(Received for publication, January 21, 1997, and in revised form, February 25, 1997)
From the Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908
cetylcholine eceptor
(AChR)-nducing ctivity (ARIA) is believed to
be the trophic factor utilized by motoneurons to stimulate AChR
synthesis in the subsynaptic area. Among the four AChR subunit genes,
the subunit gene is strictly expressed in nuclei localized to the
synaptic region of the muscle. To understand mechanisms of the
regulation of synapse-specific transcription, we studied the promoter
activity of the 5
-flanking region of the AChR
subunit gene in
response to ARIA. Transgenes containing the wild type or mutant
5
-flanking regions upstream of a luciferase gene were transfected in
C2C12 muscle cells. The promoter activity of these transgenes was
determined by assaying activity of expressed luciferase. Analyzing a
combination of 5
deletion and site-directed mutants, we identified a
10-nucleotide element (position
55/
46), which was crucial for
ARIA-induced expression from the
subunit promoter. This element was
named ARE for RIA-esponsive
lement. Mutation of ARE greatly diminished ARIA-induced
transgene expression and deletion of ARE abolished completely the ARIA
response. Electrophoretic mobility shift analyses revealed a DNA
binding activity in muscle nuclear extract that interacted with ARE.
Such interaction was enhanced by ARIA stimulation of muscle cells and
appeared to be dependent on nuclear protein phosphorylation.
The development and maintenance of a functional neuromuscular junction require that expression of all the molecular components be temporally and spatially regulated at the nerve-muscle contact. For example, the acetylcholine receptor (AChR)1 is highly enriched at the crests of the postjunctional folds at a concentration 3 orders of magnitude higher than in the extrasynaptic membrane (1, 2). There are at least three mechanisms that contribute to control of AChR subunit gene expression. Myogenic factors, including MyoD, myogenin, myf5, and MRF4, appear to play an important role in the regulation of AChR gene expression during the myogenic differentiation program by binding directly to the E-box element located in sequences upstream of the transcription initiation site of AChR subunit genes (3-6). This regulation is critical for muscle-specific expression of AChR subunit genes. Second, electrical activity of muscle fibers, resulting from motor nerve firing, appears to down-regulate transcription of AChR subunit genes in extrasynaptic nuclei (7, 8). The molecular mechanism underlying transcriptional regulation by electrical activity is unclear. It has been suggested that the reduced rates of transcription may result from suppression of the transcriptional activation mediated by members of the MyoD family (9). Third, elevated transcription in those nuclei localized to the synaptic region of the muscle contributes to the maintenance of a high level of AChR mRNAs at the neuromuscular junction (9, 10). Such synapse-specific transcription is believed to be mediated by ARIA, the trophic factor utilized by motoneurons to stimulate AChR synthesis (11-13). ARIA binds to and activates its receptor, one or two members of the ErbB family of protein tyrosine kinases (14-17). We (17) and Tansey et al. (18) recently found that ARIA-induced AChR gene expression requires activation of the MAP kinase signal pathway.
To characterize further the molecular mechanisms of synapse-specific
transcription, we have analyzed the promoter of the AChR subunit.
The mRNA coding for the
subunit is restricted to junctional
areas from the outset of its expression (19). The synaptic localization
of
mRNAs is not affected by denervation, which produces a
reappearance of other AChR subunit mRNAs in extrajunctional areas
(19, 20). Therefore a study of the
subunit promoter activity in
response to ARIA would provide a better understanding how expression of
subunit is regulated during development. In addition, the
subunit promoter is an excellent model for the study of
synapse-specific transcription. In this report, we provide evidence
that a 10-nucleotide element in the sequence upstream of the
transcription initiation site, termed ARE, is required for ARIA-induced
subunit expression in muscle cells. We also demonstrate that ARE
binds specifically to a nuclear protein(s) in a manner dependent on
ARIA stimulation of muscle cells and phosphorylation of nuclear
proteins.
The recombinant
ARIA (rHRG177-244), a peptide of HRG
1
residues 177-244) was generously provided by Dr. Mark Sliwkowski (21).
Calf intestinal phosphatase was purchased from Boehringer Mannheim. All
other chemicals, including okadaic acid, were purchased from Sigma.
Deletion mutants of the subunit
promoter were generated by polymerase chain reaction (PCR) using the
p
3500-nlacZ plasmid as a template. The upstream primers with a
HindIII site were 5
-GGG AGA AGC TTC TGA ATC TCA CTC TCA GC
(Primer 1) for
416-Luc, 5
-GGG AGA AGC TTC TCT CCT GAG ATG ACA GG for
170-Luc, 5
-GGG AGA AGC TTG GGG CAG CTG CCT CCC CC for
78-Luc, and
5
-GGG AGA AGC TTG GCA GAG GAT for
45-Luc. The downstream primer
(Primer 2) was 5
-GGG AGA AGC TTG AGG GAA CAG G with a
HindIII site. Point and internal deletion mutants of the
subunit promoter were generated by the method of PCR overlap extension
(22). The p
3500-nlacZ plasmid was used as a template in the first
PCR reaction using Vent DNA polymerase (New England Biolabs). The sense
oligonucleotides of the internal overlapping primers (with mutated
bases underlined) were 5
-GGG ACA GGG GCA GCT G
(position
89/
68) for the ets mutant, 5
-ATG GGG CAG CTG CCT
CA CCC CCA CAG CAG GGG C (position
79/
43) for the
AP-2 mutant, 5
-CTG CCT CCC CCA CCC CAG CGG CAG AGG ATT AGG TGA (position
70/
28) for the ARE mutant,
5
-CTG CCT CCC CCA CCC GGC AGA GGA TTA GGT GA (position
70/
28) for
the ARE deletion mutant, and 5
-GGT GAC AGT CCC CAA ACC TAG CCT AAC ACC CTC CTC CCC TTC ACA (position
33/+18) for the
N-box mutant. The two overlapping DNA fragments from the first PCR
reaction were used as templates in the second PCR reaction with Primers
1 and 2. After digestion with HindIII, the PCR products were
subcloned in pGL2-Basic (Promega) upstream of the luciferase gene. The
authenticity and orientation of the synthetic promoter fragments were
verified by DNA sequencing.
C2C12 cells were cultured as described
previously (17). C2C12 myoblasts at approximately 50% confluence were
co-transfected with an subunit promoter-luciferase transgene (20 µg of DNA) and a control plasmid (1 µg of DNA) (pCMV
, which
encodes
-galactosidase), by the standard calcium phosphate technique
(23). Luciferase assay was performed using a kit from Promega following
the manufacturer's instruction.
-Galactosidase activity was
determined as described previously (17). Luciferase activity of
transgenes was normalized to
-galactosidase activity to correct for
variations in transfection efficiency.
C2C12 cells were
washed in phosphate-buffered saline and homogenized in a buffer
containing 10 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.2 mM
phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol. Cell nuclei were pelleted by centrifugation at 4,000 rpm for 15 min at
4 °C. The pellets were extracted in DNA binding buffer containing
12% glycerol, 12 mM HEPES-NaOH, pH 7.9, 4 mM
Tris/HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet P-40, 0.2 mM
phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, 1 µg/ml
leupeptin, and 2 µg/ml aprotinin. Nuclear extract protein (10 µg)
was incubated with 2 µg of nonspecific competitor poly(dI·dC), 4.5 µg of bovine serum albumin, and 1 ng of 32P-labeled
double-stranded oligonucleotide probe (~10,000 cpm) in a final volume
of 15 µl of DNA binding buffer. After incubation at 30 °C for 15 min, the reaction was stopped by the addition of 5 µl of loading
buffer and run onto a 4% acrylamide gel (24). The radiolabeled probes
were 5-CCC CAC AGC AGG GG for ARE or 5
-CTA GCC CGG AAC TAA for N-box
(25). In some experiments, unlabeled double-stranded oligonucleotides
were used as a competitor, including the ARE or N-box wild type, ARE
mutant (5
-CCA AGC AGC GTT GG), or SRE wild type (5
-CCC CAT ATT AGG
GG).
To determine the minimum length of the 5 regulatory
region required for promoter activity in response to ARIA, we generated transgenes containing a series of deletion mutants in the 5
-flanking region of the
subunit gene. Transgenes were transfected in C2C12 muscle cells, and promoter activity was characterized by luciferase assay in ARIA-stimulated C2C12 myotubes. Expression of the
416-Luc transgene, which contains 416 nucleotides upstream from the
transcription initiation site, was increased by ARIA stimulation in a
concentration-dependent manner (Fig. 1). The
maximal response (around 2-fold) was achieved with 1 nM
ARIA, which agrees well the our previous observation using
p
3500nlacZ in which expression of
-galactosidase is driven by the
3.5-kilobase 5
-flanking sequence of the
subunit gene (17). These
results suggest that the promoter element(s) required for the ARIA
response is contained within 416 nucleotides upstream of the
transcription initiation site. Therefore, the
416-Luc transgene was
considered as equivalent to wild type for the purposes of this study.
The ARIA response was specific to the
subunit promoter, since the
promoter of rat skeletal muscle voltage-sensitive sodium channel
subtype 2 (skm-2) did not respond to ARIA stimulation (Fig.
1C). Promoter activities of the transgene deletion
constructs
170-Luc,
78-Luc, and
45-Luc in myotubes in response to
1 nM ARIA are shown in Fig. 1C. Seventy-eight
nucleotides upstream from the transcription initiation site of the
subunit gene was sufficient to confer an ARIA response. This is in
agreement with previous observations that 150 and 83 nucleotides
upstream of the transcription initiation site of the
subunit gene
are sufficient to respond to ARIA (15) and to confer preferential
synaptic expression (25), respectively. The transgene containing 45 nucleotides, however, failed to respond to ARIA, even at concentrations
up to 10-fold higher than required for a maximal response with the wild
type transgene (Fig. 1D). The difference in the promoter activities between
78-Luc and
45-Luc indicated that the element required for ARIA response could be localized in the 34 nucleotides between
78 and
45.
Identification of a 10-Nucleotide Element (ARE) Required for ARIA-induced
We previously demonstrated
that ARIA-stimulated AChR gene expression requires MAP kinase
activation (17). Major transcription factors mediating MAP kinase
action in mammalian cells include ternary complex factor proteins (for
the ets element) (26) and SRF (for the SRE element) (27). Anticipating
that the ARE-responsive element might be localized between 78 and
45, we identified and selectively mutated a number of sites in this
region, concentrating on elements that could potentially bind
transcription factors regulated by the MAP kinase pathway. Among the
sites mutated, a ten-nucleotide element (CCA CAG CAG G, position
55/
46) was included. This element was chosen because it resembles
SRE, having two cytidines on the 5
end and two guanosines on the 3
end (27). However, it differs from SRE in that the intervening 6 nucleotides have two cytidines and one guanosine instead of purely
adenosines or thymidines as in the consensus SRE. Replacement of
adenosines or thymidines with cytidines or guanosines in the internal 6 nucleotides dramatically decreases or diminishes SRE binding activity
(28). In addition, the putative ets site (CAG GAT), which we identified at
83/
78, the putative AP-2 element (position
64/
54), and the
N-box element (
11/
6), which was recently found to be required for
synapse-specific expression (28), were also mutated. All mutants were
made in the
416-Luc construct. While ets and AP-2 mutants diminished
basal expression, they had no effect on ARIA-stimulated expression. By
contrast, mutation of the 10-nucleotide SRE-like element attenuated
greatly or abolished the
promoter activity in response to ARIA
(Fig. 2). Myotubes possessing this mutant transgene
failed to respond to concentrations of ARIA sufficient to activate the
wild type transgene (Fig. 3A). However, an
increase in mutant transgene expression was observed at 10 nM ARIA. These data strongly suggested that this
10-nucleotide element may be required for the ARIA-induced
subunit
gene expression. We named this element ARE for
RIA-esponsive lement. To confirm
that ARE is required for the ARIA response, we tested the promoter activity of an ARE deletion mutant (ARE.
). As shown in Fig.
3B, the ARE.
mutant promoter failed to confer ARIA
response even at 10 nM of ARIA. While this work was in
progress, Duclert et al. (25) reported that the N-box is
crucial for synaptic expression of AChR
subunit gene. Using an
in vivo expression assay in intact muscles, the authors
observed that 5
deletion mutants (
83 and
75) of
subunit
promoter provided approximately the same level of expression, whereas
63 and
52 5
deletion mutants gave a decreased expression and the
36 nucleotide construct did not express. This observation prompted
that authors to suggest "the presence of an activating element" in
this region. In addition, site-directed mutation in this region
diminished synaptic expression of the
subunit gene (25). We believe
that ARE may be the activating element in this region crucial for
synaptic expression.
Interaction between ARE and a Nuclear Protein Was Enhanced by ARIA Stimulation of Muscle Cells and Dependent on Phosphorylation of Nuclear Protein
Electrophoretic mobility shift analyses were performed to
determine if ARE was able to interact with nuclear proteins in
ARIA-stimulated myotubes. Using [-32P]ATP-labeled
double-stranded oligonucleotides containing the ARE element as a probe,
we detected a prominent complex on the autoradiogram of a 4%
acrylamide gel (Fig. 4A). The interaction between the ARE probe and nuclear binding protein was specific. First,
formation of the complex was competitively inhibited by a 10- or
100-fold excess of unlabeled double-stranded wild type ARE
oligonucleotides. In contrast, ARE mutant oligonucleotides required a
much higher concentration to disrupt the complex formation. Second,
100-fold excess of unlabeled double-stranded oligonucleotides containing a typical SRE element had little, if any, inhibitory effect
on the ARE nuclear protein binding complex (Fig. 4A),
suggesting that ARE interacted with a protein different from those that
interact with SRE. Interestingly, formation of the specific complex was enhanced by ARIA stimulation of C2C12 myotubes. Treatment of myotubes with ARIA increased the binding activity by at least 2-fold (Fig. 4B). These results suggested that ARE interacted with a
nuclear protein(s).
Knowing that activation of MAP kinase is required for ARIA-induced AChR gene expression (17, 18), we attempted to determine whether the ARE nuclear binding activity was regulated by MAP kinase activity. Myotubes were treated with PD98059, an inhibitor of MAP kinase kinase, which is able to disrupt MAP kinase activation and ARIA-induced AChR gene expression in C2C12 cells (17). The ARE binding activity was decreased in PD98059-treated myotube nuclear extract (Fig. 4B). Incubation of myotube nuclear extract with calf intestinal phosphatase to dephosphorylate nuclear proteins attenuated the ARE binding activity in a concentration-dependent manner (Fig. 4C). There appeared to be a high level of endogenous phosphatase activity in myotube nuclear extract. Preincubation of myotube nuclear extract at 30 °C for 15 min alone, without the addition of exogenous phosphatase, was able to decrease ARE binding activity (Fig. 4C). Moreover, the preincubation-induced decrease in ARE nuclear binding activity was blocked by 2 nM okadaic acid, a specific inhibitor of serine/threonine phosphatases (Fig. 4C). Thus the ARE binding activity may derive from a phosphoprotein that has an enhanced DNA binding activity in the phosphorylated state. The phosphatase activity in C2C12 myotubes may be due to protein phosphatase 2A, a phosphatase which is specifically inhibited by okadaic acid at a nanomolar concentration (29).
N-box Is Not Required for ARIA-stimulatedRecently, a cis-element of 6 nucleotides (11/
6) in the
subunit promoter was identified and
implicated in compartmentalized expression of AChR genes at the
neuromuscular junction (25). We found that although mutating the N-box
dramatically decreased basal expression of the transgene, the N-box
mutant promoter was still able to respond to ARIA stimulation (Fig.
2C). Using the N-box probe, we detected a binding activity
in the C2C12 myotube nuclear extract (Fig.
5A), confirming the previous observation (25). This binding activity was specific, since it was inhibited by
excess unlabeled double-stranded N-box oligonucleotides (Fig. 5A). Stimulation of C2C12 cells with ARIA, or with ARIA in
the presence of PD98059, did not appear to affect the N-box binding activity (Fig. 5B). These results suggest that N-box may not
be crucial for ARIA-induced
subunit gene expression in C2C12
cells.
In summary, we have identified ARE, a 10-nucleotide element (CCA CAG
CAG G, position 55/
46) in the
subunit promoter, which was
required for ARIA-induced expression of
subunit gene in C2C12
muscle cells. ARE was able to interact with a nuclear protein(s). Furthermore, interaction between ARE and the nuclear protein was enhanced by ARIA stimulation of muscle cells and seemed to depend on
phosphorylation of nuclear proteins. Experiments to elucidate the
identity of the ARE binding protein are currently in progress.
We thank Dr. Wen C. Xiong for advice; Drs.
David Brautigan, Doug Bayliss, and Sheridan Swope for critical
comments; and Sandra Won, Michael Tanowitz, and Dr. Yi Sun for
discussion. Our sincere appreciation goes to Dr. Gerry Chu and Dr.
Joshua Sanes for p3500-nlacZ DNA; Dr. Evelyn Ralston for C2C12
cells; Dr. Alan Saltiel for PD98059; Dr. Mark Sliwkowski for
recombinant ARIA; and Roland Kallen for the skm-2 transgene.