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INTRODUCTION |
Opioids are widely used as analgesics following major surgery and
to alleviate the pain of terminal cancer. However, chronic administration results in the development of tolerance and dependence, which limits their usages. Opioids interact with three major types of
opioid receptors (µ,
, and
), all of which belong to the G-protein-coupled receptor superfamily (1). Based on the use of
selective agonists and antagonists, the µ-opioid receptor
(mor)1 has
traditionally been considered the main site of interaction of the major
clinically used analgesics, particularly morphine (2). The critical
role of mor in analgesia, as well as in the development of
tolerance and dependence, has been confirmed by pharmacological
analysis of "knock-out" mice, in which this receptor is eliminated
(3-6).
Mor is mainly expressed in the central nervous system;
different regions of the central nervous system have different
densities of receptors, which may play different roles (7-13).
Therefore, understanding how transcription of the mor gene
is regulated is critical. The mouse mor gene is over 53 kb
long with the coding sequence dividing into four exons (14, 15). The
existence of dual TATA-less promoters (distal and proximal) was
reported (16, 17), with the proximal promoter predominant in directing mor gene expression in mouse brain (16). Therefore, we have begun focusing on the regulatory process governing the transcription of
the mor gene within the proximal promoter.
Two cis-acting elements located in the proximal promoter
region were found to be critical for its activity: an inverted-GA (iGA)
motif and a canonical Sp1 binding site. Both elements formed complexes
with Sp factors, which were important for the proximal promoter
activity (18). In addition to these two cis-acting elements,
the mor proximal promoter region also possesses a 26-bp sequence of polypyrimidine/polypurine (PPy/u) nucleotides, which is
highly conserved in the mor gene among different species
(mouse, rat, and human) (15, 19, 20). The region encompassing the mor PPy/u sequences (
340 to
400 bp) is positioned
adjacent to the transcriptional start sites and is essential for
mor proximal promoter activity (18). Such PPy/u sequences,
varying in length, are over-represented in the genome (21-23), and
have been shown to play important functional roles in several promoters
(24-30). Several lines of evidence suggest that PPy/u sequences
possess the potential to form a non-B DNA conformation, such as the
melted DNA or H-form (an intramolecular triplex) (24, 25, 31), which
may play a role in DNA replication, transcription, and recombination in
a positive or negative manner (24-34).
Numerous studies have suggested the possible role of PPy/u sequences in
transcription. Deletion analysis of various promoters, such as those
for Drosophila hsp26 (35), c-Ki-Ras (36, 37), EGFR (38), and
c-Myc (30, 39), have shown that PPy/u sequences are essential for
promoter functioning. Several PPy/u DNA-binding proteins have been
described, including BPG1, NSEP-1, MAZ, nm23-H2, and Pur-
(40-45).
Additionally, single-stranded (ss) DNA-binding proteins, that function
as suppressers or activators, have also been identified (32-34,
46-54). For example, heterogeneous nuclear ribonucleoprotein K (hnRNP
K) specifically binds to a pyrimidine-rich ss DNA of the human
c-myc promoter and trans-activates a
heterogeneous promoter containing this binding sequence (51, 52).
Another example is a cellular nucleic acid-binding protein, a
19-20-kDa zinc finger protein, which recognizes a purine-rich ss
sequence and acts as an activator (54). Several reports have also
suggested that ss DNA-binding proteins cooperating with other
transcription factor(s) can participate and play a role in cell-type
specificity (47, 50), and are also involved in basal and drug-regulated activity (48-50). The presence of PPy/u sequences in the proximal promoter region of the mouse mor gene implies that
regulation of mor gene expression may also involve ss
DNA-binding protein.
We tested this hypothesis in the present study. We report here that the
26-bp PPy/u region of mor proximal promoter is capable of
forming ss DNA conformation (non-B form). A major ss DNA-binding protein, termed mPy protein (mor
polypyrimidine-binding protein), that is ~25 kDa in size
can bind to the sense strand of the PPy/u region with high affinity.
Electrophoretic mobility shift assay (EMSA) further revealed that mPy
protein is unrelated to Sp factors, which are known to be important for
the mor proximal promoter activity (18). Transfection and
mutation analyses suggest that mPy protein can
trans-activate the mor promoter as well as a
heterologous promoter. In summary, our results suggest that
transcription of mouse mor gene is regulated by a previously
unrecognized interplay of ss (mPy) and ds (Sps) DNA binding factors,
which interact with an overlapping region of DNA (PPy/u region).
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MATERIALS AND METHODS |
Plasmid Construction--
Luciferase fusion plasmid, pL450
construct, containing
450 to
249 bp upstream regulatory sequence
(related to the translation start site as +1) of the mouse
mor gene, was described previously (16, 18). The pL340/300
construct (18), containing
340 to
400 bp fragment, was generated by
polymerase chain reaction (PCR). The sequences of PCR products were
confirmed by sequencing, and then subcloned into polylinker sites of a
promoterless luciferase vector, pGL3-basic (Promega). The point
mutation was introduced by PCR into pL340/300 to generate pLGGT and
pLcsp. For pLGGT, a mutagenic oligonucleotide containing three base
substitutions (underlined) (5'-CTC CTT CTC TCT CCT GGT TCC
CCT CT-3') was used. For pLcsp, the mutagenic oligonucleotide
containing the consensus GC box sequences at 3'-region (underlined)
(5'-CTC CTT CTC TCT GAT CGG GGC
GGG GC-3' was used. Constructs carrying PCR
mutations were sequenced across the entire promoter region to ensure
introduction of base changes at only the intended positions. Mutant
fragments with correct sequences were then subcloned into
pGL3-basic.
To prepare a promoter construct containing the 26 bp of mor
PPy/u element positioned upstream of the SV40 promoter, pairs of
oligonucleotides containing the wild type or GGT mutation were prepared
and annealed. Wild type oligonucleotides were as follows: upper, 5'-CTC
CTT CTC TCT CCT CCC TCC CCT CT-3'; lower, 5'-AGA GGG GAG GGA GGA GAG
AGA AGG AG-3'. GGT mutation oligonucleotides were as follows: upper,
5'-CTC CTT CTC TCT CCT GGT TCC CCT CT-3'; lower, 5'-AGA GGG GAA CCA GGA
GAG AGA AGG AG-3'. The resulting double-stranded oligonucleotide was
inserted into the polylinker site of pGL3-promoter (Promega),
containing the SV40 promoter.
Cell Culture--
Human neuroblastoma SH-SY5Y cells were grown
in RPMI 1640 medium with 10% heat-inactivated fetal calf serum in an
atmosphere of 5% CO2 and 95% air at 37 °C.
Transient Transfection and Reporter Gene Activity
Assay--
SH-SY5Y cells were transfected using the Superfect (QIAGEN)
lipofection method as described previously (16). Briefly, cells with
~40% confluence were transfected with an equimolar amount of each
test plasmid. The amount of DNA used was within the linear range of the
relationship between the luciferase activity and the amount of DNA.
Forty-eight hours after transfection, cells grown to confluence were
washed and lysed with lysis buffer (Promega). To control for
differences in transfection efficiency from dish to dish, 0.2 molar
ratio of pCH110 plasmid (Amersham Pharmacia Biotech) containing the
-galactosidase gene driven by the SV40 promoter was included in each
transfection and used for normalization. All transfection experiments
were repeated three times or more with similar results, utilizing
constructs that were independently prepared at least twice. The
luciferase and
-galactosidase activities of each lysate were
determined as described by the manufacturers (Promega and Tropix, respectively).
S1 Sensitivity and S1 Fine Mapping--
The mor
proximal promoter containing plasmid was digested with various amounts
of S1 nuclease (Life Technologies, Inc.) in S1 buffer for 15 min at
37 °C. S1 nuclease digestion was terminated by phenol/chloroform
extraction and the plasmid recovered by precipitation. The resulting
S1-treated plasmid was then further digested with XbaI
restriction enzyme, and the products resolved by agarose gel
electrophoresis. The regions of S1 nuclease sensitivity were determined
by gel purification of the resulting S1 and XbaI restriction digestion fragments, following by PCR reaction using the
32P-labeled primer complementary to each strand, and then
resolved in sequencing gel.
Nuclear Extract Preparation--
Nuclear extracts were prepared
from SH-SY5Y cells using the method described by Johnson et
al. (55). Briefly, cells were grown to confluence, harvested, and
washed with phosphate-buffered saline. All of the following steps were
performed at 4 °C. The cells were resuspended in sucrose buffer
(0.32 M sucrose, 3 mM CaCl2, 2 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol (DTT),
0.5 mM PMSF, and 0.5% Nonidet P-40). The lysate was
microcentrifuged at 500 × g for 5 min to pellet the
nuclei, which were washed with sucrose buffer without Nonidet P-40. The
nuclei were resuspended in low salt buffer (20 mM Hepes, pH
7.9, 25% glycerol, 0.02 M KCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and
0.5 mM PMSF), followed by addition of high salt buffer to
extract the nuclei, with incubation for 20 min on a rotary platform.
Diluent (25 mM Hepes, pH 7.6, 25% glycerol, 0.1 mM EDTA, 0.5 mM DTT, and 0.5 mM
PMSF) was added, and the sample was microcentrifuged at 13,690 × g. Aliquots of the supernatant (nuclear extract) were stored
at
80 °C.
EMSA--
EMSA was performed with 32P-labeled
double-stranded or single-stranded oligonucleotides that were incubated
with nuclear extract in EMSA buffer, pH 7.5 (10 mM
Tris-HCl, pH 7.5, 5% glycerol, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, and 0.1 mg/ml poly(dI-dC)).
For oligonucleotide competition analysis, various amounts (as indicated in each figure legend) or 100-fold molar excess of competitor oligonucleotides were also added to the mixture. After incubation at
22 °C for 30 min, the mixture was analyzed on 5% nondenaturing polyacrylamide gels. For antibody supershift assays, 1 µl of
anti-Sp1, Sp2, Sp3, or Sp4 antibody (Santa Cruz Biotechnology, Inc.)
was added to the mixture. DNA-protein complexes and free DNA were fractionated on 5% polyacrylamide gels in 1× Tris-glycine buffer (50 mM Tris, pH 8.3, 380 mM glycine, and 2 mM EDTA) at 4 °C and were visualized by autoradiography.
Southwestern Blot Analysis--
Nuclear extracts (20 µg of
total protein) from SH-SY5Y cells were incubated with the treatment
buffer (62.5 mM Tris-HCl, pH6.8, 2% SDS, 10% glycerol,
and 5% 2-mercaptoethanol) and boiled for 5 min. The treated extracts
were electrophoresed through SDS-10% polyacrylamide gel. The gel was
electroblotted onto polyvinylidene difluoride membrane (Amersham
Pharmacia Biotech) in transfer buffer (48 mM Tris-HCl, 39 mM glycine, 20% methanol). The membrane was incubated in
blocking solution (10% dry milk, 0.1% Tween 20 in Tris-buffered
saline) overnight at 4 °C. The membrane was then incubated with
32P-labeled probe in the presence or absence of 50-fold
molar excess of unlabeled oligonucleotides. Following the probing, the
membrane was washed with binding buffer. The signals were detected
using a Molecular Dynamics Storm 840 PhosphorImager system.
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RESULTS |
Sensitivity of mor Proximal Promoter DNA to Single Strand-specific
Nuclease--
To determine whether the single-stranded (ss) DNA
conformation is present in the proximal promoter region of the
µ-opioid receptor (mor) gene, and whether a ss DNA-binding
protein participated in the regulation of mor gene
expression, we first examined the capability of the mor
proximal promoter to form a ss DNA conformation. Formation of a ss
region, resulting from the non-B DNA form (such as melting DNA or an
intramolecular triplex (H-DNA) structure), is accessible to a
ss-sensitive nuclease (S1) at low concentrations (24, 25). Accordingly,
the pL450 plasmid, containing a fragment of ~200 bp of the
mor proximal promoter inserted within the promoterless pGL3-basic plasmid (16, 18), was used to test S1 nuclease sensitivity.
As shown in Fig. 1A, the pL450
plasmid was treated with or without S1 nuclease, then digested with
XbaI enzyme. In the absence of S1 nuclease treatment
(lane 6), only an XbaI-linearized DNA band (5 kb, indicated by asterisk) was observed. In
contrast, in the presence of S1 nuclease treatment (lanes
7-9), two DNA fragments of 1.8 and 3.2 kb were generated
(indicated by arrows). The intensity of both bands increased
with increasing the amount of S1 nuclease. These results suggested the
presence of a ss region located ~1.8 kb from the XbaI site
(Fig. 1B).

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Fig. 1.
S1 nuclease sensitivity on mor
proximal promoter region. A, supercoiled
plasmids, pGL3-basic (B), or pL450 (containing
mor proximal promoter in the promoterless pGL3-basic
vector), were treated with vehicle (lanes 1 and
6) or increasing amounts of S1 nuclease (lanes
2-4 and lanes 7-9) before digestion
with XbaI restriction enzyme. The XbaI-linearized
plasmid was indicated by an asterisk (*). The linearized
pL450 (lanes 10-12) was the supercoiled pL450
plasmid digested with XbaI first, and then treated with
increasing amounts of S1 nuclease (lanes 11 and
12) or vehicle (lane 10).
Lane 5 contains molecular size markers (1-kb
ladder). The two bands observed in lanes 7-9
(indicated by arrows) are 3.2- and 1.8-kb fragments.
B, a schematic diagram representing the region accessible to
S1 nuclease (open box). The 3.2- and 1.8-kb
fragments (indicated by bold lines with
arrowhead at each end) were derived from the
pL450 plasmid via sequential digestion with S1 and then
XbaI. The proximal promoter region is indicated by
gray box.
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To confirm that the S1 nuclear accessibility of the pL450 plasmid is
due to the presence of mor proximal promoter sequences, the
promoterless pGL3-basic plasmid (B) was examined using the same
digestion procedures (Fig. 1A, lanes
1-4). Only a 5-kb linearized band was observed without S1
(lane 1) or with S1 nuclease treatment (lanes 2-4). Furthermore, formation of such a
non-B-DNA conformation was known to require a supercoiled conformation,
and does not form in a linearized construct, because the supercoiled
structure is thought to provide a ss region of DNA due to negative
supercoiling of the circular plasmid (24, 25). When the supercoiled
pL450 plasmid was linearized by XbaI digestion before S1
digestion, the 1.8- and 3.2-kb fragments were not observed (Fig.
1A, lanes 10-12).
In summary, these results suggested the mouse mor proximal
promoter has the ability to form a ss DNA conformation, and formation of the ss region is dependent on its DNA supercoiling status.
Identification of a Polypyrimidine/Polypurine (PPy/u) Region of
mor Proximal Promoter as S1 Nuclease-sensitive Region--
To
determine the exact region with a ss DNA conformation in the
mor proximal promoter, the S1 nuclease-accessible region in the pL450 plasmid was identified. Fine mapping was performed using a
PCR method with S1-digested fragments (as indicated by
arrows in Fig. 1A) as template and a
32P-radiolabeled primer complementary to either sense or
antisense strand. The individual labeled PCR fragments was then
resolved on a sequencing gel.
As shown in Fig. 2, a collection of
labeled fragments were generated from the sense (A) or
antisense strand (B) of the proximal promoter. The S1
nuclease cleaved mainly in the PPy/u region in both strands (indicated
by arrows in Fig. 2, A and B).
Additionally, some minor S1 nuclease-sensitive sites were also observed
outside of the PPy/u region at
295 and
296 bp in the sense strand
as well as at
342,
340,
339, and
335 bp in the antisense strand (indicated by asterisks). The corresponding sequences are
shown in Fig. 2C, with arrows indicating the S1
nuclease-sensitive sites located in the PPy/u region (major cleavage
sites in bold arrows and minor with
plain arrows), and asterisks
indicating the sites outside the PPy/u region. Overall, the data
suggested that PPy/u sequences were largely responsible for the ss DNA
conformation, resulting in S1 nuclease accessibility.

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Fig. 2.
High resolution mapping of PPy/u sequences of
mor proximal promoter as S1 nuclease sensitivity
region. A and B, S1-treated fragments
derived from pL450 plasmid was purified and then subjected to PCR
reaction with 32P-labeled primer complementary to either
sense strand (A) or antisense strand (B).
Lanes marked A, T, C, and
G are sequencing reactions used as markers. Corresponding
sequences of each strand are mapped and indicated on the
right. The major and minor cleavage sites by S1 nuclease in
the PPy/u region are indicated by bold and plain
arrows, respectively. The asterisks
indicate some minor S1 cleavages located outside of the PPy/u
region. C, sequences represent a portion of mor
proximal promoter from 342 to 295 bp. The PPy/u region is marked in
a gray box. The S1 cleavage sites located in the
PPy/u region are indicated by either bold (major cleavage
site) or plain (minor cleavage site) arrows. The
minor S1 cleavages located outside of the PPy/u region are indicated by
asterisks.
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Verification of the Role of the PPy/u Region in the Formation of ss
DNA Structure--
The 26-bp PPy/u fragment was subcloned into another
plasmid vector, pGL3-promoter containing the SV40 promoter, resulting in the pLPPy/u-SV plasmid. As shown in Fig.
3, the supercoiled pLPPy/u-SV plasmid was
treated with or without S1 nuclease, and then digested with
XbaI enzyme. Bands of 1.9 and 3.1 kb were generated (indicated by arrows), the intensities of which increased as
the amount of S1 nuclease increased (lanes 7-9),
whereas neither of these bands was observed in samples not treated with
S1 (lanes 1 and 6) or in S1-treated
supercoiled pGL3-promoter plasmid (lanes 2-4).
These results thus confirmed that the 26-bp mor PPy/u
sequence alone is sufficient to result in a ss DNA configuration (non-B form).

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Fig. 3.
mor PPy/u fragment as S1
nuclease-sensitive region in a heterologous promoter. Supercoiled
plasmids, pGL3-promoter (P) or pLPPy/u-SV (26 bp of
mor PPy/u fragment cloned into pGL3-promoter vector), were
treated with vehicle (lanes 1 and 6)
or increasing amounts of S1 nuclease (lanes 2-4
and 7-9) before digestion with XbaI enzyme. The
XbaI-linearized plasmid was indicated by an
asterisk (*). Lane 5 contains
molecular size markers (1-kb ladder). Two bands indicated by
arrows are 3.1- and 1.9-kb fragments.
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Nuclear Proteins Specifically Bind to Sense Strand of PPy/u Region
in mor Gene--
The preceding results suggested that sequences of
PPy/u in the mor proximal promoter could adopt a non-B DNA
structure and, in particular, a ss region, led us to examine nuclear
proteins that could specifically bind to the ss of PPy/u region.
Nuclear proteins bound to 26 bp of polypyrimidine (PPy) sense or
polypurine (PPu) antisense strand oligonucleotide of the PPy/u region
were examined using EMSA with nuclear extracts from
mor-expressing cells, SH-SY5Y.
As shown in Fig. 4A, using the
sense oligonucleotide (PPy) as the probe, a distinct ss DNA-protein
complex was observed (lane 2, indicated by
A). The amount of this major ss complex A was gradually
diminished in the presence of increasing amount (1-50-fold) of
unlabeled PPy oligonucleotide (lanes 3-6), while
two additional more slowly migrating ss DNA-protein complexes appeared,
as indicated by B and C (lanes
4-6). The major ss complex A can be completely abolished in
the presence of 10-fold excess of unlabeled PPy (lane 5), suggesting that the interaction of DNA-protein (complex
A) was sequence-specific. Further supporting this conclusion, no distinct DNA-protein complex was observed using antisense (PPu) oligonucleotide as probe (Fig. 4B, lanes
1-3).

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Fig. 4.
ss DNA-binding proteins bound to the sense
strand of mor PPy/u region. EMSAs were performed
with nuclear extracts from SH-SY5Y cells. A, the sense
strand oligonucleotide (PPy) of PPy/u region was used as the probe in
the absence (lane 2) or presence of various
concentrations (as indicated) of unlabeled PPy as competitor
(lanes 3-8). Lane 1, probe
alone; lanes 2-8, probe plus 5 µg of total
proteins of nuclear extract; lanes 3-8,
1-200-fold molar excess of unlabeled PPy as competitor. The major ss
complex is indicated by A, and the other ss complexes are
indicated by B and C. B, the antisense
strand oligonucleotide (PPu) of PPy/u region was used as the probe in
the absence (lanes 1 and 2) or
presence of 100-fold molar excess unlabeled PPu as competitor
(lane 3). Lanes 2 and
3, probe plus 5 µg of total proteins of nuclear
extract.
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Complexes B and C could also be eliminated in the presence of molar
excess unlabeled PPy oligonucleotide (lanes 7 and
8), but a greater amount was required (100- and 200-fold)
than to eliminate complex A. This result suggested that, although
binding in these complexes was also sequence-specific, it was of lower affinity than that in complex A. Since the ss complex A was predominant (lane 2), the nature of B and C complexes was not
explored further. However, there are several possible explanations for
the formation of the complexes B and C. They could result from
interactions of the original complex (complex A), producing dimers or
higher order homomeric or heteromeric complexes. An alternative
possibility is that these complexes may reflect low affinity binding of
different nuclear proteins to the probe.
In summary, these results showed that a major ss DNA-binding protein
was involved in the formation of ss complex A (as indicated by
A). This ss nuclear protein specifically bound to the sense strand (PPy), but not the antisense strand (PPu), of the mor
PPy/u region with high affinity. We therefore next identified the
binding region of this protein, and its functional role on the
mor proximal promoter.
Overlapping of DNA-binding Sites for ss and ds DNA-binding Proteins
in PPy/u Region--
To localize the exact binding region of this
major ss DNA-binding protein, two sense strand oligonucleotides, f1 and
f2, spanning the entire 26-bp PPy region were synthesized (Fig.
5A). These fragments were then
tested for their ability to inhibit the formation of ss complex A using
EMSA with SH-SY5Y nuclear extracts.

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Fig. 5.
Binding of mPy protein to f2
region. A, sequences represent the sense strand (PPy)
of PPy/u region of the mor proximal promoter. Two
oligonucleotides, f1 and f2, are indicated in
brackets. B, EMSAs were performed with nuclear
extracts from SH-SY5Y cells. The ss PPy oligonucleotide was used as the
probe (lanes 1-8) in the absence or presence of
100-fold molar excess competitor, or in the presence of various anti-Sp
polyclonal antibodies. Lane 1, probe alone;
lanes 2-8, PPy as probe plus 5 µg of total
proteins of nuclear extract; lane 3, unlabeled
PPy competitor; lane 4, f1 competitor;
lane 5, f2 competitor; lane
6, double-stranded (ds) GC-rich Sp1 competitor;
lane 7, in the presence of 1 µg of anti-Sp1 and
anti-Sp3 (lane 8) antibodies. The major ss
complex is indicated by A. C, EMSAs were
performed with SH-SY5Y nuclear extracts with ss PPy (lanes
1 and 2) or ds PPy/u oligonucleotides as the
probe (lanes 3-5). Lanes 1 and 3, probe alone; lanes 2,
4, and 5, probe plus 5 µg of total proteins of
nuclear extract; lane 5, 100-fold molar excess of
Sp1 competitor. The major ss complex is indicated by A, and
the major ds complex is indicated by .
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As shown in Fig. 5B, using PPy as the probe
(lanes 1-8), formation of the ss complex A
(lane 2, indicated by A) could be
abolished by 100-fold molar excess of PPy (lane
3) or f2 (lane 5), but not by
f1 oligonucleotide (lane 4). Results suggested
that binding of this ss DNA-binding protein confined in the f2
(
322 to
308 bp) region. To further confirm that the ss DNA-binding
protein is indeed bound to the f2 region, EMSA was performed
using f2 oligonucleotide as the probe. One major ss DNA/protein
complex was observed, and its intensity decreased in the presence of
100-fold molar excess of f2 or PPy, but not f1 oligonucleotide
(data not shown), suggesting this ss DNA-binding protein indeed bound
to the f2 region of PPy.
Interestingly, this ss DNA element (f2) resided in the same
region (
322 to
308 bp) as double-stranded (ds) mor iGA
motif, a previously identified ds motif binding to Sp1 and Sp3 (18). We
therefore examined whether Sp factors were involved in the formation of
ss complex A, using EMSA. As shown in Fig. 5B, using PPy as
the probe, the formation of the ss complex A (indicated by
A) was not abolished by 100-fold molar excess of Sp1
consensus oligonucleotide (lane 6), or by the
addition of an Sp1 (lane 7), Sp3 (lane
8), or Sp2 or Sp4 antibody (data not shown). In addition, the ss complex (indicated by A in Fig. 5C,
lane 2) exhibited a faster migration than the
complexes detected with ds PPy/u probe (indicated by
in
lane 3), which was abolished by 100-fold molar excess of Sp1 consensus oligonucleotide (lane 5),
corroborated with our previous report (18).
These results suggested that Sp proteins are not involved in the
formation of the ss complex A seen on the mor proximal
promoter. Thus, the ss DNA-binding protein, bound to the f2
region of PPy, is not the known Sp transcription factor, although the
ss binding region (f2) overlaps with that of ds mor
iGA motif, which does bind to Sp proteins. We therefore named this ss
DNA-binding protein mPy protein (mor
polypyrimidine-binding protein).
ss DNA Element Binds to a 25-kDa Protein--
To characterize the
mPy protein further, Southwestern blot analysis was performed using
SH-SY5Y nuclear extract. As shown in Fig.
6A (lane
1), a major band (indicated by arrow) with an apparent molecular mass of 25 kDa was observed by probing with 32P-labeled ss PPy oligonucleotide. A 50-fold molar excess
unlabeled ss PPy (lane 2) or f2
oligonucleotide (lane 3) significantly diminished the
binding of 32P-labeled PPy to the 25-kDa protein, but not
by the f1 oligonucleotide (lane 4). These results
indicated that mPy protein is a nuclear protein of ~25 kDa in SH-SY5Y
cells, and that it specifically binds to the f2 region of PPy
element, in agreement with our EMSA data.

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Fig. 6.
Determination of molecular weight of mPy by
Southwestern blot analysis. Twenty µg of nuclear extract from
SH-SY5Y cells were electrophoresed through 10% SDS-polyacrylamide gel
and transferred onto polyvinylidene difluoride membrane. The membrane
was then probed with 32P-labeled ss PPy oligonucleotide in
the absence (lane 1) or presence of 50-fold molar
excess of the PPy (lane 2), f2
(lane 3), or f1 (lane 4)
oligonucleotide. The sequences of these oligonucleotides are specified
in Fig. 5A. Position of concurrently electrophoresed protein
size markers are indicated.
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Contribution of mPy Protein and Its cis-Acting Element to Proximal
Promoter--
To understand functional roles of the mPy protein in the
proximal promoter, the pL340/300 construct, containing the minimum proximal promoter sequences (
340 to
300 bp), was used (18). Two
different types of transcription factors, Sps (ds DNA-binding protein)
and mPy protein (ss DNA-binding protein), bound to this fragment were
identified. Functional analysis (Fig.
7A) of mutant pLGGT and pLcsp
constructs (containing f2 region mutation within PPy/u sequences
of pL340/300 construct) was performed using transient transfection
assay in SH-SY5Y cells.

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Fig. 7.
Functional analysis of PPy/u mutations.
A, transfection analysis of PPy/u mutations in SH-SY5Y
cells. The substituted bases of each mutant construct (pLGGT or pLcsp)
are shown by bold letters and are
underlined. Relative activities were obtained by defining
the activity of wild type pL340/400 construct as 100%.
Error bars represent the standard errors.
B, EMSA was performed using SH-SY5Y nuclear extract
(lanes 1-5). The ds PPy/u was used as probe in
the absence (lane 1) or presence of 100-fold
molar excess of different competitor. Lane 2,
unlabeled ds PPy/u competitor; lane 3, GC-rich
Sp1 competitor; lane 4, mutant ds GGT
oligonucleotide; lane 5, mutant ds csp
oligonucleotide. Major ds complex is indicated by . C,
supercoiled plasmids, promoterless pGL3-basic (B), wild type
pL340/300, mutant pLcsp, or pLGGT, were treated with vehicle
(lanes 1, 4, 8, and
13) or S1 nuclease (lanes 2,
5-7, 9-11, and 14-16) before
digestion with XbaI restriction enzyme. The
XbaI-linearized plasmid is indicated by asterisk.
Lanes 3 and 12 are molecular size
makers (1-kb ladder). The 3.1- and 1.7-kb fragments were indicated by
arrows (lanes 5-7 and
14-16).
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As shown in Fig. 7A, the pLGGT mutant construct, in which
CCC in f2 region was changed to GGT (underlined),
displayed ~50% of promoter activity as compared with that (100%) of
wild type pL340/300 construct. The pLcsp mutant construct, containing
the consensus GC-rich Sp1 binding site in the f2 region
(underlined), displayed only 20% of wild type promoter activity.
Concomitantly, EMSA was performed to identify mutations resulting in
the elimination of ds Sps binding (Fig. 7B). Using ds PPy/u
as the probe, the formation of major ds complex (indicated by
in
lane 1) was inhibited by molar excess of
unlabeled ds PPy/u (lane 2), consensus Sp1
oligonucleotide (lane 3), and ds csp mutant
oligonucleotides (lane 5), but not by ds GGT
mutant oligonucleotides (lane 4). These results
suggested that the GGT mutation resulted in loss of Sp binding, while
the csp mutation retained the Sp binding.
Furthermore, the ability to form a ss DNA conformation of each mutant
plasmid was examined by S1 nuclease accessibility. As shown in Fig.
7C, the wild type pL340/300 (lanes
4-7), mutant pLcsp (lanes 8-11), and
pLGGT plasmids (lanes 13-16) were treated with
or without S1 nuclease before digestion with XbaI enzyme. In
the absence of S1 nuclease treatment, only an
XbaI-linearized DNA band (indicated by *) was observed in
either wild type (lane 4) or mutant constructs
(lanes 8 and 13), which is similar to those results using the promoterless pGL3-basic construct
(B) treated with or without S1 nuclease (lanes
1 and 2). In the presence of S1 nuclease
treatment, two DNA fragments (indicated by arrows) were
observed in the wild type (lanes 5-7) as well as
the pLGGT mutant construct (lanes 14-16), but
not in the pLcsp mutant construct (lanes 9-11).
These results showed that the pLGGT mutant construct retained a ss DNA
conformation like the wild type, but the pLcsp construct was devoid of
ss DNA conformation.
Retention of the mPy protein binding site in either of mutants (GGT and
csp) was then examined using EMSA with PPy as the probe (Fig.
8A). Formation of the ss
complex A (indicated by A in lanes 1 and 4) was inhibited by a 50- or 100-fold molar excess ss
GGT mutant oligonucleotide (lanes 2 and
3), but not by ss csp mutant oligonucleotide
(lanes 5 and 6). To further verify
that the GGT mutation still retained the ability to bind the mPy
protein, EMSA was performed using ss GGT mutant oligonucleotide as
probe (Fig. 8B). A major ss complex was observed (indicated
by A in lane 2), and its intensity
decreased in the presence of 100-fold molar excess of PPy
(lane 3), ss GGT (lane 4),
or f2 (lane 6), but not f1 oligonucleotide
(lane 5). These results suggested that the GGT
mutation resulted in the retention of mPy protein binding, whereas the
csp mutation resulted in the elimination of mPy protein binding.

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Fig. 8.
Binding of mPy protein in PPy/u
mutations. A, EMSAs were performed with nuclear extracts from
SH-SY5Y cells. The ss PPy oligonucleotide was used as probe in the
absence (lanes 1 and 4) or presence of
50 to 100-fold molar excess of ss GGT mutant (lanes
2 and 3) or ss csp mutant (lanes
5 and 6) oligonucleotide as competitor.
Lanes 1-6, probe plus 5 µg of total proteins
of nuclear extract. The mPy protein-PPy complex (ss complex A) is
indicated by A. B, EMSA was performed using the
ss GGT mutant oligonucleotide as probe with SH-SY5Y nuclear extract.
Lane 1, probe alone (indicated by F);
lanes 2-6, probe plus 5 µg of total proteins
of nuclear extract; lane 3, 100-fold molar excess
of ss PPy; lane 4, ss GGT; lane
5, f1; lane 6, f2
oligonucleotide. Major ss complex is indicated by A.
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Taken together, the above results showed that the GGT mutation retained
the mPy protein binding as well as a ss DNA conformation, but ablated
the bindings of Sp factors. The pLGGT construct still displayed 50% of
promoter activity. These results strongly suggested that the mPy
protein acts as an activator for mor proximal promoter activity. On the other hand, the csp mutation resulted in the retention
of Sps binding, but is associated with a loss of ss DNA conformation
and mPy protein binding. With this binding profile, the pLcsp construct
showed 20% of promoter activity, suggesting that Sp factors are also
important for promoter activity, in agreement with our previous report
(18). However, binding of either mPy protein or Sp factors alone cannot
result in 100% promoter activity. Therefore, these results further
suggested that a combination of both mPy protein (ss DNA-binding
protein) and Sp proteins (ds DNA-binding protein) is required for the
proximal promoter activity.
trans-Activation of a Heterologous Promoter via Binding of the ss
mPy Protein to the PPy/u Fragment of mor Gene--
To determine
whether the mPy protein could also mediate trans-activation
of a heterologous minimal promoter, we prepared the luciferase
reporters in which a 26-bp PPy/u fragment, containing GGT mutation or
wild type sequences (as depicted in Fig. 7A), was positioned
at 5'-upstream of the SV40 promoter (pGL3-promoter) in either native or
reverse orientation.
As shown in Fig. 9, the resulting wild
type construct (pLPPy/u-SV) showed significantly higher activity
(172 ± 6%) than that (100%) of SV40 promoter construct (P). The
mutant pLGGT-SV construct, retention of only the mPy protein binding
and ablation of Sp factor bindings, displayed high activity (225 ± 18%) in native orientation. Contrarily, the
pLrGGT-SV construct, in which the 26 bp of GGT
mutated PPy/u fragment was positioned in reverse orientation, displayed
no significant enhancement in promoter activity (94 ± 8%) as
compared with that (100%) of SV40 promoter construct.

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Fig. 9.
trans-Activation of a heterologous
promoter via mPy protein binding motif. SH-SY5Y cells were
transfected with luciferase reporter plasmids driven by SV40 promoter
alone (P), or SV40 promoter with 26 bp of wild type
(pLPPy/u-SV) or GGT-mutated PPy/u fragment at 5'-upstream region in
either native (pLGGT-SV) or reverse orientation
(pLrGGT-SV). Relative luciferase activities were
obtained by arbitrarily defining the activity of pGL3-promoter
construct as 100%. Error bars represent the
range of standard errors from three different experiments.
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Overall, these results suggested that binding of the mPy protein to the
sense strand (PPy) of the mor PPy/u region can mediate trans-activation not only within the context of the
mor gene promoter but also within a heterologous promoter in
orientation dependent manner.
 |
DISCUSSION |
We have begun a molecular dissection of the regulatory processes
governing the transcription of the mouse µ-opioid receptor (mor) gene, with a particular emphasis on the proximal
promoter region. Previously, we reported that Sp transcription factors are critical for mor proximal promoter activity (18). Here
we further report that the proximal promoter of the mor gene
can adopt a non-B DNA (ss) conformation, in a region localized to a
26-bp polypyrimidine/polypurine (PPy/u) sequence. The sense strand
(PPy) of mor PPy/u region is a ss cis-acting
element that specifically binds a ss DNA-binding protein (designated
mPy), which is also necessary for promoter activity. Our results
suggested that the activity of the mor proximal promoter is
dependent on not only Sp factors, bound to ds DNA elements (10), but
also on the mPy protein, bound to a ss DNA element (PPy).
Sensitivity to S1 nuclease demonstrated that the presence of the 26-bp
mor PPy/u sequence alone is sufficient to result in adoption
of the non-B-DNA form in vitro (Fig. 3), further
establishing that the mor PPy/u region can adopt the ss
conformation (Figs. 1 and 2). It is possible that S1-sensitive sites
resulted from a melted region of DNA. Furthermore, the asymmetrical
nature of the S1 cleavage pattern (Fig. 2) is suggestive of an
intramolecular triplex H-form DNA conformation (24-26). If the triplex
conformation was the only one sensitive to S1 digestion with the latter
would theoretically release only one strand. However, our in
vitro results (Figs. 1-3) showed that both of the DNA strands
were susceptible to S1 digestion. Taken together, these observations
imply that ds, melted, and/or H-DNA structures may exist in
conformational equilibrium, so that digestion on both strands can occur
during the state of transition. Furthermore, because of local
distortions of the phosphodiester backbone (in the supercoiled form), a
small portion of the triplex structure may also be accessible to S1 recognition (24, 25).
Although the precise mechanisms underlying the regulation of local
topological features of the mor PPy/u region remain unclear, we have established that a ss DNA element (PPy) in the ss configuration (non-B form), and its ss DNA-binding protein (mPy) play an important role in the mor proximal promoter. Using EMSA with ss PPy
oligonucleotide as the probe, a major ss complex A (mPy-PPy complex)
was observed (indicated by A in Fig. 4). A 15-bp fragment
located in the 3'-region of PPy (f2 oligonucleotide depicted in
Fig. 5A) appeared to be critical for the formation of ss
complex A, because this complex was specifically reduced in intensity
by unlabeled f2, but not f1 oligonucleotide located in the
5'-region of PPy (Fig. 5B). Furthermore, several lines of
evidence indicated that previously identified Sp factors, bound to a ds
iGA motif (residing in PPy/u region) (18), were not involved in
formation of the ss complex A. First, the ss complex A migrated faster
than ds complexes when the same DNA region was used in EMSA (Fig.
5B). Second, formation of the ss complex A was not
diminished in the presence of a consensus Sp1 oligonucleotide, nor
could it be supershifted with the addition of antibody against Sp1,
Sp3, Sp2, or Sp4 (Fig. 5B). Third, Southwestern analysis
indicated that ss DNA-binding protein (mPy) is an ~25-kDa nuclear
protein (Fig. 6), and that binding to its cognate DNA (PPy) does not
alter in the presence of EGTA (data not shown). In contrast, the Sp
factors are zinc-finger transcription factors with molecular mass
ranging from 80 to 140 kDa (18, 56-58).
Functional analysis suggested that the mPy protein-PPy complex (ss
complex A) plays a positive role in transcriptional regulation of the
mor gene. The pLGGT construct (containing a GGT mutation in
the mor PPy/u region; Figs. 7 and 8), which retains the mPy protein but not Sp factor binding, displayed 50% of promoter activity, suggesting that the mPy protein alone can trans-activate the
mor proximal promoter. In addition, alterations to this
region (csp mutation in Figs. 7 and 8), which abolished the ss
conformation and mPy protein binding, resulted in a significant loss
(50-80%)of promoter activity. Furthermore, a chimera formed by
placing this GGT mutated PPy/u fragment (containing only the mPy
protein binding site) upstream of the heterologous SV40 promoter, in
the native but not reverse orientation, trans-activated this
promoter (Fig. 9). It is interesting that the pLGGT construct can
display significant promoter activity in the absence of Sp1 binding.
Evidences indicate that Sp1 can tether preinitiation complexes to the
promoter by interacting with TFIID (59-61), and it is therefore
thought to be essential for transcription events at both TATA and
TATA-less promoters (62). However, recently the heterogeneous nuclear ribonucleoprotein K (hnRNP K), a ss DNA binding factor, has been suggested to interact directly with RNA polymerase II transcription machinery through TATA-binding protein (TBP)-TBP-associated factor complex (52). According to this paradigm, the mPy protein-PPy complex
(ss complex A) not only can serve as an activator, but might be able to
interact with preinitiation complexes directly to facilitate
transcription initiation of the mor gene. This is consistent
with our observation of substantial promoter activity in the absence of
Sp factors.
Nevertheless, our data suggest that both ds and ss DNA binding sites,
the ds iGA motif bound to Sps (18) and the ss PPy element bound to mPy
(this report), are required for the proximal promoter activity.
Retention of only Sps binding or only mPy protein binding did not
result in full promoter activity (Fig. 7). Furthermore, deletion of the
entire 26-bp PPy/u element, to prevent any specific protein binding,
caused the loss of promoter activity (data not shown), indicating an
overall positive regulatory function of this PPy/u region. Thus, the
ratio of Sps and mPy protein as well as the ratio of Sp1 and Sp3 in a
cell may contribute to regulation of the mor gene.
Furthermore, alteration of the DNA binding motif conformation and
fluctuation of the levels of transcription factors under various
conditions (such as variations in physiological or pathological status
or developmental stages) may also contribute to gene regulation (33,
47-58, 50). Moreover, tissue- or cell-specific regulatory factors
(63-66) presumably modulate the ability of Sp factors or ss
DNA-binding protein and of perhaps additional, as yet unidentified,
factors to regulate the promoter activity.
Although this is the first example of a ss DNA-binding protein
modulating the transcription of the opioid receptor gene, there is
increasing evidence that a non-B DNA conformation and ss DNA-binding proteins are involved in transcription regulation in either a positive
or negative manner (32-34, 45-50). In the case of the
c-myc gene, both ds (Sp factors) and ss DNA-binding proteins
(hnRNP K) bound to its PPy/u region can trans-activate the
promoter (51, 52). Although the recognition sequences (PPy/u) of
mor and c-myc are similar but not identical, our
preliminary experiments suggest that the mPy protein is not related to
the hnRNP K protein. First, the 12G4 and 4B10 anti-hnRNP K antibodies
(67, 68) did not specifically block the formation of mPy-PPy complex
(ss complex A). Second, the molecular mass of mPy protein is smaller
than hnRNP K (68). Therefore, the exact identity of the mPy protein remains to be defined, and it will ultimately require the purification, cloning, and characterization of this factor.