Characterization of a Rat Neuronal Nicotinic Acetylcholine Receptor alpha 7 Promoter*

Usha NagavarapuDagger §, Sanjay DanthiDagger , and R. Thomas Boyd||

From the Departments of  Neuroscience and Pharmacology, Dagger  The Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210

Received for publication, October 24, 2000, and in revised form, February 5, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neuronal nicotinic acetylcholine receptors (nAChRs) containing the alpha 7 subunit are expressed in the central nervous system, autonomic nervous system, retina, adrenal medulla, and PC12 cells. alpha 7 nAChRs have been implicated in several important biological activities apart from synaptic transmission such as mediating neurite growth and presynaptic control of neurotransmitter release. A 178-base pair promoter was sufficient to drive high level expression of the alpha 7 gene in PC12 cells. The alpha 7 promoter was also cell-specific, expressing in PC12 cells but not in L6 rat muscle cells. Within our minimal rat alpha 7 nAChR promoter we identified two sequences important for basal level expression. Mutation of a GC-rich sequence at -172 relative to the translational start site led to an increase in activity of the promoter, indicating the presence of a negative regulatory element. Upstream stimulatory factor-1 acted to regulate alpha 7 expression positively by binding to an E-box at -116. A site directly adjacent to the upstream stimulatory factor-1 binding site was shown to bind Egr-1. Sp1 and Sp3 binding also occurred downstream from or overlapping the Egr-1 binding site in the rat alpha 7 promoter. Several transcription factors interact in close proximity to control expression of the rat alpha 7 nicotinic receptor gene.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neuronal nicotinic acetylcholine receptors (nAChRs)1 mediate synaptic transmission in many parts of the vertebrate central nervous system, as well as in autonomic ganglia, retina, and adrenal medulla. Neuronal nAChRs are pentameric structures (1, 2) that function as ligand-gated ion channels and are composed of multiple alpha  and beta  subunits. Nine neuronal nAChR alpha  subunit genes (alpha 2-alpha 10) and three nAChR beta  subunit genes (beta 2-beta 4) have been identified (3-16).

One specific subtype of neuronal nAChR is sensitive to alpha -bungarotoxin and composed of alpha 7 subunits. alpha 7 nAChRs possess several characteristics that set them apart from most nAChRs. alpha 7 nAChRs have a high level of Ca2+ permeability, similar to that of the N-methyl-D-aspartate subtype of glutamate receptors (17). alpha 7 nAChRs also rapidly desensitize and are expressed both extrasynaptically (or perisynaptically) on neuronal soma and at presynaptic nerve terminals (17, 18).

alpha 7-containing or alpha -bungarotoxin nAChRs have been implicated in several important biological activities apart from synaptic transmission; (i) neurite outgrowth (19-22), (ii) mediating cell growth (23), (iii) neuronal development and cell death (24-26), and (iv) presynaptic control of neurotransmitter release (27). Given all of the above functions of alpha 7 nAChRs, it is clear that precise mechanisms function to control the time and place of alpha 7 gene expression. Because alpha 7 nAChRs are most likely homo-oligomeric receptors (at least some of them), regulation of alpha 7 gene expression controls the location and timing of alpha 7 nAChR functions. To examine the mechanisms that control expression of the alpha 7 gene, we have isolated a rat nAChR alpha 7 subunit promoter and identified DNA sequences and transcription factors important in regulation of the rat alpha 7 gene.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of the 5'-Flanking Region of the Rat alpha 7 nAChR Subunit Gene-- A rat genomic library in the vector EMBL SP6/T7 was purchased from CLONTECH. Approximately 106 clones were plated and lifted onto Hybond N+ filters (Amersham Pharmacia Biotech). The filters were probed using a full-length rat alpha 7 cDNA (15) labeled with [alpha -32P]dCTP using the Prime-It RmT random primer labeling kit (Stratagene) (28). Each positive clone was reprobed twice before the DNA was isolated. Several bacteriophage clones were restriction mapped and partially sequenced. DNA sequencing completed in our laboratory was performed using the dideoxynucleotide chain termination method (29) with Sequenase T7 polymerase (Amersham Pharmacia Biotech). Oligonucleotide primers (Operon; Alameda CA) were designed as needed to complete the sequence. Additional sequencing was done at the Ohio State University Sequencing facility with an Applied Biosystems model 373A. Nucleotide sequences were analyzed using GeneWorks (Intelligenetics) and MatInspector version 2.1.

Northern Analysis-- RNA was purified from PC12 cells using Trizol (Life Technologies, Inc.). Northern blot analysis was performed using 1% (w/v) agarose gels containing 7.4% (v/v) formaldehyde in 20 mM MOPS, 1 mM EDTA, and 5 mM sodium acetate at pH 7.0. 10 µg of RNA was run on each lane of the gel. After electrophoresis, the RNA was transferred to GeneScreen Plus (PerkinElmer Life Sciences) in 10 × SSC according to the manufacturer's instructions. Rat alpha 3 or alpha 7 cDNA was labeled with [alpha -32P]dCTP using the Prime-It RmT random primer labeling kit. The 32P-labeled probes (alpha 3 and/or alpha 7) were hybridized to the GeneScreen Plus membrane in 5 × SSPE, 50% deionized formamide, 5 × Denhardt's solution, 1% SDS, 10% dextran sulfate, and 100 µg/ml salmon sperm DNA at 42 °C. The filters were washed in 2 × SSPE at room temperature, 2 × SSPE, 2% SDS at 65 °C and in 0.1 × SSPE for 45 min, 0.1% SDS at room temperature for 15 min. The blots were exposed to x-ray (Kodak XAR-5) film at -70 °C with an intensifying screen. A Molecular Dynamics PhosphorImager was also used to quantify the signal intensities in each lane.

RNase Protection-- 32P-Labeled antisense RNAs were synthesized from the SstII-SstII* fragment and the BstYI-SstII fragment (see Fig. 1) using Ambion's MAXIscriptTM in vitro transcription kit according to the manufacturer's instructions. These RNA probes were hybridized to 300 pg of a synthetic sense RNA (as a positive control), 10 µg of total PC12 cell RNA, or 10 µg of total liver RNA at a molar excess using Ambion's HybSpeedTM RPA kit according to the manufacturer's instructions. Single-stranded RNA was digested by incubating for 60 min at 37 °C with a 1:50 dilution of RNase A and Tl mix (RNase A 1.0 mg/ml, 500 units/ml; and RNase Tl, 20,000 units/ml). The RNA was precipitated with ethanol and RNase HybSpeed inactivation/precipitation mix and electrophoresed in a 5% or 8% polyacrylamide gel containing 8 M urea. The gels were dried onto a Whatman 3MM paper and exposed to x-ray (Kodak XAR-5) film at -70 °C with an intensifying screen.

Analysis of Promoter Activity-- A 2.3-kb KpnI fragment isolated from the alpha 7 genomic clones was shown to contain exons 1 and 2 as well as 1.2 kb of 5'-flanking region. The 1-kb KpnI-BstYI fragment, the 178-bp BstYI-SstII fragment, and the 1.2-kb KpnI-SstII fragment from this 2.3-kb KpnI piece were subcloned into the vector pGL2-Basic (Promega) upstream from a luciferase reporter gene. Additional constructs containing a 319-bp BstYI-SstII* fragment and a 60-bp 5'-deletion of the 319-bp BstYI-SstII* fragment were made by synthesizing these fragments using the Advantage-GC cDNA polymerase chain reaction kit (CLONTECH) with appropriate oligonucleotide primers (Operon) and subcloning these products into pGL2-Basic. Point mutations were made in the 178-bp BstYI-SstII fragment using the QuikChange site-directed mutagenesis kit (Stratagene). Briefly, complementary mutagenic oligonucleotide primers containing the desired mutation were annealed to the 178-bp BstYI-SstII luciferase construct plasmid and polymerase chain reaction with Pfu Turbo DNA polymerase used to synthesize a new mutant plasmid. The complementary oligonucleotides used to create the GC mutant were 5'-GGATGCAGTTCCCTAATCGGCCACGGAGCTGCACGGC-3' and 5'-GCCGTGCAGCTCCGTGGCCGATTAGGGAACTGCATCC-3', whereas the complementary oligonucleotides used to create the E-box mutant were 5'-CGAGACGGGAGCGCGCTGTCTATTGTGGGGGCG-3' and 5'-CGCCCCCACAATAGACAGCGCGCTCCCGTCTCG-3'. The complementary oligonucleotides used to make the Egr-1 mutant were 5'-CTGTCACGTGTGAAAGCGCGCCGCGGC-3' and 5'-GCCGCGGCGCGCTTTCACACGTGACAG-3'. The sequence changes introduced are in bold and underlined. The E-box mutant plasmid was used with the mutant GC oligonucleotides to create the double mutant. The nonmutated parental plasmid DNAs were then digested with DpnI, and new mutant plasmid DNA transformed into XL-1 Blue supercompetent cells. All of the constructs used for transfection were CsCl purified and sequenced to confirm structure and sequence. PC12 cells were transfected with 3 µg of each alpha 7 promoter-luciferase construct DNA and 0.5 µg of pSV-beta -galactosidase vector DNA (Promega; internal control for transfection efficiency) using LipofectAMINE (Life Technologies, Inc.). A rat alpha 3 promoter luciferase construct (30) was also transfected into PC12 cells, and the activities of the alpha 7 constructs were compared with the activity of the alpha 3 promoter. The alpha 3 luciferase activity was thus set at 100%. All transfections were done in duplicate dishes with several experiments for each group of hybrid constructs. 2 days after transfection, extracts were harvested and luciferase activity measured using the Promega luciferase assay reagent. beta -Galactosidase assays were performed using the Galacto-Light Plus (Tropix) assay kit. Luciferase activities were normalized to the beta -galactosidase activities present in the extracts. The alpha 7 promoter plasmids were also transfected into L6 cells (rat muscle myoblasts) using the same technique as for PC12 cells.

Cell Culture-- Rat pheochromocytoma cells (PC12) and L6 myoblasts were cultured in RPMI 1640 supplemented with 10% heat-inactivated horse serum and 5% fetal calf serum (all from Life Technologies, Inc.). The medium also included 100 units/ml penicillin/streptomycin (Life Technologies, Inc.). Cells were maintained in a humidified incubator at 37 °C with a 5% C02 and 95% air atmosphere. The PC12 cells were a subclone designated N21 and were a gift from Dr. Richard Burry, Department of Neuroscience, The Ohio State University College of Medicine. The L6 myoblasts were a gift from Dr. Tony Young, Ohio State University Biotechnology Center, The Ohio State University. Drosophila melanogaster Schneider cells (SL2 cells) were a gift from Dr. Mark Seeger, Ohio State University Neurobiotechnology Center. The SL2 cells were maintained in Schneider medium supplemented with 10% fetal calf serum (both from Life Technologies, Inc.) at 25 °C.

Expression of Sp1 and Sp3 in Insect Cells-- The expression plasmids pPACSP1 and pPACuSP3 (kindly provided by Dr. Guntram Suske, Klinikum Der Philipps-Universitat At Marburg) were transfected into SL2 cells as follows. 1 day prior to transfection, the cells were plated onto 60-mm tissue culture dishes at a density of 4 × 106 cells/plate. Cells were transfected with 4 µg of plasmid DNA using LipofectAMINE in Schneider medium without serum. 12 h after the addition of DNA, the cells were placed in Schneider medium supplemented with 10% fetal calf serum at 25 °C. 24 h later the cells were washed twice with phosphate-buffered saline, and nuclear extracts were isolated (31). Briefly, the cells were washed twice in phosphate-buffered saline, pelleted, and resuspended in 400 µl of cold buffer containing 10 mM HEPES (pH 7.9), 10 mM KCI, and 0. 1 mM EDTA, 0.1 mM EGTA, 1 mM DTT and 0.5 mM phenylmethylsulfonyl fluoride. Cells were lysed by adding 25 µl of 10% Nonidet P-40, and the crude nuclei were pelleted. The nuclei were resuspended in 100 µl of buffer containing 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride. The tube was rocked vigorously at 4 °C for 15 min. The nuclear extracts were obtained by centrifugation and stored at -70 °C.

Electrophoretic Mobility Shifting and Antibody Supershifting-- PC12 nuclear extracts were prepared as described above (31). The protein concentrations of the nuclear extracts were determined using the Bio-Rad protein assay reagent. Double stranded oligonucleotides were end labeled with [32P]ATP using T4 kinase (U. S. Biochemical Corp.). For binding reactions 20,000-25,000 cpm of 32P end-labeled oligonucleotide was incubated with 5-10 µg of PC12 or SL2 nuclear extract in a 10-µl volume containing 10 mM Tris-HCI (pH 7.5), 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 1 mM MgCI2, 4% glycerol, and 0.5 µg of poly(dI-dC)·poly(dI-dC). For some experiments, the incubation buffer contained 10 mM Tris-HCI (pH 7.5), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 10% glycerol, 5 µg of bovine serum albumin, and 2 µg of poly(dA-dT)·poly(dA-dT). After incubation at 4 °C for 30 min, the complexes were separated on 4% polyacrylamide gels (80:1, acrylamide:bisacrylamide) at room temperature in 0.5 × Tris borate buffer. For some of the Sp1 and Sp3 antibody supershifting experiments a buffer containing 20 mM HEPES (pH 7.9), 100 mM KCl, 2 mM DTT, 1 mM EDTA, 20% glycerol, 0.01% Nonidet P-40, and 0.5 µg/µl bovine serum albumin was used. For competition, unlabeled oligonucleotides were added at 100-fold molar excess to each reaction for 10 min prior to the addition of the labeled probe. The gels were dried onto a Whatman 3MM paper, and were exposed to x-ray (Kodak XAR-5) film at -70 °C with an intensifying screen. Gel supershift assays were performed in the same way except that before incubation of the probes with nuclear extracts, 2.0 µl of TransCruzTM gel supershift antibody (100 µg/ml) was added to the reaction mixture and incubated for 1 h on ice. The Sp1, USF1, and USF2 supershifting antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Some of the EMSA using an anti-Egr-1 antibody were performed using the Nushift Kit (Geneka) according to the manufacturer's instructions. Briefly the nuclear extract was incubated with the binding buffer and stabilizing solution on ice for 20 min. The rabbit Egr-1 polyclonal antibody was included in supershifting reactions. Radioactively end-labeled probe was added and incubated further for another 20 min on ice. The reactions were loaded onto a 5% acrylamide:bisacrylamide gel (19:1) and electrophoresed in TGE buffer at 15 V/cm at 4 °C. The gels were exposed to x-ray film (Hyperfilm MP- Amersham) for 16-24 h. The USF consensus (5'-CACCCGGTCACGTGGCCTACACC-3') and Egr consensus (5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3') oligonucleotides were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). The Sp1 consensus (5'-ATTCGATCGGGGCGGGGCGAGC3-') oligonucleotide was purchased from Promega (Madison, WI). Double stranded oligonucleotides (sense strand shown) A (5'-AGTTCCCGGGGCGGCCACGG-3'), B (5'-CTGTCACGTGTGGGGGCGCG-3'), C (5'-CTGTCACGTGTGGGGGCGCGCCGCGGC-3'), C-m (5'-CTGTCACGTGTGAAAGCGCGCCGCGGC-3'), and D (5'-TGTGGGGGCGCGCCGCGGCTGC-3') were synthesized by Operon.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of the 5'-End of the Rat Neuronal nAChR alpha 7 Subunit Gene-- A rat genomic library (CLONTECH) was screened using a full-length rat alpha 7 cDNA (15). Six genomic clones were initially isolated. Two clones, lambda 7 1-1 and lambda 7 7-1, contained a 2.3-kb KpnI fragment that hybridized with the 5'-end of the rat alpha 7 cDNA and oligonucleotide probes from the same region. The 2.3-kb KpnI fragment from clone lambda 7 1-1 was completely sequenced using a combination of the chain termination method (29) and automated sequencing (Applied Biosystems, Inc. 373A). Parts of lambda 7 7-1 were also sequenced in the same way and had the same sequence as the corresponding region of lambda 7 1-1. Comparison of the 2.3-kb KpnI sequence with the sequence of an alpha 7 cDNA previously cloned from hippocampus (15) indicated that this fragment contained exons 1 and 2 of the rat gene and the short intervening intron as well as 1.3 kb of sequence upstream from the translational start site (Fig. 1). The sequence of the exons 1 and 2 matched the first 196 nucleotides of the rat alpha 7 cDNA sequence (15). The 2.3-kb KpnI fragment also contained 600 bp of the intron between exons 2 and 3. The sequence was analyzed for consensus transcription factor binding sites using GeneWorks and MatInspector version 2.1 (32). Consensus binding sites for Sp1, E-box (CANCTG), Ets-1, GATA factors, as well as several partial matches to the Egr-1 consensus binding site were present upstream from the first exon. The Sp1, E-box, and potential Egr-1 consensus binding sites are underlined (Fig. 1A). No consensus TATA- or CCAAT-boxes were observed. The region upstream from the first exon had a high GC content. Although the whole 2.3-kb KpnI fragment had a GC content of 54%, the region 300 bp upstream from the ATG was 68% GC, 200 bp upstream was 78% GC, and 100 bp upstream was 82% GC. The rat alpha 7 gene was similar to the bovine (33), human (34), and chick (35) alpha 7 genes in that all had a high GC content in the upstream region and lack CCAAT- or TATA-boxes. The GC content of the 400 nucleotides upstream from the rat alpha 7 translational start site is 63%. This was less than that of the human (77%), bovine (74%), and chick (83%) genes over the same region but consistent with a promoter without CCAAT- or TATA-boxes.


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Fig. 1.   Sequence of the 5'-region of the rat nAChR alpha 7 subunit gene. A, a rat genomic library was screened using a rat nAChR alpha 7 subunit cDNA, and six clones were characterized. A 2.3-kb KpnI fragment present in several clones was shown to contain exons 1 and 2 of the alpha 7 gene. The KpnI fragment was sequenced using a combination of the Sanger method and automated sequencing with an Applied Biosystems model 373A. Restriction sites used for subsequent clonings are labeled and underlined (thick lines). The downstream SstII site is noted with an asterisk (*). Exons 1 and 2 are underlined. GC-boxes (GGGCGG), E-box (CACGTG), and potential Egr-1 binding site (GCG(G/T)GGGCG) sequences are also underlined (thin lines). Approximate transcriptional start sites are noted by arrows. The ATG translation start is underlined and in bold. B, schematic representing restriction sites, restriction fragment sizes, and locations of E-box, Egr-1, and GC-rich sequences in the rat alpha 7 gene 5'-region.

Localization of Transcriptional Start Region-- DNA fragments upstream from and including the ATG translational start were used as probes for ribonuclease protection assays to locate the 5'-end of alpha 7 transcripts expressed in PC12 cells. A 139-bp SstII-SstII* fragment from within the 2.3-kb KpnI piece which contained most of the first exon was used in a ribonuclease protection assay with PC12 cell RNA (Fig. 1B). Two protected bands were observed. One was consistent with a fully protected probe, and the other was ~20 bp shorter (Fig. 2A). A 178-bp BstYI-SstII fragment from within the 2.3-kb KpnI piece and located just upstream from the 139-bp piece was also used in a ribonuclease protection assay. One small protected fragment (10 bp) was observed (Fig. 2B). This is consistent with a minor start site ~10 bp upstream from the 139-bp SstIl-SstIl* fragment. The ribonuclease protection assay results were consistent with two start sites ~30 nucleotides apart (Fig. 1A). Primer extension and 5'-rapid amplification of cDNA ends also were used to map the transcriptional start site but produced inconsistent results, most likely because of the high GC content of the promoter region.


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Fig. 2.   RNase protection assays identify the approximate transcriptional start sites. A, a 32P-labeled antisense RNA synthesized from the SstII-SstII* fragment (Fig. 1) or B, a 32P-labeled antisense RNA synthesized from the BstYI-SstII fragment (Fig. 1) was hybridized to 300 pg of a synthetic sense RNA, 10 µg of total PC12 cell RNA, or 10 µg of total liver RNA. After digestion with an RNase A/T1 mix, the protected fragments were electrophoresed into an 8% polyacrylamide gel containing 8 M urea. The gel was dried onto a Whatman 3MM paper and exposed to Kodak XAR-5 film at -70 °C with an intensifying screen. In A, two fragments are protected, one representing the complete SstII-SstII* fragment and the other one ~20 nucleotides shorter. In B, one ~10-nucleotide fragment is protected. The approximate transcriptional starts indicated by this analysis are shown in Fig. 1.

Identification of an alpha 7 Gene Promoter within the 2.3-Kb KpnI Fragment-- To identify a region upstream from the alpha 7 gene with promoter activity, we began our search within the 2.3-kb KpnI fragment from lambda 7 1-1. The 1-kb KpnI-BstYI fragment, the 178-bp BstYI-SstII fragment, and the 1.2-kb KpnI-SstII fragment upstream from the first exon were each cloned into the vector pGL2-Basic upstream from a luciferase reporter gene. Additional luciferase expression constructs containing a 319-bp BstYI-SstII* fragment and a 60-bp 5'-deletion of the 319-bp BstYI-SstII* fragment which contained most of the first exon were also made (Fig. 3). All of these plasmids were transfected into PC12 cells. A rat alpha 3 promoter-luciferase construct (30) was also transfected into PC12 cells in tandem, and the activities of the alpha 7 constructs were compared with the activity of the alpha 3 promoter. The luciferase activity produced by the alpha 3 promoter was set at 100%. All of the fragments that contained the 178-bp BstYI-SstII region had significant promoter activity in PC12 cells (Fig. 3). The 1 kb KpnI-BstYI fragment directly upstream from this region did not have any activity. The addition of the downstream 139-bp SstII-SstII* fragment to the 178-bp BstYI-SstII fragment resulted in a 38% increase in luciferase expression. A 60-bp 5'-deletion of this fragment which removed consensus GATA and Ets-1 transcription factor binding sites did not affect the promoter activity of this fragment.


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Fig. 3.   Identification of an alpha 7 gene promoter within the 2.3-kb KpnI fragment and functional analysis of sequences affecting promoter activity. A, the 1-kb KpnI-BstYI fragment, the 178-bp BstYI-SstII fragment, and the 1.2-kb KpnI-SstII fragment upstream from the first exon were each cloned into the vector pGL2-Basic upstream from a luciferase reporter gene. Additional luciferase expression constructs containing a 319-bp BstYI-SstII* fragment, both of which contained most of the first exon, were also made. PC12 cells were transfected with 3 µg of each alpha 7 promoter-luciferase construct DNA and 0.5 µg of pSV-beta -galactosidase vector DNA (internal control for transfection efficiency) using LipofectAMINE. A rat alpha 3 promoter-luciferase construct was also transfected into PC12 cells, and the activities of the alpha 7 constructs were compared with the activity of the alpha 3 promoter. The alpha 3 luciferase activity was thus set at 100%. All transfections were done in duplicate dishes with several experiments for each group of hybrid constructs. 2 days after transfection, extracts were harvested, and luciferase activity was measured using the Promega luciferase assay reagent. beta -Galactosidase assays were performed using the Galacto-Light Plus assay kit. Luciferase activities were normalized to the beta -galactosidase activities present in the extracts. B, mutations were made in the GC-box and the E-box (CACGTG) in the alpha 7 promoter contained in the 178-bp BstYI-SstII fragment. A double mutant for both the GC-box and the E-box sequences was also made. The promoter-luciferase constructs were transfected into PC12 cells, and the normalized luciferase activities present in the extracts were determined as described above. The luciferase activity of the wild-type alpha 7 promoter contained in the 178-bp BstYI-SstII fragment was set at 100%.

The smallest piece with promoter activity was the 178-bp BstYI-SstII fragment (Fig. 3). This construct was transfected into L6 cells and was not expressed, although another plasmid with the luciferase gene under SV40 promoter control (pGL2 Control, Promega) produced high levels of luciferase activity after transfection into L6 cells.2The lack of alpha 7 promoter expression in L6 cells is consistent with our Northern blot analysis, which did not detect any alpha 7 mRNA transcript in L6 cells. The lack of alpha 7 promoter activity also indicates that the 178-bp minimal promoter contains elements that provide for cell-specific expression.

Functional Analysis of Specific Sequences in the alpha 7 Promoter-- To analyze of the role of specific sequences in the activity of the alpha 7 promoter, point mutations were made in two potential transcription factor binding sites. Mutations were made in the GC-box and the E-box (CACGTG) present in the alpha 7 promoter contained in the 178-bp BstYI-SstII fragment (Fig. 1). A double mutant for both the GC-box and E-box sequences was also made. These constructs were transfected into PC12 cells, and the activities of the promoters were compared with that of the wild-type alpha 7 promoter (Fig. 3B). Mutation of the E-box (CACGTG) sequence produced a decrease in activity to 61% of the wild-type alpha 7 promoter level, whereas the GC mutation led to a greater than 2-fold increase in promoter activity. The double mutant displayed an activity similar to that of the E-box (CACGTG) mutant, a 40% decrease in promoter activity.

Relative Levels of alpha 3 and alpha 7 nAChR Subunit RNAs in PC12 Cells-- We then determined the relative amounts of alpha 3 and alpha 7 RNAs present in PC12 cells to compare the relative amounts of alpha 7 and alpha 3 RNAs with the nAChR promoter activities detected in transient transfections. To estimate the numbers of alpha 3 and alpha 7 transcripts in PC12 cells, Northern blot analysis was performed with RNA purified from PC12 cells grown under control conditions (Fig. 4). The blot was probed with 32P-labeled alpha 3 and alpha 7 cDNAs, and the amount of radioactivity associated with each species was measured with a PhosphorImager. PC12 cells expressed 5 times more of the alpha 3 3.9-kb RNA and 2 times more of the alpha 3 2.4-kb RNA than of the 6-kb alpha 7 RNA. The activity of our alpha 7 promoter was about 85% that of the rat alpha 3 promoter. The higher levels of alpha 3 RNA are consistent with the higher promoter activity of the alpha 3 promoter in PC12 cells. However, given that the alpha 3 RNA levels were several times higher than the alpha 7 RNA levels, the higher amount of alpha 3 RNA (or lower amount of alpha 7 RNA) is most likely determined by other post-transcriptional steps.


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Fig. 4.   Relative levels of alpha 3 and alpha 7 nAChR subunit RNAs in PC12 cells. 10 µg of total RNA isolated from PC12 cells was electrophoresed into each of three lanes of a 1% agarose gel containing formaldehyde and transferred to GeneScreen Plus membrane. One lane was probed with a 32P-labeled rat alpha 3 nAChR subunit cDNA, another lane probed with a 32P-labeled rat alpha 7 cDNA, and a third probed with both the alpha 3 and alpha 7 probes. After washing under stringent conditions, the membrane was exposed to Kodak XAR-5 film at -70 °C with an intensifying screen. A PhosphorImager was used to quantify the signal intensities in each lane. PC12 cells expressed about 5 times more of the alpha 3 3.9-kb RNA and 2 times more of the alpha 3 2.4-kb RNA than of the 6-kb alpha 7 RNA.

Binding of PC12 Cell Nuclear Proteins to a GC-box and an E-box within the alpha 7 Minimal Promoter-- To begin our characterization of transcription factors binding to the alpha 7 promoter, we used EMSA to determine where PC12 nuclear extract proteins bound to the minimal alpha 7 promoter. Because mutation of the upstream GC-rich sequence led to increased promoter activity, we first used an oligonucleotide containing this sequence from the promoter for EMSA using PC12 extracts. An oligonucleotide (A) containing the GC-rich sequence (Fig. 5A) was shown to produce three retarded complexes (Fig. 5B) when combined with PC12 nuclear extract.


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Fig. 5.   PC12 nuclear extract proteins bind to a GC-box in the alpha 7 promoter. A, sequence of the alpha 7 promoter with oligonucleotides used for EMSA (A-D) is overlined. B, oligonucleotide A was used for EMSA with lanes 1-5, and a consensus Sp1 oligonucleotide was used for lanes 6-11. PC12 extract was present in lanes 2, 4, 5, 7, 8, and 10. Human recombinant Sp1 was used in lanes 3, 9, and 11. Anti-Sp1 antibodies were used for supershifting in lanes 5, 10, and 11. Unlabeled competitor oligonucleotides were present in 100-fold excess in lanes 4 and 8. Shifted complexes are indicated by the ovals and antibody supershifted complexes by the box.

The upper and lower species were competed by excess unlabeled oligonucleotide A and were determined to be specific. The middle band was poorly competed and thought to be nonspecific. Because this GC-rich sequence contains a potential Sp1 binding site, human recombinant Sp1 (Promega) was used for EMSA with oligonucleotide A. Recombinant Sp1 did not retard the mobility of the oligonucleotide A (Fig. 5B, lane 3); however, recombinant Sp1 bound to an oligonucleotide containing a consensus Sp1 binding site that was used as a positive control (Fig. 5B, lane 9). Antibody supershifting of oligonucleotide A and PC12 nuclear extract with an antibody against Sp1 (Santa Cruz) had no effect. The oligonucleotide containing a consensus Sp1 binding site (Promega) produced three retarded complexes with PC12 extract in addition to binding recombinant Sp1 (Fig. 5B). Antibody supershifting with anti-Sp1 antibodies produced additional retardation of the slowest moving complex produced with the PC12 extract and the Sp1 oligonucleotide. Nuclear extracts from SL2 cells expressing mouse Sp1 and Sp3 were also used for EMSA with oligonucleotide A and did not produce retarded complexes.3Antibody supershifting with anti-Egr-1 antibodies (Geneka) also did not produce a supershift with oligonucleotide A and PC12 nuclear extract.3 Taken together these results indicate that the GC-rich sequence in the alpha 7 minimal promoter contained in oligonucleotide A binds protein(s) present in PC12 nuclear extracts which are distinct from Sp1, Sp3, or Egr-1.

Binding of PC12 nuclear proteins to a second region of the alpha 7 promoter was also examined. A consensus E-box was present in the alpha 7 promoter, and mutation of this sequence was shown to decrease promoter activity (Fig. 3B). An oligonucleotide B (Fig. 5A) containing the E-box and surrounding sequences from the alpha 7 promoter was used for EMSA and produced one retarded complex with PC12 nuclear extract (Fig. 6A). Because several transcription factors can potentially bind an E-box, we attempted antibody supershifting with antibodies to several transcription factors. Antibody supershifting with anti-USF1 antibodies revealed that USF1 binds to the alpha 7 E-box (Fig. 6A), although some of the retarded complex was not supershifted. Interestingly, the amount of retarded, nonsupershifted complex appeared to be increased in the presence of anti-USF1 (compare lane 6 with lane 8). Anti-USF2 antibodies failed to produce a supershifted complex. USF2 was also not detected in our PC12 cells using Western analysis. An oligonucleotide containing a consensus USF binding site (TCACGTG) also produced one retarded complex when incubated with PC12 nuclear extract (Fig. 6A). This band produced with the consensus oligonucleotide was completely supershifted with the same amount of anti-USF1 antiserum (Santa Cruz), which failed to supershift all of the retarded complex produced by oligonucleotide B. The failure to shift completely oligonucleotide B with the anti-USF1 antibody could be caused by technical limitations inherent to these types of experiments or was an indication that additional protein(s) bound to oligonucleotide B. However, when USF1, USF2, and Egr-1 antibodies were used together for supershifting with oligonucleotide B, all of the complex was supershifted. Because USF2 by itself did not produce a supershift, the complete supershift could be the result of antibodies to USF1 and Egr-1. Examination of oligonucleotide B revealed a sequence with homology to a potential Egr-1 binding site (36, 37). To confirm whether both USF1 and Egr-1 bound in this region, antibody supershift analysis was performed using a nuclear extract from 12-O-tetradecanoylphorbol-13-acetate-treated K-562 cells expressing high levels of Egr-1 (Geneka) and oligonucleotide B (Fig. 6B). K-562 cells also express USF1. One retarded complex is seen using the K-562 extract as seen with PC12 cell extract, whereas antibody supershifting with both anti-USF1 and anti-Egr-1 together produced two slower moving complexes, confirming that Egr-1 and USF1 were present in the same band. Thus Egr-1 binds to a site near USF1 in the alpha 7 promoter.


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Fig. 6.   USF1 and Egr-1 bind to adjacent sites on the alpha 7 promoter. A, a consensus USF1 oligonucleotide was used for EMSA in lanes 1-4. Oligonucleotide B was used for EMSA in lanes 5-10. PC12 extract was present in lanes 2-4 and 6-10. Anti-USF1 antibodies were used for supershifting in lanes 4, 8, and 10; anti-USF2 antibodies in lanes 9 and 10; and anti-Egr-1 antibodies in lanes 7 and 10. Unlabeled competitor oligonucleotide was present in 100-fold excess in lane 3. B, oligonucleotide B (lanes 1-4) was used for EMSA with a nuclear extract from 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated K-562 cells expressing a high level of Egr-1 (lanes 1-4). Unlabeled competitor oligonucleotide B was present in 100-fold excess in lane 2. An antibody to Sp3 was present in lane 3. Antibodies to USF1 and Egr-1 were used together for antibody supershifting (lane 4). C, EMSA was performed using oligonucleotide C (lanes 1 and 2) and a mutant version of oligonucleotide C (C-m) without the Egr-1 binding site. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated K-562 cells expressing a high level of Egr-1 (Geneka) were used in lanes 1-4; PC12 cell nuclear extract was used in lanes 5 and 6. Antibodies to Egr-1 were used in lanes 2 and 4; antiserum to USF1 was used in lane 6. In A-C, shifted complexes are indicated by ovals, and antibody supershifted complexes by boxes or triangles.

To study further the region of the alpha 7 promoter where USF1 and Egr-1 bound, two additional oligonucleotides were used for gel shift assays. Oligonucleotide C (Fig. 5A) contained USF1 and Egr-1 binding sites as well as additional 3'-sequence. When used for EMSA with PC12 nuclear extract two strong bands were produced, one of which was completely supershifted with the Egr-1 antibody (Fig. 6C). When a second oligonucleotide, C-m, containing a mutation that eliminates the Egr-1 binding site, was used, one strong broad band and a weaker band were produced, and no supershift was seen with the Egr-1 antiserum. This entire remaining strong band was supershifted with anti-USF1 (Fig. 6C). This confirms the Egr-1 binding to the mutated region and is consistent with the remaining binding to oligonucleotide C-m being the result of USF1. These results using the mutant oligonucleotide confirm that Egr-1 binds to the sequence directly adjacent to USF1 in oligonucleotide C.

To characterize further this region an additional oligonucleotide (D) (Fig. 5A) was constructed which lacked the nearby E-box (CACGTG). EMSAs with this oligonucleotide and PC12 extract produced one major specific species (Fig. 7A). This band was not supershifted with anti Egr-1 antibody (Fig. 7A, lane 2) as expected. A supershift also was not seen when the 12-O-tetradecanoylphorbol-13-acetate-treated K-562 cell nuclear extract expressing Egr-1 was used in place of the PC12 extract.3 Although oligonucleotide D contained the entire presumptive Egr-1 binding site, in our hands it did not bind Egr-1 using conditions under which oligonucleotides B and C bound Egr-1 (Fig. 6). Because oligonucleotide D produced a retarded complex with PC12 extract, some protein presumably bound to oligonucleotide D besides Egr-1. Binding sites for Sp1 and Egr-1 sometimes overlap, and Egr and Sp family members compete for binding to GC-rich sequences (38). To determine whether Sp1or Sp3 bound to this region, EMSAs were performed using oligonucleotide D with mouse Sp1 and mouse Sp3 expressed in SL2 cells. Oligonucleotide D produced a strong band with the Sp1 extract and a weaker one with the Sp3 extract (Fig. 7B). We next determined that Sp1 and Sp3 present in PC12 extracts bound to oligonucleotide D. Using a buffer designed to optimize binding of Sp1 and Sp3, one major and two minor retarded complexes were produced when PC12 nuclear extract was used with oligonucleotide D (Fig. 7C). Note that only one major band was observed using conditions that optimize Egr-1 binding (Fig. 7A). This points out the importance of using multiple conditions to study transcription factor binding. The addition of anti-Sp1 antiserum produced a slight supershift of the slowest complex. This is consistent with the slight supershift produced when a Sp1 consensus oligonucleotide and anti-Sp1 antiserum were used as a positive control (Fig. 7C). Anti-Sp3 did not produce a supershift with oligonucleotide D, but the two faster moving complexes disappeared as occurred with the positive control. This result is consistent with Sp1 and Sp3 binding to oligonucleotide D and supports the insect expressed Sp1 and Sp3 binding result. Interestingly, oligonucleotide C, which contained all of the sequence present in oligonucleotide D except the 3' three nucleotides, did not bind to the mouse Sp1 or Sp3, but bound USF1 and Egr-1.


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Fig. 7.   Sp1 and Sp3 bind close to the Egr-1 site in the alpha 7 promoter. A, EMSA using oligonucleotide D plus PC12 nuclear extract (lane 1) or PC12 nuclear extract and anti-Egr-1 antibodies (lane 2). B, EMSA using oligonucleotide D plus control SL2 extract (lanes 1 and 3), EMSA using oligonucleotide D plus SL2 extract expressing mouse Sp1 (lane 2), EMSA using oligonucleotide D plus SL2 extract expressing mouse Sp3 (lane 4). Nonspecific binding present in insect extracts is indicated by the arrow. C, EMSA was performed using PC12 extracts (lanes 1-4, 6-8) and oligonucleotide D (lanes 1-4) or a consensus Sp1 oligonucleotide. Anti-Sp1 antiserum (lanes 3 and 7) or anti-Sp3 antiserum (lanes 4 and 8) was used for supershifting. 100-fold excess of unlabeled oligonucleotide D was used in lane 2; no nuclear extract was present in lane 5. In A-C, shifted complexes are indicated by ovals and antibody supershifted complexes by boxes or triangles.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To examine the mechanisms that control expression of the alpha 7 gene, we isolated a 2.3-kb genomic DNA fragment containing a rat nAChR alpha 7 subunit promoter. Sequencing revealed that the region upstream from the translational start site had a high GC content and no CCAAT- or TATA-boxes. A 178-bp fragment located between -275 and -97 relative to the translational start was sufficient to drive high level expression of the alpha 7 promoter in PC12 cells. Addition of a further 1 kb of upstream DNA decreased this activity slightly, indicating that a negative regulatory element may be present in this region. This 178-bp promoter also was cell-specific, expressing in PC12 cells, but not in L6 rat muscle cells. Previously a similar sized small fragment (128 bp) was found to be sufficient to drive expression of the bovine alpha 7 promoter in SH-SY5Y cells (33). Interestingly, a 177-bp fragment from the chick alpha 7 promoter also confers tissue- and stage-specific control. Within our minimal rat alpha 7 nAChR promoter we have identified two sequences important for basal level expression. Mutation of a GC-rich sequence at -172 relative to the translational start led to an increase in the activity of the promoter, indicating the presence of a negative regulatory element. Our EMSA studies showed that this sequence does not bind Sp1 or Sp3 as might be possible given that Sp3 can act at GC-rich sequences as a negative regulatory factor (40). Another sequence, which regulated basal expression of the rat alpha 7 gene, was an E-box present at -116. Mutations in this sequence down-regulated the alpha 7 promoter, and EMSA showed that USF1 binds to this sequence. USF1 can bind to DNA as a homodimer or heterodimer with USF2. However, antibody supershifting did not show a shift with anti-USF2 antiserum, and Western blot analysis of PC12 nuclear extracts failed to detect USF2 in our PC12 cells. In our cells, USF1 up-regulates alpha 7 expression by binding to the promoter probably as a homodimer.

A site directly adjacent to the USF1 binding site was shown to bind Egr-1. Sp1 and/or Sp3 binding also occurred downstream to or overlapping the Egr-1 binding site in the rat alpha 7 promoter. Thus, two and possibly three or four transcription factors interact in close proximity on the rat alpha 7 promoter (Fig. 8). Competition for binding between Egr-1 and Sp1 can occur, and these interactions are important for the control of several genes (38). Sp1 is involved in the regulation of several nAChR genes including the bovine alpha 5 gene (41), rat alpha 3 gene (42), and rat beta 4 gene (43). Sp1 and Sp3 interact with each other and other factors to control expression of the rat beta 4 gene (44, 45). Sp1, AP2, and GC-rich sites were present in the chick alpha 7 promoter, but the role of these sequences was not defined (35, 39). The 5'-end of the human alpha 7 nAChR gene has also been cloned, and potential Sp1, AP-2, Egr-1, and cAMP response element-binding protein transcription factor binding sites were present, although the role of any of these sequences in regulation of the human alpha 7 has not been determined (34).

Before this work on the rat alpha 7 promoter, the bovine alpha 7 nAChR subunit promoter contained within a 120-bp fragment was characterized (46). Four elements contributing to promoter function in chromaffin or SHSY-5Y cells were defined. Elements 1, 2, and 4 contained GC-boxes and the other (element 3) an E-box. The transcription factor Egr-1 bound to elements 1, 2, and 4, whereas USF1 and USF2 bound to element 3. No Sp1 or Sp3 binding to the GC-boxes could be detected in gel retardation assays with nuclear extracts from chromaffin or SHSY-5Y cells. However, binding of recombinant Sp1 to element 4 was detected. Mutations of either element 1 or 2 had little effect on activity, whereas mutation of both element 1 and 2 produced dramatic decreases in promoter activity in both chromaffin and SHSY-5Y cells. Mutation of either element 3 or 4 produced a 40-50% decrease in promoter activity. Mutations of elements 1 and 4 or elements 2 and 4 did not produce effects different from the single element 4 mutation. Mutation of 1 and 3 produced more reduction in promoter activity than with 3 alone. Cotransfection experiments showed that GC element 1 was principally responsible for activation of the alpha 7 promoter by Egr-1 (46). The rat alpha 7 promoter differs from the bovine alpha 7 promoter in several significant ways. First of all, no negative element similar to the rat GC-box at -172 was seen in the bovine promoter. Although the protein(s) binding here have not been determined, we have shown that Sp1, Sp3, and Egr-1 do not bind. The rat promoter E-box binds USF1 most likely as a homodimer, as opposed to the USF1/USF2 heterodimer seen for the bovine alpha 7 promoter. In addition, the bovine E-box is about 15 bp upstream from element 2, which binds Egr-1. In the rat promoter, the USF1 and Egr-1 binding sites are directly adjacent. USF1 binding may stabilize Egr-1 binding to the alpha 7 promoter because when the USF1 binding site was removed, binding of Sp1 and Sp3 occurred downstream and close to the Egr-1 binding site, and no Egr-1 binding was detected (Fig. 8).


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Fig. 8.   Transcription factor binding to the rat alpha 7 promoter.

Thus, the regulatory elements in this region of the rat promoter are closer together than in the bovine promoter and may be more complex. Interestingly, transient transfection into PC12 cells showed that mutation of the rat alpha 7 Egr-1 site does not affect basal activity in PC12 cells.3 Egr-1 was present at low but detectable levels in our untreated PC12 cells. This difference in the role of Egr-1 in promoter regulation could be caused by species-specific differences between rat and bovine cells. It is also possible that in chromaffin cells Egr-1 plays an important role in alpha 7 expression and induction (47), whereas in a less differentiated cell line of sympathoadrenal origin, Egr-1 does not play a major role in regulating the basal level of alpha 7 expression. When our PC12 cells (48) are treated with nerve growth factor, Egr-1 RNA increased many fold while alpha 7 RNA levels decreased.3

We have identified several sequence elements that function in control of rat alpha 7 expression in PC12 cells. USF1 acts to regulate alpha 7 expression positively, whereas an unidentified factor acting at an upstream GC-box negatively affects alpha 7 promoter activity. In addition, Egr-1, Sp1, and Sp3 all bind close to USF1 and may contribute to alpha 7 expression in some cell types. Because the alpha 7 gene is regulated during development and is expressed in sympathetic neurons, multiple brain regions, chromaffin cells, muscle, and even lung (33, 49-54), additional elements beyond these influencing basal expression certainly remain to be defined.

    FOOTNOTES

* 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF321242.

§ Present address: Dept. of Dermatology, 1201 Welch Rd., MSLS Bldg., Stanford University, Stanford, CA 94305.

|| To whom correspondence should be addressed: Dept. of Neuroscience, 4068 Graves Hall, Ohio State University College of Medicine and Public Health, 333 West Tenth Ave., Columbus, OH 43210. Tel.: 614-292-4391; Fax: 614-688-8742; E-mail: boyd.16@osu.edu.

Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M009712220

2 U. Nagavarapu and R. T. Boyd, unpublished observations.

3 S. Danthi and R. T. Boyd, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: nAChR, nicotinic acetylcholine receptor; MOPS, 4-morpholinepropanesulfonic acid; kb, kilobase pair(s); bp, base pair(s); DTT, dithiothreitol; USF, upstream stimulatory factor; EMSA, electrophoretic mobility shift assay(s).

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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