Nonmyogenic Factors Bind Nicotinic Acetylcholine Receptor Promoter Elements Required for Response to Denervation*

Jean-Louis BessereauDagger , Vincent Laudenbach§, Chantal Le Poupon, and Jean-Pierre Changeux

From the Neurobiologie Moléculaire, UA CNRS D1284, Département des Biotechnologies, Institut Pasteur 25/28 rue du Dr. Roux, 75724 Paris Cedex 15, France

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 alpha -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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (alpha 2beta gamma /epsilon delta ), 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 alpha - and delta -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 alpha - (8, 9), gamma - (10), and delta - (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 alpha -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 alpha -subunit promoter (8). A similar E-box requirement for the denervation response of AChR promoters has been demonstrated for the delta -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 alpha -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 delta -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 alpha -subunit promoter. We have identified two regions in the distal part of the alpha -subunit promoter that are necessary but not sufficient to achieve the transcriptional enhancement of the alpha -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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Plasmid and Virus Construction-- 5' deletions of the chicken AChR alpha -subunit promoter have been performed with KS-842-Delta 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-Delta LA. In this latter case, the entire polymerase chain reaction fragments have been sequenced. KS-842-Delta LA contains the luciferase gene driven by an alpha -subunit promoter fragment extending from nt -842 to nt +20 relative to the transcription start site (19).

Point mutations of the alpha -subunit promoter have been introduced into KS-511-Delta LA by polymerase chain reaction and sequenced. Mutated promoter fragments have been introduced further in Ad-842-Delta 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 alpha 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-alpha 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-USFdelta B expresses a truncated form of USF1 that has been deleted from the first 207 amino acids and is similar to the Delta 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 alpha -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-alpha 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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The -842 to -110 Promoter Fragment of the AChR alpha -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 alpha -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 alpha -subunit promoter fragment together with a plasmid containing the CAT gene under the control of the promoter of the alpha -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 alpha -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 alpha -subunit promoter regions implicated in the denervation response. Luciferase expression plasmids bearing deletions of the AChR alpha -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 alpha -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-alpha 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; square , innervated muscle.

Luciferase expression remained unaffected by 5' deletions of the alpha -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 alpha -subunit promoter in denervated muscle.


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Fig. 2.   Sequence of the chicken AChR alpha -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 alpha -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 alpha -mouse extends from nt -51 to nt -70 in the mouse AChR alpha -subunit promoter (29). Previously characterized elements (19, 20) are boxed.

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 alpha -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 (alpha Sp1) or Sp3 (alpha Sp3) were used for supershift experiments.

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 alpha -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 alpha -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.

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.

Thus, at least three binding activities that are present in muscle as well as nonmuscle cells recognize the AR-160 region of the alpha -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 alpha -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 alpha -subunit (nt -45 to +3) in front of the luciferase gene was fused to four repeats of EU ((EU)4-alpha 45-luc), EB ((EB)4-alpha 45-luc), and USE-MLP ((USE-MLP)4-alpha 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 (); USFdelta B (black-square), which encodes a truncated form of USF1 and acts as a negative trans-dominant (21); and myogenin (). MSV (square ) corresponds to the parent vector. Luciferase activities have been normalized to alpha 45-luc. Results are presented as mean ± S.E. of four independent transfections, each point being determined in triplicate in each experiment.

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 alpha -subunit minimal promoter (19), giving rise to (EU)4- and (EB)4-alpha 45-luc plasmids. (USE-MLP)4-alpha 45-luc contains four repeats of the USF binding site of the adenovirus major late promoter (21). After transient transfection in fibroblasts, (EU)4-alpha 45-luc was about 3-fold more active than alpha 45-luc, while (EB)4-alpha 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-alpha 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)). USFdelta B caused a 1.7- and 2.4-fold decrease of (EU)4- and (USE-MLP)4-alpha 45-luc, respectively, while it had no effect on (EB)4-alpha 45-luc. This suggests that USFs are at least partially responsible for the activation of (EU)4-alpha 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-alpha 45-luc but strongly activated luciferase expression in (EB)4-alpha 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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -subunit promoter denervation response. Mice were infected by bilateral intramuscular injection of the posterior part of the legs with a mixture of v-alpha -luc and v-ASA-CAT recombinant adenoviruses. v-alpha -luc vectors contain the luciferase gene under the control of 842- or 110-bp promoter fragments of the AChR alpha -subunit gene as indicated below the histograms. The mutations that have been introduced in v-alpha 842-luc are described in Fig. 2. v-ASA-CAT, which contains the alpha -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-alpha 842-luc-injected innervated muscles. Indicated in parentheses are the number of individuals that have been injected with each construct. Bars, S.E.; , denervated; square , innervated.

Thus, AR-160 and AR-120 are both required to enhance the transcription of the alpha -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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our results show that different classes of cis-acting elements are involved in innervation-dependent regulation of the AChR alpha -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 alpha -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 alpha -subunit gene transcription in denervated muscle.

Interaction between Myogenic Determination Factors and Sp1-related Transcription Factors-- Deletion analysis of the alpha -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 delta -subunit promoter has recently been shown to participate in the denervation-elicited activation of the delta -subunit expression (14). The organization of the alpha -subunit promoter is slightly different from that of the delta -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 alpha -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 alpha -subunit promoter in denervated muscle. However, in this promoter as well as other genes like cardiac alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -promoter, since EU neither binds nor responds to MDFs. However, we showed that USF also binds the MDF-responsive E-boxes of the chicken alpha -subunit promoter as well as the proximal E-box of the enhancer of the mouse alpha -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 delta -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 alpha -subunit promoter transcription rate, which takes place after denervation. Similar results have been reported in the case of the AChR delta -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This study was supported by the Association Française contre les Myopathies, the Center National de la Recherche Scientifique, the Collège de France, the Institut National de la Santé et de la Recherche Médicale, and the European Community.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Biology, University of Utah, Salt Lake City, UT 84112.

§ A fellow of Assistance Publique-Hôpitaux de Paris.

To whom correspondence should be addressed: Tel.: 33 1 45 68 88 05; Fax: 33 1 45 68 88 36; E-mail: changeux{at}pasteur.fr.

1 The abbreviations used are: AChR, acetylcholine receptor; MDF, myogenic determination factor; nt, nucleotide; bp, base pair; CAT, chloramphenicol acetyltransferase; ASA, alpha -skeletal actin.

2 J.-L. Bessereau, V. Laudenbach, C. Le Poupon, and J.-P. Changeux, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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