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Volume 16, Number 11,
Issue of June 1, 1996
pp. 3641-3651
Copyright ©1996 Society for Neuroscience
Identification and Characterization of a 47 Base Pair
Activity-Dependent Enhancer of the Rat Nicotinic Acetylcholine Receptor
 -Subunit Promoter
Wade Walke,
Guozhi Xiao, and
Daniel Goldman
Mental Health Research Institute and Department of Biological
Chemistry, University of Michigan, Ann Arbor, Michigan 48109
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Nicotinic acetylcholine receptor (nAChR) genes are regulated by
muscle electrical activity. E-box sequences found in their promoters
are necessary for this regulation. However, many muscle genes contain
E-boxes, yet are not regulated by muscle depolarization. This suggests
that other elements are necessary, perhaps working in conjunction with
E-boxes, to confer depolarization-dependent control onto promoter
activity. We have used direct DNA injection into muscle as an in
vivo assay to identify and characterize these additional elements.
Mutagenesis and expression assays identified multiple elements within
the first 81 base pairs (bp) of the nAChR  -subunit promoter that
contribute to its regulation by muscle electrical activity. Within this
81 bp sequence, two regions of DNA were identified that were capable of
conferring activity-dependent regulation onto a heterologous promoter.
The stronger of these two putative enhancers was characterized further.
It is a 47 bp sequence that contains an E-box along with sequences
similar to the SV40 core enhancer and an SP1 site. Site-directed
mutagenesis identified residues within each of these sequences that
were necessary for enhancer activity. Furthermore, methylation
interference DNA footprinting assays showed increased nuclear protein
binding to sequences within both these enhancers after muscle
denervation, and this pattern of binding was very similar to that
observed with nuclear protein isolated from myotube extracts. These
latter results suggest that similar mechanisms may mediate increased
nAChR expression during muscle development and after muscle
denervation.
Key words:
gene expression;
electrical activity;
promoter;
muscle;
transcription;
denervation
INTRODUCTION
The neuromuscular junction (NMJ) provides a
relatively simple system to study the effects that presynaptic neuronal
activity have on postsynaptic gene expression. Nerve-induced muscle
electrical activity alters the distribution, composition, and
expression of muscle nicotinic acetylcholine receptors (nAChR) (Hall
and Sanes, 1993 ). Specifically, muscle electrical activity suppresses
extrajunctional expression of the embryonic-type
( 2  ) nAChRs (Goldman et al., 1988).
Characterization of the mechanisms mediating this activity-dependent
expression will not only further our understanding of synapse formation
at the NMJ, but also may identify mechanisms that contribute to
synaptic plasticity in the adult nervous system.
The control of nAChR expression by muscle activity is largely at the
transcriptional level. Both in vivo and in vitro
gene transfer experiments have shown that nAChR -, -, and
-subunit gene promoters can confer activity-dependent expression
onto a reporter gene (Merlie and Kornhauser, 1989; Chahine et al.,
1992; Simon et al., 1992; Bessereau et al., 1994; Tang et al., 1994;
Gilmour et al., 1995). Within these promoters, E-box sequences (CANNTG)
have been identified that are necessary for activity-dependent
expression (Bessereau et al., 1994; Tang et al., 1994; Su et al.,
1995).
These results suggested that trans-acting myogenic factors
may mediate activity-dependent expression via binding to their
cis-acting E-box elements. Consistent with this possibility
is the observation that the myogenic factors also are regulated by
muscle depolarization (Eftimie et al., 1991; Chahine et al., 1993) and
can regulate nAChR promoter activity (Gilmour et al., 1991; Prody and
Merlie, 1991; Berberich et al., 1993; Bessereau et al., 1993).
Therefore, muscle activity first may modulate the expression of certain
myogenic factors that subsequently bind to and regulate expression from
nAChR promoters. However, this does not explain why other
muscle-specific, E-box-containing genes are not also regulated by
muscle activity. The simplest explanation may be that additional
factors bind other nearby DNA elements and, in conjunction with
myogenic factor binding to E-boxes, confer activity-dependent
expression on the nAChR genes. Identification of these additional
sequences will not only confirm this hypothesis, but also provide the
necessary probes for identifying the proteins that bind these
sequences.
To this end, we assayed the activity of wild-type and mutant rat
-promoter expression constructs in innervated and denervated muscle
tissue. These assays identified multiple elements located throughout
the first 81 bp of the -promoter that contribute to its regulation
by muscle activity. However, no single mutation abolished this
regulation completely unless nucleotides (nt)  5 to 81 were deleted.
Within this piece of DNA, we identified two regions that appeared to
function as activity-dependent enhancers. The stronger of the two
enhancers contains an E-box that was shown previously to be necessary
for activity-dependent control of the mouse -promoter (Tang et al.,
1994). This enhancer also contains sequences similar to the SV40 core
enhancer (Khoury and Gruss, 1983) and an SP1 site. The importance of
each of these sequences in activity-dependent regulation was confirmed
by site-directed mutagenesis.
MATERIALS AND METHODS
Plasmids. For the enhancer experiments, a minimal
enkephalin promoter construct (MEK pXP2) was made by placing a 135 bp
fragment of the enkephalin promoter (Comb et al., 1986), extending from
nt 72 to +60, relative to the transcriptional start site, in the
SmaI and filled in BglII sites of the pXP2 vector
(Nordeen, 1988). Oligonucleotides containing -promoter sequences
(Chahine et al., 1992) as indicated in Figure 5 ( 81/32, 47/1) were synthesized with a BamHI restriction enzyme
site included on their 5 ends and a BglII restriction
enzyme site included on their 3 ends. These oligos then were placed in
the BamHI site of the MEK pXP2 construct. These oligos also
were subcloned into BSSK(+) for sequence determination.
Fig. 5.
Two enhancers from the 102 bp promoter can
confer activity-dependent regulation onto the MEK promoter. Diagram at
the top of the figure shows the 102 bp -promoter region
with putative regulatory elements indicated by boxes and
rectangles. Below this are diagrams of the
various constructs containing fragments of the -promoter subcloned
in front of an MEK promoter driving Luc expression. These constructs
were injected into innervated and denervated EDL muscles along
with CMVCAT for normalization. One week later, muscles were harvested
and Luc and CAT activities were measured. Results are shown with SD on
the right side of the figure along with the fold
differences. Expression levels are reported relative to the MEK Luc
construct. Numbers in parenthesis indicate the number of
animals injected. p-values (shown in brackets)
were calculated using a one-tailed t test.
[View Larger Version of this Image (17K GIF file)]
For the mutation and deletion studies on the -promoter, we used the
pXP 550/4 construct described previously (Chahine et al., 1992),
containing sequences 550 to +11 relative to the transcriptional start
site. Scanner-linker mutations (slm) were generated by creating a
series of 5 and 3 deletions on this construct using Exonuclease III.
These deleted DNAs were sequenced to define their 5 and 3 ends.
Deletions that had 3 ends exactly 17 bp upstream of an appropriate 5
deletion were ligated with a 17 bp linker inserted in between. One
exception is 550 slm 44/29, which has only a 16 bp deletion
between ends with a 17 bp linker inserted. The sequence of the linker
is CAGATCTCGAGCTCCAC. This linker contains three unique restriction
sites: BglII, XhoI, and SacI. The slm
550 slm 21/5 also was assayed in a -promoter construct
whose 5 end was at position 119. This construct was generated by PCR
amplification using an oligonucleotide with a 5 end at 119 and
another oligonucleotide complementary to plasmid sequences flanking the
3 end of the -promoter insert. The PCR fragment then was cut with
HindIII and cloned into the
SmaI-HindIII sites of BSSK(+) for sequencing and
subsequently cut with BamHI-HindIII and
subcloned into the BamHI-HindIII sites of the
pXP1 vector.
Internal deletions of the -promoter (550/+11) were created by
digestion of the 3 end of 550/+11 with Exonuclease III. After
sequence analysis to define the 3 ends, deletions were subcloned into
a pXP vector just upstream of a small -promoter fragment harboring
the -subunit gene transcription start site. All mutations and
deletions generated also were subcloned into BSSK(+) for sequence
analysis.
The mouse wild-type and E1 E-box mutant (mutation 5) -promoter
expression constructs (Simon and Burden, 1993) were kindly provided by
Dr. S. J. Burden. Because these promoters contained restriction enzyme
sequences also found in the multiple cloning site of the plasmid, they
had to be subcloned into the pXP2 expression vector in two stages.
First we isolated a KpnI-BamHI 3 promoter
fragment and subcloned it into the KpnI-BglII
sites of pXP2. Second, we isolated the remaining 5 -promoter
fragment using HindIII-KpnI and subcloned it
into the HindIII-KpnI sites of the pXP2 vector
containing the mouse 3 -promoter fragment.
Point mutations within the 47 to 1 region, used in the enhancer
assays, were created by PCR using oligonucleotides complementary to
sequences within the 47 to 1 region, containing single or multiple
point mutations, and an oligonucleotide complementary to plasmid
sequences. Amplified PCR products containing point mutations within the
47 to 1 region then were subcloned into BSSK(+) for sequence
determination and subsequently subcloned upstream of the MEK in the MEK
pXP2 construct.
The CMVCAT expression vector harbors the cytomegalovirus (CMV) promoter
(Boshart et al., 1985) driving chloramphenicol acetyltransferase (CAT)
expression. The CMVgal expression vector harbors the CMV promoter
driving -galactosidase expression (-gal). The SV40nl-gal
expression vector harbors an SV40 promoter driving expression of
-galactosidase containing a nuclear localization sequence
(SVnl-gal). RSV Luc (De Wet et al., 1987) and MCKpXP contain the
Rous sarcoma virus (RSV) and muscle creatine kinase (MCK) (Jaynes et
al., 1986) promoters, respectively, driving luciferase (Luc)
expression.
Muscle denervation and DNA injections. Rats, ~1 month old,
were anesthetized with ether, and the lower left hindlimb was
denervated by removing a 3 mm section of sciatic nerve just below the
hip. This procedure ensures that regeneration does not occur during the
course of our experiments. Four to five days after denervation, DNA
solutions were injected into innervated and denervated extensor
digitorum longus (EDL) muscles of anesthetized rats.
DNA, for injection into muscle, was purified twice on CsCl gradients.
Before injection, 150-200 µg of CMVCAT along with 150-200 µg of
one of the pXP expression constructs were mixed and precipitated two
times with 150 mM NaCl and two volumes of
ethanol. The final precipitate was rinsed two times with 70% ethanol
and dried briefly. This DNA was resuspended at 10 mg/ml in 150 mM NaCl, and ~10 µl was injected into each of
four different locations along the length of the EDL muscle.
Procedures for injecting DNA into skeletal muscle were based on those
described previously by Wolff (1991). We used a 100 µl Hamilton
syringe and a 0.5-inch-long, 27 gauge needle that was fitted with a
Williams collar exposing 2-3 mm of the end of the needle. DNA was
injected slowly into the EDL muscle through the skin.
Muscle extracts and reporter gene assays. One week after
injection, the EDL muscle was dissected out of the leg, rinsed in
ice-cold PBS, minced with a razor blade, and placed into 2 ml of
extraction buffer containing (in mM): potassium
phosphate 100, pH 7.8, EDTA 1, PMSF 1. Samples were homogenized
immediately with a polytron (setting 4) for 20 sec and placed on ice.
After homogenization, samples were placed in an ice-water bath and
sonicated twice for 10 sec using a Cole-Parmer ultrasonic homogenizer
set at 70%. Ice-cold extraction buffer (200 µl) containing 10%
Triton X-100 then was added, and the samples were mixed by vortexing.
Insoluble material was removed by centrifugation in a Ti50 rotor
(Beckman, Fullerton, CA) at 45,000 rpm for 15 min at 4°C. The
supernatant was transferred to a new tube, and 100 µl was used to
assay for Luc activity. Another 50-100 µl of the extract was used to
assay for CAT activity. Luc and CAT assays were performed as described
previously (Neumann et al., 1987; Brasier et al., 1989). CMVCAT
activity was used to normalize for differences between samples that
might result from different amounts of DNA injected into the muscle or
variability in the uptake of DNA. CMVCAT expression did not appear to
be regulated by muscle activity.
Muscle sectioning and staining. One week after injection of
-gal constructs into innervated and denervated EDL muscle, the
animals were perfused with 4% paraformaldehyde in PBS. EDL muscles
were dissected out and incubated overnight at 4°C in a 30%
sucrose/4% paraformaldehyde PBS solution. The tissue then was embedded
in OCT and frozen on dry ice. Samples were stored at 80°C until
sectioning. Cryostat sections (30 µM) were
taken, placed on polylysine-coated slides, and allowed to air dry.
Staining for -gal was performed by adding 50 µl of staining
solution (PBS containing 5 mM
K3Fe(CN)6, 5 mM
K4Fe(CN)6, 2 mM MgCl2, and 1 mg/ml
X-gal) to sections. Coverslips were added, and edges were sealed with
rubber cement. Slides then were placed in a sealed humidified container
at 37°C overnight. After -gal staining, rubber cement was removed,
and sections were soaked in PBS for 10 min to remove coverslips.
Sections then were stained with eosin (0.5%) and mounted with
permount.
Cell culture. The mouse muscle cell line C2C12 was cultured
as described previously (Evans et al., 1987).
RNase protections. RNase protections were performed on RNA
isolated from transiently transfected C2C12 myotubes by the GTC/CsCl
method as described previously (Chirgwin et al., 1979). To assay for
transcription originating at the MEK start site, an antisense probe was
generated from an 865 bp HindIII-EcoRI fragment
of the MEK pXP2 subcloned into BSSK(+). This fragment contained the 135 bp MEK promoter, including its transcriptional start site, together
with 730 bp from the 5 end of the Luc cDNA. To normalize for
variability in transfection efficiency between samples, RNase
protections also were performed on the same RNA sample using an
antisense CAT probe. The CAT probe was generated using a 270 bp
BglII-EcoRI fragment from the 5 end of the CAT
gene cloned into the BamHI-EcoRI sites of
BSSK(+). RNase protections were performed using RNase T2 as described
previously (Saccomanno et al., 1992).
Dimethylsulfate (DMS) DNA footprinting. DNA probes were made
from a -promoter clone containing 102 bp upstream of the
transcriptional start site. One end of the probe was
32P-labeled by Klenow fill-in reaction, isolated
on a polyacrylamide gel, eluted, and purified by
phenol-chloroform-isoamyl alcohol (25:24:1) extraction and ethanol
precipitation. C2C12 myotube nuclear extracts were prepared by the
method of Dignam et al. (1983). Innervated and denervated muscle
nuclear extracts were prepared by the method of Hahn and Covault
(1990). DNA-protein binding reactions and DMS footprinting were
performed by incubating either C2C12 myotube (200 µg), innervated
(160 µg) or denervated (160 µg) nuclear extracts in 125 µl of
binding buffer [25 mM HEPES, pH 7.9, 100 mM NaCl, 10% glycerol, 1 mg/ml BSA, 0.5 mM DTT, 0.2 mM PMSF, 50 µg/ml poly(dI-dC)] with 20,000 cpm of labeled probe (~0.1-0.2 ng)
for 20 min at room temperature. After the binding reaction, samples
were treated with 75 µl of DMS diluted 1:200 in DMS reaction buffer
(50 mM sodium cacodylate, pH 8.0, 1 mM EDTA) for 2 min at 25°C. DMS reaction was
terminated with DMS stop solution (1.5 mM sodium
acetate, pH 7.0, 1 M -mercaptoethanol, 250 µg/ml yeast tRNA). Samples then were phenol-chloroform extracted and
precipitated with ethanol. Strand cleavage at methylated guanine
residues then was carried out as outlined in the chemical DNA
sequencing technique of Maxam and Gilbert (1980).
RESULTS
In vivo expression of DNA injected into innervated and
denervated muscle
Before characterizing nAChR promoter activity in vivo,
we determined the number of muscle fibers and the number of nuclei
within a single fiber expressing the injected DNA. For these
experiments, we used two different -gal expression vectors.
CMV-gal was used to assay for the number and type of cells
expressing the injected DNA, and SV40nl-gal was used to determine
the distribution of nuclei expressing injected DNA within an individual
muscle fiber. Figure 1A shows a
-gal-stained muscle cross-section that was injected previously with
the CMV-gal construct. This analysis showed that expression of the
injected DNA is localized to muscle fibers in the vicinity of the
injection site. However, the level of expression is quite variable
among the expressing fibers. This likely reflects the amount of DNA
taken up by the fiber and the location of expressing nuclei in
relationship to where the cross-section was taken. Quantitation of the
number of fibers expressing this construct indicate that ~50-60 EDL
fibers were routinely able to incorporate this DNA into their nuclei
and express it.
Fig. 1.
-Galactosidase reporter genes identify muscle
fibers and nuclei expressing injected DNA. A, Cross-section
(30 µm thick) of EDL muscle injected with 150 µg of CMV-gal.
B, Longitudinal section (30 µm) of EDL muscle injected
with 150 µg of SV40nl-gal. Sections were stained for
-galactosidase activity (blue) and subsequently stained
with eosin. Photographs were taken with a Zeiss Axiophot microscope
using the 20× objective for pictures of longitudinal sections and the
10× objective for pictures of cross-sections.
[View Larger Version of this Image (77K GIF file)]
We also determined whether there were any differences in DNA uptake and
expression between innervated and denervated muscle fibers. No
significant difference in the number of fibers expressing -gal was
observed, although denervated muscle did show slightly reduced -gal
activity compared with innervated muscle (data not shown). This latter
observation is consistent with that reported by Gunderson et al. (1993)
and appears to reflect a change in -gal stability at muscle
denervation.
To determine whether the injected DNA diffuses throughout the muscle
fiber or remains localized to the site of injection, we used an
SV40nl-gal expression construct. Longitudinal sections were cut from
these muscles and stained for -gal activity (Fig. 1B).
Because the -gal product is nuclear-localized, it allowed us to
ascertain the distribution of this DNA in the injected muscle fiber.
Expression appears to be localized to a small region of the muscle
fiber (0.27 ± 0.12 mm), presumably representing the site of
injection.
We investigated next whether this system could be used to study the
effects of muscle electrical activity on nAChR promoter expression.
First, we assayed for the effects muscle denervation had on promoters
not expected to be induced by muscle denervation. Two promoters were
chosen for this analysis: (1) the MCK promoter, which is not regulated
by muscle electrical activity (Chahine et al., 1992), and (2) the Rous
sarcoma virus promoter (RSV) promoter, which is induced by a
calcium/calmodulin-dependent protein kinase (Kapiloff et al., 1991)
and, therefore, may be downregulated after denervation. Activities of
these promoters after direct injection of MCKpXP or RSV Luc into
innervated and denervated muscle are shown in Figure
2A. Consistent with our expectations, we find
that muscle denervation had little effect on the MCK promoter and
downregulated the RSV promoter.
Fig. 2.
Direct injection of expression vectors containing
RSV, MCK, or nAChR promoters shows specific induction of nAChR promoter
activity after muscle denervation. A, Expression of
the RSV Luc and MCK pXP constructs injected into innervated and
denervated EDL muscle. B, Expression of the rat 550/+11 pXP and mouse 1823/+25 pXP constructs injected into
innervated and denervated EDL muscle. Innervated and denervated EDL
muscles were injected with the expression vectors (above)
along with CMVCAT for normalization. One week later, EDL muscles were
harvested and assayed for Luc and CAT activity. Luc light units were
normalized to CAT activity. The bars indicate SD.
[View Larger Version of this Image (24K GIF file)]
To confirm that direct DNA injection into muscle was suitable for
analyzing nAChR promoter activity in response to muscle denervation, we
injected expression vectors harboring two different nAChR
-promoters. One promoter, mouse 1823/+25, previously had been
shown to be induced by muscle denervation in transgenic animals (Simon
et al., 1992) and, therefore, serves as a positive control. The other
promoter, rat 550/+11, previously had been shown to be regulated
by muscle activity in vitro (Chahine et al., 1992). Direct
injection of 1823/+25 pXP or 550/+11 pXP into innervated
and denervated muscle revealed that these promoters were induced eight-
and sixfold, respectively, in the denervated muscle fibers (Fig.
2B).
Mutational analysis of the -promoter identifies sequences within
81 to 5 that are necessary for electrical activity-dependent
regulation
Previous studies showed that activity-dependent regulation of the
rat -subunit gene was contained within a 102 bp region just upstream
of the transcriptional start site (Chahine et al., 1992). Therefore, we
mutated specific regions within the first 100 nt upstream of the start
site and assayed their effect on promoter activity, in vivo,
by direct injection of DNA into muscle.
To confirm that this system would reveal an effect, if the appropriate
mutation were created, we used a wild-type ( 1823 wt) and an E-box
mutant ( 1823 E1 mut 5) mouse -promoter. The E-box mutation
within the -promoter previously was shown to significantly decrease
activity-dependent expression in vivo (Tang et al., 1994).
Injection of these DNAs into innervated and denervated muscle revealed
significant loss in activity-dependent regulated expression from the
mutant promoter (Fig. 3).
Fig. 3.
Mutation of the E-box sequence in the mouse
-promoter suppresses activity-dependent regulation. Schematic
representation of the mouse 1823/+25 promoter is shown. Putative
transcription factor binding sites within 100 bp upstream of the
transcription start site are shown. Expression of injected 1823/+25 pXP or 1823/+25 E1 mutation 5 pXP in innervated and
denervated muscle relative to the wild-type construct, along with the
fold difference, is indicated to the right of the
constructs. Numbers in parenthesis indicate number of
animals injected. Luc light units were normalized to co-injected CMVCAT
activity. p-values (shown in brackets) were
calculated using a one-tailed t test. Putative regulatory
elements are indicated by rectangles along the sequence and
labeled at the bottom of the figure. The E-box element in
1823 E1 mut 5 is black to indicate that it was
mutated. The enhancer X is a previously identified enhancer with no
homology to known regulatory elements (W. Walke and D. Goldman,
unpublished observations) (Simon and Burden, 1993).
[View Larger Version of this Image (13K GIF file)]
We confirmed the importance of this E-box in mediating
activity-dependent expression of the rat -promoter by creating an
slm that replaced the rat -promoter's E-box and 3 flanking DNA
with a synthetic 17 bp sequence (see Materials and Methods). This
mutation ( 550 slm 21/5) reduced activity-dependent expression
by ~40% (Fig. 4). A similar but more robust effect
was observed when we used 119 as the parent for creating this
mutation (compare 119 wt with 119 slm 21/5 in Fig. 4).
Surprisingly, two other slm mutations (slm 44/29 and slm 81/65)
that altered sequences upstream of the E-box also reduced the
-promoter's response to muscle denervation (Fig. 4). It is
interesting that mutation slm 81/65 caused lower expression,
whereas mutation slm 44/29 resulted in higher expression in both
innervated and denervated muscle. This is consistent with the
disruption of a positive regulatory element in slm 81/65 and
disruption of a negative element in slm 44/29. To ensure that
specific effects were not contributed by the introduced linker,
experiments were repeated with constructs in which the linker was
removed. No significant differences were found between constructs
containing linker and those lacking the linker. These data suggest that
multiple elements contribute to activity-dependent control of
-subunit gene expression.
Fig. 4.
Elements within nt 81 to 5 of the -promoter
are necessary for activity-dependent regulation. The various slm and
internal deletions of the 550/+11 promoter are represented
schematically, with the numbers indicating the nt bordering
regions replaced or deleted. These expression vectors were co-injected
with CMVCAT into innervated and denervated EDL muscles. After 1 week,
muscles were harvested and Luc and CAT activities were determined.
Results are shown with SD and fold differences on the right
side of the figure. Values are reported as a percentage of 550 wt
expression. Numbers in parenthesis indicate the number of
animals injected. p-values (shown in brackets)
were calculated using a one-tailed t test.
[View Larger Version of this Image (34K GIF file)]
Based on these data, one would predict that a deletion spanning nt 81
to 5 would cause complete loss of activity-dependent expression.
Indeed, Figure 4 shows that such a deletion does render the
-promoter completely insensitive to muscle activity. However, if the
deletion spans nt 52 to 5, partial activity-dependent regulation is
retained.
Two regions within the -subunit 102 bp promoter can confer
electrical activity-dependent regulation onto a heterologous
promoter
Expression vector 550  81/5 (Fig. 4) showed that
elements mediating activity-dependent control of the -promoter
resided within a region of DNA spanning nt 81 to 5. However, slm
targeting specific subregions of this DNA were not able to abrogate
this regulation completely (Fig. 4). This suggested to us that there
may be redundant subdomains of the 81 to 5 region that possessed
elements both necessary and sufficient for conferring
activity-dependent regulation onto the -promoter. To identify these
putative activity-dependent regulatory subregions, we turned to
enhancer assays. For these studies, we synthesized different
oligonucleotides that spanned different regions of the 102 bp
-promoter (oligo 106/72; oligo 81/32; oligo 47/1; and
oligo 25/+10). These oligonucleotides were subcloned upstream of the
MEK promoter in the pXP expression vector. These constructs first were
transfected into C2C12 and NIH3T3 cells to evaluate whether they had
enhancer activity. Only oligos 81/32 and 47/1 were able to
confer enhanced expression on the MEK promoter. Oligo 81/32 was
shown to act as a general enhancer, whereas oligo 47/1 acted as a
muscle-specific enhancer (W. Walke and D. Goldman, unpublished
observations). Therefore, we focused on these two oligos in the
following experiments.
-Enhancer-MEK expression vectors were injected into innervated and
denervated muscle and their activity assayed 1 week later (Fig.
5). The MEK promoter alone did not show a statistically
significant response to muscle activity (Fig. 5). However, addition of
either -enhancer (oligo 81/32 or oligo 47/1) to the MEK
promoter increased its activity and conferred onto it
depolarization-dependent regulation (Fig. 5). 47/1 MEK showed
the most robust regulation on muscle denervation, with expression in
denervated muscle being approximately six- to sevenfold higher than in
innervated muscle. This change in 47/1 MEK promoter activity on
muscle denervation is similar to that observed for the 550
promoter (Figs. 2, 4). In addition, oligo 47/1 confers
activity-dependent expression onto the MEK promoter in either
orientation, albeit with only a 2.6-fold induction when it is in the 3
to 5 direction (Fig. 5). Neither the E-box nor the SVCE sequences on
their own were able to increase MEK promoter activity or confer
activity-dependent regulation onto the MEK promoter (data not
shown).
To confirm that the -promoter enhancer sequences were conferring
increased activity on the MEK promoter, we performed RNase protection
assays on transiently transfected C2C12 myotubes with probes that
allowed us to distinguish between promotion starting within the MEK
promoter versus promotion starting within the -subunit fragments.
Figure 6 shows the results of these experiments. Using
our 865 nt MEK probe, a protected band at 800 bp would indicate
transcription originating at the MEK start site, whereas protection of
a larger 865 bp band would indicate promotion originating upstream of
the MEK promoter. Cotransfection with MCKCAT and subsequent RNase
protection for CAT RNA allowed us to normalize for transfection
efficiency. Densitometric analysis showed that promoter activity
originating at the MEK start site is increased ~nine- and 15-fold by
fragments 81/32 and 47/1, respectively, located
upstream of the MEK promoter, and that promoter activity originating
from within the -promoter fragment is minimal by comparison.
Fig. 6.
The -subunit gene enhancers increase promotion
from the transcriptional start site of the enkephalin gene. C2C12
myotubes were transfected with MEK pXP2, 47/1 MEK pXP2, or 81/32 MEK pXP2 along with MCKCAT. Cells were harvested 48 hr after
transfection, and RNA was isolated for mapping the transcriptional
start site by RNase protection assays. The diagram at the
top illustrates the organization of the transfected
constructs containing the -subunit gene enhancer 5 of the MEK
promoter, which flanks the transcriptional start site of the enkephalin
gene (indicated by the arrow). The arrow above
the -enhancer is meant to indicate potential transcriptional start
sites. The thick line below the top diagram indicates the
region of DNA represented in the antisense RNA probe. Illustrated
below the probe are possible RNase-resistant products that
are expected to be generated depending on whether transcription begins
at the enkephalin or -gene start sites. The bottom half
of the figure shows results from an RNase protection assay. RNA
isolated from C2C12 cells transfected with the MEK pXP2, 47/1
MEK pXP2, or 81/32 MEK pXP2 constructs, and cotransfected with
MCKCAT DNA, was probed with the 865 nt probe and a 270 bp CAT probe.
Protection of a product at 800 nt indicates promoter activity
originating from the MEK start site, which is increased by the presence
of the -sequences. Protection of the 270 nt probe was used to assay
MCKCAT expression to account for transfection variations.
[View Larger Version of this Image (26K GIF file)]
An E-box within the 47 to 1 region is necessary but requires
adjacent sequences to confer electrical activity-dependent regulation
onto a minimal promoter
The above experiments indicated that activity-dependent regulation
of -promoter activity could be mediated by a combinatorial action of
two regions of DNA, one containing enhancer X (nt 81 to 32) and the
other containing nt 47 to 1. This latter region was a more robust
activity-dependent enhancer than the enhancer X region (Fig. 5) and,
therefore, was examined in greater detail. This sequence contains an
E-box, an SP1-like sequence, and a sequence similar to the SV40 core
enhancer (Khoury and Gruss, 1983). To determine whether residues within
each of these putative elements are necessary for mediating
activity-dependent control of promoter expression, we used
site-directed mutagenesis to change residues in each of these putative
elements. We chose to mutate specific residues that are likely to
interact with trans-acting regulatory proteins as revealed
by DNA methylation interference assays (see next section; Fig. 8).
Figure 7 shows the sequence of the mutations made in 47/1, which were placed upstream of the MEK promoter. These
constructs were injected into innervated and denervated muscle, and the
results of their expression, along with the wild-type enhancer, is
indicated to the right. These data indicate that mutation in any of
these elements abrogates 47/1 enhancer activity, in addition to
causing a loss of activity-dependent regulation.
Fig. 8.
DNA methylation interference footprinting shows
nuclear factors from denervated muscle, and C2 myotubes bind to similar
regions of the 102 bp promoter. Results from DMS footprinting
analysis of the 102 bp -promoter region (noncoding strand) using
nuclear extracts from C2C12 myotubes (C2), innervated
(Inn), and denervated (Den) muscle are shown.
Control lanes without nuclear extract are labeled con.
Regions containing guanine residues that were protected (open
circles) or hypermethylated (solid circles) are
indicated with lines and are labeled along the side.
AP, Putative AP2 element; X, enhancer X region;
SV, SV40 core enhancer-like sequence; Sp,
putative Sp1 element; and E, E-box sequence.
[View Larger Version of this Image (58K GIF file)]
Fig. 7.
Site-directed mutagenesis identifies sequences in
the 47 bp enhancer that are necessary to confer activity-dependent
regulation onto a heterologous promoter. Diagrammed are the wild-type
and various mutant constructs used in these studies. Top
diagram shows putative regulatory regions of the 47 bp
enhancer. Below this are shown the DNA sequences of the
wild-type and various mutations. Mutations are shown in bold
and lowercase type. -Enhancer MEK Luc constructs were
co-injected with CMVCAT into innervated and denervated EDL muscles and
assayed for Luc and CAT activity 1 week after injection. Results are
shown with SD on the right side of the figure along with the
fold differences. Values are represented as a percentage of wild-type
expression in denervated muscle. Results were normalized to CMVCAT
expression. Numbers in parentheses indicate
number of animals injected. p-values (shown in
brackets) were calculated using a one-tailed t
test.
[View Larger Version of this Image (17K GIF file)]
Myotubes and denervated muscle contain nuclear factors that
interact with multiple regions of the rat 102 bp promoter
Based on the above mutagenesis and enhancer studies, we predicted
that nuclear factors would bind to the 102 bp region of the -subunit
promoter in an activity-dependent manner. In addition, we were
interested in determining whether the binding of nuclear factors from
denervated muscle resembled the pattern of binding observed with that
obtained from cultured C2C12 muscle myotubes. Therefore, we performed
methylation interference DNA footprinting assays using nuclear extracts
from C2C12 myotubes and innervated-denervated muscle (Fig.
8). Innervated muscle nuclear extract had little effect
on the methylation pattern. However, denervated muscle extract revealed
multiple interactions along the whole length of the 102 bp
promoter region. Increased or decreased methylation was observed in
sequences corresponding to the E-box, Sp1, SVCE, enhancer X, and AP2
regions (Fig. 8). Most interesting is the similarity between binding of
C2C12 myotube nuclear extract and denervated muscle extract. This
latter result may indicate that similar proteins participate in
activating -gene expression in myotubes and denervated muscle.
DISCUSSION
Direct injection of plasmid DNA into skeletal muscle was used as
an in vivo expression assay to identify elements of the
nAChR -subunit gene promoter that participate in activity-dependent
control of -gene expression. Where comparisons could be made, our
results using DNA injection into muscle were comparable to those using
transgenic animals. However, one significant difference is the level of
gene induction on muscle denervation. The mouse -promoter is induced
~27-fold in transgenic animals (Tang et al., 1994), whereas direct
injection revealed only a ninefold induction (Fig. 3). One possible
explanation for this difference is damage to the innervated muscle
fibers during the injection procedure resulting in muscle fiber
regeneration and, therefore, increased transgene expression in the
regenerated myotubes. Other possible explanations may be that, unlike
transgene expression in transgenic animals, direct injection does not
result in transgene integration or a eukaryotic pattern of DNA
methylation. Nonetheless, it is clear that direct injection provides an
alternative to transgenic animals for in vivo expression
assays.
We previously had identified a 102 bp region of the rat nAChR
-subunit gene's 5 flanking DNA that contains all the necessary
elements to mediate activity-dependent control of gene expression
(Chahine et al., 1992). In the present study, scanner-linker
mutagenesis showed that multiple regions between 81 and 5 of the
rat -promoter were important for activity-dependent expression (Fig.
4). However, no single mutation completely abolished this regulated
expression, unless the region between 81 and 5 was deleted. This
suggested that multiple and perhaps redundant elements participated in
activity-dependent control of -gene expression. We reasoned that
these elements would be revealed most easily by studying them in
isolation from other -promoter sequences.
Therefore, we devised a scheme to identify these various elements using
an enhancer assay. Two -promoter sequences, 81 to 32 and 47 to
1, were found to possess activity-dependent enhancer activity (Fig.
5). Although these two enhancers share a stretch of DNA corresponding
to the SVCE, this piece of DNA on its own had no enhancer activity
(data not shown).
The relatively small increase in promoter activity associated with
enhancer X sequences 81 through 32 suggests that this enhancer may
play a more minor role in mediating activity-dependent regulation of
the -gene compared with enhancer sequences 47 to 1. Therefore,
we chose to characterize further the 47 bp enhancer.
Comparison of the mouse and rat -promoter sequences shows two nt
differences within the 47 bp enhancer. Both differences occur in the
rat -promoters Sp1-like region. Positions 27 and 30 are G
residues in the rat and A residues in the mouse. Although this is a
purine-purine substitution, it may indicate that the region labeled as
Sp1 in the rat sequence is not an Sp1 binding site. Additional studies
are needed to test this. Because site-directed mutagenesis targeted the
C residue at position 26 as necessary for mediating
activity-dependent expression (Fig. 7), we interpret this to mean that
the DNA sequence immediately flanking the E-box, which is conserved
between rat and mouse, is necessary for this type of regulation.
It is interesting that point mutations in the 47 bp enhancer
abrogate MEK promoter regulation by muscle activity (Fig. 7), yet when
these same regions of the -gene's 5 flanking DNA are disrupted by
slm (Fig. 4), they only partially block activity-dependent regulation.
This partial block is consistent with our finding that two regions of
the -promoter contribute to activity-dependent regulation, and one
cannot eliminate completely this regulation until both regions are
deleted ( 550 81/5) (Fig. 4).
This contextual effect may be illustrated further by the observation
that point mutations in the SVCE, SP1-like sequence, or E-box all
dramatically reduce MEK promoter activity in innervated and denervated
muscle (Fig. 7), yet mutations in these same regions of DNA sometimes
have little effect on -promoter activity in innervated muscle
(-550 slm 21/5; Fig. 4) or increase -promoter activity in
innervated and denervated muscle ( 550 slm 44/29; Fig. 4).
Although we favor context as an explanation for the differences
observed between the - and MEK promoters containing mutations within
the 47 bp enhancer, we cannot rule out that these differences reflect
the different types of mutations created in these two expression
constructs (therefore, scanner-linker vs point mutations).
We have formulated a model, based on the above data, to describe
-subunit gene expression in response to muscle innervation and
denervation (Fig. 9). Our methylation interference
assays define five regions of the -promoter that appear to bind more
protein in the denervated state than the innervated state (Fig. 8).
These regions correspond to the E-box, SP1-like sequence, SVCE,
enhancer X region, and an AP2 element. All these elements, except the
AP2 element, were characterized by mutagenesis and expression assays
(Figs. 4, 5, 7). The AP2 element was shown previously to be necessary
for high -promoter activity in noninnervated myotubes (Chahine et
al., 1992). Each one of these elements shows increased protein binding
on muscle denervation (Fig. 8), suggesting that they contribute to
high-level expression of the -promoter in this tissue. Therefore,
our model shows protein binding to each of these elements in the
denervated state (Fig. 9B). We have represented E-box
binding by a myogenic heterodimer, consistent with other E-box-binding
proteins (Lassar et al., 1991). The other cis-acting
elements may be bound by monomers or oligomers, and all or some of
these proteins may be interacting with each other.
Fig. 9.
Model for -promoter expression in innervated
versus denervated muscle. A, Activity of the -promoter is
low in innervated muscle because, in part, of the binding of a putative
repressor in the SVCE region, which blocks activation from a putative
activator binding to the enhancer X region. In addition, the absence of
transcription factor binding to the E-box region and AP2 element
contributes to low promoter activity in innervated muscle.
B, Expression in denervated muscle is high because of loss
of putative repressor binding at the SVCE site and the increased
binding of activating factors to the AP2 and SVCE/E-box region.
[View Larger Version of this Image (28K GIF file)]
We also have indicated that proteins binding to the E-box also interact
with residues 5 of the E-box, and comprising a portion of the SP1-like
sequence, which are conserved between rat and mouse promoters. However,
we cannot rule out the possibility that a separate protein (perhaps
SP1) or protein complex binds to the SP1-like site.
The low-level expression of the -gene in innervated muscle
correlates with a lack of detectable protein binding in methylation
interference assays (Fig. 8). However, because this assay uses a
relatively small linear piece of DNA for binding proteins in
vitro, it is possible that protein-DNA interactions normally
occurring in vivo are not observed. In addition,
low-affinity binding may not be detected in this assay. Consistent with
this is our mutagenesis and expression assays, which indicate that the
enhancer X region can function as an activator (550 slm 81/65)
and that the SVCE region can function as a repressor ( 550 slm
44/29) in innervated muscle (Fig. 4). In contrast, E-box mutations
had little effect on expression in innervated muscle (Fig. 4).
These results suggest that the level of activation via enhancer X
(represented by activator protein binding; Fig. 9A),
repression via the SVCE (represented by repressor protein binding; Fig.
9A), and the decreased binding of proteins to the E-box-SP1
and AP2 regions (represented by a lack of protein binding; Fig.
9A) play an important role in determining the level of
expression of this gene in innervated muscle. We have no information on
whether the proteins that bind these regions in the innervated state
are the same as those that bind these sequences in the denervated
state. The changes in enhancer X and SVCE binding protein illustrated
in Figure 9, A and B, are simply meant to
indicate either different proteins, protein modification or protein
conformation attributable to protein-protein interactions that would
differ in the innervated and denervated states.
Our enhancer assays and mutagenesis studies (Fig. 7) indicate that the
SVCE, SP1-like, and E-box sequences function as a single unit to confer
activity-dependent regulation onto a heterologous promoter. Mutation of
any one of these regions abrogates completely activity-dependent
control of promoter activity. These elements do not function
independently as activity-dependent enhancers and underscore the
importance of context in determining functional activity. This explains
why the same E-boxes, which are necessary for activity-dependent
regulation of the nAChR genes, don't necessarily confer
activity-dependent regulation onto other muscle specific genes.
Before this study, the only cis-acting element identified to
participate in activity-dependent control of nAChR gene expression had
been E-boxes of the -, -, and -subunit gene promoters
(Bessereau et al., 1994; Tang et al., 1994; Su et al., 1995). The
identification of additional cis-acting sequences necessary
for activity-dependent regulation reported here should facilitate the
identification of their binding proteins, which ultimately will allow
us to understand how these proteins participate in this activation
process.
FOOTNOTES
Received Dec. 13, 1995; revised March 11, 1996; accepted March 13, 1996.
This work was supported by grants from the National Institute of
Neurological Diseases and Stroke (2 RO1 NS2153-08), the Muscular
Dystrophy Association, and the Lucille P. Markey Charitable Trust. W.W.
was supported by a National Research Service Award predoctoral
fellowship from the National Institute of Mental Health (5 F31
MH10328-02). We thank Dr. S. J. Burden for providing the mouse
-promoter clones, Dr. A. F. Seasholtz for providing the enkephalin
promoter, and N. Goburdhun for technical assistance.
Correspondence should be addressed to Daniel Goldman, University of
Michigan Mental Health Research Institute, 205 Zina Pitcher Place, Ann
Arbor, MI 48109.
REFERENCES
-
Berberich C,
Durr I,
Koenen M,
Witzemann V
(1993)
Two
adjacent E box elements and a M-CAT box are involved in the
muscle-specific regulation of the rat acetylcholine receptor
-subunit gene.
Eur J Biochem
216:395-404 .
[Web of Science][Medline]
-
Bessereau JL,
Mendelzon D,
LePoupon C,
Fiszman M,
Changeux JP,
Piette J
(1993)
Muscle-specific expression of the acetylcholine
receptor -subunit gene requires both positive and negative
interactions between myogenic factors, Sp1 and GBF factors.
EMBO J
12:443-449 .
[Web of Science][Medline]
-
Bessereau JL,
Stratford-Perricaudet LD,
Piette J,
Poupon CL,
Changeux JP
(1994)
In vivo and in vitro analysis of electrical
activity-dependent expression of muscle acetylcholine receptor genes
using adenovirus.
Proc Natl Acad Sci USA
91:1304-1308 .
[Abstract/Free Full Text]
-
Boshart M,
Weber F,
Jahn G,
Dorsch-Hasler K,
Fleckenstein B,
Schaffner W
(1985)
A very strong enhancer is located upstream of an
immediate early gene of human cytomegalovirus.
Cell
41:521-530 .
[Web of Science][Medline]
-
Brasier AR,
Tate JE,
Habener JF
(1989)
Optimized use of the
firefly assay as a reporter gene in mammalian cell lines.
Biotechniques
7:1116-1122 .
[Web of Science][Medline]
-
Chahine KG,
Walke W,
Goldman D
(1992)
A 102 base pair
sequence of the nicotinic acetylcholine receptor -subunit gene
confers regulation by muscle electrical activity.
Development
115:213-219 .
[Abstract]
-
Chahine KG,
Baracchini E,
Goldman D
(1993)
Coupling muscle
electrical activity to gene expression via a cAMP-dependent second
messenger system.
J Biol Chem
268:2893-2898 .
[Abstract/Free Full Text]
-
Chirgwin JM,
Przybyla AE,
MacDonald RJ,
Rutter WJ
(1979)
Isolation of biologically active ribonucleic acid
from sources enriched in ribonuclease.
Biochemistry
18:5294-5299 .
[Medline]
-
Comb M,
Birnberg NC,
Seasholtz A,
Herbert E,
Goodman HM
(1986)
A cyclic AMP- and phorbol ester-inducible DNA
element.
Nature
323:353-356 .
[Medline]
-
de Wet JR,
Wood KV,
DeLuca M,
Helinski DR,
Subramani S
(1987)
Firefly luciferase gene: structure and expression in
mammalian cells.
Mol Cell Biol
7:725-737 .
[Abstract/Free Full Text]
-
Dignam JD,
Lebovitz RM,
Roeder RG
(1983)
Accurate
transcription initiation by RNA polymerase II in a soluble extract from
isolated mammalian nuclei.
Nucleic Acids Res
11:1475-1489 .
[Abstract/Free Full Text]
-
Eftimie R,
Brenner HR,
Buonanno A
(1991)
Myogenin and MyoD
join a family of skeletal muscle genes regulated by electrical
activity.
Proc Natl Acad Sci USA
88:1349-1353 .
[Abstract/Free Full Text]
-
Evans S,
Goldman D,
Heinemann S,
Patrick J
(1987)
Muscle
acetylcholine receptor biosynthesis.
J Biol Chem
262:4911-4916 .
[Abstract/Free Full Text]
-
Gilmour BP,
Fanger GR,
Newton C,
Evans SM,
Gardner PD
(1991)
Multiple binding sites for myogenic regulatory
factors are required for expression of the actylcholine receptor
g-subunit gene.
J Biol Chem
266:19871-19874 .
[Abstract/Free Full Text]
-
Gilmour BP,
Goldman D,
Chahine KG,
Gardner PD
(1995)
Electrical activity suppresses nicotinic
acetylcholine receptor -subunit promoter activity.
Dev Biol
168:416-428 .
[Web of Science][Medline]
-
Goldman D,
Brenner HR,
Heinemann S
(1988)
Acetylcholine
receptor - , - , - , and -subunit mRNA levels are regulated
by muscle activity.
Neuron
1:329-333 .
[Web of Science][Medline]
-
Gunderson K,
Sanes JR,
Merlie JP
(1993)
Neural regulation of
muscle acetylcholine receptor
 - and -subunit gene promoters in
transgenic mice.
J Cell Biol
123:1535-1544.
[Abstract/Free Full Text]
-
Hall Z,
Sanes JR
(1993)
Synaptic structure and development:
the neuromuscular junction.
Cell
72:99-121 .
-
Hahn C-G,
Covault J
(1990)
Isolation of
transcriptionally active nuclei from striated muscle using percoll
density gradients.
Anal Biochem
190:193-197 .
[Web of Science][Medline]
-
Jaynes JB,
Chamberlain JS,
Buskin JN,
Johnson JE,
Hauschka SD
(1986)
Transcriptional regulation of the muscle creatine
kinase gene and regulated expression in transfected mouse myoblasts.
Mol Cell Biol
6:2855-2864 .
[Abstract/Free Full Text]
-
Kapiloff MS,
Mathis JM,
Nelson CA,
Lin CR,
Rosenfeld MG
(1991)
Calcium/calmodulin-dependent protein kinase
mediates a pathway for transcriptional regulation.
Proc Natl Acad Sci USA
88:3710-3714 .
[Abstract/Free Full Text]
-
Khoury G,
Gruss P
(1983)
Enhancer elements (minireview).
Cell
33:313-314 .
[Web of Science][Medline]
-
Lassar AB,
Davis RL,
Wright WE,
Kadesch T,
Murre C,
Voronova A,
Baltimore D,
Weintraub H
(1991)
Functional activity of
myogenic HLH proteins requires hetero-oligomerization with E12/E47-like
proteins in vivo.
Cell
66:305-315 .
[Web of Science][Medline]
-
Maxam AM,
Gilbert W
(1980)
Sequencing end-labeled DNA with
base-specific chemical cleavages.
Methods Enzymol
65:499-559 .
[Medline]
-
Merlie JP,
Kornhauser JM
(1989)
Neural regulation of gene
expression by an acetylcholine receptor promoter in muscle of
transgenic mice.
Neuron
2:1295-1300 .
[Web of Science][Medline]
-
Neumann JR,
Morency CA,
Russian KO
(1987)
A novel rapid assay
for chloramphenicol acetyltransferase gene expression.
Biotechniques
5:444-447.
[Web of Science]
-
Nordeen SK
(1988)
Luciferase reporter gene vectors for
analysis of promoters and enhancers.
Biotechniques
6:454-457 .
[Web of Science][Medline]
-
Prody CA,
Merlie JP
(1991)
A developmental and
tissue-specific enhancer in the mouse skeletal muscle acetylcholine
receptor -subunit gene regulated by myogenic factors.
J Biol Chem
266:22588-22596 .
[Abstract/Free Full Text]
-
Saccomanno CF,
Bordonaro M,
Chen JS,
Nordstrom JL
(1992)
A
faster ribonuclease protection assay.
Biotechniques
13:846-850 .
[Web of Science][Medline]
-
Simon AM,
Burden SJ
(1993)
An E-box mediates activation and
repression of the acetylcholine receptor -subunit gene during
myogenesis.
Mol Cell Biol
13:5133-5140 .
[Abstract/Free Full Text]
-
Simon AM,
Hoppe P,
Burden SJ
(1992)
Spatial restriction of
AChR gene expression to subsynaptic nuclei.
Development
114:545-553 .
[Abstract]
-
Su C-T,
Huang C-F,
Schmidt J
(1995)
The depolarization
response element in acetylcholine receptor genes is a dual-function
E-box.
FEBS Lett
366:131-136 .
[Web of Science][Medline]
-
Tang J,
Jo SA,
Burden SJ
(1994)
Separate pathways for
synapse-specific and electrical activity-dependent gene expression in
skeletal muscle.
Development
120:1799-1804 .
[Abstract]
-
Wolff JA,
Williams P,
Acsadi G,
Jiao S,
Jani A,
Chong W
(1991)
Conditions affecting direct gene transfer into
rodent muscle in vivo.
Biotechniques
11:474-485 .
[Web of Science][Medline]
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H. Tang, Z. Sun, and D. Goldman
CaM Kinase II-dependent Suppression of Nicotinic Acetylcholine Receptor delta -Subunit Promoter Activity
J. Biol. Chem.,
July 6, 2001;
276(28):
26057 - 26065.
[Abstract]
[Full Text]
[PDF]
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