From the Departments of ¶ Neuroscience and
Pharmacology, 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
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ABSTRACT |
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Neuronal nicotinic acetylcholine receptors
(nAChRs) containing the 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 One specific subtype of neuronal nAChR is sensitive to
Isolation of the 5'-Flanking Region of the Rat 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 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 Analysis of Promoter Activity--
A 2.3-kb
KpnI fragment isolated from the 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 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 Isolation of the 5'-End of the Rat Neuronal nAChR 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 Identification of an
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 Functional Analysis of Specific Sequences in the Relative Levels of Binding of PC12 Cell Nuclear Proteins to a GC-box and an E-box
within the
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
Binding of PC12 nuclear proteins to a second region of the
To study further the region of the
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.
To examine the mechanisms that control expression of the 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 Before this work on the rat 7 subunit are expressed in the central
nervous system, autonomic nervous system, retina, adrenal medulla, and
PC12 cells.
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
7 gene in PC12 cells. The
7 promoter was also
cell-specific, expressing in PC12 cells but not in L6 rat muscle cells.
Within our minimal rat
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
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
7 promoter. Several
transcription factors interact in close proximity to control expression
of the rat
7 nicotinic receptor gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits. Nine
neuronal nAChR
subunit genes (
2-
10) and three nAChR
subunit genes (
2-
4) have been identified (3-16).
-bungarotoxin and composed of
7 subunits.
7 nAChRs
possess several characteristics that set them apart from most nAChRs.
7 nAChRs have a high level of Ca2+ permeability, similar
to that of the N-methyl-D-aspartate
subtype of glutamate receptors (17).
7 nAChRs also rapidly
desensitize and are expressed both extrasynaptically (or
perisynaptically) on neuronal soma and at presynaptic nerve terminals
(17, 18).
7-containing or
-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
7 nAChRs, it is clear that precise mechanisms
function to control the time and place of
7 gene expression. Because
7 nAChRs are most likely homo-oligomeric receptors (at least some of
them), regulation of
7 gene expression controls the location and
timing of
7 nAChR functions. To examine the mechanisms that control
expression of the
7 gene, we have isolated a rat nAChR
7 subunit
promoter and identified DNA sequences and transcription factors
important in regulation of the rat
7 gene.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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
7 cDNA (15) labeled with
[
-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.
3 or
7 cDNA was labeled
with [
-32P]dCTP using the Prime-It RmT random primer
labeling kit. The 32P-labeled probes (
3 and/or
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.
70 °C with an intensifying screen.
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
7 promoter-luciferase construct DNA and 0.5 µg of
pSV-
-galactosidase vector DNA (Promega; internal control for
transfection efficiency) using LipofectAMINE (Life Technologies, Inc.).
A rat
3 promoter luciferase construct (30) was also transfected into
PC12 cells, and the activities of the
7 constructs were compared
with the activity of the
3 promoter. The
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.
-Galactosidase assays were performed using the Galacto-Light Plus (Tropix) assay kit.
Luciferase activities were normalized to the
-galactosidase activities present in the extracts. The
7 promoter plasmids were also transfected into L6 cells (rat muscle myoblasts) using the same
technique as for PC12 cells.
70 °C.
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
7 Subunit
Gene--
A rat genomic library (CLONTECH) was
screened using a full-length rat
7 cDNA (15). Six genomic clones
were initially isolated. Two clones,
7 1-1 and
7 7-1, contained a
2.3-kb KpnI fragment that hybridized with the 5'-end of the
rat
7 cDNA and oligonucleotide probes from the same region. The
2.3-kb KpnI fragment from clone
7 1-1 was completely
sequenced using a combination of the chain termination method (29) and
automated sequencing (Applied Biosystems, Inc. 373A). Parts of
7 7-1 were also sequenced in the same way and had the same sequence as the
corresponding region of
7 1-1. Comparison of the 2.3-kb
KpnI sequence with the sequence of an
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
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
7 gene was similar to the bovine
(33), human (34), and chick (35)
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
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 7 subunit gene. A, a rat
genomic library was screened using a rat nAChR
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
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
7 gene 5'-region.
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.
7 Gene Promoter within the 2.3-Kb KpnI
Fragment--
To identify a region upstream from the
7 gene with
promoter activity, we began our search within the 2.3-kb
KpnI fragment from
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
3 promoter-luciferase construct
(30) was also transfected into PC12 cells in tandem, and the activities
of the
7 constructs were compared with the activity of the
3
promoter. The luciferase activity produced by the
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
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
7 promoter-luciferase
construct DNA and 0.5 µg of pSV-
-galactosidase vector DNA
(internal control for transfection efficiency) using LipofectAMINE. A
rat
3 promoter-luciferase construct was also transfected into PC12
cells, and the activities of the
7 constructs were compared with the
activity of the
3 promoter. The
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.
-Galactosidase
assays were performed using the Galacto-Light Plus assay kit.
Luciferase activities were normalized to the
-galactosidase
activities present in the extracts. B, mutations were made
in the GC-box and the E-box (CACGTG) in the
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
7 promoter contained in the 178-bp BstYI-SstII
fragment was set at 100%.
7
promoter expression in L6 cells is consistent with our Northern blot
analysis, which did not detect any
7 mRNA transcript in L6
cells. The lack of
7 promoter activity also indicates that the
178-bp minimal promoter contains elements that provide for cell-specific expression.
7
Promoter--
To analyze of the role of specific sequences in the
activity of the
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
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
7 promoter (Fig. 3B). Mutation of the E-box (CACGTG)
sequence produced a decrease in activity to 61% of the wild-type
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.
3 and
7 nAChR Subunit RNAs in PC12
Cells--
We then determined the relative amounts of
3 and
7
RNAs present in PC12 cells to compare the relative amounts of
7 and
3 RNAs with the nAChR promoter activities detected in transient transfections. To estimate the numbers of
3 and
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
3 and
7 cDNAs, and the amount of
radioactivity associated with each species was measured with a
PhosphorImager. PC12 cells expressed 5 times more of the
3 3.9-kb
RNA and 2 times more of the
3 2.4-kb RNA than of the 6-kb
7 RNA.
The activity of our
7 promoter was about 85% that of the rat
3
promoter. The higher levels of
3 RNA are consistent with the higher
promoter activity of the
3 promoter in PC12 cells. However, given
that the
3 RNA levels were several times higher than the
7 RNA
levels, the higher amount of
3 RNA (or lower amount of
7 RNA) is
most likely determined by other post-transcriptional steps.
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Fig. 4.
Relative levels of 3
and
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
3 nAChR subunit cDNA, another lane
probed with a 32P-labeled rat
7 cDNA, and a third
probed with both the
3 and
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
3 3.9-kb RNA and 2 times more of the
3
2.4-kb RNA than of the 6-kb
7 RNA.
7 Minimal Promoter--
To begin our characterization of
transcription factors binding to the
7 promoter, we used EMSA to
determine where PC12 nuclear extract proteins bound to the minimal
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 7 promoter. A,
sequence of the
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.
7 minimal promoter contained in
oligonucleotide A binds protein(s) present in PC12 nuclear extracts
which are distinct from Sp1, Sp3, or Egr-1.
7
promoter was also examined. A consensus E-box was present in the
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
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
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
7 promoter.
View larger version (55K):
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Fig. 6.
USF1 and Egr-1 bind to adjacent sites on
the 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.
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.
View larger version (41K):
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Fig. 7.
Sp1 and Sp3 bind close to the Egr-1 site in
the 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
7
gene, we isolated a 2.3-kb genomic DNA fragment containing a rat nAChR
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
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
7
promoter in SH-SY5Y cells (33). Interestingly, a 177-bp fragment from
the chick
7 promoter also confers tissue- and stage-specific
control. Within our minimal rat
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
7 gene, was an
E-box present at
116. Mutations in this sequence down-regulated the
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
7 expression
by binding to the promoter probably as a homodimer.
7 promoter. Thus, two
and possibly three or four transcription factors interact in close
proximity on the rat
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
5 gene (41),
rat
3 gene (42), and rat
4 gene (43). Sp1 and Sp3 interact with
each other and other factors to control expression of the rat
4 gene
(44, 45). Sp1, AP2, and GC-rich sites were present in the chick
7
promoter, but the role of these sequences was not defined (35, 39). The
5'-end of the human
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
7 has not been
determined (34).
7 promoter, the bovine
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
7 promoter by Egr-1 (46). The rat
7 promoter differs from the bovine
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
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
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).
View larger version (10K):
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Fig. 8.
Transcription factor binding to the rat
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 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
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
7 expression. When our PC12 cells (48) are treated with nerve
growth factor, Egr-1 RNA increased many fold while
7 RNA levels
decreased.3
We have identified several sequence elements that function in control
of rat 7 expression in PC12 cells. USF1 acts to regulate
7
expression positively, whereas an unidentified factor acting at an
upstream GC-box negatively affects
7 promoter activity. In addition,
Egr-1, Sp1, and Sp3 all bind close to USF1 and may contribute to
7
expression in some cell types. Because the
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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