5Luc (Promega), and a luciferase reporter plasmid driven by an SV40 promoter, SV40-Luc (pGL2 promoter vector), was purchased from Promega. A luciferase reporter plasmid driven by a
cytomegalovirus (CMV) promoter, CMV-Luc, was constructed by inserting a
luciferase gene downstream of the CMV promoter region of the pCS2+
vector (33, 34), and a luciferase reporter plasmid driven by a
Zic1 promoter, Zic1-Prom-Luc, was constructed as described previously (SmaI (
148); Ref. 35). The internal standard,
pRL-EF, was constructed by inserting the renilla luciferase gene of a pRL-TK vector (Promega) into a pEF-BOS vector (36).
Expression vectors were constructed by inserting the cDNA fragments
containing the entire open reading frame of mouse Zic1, Zic2, and Zic3, human GLI1, or
GLI3 into the XbaI sites of pEF-BOS (36). The
expression vector Flag-GLI3 was constructed by placing the Flag
sequence in frame to the N terminus of human GLI3. In addition to the full-length constructs, Zic1-FS and Zic3-FS were made,
which included the entire cDNA fragments of mouse Zic1
and Zic3 with frameshift mutations to produce truncated
proteins lacking all five zinc fingers and C-terminal regions (Zic1-FS,
amino acids 1- 159; Zic3-FS, amino acids 1-153).
Transfection--
293T and C3H10T1/2 cells were maintained in
Dulbecco's modified Eagle's medium and basal medium Eagle's medium,
respectively, each containing 10% fetal bovine serum. At 60-70%
confluence, the cells in a 24-well dish were transfected with Effectene
(Qiagen) or LipofectAMINE Plus (Life Technologies, Inc.) transfection
reagent according to the manufacturer's instructions. Cells were
harvested 24 h after transfection and processed for reporter assay
and Western blot analysis.
Reporter Assay--
For 293T cells, cotransfection experiments
were performed with 90 ng each of the luciferase reporters and 100 ng
each of the expression constructs, together with 5 ng of pRL-EF as an
internal standard. C3H10T1/2 cells were cotransfected with 180 ng of
the luciferase reporters, 200 ng of the expression constructs, and 10 ng of pRL-EF. When the expression vectors for Zic and GLI were cotransfected, the total amounts of expression vectors were kept constant by adding an empty vector (pEF-BOS). Luciferase activities were measured according to the manufacturer's recommendation (Promega) using a Minilumat LB 9506 luminometer (Berthold). The firefly luciferase activity was normalized to the renilla luciferase activity obtained by cotransfection. The relative fold-activation was presented as the ratio of the normalized value to that from empty vector transfectant.
Western Blot Analysis--
293T cells were transfected with 1 µg of expression vector and harvested 24 h after transfection.
The cellular proteins were separated by 5-10% SDS-polyacrylamide gel
electrophoresis and transferred to Immobilon membranes (Millipore). The
membranes were blocked in 5% skim milk overnight and incubated with
anti-Zic monoclonal antibody (ZC26; Ref. 3), anti-GLI1 polyclonal
antibody (N16; Santa Cruz Biotechnology), anti-GLI3 polyclonal antibody (C20; Santa Cruz Biotechnology), and anti-Flag monoclonal antibody (M2;
Sigma). The bound antibodies were detected using horseradish peroxidase-conjugated anti-mouse IgG (Zic and Flag) and anti-goat IgG
(GLI1 and GLI3) and ECL reagents (Amersham Pharmacia Biotech).
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RESULTS |
Binding Site Selection of Zic Family Proteins--
To determine
the DNA sequence to which Zic proteins bind, we synthesized
oligonucleotides containing 30 contiguous random nucleotides flanked on
either side by a known sequence to facilitate PCR amplification and
subsequent cloning of the selected DNA sequence. The radiolabeled
random oligonucleotides were incubated with purified GST fusion protein
containing the zinc finger domain of three mouse Zic proteins (Zic1-ZF,
Zic2-ZF, and Zic3-ZF), DNA-protein complexes were separated on native
polyacrylamide gels, and the bands shifted by the proteins were
excised. After elution, the DNA was amplified by PCR and subjected to
four additional rounds of DNA-protein binding, elution, and PCR
amplification to enrich the Zic binding sequences. After a total of
five rounds of selection, the bound oligonucleotides were cloned and
sequenced (Fig. 1). The core sequence
most favored by Zic1-ZF, Zic2-ZF, and Zic3-ZF was nearly identical to
the 9-nucleotides GLI consensus sequence (GLI-BS), i.e.
5'-TGGGTGGTC-3'. The relative frequency of the appearance of each
nucleotide in the 9 positions indicated that all 5 guanosine residues,
2 thymidine residues at the fifth and eighth positions, and a cytidine
residue at the 3'-most position were preferred in common, whereas a
thymidine residue at the 5'-most position was not.

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Fig. 1.
Compilation of the Zic1 (30 clones), Zic2 (30 clones), and Zic3 (35 clones) binding sequences. DNA sequences
that bound to the Zics were selected as described under "Experimental
Procedures." After five rounds of selection, shifted oligonucleotides
were subcloned into pBluescript, and their sequences were determined.
The cloned random sequences are aligned to conform to the GLI consensus
sequence. Compromised sequences, which involve the nonrandom flanking
sequence in the 9 positions, are removed. The table at the
bottom summarizes the frequency with which each nucleotide
is represented in the 9 positions. The bottom sequence in
each table represents the compiled most favored sequence in each of the
nine nucleotide positions.
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However, there seemed to be no absolute requirement for any specific
nucleotide position. So we asked whether a different target sequence
might exist in strongly bound sequences. To test this, we selected
target sequences that gave stronger shifted bands than GLI3-BS. Among
13 sequences with high binding affinity to Zic1-ZF, there was no
conserved sequence other than GLI-BS (data not shown). This result
indicated that the GLI-BS-like sequence was uniquely preferred by the
Zics-ZF.
Identification of the Minimal Essential Binding Sequence for the
Zic Family Proteins--
Although all of the selected sequences could
be bound by the Zics-ZF, it was unclear whether the whole 9-nucleotide
sequence was necessary for binding. To determine the minimal essential nucleotide sequence for binding, a series of mutant sequences, each of
which contained a single-base substitution, were synthesized and
analyzed by EMSA using GST-Zics-ZF or GST-GLI3-ZF (Fig.
2). The M1 mutant was efficiently bound
by the Zics-ZF, suggesting that the mutation within the oligonucleotide
was not essential for binding. In contrast, mutants M2, M4, and M5
failed to bind. M3 and M6 were bound by Zics-ZF less efficiently than
the wild-type sequence. This analysis showed that the nucleotides
altered in M2, M4, and M5 and in part in M3 and M6, i.e.
5'-GGGTGGTC-3', contained the minimal essential binding sequence for
the Zic proteins. This result was consistent with the fact that a
thymidine residue at the 5'-most position was not strongly preferred by
the binding site selection. The GLI3-ZF exhibited generally similar
binding preference for the mutant binding sequences to the Zic proteins except for reduced dependence on the 3'-most cytidine residue.

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Fig. 2.
Mutational analysis of the binding sequence
of the Zics and GLI3. EMSA was performed with 200 fmol of labeled
oligonucleotides and either 8 ng of GST-GLI3-ZF (A) or 80 ng
of GST-Zic-ZF (B-D). The boxed uppercase letters
of the wild-type (WT) oligonucleotides indicate the
consensus GLI binding sequence (GLI-BS). The underlined lowercase
letters of the M1-M6 oligonucleotides indicate the
mutated nucleotides in each mutant oligonucleotide. B,
protein-bound; F, free.
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Differential Binding Affinities between Zic Family Proteins and
GLI3 to the Selected Binding Sequence--
Although the Zics-ZF and
GLI3-ZF exhibited a similar binding preference for the target
nucleotide sequence, there was an apparent difference in binding
affinity. To determine the relative binding affinity of the Zics-ZF and
GLI3-ZF, both proteins were incubated simultaneously with the GLI3-BS
probe, and the resultant complexes were analyzed by EMSA. To
discriminate the two kinds of fusion proteins, different tags were
used. The mixture consisting of GST-GLI3-ZF (10 nM) and
His-Zic2-ZF (0-2000 nM; Fig.
3A) or His-GLI3-ZF (20 nM) and GST-Zic2-ZF (0-500 nM; Fig.
3B) was reacted with the GLI3-BS probe and analyzed by EMSA.
Even though an excess concentration of Zic2 protein was used, the
intensity of the band shifted by the GLI3 protein (the complex formed
between GLI3-ZF and GLI3-BS) was not markedly changed in both
experiments. In contrast, the bands shifted by the Zic proteins were
almost completely removed by a lower concentration of GLI3 protein
(Fig. 3C). These findings confirmed that, although the Zic
family proteins specifically bound the GLI3-BS, the binding affinity
was much lower than that of GLI3-ZF, and Zics-ZF do not compete with
GLI3-ZF for the target sequence.

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Fig. 3.
Comparison of the accessibility to the
binding site between Zics and GLI3. In one experiment, a mixture
of the Zic2 and GLI3 proteins was added to an EMSA using the GLI3-BS
probe. A fixed concentration of GST-GLI3-ZF (10 nM) was
premixed for 30 min with increasing concentrations of His-Zic2-ZF
(0-2000 nM), followed by the addition of the probe
(A). Conversely, a fixed concentration of His-GLI3-ZF (20 nM) was mixed with increasing concentrations of GST-Zic2-ZF
(0-500 nM), followed by the addition of the probe
(B). In the other experiment, 500 nM GST-GLI3-ZF
or 1000 nM His-Zics-ZF alone or a mixture of 500 nM GST-GLI3-ZF and 1000 nM His-Zic-ZF was added
to an EMSA using the GLI3-BS probe (C). Shown at the
bottom are concentrations of fusion proteins used in the
experiments. The results indicate that Zic proteins never affected the
GLI3-DNA interaction.
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To more accurately determine the binding affinities of the Zics-ZF and
GLI3-ZF, we performed EMSA with a fixed concentration of the
GLI3-BS probe (1 nM) and increasing concentrations (1 nM-1 µM) of the fusion proteins (Fig.
4). Furthermore, we performed EMSA with a
fixed concentration (20 nM) of the fusion proteins and
increasing concentrations (0.75 -200 nM) of the probe (Fig. 5). Then the intensities of the bands
corresponding to the protein-DNA complexes were measured by
densitometry, and the binding curves were plotted (Fig.
6). In these assays, the GLI3-ZF showed
considerably higher binding affinity to the GLI3-BS
(Kd, 8.5 × 10
9
M; Bmax, 9.3 × 10
9 M) than the Zics-ZF
(Kd of Zic1-ZF, 5.2 × 10
8 M; Zic2-ZF, 4.8 × 10
8 M; and Zic3-ZF, 7.1 × 10
8 M;
Bmax of Zic1-ZF, 7.9 × 10
9 M; Zic2-ZF, 5.4 × 10
9 M; and Zic3-ZF, 7.7 × 10
9 M).

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Fig. 4.
Representative EMSA experiments. A fixed
limiting concentration of the GLI3-BS probe (1 nM) was
mixed with increasing concentrations of GST-GLI3-ZF (1 nM-1 µM; A) or GST-Zics-ZF (5 nM-1 µM; B-D), incubated for
3 h at room temperature, and then electrophoresed in a native
polyacrylamide gel. After autoradiography, the bands corresponding to
free (F) and bound (B) oligonucleotides were
counted by densitometry. The concentrations of GST-GLI3-ZF in the
reaction (A) were 0 nM (lane 1), 1 nM (lane 2), 2.5 nM (lane
3), 5 nM (lane 4), 10 nM
(lane 5), 50 nM (lane 6), 100 nM (lane 7), 250 nM (lane
8), 500 nM (lane 9), and 1000 nM (lane 10). The concentrations of GST-Zics-ZF
(B-D) were 0 nM (lane 1), 5 nM (lane 2), 10 nM (lane
3), 50 nM (lane 4), 100 nM
(lane 5), 250 nM (lane 6), 500 nM (lane 7), and 1000 nM (lane
8).
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Fig. 5.
Representative EMSA experiments. A fixed
limiting concentration of GST-GLI3-ZF (A; 20 nM), GST-Zic1-ZF (B; 20 nM),
GST-Zic2-ZF (C; 20 nM), or GST-Zic3-ZF
(D; 20 nM) was mixed with increasing
concentrations of GLI3-BS probe (0.75-200 nM), incubated
for 3 h at room temperature, and then separated by native
polyacrylamide gel electrophoresis. After autoradiography, the bands
corresponding to free (F) and bound (B)
oligonucleotides were measured by densitometry. The concentrations of
GLI3-BS in the reaction were 0.75 nM (lane 1),
1.5 nM (lane 2), 3 nM (lane
3), 6.25 nM (lane 4), 12.5 nM
(lane 5), 25 nM (lane 6), 50 nM (lane 7), 100 nM (lane
8) and 200 nM (lane 9).
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Fig. 6.
Determination of Kd
values of the GLI3-BS for the GST-Zics-ZF and GST-GLI3-ZF. The
free and bound oligonucleotides in the representative experiments shown
in Fig. 5 were counted and plotted. The Kd values
were calculated from the midpoints of the titration curves according to
the equation reported under "Experimental Procedures." The values
obtained were 8.5 × 10 9 M
(GLI3; A), 5.2 × 10 8
M (Zic1; B), 4.8 × 10 8 M (Zic2; C), and
7.1 × 10 8 M (Zic3;
D). The Bmax values obtained were
9.3 × 10 9 M (GLI3;
A), 7.9 × 10 9 M
(Zic1; B), 5.4 × 10 9
M (Zic2; C), and 7.7 × 10 9 M (Zic3; D).
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To test whether some of the DNA-protein complexes might dissociate
during electrophoresis in the gel, we also electrophoresed the
DNA-protein complexes for various times. However, there was no
significant alteration in the dissociation rate during gel electrophoresis (data not shown), indicating that the different Kd values reflect the differential affinities in the binding mixture.
Different Transcriptional Activities between Zic and GLI Proteins
Mediated through the Selected Sequence--
We next asked whether the
Zic proteins were capable of activating or repressing transcription
mediated by the selected binding sequence (Fig.
7). For this purpose, we prepared Zic,
GLI1, and GLI3 expression vectors under the control of an EF1
promoter. We used the firefly luciferase genes as reporters, controlled by the major late promoter of the adenovirus (MLP-Luc), which consists
of minimal promoter elements (a TATA box and a transcription initiation
site), the Zic1 promoter (Zic1-prom-Luc), the SV40 promoter (SV40-Luc),
and the CMV promoter (CMV-Luc). Luciferase activity was measured after
cotransfection into 293T cells. The firefly luciferase activity was
normalized to the activity of the cotransfected EF1
-driven renilla
luciferase, because the Zic and GLI expression vectors failed to
transactivate the EF1
promoter (data not shown). All Zic proteins
activated the reporter genes driven by the MLP, the Zic1 promoter, and
the SV40 promoter but failed to activate that driven by the CMV
promoter (Fig. 7A). On the other hand, neither GLI1 nor GLI3
proteins activated any of the promoters.

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Fig. 7.
A, transactivation of the MLP of
adenovirus, the Zic1-promoter, the SV40 promoter, and the CMV promoter
mediated by Zic and GLI. 293T cells were transfected with GLI and Zic
expression vectors along with reporter genes. Each value for luciferase
activity was normalized to the activity of an internal control (renilla
luciferase). Normalized values for luciferase activities (arbitrary
units) in pEF-BOS-transfected cells were 14 (MLP), 1.3 (Zic1-prom), 7.4 (SV40), and 75,000 (CMV). Normalized luciferase activities were divided by the
activity obtained by a transfection of the vector control pEF-BOS.
Shown are the means of fold-activation of the reporter activity from
duplicate samples. Error bars represent S.D. B
and C, transactivation of the TK promoter mediated by
Zic and GLI. 293T or C3H10T1/2 cells were transfected with GLI and Zic
expression vectors along with the reporter genes TK-0GBS-Luc and
TK-6GBS-Luc as indicated. Normalized values for luciferase activities
(arbitrary units) in pEF-BOS transfected cells were 210 (TK-0GBS,
293T; B), 37 (TK-6GBS, 293T; B),
220 (TK-0GBS, C3H10T1/2; C), and 64 (TK-6GBS, C3H10T1/2; C).
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To examine whether the GLI-BS affected the transcriptional activation
by Zic proteins, we used the firefly luciferase genes, controlled by
the TK promoter with or without six copies of the GLI-BS (TK-6GBS-Luc
and TK-0GBS-Luc). Zics always activated transcription driven by the TK
promoter irrespective of the presence of the GLI-BS in both 293T and
C3H10T1/2 cells (Fig. 7, B and C). The presence
of GLI-BS enhanced the reporter gene expression to 1.5-2.0-fold in
C3H10T1/2 cells, whereas in 293T cells, Zic1 or Zic2 enhanced expression to a lesser extent. In contrast, GLI1 markedly enhanced reporter expression, dependent on the presence of GLI-BS in both cell
lines (14-fold (293T) or 8-fold (C3H10T1/2) elevation in comparison
with TK-0GBS-Luc). GLI3 completely failed to activate the reporter gene
expression mediated by the GLI-BS in either cell type.
The fact that Zic proteins activated reporter gene expression
independent of the GLI-BS prompted us to ask whether the Zic gene or
mRNA may indirectly affect the transcriptional activation. To
address this possibility, we transfected Zic1-FS and Zic3-FS, which
contained the entire cDNA sequence of Zic1 and
Zic3, respectively, but produced truncated proteins without
all five zinc fingers. However, these constructs were not capable of
activating any of the reporter genes tested, demonstrating that Zic
proteins themselves could activate transcription (Fig. 7).
These findings show that the Zics have transcriptional activation
capacity, but this activity is less dependent on the GLI-BS than is
GLI1 activity. The different binding affinities to the target sequence
can account for the differential dependence on GLI-BS between the Zic
proteins and GLI1.
The Zic Family Proteins Cooperate or Interfere with the GLI in
Regulating Transcription--
We investigated whether Zic proteins
could affect the GLI-BS-dependent transactivation by GLI1.
Different relative amounts of the Zic and GLI1 expression vectors were
transfected into C3H10T1/2 or 293T cells to titrate this interaction
(Fig. 8). When Zics were coexpressed with
GLI1 in C3H10T1/2 cells, the GLI-BS-TK-driven luciferase expression was
significantly increased beyond that predicted by the summed luciferase
activities induced separately by the Zics and GLI1 (Fig.
8A). Increasing amounts of cotransfected Zics enhanced
reporter gene expression in a Zic dose-dependent manner.
For example, cotransfection with 100 ng of Zic2 increased the
GLI1-mediated transactivation by 3-fold. In contrast, when Zics and
GLI1 were cotransfected into 293T cells, there was a reduction in
reporter gene expression to less than that of Zic or GLI1 single
transfectants, possibly because of the interaction between Zics and
GLI1 (Fig. 8B).

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Fig. 8.
Coactivation or suppression of reporter gene
expression by Zics and GLI1 cotransfection depending on cell
context. C3H10T1/2 cells (A) or 293T cells
(B) were cotransfected with the GLI1 expression vector and
increasing amounts of Zic expression vectors along with a reporter
gene, TK-6GBS-Luc. In C3H10T1/2 cells, 100 ng of GLI1 and 20-100 ng of
Zic expression vectors were cotransfected (A), whereas 50 ng
of GLI1 and 10-50 ng of Zic were cotransfected in 293T cells
(B). Total amounts of expression vectors were kept constant
by adding the pEF-BOS vector. Shown at the bottom are
different combinations of expression vectors. Each value for luciferase
activity was normalized to the activity of an internal control (renilla
luciferase). Normalized values for luciferase activities (arbitrary
units) in pEF-BOS-transfected cells were 44 (C3H10T1/2) and
57 (293T). Normalized luciferase activities from duplicate
samples are presented relative to the empty vector pEF-BOS. Error
bars represent S.D.
|
|
We also carried out Zic and GLI3 cotransfection, although GLI3 failed
to activate or repress the TK-6GBS-Luc reporter gene (Fig.
9). Unexpectedly, even in this case, we
obtained results essentially similar to those of Zic-GLI1
cotransfection experiments. When cotransfection was performed in
C3H10T1/2 cells, the luciferase activities were increased. However, the
increased activities were nearly equal to the addition of the
luciferase activities induced separately by the Zics and GLI3 (Fig.
9A). On the other hand, in 293T cells, luciferase activities
were lower in Zic-GLI3 cotransfectants than in Zic single transfectants
(Fig. 9B). At a certain ratio of expression vectors
(Zics:GLI3 = 10:50 ng), the activities were even lower than that
from transfection of the empty vector. As a consequence, it became
clear that the synergistic activation or repression of reporter gene
expression is dependent on cell type for both GLI1 and GLI3. Comparable
protein expression levels were confirmed by Western blot analysis for
all of the transfections (Fig. 10),
indicating that the observed transcriptional synergism and interference
were not attributable to different efficiencies of expression of the
transfected genes.

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Fig. 9.
Suppression of reporter gene expression by
Zic and GLI3 cotransfection in 293T cells. C3H10T1/2 cells
(A) or 293T cells (B) were cotransfected with the
GLI3 expression vector and increasing amounts of Zic expression vectors
along with a reporter gene, TK-6GBS-Luc. In C3H10T1/2 cells, 100 ng of
GLI13 and 20 and 100 ng of Zic expression vectors were cotransfected
(A), whereas 50 ng of GLI3 and 10 and 50 ng of Zic were
cotransfected in 293T cells (B). Total amounts of expression
vectors were kept constant by adding the pEF-BOS vector. Shown at the
bottom are different combinations of expression vectors.
Each value for luciferase activity was normalized to the activity of an
internal control (renilla luciferase). Normalized values for luciferase
activities (arbitrary units) in pEF-BOS-transfected cells were 62 (C3H10T1/2) and 54 (293T). Normalized luciferase
activities from duplicate samples are presented relative to the empty
vector pEF-BOS. Error bars represent S.D.
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Fig. 10.
Western blot analysis of Zic and GLI
proteins in 293T cells. A, 293T cells were transfected
with Zic or GLI1 expression vectors or both. A portion of cell lysates
was subjected to Western blot analysis with anti-Zic and anti-GLI1
antibodies. The positions of molecular size markers are shown on the
left. B, 293T cells were transfected with Zic or
Flag-tagged GLI3 expression vectors or both. The Western blot analysis
was performed with anti-Zic and anti-Flag antibodies.
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DISCUSSION |
Zic Proteins as General Transcription Activators--
In the
present study, we were able to establish a consensus binding sequence
for Zics by EMSA-based target selection and mutational analysis. The
Zic binding sequence was essentially identical to the GLI-BS,
5'-TGGGTGGTC -3', and had a minimum consensus sequence of
5'-GGGTGGTC-3'. The binding affinities for this sequence were very
similar among the three Zic proteins examined. However, the Zics-ZF
bound the GLI-BS much more weakly than GLI3-ZF, as shown by competition
experiments and the calculated binding constant. The
Kd values of Zics were much higher than those of other transcription factors that function in a sequence-specific manner
(37, 38). Therefore, it is unlikely that Zic proteins compete with GLI
for the GLI-BS.
The binding properties were consistent with the results of the reporter
assay, in which the dependence of Zic proteins on the GLI-BS for
transcriptional activation was much less than that of GLI1. Instead,
Zics activated transcription even in the absence of the GLI-BS via
various promoters (TK promoter, adenovirus major late promoter, SV40
early promoter, and Zic1 promoter). On the basis of these facts, rather
than being the transcription factors that regulate transcription by
direct binding to DNA, Zic family proteins may function as
transcriptional coactivators, which potentiate the activity of other
transcription regulatory factors. It is possible that Zics interact
with the transcription machinery or other factors that regulate
transcriptional efficiency. An alternative possibility is that Zic
proteins might function in the post-transcriptional gene expression
processes. Because Zic proteins are localized in the cell nuclei (1,
3), they could be involved in RNA processing or transport from the
nucleus to the cytoplasm.
Although the Zics activated a variety of promoters, they had little
activity on the EF1
and CMV promoters. This may be related to
promoter strength, because, of the six promoters we tested, these two
promoters had the strongest activities (~10-1000-fold stronger than
other promoters tested). The transcriptional machinery, which is
required for activation by Zic proteins, might have already used by the
EF1
or CMV promoters alone. Further study is needed to elucidate the
domain required for the transcriptional activation in the Zic proteins
and to identify factors that interact with that domain to understand
the regulatory mechanism of gene expression by Zic proteins.
Context-dependent Regulation of GLI Function by Zic
Proteins--
We also examined the relationship between Zic and GLI
proteins. In C3H10T1/2 cells, Zic-GLI1 or Zic-GLI3 coactivated reporter gene expression, whereas in 293T cells, coexistence of the Zic and GLI
proteins had a reverse effect. These results suggest a significant
regulatory relationship between Zic and GLI proteins; however, the
nature of this interaction remains unclear.
The interaction between Zic and GLI proteins may be entirely
independent of DNA binding. This direct or indirect interaction between
Zic and GLI proteins may be modulated by cell type-specific cofactors.
One well characterized cell type-specific cofactor is Oct-binding
factor 1 (OBF-1), which is expressed in B-lymphocyte lineages
and interacts with the POU-homeodomain proteins Oct-1 and Oct-2 to
enhance transcriptional activation in the B-cell lineage (39-41).
Similar cell type-specific cofactors might modify the GLI-Zic
interactions. It is also possible that GLI proteins may be
differentially modified post-translationally depending on the presence
of Zics in different cell types. Recently, it was shown that Gli3 was
processed depending on cAMP-activated protein kinase to generate a
phosphorylated repressor form (32, 42). Zic proteins might be involved
in this pathway.
Alternatively, the differential binding affinities of the Zic and GLI
proteins for the target sequence may underlie the regulatory relationship between these two protein families. Although Zics-ZF had
much lower binding affinities to the GLI-BS, there is a DNA binding
transcription factor that has a Kd value similar to
those of Zics (43). Moreover, the different human homeodomain proteins,
despite having similar homeodomains, bind their target sequence with
different affinities and thereby generate a complex regulatory network
in the developmental process (44). In that case, less conserved domains
other than the zinc finger may modulate binding in vivo to
determine final binding specificity, because the recombinant proteins
used in these experiments only included zinc finger domains. It is
necessary to examine the downstream target genes in the developmental
context to understand the Zic-DNA interaction in detail.
Recently, it was demonstrated that Zic1/Gli3 double mutant
mice showed severe abnormalities of vertebral arches not found in
single mutants (45), strongly suggesting that these two proteins act
synergistically in the development of the vertebral arches. On the
other hand, it was shown in Xenopus laevis that Zic2
antagonized the Gli proteins in the patterning of the neural plate
(46). These findings suggest that Zic and GLI proteins may interact to
variously repress or activate gene expression in vivo. Our results may be related to these developmental phenomena.
In conclusion, Zic1, Zic2, and Zic3-ZF specifically recognized and
bound the GLI-BS but with a much lower binding affinity than that of
the GLI3-ZF. Zic proteins activated a wide range of promoters. These
results suggest that Zic proteins may function as transcriptional
coactivators or as factors generally involved in the gene expression
process. How can such general factors regulate specific developmental
processes, including the patterning of forebrain, cerebellum, axial
skeleton, vasculature, and visceral organs? A clue to solving this
problem may be the relationship with Gli family proteins as shown in
this study. To clarify the regulatory networks under a broad range of
developmental process, the relationships between Zic proteins and other
molecules in the hedgehog signaling pathway and transforming growth
factor
superfamily, which are closely related to each other, should also be examined in both in vitro and in vivo studies.