Single-stranded DNA-binding Complex Involved in Transcriptional Regulation of Mouse µ-Opioid Receptor Gene*

Jane L. KoDagger and Horace H. Loh

From the Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455

Received for publication, May 18, 2000, and in revised form, September 20, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previously, we reported the presence of dual (distal and proximal) promoters in mouse µ-opioid receptor (mor) gene, with mor transcription in mouse brain predominantly initiated by the proximal promoter. Sp factors, bound to double-stranded (ds) cis-regulatory elements, are critical for proximal promoter activity. Here, we further report that a single-stranded (ss) cis-regulatory element and trans-acting protein factor are also important for proximal promoter activity. A 26-bp mor polypyrimidine/polypurine region (PPy/u) can adopt ss DNA conformation, as demonstrated by S1 nuclease sensitivity. Using electrophoretic mobility shift analysis with nuclear extracts from mor-expressing SH-SY5Y cells, we demonstrate that the sense strand of PPy/u interacts with a major nuclear protein, termed mor polypyrimidine-binding protein (mPy), which is not related to Sp factors. Southwestern blot analysis indicated that mPy protein is ~25 kDa in size. Functional analysis suggests that mPy protein can trans-activate mor promoter as well as a heterologous promoter. Moreover, combinatorial activation of ss (mPy) and ds (Sps) DNA binding factors, interacting with an overlapping DNA (PPy/u) region, is necessary for proximal promoter activation. Thus our results suggest that transcription of mouse mor gene is regulated by an interplay of ss and ds DNA binding factors.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, delta , and kappa ), 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-alpha (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).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.

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.

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.

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 .

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.

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).

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.

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.

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

The technical and intellectual contributions of Dr. Hsien-Ching Liu were critical to this study. We also thank Dr. Gideon Dreyfuss (University of Pennsylvania) for the kind gift of 12G4 and 4B10 anti-hnRNP K antibodies and Dr. Andrew P. Smith for editing the manuscript.


    FOOTNOTES

* This work was supported by National Institutes of Health Research Grants DA-00546, DA-01583, DA-05695, and KO5-DA-70554, and by the A. and F. Stark Fund of the Minnesota Medical Foundation.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.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church St. S.E., Minneapolis, MN 55455. Tel.: 612-626-6539; Fax: 612-625-8408; E-mail: koxxx001@maroon.tc.umn.edu.

Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M004279200


    ABBREVIATIONS

The abbreviations used are: mor, µ-opioid receptor; PPy/u, polypyrimidine/polypurine; mPy, mor polypyrimidine-binding protein; ss, single-stranded; ds, double-stranded; iGA, inverted-GA motif; EMSA, electrophoresis mobility shift assay; GGT mutation, mutant PPy/u oligonucleotide containing GGT 3-base pair mutation; csp mutation, mutant PPy/u oligonucleotide containing a consensus GC box; hnRNP K, heterogeneous nuclear ribonucleoprotein K; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride.


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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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