Use of DNA injection for identification of slow nerve-dependent regions of the MLC2s gene

Valerie A. Lupa-Kimball and Karyn A. Esser

School of Kinesiology, University of Illinois, Chicago, Illinois 60608

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

It has been well established that expression of slow contractile protein genes in skeletal muscle is regulated, in part, by activity from slow motoneurons. However, very little is understood about the mechanism by which neural activity regulates transcription of slow isoform genes. The purpose of this investigation was first to more fully define the in vivo DNA injection technique for use in both fast-twitch and slow-twitch muscles and second to use the injection technique for the identification of slow nerve-dependent regions of the myosin light chain 2 slow (MLC2s) gene. Initial experiments determined that the same amount of plasmid DNA was taken up by both the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles and that injection of from 0.5 to 10 µg DNA/muscle is ideal for analysis of promoter activity during regeneration. This technique was subsequently used to identify that the region from -800 to +12 base pairs of MLC2s gene directed ~100 times higher activity in the innervated soleus than in innervated EDL, denervated soleus, or denervated EDL muscles. Placing the introns upstream of either the MLC2s or SV40 promoter increased expression 5- and 2.7-fold, respectively, in innervated soleus but not in innervated EDL, denervated soleus, or denervated EDL muscles. These results demonstrate that 1) in vivo DNA injection is a sensitive assay for promoter analysis in both fast-twitch and slow-twitch skeletal muscles and 2) both 5' flanking and intronic regions of the MLC2s gene can independently and synergistically direct slow nerve-dependent transcription in vivo.

in vivo deoxyribonucleic acid injection; muscle regeneration; contractile protein genes

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE ACQUISITION AND maintenance of an adult skeletal muscle phenotype is regulated, in part, by the presence and activity of specific motoneurons (11, 22, 25). The presence of a slow-type nerve or chronic low-frequency electrical activity has been shown to be an important factor regulating slow-twitch skeletal muscle phenotype (13, 22). Conversely, if a slow-type nerve is removed or electrical activity is blocked, there is a concomitant transition from a slow-twitch to a fast-twitch muscle phenotype (5). These transitions in phenotype have been determined at the mRNA level, which suggests that alterations in slow neural activity transcriptionally regulate slow contractile protein genes (13, 25).

There are numerous studies characterizing the transcriptional regulation of the contractile protein isoform genes. Most of this work has focused on identifying cis-acting elements and transcription factors involved in the induction of the contractile protein genes during muscle differentiation (19). In addition, cis elements associated with muscle type specificity have been isolated in numerous contractile protein genes including the myosin light chain 1/3, troponin I, and troponin C gene families (1, 21, 23). The most common elements regulating qualitative and quantitative skeletal muscle expression include E boxes, MCAT, myocyte enhancer factor 2 (MEF2), CACC, and CArG boxes. What is still unclear is how these elements are regulated in response to specific neural cues.

Understanding how specific neural signals transcriptionally regulate muscle genes in the adult animal has been a difficult challenge because it requires the use of an in vivo model system for promoter analysis. The most-established system is the transgenic mouse in which a specific reporter construct is stably incorporated into the genome of all cells. This approach has been used to determine regions of the slow myosin heavy chain and creatine kinase genes that are responsive to chronic overload in the rat (27, 31). However, there are some difficulties with the use of transgenic animals, which include potential regulatory effects resulting from random insertion in the genome and the potential for aberrant regulation in adult tissues due to altered expression during embryonic and/or fetal development. In addition, for promoter analysis studies transgenic animals can be very time consuming and costly.

An alternative approach is the relatively new technique of in vivo plasmid DNA injection into skeletal muscle (4, 6, 16, 29). This technique has been used to identify the cis-acting element of the alpha -skeletal actin gene that is associated with transcriptional changes in response to stretch overload (4). Although the use of in vivo injection is promising, there are still technical aspects that need to be defined for its use in determining fiber type-specific and nerve-dependent regulatory elements. Therefore, the purpose of this study was twofold: first, to further optimize and define in vivo DNA injection for use in promoter/enhancer analysis with both fast-twitch and slow-twitch skeletal muscles and, second, to use the injection system to identify regions of the myosin light chain 2 slow (MLC2s) gene that are necessary for both fiber type-specific and nerve-dependent transcription in vivo.

Skeletal muscle regeneration was combined with in vivo injection of luciferase reporter gene constructs for this study. The primary rationale for using muscle regeneration is that the changes in slow nerve-dependent gene expression are large and rapid (8, 30), making analysis of promoter and enhancer elements more feasible in vivo. Additional strengths of the regeneration model include the facts that 1) the cell biology and physiology of regeneration are well defined, so that the timing of events such as reestablishment of functional innervation is known, and 2) efficiency is much higher for transfection into a regenerating muscle than into normal adult skeletal muscle (6, 7, 29). The MLC2s gene was chosen because of the specificity of its expression during muscle regeneration. The MLC2s gene is unique among most fast and slow contractile protein genes in that it is not expressed during myotube formation and is only induced in the presence of the slow nerve (8). The absence of expression of MLC2s during myotube formation and strong nerve-dependent induction make it an ideal candidate for the identification of nerve-dependent cis element(s) in vivo.

The results of this study indicate that in vivo plasmid DNA injection is a sensitive tool for the relatively rapid analysis of physiologically regulated gene regions. Analysis of luciferase constructs in vivo defined a much lower range for injection (from 0.5-10 µg DNA/muscle) than in previously published reports (4, 6, 7, 29). This is important for minimizing potential promoter competition when coinjecting reporter constructs to normalize for efficiency. It was also determined that the average amount of plasmid DNA taken up by the regenerating muscle cells was similar for both the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles, which allows for comparison between different muscles. Finally, by use of this injection system it was determined that the MLC2s gene contains two slow-twitch-muscle-specific and nerve-dependent regions within the promoter and introns 1 and 2. These regions required the presence of the slow nerve for expression, and when placed together they acted synergistically to direct high-level expression.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Generation of MLC2s plasmids. The 14.9-kilobase (kb) MLC2s genomic clone was generously provided by Dr. S. Henderson as DNA isolated from bacteriophage vector Charon 4 (10). Portions of the 14.9-kb clone were initially subcloned into plasmid vectors pGEM3z, pGEM7z, or pSP72 for subsequent construction of reporter genes. Standard procedures were followed for all subcloning steps (24).

To assess slow-twitch-muscle-specific promoter and enhancer activity of the MLC2s gene, four plasmids containing 5' flanking and/or intronic DNA ligated upstream of a luciferase expression vector (pGL3basic, pGL3promoter; Promega) were generated (Fig. 1). The first plasmid to be tested, plasmid 3800MLC2s, contains the Kpn I-to-Kpn I fragment of the MLC2s gene. This fragment includes 2.08 kb of 5' flank, exons 1 and 2, intron 1, and 83% of intron 2 [798 of 961 base pairs (bp)]. Plasmid 800MLC2s was constructed by subcloning the BamH I-to-EcoR I fragment of MLC2s (-800 to +12) from pSP72 into the Bgl II/Hind III site of the pGL3basic vector in the correct 5'-3' orientation. Plasmid IVS1,2pGL3p contains introns 1 and 2 [isolated from polymerase chain reactions (PCR)] inserted into the upstream Sac I site in the pGL3promoter vector. To test for interactions between MLC2s 5' flanking and intronic DNA, introns 1 and 2 were subcloned into the Sma I/Sac I sites upstream of the 800-bp 5' flanking MLC2s DNA, to yield the plasmid 800IVS1,2MLC2s. The plasmid pbeta gal-control was coinjected as a control for injection efficiency (Clontech). All plasmid DNA was isolated using Plasmid Maxiprep columns (Qiagen).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   A: restriction map of the rat myosin light chain 2 slow (MLC2s) gene from positions -3000 to +2700. B: individual MLC2s/luciferase gene constructs injected into rat soleus and extensor digitorum longus (EDL) muscles. IVS1, intron 1; IVS2, intron 2.

Isolation of introns 1 and 2 using PCR. PCR was utilized to isolate introns 1 and 2 of the MLC2s gene. Primers were designed using published sequence data from intron-exon boundaries of the MLC2s gene (10). The primers for intron 1 were 5'-GTGAGTGGTCAGTGGACCCT-3' and 5'-AACCGCTGGATCAGGACCTC-3' and the primers for intron 2 were 5'-AGGAGGTGAGTGTGACAGGG-3' and 5'-TAGGGAGGGTCACTGCTCAG-3' (Keystone Laboratories). Standard conditions (12) were followed for amplifying intron 1: 30 cycles with denaturation at 92°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 60 s. The conditions for intron 2 were 30 cycles with denaturation at 94°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 120 s. Amplification resulted in a 761-bp product for intron 1 and a 938-bp product for intron 2, and they were cloned using the pGEM-T vector system. Introns 1 and 2 were sequenced using Sequenase Quick-Denature plasmid sequencing kit (United States Biochemical). Sequence analyses of MLC2s promoter and intronic regions were performed using the Geneworks program (Intelligenetics).1

In vivo DNA injections. The DNA injection protocol used in this study was modified from previously published reports (6, 7, 29). To prepare the plasmid DNA for injection, 5 µg/muscle of experimental (luciferase) plasmid and 5 µg/muscle of internal control plasmid (beta gal-control) were mixed, phenol-chloroform extracted once, and ethanol precipitated overnight at -20°C. Just before injections, the combined plasmid DNA was resuspended in a 10% sucrose-phosphate-buffered saline (PBS) solution, pH 7.4, in a volume of 40 µl/muscle. All in vivo DNA injections were carried out according to the following protocol. On day 0, 7-wk-old female Wistar rats were anesthetized by an intraperitoneal injection of pentobarbital sodium (Nembutal sodium solution; 50 mg/kg body wt). Soleus and EDL muscles were isolated and pretreated by injecting with 100-150 µl of 0.75% bupivacaine hydrochloride (Marcaine) in various regions of the muscle, to induce muscle necrosis and subsequent regeneration (3). On day 3, muscles were preinjected with 50 µl of a 25% sucrose-PBS solution and, 15 min after the sucrose-PBS preinjection, muscles were injected with 40 µl of the plasmid DNA cocktail, using a Hamilton syringe. Soleus and EDL muscles that were denervated were treated as described above; however, just before preinjection on day 3, the branches of the nerves that innervate the soleus or EDL muscles were severed and trimmed back to prevent reinnervation. Eleven days post-DNA injection, muscles were excised, immediately frozen in liquid nitrogen, and stored at -80°C. Animals were cared for at the University of Illinois at Chicago animal facility according to standard institutional Animal Care and Use Committee protocols.

Luciferase and beta -galactosidase assays. Muscles were trimmed of connective tissue, weighed, homogenized in 750 µl of reporter lysis buffer (1×; Promega), and centrifuged at 5,000 revolutions/min for 20 min at 4°C. Supernatants were removed for analysis of luciferase and beta -galactosidase activity. Luciferase activity in relative light units (RLU) was measured in duplicate using the luciferase assay system from Promega. beta -Galactosidase activity was measured using the luminescent beta -galactosidase genetic reporter system II from Clontech. To reduce endogenous beta -galactosidase activity, homogenates were incubated at 50°C for 1 h before assay. Normalized luciferase activity was expressed as RLU luciferase/beta -galactosidase. Analysis of variance (Microsoft Excel program) was used to assess the differences among groups, with significance set at a level of P < 0.05.

Southern blot analysis. Southern blot analysis was utilized to determine the uptake of injected plasmid DNA in regenerating soleus and EDL muscles. Muscles were injected with 40 µg of plasmid DNA (800MCL2s) and collected as previously described. Total DNA (genomic and plasmid) was isolated from tissue homogenates using the RapidPrep genomic DNA isolation kit from Pharmacia Biotech. DNA samples digested with Hind III were electrophoresed on 1% agarose gels. As a control for quantification, we loaded 0.01 and 1.0 ng of Hind III-digested plasmid 800MLC2s. Transfer was carried out according to standard procedures (24), and the membrane was hybridized with a random prime labeled 656-bp fragment of luciferase cDNA (from pGL3basic vector). Autoradiographic signals were obtained on Kodak film and were quantified using a model GS-670 imaging densitometer from Bio-Rad.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Establishment of the in vivo DNA injection assay. The use of in vivo DNA injection for promoter analysis has only recently been developed, and thus we felt it necessary to test its sensitivity and relative efficiency between different muscles. To determine the sensitivity and dose response range of plasmid DNA injections, we injected soleus and EDL muscles with 0.5-75 µg of the plasmid 3800MLC2s. As illustrated in Fig. 2, the sensitivity of our injection technique was quite high, since we could detect significant expression down to 0.5 µg of injected DNA. There was a dose-dependent effect between 0.5 and 10 µg of injected plasmid, with saturation in luciferase activity seen above 20 µg/muscle. Subsequent experiments required the coinjection of the pbeta gal-control vector, so out of concern for promoter competition all injections of the MLC2s gene were conducted using 5 µg of plasmid DNA. Additionally, it was noted that the results of injection experiments were reproducible between trials. Although not specifically a test of reliability, the mean luciferase expression seen with 5 µg injected DNA for both soleus and EDL muscles was consistent between the two trials, and this was also seen with a different promoter vector (800MLC2s; data not shown).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   Relationship between amount of plasmid DNA injected (3800MLC2s) and luciferase activity in soleus and EDL muscles in 2 trials. RLU, relative light units.

There was some concern that the EDL and soleus muscles might take up the injected DNA with different efficiencies due to fiber type, biochemical, and/or architectural differences. We coinjected the beta gal-control vector to normalize for injection efficiencies, but we felt it was also important to quantify the range of DNA uptake into the two different muscles. Using Southern blot analysis, we determined the amount of plasmid DNA in the EDL and soleus muscles at 11 days post-DNA injection. We found that the quantity of DNA within the muscles ranged from 53 to 150 pg in the soleus muscles (mean = 110 ± 16 pg; n = 6) and from 27 to 380 pg in the EDL muscles (mean = 140 ± 76 pg; n = 5). Although the amount of plasmid DNA in the muscles varied, the average incorporation was similar for both soleus and EDL muscles. Thus we feel confident that any differences in luciferase expression detected between soleus and EDL muscles reflect transcriptional activity and are not due to dramatic differences in plasmid DNA uptake.

The 5' flank and intragenic sequences of the MLC2s gene confer slow-twitch muscle specificity. Before we tried to identify slow nerve-dependent regions of the MLC2s gene, it was necessary to first identify slow-twitch-muscle-specific regions. Because there was a report in the literature that 250 bp of 5' flanking DNA did not direct slow-twitch muscle specificity (15), the initial construct tested included more upstream sequence as well as including intronic regions. This was the 3800MLC2s construct, which contained 2088 bp of 5' flank, exons 1 and 2, intron 1, and 83% (798 of 961 bp) of intron 2. When tested in vivo (Fig. 3), 3800MLC2s conferred luciferase activity 184 times higher in soleus than in EDL muscles. Luciferase levels in the EDL muscles were similar to that seen in muscles injected with the pGL3basic vector alone. These results indicate that elements necessary for slow-twitch-muscle-specific expression of MLC2s/luciferase fusion genes are present within 2088 bp of 5' flanking DNA and/or the first two introns.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   Measurement of luciferase activity in soleus and EDL muscles injected with either 3800MLC2s or pGL3basic. Luciferase activity is expressed as RLU. Values are means ± SE. * Significantly different from all other groups shown, P < 0.0001.

The -800 to +12 region is sufficient to confer slow-twitch-muscle-specific expression. Because the 3800MLC2s construct contained both 5' flanking and intronic DNA, we next tested the 5' flanking region, independent of intronic DNA, for slow-twitch-muscle-specific regulation. The 800MLC2s construct contains MLC2s DNA from positions -800 to +12 bp. As illustrated in Fig. 4, 800MLC2s conferred luciferase activity significantly higher (115-fold) in soleus than in EDL muscles (P < 0.001). These results indicate that there is a slow-twitch-muscle-specific regulatory element(s) present within 800 bp of 5' flanking DNA of the MLC2s gene.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Slow-twitch-muscle-specific expression of MLC2s/luciferase gene constructs 800MLC2s, 800IVS1,2MLC2s, and IVS1,2pGL3p. Luciferase activity was normalized to beta -galactosidase activity and was expressed in RLU luciferase/beta -galactosidase. * Significantly different from 800MLC2s, P < 0.01; dagger  significantly different from EDL muscle injected with same MLC2s/luciferase plasmid, P < 0.001; ddager  significantly different from pGL3promoter (pGL3p), P = 0.01.

Introns 1 and 2, independent of the promoter, direct slow-twitch-muscle-specific expression. Previous studies of skeletal muscle genes have demonstrated the presence of transcriptional regulatory elements within intragenic regions (TnIs, cTnC) (1, 21). To test introns 1 and 2 of the MLC2s gene for slow-twitch-muscle-specific regulatory elements, we placed them upstream of the SV40 promoter of the pGL3promoter vector (IVS1,2pGL3p). Soleus, but not EDL muscles, injected with IVS1,2pGL3p expressed luciferase activity 2.7 times higher (P = 0.01) than those muscles injected with pGL3promoter alone, indicating the presence of slow-twitch-muscle-specific regulatory elements within introns 1 and/or 2 (Fig. 4).

Introns 1 and 2 were also placed upstream of the 800-bp MLC2s promoter (800IVS1,2MLC2s). Soleus muscles injected with 800IVS1,2MLC2s expressed luciferase activity fivefold higher than soleus muscles injected with 800MLC2s (69 RLU vs. 13.8 RLU; Fig. 4). EDL muscles injected with 800IVS1,2MLC2s also exhibited significantly higher luciferase activity compared with expression of the 800MLC2s construct; however, the level of expression was still 186-fold lower than that seen in soleus muscles (0.37 RLU vs. 69 RLU, respectively). These findings clearly indicate that elements are present within introns 1 and 2 that act to enhance transcription of the MLC2s promoter.

The 800-bp MLC2s promoter contains a slow nerve-dependent element(s). As seen in Fig. 5, the 800-bp flanking region of the MLC2s gene contains slow nerve-dependent element(s). Luciferase expression in the noninnervated soleus muscles was significantly less than expression in the innervated soleus muscles (P = 0.0001). In addition, expression in the noninnervated soleus muscles was no different from expression in the EDL muscles. These results indicate that a slow nerve-specific regulatory element is present within 800 bp of the MLC2s promoter.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   Measurement of luciferase activity in innervated (+) and noninnervated (-) soleus and EDL muscles injected with 800MLC2s or 800IVS1,2MLC2s. Luciferase activity was normalized to beta -galactosidase activity and expressed in RLU luciferase/beta -galactosidase. Values are means ± SE. * Significantly different from noninnervated soleus and innervated and noninnervated EDL muscles, P = 0.0001.

When soleus and EDL muscles were injected with the 800IVS1,2MLC2s construct in the presence or absence of the nerve (Fig. 5), results similar to that determined with the MLC2s promoter were obtained. Thus, despite the addition of introns 1 and 2 to the MLC2s promoter, expression continued to be slow nerve dependent. The introns were subsequently tested, independent of the endogenous promoter, for slow nerve specificity (Fig. 6). In the absence of the slow nerve, soleus muscles injected with IVS1,2pGL3p displayed no change in luciferase activity compared with muscles injected with the control plasmid, pGL3promoter. The 2.7-fold difference in luciferase activity seen in the innervated soleus muscles (SV40 promoter with vs. without introns) is totally abolished when innervation is removed. These results are consistent with the presence of additional slow nerve-specific regulatory elements within introns 1 and/or 2. 


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 6.   Measurement of luciferase activity in innervated and noninnervated soleus and EDL muscles injected with IVS1,2pGL3p or pGL3p. Luciferase activity was normalized to beta -galactosidase activity and expressed in RLU luciferase/beta -galactosidase. * Significantly different from pGL3p, P = 0.01.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The initial aim of this study was to more fully define the use of in vivo DNA injection for the identification of muscle type-specific and nerve-dependent transcriptional elements. By combining modifications from a number of protocols (e.g., use of muscle regeneration, sucrose preinjection), we have increased the sensitivity of the assay so that the amount of DNA injected is ~5- to 20-fold lower than for previously published reports (4, 6, 7, 29). Because the sensitivity is increased, it was also important to determine the dose-dependent range of luciferase expression, to minimize concerns about promoter competition with coinjected vectors. The information from these experiments is important, especially for injecting multiple vectors for normalization of injection efficiency. Finally, we quantified the amount of plasmid DNA taken up by both the fast-twitch EDL and slow-twitch soleus muscles. The average amount of DNA in the different muscles was the same, which indicates that there are no fiber type differences in uptake with in vivo injection. This finding strengthens any conclusions that the muscle-type specificity seen with promoter constructs is due to transcriptional regulation and not differences in DNA uptake.

Slow-twitch-muscle-specific elements are located in both 5' flanking and intronic regions of the MLC2s gene. Cis-acting elements regulating transcription of the rat myosin light chain 2 ventricular/slow (MLC2s) promoter have been well characterized in cardiac but not slow-twitch skeletal muscle (10, 14, 15). These studies have indicated that 250 bp of 5' flanking DNA is sufficient to confer cardiac-specific expression in cultured myocardial cells and transgenic mice; however, analyses of skeletal muscles from transgenic mice have not been conclusive (10, 14, 15). Lee et al. (15) suggested that the 250-bp promoter region of MLC2s was not sufficient to confer expression in slow-twitch skeletal muscle. However, more recently, work from the same laboratory has generated transgenic mice using the 250-bp region of the MLC2s gene upstream of the Cre recombinase cDNA. In these mice, the 250-bp promoter of the MLC2s gene clearly directs expression in slow-twitch skeletal muscle (K. R. Chien, Keystone Conference, April 5, 1997, unpublished observations). These recent findings in transgenic mice are consistent with the results of this study, in which the -800-bp region of the MLC2s gene directed slow-twitch-muscle-specific expression. Whether there is an additional slow-twitch-muscle-specific element(s) upstream from the -250 position of the MLC2s gene is currently being tested.

Consistent with other slow contractile protein genes (1, 21), results from this study determined that introns 1 and/or 2 of the MLC2s gene have enhancer activity. These intronic regions of the MLC2s gene have not previously been sequenced or tested for enhancer activity either in vivo or in vitro. The intronic enhancer can act upstream of the endogenous MLC2s promoter or it can act as a slow-twitch-muscle-specific enhancer independent of the MLC2s promoter. The 5-fold increase in expression when the introns are placed upstream of the endogenous promoter compared with the 2.7-fold increase when placed upstream of the SV40 promoter also suggests that there may be a synergistic interaction between the promoter and intronic enhancer elements that drives high-level expression in slow-twitch skeletal muscle.

Although the introns clearly contain enhancer activity, there is some question about the slow-twitch muscle specificity when placed upstream of the endogenous MLC2s promoter. This is due to the increased luciferase expression from the 800IVS1,2MLC2s compared with the 800MLC2s construct in the EDL muscles. Although the increase in expression was statistically significant, the relative magnitude of the increase was less in the EDL than in the soleus (3-fold vs. 6-fold, respectively), with the absolute luciferase expression in the EDL still far (186-fold) below levels in the soleus muscle. Additionally, when the introns were placed upstream of the SV40 promoter, enhancer activity was only seen in the soleus muscle. These results suggest that slow-twitch-muscle-specific enhancer activity is located within introns 1 and/or 2; however, we cannot rule out the possibility that there is an additional enhancer(s) within the introns that acts independently of muscle type.

Slow nerve-specific regulatory regions of the MLC2s gene. Expression of muscle proteins in response to specific neural activity can occur at both transcriptional and translational levels. In regenerating skeletal muscle, slow nerve-specific expression of slow contractile protein isoforms is initially regulated at the level of transcription (8, 30). For example, MLC2s mRNA is not detected in newly formed myotubes or noninnervated regenerating soleus or EDL muscles (8, 26). The MLC2s mRNA is only detected in the soleus muscle regenerating in the presence of its own "slow" nerve (8). Therefore, we assessed the expression of the MLC2s reporter constructs in innervated and noninnervated regenerating soleus and EDL muscles. This provides the ability to identify regions of the MLC2s gene that are regulated specifically in response to the slow nerve.

Slow nerve regulatory elements are present within 800 bp of the MLC2s promoter and within the intronic enhancer. This is the first report in which slow nerve-dependent regulatory regions of a contractile protein gene have been located. The 5' flanking region (-4200 to +12) of the TnIs gene was shown to be responsive to innervation during regeneration, but the specificity of the neural response was not tested (16). Most of the information available about nerve-dependent regulatory regions comes from studies of the acetylcholine receptor gene family (9, 28). The rat nicotinic acetylcholine receptor delta -subunit gene (delta -nAChR), like the MLC2s gene, contains two regions that are capable of conferring activity-dependent regulation, with one element clearly acting as the stronger enhancer (28). Thus, in the MLC2s gene, both upstream and intronic regions may have the ability to confer slow nerve-dependent regulation; however, the presence of both of these sequences may be important for full nerve-dependent activity.

What are possible sites for regulation in response to the slow nerve? Previous studies have identified E box sequences (CANNTG) as important and necessary for activity-dependent expression of several muscle-specific genes, including nAChR alpha -, gamma -, and delta -subunit promoters (9, 28). Therefore, the DNA sequences of the 800-bp intron 1 and intron 2 were analyzed for E box sequences and other known muscle regulatory elements. These are illustrated in Fig. 7, with each region containing multiple E box sequences. However, it is unlikely that E boxes alone are responsible for slow nerve-specific expression because the MLC2s gene is not expressed in muscle cells in vitro or in vivo at times during myogenesis in which the expression levels of the E box-binding muscle regulatory factors (MyoD, myogenin, Myf-5, and muscle regulatory factor 4) are high (19). Thus, if E boxes are important for MLC2s gene expression, then other trans-acting factors must bind in addition to, in conjunction with, or instead of the myogenic factors.


View larger version (7K):
[in this window]
[in a new window]
 
Fig. 7.   Common skeletal muscle regulatory elements mapped on MLC2s promoter, intron 1, and intron 2. MEF2, myocyte enhancer factor 2.

Other sites of interest within the MLC2s gene are the CACC boxes and MEF2 sites in the promoter and in introns 1 and 2 (Fig. 7). A CACC box will bind specific nuclear factors present within cardiac and skeletal muscle myocytes (2). An MEF2 site has been described as a sequence containing a 9-bp AT-rich core and is associated with the transcriptional regulation of muscle-specific genes (20). What is most interesting when comparing flanking and intronic sequences is the close proximity of the MEF2 sites to E box and CACC box sequences. This combination of sites is also seen within the slow upstream regulatory element in the TnIs gene (18) and, in general, is consistent with a potential role for MEF2 as a transcriptional cofactor with either the muscle regulatory factors or CACC box binding proteins (2, 17).

In summary, the results of this study further define the use of the in vivo plasmid DNA injection technique as a tool for the analysis of physiologically important promoter and enhancer elements of skeletal muscle genes. With this technique, both slow-twitch-muscle-specific and slow nerve-dependent regulatory regions within the MLC2s gene were identified. These regions work independently and synergistically in the presence of the slow nerve to direct high-level expression. This is the first study in which nerve-dependent regions of a slow contractile protein gene have been identified. Additional work is required to identify the specific DNA elements and trans-acting factors required for nerve-dependent regulation.

    ACKNOWLEDGEMENTS

We thank Dr. Scott Henderson for providing us with the MLC2s genomic clone. We gratefully acknowledge the technical assistance of Keith Baar and Bryce Bederka.

    FOOTNOTES

This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-43349 and University of Illinois at Chicago Campus Research Board Grant F93-111.

1 The nucleotide sequences for introns 1 and 2 have been submitted to GenBank with accession nos. AFO16324 and AFO16325, respectively.

Address for reprint requests: K. A. Esser, School of Kinesiology (m/c194), University of Illinois at Chicago, 901 W. Roosevelt Rd., Chicago, IL 60608.

Received 8 May 1997; accepted in final form 24 September 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Banerjee-Basu, S., and A. Buonanno. Cis-acting sequences of the rat troponin I slow gene confer tissue- and development-specific transcription in cultured muscle cells as well as fiber type specificity in transgenic mice. Mol. Cell. Biol. 13: 7019-7028, 1993[Abstract].

2.   Bassel-Duby, R., M. D. Hernandez, Q. Yang, J. M. Rochelle, M. F. Seldin, and R. S. Williams. Myocyte nuclear factor, a novel winged-helix transcription factor under both developmental and neural regulation in striated myocytes. Mol. Cell. Biol. 14: 4596-4605, 1994[Abstract].

3.   Carlson, B. Regeneration of entire skeletal muscles. Federation Proc. 45: 1456-1460, 1886.

4.   Carson, J., R. Schwartz, and F. Booth. SRF and TEF-1 control of chicken skeletal alpha -actin gene during slow-muscle hypertrophy. Am. J. Physiol. 270 (Cell Physiol. 39): C1624-C1633, 1996[Abstract/Free Full Text].

5.   Condon, K., L. Silberstein, H. M. Blau, and W. J. Thompson. Differentiation of fiber types in aneural musculature of the prenatal rat hindlimb. Dev. Biol. 138: 275-295, 1990[Medline].

6.   Danko, I., J. D. Fritz, S. Jiao, K. Hogan, J. S. Latendresse, and J. A. Wolff. Pharmacological enhancement of in vivo foreign gene expression in muscle. Gene Ther. 1994: 114-121, 1994.

7.   Davis, H. L., R. G. Whalen, and B. A. Demeneix. Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression. Hum. Gene Ther. 4: 151-159, 1993[Medline].

8.   Esser, K. A., P. Gunning, and E. Hardeman. Nerve-dependent and -independent patterns of mRNA expression in regenerating skeletal muscle. Dev. Biol. 159: 173-183, 1993[Medline].

9.   Gilmour, B. P., D. Goldman, K. G. Chahine, and P. D. Gardner. Electrical activity suppresses nicotinic acetylcholine receptor gamma subunit promoter activity. Dev. Biol. 168: 416-428, 1995[Medline].

10.   Henderson, S. A., M. Spencer, A. Sen, C. Kumar, M. A. Q. Siddiqui, and K. R. Chien. Structure, organization, and expression of the rat cardiac myosin light chain-2 gene. J. Biol. Chem. 264: 18142-18148, 1989[Abstract/Free Full Text].

11.   Hoh, J. F. Y. Neural regulation of mammalian fast and slow muscle myosins: an electrophoretic study. Biochemistry 14: 743-746, 1975.

12.   Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J. White. PCR Protocols: A Guide to Methods and Applications. San Diego, CA: Academic, 1990.

13.   Kraus, W., C. Torgan, and D. Taylor. Skeletal muscle adaptation to chronic low-frequency motor nerve stimulation. In: Exercise and Sport Sciences Reviews, edited by J. O. Holloszy. Baltimore, MD: Williams and Wilkins, 1994, p. 313-360.

14.   Lee, K. J., R. Hickey, H. Zhu, and K. R. Chien. Positive regulatory elements (HF-1a and HF-1b) and novel negative regulatory element (HF-3) mediate ventricular muscle-specific expression of myosin light-chain 2-luciferase fusion genes in transgenic mice. Mol. Cell. Biol. 14: 1220-1229, 1994[Abstract].

15.   Lee, K. J., R. S. Ross, H. A. Rockman, A. N. Harris, T. X. O'Brien, M. van Bilsen, H. E. Shubeita, R. Kandolf, G. Brem, J. Price, S. M. Evans, H. Zhu, W.-M. Franz, and K. R. Chien. Myosin light chain-2 luciferase transgenic mice reveal distinct regulatory programs for cardiac and skeletal muscle-specific expression of a single contractile protein gene. J. Biol. Chem. 267: 15875-15885, 1992[Abstract/Free Full Text].

16.   Levitt, L. K., J. V. O'Mahoney, K. J. Brennan, J. E. Joya, L. Zhu, R. P. Wade, and E. C. Hardeman. The human troponin I slow promoter directs slow fiber-specific expression in transgenic mice. DNA Cell Biol. 14: 599-607, 1995[Medline].

17.   Molkentin, J., B. Black, J. Martin, and E. Olson. Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins. Cell 83: 1125-1136, 1995[Medline].

18.   Nakayama, M., J. Stauffer, J. Cheng, S. Banerjee-Basu, E. Wawrousek, and A. Buonanno. Common core sequences are found in skeletal muscle slow and fast-fiber type regulatory elements. Mol. Cell. Biol. 16: 2408-2417, 1996[Abstract].

19.   Olson, E. N., and W. H. Klein. bHLH factors in muscle development: dead lines and commitments, what to leave in and what to leave out. Genes Dev. 8: 1-8, 1994[Medline].

20.   Olson, E. N., M. Perry, and R. A. Schulz. Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors. Dev. Biol. 172: 2-14, 1995[Medline].

21.   Parmacek, M. S., H. Ip, F. Jung, T. Shen, J. F. Martin, A. J. Vora, E. N. Olson, and J. M. Leiden. A novel myogenic regulatory circuit controls slow/cardiac troponin C gene transcription in skeletal muscle. Mol. Cell. Biol. 14: 1870-1885, 1994[Abstract].

22.   Pette, D., and G. Vrbova. Neural control of phenotypic expression in mammalian muscle fibers. Muscle Nerve 8: 676-689, 1985[Medline].

23.   Rosenthal, N., J. Kornhauser, M. Donoghue, K. Rosen, and J. Merlie. The myosin light chain enhancer activates muscle-specific, developmentally regulated expression in transgenic mice. Proc. Natl. Acad. Sci. USA 86: 7780-7784, 1989[Abstract].

24.   Sambrook, J., E. F. Fritsch, and T. Maniatis. Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.

25.   Schiaffino, S., and C. Reggiani. Molecular diversity of myofibrillar proteins: gene regulation and functional significance. Physiol. Rev. 76: 371-423, 1996[Abstract/Free Full Text].

26.   Sutherland, C., K. Esser, V. Elsom, M. Gordon, and E. Hardeman. Identification of a program of contractile protein gene expression initiated upon muscle differentiation. Dev. Dyn. 196: 25-36, 1993[Medline].

27.   Tsika, R., S. Hauschka, and L. Gao. M-creatine kinase gene expression in mechanically overloaded skeletal muscle of transgenic mice. Am. J. Physiol. 269 (Cell Physiol. 38): C665-C674, 1995[Abstract].

28.   Walke, W., G. Xiao, and D. Goldman. Identification and characterization of a 47 base pair activity-dependent enhancer of the rat nicotinic acetylcholine receptor delta-subunit promoter. J. Neurosci. 16: 3641-3651, 1996[Abstract/Free Full Text].

29.   Wells, D. J. Improved gene transfer by direct plasmid injection associated with regeneration in mouse skeletal muscle. FEBS Lett. 332: 179-182, 1993[Medline].

30.   Whalen, R. G., J. B. Harris, G. S. Butler-Browne, and S. Sesodia. Expression of myosin isoforms during notexin-induced regeneration of the rat soleus muscles. Dev. Biol. 141: 24-40, 1990[Medline].

31.   Wiedenman, J. L., I. Rivera-Rivera, D. Vyas, G. Tsika, L. Gao, K. Sheriff-Carter, X. Y. Wang, L. Kwan, and R. W. Tsika. beta -MHC and SMLC1 transgene induction in overloaded skeletal muscle of transgenic mice. Am. J. Physiol. 270 (Cell Physiol. 39): C1111-C1121, 1996[Abstract/Free Full Text].


AJP Cell Physiol 274(1):C229-C235
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society