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Originally published In Press as doi:10.1074/jbc.M104793200 on October 2, 2001

J. Biol. Chem., Vol. 276, Issue 48, 45462-45469, November 30, 2001
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Transcriptional Regulation of Mouse delta -Opioid Receptor Gene

ROLE OF Ets-1 IN THE TRANSCRIPTIONAL ACTIVATION OF MOUSE delta -OPIOID RECEPTOR GENE*

Ping SunDagger and Horace H. Loh

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

Received for publication, May 25, 2001, and in revised form, September 20, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previously, we identified a minimum core promoter of the mouse delta -opioid receptor (DOR) gene. The DOR promoter contains an E-box that binds upstream stimulatory factor and is crucial for the DOR promoter activity in NS20Y cells, a mouse neuronal cell line that constitutively expresses DOR. In the present study, we further analyzed the DOR promoter in NS20Y cells and have demonstrated that transcription factor Ets-1 binds to an Ets-1-binding site overlapping the E-box and trans-activates the DOR promoter by synergizing with upstream stimulatory factor in specific DNA binding. In addition, the Ets-1 DNA-binding domain is sufficient to play the functional role of Ets-1 in trans-activating the DOR promoter. Furthermore, through in vivo cross-linking assays and Northern blot analyses, we have demonstrated that Ets-1 binds to the DOR promoter in the neonatal mouse brain and that overexpressed Ets-1 can significantly enhance the expression of DOR mRNA in primary neonatal mouse neuronal cells. Collectively, our data suggest that Ets-1 functions as a trans-activator of the DOR promoter in the neonatal mouse brain and thus may contribute to the development of the mouse brain DOR system.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Opioids are the preferred clinical analgesics for severe pain. Three major types of opioid receptors (ORs),1 µ, delta , and kappa , have been cloned and shown to belong to the G protein-coupled receptor superfamily (1). Although all three ORs mediate opioid-induced analgesia, each receptor type exhibits a distinct pharmacological profile as well as a unique pattern of temporal and spatial expression (2-3).

The localization of the delta -opioid receptor (DOR) generally matches the pharmacological action sites of delta -opioids (2). In addition, it has been reported that the expression levels of DOR can determine the DOR agonist's activities (4). Thus, understanding the molecular mechanism underlying the spatiotemporal expression of DOR may raise the possibility of maximizing the pharmacological benefits of delta -opioids by manipulation of the DOR expression levels.

DOR is mainly confined to the central nervous system. There is a strong correlation between the spatiotemporal presence of DOR mRNA and delta -opioid-binding sites (5). In addition, levels of DOR mRNA as well as delta -opioid-binding sites can be regulated by various agents in some neuronal cell lines. For example, nerve growth factor (6), ethanol (7), or retinoic acid (8) can up-regulate DOR mRNA and delta -opioid-binding sites. On the other hand, activation of the protein kinase A pathway by forskolin or cyclic AMP analogues results in down-regulation of DOR mRNA and delta -opioid-binding sites (9). All of these studies suggest that the expression of DOR is subjected to several regulation mechanisms at the transcriptional level. Therefore, study of the transcriptional regulation of DOR gene will provide insights into the spatiotemporal expression of DOR.

Previously, we identified a minimum core promoter of the mouse DOR gene. We found that an E box and a GC box in the DOR promoter are crucial for the DOR promoter activity in NS20Y cells, a mouse neuronal cell line that constitutively expresses DOR. In addition, we demonstrated that upstream stimulatory factor (USF) and Sp family proteins bound to and trans-activated the DOR promoter via the E box and the GC box, respectively (10-11).

In the present study, we further analyzed the DOR promoter in NS20Y cells and have demonstrated that transcription factor Ets-1 binds to an Ets-1-binding site overlapping the E-box and trans-activates the DOR promoter by synergizing with USF in specific DNA binding. In addition, the Ets-1 DNA-binding domain is sufficient to play the functional role of Ets-1 in trans-activating the DOR promoter. Finally, through in vivo cross-linking assays and Northern blot analyses, we have demonstrated that Ets-1 can bind to the DOR promoter in the neonatal mouse brain and enhance the expression of DOR mRNA in primary neonatal mouse neuronal cells. These results suggest that Ets-1 functions as a trans-activator of the DOR promoter in the neonatal mouse brain and thus may contribute to the development of the mouse brain DOR system.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmid Construction-- The luciferase fusion plasmids pD262 and pDm184 were constructed as previously described (11). Plasmid pDm192 was generated by oligonucleotide-directed mutagenesis to replace the sequence at positions -192 to -187 of pD262 with AAGCTT. The double mutation construct, pD262Ets1*/E*, was created by polymerase chain reaction (PCR) to replace the sequence at positions -192 to -180 of pD262 with a HindIII site (AAGCTT). All mutation constructs were confirmed by DNA sequencing.

Cell Line Culture-- Mouse neuroblastoma NS20Y cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum. The cells were incubated at 37 °C in an atmosphere of 10% CO2 and 90% air.

Transient Transfection and Reporter Gene Assay-- NS20Y cells were transfected using the DOTAP (Roche Molecular Biochemicals) lipofection method as described previously. Briefly, cells at ~40% confluence were transfected with an equal amount of test plasmids. Forty-eight hours after transfection, cells grown to confluence were washed and lysed with lysis buffer (Promega). To control for differences in transfection efficiency, a one-fifth 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.

DNase I Footprint Assay-- Bacterially expressed recombinant human USF1, p68 chicken Ets-1, and mouse USF2Delta b were prepared as described by Pognonec et al. (12). Plasmid pD262, pDm192, or pDm184 was digested with KpnI, dephosphorylated with calf intestinal alkaline phosphatase, end-labeled with [gamma -32P]ATP, and then digested with NcoI to create the 5'-labeled 262-bp probes. The probes were then purified by polyacrylamide gel electrophoresis. Binding reactions were carried out for 40 min at 0 °C in a final volume of 50 µl. The binding solutions contained 100 fmol of labeled probe and 16 µg of NS20Y nuclear extracts or indicated amounts of recombinant Ets-1 or USF1 in a final buffer concentration of 10 mM Tris-HCl (pH 7.5), 40 mM NaCl, 1 mM dithiothreitol, and 1 µg of poly(dI-dC). After incubation, the concentrations of MgCl2 and CaCl2 were adjusted to 5 and 2.5 mM, respectively. Then 1 unit of DNase I (Promega) was added. The incubation was continued for 1 min at room temperature. The digestion was stopped by using 100 µl of stop solution containing 20 mM EDTA, 1% sodium dodecyl sulfate (SDS), 0.2 M NaCl, and 250 µg/ml tRNA. DNA was extracted by using phenol-chloroform (1:1) and ethanol precipitation, before loading onto a 6% sequencing polyacrylamide gel for electrophoresis.

Primary Neuronal Cell Culture and Transient Transfection-- Cultures were prepared according to Yavin and Yavin (13). Briefly, cerebral hemispheres from 2-day-old neonatal mice were pooled and dissociated mechanically in Eagle's basal medium supplemented with 20% fetal calf serum, 2 mM glutamine, and 6 mg/ml glucose. The cells were seeded on poly-L-lysine-coated culture dishes (one hemisphere per 60-mm dish) and incubated for 2 h at 37 °C and 5% CO2, then the medium was removed and 8 ml of the same medium, without serum, was added. The cell cultures were maintained at 37 °C and 5% CO2 for 10 days without changing the medium. To determine the proportion of neurons in the primary cultures, one-third of the primary culture dishes were randomly selected for immunocytochemical analysis using specific antibody against mouse neuron-specific enolase (Polysciences) and the vectastatin ABC kit (Vector Lab). The cultures were found to contain an average of 85% neurons. The rest of the primary culture dishes were transfected with the pSG5-Ets-1 expression vector or the empty pSG5 vector (Stratagene) using DNA/Ca2+-phosphate co-precipitation, as described by Sambrook and Russel (14). Briefly, the calcium phosphate-DNA co-precipitate was prepared by combining 100 µl of 2.5 M CaCl2 with 25 µg of plasmid. One volume of this 2 × calcium-DNA solution was mixed with an equal volume of 2 × HEPES-buffered saline at room temperature. One min later, the calcium phosphate-DNA suspension was transferred into the medium. 0.1 ml of suspension was used for each 1 ml of medium in a 60-mm dish and incubated in a 2.5% CO2 atmosphere at 37 °C for 3 h. The medium was changed after 3 h and the cells were harvested after 48 h.

In Vivo Formaldehyde Cross-linking and Immunoprecipitation of Chromatin-- Cerebrums from 10 2-day-old neonatal mice were isolated, homogenized, and washed in cold phosphate-buffered saline containing 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 100 ng/ml leupeptin, and 100 ng/ml aprotinin. Then, formaldehyde (Fisher Scientific) was added to the homogenate at a final concentration of 1%. Fixation proceeded at room temperature for 15 min and was stopped by the addition of glycine to a final concentration of 0.125 M. Nuclei were collected as described by Ausubel et al. (15). The following chromatin immunoprecipitation was performed according to the protocols described by Farnham and co-workers (16). Briefly, the nuclei were sonicated on ice to shear the chromatin to an average length of 400 bp. The chromatin solutions were precleared with the addition of Staph A cells for 15 min at 4 °C. Precleared chromatin was incubated with 2 µg of rabbit polyclonal antibody (Santa Cruz: anti-Ets-1 N-276 or anti-USF1 C-20), 1 µl of rabbit preimmune control serum, or no antibody at 4 °C overnight. After immunoprecipitation, washing, and elution (17), cross-links were reversed by addition of NaCl to a final concentration of 200 mM; RNA was removed by addition of 10 µg of RNase A per sample followed by incubation at 65 °C for 5 h. Samples were ethanol-precipitated and resuspended for incubation with proteinase K. Then DNA was extracted and precipitated by standard protocols. DNA pellets were collected and analyzed through PCR. Approximately 2 ng of DNA was used as a template in a 50-µl PCR reaction mixture using 1 unit of pfu polymerase. For DOR amplification, a sense primer corresponding to the DOR promoter sequence from -262 to -243 and an antisense primer corresponding to the DOR promoter sequence from -160 to -141 were used. A set of primers specific to the GAPDH exon 8 was used for GAPDH amplification. The amplification was performed using one cycle at 95 °C for 2 min, 35 cycles at 95 °C for 40 s, 68 °C (for DOR) or 64 °C (for GAPDH) for 30 s, and 72 °C for 30 s.

Northern Blot Analysis-- Poly(A)+ RNA was isolated from primary cultures of mouse neuronal cells using the Micro-FastTrackTM 2.0 Kit (Invitrogen) according to the instructions of the manufacturer. The poly(A)+ RNA (1.6 µg per lane) was separated on a 1% agarose gel containing 2.2 M formaldehyde and transferred to a nylon membrane (Hybond-N+) according to the manufacturer's instructions (Amersham Pharmacia Biotech). UV cross-linking was carried out for fixation of the RNA to the membrane. RNA blots were prehybridized at 58 °C for 1 h in a hybridization solution of the following composition: 5 × Denhardt's solution, 5 × SSC, 10 mM sodium phosphate buffer, 1 mM EDTA, 0.5% SDS, 100 µg/ml sonicated and denatured salmon sperm DNA, and 50% formamide. A 1.8-kilobase cDNA probe was radiolabeled by PCR (14), using the mouse DOR cDNA as a template. Northern blots were hybridized with the DOR cDNA probe at 58 °C overnight. Blots were then washed with 2 × SSC, 0.1% SDS (three times for 15 min at 50 °C) and 0.1 × SSC, 0.1% SDS (two times for 15 min at 58 °C). All Northern blots were subsequently stripped of the DOR cDNA probe and rehybridized with a mouse beta -actin cDNA probe. The mRNA levels were visualized using a Storm 860 PhosphorImager (Molecular Dynamics). The total density of the DOR mRNA bands in each lane on the membrane was calculated using ImageQuant software (Molecular Dynamics) and normalized to the density of the beta -actin mRNA band. The resultant relative DOR mRNA levels were analyzed using analysis of variance (ANOVA) followed by post-hoc comparisons of means by the least-significance differences method. alpha  Level was set at 0.05 for each analysis. All analyses were performed using SPSS for Windows release 8.0.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Functional Identification of the Ets-1-binding Site in the DOR Promoter-- It has been reported that an E-box in the minimum core promoter of the mouse DOR gene binds USF and is essential for the DOR promoter activity in NS20Y cells, a mouse neuroblastoma cell line that constitutively expresses DOR (11). Subsequent sequence analysis revealed a potential Ets-1-binding site (EBS) that overlaps the 5' end of the E box (Fig. 1A). Ets-1 has been reported to be present in neuroblastoma cells (18). In addition, Ets-1 is able to synergize with basic helix-loop-helix leucine zipper (bHLHZip) proteins such as USF and TFE3 via binding to composite EBS/E box in various promoters (19-21). Thus, we first determined whether the putative EBS had any function in the DOR promoter.


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  		Fig. 1.   Functional identification of the Ets-1-binding site (EBS) in the mouse DOR promoter. A, schematic drawing of the minimum core promoter of the mouse DOR gene (-262 to -141) with a detailed view of base pairs from -194 to -179, containing the putative EBS and the E-box. The translation start site (ATG) is designated as +1. B, the effect of the EBS mutation. The luciferase reporter construct pD262 contains the wild-type DOR promoter sequence from -262 to +1. The mutant construct pDm192 was made by introducing a 6-bp mutation into the core binding sequence of the putative EBS in pD262. The composite EBS/E box in pD262 are underlined, and the mutated sequence in pDm192 is shown in boldface. Transient transfection and luciferase assays of these constructs were performed using NS20Y cells. Luciferase activities were normalized to beta -galactosidase activity from a co-transfected LacZ vector (pCH110) and expressed as percentages of the luciferase activity of pD262, which is arbitrarily defined as 100%. All values are expressed as mean ± S.E. C, NS20Y cells were co-transfected with 1.0 µg of pD262 or pDm192 and 1.0 µg of expression vector for p68 chicken Ets-1. Luciferase activities, normalized to beta -galactosidase activity, are expressed as fold activation to the luciferase activity of pD262, which is arbitrarily defined as 1. Results are means of three independent experiments. Error bars indicate the range of standard errors. The empty expression vector (pSG5, Stratagene) was added to make an equal amount of 5 µg of DNA for each transfection.

The luciferase fusion plasmid pD262 containing the DOR promoter sequence from -262 to +1 (the translation start site is designated as +1) was used as the primary construct. The mutant construct pDm192 was made by introducing a 6-bp mutation into the core binding sequence of the putative EBS in pD262, while preserving the E box that was sufficient for USF binding (22) (Fig. 1B). The pGL3-basic plasmid (designated as basic) containing neither promoter nor enhancer was included as a negative control. The promoter activity of each construct was tested by transient transfection assays in NS20Y cells.

As shown in Fig. 1B, pDm192 (EBS mutated) exhibited only 47% of the pD262 luciferase activity. This result indicates that the putative EBS contributes to the DOR promoter activity. To confirm this conclusion, we co-transfected NS20Y cells with pD262 and an expression vector encoding full-length p68 chicken Ets-1, which shows over 95% amino acid identity to murine Ets-1 (23, 24). As shown in Fig. 1C, the transfected Ets-1 elevated the promoter activity of pD262 by more than 3-fold. In contrast, co-transfection of pDm192 and Ets-1 resulted in only 0.4-fold (40%) of the pD262 promoter activity, showing no increase over the promoter activity of pDm192. Taken together, these results suggest that Ets-1 might trans-activate the DOR promoter via the putative EBS.

Co-binding of Ets-1 and USF to the Composite EBS/E Box in the DOR Promoter-- To determine whether Ets-1 could specifically bind to the putative EBS, we performed DNase I footprint assays using DOR promoter sequence from -262 to +1 as the wild type probe. Mutated probes M192 and M184, which contain the same sequence as the wild type probe except for a 6-bp mutation at -192 to -187 in the EBS or at -184 to -179 in the E box (Fig. 2A), were also used. In addition, as both chicken Ets-1 and human USF1 show high homology to their murine counterparts, especially in the DNA-binding domains (100% amino acid identity) (23-25), DNase I footprint assays using recombinant chicken Ets-1 or human USF1 were included as positive controls.


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  		Fig. 2.   Co-binding of Ets-1 and USF to the composite EBS/E box in the DOR promoter. A, three different probes were used in DNase I footprint assays. The probe -262/+1 contained the DOR promoter sequence from -262 to +1 including the putative EBS and the E box (underlined). Two other probes, M192 and M184, contained the same sequence as -262/+1 except for a 6-bp mutation in the core binding sequence of the putative EBS or the E box as indicated in boldface. B, the probe -262/+1 was used in DNase I footprint assays with NS20Y nuclear extracts (lanes 5 and 6), or bacterially expressed recombinant p68 chicken Ets-1 (lanes 2 and 3) or human USF1 (lane 4) as indicated. Lane 1, no protein added; lanes 2 and 3, 30 and 300 ng of recombinant chicken Ets-1, respectively; lane 4, 30 ng of recombinant human USF1. NS20Y nuclear extracts were used in lanes 5 and 6 in the absence (lane 5) or presence of 50-fold excess of unlabeled -262/+1 fragment as a competitor (lane 6). The Ets-1- or USF-protected region (EBS or UBS) is marked by a bar on the right. C, DNase I footprint assays were performed in the absence (lane 1) or presence of NS20Y nuclear extracts (lanes 2-7). -262/+1 was used as the probe in lanes 1-5. Lane 1, no protein added; lane 2, normal reaction; lane 3, 1 µl of preimmune control serum; lane 4, 1 µl of anti-Ets-1 Ab; lane 5, 1 µg of recombinant USF2Delta b. M192 and M184 were used as the probes in lanes 6 and 7, respectively. The Ets-1/USF-protected region (EBS/UBS) is marked by a bar on the right.

As shown in Fig. 2B, a very high concentration of recombinant Ets-1 (300 ng) generated a characteristic DNase I-hypersensitive site at -201 and a protected region (-201 to -180) against DNase I digestion. In contrast, a relatively lower concentration of Ets-1 (30 ng) hardly altered the DNase I digestion pattern of the probe (Fig. 2B, compare lane 2 with lane 3). This may be explained by the known low affinity of Ets-1 for its own recognition site in the absence of other factors (26). Recombinant USF1 (30 ng) protected the region from -193 to -173, which overlaps the area protected by Ets-1 (lane 4). Interestingly, NS20Y nuclear extracts generated a large protected region (-201 to -173) equivalent to a combination of the respective areas protected by Ets-1 and USF1. In addition, as with the recombinant Ets-1, the nuclear extracts also generated a DNase I-hypersensitive site at -201 (compare lane 5 with lanes 3 and 4). These results suggest that the endogenous transcription factor(s) binding this area might include both Ets-1 and USF. A 50-fold excess of unlabeled -262/+1 fragment abolished the protection (lane 6), demonstrating the specificity of this protection.

The anti-Ets-1 Ab (C-20, Santa Cruz Biotechnology) used in this study has been reported to prevent the formation of Ets-1·DNA complex (19). To determine whether the endogenous Ets-1 in the nuclear extracts bound to the composite EBS/E box, we used the anti-Ets-1 Ab in DNase I footprint assays. As shown in Fig. 2C, incubation of NS20Y nuclear extracts with the anti-Ets-1 Ab prior to adding the -262/+1 probe abolished the DNase I-hypersensitive site at -201 and the protection over the region from -201 to -193 (lane 4), both of which were characteristic of the protection pattern shown by Ets-1 (Fig. 2B, lane 3); the antibody did not abolish the protection at -193 to -180, consistent with our prior observation that this region could also be protected by USF (Fig. 2B, compare lane 4 with lane 3). In contrast, pretreatment of the nuclear extracts with the preimmune control serum did not change the DNase I digestion pattern of the probe (Fig. 2C, lanes 3). These results indicate that endogenous Ets-1 in the NS20Y nuclear extracts binds to the composite EBS/E box in the DOR promoter.

Due to its lack of the basic region, USF2Delta b is a mutant form of USF. Since the basic region of USF is required for its binding of DNA, USF2Delta b is unable to bind DNA, but still able to dimerize with wild type USF through its leucine zipper dimerization domain. Thus, USF2Delta b is expected to act as a dominant negative form of USF by dimerizing with the wild type USF and preventing it from binding DNA (11, 22). To confirm that the endogenous protein binding to the DOR promoter sequence at -193 to -173 was USF, NS20Y nuclear extracts was incubated with recombinant mouse USF2Delta b before DNase I footprint assay. USF2Delta b abolished all the protection generated by NS20Y nuclear extracts over the composite EBS/E box (Fig. 2C, lane 5), indicating that endogenous USF in the nuclear extracts binds to the composite EBS/E box. In addition, this result indicates that USFs binding to the E box is required for the binding of Ets-1 to the EBS, because when USF2Delta b prevented USF from binding to the E box, the protection generated by Ets-1 over the EBS was abolished. This result is in agreement with evidence from other groups that the DNA binding affinity of Ets-1 can be significantly enhanced by association with certain protein partner(s) binding at adjacent site(s) (27-30). Moreover, mutation of the core binding sequence of the EBS (M192) abolished the protection at -201 to -193 and the DNase I-hypersensitive site at -201 in exactly the same pattern as the anti-Ets-1 Ab (Fig. 2C, compare lane 6 with lane 4). Similarly, mutation of the E box (M184) abolished all the protection at -201 to -173 in the same way as USF2Delta b (Fig. 2C, compare lane 7 with lane 5). These results indicate that the integrity of the EBS and the E box is necessary for the co-binding of Ets-1 and USF to the composite EBS/E box in the DOR promoter.

Ets-1 and USF Differentially Contribute to the DOR Promoter Activity-- In light of our observation that the integrity of their individual sites was necessary for endogenous Ets-1 and USF binding to the composite EBS/E box (Fig. 2C), we expected that the contribution of endogenous Ets-1 or USF to the DOR promoter activity could be determined by examining the effects of mutations of their respective binding sites. Wild type construct pD262 and constructs with mutations either in the EBS or the E box, or both, were used to transfect NS20Y cells. As shown in Fig. 3, a mutation in the EBS element (pDm192) caused a 57% reduction in the promoter activity, confirming the results in Fig. 1B. The mutation of the E box (pDm184) resulted in a 90% reduction in the promoter activity, almost the same as the double mutation (pDm262Ets1*/E*). These results confirm that the DNA binding activity of USF is essential for the DOR promoter activity (11) and suggest that Ets-1 functions through DNA-bound USF. This is in agreement with our previous conclusion that the DNA binding activity of USF is required for the binding of Ets-1 to the composite EBS/E box in the DOR promoter (Fig. 2C).


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  		Fig. 3.   Effects of the EBS and the E box mutations on the DOR promoter activity. NS20Y cells were transfected with various DOR promoter/luciferase constructs containing wild type DOR promoter (pD262), or DOR promoter with mutated EBS (pDm192), or DOR promoter with mutated E box (pDm184), or DOR promoter with the composite EBS/E box replaced by a HindIII linker (pD262 Ets1*/E*). Luciferase activities were normalized to beta -galactosidase activity from a co-transfected LacZ vector (pCH110) and expressed relative to the luciferase activity of pD262, which is arbitrarily defined as 100%. The underlined sequences represent either the EBS or the E box as indicated. Mutant sequences are shown in boldface.

Ets-1 and USF Synergistically Trans-activate the DOR Promoter-- To characterize the functional interaction between Ets-1 and USF in the DOR promoter, plasmid pD262, pDm192 (EBS mutated), or pDm184 (E box mutated) was co-transfected with Ets-1 and USF1 into NS20Y cells. As shown in Fig. 4, overexpression of either Ets-1 or USF1 alone elevated the promoter activity of pD262 ~3-fold, while the combined overexpression of Ets-1 and USF1 resulted in a 9-fold activation. The latter activation was abolished either by mutation of the EBS (pDm192) or the E box (pDm184) in the DOR promoter. In addition, similar results were observed in co-transfection assays using Ets-1 and USF2 (data not shown). Taken together, these data demonstrate that Ets-1 synergizes with USF in trans-activating the DOR promoter via the composite EBS/E box.


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  		Fig. 4.   Ets-1 and USF synergistically trans-activate the DOR promoter. Plasmid pD262, pDm192, or pDm184 was co-transfected with the Ets-1 expression vector and/or the USF1 expression vector into NS20Y cells. Luciferase activities were normalized to beta -galactosidase activity from a co-transfected LacZ vector (pCH110) and expressed as fold activation to the luciferase activity of pD262, which is arbitrarily defined as 1. The results shown are based on three transfection experiments carried out in duplicate. Error bars represent standard errors. The empty expression vector (pSG5, Stratagene) was added to make an equal amount of 5 µg of DNA for each transfection.

The DNA-binding Domain of Ets-1 Is Sufficient to Play the Functional Role of Ets-1 in Trans-activating the DOR Promoter-- To investigate the mechanism underlying the trans-activation of the DOR promoter by Ets-1, we employed an expression vector encoding the functional DNA-binding domain of p68 chicken Ets-1 (Ets1-DBD), which is identical with the minimal fragment of murine Ets-1 that is fully functional for DNA binding (23, 31-32). First, NS20Y cells were co-transfected with pD262 and Ets1-DBD. Surprisingly, Ets1-DBD elevated the promoter activity of pD262 more than 3-fold, just like the full-length Ets-1 (Fig. 5A). In addition, mutation in the EBS (pDm192) abolished the effect of Ets1-DBD. These data indicate that the DNA-binding domain of Ets-1 is sufficient to trans-activate the DOR promoter via the EBS.


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  		Fig. 5.   The DNA-binding domain of Ets-1 is sufficient to play the functional role of Ets-1 in the DOR promoter. A, NS20Y cells were co-transfected with 1.0 µg of pD262 or pDm192 and 1.0 µg of expression vector encoding the Ets-1 DNA-binding domain (Ets1-DBD). B, plasmid pD262, pDm192, or pDm184 was co-transfected with the Ets1-DBD and/or the USF1 expression vector into NS20Y cells. Luciferase activities were normalized to beta -galactosidase activity from a co-transfected LacZ vector (pCH110) and expressed as fold activation to the luciferase activity of pD262, which is arbitrarily defined as 1. The results shown are based on three transfection experiments carried out in duplicate. Error bars represent standard errors. The empty expression vector (pSG5, Stratagene) was added to make an equal amount of 5 µg of DNA for each transfection.

Further experiments were carried out to determine whether Ets1-DBD could synergize with USF in trans-activating the DOR promoter. As shown in Fig. 5B, co-transfected Ets1-DBD and USF1 activated the DOR promoter activity about 9-fold, which closely resembles the synergy between the full-length Ets-1 and USF1. Mutations in either the EBS or the E box abolished the functional synergy. Based on these results, we conclude that the DNA-binding domain of Ets-1 alone is sufficient to play the functional role of Ets-1 in trans-activating the DOR promoter.

Ets-1 Enhances the Binding of USF to the Composite EBS/E box in the DOR Promoter-- It has been reported that Ets-1 synergizes with USF in specific DNA binding (19-21). In this study, we have noted that USF is indispensable for the effective binding of Ets-1 to the composite EBS/E box in the DOR promoter. To examine whether Ets-1 could affect the binding of USF to the composite EBS/E box, different concentrations of unlabeled DNA fragment M192 were used (Fig. 2A) to compete with the wild type probe for binding USF in DNase I footprint assays. As shown in Fig. 6, when Ets-1 was prevented from binding DNA by pretreatment of NS20Y nuclear extracts with the anti-Ets-1 Ab, unlabeled M192 at a concentration 30-fold higher than that of the -262/+1 probe completely abolished the protection at the composite EBS/E box (lane 4). In contrast, when NS20Y nuclear extracts were pretreated with the preimmune control serum (lane 3) or with no serum (lane 5), the 30-fold excess of M192 only slightly diminished the protection at the composite EBS/E box; a 60-fold excess of M192 was required to completely abolish the protection (lane 6). These results indicate that the DNA binding activity of Ets-1 enhances USF's binding affinity for the composite EBS/E box in the DOR promoter.


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  		Fig. 6.   Ets-1 enhances USF binding to the composite EBS/E box in the DOR promoter. DNase I footprint assays were carried out by using -262/+1 as the probe in the absence (lane 1) or presence of NS20Y nuclear extracts (lanes 2-6). Lanes 3 and 4, 30-fold excess of unlabeled M192 as the competitor in the presence of 1 µl of control serum and 1 µl of anti-Ets1 Ab, respectively. Lanes 5 and 6, 30- and 60-fold excess of unlabeled M192 as the competitor, respectively. The Ets-1/USF-protected region (EBS/UBS) and USF-protected region (UBS) are marked by bars on the right.

Ets-1 Binds to the DOR Promoter in the Neonatal Mouse Brain-- The DOR system in the mouse brain is markedly developed at the neonatal stage, when Ets-1 is highly expressed in the mouse brain (5, 33). To determine whether Ets-1 could affect the transcription of DOR gene in the neonatal mouse brain, we first adapted an in vivo formaldehyde cross-linking procedure to determine whether Ets-1 could bind to the DOR promoter in the neonatal mouse brain. Brains from 2-day-old neonatal mice were homogenized and treated with 1% formaldehyde. Then the cross-linked chromatin was immunoprecipitated by using antibodies against Ets-1 (N-276, Santa Cruz Biotechnology) or USF1. As negative controls, we included a sample without the addition of antibody, a sample with the addition of normal rabbit serum, and a noncross-linked sample. After immunoprecipitation and reversal of the cross-links, enrichment of the endogenous DOR promoter fragment in each sample was monitored by PCR amplification using primers specific for the DOR promoter (as described under "Materials and Methods"). As shown in Fig. 7, the PCR-amplified DOR promoter fragment was readily detectable in samples with the addition of anti-Ets-1 Ab or anti-USF1 Ab, while no detectable PCR products were observed in the negative controls. The binding detected at the DOR promoter is specific, because the antibodies against Ets-1 or USF1 do not enrich the GAPDH gene fragments to the same level as they enrich the DOR promoter fragments (Fig. 7, lower). In addition, in vivo formaldehyde cross-linking assays using the 10-day-old neonatal mouse brain showed similar results (data not shown). These results demonstrate that both Ets-1 and USF can bind to the DOR promoter in the neonatal mouse brain, which confirms our findings in NS20Y cells (Fig. 2).


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  		Fig. 7.   Analysis of Ets-1-DNA interaction in the neonatal mouse brain using in vivo formaldehyde cross-linking assays. Formaldehyde cross-linked chromatin was prepared from brains of 2-day-old neonatal mice. The cross-linked chromatin was incubated with anti-Ets-1 Ab (lane 7) or anti-USF1 Ab (lane 8), with rabbit preimmune control serum (lane 4), or with no Ab (lane 3). A noncross-linked sample was also incubated with anti-Ets-1 Ab (lane 2). Immunoprecipitates from each sample was aliquoted and then analyzed through PCR with primers specific for the DOR promoter. Several other controls were included in the PCR: lane 1, a negative control of PCR in the absence of DNA; lane 5, a positive control of PCR in the presence of 10 ng of genomic DNA; lane 6, DNA marker. Background was measured in all samples after immunoprecipitation, using specific primers for exon 8 of mouse GAPDH gene.

Ets-1-transfected Primary Neonatal Mouse Neuronal Cells Show Enhanced Expression of DOR mRNA-- To examine the function of Ets-1 in the transcription of DOR gene in the neonatal mouse brain, primary cultures of neonatal mouse neuronal cells were transfected with the Ets-1 expression vector, followed by Northern blot analysis to determine the expression of DOR mRNA. As a compromise between cell viability and suitable cell size for efficient transfection, cerebrums from 2-day-old neonatal mice were chosen for primary neuronal cell culture. The primary cultures of neonatal mouse neuronal cells employed in this study were determined to contain 85% neuronal cells (as described under "Materials and Methods"). The Ets-1 expression vector was introduced into the primary cultures by transient transfection. Primary cultures transfected with the empty expression vector or with no plasmids were included as controls. 48 h later, poly(A)+ RNA were isolated and subjected to Northern blot analysis. As shown in Fig. 8A, three main bands corresponding to DOR mRNA of 8.5, 6.5, and 4.5 kilobases were revealed in Northern blot analysis, consistent with the reported presence of multiple transcripts of the DOR gene (34). Quantitation of the DOR mRNA was performed with PhosphorImager and ImageQuant software (Molecular Dynamics). As all of the three DOR transcripts encode the full-length DOR (34), the total density of the three DOR mRNA bands in each lane was calculated and normalized to the density of the beta -actin mRNA band (Fig. 8B). One-way ANOVA revealed significant between group differences in the relative DOR mRNA levels (F = 9.208, p < 0.05). The post-hoc testing using the least-significance differences method showed that the DOR mRNA level in the Ets-1-transfected primary neuronal cell cultures was significantly higher than that in the controls (p < 0.05), while no significant difference was noted between the control groups. These results indicate that Ets-1 functions to enhance DOR mRNA expression in the neonatal mouse brain.


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  		Fig. 8.   Transfected Ets-1 enhances the expression of DOR mRNA in primary neonatal mouse neuronal cells. A, the primary cultures of neonatal mouse neuronal cells were transfected with the Ets-1 expression vector (pSG5-Ets-1) or the empty expression vector (pSG5, Stratagene), as described under "Materials and Methods." 48 h after transfection, cells were harvested for poly(A)+ RNA isolation. 1.6 µg of poly(A)+ RNA from each sample was subjected to Northern blot analysis using a mouse DOR cDNA probe. The positions of molecular weight size standards are indicated on the left. All Northern blots were subsequently stripped of the mouse DOR cDNA probe and rehybridized with a mouse beta -actin cDNA probe to check levels of RNA between lanes. B, the relative DOR mRNA level was calculated as described under "Materials and Methods." The histograms represent the means of relative DOR mRNA level from three independent experiments. The error bars indicate the range of standard deviations. One-way ANOVA revealed significant between groups differences (F = 9.208, p < 0.05), and post-hoc testing using the least-significance differences method showed significant differences between "No transfection" and "pSG5-Ets-1" (p < 0.05), and between "pSG5" and "pSG5-Ets-1" (p < 0.05), while no significant difference was found between "No transfection" and "pSG5" (p = 0.826).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previously, an E box that binds USF in the mouse DOR promoter was reported to be crucial for the DOR promoter activity in NS20Y cells, a mouse neuronal cell line that constitutively expresses DOR (11). Through further analysis of the DOR promoter in NS20Y cells, we have demonstrated that transcription factor Ets-1 binds to an Ets-1-binding site overlapping the E-box and trans-activates the DOR promoter by synergizing with USF in specific DNA binding.

Ets-1 is the prototypic member of the Ets family of transcription factors. It has been documented that Ets-1 binds as a monomer to specific DNA sequences characterized by an invariant GGA core sequence and functions in association with other transcription factors binding at adjacent sites or even at the same site (35-39). The DNA binding activity of Ets-1 is negatively regulated by two autoinhibitory domains flanking the minimal DNA-binding domain. However, the autoinhibition of Ets-1 can be relieved by protein partner(s), which will greatly enhance the DNA binding affinity of Ets-1 (38, 41-43). This is in agreement with our observation that a very high concentration of recombinant Ets-1 was required for its binding to the composite EBS/E box in the DOR promoter (Fig. 2B), whereas lower concentrations of Ets-1 in NS20Y nuclear extracts effectively bound to the composite EBS/E box in the presence of USF (Fig. 2C). As the bHLHZip domain of USF can specifically bind to the DNA-binding domain of Ets-1 (19), it appears that the interaction between the DNA-binding domains of Ets-1 and USF relieves the autoinhibition of Ets-1 and enhances Ets-1's binding affinity for the composite EBS/E box.

An apparent functional synergy between Ets-1 and human USF1 or mouse USF2 in trans-activating the DOR promoter was seen in co-transfection assays (Fig. 4). Since chicken Ets-1 and human USF1 show 95 and 98% amino acid identity to their murine counterparts (23-25), respectively, the interaction between them is expected to well represent the interplay between their murine counterparts. To uncover the mechanism underlying the functional synergy between these factors, we employed an expression vector encoding the functional chicken Ets-1 DNA-binding domain (Ets1-DBD), which consists of 110 amino acids and is identical with the minimal fragment of murine Ets-1 that is fully functional for DNA binding (23, 31, 32). This 110-residue protein contains the highly conserved 85-residue ETS DNA-binding domain and a 25-residue extension that ends at the native C terminus of chicken or murine Ets-1 (23, 24, 31, 32). While not containing any possible trans-activation domain, this deletion mutant of Ets-1 shows similar DNA binding activity to that observed for the full-length protein (32). Thus, the 110-residue Ets1-DBD has been employed as a dominant negative form of Ets-1 to interfere with the effects of wild type Ets-1 (44). Interestingly, in our study, the Ets1-DBD and the full-length Ets-1 showed almost the same synergy with USF in trans-activating the DOR promoter (Figs. 4 and 5), which indicates that the DNA-binding domain of Ets-1 is sufficient to play the functional role of Ets-1 in trans-activating the DOR promoter. This is the first report that Ets-1 can function only through its DNA-binding domain.

USF consists of two ubiquitous polypeptides, USF1 (43 kDa) and USF2 (44 kDa), which play essential roles in the development of the mouse brain and have pleiotropic effects in adult mice (45). Both of the polypeptides have a highly conserved bHLHZip domain, which enables them to display identical dimerization and DNA-binding specificities (46-48). It has been reported that the 110-residue Ets1-DBD can bind to the bHLHZip domain of USF and enhance USF's binding affinity for DNA (19). This is consistent with our observation that when Ets-1 was prevented from binding to the EBS, USF's binding affinity for the composite EBS/E box was significantly reduced (Fig. 6). Since the DNA binding activity of USF is essential for the DOR promoter activity (11), the enhancement of USF's DNA binding affinity by Ets-1 provides a mechanistic basis for the ability of Ets-1 to trans-activate the DOR promoter. In addition, Ets-1 generated a characteristic DNase I-hypersensitive site at the 5' end of the protected region in DNase I footprint assays (Fig. 2), which suggests that the binding of Ets-1 resulted in a conformational change of the DNA. This is in agreement with the report that Ets-1 contacts DNA in a unique pattern and generates a distinct conformational change of DNA (49, 50). Thus, we suggest that in addition to its cooperativity with USF in DNA binding, Ets-1 may cause some unique conformational change of the DNA, so that the trans-activation domains of USF and its associated factors can be better organized and make optimal contacts with the basal transcriptional machinery to result in an efficient transcription initiation.

Although not expressed in the adult brain, Ets-1 is highly expressed in the developing mouse brain (33). In addition, the distribution of Ets-1 overlaps that of DOR in the developing cerebral cortex of mice (5, 33). The mouse brain DOR system is mainly developed at postnatal stages and exhibits a rapid maturation process during the neonatal development (5). In the present study, we have demonstrated that Ets-1 can bind to the DOR promoter in the neonatal mouse brain (Fig. 7) and significantly enhance DOR mRNA expression in primary neonatal mouse neuronal cells (Fig. 8). These results indicate that Ets-1 can bind to and trans-activate the DOR promoter in the neonatal mouse brain. Interestingly, the expression of the DOR gene is rarely detected in the mouse brain throughout the prenatal development, although Ets-1, USF, and the Sp family proteins are present in the prenatal mouse brain (5, 33, 45, 51). This phenomenon suggests that other uncovered factors or regulation mechanisms that appear during the neonatal development are also required for the rapid development of the DOR system in the neonatal mouse brain. Further studies will be needed to identify the underlying mechanism(s). Nevertheless, in light of our findings in NS20Y cells and in the neonatal mouse brain cells, it is evident that Ets-1 can transcriptionally activate the DOR gene in the neonatal mouse brain. Thus, Ets-1 may contribute to the development of the mouse brain DOR system.

Endogenous opioid systems are present during development and serve to modify nervous system maturation in the brain through the opioid receptor systems (52-55). Since Ets-1 may contribute to the development of the mouse brain DOR system, it is implicated in the regulation the mouse brain development, which provides insight into the functions of Ets-1 in the developing mouse brain. In addition, as DOR mediates the DOR agonist-induced adaptation to hypoxia (56-58), manipulation of the DOR expression levels in the neonatal brain may have therapeutic potential in the management of neonatal brain hypoxia injury that may occur in clinical settings such as child birth complication and hypoxia associated with Caesarean section (C-section) (40, 58, 59). Since the expression of DOR can be considerably up-regulated by increasing the expression of DOR mRNA (6, 7), our identification of Ets-1 as an effective trans-activator of the DOR promoter in the neonatal mouse brain suggests a novel target for the research aimed at seeking therapeutic interventions for neonatal brain hypoxia injury.

    ACKNOWLEDGEMENTS

We thank Dr. Andrew P. Bradford (University of Colorado Health Sciences Center) for the generous gifts of p68 chicken Ets-1 and dominant negative Ets-1 expression vectors. We thank Dr. Michéle Sawadogo (University of Texas Cancer Center) for the kind gifts of human USF1, mouse USF2, and mouse USF2Delta b expression vectors. We thank Dr. Hsien-Ching Liu (University of Minnesota) for the kind gift of the pDm184 plasmid. We thank Dr. Andy Smith for helping with the preparation of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DA-00546, DA-01583, DA-11806, and KO5-DA-70554 and by the A. & 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: sunx0078@tc.umn.edu.

Published, JBC Papers in Press, October 2, 2001, DOI 10.1074/jbc.M104793200

    ABBREVIATIONS

The abbreviations used are: OR, opioid receptor; DOR, delta -opioid receptor; USF, upstream stimulatory factor; bHLHZip, basic helix-loop-helix leucine zipper; PCR, polymerase chain reaction; Ab, antibody; bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EBS, Ets-1-binding site.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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