Proximal Sequences of the Aldolase A Fast Muscle-specific Promoter Direct Nerve- and Activity-dependent Expression in Transgenic Mice*

Muscle activity is known to modulate the muscle fiber phenotype. Changes in muscle activity (normal or experimentally induced) lead to modifications of the expression status of several muscle-specific genes. However, the transcription regulatory elements involved in the adaptative response are mainly unknown. The aldolase A muscle-specific promoter, pM, is expressed in adult fast twitch muscle with a preferential expression in fast glycolytic-2B fibers. Its activity is induced during postnatal muscle maturation, suggesting a role of nerve and/or muscle activity. Indeed, denervation of gastrocnemius in newborn mice prevented the activation of the promoter in this muscle, despite the nerve-independent formation of 2B fibers. Although the nerve was necessary for pM onset during development, denervating the gastrocnemius in adults had only mild effects on pM activity. By contrast, a transgene including the pM proximal regulatory sequences that are sufficient to reproduce the 2B fiber-specific expression of the endogenous promoter was shown to be highly sensitive to both neonatal and adult denervation. Transgenes containing muscle-specific pM proximal promoter elements were used to delineate the regulatory elements involved in this response to innervation and changes in the contractile activity pattern. Nerve- and activity-dependent elements could be localized in the 130-base pair-long proximal promoter region of the human aldolase A gene.

glycolytic), and type 2X (fast twitch, mixed oxidative-glycolytic). Muscle differentiation first proceeds independently of the nerve, and fiber diversity is thought to arise from distinct myoblast populations (3). However, accumulating evidence shows that later during development, nerve instructions modulate the specialization of muscle fibers. Moreover, adult muscle fibers are plastic entities whose phenotype can change and adapt in response to external influences including relative muscle usage, motor neuron activity, energy source availability, and hormonal status (4). The neuronal influence seems to be transmitted to skeletal muscle fibers mainly via contractile activity, and chronic low frequency electrical stimulation of muscle was shown to cause a transformation of fast fibers into slow fibers (5)(6)(7). These data suggest that nerve and muscle contractile activity play a major role in the final specialization of adult muscle fibers. Adaptative changes to extrinsic factors are accompanied by modifications of the expression patterns of multiple sarcomeric proteins and metabolic enzymes, which are controlled in part at the transcriptional level (8). The precise transduction pathways and regulatory mechanisms coupling external stimuli and muscle phenotypic plasticity are still poorly understood. As a first step to investigate such mechanisms, it is necessary to characterize the regulatory sequences and corresponding transcription factors involved in the response of genes whose transcription is affected by these stimuli.
Aldolase A is a glycolytic enzyme that is highly expressed in skeletal muscle and whose expression was shown to be modulated in response to alterations in contractile activity in the chicken (9), in the rat (10), and in the rabbit (11). Aldolase A expression is achieved through the use of alternative promoters that have distinct tissue and developmental specificities (12)(13)(14)(15)(16)(17). The muscle-specific promoter, pM, is highly activated during postnatal muscle maturation, and in adults, pM-derived transcripts accumulate in fast twitch glycolytic muscles and to a much lower level in slow twitch muscles (14,18,19). We have previously shown that the proximal regulatory sequences (bp Ϫ310 to ϩ45 relative to the transcription start site) of the human aldolase A muscle-specific promoter are sufficient to target the expression of a chloramphenicol acetyltransferase reporter gene (pM310CAT transgene) to fast twitch muscles of transgenic mice, in a pattern mimicking its preferential expression in 2B fibers (20).
In this paper we have investigated the role of innervation and muscle activity on both the murine endogenous muscle pM promoter and the pM310CAT transgene and define the regulatory sequences responsive to these stimuli. To examine the role of the nerve, sections of the sciatic nerve were performed * These studies were supported by the Institut National de la Santé et de la Recherche Médicale and by the Association Française contre les Myopathies. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  either on newborn mice (before the induction of the promoter) or on adult mice (when the promoter is fully active); whereas neonatal denervation drastically affects the endogenous murine pM activity, only a mild effect is observed after adult denervation, suggesting that the nerve is required during a critical period of neonatal development. However, in both cases, the expression of the transgene is highly sensitive to denervation, showing that the proximal sequences of the human pM promoter contain a very efficient nerve-response element (NRE). To further investigate the influence of muscle activity on aldolase A expression, we crossed our transgenic mice with ADR mice that were affected by a recessive myotonia (21). ADR mice constitute a model of muscle hyperactivity (22,23) showing biochemical changes reminiscent of those observed in chronically stimulated muscle (24). Expression of the pM310CAT transgene is severely reduced in ADR both in formerly fast and slow muscles, suggesting that some elements of the pM promoter respond to muscle activity regardless of the fiber type. Studies of transgenes harboring shorter portions of the pM regulatory sequences enabled us to further delineate the sequences responsible for the nerve-response and the contractile activity-dependent expression elements to a small DNA region of 130 bp.

EXPERIMENTAL PROCEDURES
Transgenic and ADR Mice-The transgenic mice pM310CAT, M-tkCAT, and ⌬AT-tkCAT have been described elsewhere (20,25). The mice were propagated in the B6/CBA background, and positive carriers of the transgene were identified by Southern blot analysis as described previously (20). CAT activity was assayed as described previously (20), using various amount of protein extracts and different times of incubation to keep the enzyme activity in a linear range and to ensure detection of activities ranging from 0.1 to 10,000 cpm/g/min of reaction.
The ADR mouse colony originated from the kind gift of a couple of ADR heterozygote mice by Dr. Harald Jockusch. The mutation was originally in the A2G background, but this background was lost as the mice were bred with the aldolase A transgenic mice. Comparison of ADR and wild-type mice was always carried on littermate animals. Detection of the adr allele was performed by polymerase chain reaction on about 500 ng of tail genomic DNA using a specifically designed couple of oligonucleotides (one in the muscle chloride channel CPC1 gene, and the other in the transposon whose insertion has disrupted the gene (21)). The sequences of the primers used were: adr sense, 5Ј-CTGTCCAACCTAAACTCTCAAGC-3Ј; and adr antisense, 5Ј-TCCTAC-CGCATCCTCAGCAA-3Ј. Denaturation was performed at 95°C for 2 min followed by 30 cycles of polymerase chain reaction (1 min at 94°C, 45 s at 59°C, 2 min at 72°C). The reaction was performed using 50 pmol of each primer, 250 M of each dNTPs in Taq polymerase buffer (Eurogentec) supplemented with 1.5 mM MgCl 2 . The specific amplified DNA fragment (591 bp) was then detected by migration of the polymerase chain reaction product on an 1% agarose gel. Heterozygous and homozygous mice were easily distinguished by the evident myotonic phenotype.
Denervation of Mice-Newborn mice were anaesthetized by brief exposure in ice, whereas intraperitoneal injection of avertin was used for older mice. The sciatic (or femoral) nerve was then exposed and surgically cut at two locations, and a section of the nerve was removed to prevent reinnervation during the time of the experiment. In all cases, for surgical and controls procedures, mice were handled in accordance with French and European laws governing animal use for experimental purposes.
Northern Blots-Total RNAs were prepared by the guanidium thiocyanate single-step procedure (26). Northern blot analysis was done as described previously (14). The murine aldolase A pM-derived mRNAs (M-mRNA)-specific probe corresponds to the M-specific exon of the mouse aldolase A gene (18). The myoglobin probe was synthesized by reverse transcription-polymerase chain reaction and corresponds to the third exon of the gene (27). MyHC-2B transcripts were detected using a 76-bp fragment of the 3Ј-untranslated region of the rat gene (28); in this case, hybridization was performed at 55°C (in these conditions, there is no cross-hybridization with other MyHC as shown by the absence of signal in soleus (see Fig. 5) and diaphragm (not shown), which are devoid of 2B fibers). Normalization was performed by hybridization with the ribosomal (18 S) probe R45 (14) or by staining the membrane with methylene blue (29).

Innervation Is Required for pM Activation during
Development-In mouse, pM is activated late during postnatal development (after day 10 after birth) (14), during a period that coincides with the end of polyinnervation and establishment of adult contractile activity (32). We first tested whether innervation is required for this postnatal pM activation. For this purpose, distal hindlimb muscles of newborn mice including gastrocnemius (a fast muscle mainly composed of 2B fibers in the adults) were denervated by section of the sciatic nerve. Six weeks later, we compared the amount of M-mRNAs in the denervated gastrocnemius and in the nonoperated contralateral muscle. As shown in Fig. 1, although M-mRNAs were easily detected in the innervated gastrocnemius, they were only barely detectable in the denervated gastrocnemius. This down-regulation of pM activity could result from muscle atrophy and/or improper fiber specialization caused by denervation. Because M-mRNAs are mostly expressed in 2B fibers (18) and thus coexpressed in adults with transcripts of the MyHC-2B gene, we checked by Northern blots (Fig. 1) and immunohistochemistry (not shown) whether the formation of 2B fibers and concomitant accumulation of MyHC-2B transcripts is altered following denervation. In agreement with previous works on rats (33,34) we found that denervation leads only to a small decrease of the amount of MyHC-2B transcripts. Thus muscle atrophy induced by denervation did not prevent appearance of the 2B fibers in which pM is normally active. Therefore, contrary to postnatal accumulation of MyHC-2B transcripts, which is mostly innervation-independent, the presence of the nerve is required for the strong activation of pM.
The Activation of the pM310CAT Transgene Is Greatly Impaired by Denervation-The regulatory elements of the proximal region of the human and the mouse promoters are highly conserved (17). We have previously shown that the pM310CAT transgene containing 355 bp of the human aldolase A pM promoter (bp Ϫ310 to ϩ45 relative to the transcription start FIG. 1. Innervation is required for the developmental accumulation of aldolase A M-mRNAs but not MyHC-2B mRNAs. Transcripts derived from the mouse endogenous pM promoter or from the MyHC-2B gene were detected by Northern blot in gastrocnemius of mice that have suffered from a section of the sciatic nerve on day 1 postpartum. Both denervated gastrocnemius (lane D) and contralateral nonoperated muscles (lane C) were analyzed 6 weeks after section of the nerve. 10 g of total RNA from pooled muscles of five independent mice have been used. The membrane was stained with methylene blue to control RNA amount and quality and sequentially hybridized with probes for M-mRNAs, MyHC-2B mRNAs, and ribosomal 18 S RNA (R45). site) reproduced the fast muscle specificity of the endogenous gene in all transgenic lines analyzed (20). To see whether this transgene can also reproduce the nerve-dependent activation of the murine pM promoter, the sciatic nerve of newborn transgenic pM310CAT mice (line 98) was sectioned, and CAT reporter activity was measured 6 weeks later in both denervated gastrocnemius and soleus (a slow muscle devoid of 2B fibers) and in control contralateral muscles ( Fig. 2A). Denervation led to an about 100-fold decrease of CAT activity in the denervated gastrocnemius, showing that the pM310CAT transgene activation in this fast muscle requires the presence of the nerve as was observed for the endogenous murine pM promoter. Interestingly, although transgene expression was much less affected by denervation in the soleus (a less than 10-fold decrease is observed), a preferential expression still persisted after denervation in the (fast) gastrocnemius muscle compared with soleus.
Taking advantage of the great sensitivity of the CAT enzymatic assay, we could follow transgene activation in denervated gastrocnemius and control contralateral at various times of development after neonatal denervation (Fig. 2B). Denervation-related down-regulation of transgene expression was already effective at 15 days, suggesting that innervation is required for pM expression even before its full activation. Surprisingly, a first step of this activation (between 15 and 25 days) was observed in denervated gastrocnemius with a fold of increase similar to what is observed in the control innervated muscles. However, denervation prevented further activation of the transgene after day 25, leading to the 100-fold difference between denervated and contralateral muscle at day 40 -50 after birth.
Taken together, these observations show that innervation is crucial for the high level postnatal expression of the aldolase A muscle-specific promoter in hindlimb fast twitch muscles and that the 355 bp of the human pM promoter are sufficient to reproduce this nerve-dependent activation. In addition, it suggests that distinct pathways are involved in the activation of different genes in the same type of myofibers, with some pathways depending on innervation (early pM developmental onset) while others seems to be innervation-independent (activation of the MyHC-2B gene).
Denervation of Adult Transgenic Mice Induces an Early Shut-down of the pM310CAT Transgene Expression-Then we examined whether nerve activity is also required for the maintenance of pM expression in an adult muscle. Hindlimb muscles of adult transgenic mice from two independent pM310CAT lines (lines 20 and 98) were denervated by section of the sciatic nerve. Because the great stability of the CAT protein could mask a rapid effect of denervation, we performed Northern blots experiments to detect CAT mRNAs in denervated and innervated muscles at various times after denervation (Fig. 3).  ). B, at various times in postnatal development, CAT enzymatic activity was measured in denervated gastrocnemius (white symbols) and in the contralateral innervated muscle (black symbols). For a given time, symbols of the same shape correspond to muscles from the same animal (at least three different animals were assayed for a given time). The mean CAT activities in denervated and control muscles were represented by curves. A log scale was used on the y axis.
In control nondenervated muscles, two CAT mRNAs could be observed that correspond to the use of different polyadenylation sites in the SV40 3Ј end of the pM310CAT construct. As illustrated in Fig. 3, no more CAT transcripts could be detected in muscles 3 or 7 days after denervation. The diminution of CAT transcripts was faintly detectable as early as 12 h after the nerve section (not shown) and was already marked 24 h after denervation of the gastrocnemius for one of the two CAT mRNAs, which is probably the less stable one (Fig. 3A). The induction of the myogenin gene, which is strongly induced after muscle denervation (35,36), was used here as a control of effective denervation. The induction of myogenin after denervation could be reproduced with the CAT gene under the control of myogenin regulatory sequences (37), strenghtening the idea that the loss of CAT mRNAs that we observed in our denervation experiments is due to a transcriptional control of pM rather than changes in CAT mRNA stability. Thus denervation of gastrocnemius in adult mice leads to a dramatic and nearly immediate shut-down of the pM310CAT transgene expression.
The activity of the endogenous murine aldolase A muscle promoter was affected by neonatal denervation. Surprisingly, in adult mice, the section of the sciatic nerve had only a minor effect on the amount of M-mRNAs (2-4-fold decrease after 7 days of denervation) as for MyHC-2B mRNAs (Fig. 3B). This could result from a very high stability of M-mRNAs. Alternatively, and perhaps more probably, this could result from the influence of additional regulatory sequences that could maintain the expression of the endogenous pM promoter in absence of innervation and that are not present in the pM310CAT transgene.
Use of the adr Mutation to Test the Role of Muscle Hyperactivity on pM Expression-Electrical stimulation of muscle was shown to lead to a lowered expression of aldolase A in rat and rabbit (10,11). In rabbit, a qualitative modification in the pattern of aldolase A transcripts was also observed (11). Through the use of alternative promoters, aldolase A gene gives rise to multiple transcripts of different sizes (alternative first exons have different lengths) (12,16,19,38). The changes observed by Williams et al. (11), before the identification of the different promoters, probably reflected a decline in M-mRNAs, the remaining transcripts corresponding to longer mRNAs controlled by the other ubiquitous promoter.
Muscle stimulation is rather difficult to perform on mice and usually results in milder changes in these animals than in other species (rats or rabbits). To overcome this difficulty, we have taken advantage of the ADR mutant strain. The ADR mice carry a mutation in the muscle chloride channel ClC1 gene (21), which causes a reduction of the chloride conductance of their muscles. As a consequence, voluntary movements are followed by electrical after-activity leading to a series of "aftercontractions" independent of neural transmission (24). The chronic electrical activity of this myotonia is similar to a chronic stimulation at low frequency (as normally observed in slow twitch fibers). Indeed, the expression pattern of several genes is modified in the ADR mice in a similar fashion to those in chronically stimulated muscles (22)(23)(24). In particular, the expression pattern of MyHC isoforms is changed in ADR mice, with a disappearance of MyHC-2B replaced by the 2X, 2A, and 1 isoforms and a shift to an oxidative phenotype (22,24,39).
By successive breeding, we transferred the pM310CAT transgene from line 20 and line 98 onto the adr background. Then we compared the transgene activity in ADR homozygous mice to their wild-type (or heterozygous) littermates and investigated how the expression pattern of fiber-specific genes is changed by the adr mutation (Fig. 4).
A comparative immunohistochemical analysis of the gastrocnemius of wild-type and ADR/transgenic mice (Fig. 4) illustrated the almost complete disappearance of 2B fibers in ADR gastrocnemius (rare 2B fibers were observed (data not shown), but they represented less than 1% of their normal proportion), which were replaced mostly by 2X fibers. By contrast, the fiber composition of the ADR/transgenic soleus was not significantly modified (Fig. 4). Northern blot analysis of the normally glycolytic muscles (vastus lateralis and gastrocnemius) in ADR/ transgenic mice confirmed the disappearance of MyHC-2B transcripts with a concomitant increase of myoglobin expression (marker of oxidative fibers) (Fig. 5). The endogenous murine pM promoter was only weakly (2-3-fold) affected by the ADR phenotype (Fig. 5). Previous studies of the pattern of expression of the endogenous pM have suggested that it is expressed both in 2B and 2X fibers rather than in 2B only as is the case for the pM310CAT transgene. The observation that pM expression level is only slightly modified in the ADR mice is consistent with this point. In contrast, the proximal regulatory sequences of pM were highly sensitive to ADR-linked changes in muscle activity, because the activity of the pM310CAT transgene is reduced by more than 100-fold in ADR mice (observation made on two independent transgenic lines) (Fig. 6). This

FIG. 3. Denervation in adult mice induced a fast diminution of transgene expression.
Northern blots were used to follow the time course effect of denervation on pM310CAT transgene activity at the mRNA level. For each time following denervation, three adult mice (6 -8 weeks old) from the two independent transgenic lines 20 and 98 were analyzed, and 10 g of total RNAs from denervated (denerv.) or contralateral gastrocnemius (control.) were used. A, 3 days after denervation. Hybridization was performed successively with a CAT probe (giving two bands corresponding probably to alternative polyadenylation sites) and with a myogenin-specific probe to control denervation efficiency. The diminution of the CAT mRNAs was already detectable as early as 24 h after denervation on one of the two CAT transcripts (probably the less stable, marked by an arrowhead). B, 7 days after denervation: the blot was successively hybridized with a CAT probe, a murine aldolase A M-mRNA-specific probe, and a MyHC-2B probe. Normalization of the RNAs was performed by staining the membrane with methylene blue. drastic decrease of transgene activity could parallel the transition of the 2B fibers to 2X fibers. However, interestingly, pM310CAT activity is also strongly diminished in the ADR soleus, a slow twitch muscle that is devoid of 2B fibers and whose fiber composition was found unchanged in our conditions. This suggests that hyperactivity-induced changes are not only related to fiber type transition and that the contractile hyperactivity also acts directly on pM310CAT transcription level.
The Regulatory Sequences Necessary for Contractile Activityinduced Activation of pM Were Delineated to the Ϫ164 to Ϫ35 bp Promoter Fragment-The data presented here showed that the Ϫ310/ϩ45 sequences of pM contained a NRE and an activitydependent response element (ADRE). To further shorten the region containing the NRE and the ADRE (which may be the same sequence), we studied the sensitivity to denervation of transgenic mice harboring smaller fragments of the pM regulatory sequences. Fragments encompassing the Ϫ235/Ϫ35 and Ϫ160/Ϫ35 promoter fragments cloned upstream of the herpesvirus simplex thymidine kinase Ϫ105 promoter and the CAT gene gave the M-tk and ⌬AT-tk transgenes, respectively (depicted in Fig. 7), which display the same pattern of expression than pM310CAT (25). As for the pM310CAT transgene, adult denervation of gastrocnemius caused a rapid and strong decrease of the CAT mRNAs in both M-tk and ⌬AT-tk transgenic lines compared with mRNAs from the contralateral muscle (Fig. 7). These results demonstrate that the NRE is included in the DNA fragment extending from bp Ϫ164 to bp Ϫ35 relative to the pM transcription start site. In addition, the ⌬AT-20 transgene activity was also strongly reduced by at least 100fold in the ADR background compared with wild-type mice (not shown), suggesting that the ADRE is also included in this short DNA fragment.

DISCUSSION
Nerve-and Activity-dependent Specialization of Muscle Fibers-Nerve and muscle activity not only exert a trophic effect on muscle fibers but are also involved in their final specialization by modulating the expression of sarcomeric proteins and metabolic enzymes (1,2). To obtain new insights in this process, we have investigated the effects of modifications in the contractile activity on the fast muscle-specific promoter (pM) of the aldolase A gene. For this purpose, we used transgenic mice harboring a transgene (pM310CAT) that reproduces this preferential expression in 2B fibers (20). 2 The consequences of modifications in the contractile activity resulting either from the absence of the nerve or from a genetically induced hyperexcitability could thus be studied on both the murine endogenous pM promoter and transgene expression.
Although MyHC-2B mRNAs accumulated in gastrocnemius even in the absence of innervation ( Fig. 1 and Refs. 33 and 34), denervation prevented the developmental increase of murine aldolase A M-mRNAs. Thus, postnatal activation of the murine aldolase A muscle-specific promoter in fast muscles requires a nerve-derived signal, whereas MyHC-2B expression is mostly nerve-independent. However, although activation of murine FIG. 4. Fiber type transitions induced by the adr mutation. A comparative immunohistochemical analysis of the fiber types composition was performed on the gastrocnemius (Gastroc.) and soleus from adult wild-type (wt) or ADR mice in the adr/transgenic background. Serial sections from comparable areas allowing visualization of both muscles were stained immunohistochemically with a panel of MyHCspecific monoclonal antibodies that stain fibers expressing respectively MyHC-1 (A and E), MyHC-2A (B and F), and MyHC-2B (D and H), or all fibers except those expressing MyHC-2X (C and G). In the ADR background, the gastrocnemius became almost homogeneously composed of 2X fibers, with some rare 2B fibers.

FIG. 5. Endogenous pM activity is weakly affected by the ADR phenotype.
To monitor the phenotypic changes induced by the adr hyperactivity, a Northern blot analysis was performed, using 10 g of total RNA prepared from vastus lateralis (VL), gastrocnemius (G), or soleus (SOL) from four pooled ADR (adr) or wild-type (wt) mice. The blot was successively hybridized with MyHC-2B, endogenous aldolase A (M), myoglobin, and 18 S specific (R45) probes.

FIG. 6. pM310CAT transgenes activity is altered in ADR mice.
Transgene activity was measured in normally fast twitch (gray) or slow twitch (white) muscles of pM310CAT transgenic mice homozygotes for the adr allele (adr) and compared with activity in wild-type (wt) littermates. Muscles used were the gastrocnemius (G), vastus lateralis (VL), tibialis anterior (TA), and soleus (SOL). The histograms show for two pM310CAT lines, on a logarithmic scale, means and standard deviations of CAT activity (three to four animals for each bar). pM was prevented by denervation of newborn hindlimb muscles, its expression was only moderately affected by denervation of adult muscles. Therefore, innervation seems to be crucial for only a short period of time during the activation of promoter activity. Similar observations had already been reported for the MyHC-2A gene (34).
The proximal regulatory sequences of human pM (transgene pM310CAT) were sufficient to reproduce the nerve-dependent onset of the endogenous gene but do not maintain expression when innervation is suppressed in adult muscle. This suggests that different regulatory regions are involved in the nerve-dependent stimulation during development and in the nerveindependent maintenance of the promoter activity and that the latter sequences are missing in the transgene. In the vicinity of the pM promoter lies a strong ubiquitous enhancer that is associated to the other alternative aldolase A pH promoter. This enhancer was previously shown to spread pM activity in a broader range of fast fibers than what is observed when the transgene includes only the 2B-specific proximal pM regulatory sequences (14,20). This ubiquitous enhancer could be able, as well, to stimulate an already active pM promoter in fast twitch muscles even in the absence of innervation (denervation of adult mice) but would be unable to participate to its developmental onset.
Interestingly, we observed that denervation does not prevent an induction of pM310CAT during development but rather acts by strongly diminishing its activity. Thus, the increased expression of pM that takes place during postnatal muscle maturation is only partially controlled by the nerve. This developmental increase coincides with a peak in thyroid hormone that is known to promote muscle maturation (40) and favor the fast twitch glycolytic phenotype maturation (41). Action of thyroid hormone is nerve-independent (33), and thus activation of pM in fast twitch muscle could result from the combined effects of innervation and thyroid hormone.
Muscle hyperactivity (as observed in ADR mutant mice) has been described to cause a transition toward an oxidative phenotype and a disappearance of the fast 2B fibers (42)(43)(44). Indeed we observed that in ADR/transgenic mice, the gastrocnemius is now composed almost exclusively of MyHC-2X fibers. In contrast to the endogenous pM promoter activity, which is moderately affected by the adr mutation, pM310CAT expression is highly diminished in all ADR muscles that we analyzed, including the slow twitch soleus. This discrepancy could result, here too, from the presence in the vicinity of pM proximal sequences of regulatory elements that are missing in the transgene (and as previously suggested for denervation sensitivity, the ubiquitous H enhancer is a likely candidate). These sequences that were shown to spread pM expression to 2X fibers (20) could thus attenuate the effects observed when only the proximal 2B-specific promoter sequences are present, as in the pM310CAT transgene. However, the observation that the transgene expression is also greatly diminished in the soleus supports a fiber type-independent effect of the contractile hyperactivity.
Taken together, these results highlight the major but complex influence of nerve and muscle contractile activity on the maturation of skeletal muscle. Genes expressed within the same fibers may respond differentially to those extrinsic factors. Our observations support the idea that multiple transcription programs are involved in fiber specificity, each responding probably to different clues that could be extrinsic (nerve and contractile activity) but also intrinsic to the fiber (contribution of myoblasts potentially committed to form a particular type of myofiber). This has been previously suggested by the studies of the expression patterns of muscle sarcomeric genes during regeneration in the presence or the absence of nerve (45) or after muscle stimulation (43,44). Recently, it has been shown that in chicken myotubes, the expression of the myosin heavy chain 2 slow gene depends on both intrinsic (type of myoblasts) and extrinsic (nerve-induced contraction) factors (46). The present study shows that the precise regulation of pM results from the combination of different transcriptional regulatory elements associated with these different programs whose relative contribution may evolve during muscle maturation.
Although pM310CAT transgene could reproduce most aspects of the endogenous pM control (fiber 2B preferential expression and nerve-dependent activation during development), it appeared much more sensitive to modifications in nerve and contractile activity in adults. These discrepancies illustrate the composite nature of the elements participating to the control of a muscle gene, each of them directing a portion of the overall expression pattern of the gene (for a review see Ref. 47). This study in transgenic mice identifies in the proximal regulatory sequences of the pM promoter a regulatory module that was both sufficient to direct 2B fiber-specific expression and to integrate signals originating from the nerve and from the pattern of contractile activity that modulates its level of activity.
Transcriptional Response to Nerve and Contractile Activity-The finding that the pM310CAT transgene expression is regulated by contractile activity is supported by the observation that denervation and hyperactivity in the ADR mice caused a dramatic decrease of its expression. Therefore, in the short Ϫ310/ϩ45 fragment lie a NRE and an ADRE, and our results suggest that both elements are included in the Ϫ164/Ϫ35 region. Although denervation and hyperactivity lead to different alterations in the expression of a battery of muscle proteins, Activity of two transgenes containing only subfragments of the pM regulatory region cloned upstream the thymidine kinase minimum promoter (tk-105) was measured in control contralateral (lanes C) and denervated (lanes D) gastrocnemius 3 days after surgical section of the sciatic nerve on adult transgenic mice. Northern blot was performed with 10 g of total RNA from three different mice for each line and hybridized with a CAT-specific probe. Normalization of the RNAs was obtained by staining the membrane with methylene blue. The absolute activity is different for the pM310CAT, M-tk, and ⌬AT-tk lines, but in each case, no signal was found in denervated muscle. A schematic representation of the transgenes used and of the regulatory motifs found within pM promoter is given. some similar changes were also observed, as increased amounts of myogenin mRNAs (22), and we cannot exclude that their similar effects on pM310CAT transgene transcription results from the triggering of a common transduction pathway; the NRE and the ADRE could therefore correspond to one single DNA element. Among the other genes whose activity is controlled by nerve-derived electrical activity, attention has been mostly focused on those that were induced by denervation, like the acetylcholine receptor subunits genes or the myogenin gene (37, 48 -52). The NRE of the genes encoding acetylcholine receptor subunits has been delineated, and it was found that some E-boxes were necessary for denervation-induced expression of those genes (48,51,52). This importance is supported by the fact that the myogenic basic helix-loop-helix factors that bind E-boxes are induced by denervation (35,36). However, E-boxes are not sufficient to give denervation responsiveness because many genes that are regulated by E-boxes are not controlled by nerve activity (48). The bp Ϫ164 to Ϫ35 pM region contains an E-box that is bound mostly by USF/MLTF but not MyoD proteins in gel mobility shift assays using skeletal muscle nuclear extracts (53). Mutation of this E-box has been shown to reduce pM310CAT activity in transgenic mice but does not alter its fiber-specific expression (25,54); one can imagine that the myogenic basic helix-loop-helix factors that are overexpressed after denervation could compete with USF/ MLTF for their common binding site and cause a reduction of promoter activity. Nevertheless, this region contains other binding sites that have been shown to be important for promoter activity (including a MEF3 motif, overlapping MEF2 and NFI sites, and a M1 sequence bound by nuclear receptors (25,31,54) and that could serve as targets of the nerve-and activitydependent pathways. The demonstration that the small Ϫ164/ Ϫ35 regulatory region is sufficient to confer both fiber specificity and nerve-and contractile activity-dependent expression in skeletal muscles should facilitate the identification of the key elements involved in nerve-and activity-dependent regulation of muscle-specific genes.