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INTRODUCTION |
The acquisition and maintenance of an adult skeletal muscle
phenotype is regulated, in part, by the activity of specific motor neurons (1-3). An adult skeletal muscle fiber normally expresses a
relatively homogeneous pattern of either fast or slow contractile protein isoforms consistent with its type of innervation. These proteins include fast and slow isoforms from the myosin heavy chain,
myosin light chain (MLC),1
tropomyosin, and the troponin I, T, and C gene families (3, 4).
It has been well established that chronic low frequency electrical
stimulation of fast muscle fibers, by stimulating either through the
nerve or by direct stimulation of the muscle, induces the slow skeletal
muscle phenotype (2-4). This phenotype is characterized by an
increased expression of the slow contractile protein isoforms. Conversely, if a slow nerve is removed or electrical activity is
blocked, there is a transition from a slow to fast muscle phenotype. These transitions in phenotype have been determined at the mRNA level, which suggests that alterations in slow neural activity transcriptionally regulate slow isoform gene expression (3, 5, 6).
Regions of the MLC1slow, TnIslow, and
MLC2slow genes have been identified that are responsive to
slow innervation; however, the specific elements involved have not been
isolated (7-10). Potential cis-acting elements include those that are
known to regulate qualitative and quantitative skeletal muscle gene expression. Some examples are E-box, MCAT, MEF2, CACC, and CArG box
sequences (11-16).
The MLC2slow gene, in particular, is one of the contractile
protein isoform genes that is largely regulated by slow innervation. Unlike many of the other contractile proteins genes, the
MLC2slow gene is not expressed in newly formed myotubes
either in vivo or in vitro (17-20). During
skeletal muscle development, in humans and rats, induction of MLC2slow
expression occurs late and correlates with the establishment of mature
innervation patterns. The requirement of specific neuromuscular
activity for MLC2slow gene expression is most evident when studying
skeletal muscle regeneration in vivo. Expression of the
MLC2slow gene is only detected in the soleus muscle
regenerating in the presence of its own nerve (6). In this system,
induction of MLC2slow mRNA occurs at a time that is coincident with
the establishment of functional neuromuscular junctions (21). Because
of this relatively clear on/off pattern of control, the
MLC2slow promoter serves as a good molecular tool for the
identification of promoter elements responsible for slow nerve-dependent regulation of transcription.
Recently, it was determined that the 800-bp promoter of the rat
MLC2slow gene was capable of directing slow
nerve-dependent expression in regenerating muscles in
vivo (9). As in the previous work, this study combined skeletal
muscle regeneration with in vivo injection of plasmid DNA
constructs into muscle. The main objectives were 1) to find a smaller
region of the 800-bp MLC2slow promoter capable of directing
slow nerve-dependent expression, 2) to determine specific
cis-acting regulatory sites, and 3) to determine site-specific factor
binding and assess potential transcription factor involvement in
regulation of the MLC2slow promoter. Using site-directed
mutagenesis, three sites (E-box, MEF2, and CACC box) were mutated to
investigate their functional role in MLC2slow expression. It was
determined that both the CACC box and MEF2 sites were important for
appropriate slow nerve-dependent promoter activity during
regeneration. These data suggest that the slow nerve acts through the
CACC box to derepress the promoter. Once repression is lifted, then
high level expression is regulated by MEF2. Interestingly, the proximal
combination of the CACC box and MEF2-like sites is seen in other
previously defined slow muscle specific regions of contractile protein
genes, including TnIslow, TnCslow, MLC1slow, myoglobin, and
slow myosin heavy chain. This suggests that a conserved mechanism may
exist by which the CACC box and MEF2 sites contribute to slow
nerve-dependent regulation of the subset of slow
contractile protein isoform genes.
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EXPERIMENTAL PROCEDURES |
Generation of the
270MLC2slow Construct--
Previous work had
generated the
800MLC2slow construct that contains from
800 to +12
of the rat MLC2slow gene inserted upstream of the luciferase
cDNA in the pGL3basic vector (Promega Corp.). This template was
used to amplify a shortened promoter region from
270 to +12 of the
MLC2slow gene. For the polymerase chain reaction, the 3'
primer overlapped a natural EcoRI site at position +12
(5'-CTGCTGCCTCTGAAGAATTC-3'). The 5' primer at position
270 contained
a 5' KpnI restriction site
(5'-GGGGTACCCCATTAGACAATGGCAGG-3'). Standard polymerase chain reaction
conditions were followed (22, 23), and the product was ligated into a
modified pGL3basic luciferase expression vector (Promega Corp.)
containing the desired restriction sites. Identification and
orientation of the
270MLC2slow clone was verified by restriction
digest analysis.
Generation of the Mutated Constructs--
The QuikChange
site-directed mutagenesis kit (Stratagene) was utilized to mutate the
MEF2 site, E-box, and CACC box of
270MLC2slow(see Figs. 1 and 3).
Complementary primers (mutated bases underlined) containing the MEF2
mutation (sense,
5'-GCCAAAAGTGGTCATGGGGTTAGGTTTAACCCCAGGGAAGAGG-3'), the E-box mutation (sense,
5'-GGCCTCTGCCTCACCTAGTACGTCCAAAAGTGGTCATGGGG-3'), or the CACC box mutation (sense,
5'-CAGAAGAACGGCATTACCGGGGGGGCTTAGGTGGCCTCTGCCTCA-3') were designed. Polymerase chain reaction conditions followed the protocol provided by Stratagene. The polymerase chain reaction was used
to transform XL1-Blue supercompetent cells (Stratagene), colonies were
selected, and plasmid DNA sequenced using standard protocols with the
Sequenase Quick-Denature plasmid sequencing kit (Amersham Pharmacia Biotech).
In Vivo DNA Injection--
For promoter and overexpression
assays, in vivo DNA injection during muscle regeneration was
utilized following the protocol described previously (9). Briefly, 5 µg of the promoter construct (
270MLC2s,
270MEF2,
270Ebox, or
270CACC) and 3 µg of
-galactosidase control plasmid DNA (vector:
SV40 promoter upstream of the
-galactosidase cDNA;
CLONTECH Laboratories, Inc.) were mixed in a 10%
sucrose-phosphate-buffered saline solution, pH 7.4. For the
overexpression studies, 3 µg of the expression vector MEF2A (24),
MEF2C (25), or myogenin (26) and 5 µg of the promoter vector were
prepared for injection. At day 0, muscle regeneration was induced by
injecting bupivacaine hydrochloride (Marcaine®). On day 3, plasmid DNA
was injected (40 µl/muscle) with a disposable 0.3cc syringe (28G1/2
needle). At this time, the nerve to the soleus was cut (noninnervated
SOL (SOL
)) or left intact (innervated SOL (SOL+)). On day 14, the SOL
and EDL muscles were collected, immediately frozen in liquid N2, and stored at
80 °C.
Luciferase and
-Galactosidase Assays--
Muscles were
homogenized in 750 µl of Reporter Lysis buffer (1×, Promega Corp.)
and centrifuged at 5000 × g for 20 min at 4 °C.
Supernatants were removed for analysis of luciferase and
-galactosidase activity. Luciferase activity was measured using the
luciferase assay system from Promega Corp. Each homogenate was tested
in duplicate, using 100 µl per reaction. Relative Light Units (RLUs)
were integrated over 10 s and were within the linear range.
-Galactosidase activity was measured using the
Glacto-StarTM luminescent
-galactosidase reporter system
II (Tropix). Homogenates were tested in duplicate, using 10 µl per
reaction. To reduce endogenous
-galactosidase activity, homogenates
were incubated at 50 °C for 1 h prior to assay. RLUs were
integrated over 10 s and were within the linear range. Normalized
luciferase activity was expressed as RLUs luciferase/
-galactosidase.
All results were statistically analyzed using analysis of variance with
significance set a priori at p < 0.05.
Preparations of Nuclear Extracts, Western Blots, and
Electrophoretic Mobility Shift Assays--
Nuclear extracts were
prepared according to recently published techniques (27). Frozen
skeletal muscle tissue was minced on ice, homogenized (10 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM KCl, 0.1 mM EDTA, pH 8.0, 0.1% Triton
X-100, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, aproprotin (2 µg/ml), and leupeptin (2 µg/ml)) and spun down for 5 min at 3000 × g. The
pellet was resuspended in ice cold lysis buffer (20 mM
HEPES, pH 7.9, 25% glycerol, 500 mM NaCl, 1.5 mM MgCl2, 0.2 EDTA, pH 8.0, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride,
aproprotin (2 µg/ml), and leupeptin (2 µg/ml)) and incubated for 30 min on ice. To exchange the high salt buffer, an equal volume (~500
µl) of binding buffer (20 mM HEPES, pH 7.9, 40 mM KCl, 2 mM MgCl2, 10% glycerol,
0.5 mM dithiothreitol, 0.2 mM
phenylmethylsulfonyl fluoride, aproprotin (2 µg/ml), and leupeptin (2 µg/ml)) was added, and this was spun using filters from Millipore
(Ultrafree 5K NMWL membranes). This centrifugation step was repeated
two times. The protein concentrations of the nuclear enriched extracts
were determined using the Bradford protein assay.
Western blots were performed as described elsewhere (28). Briefly,
equivalent amounts of nuclear enriched protein (from 15 to 50 µg)
were separated by 7% SDS-polyacrylamide gel electrophoresis and
transferred to polyvinylidene difluoride membranes. MEF-2 protein
levels were detected using the polyclonal rabbit anti-MEF2 antibody
(Santa Cruz Biotechnology) with an enhanced chemiluminescence kit
(Amersham Pharmacia Biotech).
For the electrophoretic mobility shift assays (EMSAs), standard
conditions were used (23). Double stranded oligonucleotides specific
for the MEF2 and CACC box sites within the MLC2slow promoter were
end-labeled using T4 polynucleotide kinase (Promega, Madison, WI).
Binding reactions were performed using 5 µg of nuclear extracts, 1 µg of poly(dI-dC)·poly(dI-dC), and 1 ng of labeled probe (40,000 cpm/ng) in binding buffer as outlined elsewhere (23). Cold mutant specific or nonspecific (mutated E-box) oligos were added at a 200-fold
molar excess, and DNA binding reactions were carried out at 4 °C.
For the supershift experiments, preimmune serum or antibody (1 µl)
was added 30 min after the initial incubation, and these samples were
incubated for an additional 30 min. The Sp1 and MEF2 antibodies were
obtained from Santa Cruz Biotechnology. MEF2A- and MEF2D-specific
antibodies were generously provided by Dr. R. Prywes (29), the muscle
LIM antibody was provided by Dr. P. Caroni (30), the Oct-1 antibody was
provided by Dr. R. Roeder (31), the myocyte nuclear factor (MNF)
antibody was provided by Dr. Rhonda Bassel-Duby (32), and the antibody
to 4E-BP1 was generated in this laboratory. Protein-DNA complexes were
separated by electrophoresis in 7% nondenaturing polyacrylamide gels
in 0.5× TBE (90 mM Tris-base, 90 mM boric
acid, 2 mM EDTA, pH 8.0) buffer at 4 °C. Following
electrophoresis at 30 mA for 3-4 h, gels were dried and exposed to
film overnight at
80 °C.
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RESULTS |
Deletion and Mutational Analyses of the MLC2slow
Promoter--
Previous studies determined that the
800-bp promoter
of MLC2slow was sufficient to elicit slow
nerve-dependent expression in regenerating skeletal muscle
(9). Approximately 100-fold higher expression was detected in the SOL+
compared with SOL
or innervated EDL muscles. As can be seen in Fig.
1 (bottom panel), the
270-bp
promoter of the MLC2slow gene maintains slow
nerve-dependent expression in vivo. Relative
luciferase activity for
270MLC2slow was no different compared with
that for
800MLC2slow across all SOL and EDL muscles studied. This
promoter region was then used to identify potential
nerve-dependent regulatory elements using site-specific
mutations.

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Fig. 1.
Sequence and expression of the wild type
E-box, MEF2, and CACC box mutations of the rat MLC2slow promoter.
Top, the diagram of the rat 270MLC2slow luciferase vector
with E-box, MEF2, and CACC sites identified. The specific mutations
(MUT) introduced into the wild-type sequence
(WT) for each site are listed. Bottom, expression
of MLC2slow wild-type ( 800MLC2slow and 270MLC2s), E-box mutant
( 270Ebox), MEF2 mutant ( 270MEF2), and CACC mutant ( 270CACC)
promoter-luciferase constructs (mean ± S.E.) in regenerating
muscles in vivo. Luciferase activity in RLUs is normalized
to luminescent -galactosidase activity (RLUs) for the SOL+, SOL ,
and EDL muscles. Regenerating muscles (n = 6-8/group)
were injected with 5 µg of the luciferase construct and 3 µg of the
SV40- gal construct. Statistical significance was set at
p < 0.05; * denotes that the normalized luciferase
activity for that group was statistically different from that
determined with the 270MLC2slow construct for the same group.
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Work on other slow isoform contractile protein genes, such as the
TnIslow and TnCslow genes, has demonstrated that
MEF2, E-box, and CACC box sites are important in directing slow
skeletal muscle specific expression (15, 16, 33, 34). To address
potential functional roles of these three sites within the 270-bp
MLC2slow promoter, we individually mutated the E-box, MEF2,
and CACC sites (Fig. 1). As can be seen in Fig. 1, mutation of the
E-box (
270Ebox) within the MLC2slow promoter did not
result in a change in expression in the SOL+ and EDL muscles.
Specifically, relative luciferase expression in the SOL+ muscles was
10.3 ± 1.5 RLUs for
270MLC2slow compared to 8.12 ± 1.1 RLUs for
270Ebox construct. In EDL muscles, expression of the
270MLC2slow (0.23 ± 0.05) versus
270Ebox
(0.12 ± 0.04) construct was also not significantly different.
These results suggest that the E-box is not required for
MLC2slow promoter activity or slow nerve specificity. The
decrease in expression with the
270Ebox construct in the SOL
muscles was statistically significant (0.135 ± 0.03 versus 0.06 ± 0.005 RLUs; p < 0.05). However, expression of
270MLC2slow construct is already very low in
the SOL
muscles (75-fold lower then SOL+ muscles), so it is not clear
what the functional significance of this drop in expression means.
In contrast to the E-box mutation, mutation of the MEF2 site
(
270MEF2) does result in a significant reduction in relative luciferase expression in all the innervated and noninnervated muscles
(Fig. 1). In the SOL+ muscles, the average luciferase/
-galactosidase ratio decreased 30-fold between the
270MLC2slow construct and the
270MEF2 construct (10.3 ± 1.5 versus 0.36 ± 0.05 respectively; p < 0.01). The MEF2 mutation also
resulted in significant decreases in relative luciferase levels in the
SOL
(0.135 ± 0.03 versus 0.024 ± 0.005 RLUs;
p < 0.01) and EDL muscles (0.23 ± 0.05 versus 0.024 ± 0.006 RLUs; p < 0.01).
The next site within the MLC2slow promoter chosen for
mutation was the CACC box because it has been implicated in slow muscle specific expression of the TnIslow and TnCslow
gene (7, 15, 34). As can be seen in Fig. 1, expression of the mutated
CACC box construct results in no change in expression compared with the
intact
270-bp MLC2slow promoter in SOL+ muscles (7.4 ± 0.83 versus 10.33 ± 1.5 RLUs, respectively). Compared with
270MLC2s, the CACC mutation resulted in a nonsignificant increase in
expression in the SOL
muscles (0.4 ± 0.095 versus
0.135 ± 0.03 RLUs; p = 0.07). A dramatic increase
in luciferase expression was evident in the EDL muscles, with the
270CACC construct expressing 18-fold higher compared with the
270MLC2slow construct (4.08 ± 1.1 versus 0.23 RLUs
respectively; p < 0.01). These results indicate that the CACC box site on the MLC2slow promoter acts as a repressor element
in fast EDL muscle and possibly in the SOL
muscle as well. The lack
of repressor activity detected in the SOL+ muscle also suggests that
the slow nerve may regulate the MLC2slow promoter, in part,
by derepression at the CACC box site.
Transactivation of the MLC2slow Promoter in
Vivo--
Because the MEF2 site is important for high level promoter
activity in SOL+ muscle, we tested whether forced overexpression of
MEF2 could transactivate the MLC2slow promoter in SOL
or
EDL muscles in vivo. For these experiments, the
270MLC2slow construct was co-injected with either a MEF2C, MEF2A, or
myogenin expression vector. As can be seen in Fig.
2, overexpression of either MEF2A or
MEF2C was not sufficient for transactivation of the
270MLC2slow promoter in the SOL
or EDL muscles. However, in the SOL+ muscles, overexpression of MEF2A or MEF2C increased luciferase expression 3-4-fold (
270MLC2slow 676,340 ± 105,169 RLUs; +MEF2C
2,225,957 ± 346,793 RLUs; +MEF2A 2,104,446 ± 793,125 RLUs;
p < 0.01). This enhanced expression was specific for
MEF2 as the overexpression of myogenin did not result in any change in
luciferase activity in either the SOL+ or EDL muscles (Fig. 2).
Co-injection of the MEF2 expression vector with the
270MEF2 mutant
construct did not enhance luciferase activity in SOL+ muscles (Fig. 2),
which argues that the MEF2 protein, through binding at the MEF2 site, is functionally important for MLC2slow promoter activity
in vivo.

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Fig. 2.
Overexpression of MEF2, but not myogenin,
enhances transcription of the 270MLC2slow construct through the MEF2
site. Luciferase expression (mean ± S.E.) from regenerating
soleus and EDL muscles injected with the 270MLC2slow or the mutated
270MEF2 construct with either myogenin (Myog) (26), MEF2A
(24), or MEF2C (25) expression vectors. Statistical significance was
set at p < 0.05; * denotes that the luciferase
activity for that group was statistically different from that
determined with the 270MLC2slow or 270MEF2 construct within the
same experimental group.
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Because overexpression of MEF2 was not sufficient by itself to induce
MLC2slow promoter activity in the SOL
and EDL muscles, we
next tested whether derepression, through mutation of the CACC box,
would permit MEF2 transactivation. SOL
and EDL muscles were co-injected with the
270CACC construct and the MEF2C expression vector. As can be seen in Fig. 3, once
the promoter is derepressed in the SOL
muscle, it can be strongly
transactivated by overexpressing MEF2C. Luciferase activity was
increased almost 70-fold when MEF2C was overexpressed with the
270CACC construct in SOL
muscles (1,605,797 ± 582,998 RLUs
for
270CACC+MEF2 versus 27,536 ± 6269 RLUs for
270CACC; p < 0.05). It was surprising that
overexpressing MEF2C with the
270CACC construct in the EDL did not
result in a significant increase in luciferase activity. The luciferase activity was about 1.6-fold higher with the overexpression of MEF2C,
but this difference was not statistically significant. This suggests
that either MEF2 levels are not limiting for transcription in the EDL
or that the EDL regulates transcription of the
270CACC construct
through a different mechanism compared with that in the SOL muscle.

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Fig. 3.
Overexpression of MEF2 enhances transcription
of the 270CACC construct in SOL muscles. Luciferase expression
(mean ± S.E.) from regenerating SOL and EDL muscles injected
with the 270MLC2slow or 270CACC construct. Some of the muscles were
also co-injected with the MEF2C expression vector (+MEF2). Statistical
significance was set at p < 0.05; * denotes that the
luciferase activity for the co-injected group was statistically
different from the expression of the promoter vector injected
alone.
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Analysis of MEF2 Protein Levels and EMSA with Extracts from
Regenerating SOL and EDL Muscles--
Because MEF2 levels were
implicated in the transcriptional regulation of the MLC2slow
promoter, quantitative assessment of MEF2 protein levels in nuclear
extracts from SOL+, EDL, and SOL
muscles were made by Western blot
analysis. A representative blot is presented in Fig.
4 using the polyclonal MEF2 antibody from Santa Cruz Biotechnology that was raised against human MEF2A but does
recognize both MEF2C and MEF2D isoforms. As can be seen from Fig. 4,
EDL and SOL+, MEF2 protein levels are similar
between SOL+ and EDL nuclear extracts (15 µg of total protein
loaded). However, there was a dramatic decrease in MEF2 levels in the
extracts from SOL
muscles. When analyzing 15 µg of nuclear extract,
MEF2 levels were undetectable in the SOL
extracts (Fig. 4,
SOL
). MEF2 protein levels were detected in the SOL
extracts when the total protein loaded was increased over 3-fold (to 50 µg). Although not conclusive, this finding suggests that MEF2 levels
are lower in SOL
extracts when compared with levels in SOL+ and EDL
extracts. Extracts obtained from SOL
muscles transfected with the
MEF2 expression vector were also analyzed on Western blots (Fig. 4). When comparing the right lane of Fig. 4 to the lane just to
the left, it is evident that in vivo transfection of SOL
muscles with the MEF2 expression vector does result in detectable
increases in MEF2 protein levels. This observation was also verified in the extracts from in vivo transfected EDL muscles (data not
shown).

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Fig. 4.
MEF2 protein levels are depressed in nuclear
extracts from noninnervated soleus muscles compared with either
innervated soleus or EDL muscles. Western blot analysis of MEF2
levels from nuclear extracts (15 µg) of SOL and EDL muscles
(left lanes, 15 µg). Nuclear extracts were prepared from
regenerating SOL+, SOL m, and EDL muscles. Proteins were separated
using SDS-polyacrylamide gel electrophoresis, transferred to
polyvinylidene difluoride membrane, and incubated against the MEF2
antibody. In the right lanes (50 µg), 50 µg
of nuclear extracts from SOL and SOL muscles injected with the
MEF2C expression vector were analyzed to verify that transfection with
the MEF2 expression vectors does increase MEF2 protein levels in
vivo.
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To assess transcription factor binding to the MEF2 and CACC sites
within the MLC2slow promoter, EMSA and supershift assays were performed with nuclear extracts from the regenerating SOL+, SOL
,
and EDL muscles (Figs. 5 and
6). As can be seen in Fig. 5A
(lanes 1, 4, and 7), there was one primary
complex formed with the labeled MLC2slow MEF2 oligonucleotide and
nuclear extracts from either SOL+, EDL, or SOL
muscles. This complex
was specific for the MEF2 site as it was competed off by excess cold
MEF2 oligo (lanes 2, 5, and 8) but was not
affected by excess nonspecific oligo (E-box mutant; Fig. 5A,
lanes 3, 6, and 9) or the mutant MEF2 oligo (Fig.
5B, compare lanes 1-4). Supershift assays using extracts from SOL+ muscles are seen in Fig. 5B with
antibodies to transcription factors MEF2A and MEF2D, Sp1, and a
translational regulatory factor, 4E-BP1. Results from this series of
experiments confirmed that MEF2 and Sp1 proteins contribute to complex
binding at the MEF2 site. Interestingly both MEF2 antibodies, which are specific to the 2A and 2D forms of MEF2, were very effective in competing away the binding activity in these samples. This suggests that both MEF2A and MEF2D contribute to complex formation in extracts from skeletal muscle. Addition of the Sp1 antibody resulted in a
classic supershift of the MEF2 complex as seen in Fig. 5B, lane 7. The competition and supershift activities with the MEF2 and Sp1
antibodies were specific because addition of an antibody to a protein
that regulates translation, 4E-BP1/PHAS-I, did not affect DNA binding
(Fig. 5B, lane 8). We also saw no effect on DNA binding to
the MEF2 site with an antibody to another A+T-rich binding transcription factor, Oct-1 (35) (data not shown). It is important to
note that there was no muscle specific or nerve specific binding to the
MEF2 DNA because we saw the same mobility shift and supershift pattern
when extracts from the SOL
and EDL muscles were used (data not
shown).

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Fig. 5.
EMSA analysis of the MEF2 binding sites in
the 270-bp MLC2slow promoter. A, competition
experiments demonstrate specificity of MEF2 protein binding to the
MLC2slow MEF2 site in SOL+ and SOL and EDL muscles. Lanes
1-3, SOL+; lanes 4-6, SOL ; lanes 7-9,
EDL extracts. B, supershift assays using antibodies to
either MEF2A, MEF2D, Sp1, or 4E-BP1 identify that MEF2 and Sp1
contribute to the complex formed with the MLC2slow MEF2 oligonucleotide
(SOL+, lanes 1-8). Competition experiments demonstrate that
the complex formed is specific to the intact MEF2 site as it is
competed off by cold MEF2 but it is not effectively competed off by
either excess cold mutated Ebox or the mutated MEF2 oligos (lanes
3 and 4).
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Fig. 6.
Mobility shift and supershift analyses of
CACC box binding with extracts from regenerating SOL+ and EDL
muscles. Two protein-DNA complexes are seen using extracts from
SOL+ and EDL muscles and they are similar for both muscles (compare
lanes 2 and 12). The complexes for all extracts
are specific to the CACC box sequence as excess cold CACC sequence does
compete for binding but neither mutated CACC or mutated Ebox oligos
effectively compete for binding (lanes 2-4 and
9-12). Supershift experiments using an antibody to MNF and
muscle Lim protein indicate that neither of those proteins is part of
the complexes formed with the CACC oligo (lanes 5-8).
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Binding to the CACC box site of the MLC2slow promoter was
also analyzed using EMSA and supershift assays (Fig. 6). As with the
MEF2 probe, the same pattern of binding was seen with extracts from
SOL+, SOL
, and EDL muscles (Fig. 6, lanes 1-12). At least two complexes were apparent, with the faster migrating complex being
most predominant. Both complexes, however, were specific to the intact
CACC site as competition for binding was seen with excess cold CACC
oligo but not with either excess mutated E box or the mutated CACC box
oligos. This pattern of protein-DNA binding was also seen with extracts
from the SOL
muscles (data not shown). Supershift experiments (Fig.
6, lanes 5-8) using antibodies to the MNF or muscle Lim
protein suggest that neither of those proteins are part of the
complexes binding to the MLC2slow CACC sequence (compare lanes 2, 5, and 6). This was seen using extracts from SOL+ and
EDL muscles as well as SOL
muscles (data not shown). This suggests
that even though MNF was originally isolated by its binding to the CACC
sequence of the myoglobin promoter, it is not part of the complex
binding at the MLC2slow CACC box site.
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DISCUSSION |
From the results of this study, we describe a two-step model for
slow nerve-dependent regulation of the MLC2slow
promoter. The first step of the model is derepression at the CACC box
followed by high level expression regulated through the MEF2 site.
Because overexpressing MEF2 is not sufficient to enhance transcription in SOL
and EDL muscles, this suggests that repression of the MLC2slow promoter may be a dominant effect. The appearance
of closely linked CACC box and MEF2-like sites within the regulatory regions of a number of previously identified slow contractile protein
genes raises the possibility that this two-step model for slow nerve
regulation of the MLC2slow promoter may represent a general
mechanism for controlling slow isoform contractile protein gene expression.
A two-step model of gene regulation has been previously proposed for
-actin and keratin genes (36-38). With this model, the activation
of transcription is controlled by one set of transcription factors and
high level expression is regulated by a different set of transcription
factors. When applying this model to the MLC2slow promoter,
derepression is the first step, and it is mediated by the slow nerve
through binding at the CACC box. However, because there is no
difference in EMSA results with SOL+, SOL
, or EDL extracts, that
would argue that the slow nerve does not act by altering the number of
factors binding to the CACC box. This suggests either that slow
innervation leads to derepression through posttranslationally modifying
the existing protein(s) binding to the CACC box or that the CACC box
site, in the context of the full
270-bp promoter, has a different
binding behavior compared with what was seen with the oligonucleotide
in an in vitro assay.
To date, there is very little known about CACC box-binding proteins.
One candidate protein that was considered is the winged-helix transcription factor, MNF. This factor was initially isolated using the
CACC box sequence of the human myoglobin gene (32), and its expression
is up-regulated in muscles subjected to chronic motor nerve stimulation
(32). However, in this study, negative results from supershift assays
suggest that MNF is not part of the complex binding to the CACC box
sequence as either a repressor or derepressor.
It was interesting to find that derepression through the CACC site is
not sufficient for high level expression in SOL
muscles (see Fig. 1).
This finding led to the identification of the second step of regulation
in which activation through the MEF2 site is necessary for high level
expression of the MLC2slow promoter. The findings from the
in vivo transfection experiments (mutational and
overexpression experiments) were consistent with those from the
in vitro DNA binding and supershift assays. This strongly suggests that MEF2 proteins are specifically binding at the MEF2 site
within the MLC2slow promoter and that this binding is
critical for complex formation and subsequent transcriptional activity. The supershift experiments also indicated that the ubiquitous factor,
Sp1, but not Oct-1 contributes to the binding activity on the MEF2
site. This is seen with extracts from all muscles studied and is
consistent with reported Sp1 and MEF2 interaction seen with nuclear
extracts from myotubes in culture (39) and nerve cells (40).
Another potential factor that has been implicated in regulating
transcriptional activity through the MEF2 site of muscle specific promoters is the nuclear factor of activated T cells, NFAT. In a recent
study, Chin et al. (41) demonstrated that overexpression of
activated calcineurin, which leads to increased NFAT translocation to
the nucleus, can enhance transcription of slow muscle type genes. They
propose that NFAT and MEF2 are involved in a combinatorial mechanism to
regulate slow muscle type genes in skeletal muscle. Although
intriguing, the results from this study are not consistent with that
NFAT-MEF2 model. If the slow nerve does activate transcription of slow
muscle type genes through a calcineurin/NFAT mechanism, then it would
be predicted in our experiments that the increase in nuclear NFAT and
its interaction with MEF2 should be evident in EMSA experiments with
SOL+ versus SOL
nuclear extracts. However, results from
electrophoretic mobility shift assays did not detect any differences
between the MEF2 complexes in SOL+ and SOL
extracts. In addition, if
NFAT translocation to the nucleus was required for transcriptional
activation, then overexpressing MEF2 in the SOL
muscles would not be
effective in transactivating the promoter. However, overexpression of
MEF2 did induce significant transactivation in the SOL
muscles when
the CACC site was mutated within the MLC2slow promoter. This
indicates that there are multiple levels of nerve-dependent
regulation, of which modulation of repressor/derepressor activity
through the CACC site is, in part, a critical step. Finally, unlike the
promoters discussed by Chin et al. (41), the
MLC2slow promoter does not contain a consensus NFAT site.
These arguments do not rule out NFAT involvement in some aspect of
regulation, but clearly, further experiments must be done to determine
its function in this system.
A unique aspect of this study came from the in vivo
transfection studies that provided important insight into potential
mechanisms by which MEF2 transcriptional activity is regulated in adult
skeletal muscle in vivo. To date, most of the information
regarding regulation of contractile proteins genes by either MEF2 or
the bHLH myogenic regulatory factors have been made during embryonic
development or in cell culture (11, 26, 42-45). In this in
vivo study, overexpressing MEF2 (both MEF2A and MEF2C) enhanced
MLC2slow promoter activity in the SOL+ muscles. This suggests that MEF2
levels are limiting for quantitative transcriptional output from the
MLC2slow promoter during skeletal muscle regeneration and
that the MLC2slow promoter does not seem to discriminate between these
two MEF2 isoforms. The additional observation from Western blots that
MEF2 protein levels are significantly decreased in the SOL
muscles compared with either the SOL+ or EDL muscles also suggests that innervation is important for appropriate regulation of MEF2 levels in
adult skeletal muscle.
In contrast to the regulation of other skeletal muscle specific
contractile protein genes (7, 15, 16, 44, 46), neither the E-box site
nor one of the basic helix loop helix myogenic regulatory proteins,
myogenin, was important for expression of the MLC2slow
promoter. This is consistent with the lack of MLC2slow gene
expression in myotubes at times when expression of the myogenic regulatory factors are high (18). However, recent in vitro
work has elegantly shown that the basic helix loop helix myogenic
regulatory factors (myoD and myogenin) can cooperate with MEF2 proteins
and transactivate muscle specific genes (47). Therefore, it was somewhat surprising in this study that MEF2 overexpression could only
enhance transcription in SOL+ muscles through the MEF2 site and that
overexpression of myogenin did not enhance MLC2slow promoter activity.
These results suggest that within the MLC2slow promoter, myogenin (and perhaps the other basic helix loop helix proteins) is not
transcriptionally important for either induction or high level expression.
Results from this study have implicated the CACC box of the
MLC2slow promoter as a dominant site through which the slow
nerve mediates expression. The identification of the CACC box as a
repressor site within the MLC2slow promoter was a surprise
because mutational studies of the CACC box within the SURE element of
the TnIslow gene suggested that this site acted as an
activator in slow skeletal muscle of transgenic mice (48). However,
more recent in vitro transfection studies showed that
mutation of the SURE element CACC box only resulted in a slight
decrease in promoter activity (7). These contrasting results between
transgenic mice and in vitro transfection studies suggests
that the CACC box of the SURE element may provide different
transcriptional functions depending on the cellular environment or
developmental stage. Whether the CACC box of the MLC2slow
promoter and the CACC box of the TnIslow element share
common roles in regulating slow nerve specific expression in adult
animals is not clear at this time.
It was not surprising, however, that a repressor element was identified
within the rat MLC2slow promoter. Previous reports had noted
that the rat MLC2slow gene had an upstream repressor site
(HF3) that when mutated resulted in up-regulation in nonmuscle tissues
(49). In addition, two separate repressor elements have been identified
in the chicken MLC2slow promoter: the CSS and NMS sites (50). These two
sites within the chicken gene share a common sequence motif, GAAG/CTTC,
which is associated with the repressor activity. Whereas the HF3 site
of the rat MLC2slow gene does contain a GAAG sequence, the
CACC box of the same gene does not. This suggests that the use of the
CACC box within the MLC2slow promoter may be critical for
fiber type specificity in skeletal muscle, whereas the HF3/CSS/NMS type
site may be important for providing striated muscle restriction.
The induction of slow contractile protein isoforms in response to slow
innervation or low frequency chronic stimulation in vivo has
been well characterized (3, 5, 51). These studies have contributed to
the concept of a coordinated pattern of isoform gene expression and
have raised the possibility of a shared mechanism of gene regulation.
Within the last 4 years, slow nerve-dependent regions of
the MLC1slow, TnIslow, and MLC2slow genes have
been identified (7-10). Therefore, we compared the DNA sequences of previously identified slow muscle specific regions of slow contractile protein genes (Fig. 7). What is apparent
is that each of these regions contains a CACC box site with a MEF2-like
site in close approximation, within 50 base pairs (33, 34, 53-55).
Although speculative, this raises the possibility that the two-step
model for slow nerve-dependent regulation of the
MLC2slow promoter, proposed in this paper, may be shared by
other slow isoform genes. Future experiments are required to test the
applicability of this two-step model to other slow isoform genes.

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Fig. 7.
DNA motif similarities among slow muscle/slow
nerve specific regions of mammalian contractile protein genes. DNA
sequence for the slow muscle/slow nerve regulatory regions of rat
MLC2slow (42), human MLC2slow (GenBankTM
accession number Z15030; Ref. 41), rat TnIslow (39), human
TnIslow (25), mouse MLC1slow (44), mouse
TnCslow (26), and rat slow/ -myosin heavy chain
(slow/ MHC) (43) genes are presented with the CACC box
and MEF2-like sequences in boldface and
underlined. As can be seen, each region contains a CACC box
and MEF2-like site within 50 bp of each other.
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