School of Kinesiology, University of Illinois, Chicago, Illinois 60608
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ABSTRACT |
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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
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INTRODUCTION |
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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 -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.
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MATERIALS AND METHODS |
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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 (
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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 (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 -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
-galactosidase activity. Luciferase
activity in relative light units (RLU) was measured in duplicate using
the luciferase assay system from Promega.
-Galactosidase activity
was measured using the luminescent
-galactosidase genetic reporter
system II from Clontech. To reduce endogenous
-galactosidase activity, homogenates were incubated at 50°C for 1 h before assay. Normalized luciferase activity was expressed as RLU
luciferase/
-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.
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RESULTS |
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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 pgal-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).
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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.
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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.
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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.
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DISCUSSION |
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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.
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 (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
-,
-, and
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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