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CP3 as a transcriptional repressor of the mu opioid receptor geneDepartment of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota, USA
1Correspondence: Department of Pharmacology, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455, USA. E-mail: choix074{at}umn.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CP) are generally known as RNA-binding proteins that interact in a sequence-specific fashion with single-stranded poly(C). These proteins are mainly involved in various post-transcriptional regulations (e.g., mRNA stabilization or translational activation/silencing). Here we report a novel function of CP3, a member of the CP family. CP3 bound to the double-stranded poly(C) element essential for the mu opioid receptor (MOR) promoter and repressed the promoter activity at the transcriptional level. We identified CP3 using affinity column chromatography containing the double-stranded poly(C) element and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. CP3 binding to the poly(C) sequence of the MOR gene was sequence specific, as confirmed by the supershift assay. In cotransfection studies, CP3 repressed the MOR promoter only when the poly(C) sequence was intact. Ectopic expression of CP3 led to repression of the endogenous MOR transcripts in NS20Y cells. When CP3 was disrupted using small interfering RNA (siRNA) in NS20Y cells, the transcription of the endogenous target MOR gene was increased significantly. Our data suggest that CP3 can function as a repressor of MOR transcription dependent on the MOR poly(C) sequence. We demonstrate for the first time a role of CP3 as a transcriptional repressor in MOR gene regulation.—Choi, H. S., Kim, C. S., Hwang, C. K., Song, K. Y., Law, P.-Y., Wei, L.-N., and Loh, H. H. Novel function of the poly(C)-binding protein CP3 as a transcriptional repressor of the mu opioid receptor gene.
Key Words: double-stranded poly(C) element • MOR transcription
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CPs) (1)
. The PCBPs studied in greatest detail are hnRNP K, CP1, and CP2. The latter two proteins are alternatively referred to as PCBP1 and PCBP2 or hnRNPE1 and hnRNPE2 (2)
. Recently, two other members of the CP family were discovered: CP3 and CP4 (3)
.
All members of the PCBP family are evolutionarily related. The common feature of all PCBPs is the presence of three KH (hnRNP K homology) domains: two of them located at the N terminus and a third located at the C terminus (1)
. The KH domains are RNA binding modules of 
70 amino acids in length. Members of this PCBP family are involved in multiple functions through their poly(C)-binding nature, such as mRNA stabilization (4)
, translational silencing (5)
, and translational enhancement (6)
. The hnRNP K also appears to be involved as a transcriptional factor (7
, 8)
and CP4 (MCG10) is capable of inducing apoptosis (9)
. These proteins are broadly expressed in human and mouse tissues and demonstrate poly(C) binding specificity (10)
. CP1 and CP2 share the highest level of amino acid sequence similarity at 89% (11)
. CP3 is more divergent, and CP4 is most distantly related (52% divergence from CP2) (3)
. Post-translational modifications can regulate the binding of CPs to RNA. For example, phosphorylation of CP1 and CP2 results in a marked decrease in RNA-binding activity (2)
. An additional major determinant of CP isoform function may relate to subcellular localization (12)
.
Opioids are used as potent clinical analgesics for pain but have serious limitations such as tolerance and dependence. The opioid receptors are classified into three major types (
, 
, and µ), studied by numerous pharmacological reports and molecular cloning (13
, 14)
. All three types of opioid receptors belong to the superfamily of G-protein-coupled receptors. The mu opioid receptor (MOR) is known to play roles in morphine-induced analgesia, tolerance, and dependence, as indicated by pharmacological studies and analyses of MOR knockout mice (15
, 16)
. Upon the binding of opioids, MOR is able to couple to G-proteins and to regulate adenylyl cyclase, intracellular calcium, inwardly rectifying potassium channels, MAP kinase, and other messengers, which further trigger a cascade of intracellular events (17)
. MOR is mainly expressed in the central nervous system, with receptors varying in densities in different regions (and perhaps playing different roles; refs 18
, 19
). To achieve its unique spatial expression pattern, expression of MOR must be tightly regulated. The mouse MOR gene spans 250 kb and consists of multiple exons (20)
. Several MOR isoforms have been reported (21
, 22)
.
Two different promoters (distal and proximal) of the mouse MOR gene have been reported, which are located within 1 kb upstream of the ATG translational start site (23)
. The distal promoter initiates MOR transcription from a single transcription initiation site located 794 bp upstream of the translation start site. The proximal promoter initiates MOR transcription from four major transcription initiation sites located in a region ranging from 291 to 268 bp upstream of the translation start site. The mouse MOR promoter contains a 5'-distal promoter regulatory sequence: a 34-bp cis-acting element that possesses a strong inhibitory effect against the transcriptional function of the distal promoter (24
, 25)
. Both promoters exhibit characteristics of housekeeping genes lacking a TATA box. The distal promoter is known to be 20-fold less active than the proximal promoter, based on quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using adult and embryonic mouse brains (26)
.
In this study, we report identification and characterization of a poly(C) sequence binding protein that regulates mouse MOR gene regulation. We used affinity column chromatography containing a specific competitor and mass spectrometry to purify and identify the specific interacting factor between the poly(C) sequence of the MOR proximal promoter region and its binding protein(s) in mouse neuronal NS20Y cells. A poly(C)-binding protein, CP3, was identified. CP3 was able to bind specifically to the mouse MOR poly(C) sequence. This protein served as a transcription repressor in the proximal promoter of the mouse MOR gene. This study demonstrates a novel function for the CP3 protein: it serves as a transcription repressor via specific interaction with the double-stranded poly(C) sequence of the mouse MOR promoter.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For cloning the CP3 gene, total RNA was isolated from mouse NS20Y cells. The RNA was treated with RNase-free DNase (Promega), according to the manufacturer’s instructions. RT-PCR was performed using a OneStep RT-PCR kit (Qiagen, Valencia, CA, USA). The PCR reaction was performed with the following primers for CP3, designed using the gene sequence information from GenBank (GeneID: 59093): 5'-AAAATGGAATCTAAGGTCTCGGAAG-3' (sense); 5'-GAGTGCACCCATCCCGGTGACCTC-3' (antisense). The PCR conditions were as follows: 94°C for 5 min; 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min; and 72°C for 10 min. The RT-PCR product (1022 bp) was excised from a 1% agarose gel, purified using a QiaQuick gel extraction kit (Qiagen), and cloned in a pCRII-TOPO vector (Invitrogen, Carlasbad, CA, USA). Candidate plasmids containing the correct size inserts were confirmed by restriction enzyme digestions and DNA sequencing on an ABI 3100 sequencer (Applied Biosystems, Foster City, CA, USA).
For transient expression studies, the CP3 gene was cloned by digesting the above pCRII-TOPO-CP3 clone with 5'-HindIII and 3'-XhoI into the same sites of a pcDNA4 vector (Invitrogen), generating a pcDNA4-CP3 plasmid. The DNA sequences of all constructs were confirmed by DNA sequencing. In vitro translations were carried out with Myc-tagged pcDNA4-CP3 in a reaction mixture containing [35S]methionine (Amersham, Piscataway, NJ, USA) using a TNT quick-coupled transcription/translation system (Promega). The labeled proteins were then analyzed via SDS-PAGE on a 12% gel, and their sizes were compared with the predicted sizes.
Transient transfection and reporter gene assay
Mouse neuroblastoma NS20Y cells were routinely grown in Dulbecco’s minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2. The NS20Y cells were plated in 6-well dishes at a concentration of 0.5 x 106 cells/well and cultured overnight before transfection. Various plasmids at equimolar concentrations were used with Effectene transfection reagent (Qiagen) as described previously (27)
. Briefly, for luciferase analysis of MOR promoters, 0.5 µg of the reporter plasmids were mixed with the Effectene transfection reagent for 10 min before being added to the NS20Y cells. Forty-eight hours after transfection, cells grown to confluence were washed once with 1x phosphate-buffered saline and lysed with lysis buffer (Promega). To correct for differences in transfection efficiency, a one-fifth molar ratio of pCH110 (Amersham) containing the β-galactosidase gene under the SV40 promoter was included in each transfection for normalization. The luciferase and β-galactosidase activities of each lysate were determined according to the manufacturer’s recommendations (Promega and Tropics).
Nuclear extract preparation
Nuclear extracts were prepared from NS20Y cells as described previously (28)
. 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 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. The nuclei were resuspended in low-salt buffer (20 mM HEPES pH 7.9, 25% glycerol, 20 mM 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. A diluent (2.5 vol of 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 13,000 g. Aliquots of the supernatant containing the nuclear extracts were stored at –80°C.
Electrophoretic mobility shift assay (EMSA)
The EMSA was performed as described previously (29)
. The upper and lower strands of each probe (5'-CTTCTGCTCCCCCCCCCCCTACCC-3' and 5'-GGGTAGGGGGGGGGGGAGCAGAAG-3') were annealed, and the double-strand oligonucleotides were then end-labeled with [
-32P] dATP. Free nucleotides were separated by centrifugation through a G-25 column (Roche). The end-labeled DNA probes were incubated with in vitro-translated protein in a final volume of 20 µl EMSA buffer (10 mM Tris pH 7.5, 5% glycerol, 1 mM EDTA pH 7.1, 50 mM NaCl, 1 mM DTT, and 0.1 mg/ml poly [dI-dC]) at room temperature for 20 min. For oligonucleotide competition analyses, a 100-fold molar excess of cold competitor oligonucleotide was added to the mixture before adding the probe. For antibody supershift assays, 0.5 µg of antic-Myc (sc-40; Santa Cruz, Santa Cruz Biotechnology, CA, USA) or anti-FLAG M2 (Sigma, St. Louis, MO, USA) antibodies were added to the mixture. The reactions were then incubated at 4°C for 30 min. The reaction mixtures were electrophoresed on a 4% polyacrylamide nondenaturing gel in 0.5x TBE (45 mM Tris borate and 1 mM EDTA) at 4°C and visualized by autoradiography.
DNA-affinity purification of poly(C)-binding protein using a competitor
The following procedure is based on the interaction between biotin and streptavidin. Oligonucleotides were synthesized and purified using HPLC. In a sterile tube, 500 pmol of biotinylated sense oligonucleotide (5'-CTTCTGCTCCCCCCCCCCCTACCC-3') and 500 pmol of nonbiotinylated antisense oligonucleotide (5'-GGGTAGGGGGGGGGGGAGCAGAAG-3') were combined. The two oligonucleotides were annealed in a total volume of 20 µl by incubating in a heating block at 95°C for 10 min. They were subsequently allowed to cool completely to room temperature. Five hundred microliters of 0.5x saline-sodium citrate (SSC) solution were added to 500 pmol of the 5'-terminal-biotinylated double-stranded DNA. Meanwhile, 500 pmol of streptavidin-paramagnetic particles (Promega) were resuspended by gently flicking the bottom of the tube until they were completely dispersed and then captured by placing the tube in a magnetic stand. The supernatant was carefully removed. The magnetic particles were washed three times with 0.5x SSC and resuspended in 100 µl of 0.5x SSC.
Five hundred picomoles of biotinylated double-stranded DNA and 500 pmol of the streptavidin-Paramagnetic particles were combined and incubated for 15 min at room temperature. Samples were mixed by gentle inversion every 2 min. The magnetic beads were captured using a magnetic stand. The particles were washed three times with 300 µl of buffer A (5 mM HEPES, 26% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 300 mM NaCl), pH 7.9. One milligram of nuclear proteins was added to the affinity particles and incubated for 1 h at 4°C. The particles were washed three times with buffer A, buffer B (5 mM HEPES, 26% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 100 mM NaCl), pH 7.9, and buffer C (5 mM HEPES, 26% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF), pH 7.9. Proteins bound to the particles were released by incubation for 10 min at 95°C in a heating block in 50 µl of 1x SDS sample buffer.
To eliminate nuclear proteins that might bind nonspecifically, control experiment were performed as follows: 2500 pmol of nonbiotinylated double-stranded DNA (5X competitor) were mixed with 1 mg of nuclear proteins for 15 min on ice. The nuclear extracts containing the 5x competitor were added to the affinity particles and incubated for 1 h at 4°C. The remainder of the procedure was performed as above. The resultant protein solutions with (Fig. 1
B; control) and without (Fig. 1C
; sample) competitor were electrophoresed on a 12% SDS-PAGE gel and stained with Coomassie blue.
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SDS-PAGE, ingel tryptic digestion, and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of poly(C)-binding protein
Purified proteins were resolved by 12% SDS-PAGE. Coomassie blue-stained gels were destained, and gel slices of interest (differential bands) were subjected to ingel tryptic digestion as described previously (30)
. Tryptic peptides were extracted with 5% acetic acid, followed by 5% acetic acid and 50% acetonitrile. Samples were dissolved in 5% acetic acid and desalted using ZipTip C18 reverse-phase desalting Eppendorf tips (Millipore, Bedford, MA, USA). The peptides were eluted with 2% acetonitrile containing 0.1% trifluoroacetic acid (TFA) in a volume of 20 µl. Sample were analyzed using a MALDI-TOF mass spectrometer (Applied Biosystems). The masses of monoisotopic peaks were used for comparison to a theoretical digestion of the protein by trypsin. The Mascot database-searching software (Matrix Science, Boston, MA, USA; http://www.matrixscience.com) was used for the identification of binding proteins.
Western blot analysis
Purified proteins from NS20Y cells were incubated with treatment buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol) and boiled for 5 min. Treated extracts were resolved by SDS-PAGE using a 12% polyacrylamide gel. Gels were electroblotted onto polyvinylidene difluoride membranes (Amersham) in transfer buffer (48 mM Tris-HCl, 39 mM glycine, and 20% methanol). Membranes were blocked in blocking solution (10% dry milk and 0.1% Tween-20 in Tris-buffered saline) overnight at 4°C. Western blotting with anti-Sp1 and anti-Sp3 antibodies (Santa Cruz) was performed according to the manufacturer’s instructions (Amersham Biosciences). The signals were detected using a Storm 840 PhosphorImager system (Amersham Biosciences).
RT-PCR, real-time PCR, and heterologous expression of CP3
Total RNA was isolated according to the supplier’s protocol (TRI reagent; Molecular Research Center, Inc., Cincinnati, OH, USA). For RT-PCR, 2 µg of total RNA were used for the RT-PCR reaction using the OneStep RT-PCR reagent (Qiagen). The PCR cycle conditions consisted of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min, followed by a 10 min extension at 72°C. Primers for mouse CP3 were as follows: 5'-AACTGACGCCATCTTCAAGG-3' (sense) and 5'-ATAGTCACTGCCCGCTCTGT-3' (antisense). Similar reactions were carried out using 5'-TGGCCTTAGGGTGCAGGGGG-3' (sense) and 5'-GTGGGCCGCTCTAGGCACCA-3' (antisense) primers for β-actin as an internal control.
For real-time quantitative RT-PCR, 5 µg of total RNA were treated with DNase I and reverse-transcribed using reverse transcriptase (Roche) and primers combined with oligo (dT) and random hexamer. One-fortieth of this reaction was used for Real-time qRT-PCR analysis of gene expression, using SYBR Green I dye. Quantitative Real-time PCR was performed in an iCycler (Bio-Rad, Hercules, CA, USA) using SYBR-green (Quantitect SYBR Green PCR kit; Qiagen). The PCR cycle conditions consisted of 95°C for 20 s, 60°C for 30 s, and 72°C for 30 s, followed by a 10 min extension at 72°C. To calculate relative mRNA gene expression, amplification curves of a test sample and standard samples that contained 101 to 108 molecules of the gene of interest (e.g., the MOR expression plasmid pmMuEG constructed in our lab) were monitored and the number of target molecules in the test sample was analyzed using qCalculator ver. 1.0 software (http://www.gene-quantification.de/download.html#qcalculator). The number of target molecules was normalized against that obtained for β-actin, used as an internal control. Primers for mouse MOR were as follows: 5'-CATGGCCCTCTATTCTATCGTGT-3' (sense) and 5'-CAGCGTGCTAGTGGCTAAGG-3' (antisense). The specificity of real RT-PCR primers was determined using a melt curve after the amplification to show that only a single species of qPCR product resulted from the reaction. Single PCR products were also verified on a 2% agarose gel.
For heterologous expression of CP3, the pcDNA4-CP3 plasmid harboring the coding sequences of CP3 was transfected into mouse NS20Y cells using Effectene transfection reagent (Qiagen). To test the endogenous MOR gene regulation by CP3, total RNAs were isolated from NS20Y cells transfected with CP3. Real-time PCRs were performed using SYBR-green (Quantitect SYBR Green PCR kit; Qiagen), mouse MOR PCR primers, β-actin primers, and experimental conditions were as described above for the Real-time PCR experiments. PCR products were also verified on a 2% agarose gel.
Up-regulation of the MOR gene by silencing CP3 with small interfering RNA (siRNA)
To confirm the role of CP3 in MOR regulation, we used siRNAs to inhibit the expression of CP3 and examined the subsequent effects on MOR expression levels. One hundred picomoles of siRNA duplexes for PCBP3 (r[GAGGGAAGAUGGAAUCUAA]dTdT [sense] and r[UUAGAUUCCAUCUUCCCUC]dCdA [antisense]; Qiagen, HP Genomewide siRNA) were transfected into NS20Y cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Control and scramble (i.e., nontargeting oligos designed by Qiagen) transfections were included as negative controls. RNA was isolated 48 h after the transfections, and the expression levels of CP3 and MOR were determined by Real-time PCR using the same PCR primers and conditions as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Previously, we reported that MOR transcription is regulated by a cis-acting poly(C) sequence in the mouse MOR promoter through the binding of Sp1 and Sp3 (31)
. The poly(C) sequence (Fig. 1A
) is essential for promoter activity of mouse MOR. We report here the discovery of another regulator for this MOR promoter through binding to the poly(C) sequence, identified using the following new procedure.
In general, purification of transcription factors require many cells, several different types of columns (e.g., ion exchange chromatography, gel chromatography, and DNA affinity chromatography) and repeated experiments (32
, 33)
. It is essential to eliminate nuclear proteins that bind nonspecifically. We have developed a new one-step method for purification of transcription factors that bind to specific DNA sequences without contamination by nonspecifically binding nuclear proteins using a competitor. First, biotinylated double-stranded DNA and streptavidin-coupled paramagnetic particle were prepared and mixed. The affinity particles were immobilized using a magnetic stand. Nuclear proteins were added to the affinity particles and washed three times each with buffers A, B, and C. Proteins bound to the particles were released by heating in 50 µl of 1x SDS sample buffer (Fig. 1C
). To eliminate nonspecific binding of nuclear proteins, control experiments were performed in parallel as follows. A five-fold excess of nonbiotinylated double-stranded DNA was mixed with the nuclear proteins on ice. The nuclear extract containing the competitor was added to the affinity particles and washed and eluted as above (Fig. 1B
). The resulting protein solutions, with and without competitor, were electrophoresed on a 12% SDS-PAGE gel and stained with Coomassie blue.
Using this protocol, we were able to purify and identify a protein from NS20Y nuclear extracts that binds specifically to the poly(C) sequence. A unique band migrating at 36 kDa was visualized by Coomassie staining (Fig. 2
A), cut out of the gel, and identified by MALDI-TOF mass spectrometric analysis. Based on its high score (121) on the Mascot search results (Fig. 2C
), the protein was identified as poly(rC)-binding protein 3 (CP3). Western blot analyses were carried out using purified proteins from NS20Y cells. Although Sp1 and Sp3 proteins were identified on the blots (Fig. 2B
), the quantity of Sp1 and Sp3 binding to the poly(C) region of the proximal promoter was very small; the major binding protein identified was CP3. Nevertheless, it is known that Sp1 and Sp3 bind to poly(C) region of proximal promoter and regulate the transcription of MOR gene (34)
. These data indicate that they might also serve as a useful positive control for this purification assay.
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CP3 specifically binds to poly(C) DNA sequences of the mouse MOR proximal promoter
To determine the physical interaction of CP3 with the mouse MOR promoter, EMSA was performed using [35S]methionine-labeled CP3 and double-stranded oligonucleotide (NS; Fig. 3
A) on the MOR poly(C) sequence. An CP3 protein of the expected size (36 kDa) was produced using an in vitro translation system with [35S]methionine(Fig. 3B
). The formation of a major complex was observed by using the NS sequence with [35S]-labeled CP3 (Fig, 3C, lane 2). In contrast, only non-specific complexes were observed in the control lane without the NS sequence (Fig. 3C
, lane 1).
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To confirm the interaction of the in vitro-translated CP3 protein with the MOR poly(C) sequence (NS), EMSAs were carried out using 32P-labeled NS probe and unlabeled CP3 protein (Fig. 3D
). The CP3 protein was able to shift the target NS oligonucleotide probe (Fig. 3D
, lane 3). The specificity of this DNA-protein interaction was verified by competitive inhibition in the presence of a 100-fold excess of unlabeled self-competitor (Fig. 3D
, lane 4). We also used anti-Myc antibody with the same in vitro-translated CP3 protein, which was Myc-tagged from the pcDNA4-CP3 plasmid. The CP3-DNA complex formation was abolished by addition of the anti-Myc antibody (Fig. 3D
, lane 5) but was retained after the addition of preimmune serum or anti-FLAG antibody as negative controls (Fig. 3D
, lanes 6 and 7), indicating a specific interaction between CP3 and the poly(C) sequence DNA. Reticulocytes exhibited either nonspecific interaction, or possibly the presence of an endogenous poly(C)-binding protein (Fig. 3D
, lane 2). These results demonstrate that CP3 binds specifically to the MOR poly(C) sequence.
Defining the core binding motif of the poly(C) sequence for the CP3 protein
To determine the CP3 binding motif within the poly(C) sequence of the proximal promoter, the EMSA was carried out using the [35S]methionine-labeled CP3 with unlabeled NS sequence and mutated sequences as indicated (Fig. 4
A; NS-m1–NS-m5). The formation of major complexes was observed using NS, NS-m1 (although reduced), NS-m2, and NS-m4 (Fig. 4B
, lanes 2, 3, 4, and 6, respectively) with the [35S]-labeled CP3. No CP3 complexes were observed using the NS-m3 or NS-m5 sequences (Fig. 4B
, lanes 5 and 7, respectively). The flanking sequence overlapping the mutated site of NS-m1 could affect the CP3 protein binding to the core motif, as shown by decreasing CP3 complex formation (Fig. 4B
, lane 3). Based on the above observations, we determined that the poly(C) sequence 5'-CCCC-3' (underlined in Fig. 4C
) serves as the CP3 binding motif within poly(C) sequence of the mouse MOR proximal promoter.
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CP3 protein represses mouse MOR proximal promoter activity through the CP3 binding motif
To examine the functional role of the CP3 protein in mouse MOR gene regulation, we used the MOR proximal promoter fused with a luciferase reporter and an CP3 expression plasmid. This CP3 plasmid was cotransfected with the mouse proximal promoter construct pGL450 into neuronal NS20Y cells. When the CP3 plasmid was cotransfected into the NS20Y cells, the CP3 protein repressed 80% of the MOR proximal promoter activity, compared to vector pcDNA4-only transfected cells (Fig. 5
B). However, the CP3 protein could not repress the promoter activity of the construct pGL450mpolyC (containing a mutated CP3 binding motif; Fig. 5C
). The results suggest that the CP3 protein regulates MOR transcription through the CP3 binding motif of the MOR proximal promoter.
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Silencing of CP3 in CP3-expressing NS20Y cells with siRNA results in an increase in endogenous MOR mRNA transcription levels
The role of CP3 in the regulation of the endogenous MOR gene was tested by using a siRNA, RT-PCR, and real-time RT-PCR analysis (Fig. 6
B). We used siRNA to silence CP3 expression in NS20Y cell lines that express CP3 endogenously. The mouse NS20Y cells were transfected with 100 pmol of mouse CP3 siRNA or scrambled (scb) siRNA (Fig. 6A
). After total RNA was isolated from the transfected cell lines, RT-PCR and real-time RT-PCR were performed. Each mRNA signal was quantified using ImageQuant 5.2 software and normalized against β-actin (Fig. 6A
). The siRNA of CP3 effectively repressed the target CP3 expression to 50% of the level seen in untreated controls or scramble-transfected samples. Real-time RT-PCR revealed that in the presence of CP3 siRNA, MOR mRNA levels increased to 70% compared to the controls, whereas β-actin mRNA levels were not changed (Fig. 6B, C). These results indicate that the CP3 protein regulates endogenous MOR gene expression at thetranscriptional level.
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Effect of exogenous CP3 protein on mouse MOR gene expression
To evaluate whether transiently overexpressed CP3 can result in down-regulation of endogenous MOR transcript, real-time RT-PCR analysis using MOR-specific primers was performed with total RNA from NS20Y cells transfected with varying amounts (0–2 µg) of pcDNA4-CP3, as well as with pcDNA4 vector control. As shown in Fig. 7
A, B), CP3 down-regulated endogenous MOR gene expression in a dose-dependent manner. These results indicate that the CP3 protein plays an important role in the regulation of mouse MOR gene expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Our earlier studies showed that mouse MOR transcription was regulated by a cis-acting element: a poly(C) sequence that was essential for the activity of the mouse MOR promoter through the binding of Sp1 and Sp3 (31)
. We have developed an efficient method to purify transcription factors (Fig. 1B, C
). This simple method has many advantages, including a smaller population of cells required for analysis, rapidity (<5 h), and a one-step process that eliminates the need for additional column chromatography steps. Additionally, this method is very effective at removing nuclear proteins that bind nonspecifically. We have used this new procedure to purify new transcription factors that bind to single-stranded DNA of the MOR proximal promoter (34
and unpublished data). In this study, we use a combination of the one-step purification method and MALDI-TOF mass spectrometry to identify an CP (CP3) that binds to the poly(C) sequence of the MOR promoter (Fig. 2)
.
The CP3 contains three KH domains. The 70-amino acid KH domain is comprised of a triple-β-sheet platform supporting three -helical segments (35)
. Cocrystal structures revealed that the KH domain can interact in a highly specific manner with four to five contiguous bases in a target RNA (12)
. Poly(C)-binding proteins have diverse functions, including viral or nonviral mRNA stability, translational silencing, translational enhancement, transcriptional activation, transcriptional inhibition, and induction of programmed cell death. Indeed, the related CP1 and CP2 proteins exhibit stabilization of cellular and viral mRNA. The binding of CP1 and CP2 to the polioviral 5' UTR can enhance polio viral mRNA stability and also coordinates the switch from translation to replication of the polio viral genome (36
, 37)
. The binding of CP1 and CP2 to 3' UTR C-rich elements can also stabilize human β-globin mRNA (4)
. Translational silencing of 15-lipoxygenase mRNA is tightly linked to formation of a RNP complex at an evolutionarily conserved, CU-rich, repeated differentiation control element within the 3' UTR. The complex that forms at this site contains two PCBPs: hnRNP K and one of the two major CPs (CP1 or CP2; 38
, 39
). CP2 also plays a role in translational enhancement of polio viral mRNA. Binding of CP2 to stem-loop IV within the central region of the internal ribosome entry site is essential to efficient poliovirus translation in HeLa cell extracts (36)
.
In addition to their roles in mRNA stability and translational control, PCBPs have transcriptional regulatory functions. The hnRNP K has a specific binding site on the SV40 early promoter (40)
and in the pyrimidine-rich strand of the CT element in the promoter of the human c-Myc gene (41)
. In the case of the thymidine kinase promoter, hnRNP K cannot physically interact with the promoter but can repress transcription by inhibiting the binding of other trans-factors to cell-cycle-regulatory determinants for this promoter (42)
. Finally, the CP4 isoform MCG10 is a potential mediator of p53 tumor suppression (9)
. Although detailed studies have been performed on other PCBPs (e.g., hnRNP K, CP1, and CP2), the biological function of CP3 has not been studied.
Here we report that CP3, a member of the CP family, binds to the double-stranded poly(C) element essential for activity of the MOR promoter and repressed the promoter activity at the transcriptional level. Specific interaction between CP3 and the poly(C) sequence of the mouse MOR promoter was first observed during one-step purification using an affinity column. EMSA further revealed the characteristics of the sequence-specific interaction between CP3 and the poly(C) sequence of the mouse MOR promoter (Fig. 3)
. In particular, the four base sequence (–418 5'-CCCC-3' –415) located in the poly(C) sequence is critical for CP3-poly(C) complex formation (Fig. 4)
. Functional analyses suggest that CP3 specifically binds to the MOR poly(C) sequence and is able to repress MOR proximal promoter containing the wild type poly(C) sequence but not mutated poly(C) sequences (Fig. 5B
). In addition, transfection with CP3 siRNA led to a remarkable increase in endogenous MOR transcription in NS20Y cells (Fig. 6)
. These data suggest that the CP3 acts as a transcription repressor. Finally and most importantly, an increase in the exogenous expression of CP3 in NS20Y cells (a mouse neuroblastoma cell line endogenously expressing MOR) correspondingly results in down-regulation of endogenous MOR transcripts in vivo in a dose-dependent manner (Fig. 7)
. It has been reported that MOR gene regulation is regulated by the poly(C) sequence of the mouse MOR promoter through the binding of Sp1 and Sp3 (34)
. However, in this study, the quantity of Sp1 and Sp3 binding to the poly(C) region of the proximal promoter was very small. Indeed, the major protein binding to the poly(C) region was CP3. It is possible that binding of Sp proteins to the poly(C) sequence is regulated by competition for binding sites with the CP3 protein and that such competition is important for negative regulation of the mouse MOR gene. We conclude that CP3 acts as a repressor of MOR transcription in neuronal cells, via a mechanism dependent on the poly(C) site of the MOR proximal promoter. We demonstrate for the first time the mechanism of transcriptional regulation of the MOR gene through CP3 in neuronal cells.
Several G-protein-coupled receptor genes, including the MOR gene, are also controlled by a promoter with constitutive activity. Thus, gene activity must be modulated via sequence-specific enhancer and/or silencer binding proteins in to produce restricted patterns of expression in the nervous system. Tissue- or cell-specific regulatory factors (28
, 43
44
45)
presumably modulate the ability of ubiquitous factors (such as CP3 or other, as yet unidentified, factors) to regulate the MOR gene promoter activity. In summary, our findings may promote a better understanding of molecular mechanisms underlying MOR gene expression.
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ACKNOWLEDGMENTS |
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Received for publication March 12, 2007. Accepted for publication June 7, 2007.
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