1 Department of Biochemistry, School of Medicine, 2 Department of Biomedical Sciences, School of Veterinary Medicine, and 3 Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, Missouri 65211
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ABSTRACT |
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We examined the functional role
of distinct muscle-CAT (MCAT) elements during non-weight-bearing (NWB)
regulation of a wild-type 293-base pair -myosin heavy chain
(
MyHC) transgene. Electrophoretic mobility shift assays (EMSA)
revealed decreased NTEF-1, poly(ADP-ribose) polymerase, and Max binding
at the human distal MCAT element when using NWB soleus vs. control
soleus nuclear extract. Compared with the wild-type transgene,
expression assays revealed that distal MCAT element mutation decreased
basal transgene expression, which was decreased further in response to
NWB. EMSA analysis of the human proximal MCAT (pMCAT) element
revealed low levels of NTEF-1 binding that did not differ between
control and NWB extract, whereas the rat pMCAT element displayed robust
NTEF-1 binding that decreased when using NWB soleus extracts.
Differences in binding between human and rat pMCAT elements were
consistent whether using rat or mouse nuclear extract or in vitro
synthesized human TEF-1 proteins. Our results provide the first
evidence that 1) different binding properties and likely
regulatory functions are served by the human and rat pMCAT elements,
and 2) previously unrecognized
MyHC proximal promoter
elements contribute to NWB regulation.
skeletal muscle hypertrophy; skeletal muscle atrophy; fiber-type transitions; chloramphenicol acetyltransferase
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INTRODUCTION |
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ADULT MOUSE HINDLIMB
skeletal muscles express four major myosin heavy chain (MyHC) isoforms
(fast IIb, IIx/d, IIa, and slow type I) whose differential expression
pattern has contributed to the broad classification scheme that
distinguishes four primary fiber types termed fast type IIb, IIx/d,
IIa, and slow type I (or ). Each MyHC isoform is thought to serve a
specific physiological role; therefore, variation in the proportion and
spatial arrangement of each fiber type contributes to the biochemical
and functional specialization of each muscle. The notion that each MyHC
serves a physiological role is supported by the classic findings that actin-activated myosin ATPase and unloaded shortening velocity (Vmax) are highly correlated to the amount and
type of native isomyosin or MyHC comprising a given muscle or muscle
fiber (2). For example, type I fibers primarily populate
slow-twitch muscles, rely on oxidative metabolism, and express the
MyHC, which is highly efficient in energy utilization while
maintaining tension. Thus slow-twitch muscles are primarily used in
chronic activities such as posture maintenance and for sustained
locomotor activity. On the other hand, fast type IIb and IIx/d fibers
are used for high-force burst activities, primarily populate
fast-twitch muscles, rely on glycolytic energy production, and are less
efficient in energy utilization while maintaining tension (4,
36).
Once established, the adult skeletal muscle phenotype is not static but
instead retains the ability to adjust to variations in load bearing and
contractile usage patterns, resulting in profound adaptations in
morphology, phenotype, and contractile properties (1, 4).
The removal of body loading in the microgravity environment of space
flight results in decreased bone density, a marked degree of muscle
atrophy, and an altered protein phenotype that correlates with a
slow-to-fast change in contractile and metabolic properties for both
rodents and humans alike (1, 10, 11, 14). Likewise,
qualitatively comparable results have been obtained from animal studies
by using a rodent ground-based model of simulated microgravity imposed
by hindlimb unloading [non-weight bearing (NWB)] (1, 10, 11,
14). In addition to altered muscle mass, strength, and
endurance, alterations in the pattern of motor nerve activity have been
reported (3, 34). Thus it is not surprising that
chronically innervated postural muscles such as the slow-twitch soleus
are most susceptible to the effects of a microgravity or simulated
microgravity environment. In agreement with the aforementioned
findings, our studies on the NWB mouse soleus muscle have documented a
loss in mass, a histochemical slow-to-fast fiber type shift, and a
decrease in endogenous MyHC mRNA expression (29).
Transcriptional regulation is a fundamental mechanism by which
adult-stage skeletal muscle phenotype and its adaptation are controlled. To identify regulatory elements that control MyHC gene
transcription during NWB activity, we have performed a transgenic deletion and mutational analysis of the
MyHC promoter. An expression analysis of transgenes comprised of either 5,600 or 600 base pairs (bp)
of wild-type
MyHC promoter revealed that expression had significantly decreased in response to NWB; however, this response was
found to be substantially blunted when the
MyHC proximal promoter
control region (
300/
170; Fig.
1A) distal muscle-CAT (dMCAT),
C-rich, and proximal MCAT (pMCAT) elements were simultaneously mutated
(29). Further analysis of transgenes comprising either 350 or 293 bp of wild-type
MyHC promoter revealed that transgene
350wt, which contains a negative element at its 5'-terminal end, did
not express under basal or malleable (NWB or functional overload) conditions, whereas transgene
293wt was unexpectedly upregulated in
response to NWB activity (30). The latter observation was of particular importance because it revealed that the
600/
294 region of the
MyHC promoter contained a NWB responsive element(s), a
regulatory role possibly served by the negative (
350/
294) element.
Our subsequent biochemical analysis of this sequence led to the
elucidation of the first putative NWB element, termed d
NRE-S
(
332/
311), that bound two distinct proteins highly enriched in NWB
soleus (NWB-S) nuclear extract (Fig. 1A; Ref. 30).
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Our current questions regarding MyHC NWB regulation arise from two
intriguing observations obtained from our above-cited transgenic
analysis. First, the loss of normal NWB regulation of wild-type
transgene
293wt occurred when the upstream NWB-responsive region
(
600/
294, containing d
NRE-S) was eliminated. Thus it is
conceivable that deletion of this NWB-responsive region may have
disrupted critical interactions between this element and those elements
within the
MyHC control region. In support of this notion, numerous
studies using transgenic mice have shown that distinct regulatory
modules (enhancers) and/or individual cis-acting elements are required
to accurately duplicate diverse aspects of endogenous muscle-specific
gene expression (9, 13, 16, 20, 25, 26, 32). Second, the
partial loss of NWB regulation of the 5,600-bp
MyHC promoter
occurred when the
MyHC control region (
300/
170) dMCAT, C-rich,
and pMCAT elements were simultaneously mutated, indicating that
these elements, or some combination of these elements, may provide key
regulatory contributions necessary for complete NWB responsiveness.
Because previous work has shown the involvement of MCAT elements in
mediating both muscle-specific and inducible (muscle loading and
hormonal stimuli) gene expression, they represent likely candidate
elements for a regulatory role during NWB (6, 23, 24, 28,
44). Furthermore, an extensive electrophoretic mobility shift
assay (EMSA) analysis to determine whether MCAT elements might be
involved in NWB regulation has not been investigated. Therefore, one
aim of this study was to investigate by EMSA analysis the potential
contribution of the distal and proximal MCAT elements to the regulation
of
MyHC expression in response to NWB activity. Another goal was to
determine the mechanistic basis underlying the unexpected upregulation
of transgene
293wt after NWB. Thus we performed a transgenic
mutagenesis analysis to test the hypothesis that the strong positive
dMCAT element drives upregulation of transgene
293wt in response to
NWB in the absence of the upstream NWB-responsive region (Fig.
1A).
Our transgenic analyses demonstrate conclusively that the dMCAT element
underlies the atypical NWB response of transgene 293wt and,
importantly, reveal that previously unidentified sequence(s) located
downstream from the dMCAT element participate in NWB regulation. In
addition, our EMSA analyses suggest that the dMCAT element contributes,
at least in part, to NWB regulation of the
MyHC gene by incurring
decreased binding of transcriptional activator proteins, thus allowing
negative regulation to proceed in the presence of other intact
NWB-responsive elements. As regards the proximal MCAT element, our EMSA
results indicate that the rat pMCAT element, but not the human pMCAT
element, is likely involved in NWB regulation. Our EMSA analyses also
reveal that the human and rat
MyHC pMCAT elements display very
different nuclear protein binding properties that are likely due to
differences in nucleotide composition, because these differences were
consistently observed whether using mouse or rat nuclear extract or in
vitro synthesized human TEF-1 proteins. These data provide the first
evidence that combinatorial interactions are required to confer
complete NWB regulation of the human
MyHC gene and that specific
combinatorial interactions may differ with species.
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EXPERIMENTAL PROCEDURES |
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Transgenes and site-directed mutagenesis.
Wild-type transgene 293wt consists of 293 bp of human
MyHC
promoter sequence and 120 bp of 5'-untranslated region (UTR; includes
exon 1), fused to the 5'-end of the bacterial chloramphenicol acetyltransferase (CAT) gene (Fig. 1B). The distal MCAT
element within the human
293 promoter was mutated within the plasmid p
293CAT using the QuikChange site-directed mutagenesis kit
(Stratagene) according to the manufacturer's instructions. Detailed
procedures for PCR-mediated incorporation of mutant distal MCAT
sequence have been described elsewhere (44). Briefly,
complementary oligonucleotide primers harboring mutations (bold
lowercase) within the distal MCAT were designed as follows:
5'-GCATAGTTAAGCCAGCCAGCTGcGtctTagGAGGCCTGGCCTGGG-3. Bp substitutions were incorporated at nucleotides determined by our
previous diethylpyrocarbonate interference footprinting to be crucial
DNA protein contact sites (45). Unintentional
transcription factor recognition sites were not created by these
mutations as assessed by cross-referencing the mutated primers against
the Eukaryotic Transcription Factor database (TFD) available on the Wisconsin Package [version 10.0; Genetics Computer Group (GCG), Madison, WI]. Successful incorporation of the mutation was verified via automated sequencing of both strands (Applied Biosystems, model
377). Wild-type (
293wt) and mutant (
293Mm) transgenes were
isolated and purified as described previously (44).
Transgenic mice. Transgenic mice were generated by microinjection of purified transgene DNA into pronuclei of zygotes as described previously (40). Transgenic founder mice were identified by Southern blot analysis, and copy number was then estimated (47). Transgene-positive offspring were identified by PCR amplification by using primers specific for the CAT gene (47). All lines were maintained in a heterozygous state by backcrossing to the nontransgenic FVB/N parental mouse strain.
Animal care and NWB procedure.
The Animal Care Committee for the University of Missouri-Columbia
approved the NWB procedure used in this study, and the NWB mice were
housed in an Association for the Assessment and Accreditation of
Laboratory Animal Care International-accredited animal facility. All
animals were provided with food and water ad libitum and were housed at
room temperature (24°C) with a 12:12-h light-dark cycle in standard
rodent cages (control animals) or in cages designed for head-down tilt
hindlimb suspension (NWB) as described in detail previously
(29). Adult female wild-type 293 (
293wt) transgenic mice from line 2 are used in this study to show 1) control
vs. NWB body and muscle weight and 2) basal and NWB
expression levels (Tables 1 and 2). Basal
and NWB expression levels for transgenic mice lines 99, 96, and 5 have
been reported previously (30) and are used here to
facilitate comparisons against basal and NWB expression data collected
for transgenic mice carrying mutant transgene
293Mm (Table
2). Adult female
293Mm transgenic mice from lines 4, 6, 7, and 9 (
22 g) were assigned to one of two groups:
1) a NWB group that used 2 wk of hindlimb suspension to impose NWB conditions (NWB; n = 8), and 2) a
group that served as cage ambulatory controls for the NWB group
(control; n = 8). After a 2-wk experimental period,
both cage control and NWB mice were anesthetized and weighed, and their
control soleus (CS) and NWB-soleus (NWB-S) muscles were collected for
further study (Tables 1 and 2). At the same time, we collected
gastrocnemius muscle (
123 mg) from 40 adult (
22g) control mice to
be used for the isolation of nuclear extract. The gastrocnemius muscle
was used for the isolation of nuclear extract because of the small size of the CS (7-8 mg) muscle. All control and NWB muscles were
trimmed clean of fat and connective tissue, weighed, and stored at
80°C until assayed for CAT-specific activity (CS and NWB-S) or used for the isolation of nuclear extract (control gastrocnemius). All mice
designated for terminal sample collection were anesthetized by using
2.5% avertin at a dosage of 0.017 ml/g of body wt. Mice were
euthanized by cervical dislocation while under anesthesia.
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CAT assays. CAT assays were performed as previously described (41). Muscle extracts were prepared from transgenic tissues by using a glass tissue homogenizer to disrupt tissues in 250 mM Tris · HCL (pH 7.8) and 5 mM EDTA. All muscle extracts were prepared from frozen tissue, and each n value represents the number of pooled soleus muscles from one mouse. The protein concentration of the extracts was determined by the method of Bradford (5). Muscle extracts were heated at 65°C for 10 min, followed by centrifugation at 10,000 g for 20 min. Because different transgenic lines exhibit inherently different transgene expression levels, it was necessary to use different amounts of tissue extract and variable incubation times so that the CAT enzyme activities could be determined within a linear range (30% conversion) as described previously (30, 39, 41, 48). The percent conversion of [14C]chloramphenicol to the acetylated form was quantified by using a PhosphorImager (Storm860) with ImageQuant version 5.1 software. Direct comparisons between and within transgenic lines representing both control and experimental groups (CS and NWB-S) were facilitated by presenting the data as specific CAT activity (picomoles per microgram of protein per minute) (Table 2).
Preparation of nuclear protein extract from adult skeletal muscle. Nuclear extracts (NE) were isolated from adult rat CS and NWB-S muscle and from mouse control gastrocnemius muscle as previously described (30). Three independent batches of CS, NWB-S, and control gastrocnemius nuclear extract were isolated and used in EMSA analysis and yielded the same results. Protein concentration was determined according to Bradford (5).
EMSA.
All oligonucleotide probes used in this study are listed in Table
3. EMSAs were carried out as previously
described (30, 43, 44). The double-stranded human and rat
proximal MCAT oligonucleotide probes were labeled by fill-in reaction
using Klenow fragment pf Escherichia coli DNA polymerase I
(Stratagene, La Jolla, Ca) and [-32P]dCTP
(3,000Ci/mmol). The human distal MCAT oligonucleotide probe was
end-labeled by T4 polynucleotide kinase (New England Biolabs, Beverly,
Ma) and [
-32P]dATP (6,000Ci/mmol). All probes were
purified by polyacrylamide gel electrophoresis before use in EMSAs.
Binding reactions were performed by using either rat CS or NWB-S
nuclear extract (dMCAT probe = 4.0 µg, pMCAT probe = 5.0 µg) or mouse control gastrocnemius nuclear extract (pMCAT probe = 5.5 µg) and 20,000 cpm of labeled probe for 20 min at room
temperature in a 25-µl total volume. Where indicated, binding
reactions contained 1 µl (rat pMCAT element) or 5 µl (human pMCAT
probe) of in vitro-translated human NTEF-1 protein in place of muscle
nuclear extract. The binding reactions were resolved on a 5%
nondenaturing polyacrylamide gel at 220 volts for 2.5 h at 4°C.
Supershift EMSAs were performed by first preincubating skeletal muscle
nuclear extract with 2 µl of either preimmune serum (PI) or the
corresponding antibody for 30 min at room temperature, followed by the
addition of the 32P-labeled DNA probe. After
electrophoresis, the gels were dried, and DNA protein complexes were
visualized by autoradiography at
80°C.
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In vitro transcription/translation. In vitro-translated protein was produced by using 1 µg of TEF-1 expression plasmid in the T7 transcription/translation (TNT) rabbit reticulocyte-coupled TnT system according to the instructions provided by the manufacturer (Promega). The expression plasmid corresponded to NTEF-1 [pXJ40-TEF-1, open reading frame (ORF) of human TEF-1] (21, 51). Parallel TNT reactions were performed in the presence of [35S]methionine (Perkin Elmer). Efficient translation and expected molecular weights of the protein products were verified by resolving the radiolabeled reaction products on a sodium dodecyl sulfate-12% polyacrylamide gel (SDS-PAGE). Equivalent reactions of lysate not programmed with plasmid DNA were used as negative controls (unprogrammed lysate, UL).
Antibodies. The antibodies used in this study were as follows: NTEF-1, mouse polyclonal antibody raised against amino acids 86-199 of human TEF-1 (BD Transduction Laboratories); poly(ADP-ribose) polymerase (PARP), rabbit polyclonal antibody raised against full-length human PARP (Boehringer Mannheim); and Max, rabbit polyclonal antibody raised against full-length mouse Max (Upstate Biotechnology). All antibodies listed hereafter were purchased from Santa Cruz Biotechnology and include MyoD, rabbit polyclonal antibody raised against full-length (amino acids 1-318) mouse MyoD; myogenin, rabbit polyclonal antibody raised against full-length (amino acids 1-225) rat myogenin; upstream stimulatory factor-1 (USF-1), rabbit polyclonal antibody raised against carboxy-terminal amino acids 291-310 of human USF-1; E2A.E12, rabbit polyclonal raised against a peptide in the carboxy terminus of human E47 and corresponding to amino acids 422-439 of human E12; and HEB, rabbit polyclonal antibody raised against a peptide in the carboxy-terminal domain of human HEB (HTF 4).
Statistical analysis.
All statistical analyses were performed by using SPSS Graduate Pack
10.0 program (SPSS, Chicago, IL). A Levene's test for equality of
variances was performed, followed by a two-tailed independent-samples t-test used to assess differences
between group means. Where the Levene's test was rejected
(significance 0.05), the separate variance t-test
for means was used, where equal variances were not assumed. All data
are reported as means ± SE. The lowest significance level
accepted was P < 0.05.
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RESULTS |
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The formation of a highly specific and enriched low-mobility
complex at the MyHC dMCAT decreases after 2 wk of NWB.
To determine whether the binding properties of the
MyHC dMCAT
element differed between control and NWB conditions, we performed a
direct and competition EMSA analysis using either CS or NWB nuclear
extract. Binding reactions containing the 32P-labeled
wild-type dMCAT probe (Table 3) and either CS or NWB-S nuclear extract
showed the formation of multiple binding complexes termed low-mobility
complex (LMC), intermediate-mobility complex (IMC), and high-mobility
complex (HMC) (Fig. 2). The formation of
a LMC when using NWB-S nuclear extract was substantially reduced compared with the LMC formed when using CS nuclear extract, whereas those comprising the HMC increased (Fig. 2, lane 1 vs.
6). The addition of 100-fold molar excess cold wild-type
MyHC dMCAT probe to binding reactions containing either CS or NWB-S
nuclear extract completely abolished complex formation, indicating that
these complexes are specific (Fig. 2, lanes 2 and
7). Interestingly, the addition of 100-fold molar excess
cold cTnT MCAT1 probe [an element previously shown to form LMC, IMC,
and HMC binding complexes (28)] to binding reactions
containing either CS or NWB-S nuclear extract prevented IMC and HMC
formation and partially competed away the LMC formation (Fig. 2,
lane 3 vs. 8). The addition of 100-fold molar
excess cold muscle creatine kinase (MCK) transcriptional regulatory
factor x (Trex) element [an element that has relative sequence
similarity to consensus MCAT site but that is shown not to bind TEF-1
(12)] to binding reactions containing either CS or NWB-S
nuclear extract did not effectively compete for complex formation (Fig.
2, lane 4 vs. 9). Because the
immediate 5'-flanking region of the
MyHC dMCAT element contains a
consensus E-box element, we examined whether E-box binding proteins
were components of the LMC. The addition of 100-fold molar excess
high-affinity MCK E-box as competitor to binding reactions containing
either CS or NWB-S nuclear extract did not alter complex formation
(Fig. 2, lane 5 vs. 10). These data show that
specific binding complex formation at the dMCAT element differed when
using CS vs. NWB-S nuclear extract, and this difference was
characterized by a striking reduction in LMC formation only when using
NWB extract. Also, known muscle regulatory factors (MyoD, myogenin)
previously shown to bind the MCK E-box are likely not components of the
specific binding complex formed at the
MyHC dMCAT element.
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dMCAT element core nucleotides are required for DNA-protein binding
complex formation.
To determine whether different nucleotides within the 21-bp MyHC
dMCAT element interact with CS vs. NWB-S nuclear protein, we performed
competition EMSA analysis using scanning mutagenesis. In these
experiments, we introduced nucleotide substitutions 2 bp at a time,
starting within the immediate 5'-flanking region (E-box) and extending
throughout the core MCAT element and its immediate 3'-flanking region
(Fig. 3, A and B).
The resulting unlabeled oligonucleotides were then added in 100-fold
molar excess to binding reactions containing either CS or NWB-S nuclear
extract or the 32P-labeled wild-type dMCAT probe (Fig.
3A). As shown previously, the addition of excess wild-type
MyHC dMCAT probe to binding reactions containing either CS or NWB-S
nuclear extract completely abolished the formation of all binding
complexes (Fig. 3A, lanes 2 and 12).
Similarly, all binding complexes were effectively competed away by the
addition of either dMCAT mut-1, mut-2 (mutations within E-box), or
mut-7 (3'-flanking region) to binding reactions containing either CS
(Fig. 3A, lane 1 vs. 3, 4,
and 9) or NWB-S (Fig. 3A, lane 11 vs.
13, 14, and 19) nuclear extract.
Importantly, mutant MCAT probes carrying nucleotide substitutions
within the core MCAT element (mut-3, 4, 5, and 8) did not act as
effective competitors of complex formation when added to binding
reactions containing CS (Fig. 3A, lane 1 vs.
5-7 and 10) or NWB-S (Fig.
3A, lane 11 vs. 15, 16,
18, and 20) nuclear extract. Interestingly, the
addition of dMCAT mut-6 probe to binding reactions containing CS (Fig. 3A, lane 1 vs. 8) or NWB-S (Fig.
3A, lane 11 vs. 18) nuclear extract partially competed for LMC formation, but not IMC or HMC. These data
show that nucleotides comprising the core
MyHC dMCAT element are
critical for the formation of all binding complexes when using either
CS or NWB-S nuclear extract (Fig. 3C).
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NTEF-1, PARP, and Max comprise the LMC formed at the MyHC distal
MCAT element.
Although we have shown previously that NTEF-1, PARP, and Max comprise
the LMC formed at the dMCAT element when using CS nuclear extract, it
is possible that different nuclear proteins form the LMC under NWB
conditions (44). Thus we performed antibody EMSAs using
polyclonal antibodies that recognize NTEF-1, PARP, or Max to determine
whether these proteins comprised the LMC formed at the
MyHC dMCAT
element when using NWB-S nuclear extract or whether other proteins were
components of this complex. The specific binding complexes formed at
the 32P-labeled human
MyHC dMCAT element when reacted
with NWB-S nuclear extract were not altered by preincubation with PI
(Fig. 4, lane 2 vs.
3). The addition of polyclonal
NTEF-1 antibody to binding reactions containing NWB-S nuclear extract
supershifted only the top band of the IMC doublet and the prominent HMC
band, whereas the LMC was partially abolished (Fig. 4, lane
2 vs. 4). Interestingly, the addition of either
polyclonal PARP or Max antibody to binding reactions using NWB-S
extract essentially immunodepleted the LMC, which resulted in an
embellishment of the top band of the IMC, which entirely comprises
NTEF-1 protein (Fig. 4, lane 2 vs. 5 and
6). When all combinations of the three polyclonal antibodies were added to binding reactions using NWB-S nuclear extract, the LMC
was completely immunodepleted (Fig. 4, lane 2 vs.
7). The preincubation of binding reactions containing either
CS or NWB-S nuclear extract with antibodies recognizing other
E-box-binding bHLH (basic helix-loop-helix) and bHLH-Zip proteins
(MyoD, myogenin, E2A, HEB, and USF) did not alter
MyHC dMCAT element
binding complex formation or mobility (unpublished observation). These
data support several noteworthy conclusions: 1) proteins
antigenically related to NTEF-1, PARP, and Max comprise the LMC formed
at the human
MyHC dMCAT element; 2) protein
antigenically related to NTEF-1 likely constitutes the prominent HMC
band and the top band of the IMC doublet; and 3) decreased binding of
NTEF-1, PARP, and Max at the dMCAT element under NWB conditions may, in
part, contribute to NWB decreases in
MyHC gene expression.
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Morphological changes after NWB.
Body and soleus muscle weight data reported for transgenic mice
harboring mutant transgene 293Mm represent pooled weights from
transgenic lines 4, 6, 7, and 9 (Table 1). Compared with control
values, 2 wk of NWB activity imposed by hindlimb suspension resulted in
a significant decrease in the body wt of
293Mm (
15.8%) and
293wt (
19.3%) transgenic mice (Table 1). Similarly, significant decreases were measured in both absolute (
293Mm =
27.1%;
293wt =
31.9%) and normalized (
293Mm =
17.1%;
293wt =
16%) NWB-S muscle weight compared with control
values (Table 1). These data are consistent with our previous findings
showing that a 2-wk NWB treatment produces statistically significant
decreases in both mouse body and muscle mass (29, 30).
Mutation of distal MCAT element restores NWB regulation and alters
basal slow muscle expression of wild-type transgene 293wt.
We examined the hypothesis that the dMCAT element is responsible for
the unexpected upregulation of transgene
293wt in the absence of the
upstream NWB-region by studying multiple independent lines of two
distinct classes of transgenic mice (Fig. 1B). The first
class of transgenic mouse carried a wild-type transgene comprised of
293 bp of human
MyHC promoter fused to the CAT reporter gene
(
293wt). The second class carried the same transgene, except that
the highly conserved dMCAT element was mutated (
293Mm). Importantly,
if this mutation restores NWB regulation, we will have elucidated the
mechanistic basis underlying the atypical response of transgene
293wt after NWB and revealed that previously unidentified
sequence(s) located downstream of the dMCAT element participate in NWB regulation.
Dissimilar binding of rat nuclear extract between human and rat
MyHC pMCAT elements suggests a species difference in regulatory
roles.
The pMCAT element (
210/
203) resides downstream from the dMCAT
element (
290/
284) and has been shown to play a regulatory role in
the downregulation of injected rat
MyHC reporter plasmids in the
soleus muscle of spinal cord isolated rats (19). Because spinal cord isolation and NWB are associated with decreased soleus muscle
MyHC gene expression, we have used an EMSA analysis to evaluate whether the human pMCAT element (
210/
203) serves a regulatory role in response to NWB. Incubation of
32P-labeled human pMCAT element with rat CS nuclear extract
resulted in the formation of three binding complexes (termed C1, C2,
C3) that did not change in intensity when rat NWB-S nuclear extract was
used (Fig. 5A, lanes 1 and
6). The addition of 100-fold
molar excess cold wild-type human pMCAT probe to binding reactions
containing rat CS nuclear extract competed away complex C2 only,
whereas competition with 100-fold molar excess of mutant human pMCAT
(pMCATm) element did not compete away complex C2 but did compete away
complexes C1 and C3, indicating that only complex C2 represents
specific binding (Fig. 5A, lanes 2 and
3). When binding reactions containing rat CS or NWB-S
nuclear extract were preincubated with PI, complex formation at the
human pMCAT element was not altered (Fig. 5A, lanes
1 vs. 4 and 6 vs. 7), whereas
addition of polyclonal NTEF-1 antibody led to a supershift (SS) of only
complex C2 (Fig. 5A, lanes 1 vs. 5 and
6 vs. 8). These data indicate that a nuclear protein antigenically related to NTEF-1 forms the low-intensity C2
specific binding complex at the human pMCAT element. However, because
the intensity of this complex did not differ between control and NWB
conditions, it is unlikely that the human pMCAT element plays a
regulatory role under NWB conditions.
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Use of mouse nuclear extracts or in vitro-synthesized human TEF-1
proteins confirms a species difference in binding properties of TEF-1
at the human vs. rat MyHC pMCAT elements.
To clarify whether the dissimilar binding of nuclear TEF-1 at the human
vs. rat pMCAT element was due to differences in nucleotide composition
and not species difference in nuclear TEF-1 protein, we performed an
EMSA analysis using either mouse nuclear extract or in vitro
synthesized human TEF-1 protein. EMSA analysis of binding reactions
containing mouse control gastrocnemius nuclear extract and a
32P-labeled human pMCAT element revealed three binding
complexes (C1, C2, C3; Fig. 6) that were
of very low intensity like those obtained when using rat nuclear
extract. When 100-fold molar excess cold wild-type pMCAT element was
added to the binding reaction, only complex C2 was abolished (Fig. 6,
lanes 1 vs. 2). The addition of PI to the binding reaction
did not alter the complex formation (Fig. 6, lane 3),
whereas addition of polyclonal NTEF-1 antibody resulted in a SS of
complex C2 only (Fig. 6, lane 4). Similarly, three binding
complexes formed at the 32P-labeled rat pMCAT element when
mouse control gastrocnemius nuclear extract was used, and only complex
C2 was competed away with the addition of 100-fold molar excess
unlabeled wild-type pMCAT probe (Fig. 6, lane 5 vs.
6). In contrast, formation of complex C2 at the rat pMCAT
element was highly enriched compared with the formation of complex C2
when using the human pMCAT element (Fig. 6, lane 1 vs.
5). Preincubation of binding reactions with PI did not alter complex formation at the rat pMCAT element (Fig. 6, lane 7),
whereas addition of polyclonal NTEF-1 antibody resulted in a SS of
complex C2 (Fig. 6, lane 8).
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DISCUSSION |
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The genetic regulatory programs that control the adaptation of
adult-stage skeletal muscle phenotype in response to decreased weight
bearing are complex and remain incompletely understood. To gain insight
into this process, our current study utilizes a transgenic mutagenesis
and EMSA analysis to investigate whether two distinct MyHC proximal
promoter MCAT elements (dMCAT and pMCAT) serve regulatory roles during
NWB. Our transgenic study provides the first in vivo evidence that:
1) the dMCAT element is responsible for the unexpected
upregulation of human transgene
293wt in response to NWB, and
2) regulatory sequence(s) located downstream from the dMCAT
element are sufficient to direct NWB responsiveness. Our EMSA
experiments are the first to reveal the existence of a species (human
vs. rat) difference in
MyHC pMCAT element binding of NTEF-1 under
both control and NWB conditions, suggesting a possible species
divergence in functional roles served by this element.
Transgenic mutagenesis analysis reveals that the dMCAT element is
required to achieve high-level basal expression of transgene 293wt.
MCAT regulatory elements are frequently found in the control region of
numerous muscle genes and have been shown to mediate muscle-specific
and inducible gene transcription. In our previous transgenic
mutagenesis study, we found that mutation of the dMCAT element did not
abolish induction of transgene
293Mm expression in the functionally
overloaded adult plantaris muscle; however, basal expression levels of
this transgene were significantly reduced (44). Likewise,
our current transgenic analysis shows that mutation of the dMCAT
element significantly decreases transgene
293Mm expression in the
adult CS muscle, indicating that in the context of a 293-bp
MyHC
promoter, this element acts as a strong positive cis-acting sequence
(Table 2).
Transgenic mutagenesis analysis reveals that the dMCAT element is
responsible for the increase in transgene 293wt expression after
NWB.
Our observation that the dMCAT element acts as a strong positive
cis-acting sequence offers insight into how the dMCAT element mediates
the uncharacteristic upregulation of transgene
293wt in response to
NWB. In this regard, it is plausible that in the absence of the
upstream NWB-responsive region (
600/
294; Fig. 1A),
interactions required for NWB downregulation would be disrupted, thereby allowing the dMCAT element (
290/
284) to exert a dominant positive effect on transgene
293wt expression in response to NWB.
The transgenic results presented here support this notion by showing
NWB decreased expression of a transgene containing a mutated dMCAT
element (
293Mm). In fact, decreases in transgene
293Mm expression
were similar to those previously reported by us for larger (5,600 or
600 bp)
MyHC promoter/transgenes (Table 2; Ref. 29), decisively
demonstrating in vivo that the dMCAT element is responsible for the
unexpected upregulation of wild-type transgene
293wt in response to NWB.
EMSA analysis suggests a role for the dMCAT element during NWB
regulation of the MyHC gene.
In this study, we have used EMSA analysis to investigate how a strong
positive cis-acting regulatory element might function during a
physiological process that normally results in negative regulation. One
conceivable mechanism by which a strong positive cis-acting element can
contribute to decreased expression levels of a given gene is by
incurring changes in the type and/or amount of transcription factor
binding. As concerns change in the type of transcription factor
binding, our current antibody EMSA experiments show that the proteins
interacting at the dMCAT element do not differ between control and NWB
conditions. Specifically, antibody EMSA analysis determined that
proteins antigenically related to PARP, NTEF-1, and Max comprised the
LMC that formed at the
MyHC dMCAT element when using either control
or NWB nuclear extract (Fig. 4). This analysis also showed that other
factors, such as the E-box binding proteins MyoD, myogenin, E2A, HEB,
and USF, were not components of the binding complexes formed at the
dMCAT element (data not shown). Furthermore, we showed by EMSA scanning mutagenesis that no difference existed in protein-DNA interactions at
the dMCAT element because the same nucleotides comprising only the core
MCAT element were involved in both control and NWB nuclear protein
binding (Fig. 3).
Previously unidentified NWB element(s) reside downstream of the
dMCAT element.
In addition to determining that the dMCAT element directs the
uncharacteristic upregulation of transgene 293wt, our transgenic analysis showing downregulation of transgene
293Mm expression after NWB provides the first in vivo evidence that previously unidentified NWB responsive element(s) reside downstream from the dMCAT
element (Fig. 1A). Candidate elements that may fulfill this
role that are located downstream from the dMCAT element (
290/
284) are the A/T-rich (
269/
258), C-rich (
242/
231), pMCAT
(
210/
203, examined herein), and E-box/NFAT (
179/
171) elements.
Our previous transgenic analysis revealed that the independent mutation
of the A/T-rich element in the context of a 293-bp
MyHC
promoter/transgene resulted in the complete loss of expression in all
21 independent lines examined (CAT = 15 lines, luciferase = 6 lines) (44, 46). Similarly, E-box/NFAT mutation in 11 independent lines resulted in 4 lines that did not express and 7 lines
whose basal expression levels were very low-rendering results, with NWB
being unreliable (unpublished observation). Interestingly, a molecular
model that accounts for slow fiber-specific gene expression and
involves both A/T-rich and NFAT elements has been recently proposed.
This model suggests that sustained elevations of intracellular calcium coactivate the calcineurin and calmodulin-dependent protein kinase signaling pathways and the subsequent transcriptional activation of
slow fiber genes by various members of the myocyte enhancer factor-2
(MEF-2) and/or nuclear factor of activated T-cell (NFAT) transcription
factor families (31, 33, 49, 50). Because NWB has been
shown to result in a decrease in total muscle electrical activity and a
slow-to-fast fiber-type transition (3, 34), it is
conceivable that a decrease in nuclear protein binding at the A/T-rich
and/or NFAT elements may contribute to reduced
MyHC expression under
NWB conditions. In fact, we have previously reported reduced levels of
NFAT protein binding at the
MyHC NFAT element (46).
Although not all agree with the proposed model of slow fiber-specific
expression (7), it will be instructive to reevaluate the
involvement of the A/T-rich and E-box/NFAT elements in regulating
MyHC expression during the NWB induced slow-to-fast fiber-type by
using larger
MyHC promoter/transgenes.
Divergence in pMCAT element sequences underlies species difference
in NTEF-1 binding.
The pMCAT element (210/
203) is located downstream from the dMCAT
element and represents a viable element that could confer NWB
regulation. Previous EMSA analysis has revealed robust binding at this
element when reacted with nuclear extracts isolated from a variety of
differentiated myogenic cell lines (37, 38). In addition,
mutation of this element within a 600-bp
MyHC promoter representing
several species (rat, human, rabbit, mouse) resulted in a significant
reduction and/or abolished
MyHC reporter gene expression when
assayed in the in vitro context (37, 38). The pMCAT
element has also been shown to confer inducible expression of the rat
MyHC gene in response to
1-adrenergic treatment of primary rat neonatal cardiomyocytes (23, 24).
Mechanistically, the latter was shown by immunological techniques to
involve the formation of a single binding complex (termed C2) that was
comprised of TEF-1 protein. More recently, it was reported that the
pMCAT element plays a role in the downregulation of rat
MyHC
plasmids injected into the soleus muscle of spinal cord isolated rats
(19).
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ACKNOWLEDGEMENTS |
---|
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants R01-AR-41464 and R01-AR-47197 to R. W. Tsika.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: R. W. Tsika, Univ. of Missouri-Columbia, Dept. of Veterinary Biomedical Sciences and Dept. of Biochemistry, 1600 E. Rollins St., W112 VET Medicine Bldg., Columbia, MO 65211 (E-mail: tsikar{at}missouri.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 22, 2002;10.1152/ajpcell.00278.2002
Received 18 June 2002; accepted in final form 15 August 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Baldwin, KM,
and
Haddad F.
Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle.
J Appl Physiol
90:
345-357,
2001
2.
Barany, M.
ATPase activity of myosin correlated with speed of muscle shortening.
J Gen Physiol
5:
197-218,
1967.
3.
Blewett, C,
and
Elder GCG
Quantitative EMG analysis in soleus and plantaris during hindlimb suspension and recovery.
J Appl Physiol
74:
2057-2066,
1993[Abstract].
4.
Booth, FW,
and
Baldwin KM.
Muscle plasticity: energy demand and supply processes.
In: Handbook of Physiology. Exercise: Regulation and Integration of Multiple Systems. Bethesda, MD: Am. Physiol. Soc, 1996, sect. 12, chapt. 24, p. 1075-1123.
5.
Bradford, MM.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1976[ISI][Medline].
6.
Butler, AJ,
and
Ordahl CP.
Poly(ADP-Ribose) polymerase binds with transcription enhancer factor 1 to MCAT1 elements to regulate muscle-specific transcription.
Mol Cell Biol
19:
296-306,
1999
7.
Calvo, W,
Venepally P,
Cheng J,
and
Buonanno A.
Fiber-type-specific transcription of the troponin I slow gene is regulated by multiple elements.
Mol Cell Biol
19:
515-525,
1999
8.
Chen, JCJ,
Ramachandran R,
and
Goldhamer DJ.
Essential and redundant functions of the MyoD distal regulatory region revealed by targeted mutagenesis.
Dev Biol
245:
213-223,
2002[ISI][Medline].
9.
Donoghue, MJ,
Alvarez JD,
Merlie JP,
and
Sanes JR.
Fiber type- and position-dependent expression of a myosin light-chain CAT transgene detected with a novel histochemical stain for CAT.
J Cell Biol
115:
423-434,
1991[Abstract].
10.
Edgerton, VR,
and
Roy RR.
Neuromuscular adaptations to actual and simulated spaceflight.
In: Handbook of Physiology, Environmental Physiology Bethesda, MD: Am. Physiol. Soc, 1996, sect. 4, chapt. 32, p. 721-763.
11.
Edgerton, VR,
and
Roy RR.
Gravitational biology of the neuromotor systems: a perspective to the next era.
J Appl Physiol
89:
1224-1231,
2000
12.
Fabre-Suver, C,
and
Hauschka SD.
A novel site in the muscle creatine kinase enhancer is required for expression in skeletal but not cardiac muscle.
J Biol Chem
271:
4646-4652,
1996
13.
Firulli, AB,
and
Olson EN.
Modular regulation of muscle gene transcription: a mechanism for muscle cell diversity.
Trends Genet
13:
364-369,
1997[ISI][Medline].
14.
Fitts, R,
Riley DR,
and
Widrick JJ.
Physiology of a microgravity environment. Invited Review: microgravity and skeletal muscle.
J Appl Physiol
89:
823-839,
2000
15.
Giger, JM,
Haddad F,
Qin AX,
and
Baldwin KM.
In vivo regulation of the -myosin heavy chain gene in soleus muscle of suspended and weight-bearing rats.
Am J Physiol Cell Physiol
278:
C1153-C1161,
2000
16.
Grayson, J,
Williams Yu RS, YT,
and
Bassel-Duby R.
Synergistic interactions between heterologous upstream activation elements and specific TATA sequences in a muscle-specific promoter.
Mol Cell Biol
15:
1870-1878,
1995[Abstract].
17.
Gupta, MP,
Amin CS,
Gupta M,
Hay N,
and
Zak R.
Transcription enhancer factor 1 interacts with a basic helix-loop-helix zipper protein, max, for positive regulation of cardiac -myosin heavy chain gene expression.
Mol Cell Biol
17:
3924-3936,
1997[Abstract].
18.
Gupta, MP,
Kogut P,
and
Gupta M.
Protein-kinase-A dependent phosphorylation of transcription enhancer factor-1 represses its DNA-binding activity but enhances its gene activation ability.
Nucleic Acids Res
28:
3168-3177,
2000
19.
Huey, KA,
Roy RR,
Haddad F,
Edgerton VR,
and
Baldwin KM.
Transcriptional regulation of the type I myosin heavy chain promoter in inactive rat soleus.
Am J Physiol Cell Physiol
282:
C528-C537,
2002
20.
Hallauer, PL,
Bradshaw HL,
and
Hastings KE.
Complex fiber-type-specific expression of fast skeletal muscle troponin I gene constructs in transgenic mice.
Development
119:
691-701,
1993
21.
Jacquemin, P,
Hwang JJ,
Martial JA,
Dolle P,
and
Davidson I.
A novel family of developmentally regulated mammalian transcription factors containing the TEA/ATTS DNA binding domain.
J Biol Chem
271:
21775-21784,
1996
22.
Jiang, P,
Song J,
Gu G,
Slonimsky E,
Li E,
and
Rosenthal N.
Targeted deletion of the MLC1f/3f downstream enhancer results in precocious MLC expression and mesoderm ablation.
Dev Biol
243:
281-293,
2002[ISI][Medline].
23.
Kariya, K,
Farrance IK,
and
Simpson PC.
Transcriptional enhancer factor-1 in cardiac myocytes interacts with an 1-adrenergic- and
-protein kinase C-inducible element in the rat
-myosin heavy chain promoter.
J Biol Chem
268:
26658-26662,
1993
24.
Kariya, K,
Karnes LR,
and
Simpson PC.
An enhancer core element mediates stimulation of rat -myosin heavy chain promoter by an
1-adrenergic agonist and activated
-protein kinase C in hypertrophy of cardiac myocytes.
J Biol Chem
269:
3775-3782,
1994
25.
Kelly, R,
Alonso S,
Tajbakhsh S,
Cossu G,
and
Buckingham M.
Myosin light chain 3F regulatory sequences confer regionalized cardiac and skeletal muscle expression in transgenic mice.
J Cell Biol
129:
383-396,
1995[Abstract].
26.
Kelly, RG,
and
Buckingham M.
Modular regulation of the MLC1F/3F gene and striated muscle diversity.
Microsc Res Tech
50:
510-521,
2000[ISI][Medline].
27.
Knotts, S,
Rindt H,
and
Robbins J.
Position independent expression and developmental regulation is directed by the myosin heavy chain gene's 5' upstream region in transgenic mice.
Nucleic Acids Res
23:
3301-3309,
1995[Abstract].
28.
Larkin, SB,
Farrance IK,
and
Ordahl CP.
Flanking sequences modulate the cell specificity of M-CAT elements.
Mol Cell Biol
16:
3742-3755,
1996[Abstract].
29.
McCarthy, JJ,
Fox AM,
Tsika GL,
Gao L,
and
Tsika RW.
-MHC transgene expression in suspended and mechanically overloaded/suspended soleus muscle of transgenic mice.
Am J Physiol Regul Integr Comp Physiol
272:
R1552-R1561,
1997
30.
McCarthy, JJ,
Vyas DR,
Tsika GL,
and
Tsika RW.
Segregated regulatory elements direct -myosin heavy chain expression in response to altered muscle activity.
J Biol Chem
274:
14270-14279,
1999
31.
Naya, FJ,
Mercer B,
Shelton J,
Richardson JA,
Williams RS,
and
Olson EN.
Stimulation of slow skeletal muscle fiber gene expression by calcineurin in vivo.
J Biol Chem
275:
4545-4548,
2000
32.
Neville, C,
Gonzales D,
Houghton L,
McGrew MJ,
and
Rosenthal N.
Modular elements of the MLC 1f/3f locus confer fiber-specific transcription regulation in transgenic mice.
Dev Genet
19:
157-162,
1992.
33.
Olson, EN,
and
Williams RS.
Calcineurin signaling and muscle remodeling.
Cell
101:
689-692,
2000[ISI][Medline].
34.
Riley, DA,
Slocum G,
Bain JL,
Sedlak FR,
Sowa TE,
and
Mellender JW.
Rat hindlimb unloading: soleus histochemistry, ultrastructure, and electromyography.
J Appl Physiol
69:
58-66,
1990
35.
Rulicke, T,
and
Hubscher U.
Germ line transformation of mammals by pronuclear microinjection.
Exp Physiol
85:
589-601,
2000[Abstract].
36.
Schiaffino, S,
and
Reggiani C.
Molecular diversity of myofibrillar proteins: gene regulation and functional significance.
Physiol Rev
76:
371-432,
1996
37.
Shimizu, N,
Dizon E,
and
Zak R.
Both muscle-specific and ubiquitous nuclear factors are required for muscle-specific expression of the myosin heavy chain- gene in culture cells.
Mol Cell Biol
12:
619-630,
1992[Abstract].
38.
Thompson, WR,
Nadal-Ginard B,
and
Mahdavi V.
A MyoD1-independent muscle-specific enhancer controls the expression of the -myosin heavy chain gene in skeletal and cardiac muscle cells.
J Biol Chem
266:
22678-22688,
1991
39.
Tsika, GL,
Wiedenman JL,
Gao L,
McCarthy JJ,
Sheriff-Carter K,
Rivera-Rivera ID,
and
Tsika RW.
Induction of -MHC transgene in overloaded skeletal muscle is not eliminated by mutation of conserved elements.
Am J Physiol Cell Physiol
271:
C690-C699,
1996
40.
Tsika, RW.
Transgenic animal models.
Exerc Sport Sci Rev
22:
361-388,
1994[Medline].
41.
Tsika, RW,
Hauschka SD,
and
Gao L.
M-creatine kinase gene expression in mechanically overloaded skeletal muscle of transgenic mice.
Am J Physiol Cell Physiol
269:
C665-C674,
1995[Abstract].
42.
Ueyama, T,
Zhu C,
Valenzuela YM,
Suzow JG,
and
Stewart AFR
Identification of the functional domain in the transcription factor RTEF-1 that mediates 1-adrenergic signaling in hypertrophied cardiac myocytes.
J Biol Chem
275:
17476-17480,
2000
43.
Vyas, DR,
McCarthy JJ,
and
Tsika RW.
Nuclear protein binding at the -myosin heavy chain A/T-rich element is enriched following increased skeletal muscle activity.
J Biol Chem
274:
30832-30842,
1999
44.
Vyas, DR,
McCarthy JJ,
Tsika GL,
and
Tsika RW.
Multiprotein complex formation at the myosin heavy chain distal muscle CAT element correlates with slow muscle expression but not mechanical overload responsiveness.
J Biol Chem
276:
1173-1184,
2001
45.
Vyas, DR,
McCarthy JJ,
Tsika GL,
and
Tsika RW.
Dissimilar nuclear protein binding at human -myosin heavy chain proximal and distal MCAT elements in response to increased skeletal muscle activity.
Basic Appl Myol
10:
5-16,
2000.
46.
Vyas, DR,
McCarthy JJ,
Tsika GL,
and
Tsika RW.
Muscle-specific transcription of -myosin heavy chain transgene requires an A/T-rich element.
Basic Appl Myol
10:
87-88,
2000.
47.
Wiedenman, JL,
Rivera-Rivera I,
Vyas D,
Tsika G,
Gao L,
Sheriff-Carter K,
Wang X,
Kwan LY,
and
Tsika RW.
-MHC and SMLC1 transgene induction in overloaded skeletal muscle of transgenic mice.
Am J Physiol Cell Physiol
270:
C111-C1121,
1996.
48.
Wiedenman, JL,
Tsika GL,
Gao L,
McCarthy JJ,
Rivera-Rivera ID,
Vyas D,
Sheriff-Carter K,
and
Tsika RW.
Muscle-specific and inducible expression of 293-base pair -myosin heavy chain promoter in transgenic mice.
Am J Physiol Regul Integr Comp Physiol
271:
R688-R695,
1996
49.
Wu, H,
Naya FJ,
McKinsey TA,
Mercer B,
Shelton JM,
Chin ER,
Simard AR,
Michel RN,
Bassel-Duby R,
Olson EN,
and
Williams RS.
MEF2 responds to multiple calcium-regulated signals in the control of skeletal muscle fiber type.
EMBO J
19:
1963-1973,
2000
50.
Wu, H,
Rothermel B,
Kanatous S,
Rosenberg P,
Naya FJ,
Shelton JM,
Hutcheson KA,
DiMaio JM,
Olson EN,
Bassel-Duby R,
and
Williams RS.
Activation of MEF2 by muscle activity is mediated through a calcineurin-dependent pathway.
EMBO J
20:
6414-6423,
2001
51.
Xiao, JH,
Davidson I,
Matthes H,
Garnier JM,
and
Chambon P.
Cloning, expression, and transcriptional properties of the human enhancer factor TEF-1.
Cell
65:
551-568,
1991[ISI][Medline].