From the Divisions of Hazard Assessment and
¶ Health Effects Research, National Institute of Industrial
Health, 6-21-1, Nagao, Tama-ku, Kawasaki 214-8585 and the ** Department
of Environmental Toxicology, Faculty of Pharmaceutical Sciences, Teikyo
University, Sagamiko, Kanagawa 199-0195, Japan
Received for publication, January 22, 2001
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ABSTRACT |
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Transcription of mammalian metallothionein
(MT) genes is activated by heavy metals via multiple copies of a
cis-acting DNA element, the metal-responsive element (MRE).
Our previous studies have shown that certain MREs of the human MT-IIA
gene (MREb, MREc, MREd, and MREf) are less active than the others
(MREa, MREe, and MREg). Gel shift analysis of HeLa cell nuclear
proteins revealed that whereas the active MREs strongly bind the
transcription factor MTF-1 essential for metal regulation, the less
active MREs bind another distinct protein, MREb-BF. This protein
recognizes the GC-rich region of MREb rather than the MRE core required
for MTF-1 binding. All the MREs recognized by MREb-BF contain the CGCCC and/or CACCC motif, suggesting that the MREb-BF·MRE complex contains Sp1 or related proteins. Supershift analysis using antibodies against
Sp1 family proteins as well as gel shift analysis using the recombinant
Sp1 demonstrated that Sp1 represents the majority of MREb-BF activity.
An MREb mutant with reduced affinity to Sp1 mediated
zinc-inducible transcription much more actively than the
wild-type MREb. Furthermore, when placed in the native promoter, this mutant MREb raised the overall promoter activity. These
results strongly suggest that Sp1 acts as a negative regulator of
transcription mediated by specific MREs.
Heavy metal-induced transcriptional activation of the genes coding
for metallothioneins (MTs)1
is mediated by multiple copies of a cis-acting DNA element,
the metal-responsive element (MRE; Refs. 1-3). It has been shown that
the mouse MRE-binding transcription factor-1 (MTF-1) is essential for
MRE-mediated transcription from gene knockout (4) and antisense RNA
expression studies (5). Nevertheless, it remains possible that metal
regulation of MT genes involves additional transcriptional regulators.
A number of MRE-binding proteins have been reported (reviewed in Ref.
6), and cDNAs encoding MRE-binding proteins distinct from MTF-1
have been cloned (7, 8). However, no protein except MTF-1 has been
demonstrated to be functionally relevant to metal regulation.
MREs were first identified by deletion analysis in duplicated sites
upstream of the mouse MT-I (mMT-I) and human MT-IIA
(hMT-IIA) genes (1, 2, 9). After that, a search for
MRE-related sequences revealed that there are multiple MRE homologs in
the upstream region of all the mammalian MT genes reported so far (3,
10). These facts imply that such a redundancy is probably essential for
the high metal inducibility of MT gene promoters. However, MREs are not
always functionally equivalent. In transfection assays using reporter
genes driven by synthetic MRE-containing promoters, striking
differences in zinc-induced transcriptional activity were observed
among MREs of the mMT-I (3) and hMT-IIA (11)
genes, despite the observation that all these sequences share the
highly conserved MRE core (whose functional importance has been
demonstrated; Refs. 12, 13) and the flanking semi-conserved GC-rich
sequence. In addition, we have observed that the transcriptional activity of MRE does not always reflect its binding affinity to MTF-1;
certain MREs of the hMT-IIA gene are able to bind the
purified human MTF-1 in vitro, although they show no or very
low transcriptional activity in vivo (11). We had assumed
that additional transcriptional regulators might be responsible for
such a discrepancy and explored HeLa cell crude nuclear extracts for
another MRE binding activity. Here we show that a nuclear protein
distinct from MTF-1, which was finally proven to be the transcription
factor Sp1 (SV40 protein 1), binds to the less active MREs and probably
acts as a negative regulator of MRE-mediated transcription.
Cell Culture and Preparation of Extracts--
HeLa S3 cells
(CCL2.2) were cultured in Eagle's minimum essential medium
supplemented with 10% calf serum and non-essential amino acids at
37 °C under a 5% CO2 atmosphere. In experiments, medium
was supplemented with streptomycin (100 µg/ml) and penicillin (100 units/ml). Crude HeLa cell nuclear extracts (NEs) were prepared as
described previously (14), except for the following modifications. For
all buffer solutions, Hepes-Na, pH 8.0, and NaCl were replaced with
Hepes-K, pH 7.9, and KCl, respectively (KCl was adjusted to 0.1 M in Buffer 3), and dithiothreitol was added to 1 mM.
Proteins--
The human MTF-1 was purified ~2,000-fold from
HeLa cell NEs by the biotin-streptavidin affinity purification
procedure as previously described (15). Affinity-purified recombinant
Sp1 was purchased from Promega Japan, Tokyo.
Synthetic Oligonucleotides--
Preparation of double-stranded
synthetic oligonucleotides containing the hMT-IIA MREs were
described previously (11). Mutant MREb oligonucleotides (Figs.
4a and 8a, sequences) were prepared in the same
manner. All double-stranded oligonucleotides carry BamHI and
BglII sites (at the 5'- and 3'-ends, respectively), which
are convenient for multimerization and subcloning (11). An
oligonucleotide containing two copies of the Sp1 binding site was
previously described (16).
Electrophoretic Mobility Shift Assay (EMSA)--
DNA binding
activity of proteins was determined by EMSA. The MRE oligonucleotides
described above were end-labeled with T4 polynucleotide kinase and
[ Supershift Assay--
The affinity-purified rabbit polyclonal
antibody against human Sp1, Sp2, Sp3, and Sp4 were obtained from Santa
Cruz Biotechnology, Inc., Santa Cruz, CA. Each of these antibodies does
not cross-react with other members of the Sp1 family proteins. The
antibodies were added to EMSA reaction mixtures (2 µg immunoglobulin
G/reaction) prior to or after binding reaction and incubated at
25 °C for 20 to 30 min. Reactions were then analyzed by the standard
EMSA procedure as described above.
Plasmid Construction--
Plasmids with MRE-containing model
promoters were derived from pTKprCAT, which carries the bacterial
chloramphenicol acetyltransferase (CAT) gene driven by the
herpes simplex virus thymidine kinase (HSV-TK) gene promoter (17).
Construction of pTKprCAT variants with MREa or MREb tetramers placed
upstream of the TK promoter was previously described (11). The plasmid
containing the tetramer of an MREb mutant was prepared in the same manner.
A CAT reporter plasmid driven by the native hMT-IIA promoter
was constructed as follows. The HindIII/BamHI
fragment of pSV2CAT containing the CAT gene (18) was
subcloned between the HindIII and BamHI sites of
pUC19 to generate a plasmid pUC-CAT. The plasmid phMT-IIA, in which the
3-kilobase HindIII fragment containing the entire
hMT-IIA gene (19) had been subcloned, was linearized by
NcoI digestion and blunt-ended by Mung bean nuclease and
Klenow polymerase. HindIII linkers were then ligated to the
blunt ends. A fragment containing the hMT-IIA promoter
( Site-directed Mutagenesis--
Under the background of
pUC-MTCAT, the MREb sequence was converted to the mutant MREb m3
sequence (see Fig. 8a) using the Mutan-Express Km kit
(Takara Shuzo, Osaka, Japan). The PstI/HindIII fragment containing bases CAT Assay--
Plasmid constructs to be tested (10 µg
each/10-cm plate or 4 µg each/6-cm plate) and a reference plasmid
pRSVL (2.5 µg/10-cm plate or 1 µg/6-cm plate) were transfected into
HeLa cells by the standard calcium phosphate transfection procedure.
After 28 h, cells were further incubated with or without 100 µM ZnSO4 for 20 h. Cell extracts were
prepared, and CAT levels were determined by an enzyme-linked
immunosorbent assay (ELISA). CAT values were normalized relative to the
luciferase activity expressed by the reference pRSVL. Details of this
procedure were previously described (11).
A Nuclear Protein Distinct from MTF-1 Binds to Less Active
MREs--
In the upstream region of the hMT-IIA gene, there
are seven sites that contain sequences perfectly matching the MRE core
consensus (10) as indicated in Fig.
1a. Three of these MREs (MREa,
MREe, and MREg) efficiently mediate metal response of a reporter gene in transient transfection experiments and strongly bind the purified human MTF-1 in vitro (11), consistent with the idea that
MTF-1 plays the major role in metal-induced transcription (4, 5, 11).
However, the properties of certain MREs are quite different from these
active MREs (see Fig. 1a). In particular, MREb has extremely
low activity when compared with the three active MREs, although it is
responsive to heavy metals and is able to bind MTF-1 (11). On the other
hand, MREd and MREf do not respond to metals, although they appear to
bind MTF-1 weakly (11). To unambiguously determine the affinity of
MTF-1 with these less active MREs, we carried out competitive EMSA
using the affinity-purified human MTF-1 protein (15), a
32P-labeled MREa probe, and varied amounts of unlabeled
competitors each containing one of the MRE sequences. As shown in Fig.
1b, MREb competed with the MREa probe as efficiently as the
unlabeled MREa competitor, confirming that MTF-1 has a high binding
affinity to MREb, comparable with that of MREa. MREd and MREf also
competed with the MREa probe, although less efficiently than MREb.
These results demonstrated that MREb, MREd, and MREf are able to
interact with the purified human MTF-1 in vitro, despite low
or no activity for mediating metal-dependent transcription
in vivo. MREc, another MRE not responsive to zinc, showed no
affinity to human MTF-1 in similar competition experiments (data not
shown).
Based on these observations, we assumed that additional regulator
proteins could possibly interact with the less active MREs in
vivo, resulting in their unusual properties. To detect additional MRE binding activity, we analyzed HeLa cell crude nuclear extract by
EMSA using 32P-labeled probes, each containing one of the
seven hMT-IIA MREs (Fig. 2).
MREe and MREg formed zinc-inducible complexes co-migrating with the
MTF-1·MREa complex (lanes 10 and 14; compare
with uninduced controls in lanes 9 and 13; also
compare with MTF-1·MREa complex in lane 2), as expected
from our previous competition experiments (11). However, a strong band
migrating slightly slower than the MTF-1 complex was detected with the
MREb probe (lanes 3 and 4). Formation of this
complex was not affected by zinc. Complexes with identical mobility
were also detected for MREd (lanes 7 and 8) and
MREf (lanes 11 and 12), although the levels of
complex formation were lower than that observed for MREb. The protein that binds to MREb was designated MREb-BF and was analyzed further. Neither MTF-1 nor MREb-BF bound to MREc (lanes 5 and
6).
Properties of MREb-BF--
To determine the sequence recognition
specificity of MREb-BF, competition experiments were done using the
[32P]MREb probe and unlabeled oligonucleotides containing
the seven MREs (Fig. 3). In addition to
MREb (lanes 4 and 5), MREd (lanes 8 and 9), and MREf (lanes 12 and 13)
competed out the MREb-BF·[32P]MREb complex, suggesting
that an identical factor binds to these three MREs. Binding affinity to
MREb-BF was higher in the order of MREb, MREf, and MREd, consistent
with the levels of complex formation observed in Fig. 2. By contrast,
MREa (lanes 2 and 3), MREc (lanes 6 and 7), MREe (lanes 10 and 11) and
MREg (lanes 14 and 15) did not compete with the
MREb probe, indicating that these are not the target sites of MREb-BF.
We then tested two additional competitors containing mutated MREb (Fig.
4a), to locate the bases essential for MREb-BF binding within the MREb sequence. The mutant m1
has clustered base substitutions within the MRE core sequence that is
important for MRE function and MTF-1 binding (11, 13). The other mutant
m2 has mutations in the adjacent GC-rich region. As shown in Fig.
4b, m1 competed with the MREb probe as efficiently as the
wild-type MREb (lanes 4 and 5; compare with
lanes 2 and 3), but m2 showed no competition
(lanes 6 and 7). These results indicate that the
GC-rich region of MREb, not the MRE core, is recognized by MREb-BF. We
noted that the GC-rich region of MREb is quite similar to the consensus
recognition site of the transcription factor Sp1 (Ref. 20; see Fig.
5). Therefore we also tested a competitor
containing two copies of the Sp1 binding site (GC box). In fact, this
oligonucleotide strongly competed with the MREb probe (lanes
8 and 9).
In addition to MREb, MREd and MREf also contained subregions that
resemble the Sp1 consensus sequence, although they are less similar
than that in MREb (Fig. 5). Moreover, MREd and MREf contain the CACCC
motif that is known as another recognition site for the Sp1 family
proteins (21, 22); these nucleotides are marked by asterisks
in Fig. 5. To confirm the binding of Sp1 to these MREs, we carried out
a supershift analysis using an Sp1-specific antibody (Fig.
6a). The human MTF-1·MREa
complex was not affected by this antibody (lane 2; compare
with lane 1). By contrast, the MREb-BF complexes with MREb,
MREd, and MREf probes were all supershifted (lanes 4,
6, and 8; compare with lanes 3,
5, and 7, respectively). These results strongly
suggest that Sp1 is responsible for the MRE-BF DNA binding activity.
Because Sp1 family proteins are known to have recognition sequence
specificity similar to that of Sp1 (21, 22), we further tested
reactivity of the MREb-BF complex with antibodies against other Sp1
family proteins including Sp2, Sp3, and Sp4 (Fig. 6b).
Antibodies against Sp2 and Sp4 did not supershift the MREb-BF complex
(lanes 3 and 5, respectively). Anti-Sp1 antibody
supershifted the majority of the MREb-BF complex (lane 2),
but a part of the complex always remained unshifted. Even when the
antibody was added in a large excess, this population was not shifted
(data not shown). We observed that anti-Sp3 antibody supershifted a
small part of the MREb-BF complex (lane 4). In addition,
when both anti-Sp1 and anti-Sp3 antibodies were simultaneously added to
the binding reaction, almost all of the MREb-BF complex was
supershifted. These results indicate that the MREb-BF complex contains
Sp1 as a major component and Sp3 as a minor component.
We further examined direct interactions of the purified MTF-1 and Sp1
proteins with the MREa and MREb sequences. As shown in Fig.
7, the purified human MTF-1 bound both
MREa and MREb only in the presence of zinc (lane 4 in the
upper and lower panels, respectively; compare
with lane 3). The level of complex formation was similar for
both complexes, consistent with the results of competition experiments
shown in Fig. 1b. On the other hand, the affinity-purified
recombinant Sp1 protein binds to MREb, either in the absence or
presence of zinc (lanes 5 and 6 in the
lower panel, respectively). The mobility of this complex was
identical with that of MREb-BF (Fig. 2). By contrast, Sp1 formed no
complex with MREa (lanes 5 and 6 in the
upper panel). These results demonstrate that Sp1
specifically recognizes the MREb sequence in the same manner as MREb-BF
does (Fig. 3).
Sp1 Probably Acts as a Negative Regulator of MRE-mediated
Transcription--
Competition between Sp1 and MTF-1 for binding to
certain MREs could possibly be an important mechanism involved in the
metal-regulated transcription of MT genes. To approach this
possibility, we prepared and screened several MREb mutants to obtain
one that is normal in MTF-1 binding but defective in Sp1 binding.
Consequently, we were able to obtain a mutant satisfying those
criteria, m3, that carries a single C
The mutant m3 was then assayed for the ability to mediate
zinc-inducible transcription (Fig. 9).
Four direct repeats of MREa, wild-type MREb, and m3 oligonucleotides
were inserted upstream of the HSV-TK promoter in the pTKprCAT plasmid,
respectively. These constructs were introduced into HeLa cells, and
reporter CAT gene expression was monitored after incubation
with or without 100 µM ZnSO4 for 20 h.
The wild-type MREb responded to zinc, but the level of CAT expression
was extremely low when compared with MREa both in the absence and
presence of zinc. However, the single base change in the mutant m3
dramatically increased both induced and uninduced reporter gene
expression, the level of which is almost comparable with that of MREa.
These results strongly suggest that the defect in Sp1 binding confers
high transcriptional activity on MREb.
We further examined the effect of m3 mutation on the overall
hMT-IIA promoter activity (Fig.
10). Plasmids that carry the wild-type or mutated hMT-IIA gene upstream sequence ( Binding of Sp1 to MREs--
Our data demonstrate that Sp1
represents the majority of the MREb-BF DNA binding activity. Sp1 was
originally identified as a transcriptional activator that recognizes
the sequence containing the core CGCC motif (GC box) located in the
early promoter of SV40 as well as many other viral and cellular
promoters (23-26). However, the target site of Sp1 is not restricted
to the GC box; Sp1 can bind to the CACCC motif (21, 22, 27), which is
known as a regulatory element in a variety of promoter and enhancer sequences (28-31) as well as other sequences (32-34). MREb contains a
sequence highly homologous to the GC box consensus sequence (Fig. 5),
and mutations within this site abolish Sp1 binding (Figs. 4 and 8),
indicating that Sp1 recognizes the GC box region of MREb. The other two
Sp1-binding MREs, MREd and MREf, also have GC box-like sequences, but
they are less similar to the GC box consensus sequence (Fig. 5).
However, these two MREs also contain another Sp1 recognition site, the
CACCC motif. It remains to be determined which motif in these MREs is
important for recognition by Sp1.
Negative Transcriptional Regulation by Sp1--
The MREb mutant
defective in Sp1 binding exhibited a much higher transcriptional
activity than wild-type MREb in a simple model promoter (Fig. 9).
Furthermore, when placed under the native hMT-IIA promoter
background, this mutant MREb raised the overall promoter activity (Fig.
10). These findings strongly suggest that Sp1 could act as a negative
regulator by competing with the positive regulator MTF-1 for binding to
particular MREs. Although the Sp1 recognition site was originally
regarded as a positive regulatory element (23, 26, 35), reports
suggesting their negative regulatory roles have recently been
published. Several DNA elements identified as those responsible for
negative gene regulation contain GC-rich motifs, and the binding of Sp1
or closely related proteins to them is likely to be involved in
down-regulation (34, 36-40). Transcriptional repression mediated by
the Sp1 binding sites appears to occur through several different
mechanisms. Proteins that bind to the sequences overlapping certain Sp1
sites appear to competitively inhibit Sp1 binding thereby abolishing
transactivation by Sp1 (37, 41, 42). Alternatively, Sp1 transactivation
could be inhibited by proteins that directly interact with Sp1
(43-45). In either case, Sp1 acts as a positive regulator, and other
proteins appear to play roles in negative regulation. In contrast to
this, DNA-binding of Sp1 itself could exert a negative effect (34). In
MREb-mediated transcription, Sp1 appears to act in such a manner, namely its binding to MREb directly leads to transcriptional
repression. In this particular case, however, the negative effect is
likely to result from the competition between Sp1 and MTF-1. This is the first example that suggests the negative regulatory role of Sp1
through direct competition with another transcriptional activator.
In the hMT-IIA gene promoter, an Sp1 site located between
MREa and MREb is known to be essential for promoter activity, both in vivo and in vitro (35, 46). These observations
together with our present results suggest that Sp1 could act either
positively or negatively, depending on its binding sites within the
same promoter. There have been reports on the dual function of Sp1 that
are similar to this (34, 39). It is curious to consider why DNA binding
of a single transcription factor could result in apparently opposite
effects. Positional effects exerted by surrounding DNA sequences might
be important. Alternatively, the DNA sequence itself could directly
affect the function of DNA-binding proteins through allosteric effects
(47). It has been reported that the phosphorylated form of Sp1 is
transcriptionally active (48). This finding raised the possibility that
the non-phosphorylated, inactive form of Sp1 could act negatively on
MRE-mediated transcription. This point remains to be studied.
It has been suggested that the level of Sp1 expression could directly
affect the tissue-specific expression of particular genes (49, 50). It
is possible that in tissues with high levels of Sp1 expression or in
cells with increased Sp1 levels induced by certain extracellular
stimuli, Sp1 displaces MTF-1 at certain MREs such as MREb, and
consequently represses MT gene expression. Conversely, at low Sp1
levels MTF-1 might bind to these MREs, thereby increasing the level of
MRE-mediated transcription. Functional modification of pre-existing Sp1
could also yield similar results. However, it remains to be
demonstrated whether differential Sp1 levels among tissues or during
induction could directly modulate MT gene transcription. Because
multiple copies of MRE in model promoters act synergistically in
transcriptional activation (11), the binding of MTF-1 to particular
MREs could greatly affect the overall promoter activity and could be a
regulatory switch. The data shown in Fig. 10 are consistent with this idea.
Several proteins related to Sp1 have been cloned (21, 22). Sp3 and Sp4
bind to both the CACCC motif and GC box with similar affinity and
specificity (21, 22). Sp4 was shown to be a transcriptional activator
(51), whereas Sp3 appears to be a transcriptional repressor that binds
competitively to common target sites with Sp1 or Sp4 (41, 42, 51). It
was also suggested that the relative levels of Sp1 and Sp3 could
determine specific gene expression (49, 50). From our supershift
analysis using specific antibodies against Sp1 family proteins, the
majority of protein components contained in the MREb-BF complex is Sp1,
and Sp3 was detected as only a minor component. These findings suggest
a major role for Sp1 in negative regulation, although Sp3 may also have
a contribution.
Although a lot of MRE-binding proteins have been reported since MRE was
recognized as an element mediating heavy metal-activated transcription
(6-8), no proteins with the exception of MTF-1 have been demonstrated
to be involved in MRE-mediated gene regulation (4, 5). Our present
results strongly suggest that Sp1 acts as the second regulator of
MRE-mediated transcription by recognizing only particular MREs and
repressing MTF-1 transactivation through competition for binding to
the overlapping recognition sites.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and used as probes. The standard reaction
mixture (12.5 µl) contained 10 mM Hepes-K, pH 7.9, 2 mM MgCl2, 50 mM KCl, 16 mM NaCl, 10 mM dithiothreitol, 10% glycerol,
0.2 mg/ml bovine serum albumin, 80 µg/ml poly(dI-dC), 15 fmols
[32P]MRE probe. Proteins (NE or purified proteins;
amounts are indicated in the figure legends) were incubated in the
reaction mixture at 25 °C for 20 min and were electrophoresed in a
5% polyacrylamide gel in a buffer containing 22 mM Tris
and 22 mM boric acid for 1.5 h. Protein/DNA complexes
were detected by autoradiography.
767 to +71) was excised with HindIII and cloned into the
HindIII site of pUC-CAT. The upstream HindIII
site was eliminated by end-filling and religation to facilitate later
mutagenesis manipulations. This plasmid was designated pUC-MTCAT.
624 to +71 of the hMT-IIA gene
was excised and subcloned into a mutagenesis vector pKF-19k, and was subjected to in vitro mutagenesis according to the
manufacturer's instructions. The mutated MRE-containing fragment was
cloned back to the original pUC-MTCAT, and was inspected by dideoxy sequencing.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Binding of human MTF-I to MREs of the
hMT-IIA gene. a, MREs located upstream of
the hMT-IIA gene. The location and orientation of MREs are
indicated by arrows: black arrow, zinc responsive
MRE with high activity; gray arrow, zinc-responsive MRE with
low activity; white arrow, MRE not responsive to zinc (11).
MRE and flanking sequences contained in the oligonucleotides used as
the probe and/or competitors in b are shown below the map.
The MRE sequences are underlined. b, interactions
of human MTF-1 with the less active MREs. Binding of the purified human
MTF-1 (1 µl; Ref. 15) to MREb, MREd and MREf was estimated by
competitive EMSA using a [32P]MREa oligonucleotide probe.
Competitor oligonucleotides containing the MREa, MREb, MREd, or MREf
sequence (labeled a, b, d, and
f, respectively) were added to binding reactions containing
100 µM ZnSO4 as indicated (in a 20-, 30- or
50-fold molar excess). The MTF-1 complex bands on the autoradiogram
were quantified by densitometry, and each value was normalized relative
to the control included in every assay (with zinc and without
competitor; taken as 100). Data compiled from six independent
experiments are shown. Controls without competitor are indicated at the
left; , without metal; +, with 100 µM
ZnSO4. Each column represents the average of
2-4 values from independent experiments with S.E.
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Fig. 2.
Detection of MRE-binding proteins in HeLa
cell nuclear extract. HeLa cell NE (2 µg) was analyzed by EMSA
using 32P-labeled oligonucleotide probes each containing
one of the seven MRE sequences (MREa to MREg; labeled a to
g, respectively) in the absence ( ) or presence (+) of 100 µM ZnSO4. F indicates the free
probe.
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Fig. 3.
Interaction of MREb-BF with
hMT-IIA MREs. The affinity of MREb-BF to each of
the seven hMT-IIA MREs was estimated by competitive EMSA
using HeLa cell NE (2 µg), [32P]MREb probe and
unlabeled competitors each containing one of the MRE sequences (MREa to
MREg, labeled a to g, respectively; in a 30- or
100-fold molar excess). F indicates the free probe.
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Fig. 4.
Nucleotides required for MREb-BF binding.
a, sequences of oligonucleotides containing MREb mutants.
Clustered base substitutions introduced into MREb mutants m1 and m2 are
indicated below the wild-type MREb sequence. b, competitive
EMSA. Binding affinity of MREb-BF to the MREb mutants and GC box was
examined by competitive EMSA as in Fig. 3, using HeLa cell NE (2 µg),
[32P]MREb probe, and the competitors (in a 30- or
100-fold molar excess). The DNA sequence of the oligonucleotide
containing two copies of the GC box was previously described (16).
F indicates the free probe.
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Fig. 5.
Sequence similarity of MREb, MREd, and MREf
to the Sp1 recognition sites. The consensus Sp1 binding sequence
(20) is indicated at the top. MREs are
underlined. Bases matching the Sp1 consensus are indicated
by dots. Asterisks indicate the CACCC
motif.
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Fig. 6.
a, supershift analysis of interactions
between Sp1 and MREs. EMSA reaction mixtures containing HeLa cell NE (2 µg) were incubated with (+) or without ( ) an anti-Sp1 antibody for
25 °C, 30 min followed by a binding reaction with the addition of
32P-labeled oligonucleotide probes. MREa, MREb, MREd, and
MREf probes were added as indicated (labeled a,
b, d, and f, respectively). The
arrowhead indicates the supershifted band. The
arrow indicates the slot position. F indicates
the free probe. b, interaction of Sp1 family proteins with
MREb. After the standard EMSA binding reaction with
[32P]MREb probe and HeLa cell NE (2 µg), reaction
mixtures were further incubated without antibody (control)
or with antibody against Sp1, Sp2, Sp3, or Sp4, or their combination at
25 °C for 20 min before electrophoresis. The arrowhead
shows the supershifted band. The arrow shows the slot
position; note that supershifted signals can be seen also at this
position. F indicates the free probe.
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Fig. 7.
Binding of purified MRE-binding proteins to
MREa and MREb. Interactions of the purified human MTF-1 (1 µl/reaction) and Sp1 (0.125 footprint units/reaction) with
32P-labeled MREa and MREb oligonucleotides were examined by
EMSA in the absence ( ) or presence (+) of 100 µM
ZnSO4. Only the regions of the autoradiogram containing the
MTF-1 and Sp1 complexes are shown; no other complex was detected by
this assay.
G base change within the
GC-rich region (Fig. 8a) When
assayed by competitive EMSA (Fig. 8b), m3 failed to compete with the [32P]MREb probe (lanes 4 and
5), under a condition where the wild-type MREb competitor
almost completely competed out the labeled MREb·Sp1 complex (Fig.
8b, lane 3; compare with control without
competitors in lane 1). This result indicates that m3 almost
completely lacks affinity for Sp1. By contrast, when assayed for
MTF-1-binding with the [32P]MREa probe (Fig.
8c), m3 competed with the probe (lanes 4 and 5) as efficiently as MREa and as wild-type MREb (lanes
2 and 3, respectively), indicating that the affinity of
m3 for MTF-1 remains intact. These data demonstrate that the MREb
mutant m3 has a defect in Sp1 binding but not in MTF-1 binding.
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Fig. 8.
Binding of Sp1 and MTF-1 to an MREb mutant
with a single base change in the GC-rich region. a,
nucleotide sequence of the MREb mutant m3. The mutated base in the MREb
variant is indicated below the sequence of the wild-type
MREb. b, binding affinity of Sp1 to m3. Protein-DNA
interactions were assayed by competitive EMSA as described in the
legend to Fig. 3. Reactions contained HeLa cell NE (2 µg) and
32P-MREb probe. Competitors (MREa, MREb, and m3; labeled
a, b, and m3, respectively) were added
to the reactions as indicated. F indicates the free probe.
c, binding affinity of human MTF-1 to m3. Protein-DNA
interactions were assayed as in b except that reactions
contained [32P]MREa probe and 100 µM
ZnSO4.
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Fig. 9.
Transcriptional activity of the MREb mutant
m3. Four direct repeats of MREa, MREb, and m3, respectively, were
inserted upstream of the HSV-TK gene promoter in the pTKprCAT reporter
plasmid. These constructs were introduced into HeLa cells (in 10-cm
dishes) by calcium phosphate transfection in duplicates. After
incubation with (open columns) or without (filled
columns) 100 µM ZnSO4 for 20 h, CAT
levels in cell extracts were determined by ELISA. Values were
normalized relative to the CAT level in cells transformed with the MREa
construct and induced by zinc (taken as 100). The average values with
S.E. from four independent experiments are shown.
767 to +71)
linked to the CAT reporter gene were constructed, and tested
for their transcriptional activity as in Fig. 9. In the mutant plasmid, MREb was replaced with m3 by site-directed mutagenesis. The wild-type promoter showed a typical zinc-inducible nature of the MT gene promoter
(left columns), which represents the combined effect of
multiple MREs. The transcriptional activity of the mutant promoter was
significantly higher than the wild type both under uninduced (3.1-fold)
and zinc-induced (2.2-fold) conditions (right columns). These results demonstrate that Sp1 binding to MREb clearly has a
negative effect under the native promoter background.
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Fig. 10.
Effect of MREb mutation on the overall
transcriptional activity of the hMT-IIA promoter.
Plasmids carrying the wild-type or mutant hMT-IIA promoter
( 767 to +71) linked to the CAT gene were constructed. The
mutant promoter carried MREb mutant m3 (see Fig. 8) instead of the
wild-type MREb. These plasmids were tested for transcriptional activity
as in Fig. 9, except that cells plated in 6-cm dishes were used. Values
were normalized relative to the CAT level in cells transformed with the
wild-type construct and induced by zinc (taken as 100). The average
values with S.E. from three independent experiments are shown.
Constructs: w, wild type; m, mutant.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid from the Science and Technology Agency, Japan.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.
§ Present address: Faculty of Pharmaceutical Sciences, Chiba University, 1-33, Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Present address: Dept. of Molecular Genetics, Alton Ochsner
Medical Foundation, 1516 Jefferson Highway, New Orleans, LA 70121.
To whom correspondence should be addressed. Tel.:
81-44-865-6111; Fax: 81-44-865-6116; E-mail: koizumi@niih.go.jp.
Published, JBC Papers in Press, February 6, 2001, DOI 10.1074/jbc.M100570200
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ABBREVIATIONS |
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The abbreviations used are: MT, metallothionein; MRE, metal-responsive element; MTF-1, MRE-binding transcription factor-1; mMT-I, mouse MT-I; hMT-IIA, human MT-IIA; NE, nuclear extract; EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol acetyltransferase; HSV-TK, herpes simplex virus thymidine kinase.
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REFERENCES |
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