From the Neurobiologie Moléculaire, UA CNRS D1284,
Département des Biotechnologies, Institut Pasteur 25/28 rue du
Dr. Roux, 75724 Paris Cedex 15, France
Nicotinic acetylcholine receptors (AChRs) belong
to a class of muscle proteins whose expression is regulated by muscle
electrical activity. In innervated muscle fiber, AChR genes are
transcriptionally repressed outside of the synapse, while after
denervation they become reexpressed throughout the fiber. The myogenic
determination factors (MDFs) of the MyoD family have been shown to play
a central role in this innervation-dependent regulation. In
the chicken AChR
-subunit gene promoter, two E-boxes that bind MDFs
are necessary to achieve the enhancement of transcription following
muscle denervation. However, the deletion of promoter sequences located
upstream to these E-boxes greatly impairs the response to denervation
(Bessereau, J. L., Stratford- Perricaudet, L. D.,
Piette, J., Le Poupon, C. and Changeux, J. P. (1994)
Proc. Natl. Acad. Sci. U. S. A. 91, 1304-1308). Here we
identified two additional cis-regulatory elements of the
-subunit gene promoter that cooperate with the E-boxes in the
denervation response. One region binds the Sp1 and Sp3 zinc finger
transcription factors. The second region binds at least three distinct
factors, among which we identified an upstream stimulatory factor, a
b-ZIP-HLH transcription factor. We propose that among MDF-responsive
muscle promoters, a specific combination between myogenic and
nonmyogenic factors specify innervation-dependent versus innervation-independent promoters.
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INTRODUCTION |
In skeletal muscle, action potential frequency affects the
properties of the extrasynaptic membrane and controls the repertoire of
contractile proteins expressed in the fiber. This provides a relatively
simple system to analyze the cascade of events that couple regulation
of gene expression to electrical activity in excitable cells. One of
the best characterized examples of electrical activity-dependent regulation in muscle is the expression
of the nicotinic acetylcholine receptor
(AChR).1 AChR is a
heteropentameric transmembrane protein made up of four different types
of subunits (
2
/
), which are encoded by
distinct genes (reviewed in Refs. 1 and 2). In innervated muscle, AChRs
are almost exclusively detected at the postsynaptic membrane of the
neuromuscular junction, which represents less than 0.1% of the total
surface of the muscle fiber in mammals. Silencing electrical activity
either by surgical denervation or by chemical blockade of
neurotransmission causes a dramatic increase of AChR expression in the
extrasynaptic regions of the muscle fibers. Electrical stimulation of
denervated muscle represses the de novo expression of
extrasynaptic AChRs, demonstrating that electrical activity is the
first signal responsible for the down-regulation of AChR expression
outside of the synapse (reviewed in Refs. 1 and 3).
Muscle activity controls AChR gene expression primarily at the
transcriptional level. The use of transgenic mice demonstrated that
promoter fragments of the AChR
- and
-subunit genes confer denervation-responsive expression to a reporter gene (4-7). Either by
manipulating the spontaneous electrical activity of primary cultures of
muscle cells or by using in vitro stimulation techniques, promoter fragments of the
- (8, 9),
- (10), and
- (11, 12)
subunit genes have been shown to contain electrical activity-responsive sequences. By using adenovirus-mediated gene transfer of point-mutated fragments of the
-subunit promoter in vitro and in
vivo, we have shown that the E-box DNA motifs (CANNTG) that bind
the myogenic differentiation factors (MDFs) of the MyoD family are
necessary for activity-dependent regulation of the
-subunit promoter (8). A similar E-box requirement for the
denervation response of AChR promoters has been demonstrated for the
-subunit promoter either in transgenic mice (13) or by using naked
DNA injection (14).
MDFs (that include MyoD, myogenin, Myf-5, and MRF4) are basic
helix-loop-helix transcription factors that are critical for muscle-specific expression of several genes including AChR
subunit-encoding genes (reviewed in Ref. 1). Interestingly, MyoD and
myogenin expression has been shown to be regulated by electrical
activity. Denervation of adult muscle strongly enhances MyoD and
myogenin mRNA levels, while exogenous stimulation of denervated
muscle reverses this increase (15-17). Results obtained in primary
cultures of muscle cells also suggest that electrical activity
stimulates a serine/threonine kinase that, in turn, down-regulates
myogenin transactivation potency by phosphorylation of the protein
(18). Together with the identification of E-boxes as target elements of
the activity-dependent regulation of AChR gene
transcription, these results support the hypothesis that electrical
activity modulates MDF transactivation potency at transcriptional and
post-transcriptional levels and in turn controls AChR gene
transcription.
The question remains, however, why only a subset of the muscle genes
that contain MDF-responsive E-boxes are regulated by electrical
activity. We have shown previously that the EB and EP E-boxes that are located in the proximal part of the
chicken AChR
-subunit promoter (see Fig. 2) are necessary but not
sufficient for activity-dependent transcription (8).
Similar results have been recently reported with the proximal E-box of
the rat
-subunit promoter (14). This suggests that, in addition to
MDFs, other trans-acting factors that bind AChR promoters
are involved in electrical activity-dependent transcription
of AChR genes. In order to identify these factors, we have mapped the
cis-regulatory sequences that cooperate with E-boxes in the
-subunit promoter. We have identified two regions in the distal part
of the
-subunit promoter that are necessary but not sufficient to
achieve the transcriptional enhancement of the
-subunit promoter
after denervation. One region binds Sp1-related transcription factors.
The other one is recognized by several nuclear proteins including
"upstream stimulatory factor" (USF) transcription factors.
Functional implications of these results are discussed.
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MATERIALS AND METHODS |
Plasmid and Virus Construction--
5' deletions of the chicken
AChR
-subunit promoter have been performed with KS-842-
LA as
parent vector either by using available restriction sites in the
promoter or by subcloning polymerase chain reaction fragments between
the SpeI and XbaI unique sites of KS-842-
LA.
In this latter case, the entire polymerase chain reaction fragments
have been sequenced. KS-842-
LA contains the luciferase gene driven
by an
-subunit promoter fragment extending from nt
842 to nt +20
relative to the transcription start site (19).
Point mutations of the
-subunit promoter have been introduced into
KS-511-
LA by polymerase chain reaction and sequenced. Mutated
promoter fragments have been introduced further in Ad-842-
LA (8).
Recombinant viruses were constructed and propagated as described (8).
Four copies of double-stranded oligonucleotide EB (see Fig.
2), EU (GGAGCCATCTGAGCGG), and USE-MLP
(TAGGCCACGTGACCGGGT) have been subcloned in the BglII
site of
45-luc (referred to as construct I in Ref. 19 and contain a
minimal promoter fragment extending from nt
45 to nt +3 relative to
the transcription start site) to give rise to (EU)4-,
(EB)4-, and (USE-MLP)4-
45-luc, respectively.
Expression vectors have been constructed by using pEMSV-myogenin as
parent vector (20). The coding region of human USF1 (kind gift from
Michel Raymondjean (21)) was polymerase chain reaction amplified and
subcloned into the EcoRI site of pEMSV-myogenin giving rise
to MSV-USF. MSV-USF
B expresses a truncated form of USF1 that has
been deleted from the first 207 amino acids and is similar to the
bTDU1 construct that was described by Lefrancois-Martinez et
al. (21).
Transfection Experiments--
1 µg of expression vector and 1 µg of reporter construct were mixed with 5 µl of
LipofectAMINE® (Life Technologies, Inc.) in
Opti-MEM® medium (Life Technologies) for 45 min at room
temperature. Cells were rinsed and incubated with the DNA-LipofectAMINE
solution for 5 h at 37 °C. Transfection medium was then
replaced by 4% fetal calf serum-enriched medium. Cells were harvested
48 h after the transfection and processed as described in Ref.
19.
Naked DNA Injections--
Tibialis anterior muscles of
6-8-week-old mice were injected with 50 µl of a 10 µM
cardiotoxin solution (Latoxan, France) in phosphate-buffered saline.
Five days later, each regenerating muscle was injected with 40 µg of
plasmid DNA of the
-promoter construct to be tested together with 40 µg of the KS-ASA-CAT plasmid. DNA were diluted in phosphate-buffered
saline. After 2 weeks, mice were unilaterally denervated. 48 h
after denervation, animals were sacrificed, and muscles were processed
as described (8).
Adenovirus Infections--
Infections were performed as
described in Ref. 8 with minor modifications. The posterior part of
each mouse leg was injected by a mixture containing 2 × 109 plaque-forming units of v-
luc and 1.5 × 109 plaque-forming units of v-ASA-CAT. Before injection,
the virus-containing solution was dialyzed for 3 h against storage
buffer without glycerol (135 mM NaCl, 1 mM
MgCl2, 10 mM Tris·Cl, pH 7.4).
Mobility Shift and Supershift Assays--
Gel shift experiments
were performed as described (19). The antibodies that were used for
supershift experiments were all from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA) and were tested in Western blot experiments (data not
shown).
UV Cross-linking--
The binding of nuclear proteins to the
probe and the electrophoresis of the resulting complexes were performed
as in a classical gel shift experiment. After electrophoresis, the gel
was UV-irradiated at 320 nm (128 watts/cm2 for 15 min) and
autoradiographed. Gel slices containing the retarded complexes were
subsequently excised. DNA-protein complex was eluted overnight from the
gel in 50 mM Tris·Cl, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 5 mM dithiothreitol, 0.1% SDS.
After trichloroacetic acid precipitation, complexes were migrated in a
denaturating polyacrylamide-SDS gel. The gel was dried and
autoradiographed.
Size Fractionation of DNA-binding Proteins--
150 µg of C2
myotube nuclear extracts were denatured and electrophoresed on 8%
SDS-PAGE. Gel slices were cut, and proteins were eluted in the same
buffer as described above. Proteins were precipitated by the addition
of 3 volumes of cold acetone and kept at
20 °C for 4 h. After
centrifugation, the pellet was resuspended in buffer A (10 mM Hepes, pH 8.0, 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 17.5% glycerol (v/v))
containing 6 M guanidinium isothiocyanate. Samples were dialyzed at 4 °C against buffer A without guanidinium and tested for
DNA binding activity in gel shift experiments.
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RESULTS |
The
842 to
110 Promoter Fragment of the AChR
-Subunit
Promoter Contains Two Regions That Activate Transcription in Denervated
Muscle--
By using recombinant adenoviruses, we reported in a
previous work that regulatory elements that cooperate with the proximal E-boxes of the AChR
-subunit promoter for the denervation response are located in a region ranging from nucleotides
842 to
110 relative to the transcription start site (8). Systematic analysis of a
750-bp promoter region by using adenoviruses would have required several rounds of recombinant virus construction. To save time, we
based our initial strategy on the injection of naked DNA into regenerating muscle.
The expression of exogenous gene can be achieved in a small percentage
of myofibers after injection of plasmid DNA into adult muscle (22, 23).
By using this technique, however, we were not able to reliably quantify
the transcriptional enhancement of an AChR gene promoter after
denervation (data not shown). Davis et al. (24) have
reported that the efficiency of this method is strongly increased when
injected muscles are in a process of regeneration. Therefore, adult
mouse muscles were first injected with cardiotoxin to elicit
degeneration. Five days later, regenerating muscles were injected with
a plasmid containing the luciferase gene driven by an
-subunit
promoter fragment together with a plasmid containing the CAT gene under
the control of the promoter of the
-skeletal actin gene. Since this
promoter has been previously shown to be unaffected by short-term
denervation (8), CAT activity could be used to normalize luciferase
levels (for details, see Ref. 8 and "Materials and Methods").
Mice were unilaterally denervated after 2 weeks. When the 842-bp
fragment of the
-subunit promoter was used to drive luciferase
expression, luciferase activity was increased 5-fold in denervated
versus innervated muscles (Fig. 1).

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Fig. 1.
Identification of -subunit promoter
regions implicated in the denervation response. Luciferase
expression plasmids bearing deletions of the AChR -subunit promoter
(numbers below histogram bars indicate 5'-boundaries
relative to the transcription start site) were injected into
regenerating tibialis anterior muscles of adult mice. For
normalization, luciferase constructs were coinjected with ASA-CAT,
which contains the -skeletal actin promoter in front of the CAT
gene. 2 weeks after DNA injection, mice were unilaterally denervated.
Muscles were harvested 48 h after denervation. Luciferase
activities were normalized to CAT activities. Results are presented as
the mean expression relative to the activity of KS- 842-luc-injected
innervated muscles. Numbers in brackets indicate
the number of individuals that have been injected with each construct.
Bars, S.E; , denervated muscle; , innervated
muscle.
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Luciferase expression remained unaffected by 5' deletions of the
-subunit promoter up to nt
166 relative to the start site (Fig.
1). Removing the
166 to
150 region caused a 2-fold decrease of
luciferase activity in denervated muscle. The deletion of the promoter
from nt
122 to
110 further reduced the expression of the luciferase
both in innervated and denervated muscle. These results show that two
activating regions (called AR-160 and AR-120) (Fig.
2) are located upstream to the proximal
EB and EP E-boxes and contribute to the
transcriptional enhancement of the AChR
-subunit promoter in
denervated muscle.

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Fig. 2.
Sequence of the chicken AChR -subunit
promoter. The sequence is numbered relative
to the transcription start site that had been initially determined by
Klarsfeld et al. (47). Regions AR-160 and AR-120, whose
deletion decreases the -subunit promoter transcription in denervated
muscle, are underlined. Oligonucleotides that have been used
in gel shift experiments are indicated above the promoter
sequence. Dashes correspond to unchanged positions compared
with wild-type sequence. Each underlined A indicates a
position that has been bromodeoxyuridine-substituted in the
complementary strand for UV cross-linking experiments. Oligonucleotide
-mouse extends from nt 51 to nt 70 in the mouse AChR -subunit
promoter (29). Previously characterized elements (19, 20) are
boxed.
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Sp1-related Proteins Bind the AR-120 Region--
The binding of
transcription factors to AR-120 was checked in gel mobility shift
assays. The incubation of muscle cell nuclear extracts with a probe
extending from nt
122 to nt
105 in the AChR
-subunit promoter
(WT-120, Fig. 2) generated two retarded bands (I and II) that were
competed by an excess of unlabeled probe (Fig.
3). A similar pattern was obtained with
fibroblast extracts (data not shown). AR-120 contains a GC-box that
resembles the canonical binding site of the zinc finger Sp1
transcription factor. Mutation of this GC-box (mutS, Fig. 2)
prevented the formation of complexes I and II. To test whether AR-120
binding activities have the same sequence recognition specificity as
Sp1, we used an oligonucleotide bearing an Sp1 binding site from SV40.
This oligonucleotide indeed competed the formation of complex I and, to
a lesser extent, complex II (Fig. 3, lane 3).

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Fig. 3.
Binding of Sp1-related proteins to
AR-120. 0.1 pmol of oligonucleotide WT-120 (see Fig. 2) was used
as a probe in gel retardation experiments with nuclear extracts of C2
myotubes. The unlabeled competitors (Comp) described in Fig.
2 were added in a 50-fold molar excess. 2.5 µg of polyclonal
antipeptide antibodies against Sp1 ( Sp1) or Sp3
( Sp3) were used for supershift experiments.
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In order to check whether Sp1 is involved in the formation of complex I
and/or II, we performed supershift experiments. Incubation of muscle
cell extracts with an anti-Sp1 antibody decreased the intensity of band
I and generated a supershifted complex (Fig. 3, lane 5).
However, even by increasing the amount of anti-Sp1 antibody, the effect
remained partial with respect to band I, while the intensity of band II
was unchanged. Proteins have been cloned that share homologies with Sp1
and bind similar DNA motifs (25, 26). Among the different Sp1-related
proteins, we tested whether Sp3 could contribute to AR-120 binding
activities, since Sp3 is ubiquitously expressed and generates gel shift
patterns that resemble those obtained with WT-120 (see for example
Refs. 27 and 28). The addition of an anti-Sp3 antibody to the muscle cell extract caused the decrease of band I intensity and the
disappearance of band II. Consistent with these results, Sp3 has been
previously shown by Western blot to exist as at least three
polypeptides of 97, 60, and 58 kDa and to form protein-DNA complexes of
different mobilities in gel shift assays (27, 28). The anti-Sp3
antibody that we used similarly detected three polypeptides of the same size in our nuclear extracts (data not shown). The anti-Sp3 antibody most completely disrupts the binding of Sp3 to the probe, since the
intensity of the supershifted complex is very faint compared with the
signal obtained in the absence of antibody. Nuclear extract incubation
with both anti-Sp1 and anti-Sp3 antibodies caused the disappearance of
band I and band II (Fig. 3, lane 7) suggesting that no other
protein than Sp1 and Sp3 binds AR-120.
Nuclear Extracts from Muscle and Nonmuscle Cells Contain Several
AR-160 Binding Activities--
To identify AR-160 binding activities,
we performed gel shift analysis using myotube nuclear extracts and a
probe encompassing the nt
169 to
145 region of the
-subunit
promoter (WT-160, Fig. 2). Three major complexes could be detected that
were specifically competed by an excess of unlabeled probe (Fig.
4, lanes 1 and 2).
This pattern remained identical when the probe was incubated with
nuclear extracts of fibroblasts (lanes 3 and 4).
No additional complex was detected with a probe extending to nt
135
(data not shown). To characterize further the interactions of the
different binding activities with the AR-160 region, we introduced
mutations at different positions of the WT-160 oligonucleotide (Fig. 2
and data not shown). A DNA fragment containing mutation U was no longer able to compete for the formation of any of the two upper complexes (Fig. 4, lane 4), while mutation T specifically prevented
the competition of the third complex (lane 5).
Interestingly, the enhancer of the mouse AChR
-subunit promoter
contains a region that is highly homologous to the right part of AR-160
(Ref. 29; see Fig. 2). An oligonucleotide containing the mouse sequence efficiently competes the formation of complexes I and II (lane 6).

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Fig. 4.
Gel retardation experiments with region
AR-160. Oligonucleotide WT-160 (see Fig. 2) was used as a probe
and incubated with nuclear extracts of C2 myotubes (lanes 1,
2, and 5-7) or 3T6 fibroblasts (lanes
3 and 4). The unlabeled competitors (Comp)
described in Fig. 2 were added in a 50-fold molar excess.
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We then started to characterize the proteins that bind AR-160.
DNA-protein cross-linking experiments have been performed with a
thymidine to bromodeoxyuridine-substituted probe (Fig. 2,
underlined positions). Bromodeoxyuridine did not change the
pattern of the retarded complexes but increased the affinity of the
protein(s) that participates in the formation of complex III (data not
shown). Cross-linking of AR-160 binding proteins to the radioactively labeled DNA was achieved by UV irradiation. Gel pieces corresponding to
the different retarded bands were cut, and DNA-protein complexes were
recovered by elution and loaded on an SDS-PAGE gel. Complex III
migrated with an apparent mobility of 77 ± 2 kDa (Fig.
5A). The oligonucleotide
contributes for 14 kDa to the complex mass. A slight band corresponding
to complex I was detected at a position corresponding to a molecular
mass of 120 kDa. In some experiments, two fainter bands were detected
at lower positions. We have not been able to obtain a signal with
complex II by following the same strategy. In a second set of
experiments, nuclear extracts were initially size-fractionated on
SDS-PAGE. The gel was cut into pieces corresponding to different
molecular weight ranges. After elution from the gel, proteins were
renatured and tested for binding in a gel shift assay. The activity
corresponding to complex I could be recovered from the fraction that
contained proteins ranging from 95 to 70 kDa (Fig. 5B). The
lower size limit has been further restricted to 83 kDa using the same
experiments (data not shown). Together with the cross-linking results,
it suggests that a single protein of approximately 95 kDa is involved in the formation of complex I and that the faster migrating complexes detected in cross-linking experiments most probably correspond to
degradation products. The activity corresponding to complex II was
recovered in the fraction ranging from 48 to 33 kDa. The binding
activity participating in complex III formation could not be renatured
from denatured nuclear extracts (not shown).

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Fig. 5.
Characterization of AR-160 binding factors.
A, cross-linking experiments. A thymidine to
bromodeoxyuridine-substituted WT-160 oligonucleotide
(underlined positions in Fig. 2) was incubated with nuclear
extracts of C2 myotubes and electrophoresed in the same conditions as
classical gel shift experiments. After UV irradiation, proteins were
eluted from gel pieces corresponding to complex I (lane 1)
and III (lane 2) and loaded on an 8% polyacrylamide-SDS
gel. The gel autoradiogram is presented. Molecular masses (kDa) are
indicated on the left. B, size fractionation.
Nuclear extract of C2 myotubes was denatured and run on an 8%
polyacrylamide-SDS gel. The gel was cut into slices corresponding to
molecular masses whose boundaries are indicated at the top.
Proteins were eluted, renatured according to the procedure described
under "Material and Methods," and used in gel shift experiments
with WT-160 as a probe. Native extract has been used in lane
1. Positions of the different complexes are indicated on the
left. NS corresponds to a nonspecific band that
is occasionally observed with WT-160.
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Thus, at least three binding activities that are present in muscle as
well as nonmuscle cells recognize the AR-160 region of the
-subunit
promoter but contain distinct proteins according to biochemical
criteria.
AR-160 Contains an E-box, Which Is a Target of USF and Not of
MDFs--
The 5'-CATCTG-3' sequence that is contained in AR-160 fits
the CANNTG minimal consensus motif of an E-box (Fig. 2). To check whether this E-box (that we called EU for E-upstream) is
the target of some of the previously characterized binding activities,
we used the proximal EB box of the
-subunit promoter as
a competitor of the AR-160 labeled probe in a gel shift assay.
EB efficiently competed the formation of complex II, while
a mutated form of EB (20) that no longer contained the
CANNTG motif was ineffective (Fig.
6A, lanes 3 and
4). Similar results have been obtained with the
EP box (not shown). Thus, complex II contains proteins that are present in muscle as well as nonmuscle cells and have an E-box binding specificity. Moreover, we knew from the experiments presented above that these proteins have molecular masses between 48 and 33 kDa.
According to these criteria, the USF was a plausible candidate for
participating in complex II. USF is a member of the
basic-helix-loop-helix leucine zipper family of transcription factors
that contain both helix-loop-helix and leucine zipper structural
domains (30). The usf1 gene encodes a single factor of 43 kDa, while a second gene, usf2, generates by mRNA
alternative splicing two distinct proteins, USF2a and USF2b, of 44 and
38 kDa, respectively (31-34). These factors bind DNA as USF homo- or
heterodimers but do not associate with MDFs.

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Fig. 6.
Interaction of USF with EU and
EB E-boxes. A, gel retardation experiments.
Oligonucleotide WT-160 or EB (described in the legend to
Fig. 2) was used as a probe as indicated at the bottom in
gel shift experiments using nuclear extracts of C2 myotubes.
Competitors and antibodies were used as in Fig. 3. Antibodies were
against USF1 and E2A proteins as indicated. The arrowheads
indicate the complexes that are not in common between WT-160 and
EB. B, transfection experiments. The minimal
promoter of the -subunit (nt 45 to +3) in front of the luciferase
gene was fused to four repeats of EU
((EU)4- 45-luc), EB
((EB)4- 45-luc), and USE-MLP ((USE-MLP)4- 45-luc),
which is the USF binding site of the adenoviral major late promoter
(see "Materials and Methods" for oligonucleotide sequences). These
constructs were transfected into 3T6 fibroblasts together with
expression vectors for USF1 ( ); USF B ( ), which encodes a
truncated form of USF1 and acts as a negative trans-dominant
(21); and myogenin ( ). MSV ( ) corresponds to the parent vector. Luciferase activities have been
normalized to 45-luc. Results are presented as mean ± S.E. of
four independent transfections, each point being determined in
triplicate in each experiment.
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The binding of USF to WT-160 was tested in supershift experiments.
Incubation of muscle cell nuclear extracts with an antibody raised
against USF1 caused a supershift of complex II (Fig. 6A, lanes 5 and 6). This antibody interferes with the
binding of USF to DNA, since the supershifted band was much less
intense than band II. In contrast, an antibody that recognizes the
basic helix-loop-helix E2A proteins, the major partners of MDFs in
muscle cells, did not affect the pattern of the retarded bands. Similar
experiments were performed with EB as a probe. Several
complexes were detected with muscle cell nuclear extracts. One of them
has the same mobility as complex II. The slower migrating complexes
were not detected with fibroblast nuclear extracts (data not shown).
The anti-USF antibody supershifted complex II, while the anti-E2A
protein antibody supershifted the upper complexes (Fig. 6A,
lanes 12-14). These complexes were also shifted by
antibodies raised against myogenin or MyoD (not shown). Therefore, the
EB box is bound by USF and MDFs while EU is
recognized by USF only.
In order to evaluate putative functional differences between
EU and EB, four copies of each site were placed
in front of an AChR
-subunit minimal promoter (19), giving rise to
(EU)4- and (EB)4-
45-luc plasmids.
(USE-MLP)4-
45-luc contains four repeats of the USF binding site of
the adenovirus major late promoter (21). After transient transfection
in fibroblasts, (EU)4-
45-luc was about 3-fold more
active than
45-luc, while (EB)4-
45-luc activity was
not significantly different from that of the parent vector (Fig.
6B). Cotransfection of a USF1 expression vector did not
significantly stimulate luciferase expression from any of the plasmids,
suggesting that endogenous USF1 expression was saturating in the cell
line we used. To assess the contribution of USFs to the transcriptional
enhancement of (EU)4- and (USE-MLP)4-
45-luc, we used a
truncated form of USF1 that dimerizes with USF proteins but is no
longer able to bind DNA and acts as a negative
trans-dominant (as previously characterized by
Lefrancois-Martinez et al. (21)). USF
B caused a 1.7- and
2.4-fold decrease of (EU)4- and (USE-MLP)4-
45-luc, respectively, while it had no effect on (EB)4-
45-luc.
This suggests that USFs are at least partially responsible for the
activation of (EU)4-
45-luc although factor I remains
able to bind the EU oligonucleotide with low affinity in
gel shift assays (data not shown). We also tested the responsiveness of
the different E-boxes to MDFs. Cotransfection of a myogenin expression
plasmid had no effect on (EU)4- and (USE-MLP)4-
45-luc
but strongly activated luciferase expression in
(EB)4-
45-luc transfected cells.
Hence, we conclude that EU does not belong to the same
class of E-boxes as do the more proximal EB and
EP sites, since it only binds and responds to USF and not
to MDFs.
The Denervation-dependent Activation of the AChR
-Subunit Promoter Requires Both AR-160 and AR-120 cis-Acting
Elements in the Adenoviral Context--
To determine the relative
contribution of AR-160 and AR-120 to the
innervation-dependent regulation of the
-subunit gene transcription, we decided to return to an assay based on the in vivo utilization of recombinant adenoviruses, since such vectors can be used with high efficiency in nonregenerating muscle of adult
animals, in contrast to the DNA injection technique.
As previously reported (8), luciferase expression is strongly enhanced
in denervated muscle when the luciferase gene is driven by the 850-bp
-subunit promoter fragment, while there is no significant increase
when the shorter 110-bp fragment is used (Fig.
7). Point mutations of AR-160 and AR-120
have been introduced in the 850-bp fragment of the
-subunit promoter
and challenged for denervation responsiveness. Mutating AR-160 to mutU, which prevents the formation of complex I and II in
EMSAs, or introducing mutT, which disrupts complex III,
reduced luciferase expression in denervated muscle by about 2-fold but
did not affect expression in innervated muscle. The introduction of
mutation S in the AR-120 region caused a 3-fold decrease of the
denervation response. v-850-mutUS and v-850-mutTS, which are doubly
mutated in AR-160 and AR-120, failed to respond to denervation although the mutated promoters kept intact the proximal EB and
EP boxes (Fig. 7).

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Fig. 7.
Point mutations of AR-160 and AR-120 abolish
-subunit promoter denervation response. Mice were infected by
bilateral intramuscular injection of the posterior part of the legs
with a mixture of v- -luc and v-ASA-CAT recombinant adenoviruses.
v- -luc vectors contain the luciferase gene under the control of 842- or 110-bp promoter fragments of the AChR -subunit gene as indicated
below the histograms. The mutations that have
been introduced in v- 842-luc are described in Fig. 2. v-ASA-CAT,
which contains the -skeletal actin promoter in front of the CAT
gene, has been used for normalization. 8 days after injection, mice
were unilaterally denervated. Muscles were harvested 48 h after
denervation. Luciferase activities were normalized to CAT activities.
Results are presented as the mean expression relative to the activity
of v- 842-luc-injected innervated muscles. Indicated in
parentheses are the number of individuals that have been
injected with each construct. Bars, S.E.; , denervated;
, innervated.
|
|
Thus, AR-160 and AR-120 are both required to enhance the transcription
of the
-subunit promoter in denervated muscle. These cis-acting elements are, however, not sufficient, since the
mutation of the proximal E-boxes similarly abrogated denervation
responsiveness (8).
 |
DISCUSSION |
Our results show that different classes of cis-acting
elements are involved in innervation-dependent regulation
of the AChR
-subunit gene. In addition to the previously
characterized EB and EP boxes that are the
target of MDFs, we have characterized two new cis-acting
elements of the chicken AChR
-subunit promoter that bind proteins
that are not muscle cell-specific: Sp1, Sp3, USF and at least two other
not yet identified factors. Both E-box and non E-box elements are
necessary albeit not sufficient to fully enhance AChR
-subunit gene
transcription in denervated muscle.
Interaction between Myogenic Determination Factors and Sp1-related
Transcription Factors--
Deletion analysis of the
-subunit
promoter led to the identification of a new binding site for
Sp1-proteins. Point mutation of this site alters the denervation
response in the context of adenoviral vectors. Interestingly, an
Sp1-like sequence of the rat AChR
-subunit promoter has recently
been shown to participate in the denervation-elicited activation of the
-subunit expression (14). The organization of the
-subunit
promoter is slightly different from that of the
-subunit promoter
since it contains a second Sp1 binding site in its very proximal part,
downstream to the MDF binding sites (35, 19). The two proximal and
distal Sp1 sites are, however, not redundant since an
-subunit
promoter fragment that retains intact the 5' Sp1 site is expressed in
denervated muscle at lower level after deletion or disruption of the 3'
Sp1 binding site. This could reflect that MDFs requires at least two Sp1 sites to activate the
-subunit promoter in denervated muscle. However, in this promoter as well as other genes like cardiac
-actin
(36) and troponin I (37), only one site is sufficient for MDFs to
activate transcription at high level in transfected muscle cells. In
the AChR
-subunit promoter, the two Sp1 sites could have distinct
functions. It is tempting to speculate that the proximal site that is
located close to the initiation site helps mostly in recruiting
components of the basal machinery (38) while the distal one cooperates
with MDFs by different molecular mechanisms.
Interestingly, both sites bind a set of proteins with antagonistic
properties. The proximal site overlaps a GC-stretch that recognizes the
so-called GBF factor (19). When GBF is bound on its site, it
subsequently prevents in vitro the binding of Sp1. In
vivo, GBF is expressed in muscle as well as in non muscle cells
and represses the residual transcription activity of the
-subunit
promoter observed in non-muscle cells. The distal site that we
characterized in this study binds two related proteins: Sp1, the first
protein of the family to be cloned, and Sp3 that has been cloned by
recognition screening (25) and by low-stringency screening using the
Sp1 zinc finger encoding region as a probe (26). Sp3 contains modular
activation and repression domains (39, 40) and is able to repress
Sp1-mediated transcriptional activation (41, 27). Therefore, Sp3 is
considered as an inhibitory member of the Sp1 transcription factor
family, even though recent evidences suggest that Sp3 can also act as a
weak activator depending on the promoter and cellular context (40).
Depending on the ratio between Sp1 and Sp3, an Sp1 binding site can
therefore act as a positive as well as a negative cis-acting
regulatory element. Preliminary results suggest that the Sp1/Sp3 ratio
increases during muscle differentiation, in parallel with the
activation of the AChR
-subunit gene expression (data not shown). It
will be of particular interest to analyze in detail the expression of
these factors in vivo during development and after
denervation.
Contribution of USF to the Expression of AChR Genes--
AR-160 is
the second cis-regulatory element that we have shown to
participate in the denervation-elicited enhancement of the
-subunit
promoter activity. It binds at least three different factors which
exhibit distinct DNA-binding properties. Mutating the left part of
AR-160 specifically disrupts the binding of the faster migrating
complex in gel shift assays while mutation of the right part, namely
mutU, prevents the formation of the two upper complexes I
and II. Since the EB oligonucleotide only competes for the
formation of complex II, the factors involved in complexes I and II
have distinct but most probably overlapping DNA-binding sites. To
evaluate the respective requirement of these different factors for
denervation response, we have introduced mutU and mutT in the 850-bp promoter fragment of the
-subunit
promoter. Both mutations decrease the transcription rate of the
promoter in denervated muscle to similar extents. This suggests that
either complex I or II and complex III both participate in the
denervation response. However, the binding sites of these different
factors are close or even overlapping, and a mutation that disrupts a binding site most probably modifies the flanking regions of the neighboring binding site. Since it is well known that there is not
always a direct correlation between in vitro binding
experiments and in vivo transactivation by a specific factor
(see for example Ref. 42), the possibility cannot be ruled out that
only one of the factors that bind AR-160 is critically involved in the denervation response. In keeping with this possibility, we have shown
that the enhancer fragment of the mouse AChR
-subunit promoter contains a motif that shares striking homology with AR-160 (10/12 identical bases). This region only binds complexes I and II, suggesting that these factors could be privileged actors of the denervation response.
We have identified the proteins that generate complex II as the USF
transcription factors. In AR-160, USF binds an E-box, namely
EU, that differs from the previously characterized
EB and EP E-boxes of the chicken
-promoter,
since EU neither binds nor responds to MDFs. However, we
showed that USF also binds the MDF-responsive E-boxes of the chicken
-subunit promoter as well as the proximal E-box of the enhancer of
the mouse
-subunit promoter (this
work).2 According to the
pattern of migration of previously published gel shift assays (43), it
is also probable that USF binds the proximal E-box of the mouse
-subunit promoter (USF most probably corresponds to the binding
activity referred to as "3" in the work of Simon and Burden (43)).
What is the significance of USF binding to various AChR subunit
promoters? By transfection experiments in fibroblasts, we have observed
that the multimerization of EU in front of a minimal
promoter only activates slightly the transcription rate from this
promoter, while in the same conditions EB has no effect.
Either some other factor(s) interfere with the binding of USF on these
sites or, more probably, USF is unable to activate transcription
through these E-boxes in vivo. A similar discrepancy between
the binding of USF to promoter elements and the lack of transactivation
of the promoter by USF has been reported with other genes including
L-type pyruvate kinase (44) and immunoglobulin heavy chain (45). In the
IgH enhancer, USF has even been demonstrated to play the role of a
repressor by preventing the binding of the basic-helix-loop-helix
leucine zipper TFE3 factor, which is the relevant activator of the IgH
promoter (45). In keeping with these data, USF factors could similarly
interfere with the binding of MDFs on AChR subunit promoters and
contribute to the down-regulation of AChR expression in innervated
muscle when MDF levels decrease. The same kind of antagonistic
interactions could occur at the level of AR-160 between USF and the
factor I that we are now actively trying to characterize.
Role of MDFs in the Denervation Response--
The
cis-acting elements that bind ubiquitous factors, as well as
the previously characterized MDF binding sites EB and
EP, are all necessary albeit not sufficient to fully
achieve the enhancement of the
-subunit promoter transcription rate,
which takes place after denervation. Similar results have been reported
in the case of the AChR
-subunit promoter, where an Sp1-like site
and an SV40 core element are involved in the denervation response (14) in addition to the proximal E-box (13). The question remains whether
all of the transcription factors that bind these elements are
equivalent mediators of the innervation-dependent
regulation of AChR gene expression or whether some of them are limiting
components of this regulation.
Interestingly, the
-subunit promoter has been studied in various
systems and, under certain experimental conditions, the promoter region
upstream of the
110 position seems dispensable for the denervation
response. In the DNA injection experiments performed in this study, the
110-bp promoter fragment was still responsive to denervation although
less active than a longer fragment. However, we used
cardiotoxin-treated regenerating muscles that were not fully
differentiated at the time of denervation. The high levels of
expression of myogenic factors which are observed in regenerating
muscles could possibly render the interaction of MDFs with other
factors less critical for the activation of the AChR
-promoter.
Moreover, expressing nuclei most probably contained a very high
number of plasmid copies (23), which are known to perturb some
transcriptional regulations. Therefore, this result must be interpreted
cautiously. The
-subunit promoter has also been analyzed in
transgenic mice (5, 46). Shortening the promoter fragment from 850 to
110 bp causes a drastic drop in the overall activity of the promoter
and a 5-20-fold reduction of the denervation response, although a bp
111 promoter fragment remains denervation-responsive in the extensor
digitorum longus and tibialis anterior muscles (46). In transgenic
mice, a large number of transgene copies are inserted at the same
locus in the chromatin (more than 100 copies in the case of the mice
described by Merlie and Kornhauser (4)). Since E-boxes can act
synergistically, positive interactions between E-boxes located in
different transgene copies could substitute for the interactions
between myogenic and nonmyogenic factor binding elements that are
required for full denervation response in the context of adenovirus
vectors and possibly in the endogenous promoter. Finally, although the adenovirus vector used in this study has been designed to be as neutral
as possible (8), it is not possible to definitely rule out the
possibility that in a particular context some adenoviral sequence
interacts with the promoter under study. The variation in the results
depending on the experimental strategy shows the importance of using
different methodologies to analyze a promoter, especially in
vivo. However, all these data consistently support the hypothesis
that the proximal MDF responsive E-boxes are central in the regulation
of the AChR
-subunit promoter by electrical activity and that they
interact with the regulatory elements that we have characterized in
this study.
Promoter context plays a critical role in the ability of E-boxes to
mediate MDF transactivation. For example, a defined E-box motif is
unable to transduce MyoD transactivation in the cardiac
-actin
promoter, while the same sequence will be extremely potent in the
muscle creatine kinase promoter (42). In AChR promoters, positive and
negative interactions have been demonstrated (i) between E-box and
non-E-box cis-acting elements and (ii) between distinct
trans-acting factors for binding to the same site. We propose that these layers of interactions help specify
innervation-dependent versus innervation-independent
promoters in muscle. Modulations of MDF transactivation potency that
are caused by muscle innervation will be amplified by a specific
network of interactions at the level of AChR gene promoters, while in
innervation-independent genes these variations will be neutralized by
the contribution of other transcription factors. Such networks help us
to understand how a limited number of factors can achieve the
independent tuning of several genes in response to subtle changes of
the physiological status of the cell.
We thank Alban de Kerchove and Elisabeth
Brown for critical reading of the manuscript. Nathalie Duclert and
Laurent Schaeffer are acknowledged for sharing reagents and technical
expertise.