Segregated Regulatory Elements Direct
-Myosin Heavy Chain
Expression in Response to Altered Muscle Activity*
John J.
McCarthy
,
Dharmesh R.
Vyas
,
Gretchen L.
Tsika
, and
Richard W.
Tsika
§¶
From the
Department of Veterinary Biomedical
Sciences, School of Veterinary Medicine, the § Department of
Biochemistry, School of Medicine, and ¶ Dalton Cardiovascular
Research Center, University of Missouri, Columbia, Missouri 65211
 |
ABSTRACT |
Our previous transgenic analyses revealed that a
600-base pair
-myosin heavy chain (
MyHC) promoter conferred
mechanical overload (MOV) and non-weight-bearing (NWB) responsiveness
to a chloramphenicol acetyltransferase reporter gene. Whether the same
DNA regulatory element(s) direct
MyHC expression following MOV or
NWB activity in vivo remains unknown. We now show that a
293-base pair
MyHC promoter fused to chloramphenicol
acetyltransferase (
293) responds to MOV, but not NWB activity,
indicating a segregation of these two diverse elements. Inclusion of
the
MyHC negative regulatory element (
332 to
300;
NRE) within
transgene
350 repressed expression in all transgenic lines.
Electrophoretic mobility shift assays showed highly enriched binding
activity only in NWB soleus nuclear extracts that was specific to the
distal region of the
NRE sense
strand (d
NRE-S;
332 to
311). Supershift electrophoretic mobility
shift assay revealed that the binding at the distal region of the
NRE sense strand was antigenically distinct from cellular nucleic
acid-binding protein and Y-box-binding factor 1, two proteins shown to
bind this element. Two-dimensional UV cross-linking and shift
Southwestern blotting analyses detected two proteins (50 and 52 kDa)
that bind to this element. These in vivo results
demonstrate that segregated
MyHC promoter elements transcriptionally
regulate
MyHC transgene expression in response to two diverse modes
of neuromuscular activity.
 |
INTRODUCTION |
The sarcomeric myosin heavy chain
(MyHC)1 is a major
contractile protein that is encoded by a multigene family constituted by eight members (1). Each member has been shown to be responsive to a
complex set of intrinsic and extrinsic signals that collectively regulate their expression in a defined developmental stage- and muscle
fiber type-specific pattern. Within the MyHC gene family, the
MyHC
is the one member whose regulated expression in both cardiac and
skeletal muscle has been most extensively studied. Expression of the
MyHC gene has been detected in the ventricular myocardium and
skeletal muscles comprising the hind limb of fetal mice, whereas in the
adult, its expression is primarily restricted to skeletal muscles or
muscle regions composed predominately of slow twitch type I fibers (2,
3). In vitro investigations of the control of
MyHC gene
transcription have led to the identification of a control region
located within the proximal promoter (nucleotides
300 to
188 of the
human
MyHC gene) region (4-7). This control region was found to be
composed of three discrete DNA regulatory elements termed MCAT-like,
C-rich and
e3, which are highly conserved in location and sequence
across species (4-7). The absolute requirement for these elements to
obtain high levels of gene expression was demonstrated in transient
transfection studies where the independent disruption of any of these
conserved elements decreased and/or abolished muscle-specific
expression of
MyHC reporter genes (4-7).
Located just upstream from the
MyHC control region is a highly
conserved negative regulatory element (
NRE; nucleotides
332 to
300) that represses transcription of
MyHC reporter genes and
heterologous promoters in transient expression assays (4, 8). Several
recent studies have provided evidence that two distinct proteins
interact with this element: cellular nucleic acid-binding protein
(CNBP) and the Y-box binding factor, YB-1 (9, 10). In co-transfection
experiments, CNBP
, but not CNBP
, was shown to repress
MyHC
reporter gene expression in rat fetal heart cells. Although the
regulatory effects of YB-1 binding at the
NRE have not been
determined, existing evidence suggests that YB-1 plays a role in
mediating tissue-specific gene expression in a variety of tissue types
including striated muscle (11-14). While the
NRE has been shown to
regulate
MyHC gene expression in primary cardiocytes (9), its
regulatory role in vivo has not been identified, nor has
this element been shown to play a regulatory role in mediating
MyHC
gene expression in skeletal muscle.
A more complex picture of the regulatory mechanisms directing
MyHC
gene expression has emerged from recent transgenic investigations. These investigations revealed that decreased muscle-specific
MyHC transgene expression was observed only when all three DNA regulatory elements comprising the
MyHC control region were simultaneously mutated (15, 16). Furthermore, it was also shown that additional sequences located upstream of nucleotide
600 are required for high
levels of muscle-specific
MyHC transgene expression and that these
element(s) can compensate for the loss of any one or two control region
elements (15, 16). Clearly, these in vivo findings indicate
that complex combinatorial interactions between distal and proximal DNA
regulatory elements are required for high levels of striated
muscle-specific expression.
An important and relatively unexplored area concerns the malleable
nature of
MyHC gene expression in response to a broad spectrum of
physiological perturbations including changes in the levels of
circulating thyroid hormone, modified patterns of electrical stimulation, and altered mechanical work loads. We have focused our
efforts on delineating DNA regulatory element(s) that direct
MyHC
gene expression in response to diverse modes of neuromuscular activity
such as mechanical overload (MOV) and non-weight-bearing (NWB) activity
(17, 18). Our transgenic deletion analysis revealed that 293 base pairs
(bp) of proximal promoter region is minimally sufficient to confer MOV
induction to a CAT reporter gene, while 600 bp of proximal promoter
region is sufficient to direct NWB responsiveness (17, 18). The
regulation of
MyHC expression in response to MOV and NWB is
correlated with a specific fiber phenotype change, where under normal
conditions
MyHC expression is primarily restricted to slow twitch
type I fibers but can be induced in fast twitch type II fibers
following MOV and decreased in slow twitch type I fibers by NWB
activity. Given the associated fiber type shift with the antithetic
nature of
MyHC expression in response to these two diverse
perturbations, several queries arise. First, do MOV and NWB activity
direct
MyHC gene transcription via the same or distinct cis-acting
element(s)? Second, do MOV and/or NWB element(s) serve an independent
or overlapping role in specifying fiber type-specific expression?
Third, does the
NRE contribute to the decrease in
MyHC expression
seen under NWB conditions in vivo? As an initial step toward
resolving these critical regulatory mechanism(s), we have set out to
delimit a
MyHC gene promoter region that is responsive to NWB
activity by generating transgenic lines harboring transgenes composed
of either 293 or 350 bp of the human
MyHC gene proximal promoter region (
293,
350) fused to the 5'-end of the CAT gene. Notable findings resulting from this transgenic study are that the DNA regulatory sequence(s) that mediate
MyHC gene expression in response to either MOV or NWB activity are segregated to different regions within the proximal promoter of the
MyHC gene and that a negative regulatory element (d
NRE-S) that interacts with two unidentified protein(s), may play an in vivo role in NWB regulation of
MyHC.
 |
EXPERIMENTAL PROCEDURES |
Generation and Screening of Transgenic Mice--
Transgenic mice
were generated by microinjection of purified transgene DNA into
pronuclei of single cell fertilized embryos as described previously
(19). Transgene-positive mice (founders) were identified by Southern
blot analysis. Subsequent offspring derived from mating the founders
were screened for transgene incorporation using the polymerase chain
reaction. In the present study, multiple independent transgenic lines
representing transgenes
350 and
293 (Fig. 1) were studied, and
all lines were maintained in a heterozygous state by continual
outbreeding to nontransgenic FVB/n mice.
Animal Care and MOV and NWB Procedures--
The NWB and MOV
procedures used in this study were approved by the Animal Care
Committee for the University of Missouri-Columbia, and the NWB and MOV
mice were housed in an AAALAC (Association for the Assessment and
Accreditation of Laboratory Animal Care International) accredited
animal facility. All mice were provided with food and water ad
libitum and were housed at room temperature (24 °C) with a 12-h
light-dark cycle in either standard filter top cages (control and MOV
mice) or cages designed for head-down tilt suspension (hind limb
suspension), as described previously (18). Adult
293 transgenic
mice, 12 weeks of age, were randomly assigned to one of four groups: 1)
a NWB group that used hind limb suspension to impose NWB conditions
(NWB; n = 46); 2) a NWB control group that served as
cage ambulatory controls for the NWB group (C, n = 46);
3) a MOV group where MOV was imposed on the fast twitch plantaris
muscle bilaterally by surgically removing the gastrocnemius and soleus
muscles (17, 20) (MOV; n = 17); and 4) a MOV control
group that represented a sham-operated control for the MOV group (C;
n = 17). Mice were prepared for the NWB experiment by
an inexpensive modification of the noninvasive tail traction procedure,
as described previously (18). For MOV studies, mice weighing an average
of 25.7 g (see Tables II and III) were anesthetized with 0.017 ml
of 2.5% (w/v) avertin/g of body weight (17, 20). Following a 2-week
NWB period and an 8-week MOV period, control (NWB-C, MOV-C) and
experimental (NWB, MOV) animals were anesthetized and weighed, and
control-soleus (CS), NWB-soleus (NWB-S), control-plantaris (CP), and
MOV-plantaris (MOV-P) muscles were collected for further study. All
muscles were trimmed clean of fat and connective tissue, weighed, and
stored at
80 °C until assayed. Prior to the initiation of the NWB
study, a 4-week time course analysis was undertaken to establish the
time period over which the greatest decrease in mouse muscle mass,
transgene expression (composed of 5600 bp of
MyHC 5' promoter
sequence upstream of the CAT gene (15-17)), and endogenous
MyHC
mRNA and protein levels occurs post-NWB activity. Our results
indicated that the greatest magnitude of change for each of the
aforementioned parameters occurred after 2 weeks of NWB activity;
therefore, the present NWB study was conducted with a 2-week
termination time point.2
Isolation and Analysis of RNA--
Isolation of total
cellular RNA was performed as described previously (20) using the acid
guanidinium thiocyanate phenol-chloroform method as described by
Chomczynaski and Sacchi (22). The purity of RNA was judged by the ratio
of the absorbances (260/280 nm). Due to the small size of mouse muscle,
it was necessary to pool muscle from 3-5 mice (CS = 6-8 mg;
NWB-S = 5-6 mg; CP = 16 mg; MOV-P = 30 mg). Six
micrograms of total RNA from CS and NWB-S or 8 µg from CP and MOV-P
muscles was denatured at 65 °C in a solution containing 5 µl of
deionized formamide and 1 µl of 10× running buffer. The inclusion of
ethidium bromide allowed visualization of the RNA samples. Total RNA
was fractionated on a 1.5% (w/v) agarose, 2.2 M
formaldehyde-containing gel, transferred to a nylon filter (Duralon,
Stratagene) by capillary blotting, and immobilized on the membrane by
ultraviolet (UV) irradiation (UV Stratalinker, Stratagene).
The specific cDNA probes used in this study were as follows:
mouse-specific CNBP probe (23), rat-specific YB-1 (10), and human 18 S
rRNA (24). For detection of CAT mRNA, a
HindIII/BamHI fragment from pSVOCAT was used. All
cDNA probes were labeled with [
-32P]dCTP (3000 Ci/mmol) using a random primer kit (Stratagene) to a specific activity
of 7-8 × 108 cpm/µg of DNA. An oligonucleotide
probe corresponding to the mouse
MyHC 3'-untranslated region was
used to detect mouse
MyHC-specific transcripts (20) and was
end-labeled to a specific activity of 6 × 108 cpm/ng
of DNA with
-32P (6000 Ci/mmol)-labeled ATP using a
KinAse-It kit (Stratagene). Filters were washed twice in 2× sodium
chloride/sodium citrate, 0.1% (w/v) SDS for 15 min at room temperature
followed by two 30-min washes in 0.1× SSC, 0.1% (w/v) SDS at
60 °C. Filters were dried and exposed to x-ray film (XAR-5, Eastman
Kodak Co.) overnight at
80 °C. Differences between intensities of
signal in control and experimental lanes were quantified using a
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and expressed
as relative percentage difference of arbitrary units. All Northern
blots were hybridized with an 18 S rRNA cDNA probe to verify that
each lane contained equal (±5%) amounts of RNA and for the purpose of
normalizing all RNA values.
CAT Assay--
CAT assays were performed as described by Tsika
et al. (20) with the following modifications. Extracts were
prepared from transgenic tissues 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 pooled muscles from one
mouse. The protein concentration of each extract was determined by the
method of Bradford (21). Tissue extracts were heated at 65 °C for 10 min followed by centrifugation for 10 min at 10,000 × g. Since each transgenic line exhibits 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 (17, 20). Direct comparisons between and within
transgenic lines representing both control and experimental groups (CS,
NWB-S, CP, and MOV-P) were facilitated by presenting the data as
specific CAT activity (pmol/µg of protein/min).
Isolation of Nuclear Protein Extracts--
Nuclear extracts were
isolated from adult rat CS and NWB-S muscle. The sequential isolation
of myonuclei and nuclear extract protein was performed according to the
protocol of Lichtstein et al. (25) with minor modifications
as described by Mar et al. (26) and Larkin et al.
(27). All procedures were carried out on ice. All buffers contained 2 µg/ml of each protease inhibitor: aprotinin and leupeptin and 0.5 mM phenylmethylsulfonyl fluoride. Eight grams of either CS
or NWB-S muscle, harvested from adult female Sprague-Dawley rats (200 g), were minced in phosphate-buffered saline. Minced muscle tissue was
incubated for two 15-min intervals in relaxation buffer 1 (100 mM KCl, 5 mM MgCl2, 5 mM EGTA, 5 mM sodium pyrophosphate, pH 6.8),
followed by two 10-min washes in RBII (50 mM KCl, 5 mM MgCl2, 1 mM EGTA, 1 mM sodium pyrophosphate, pH 6.8). The muscle tissue was
then washed with 100 ml of homogenization buffer A (0.3 M
sucrose, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 15 mM HEPES, pH 7.5, 0.5 mM EGTA, 2 mM EDTA, 10 mg/ml bovine serum albumin, 1 mM dithiothreitol (DTT)), followed by
homogenization in 300 ml of fresh homogenization buffer A. Muscle
tissue was disrupted with an Omni homogenizer (20-mm saw-tooth
generator) at 60% power for three 20-s pulses. Muscle homogenates were
then centrifuged (1000 × g, 4 °C, 10 min) in a
Beckman JA-25-50 rotor, and the pellets were solubilized in
homogenization buffer B (0.3 M sucrose, 60 mM
KCl, 0.15 mM spermine, 0.5 mM spermidine, 15 mM HEPES, pH 7.5, 0.1 mM EGTA, 0.1 mM EDTA, 10 mg/ml bovine serum albumin, 1 mM
DTT) containing 0.5% (v/v) Triton X-100. The solubilized pellet was
homogenized (7-mm saw-tooth generator) at 60% power for one 15-s
pulse, followed by homogenization with a hand-held Teflon glass Dounce
homogenizer. Percoll (Amersham Pharmacia Biotech) was added to the
homogenate to a final concentration of 27% (v/v), and the mixture was
centrifuged at 27,000 × g at 4 °C for 15 min. The
nuclear pellet was solubilized in 10 ml of lysis buffer (10 mM HEPES, pH 7.5, 100 mM KCl, 3 mM
MgCl2, 0.1 mM EDTA, 10% (w/v) glycerol).
Nuclei were lysed by dropwise addition of 3 M
NH4SO4 (pH 7.9) to a final concentration of 0.4 M, and the resulting extract was gently shaken for 30 min
at 4 °C. The lysate was ultracentrifuged at 126,000 × g for 1 h using a Beckman Ti-70 rotor, and the
supernatant was collected. Solid NH4SO4 (0.3 g/ml) was added slowly to the supernatant, and nuclear proteins were
allowed to precipitate on ice for 30 min. Precipitated proteins were
pelleted by ultracentrifugation at 126,000 × g for 30 min and resuspended in 500 µl of dialysis buffer (25 mM
HEPES, pH 7.6, 40 mM KCl, 0.1 mM EDTA, 10%
(w/v) glycerol, 1 mM DTT). The muscle nuclear protein
extract was dialyzed twice for 1 h each time against dialysis
buffer, and aliquots were quick frozen and stored at
80 °C. Once
thawed, nuclear protein extracts were not refrozen for use. Protein
concentration was determined according to Bradford (21).
Electrophoretic Mobility Shift Assay--
All oligonucleotide
probes used in this study are listed in Table
I. The double-stranded
NRE
oligonucleotide probe (nucleotides
332 to
300) was labeled by
fill-in reaction using the Klenow fragment of Escherichia
coli DNA polymerase I and [
-32P]dCTP.
Single-stranded
NRE oligonucleotide probes (antisense, sense,
sense-proximal, and sense-distal) were end-labeled by T4 polynucleotide
kinase (New England Biolabs, Beverly, MA) and
[
-32P]dATP (6000 Ci/mmol) and gel-purified. Binding
reactions were performed for 20 min at room temperature in a 25-µl
total volume. The binding reactions contained binding buffer (50 mM Tris-HCl, pH 7.9, 50 mM KCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, and 5% (w/v) glycerol), 20,000 cpm of labeled DNA
probe, 1 µg of either CS or NWB-S nuclear extract, and 0.1 µg of
poly(dI·dC) (Amersham Pharmacia Biotech) as a nonspecific competitor.
Specificity of DNA binding was assessed by the addition of a 100-fold
molar excess of unlabeled homologous or heterologous competitor DNA to
the binding reaction. In some binding reactions, either HeLa nuclear extract (250 ng) or partially purified recombinant rat YB-1 protein extract (100 ng) was used in place of soleus nuclear extracts. Following incubation, DNA-protein complexes were electrophoretically resolved from unbound oligonucleotide probe on a 5% (w/v)
nondenaturing polyacrylamide gel using 0.5% (w/v) Tris borate/EDTA
buffer, pH 8.3, at 220 V for 2.5 h at 4 °C. Supershift
experiments were performed by preincubation of nuclear extracts or
partially purified YB-1 protein with polyclonal antibody to human CNBP
or Xenopus YB-3 (contains 89% overall amino acid identity
with rat YB-1 (14)) for 30 min at 4 °C prior to the addition of the
labeled DNA probe. Following electrophoresis, gels were dried, and
DNA-protein complexes were visualized by autoradiography.
Western Analysis--
Muscle extracts, nuclear (50 µg) or
cytoplasmic (50 µg), were fractionated by 10% (w/v)
SDS-polyacrylamide gel electrophoresis. Proteins were
electrophoretically transferred to a nitrocellulose membrane in
transfer buffer containing 25 mM Tris-HCl, pH 8.3, 192 mM glycine, and 20% (v/v) methanol. The membrane was
blocked by incubation for 16 h at 4 °C in Tris-buffered
saline/Tween (TBS-T; 10 mM Tris-HCl, pH 7.9, 150 mM NaCl, 0.05% (v/v) Tween 20) containing 5% (w/v) nonfat
milk. The filter was then incubated for 2 h at 25 °C with a
1:1000 dilution of primary antibody, corresponding to either polyclonal
human anti-CNBP or rat anti-YB-1. The filter was then washed three
times for 5 min each with TBS-T and incubated for 1 h at 25 °C
with a 2000-fold dilution of donkey anti-rabbit IgG horseradish
peroxidase-linked secondary antibody (Amersham Pharmacia Biotech) in
TBS-T containing 5% (w/v) nonfat milk. Following three 5-min washes
with TBS-T, immunocomplexes were visualized using a chemiluminescent
detection kit (Amersham Pharmacia Biotech) according to the
manufacturer's recommendation.
Two-dimensional UV Cross-linking Analysis--
The first
dimension of this assay involved EMSA using the d
NRE-S probe and
NWB-S nuclear extracts, since only this reaction revealed the formation
of a highly enriched DNA-protein complex. EMSA was performed as
described above, except that the reaction mixture was scaled up 10-fold
and run on a 0.75-mm-thick nondenaturing gel. Immediately following
electrophoresis, the top gel glass plate was removed, and the gel was
covered with Saran Wrap and placed on a transluminator (312 nm) for 30 min at 4 °C. The gel was dried for 20 min and exposed to film for an
additional 30 min. Following autoradiography, the specific band
corresponding to the cross-linked DNA-protein complex was excised and
soaked in 2× sample buffer (125 mM Tris-HCl, pH 6.8, 4%
(w/v) SDS, 20% (w/v) glycerol, 28 mM
-mercaptoethanol,
0.01% (w/v) bromphenol blue) for 30 min at room temperature. The gel
slice was transferred to a sample well of a 1.5 mm SDS-polyacrylamide
gel (4% (w/v) stacking gel, 12% (w/v) resolving gel) and
electrophoresed at constant voltage (125 V) through the stacking gel
and at 200 V through the resolving gel for 90 min at room temperature
(28). Following electrophoresis, the gel was placed on Whatman filter paper and dried, and DNA-protein complexes were visualized by autoradiography.
Shift Southwestern Analysis--
The specific DNA-protein
complex formed when the distal portion of the
NRE sense strand
(
332 to
311) was incubated in a binding reaction with NWB soleus
nuclear extracts and was separated by EMSA. EMSA was performed
essentially as described above except that the binding reaction was
scaled up 10-fold, and 13 independent reaction mixtures were
electrophoresed in a 0.75-mm-thick gel. Following EMSA, the section of
the gel containing the DNA-protein complex was electrophoretically
transferred to membranes (nitrocellulose and DEAE) placed in series
using conditions described above for Western blot analysis. During
transfer, the protein component of the DNA-protein complex bound to the
nitrocellulose membrane and the d
NRE-S DNA probe bound to the DEAE
membrane. Following localization of the bound protein using the DEAE
membrane, the protein was eluted from the nitrocellulose membrane by
incubation in a 20% (v/v) acetonitrile solution for 3 h at
37 °C. The eluate was centrifuged for 10 min to remove particulate
material, lyophilized to remove solvent, and resuspended in 20 µl of
50 mM Tris-HCl, pH 7.5. The recovered protein was
solubilized in 6× sample buffer (350 mM Tris-HCl, pH 6.8, 30% (w/v) glycerol, 10% (w/v) SDS, 0.93 mM DTT, 0.012%
(w/v) bromphenol blue) and electrophoretically resolved by 12% (w/v)
SDS-polyacrylamide gel electrophoresis at constant voltage (200 V) for
45 min at room temperature. The protein was electrophoretically
transferred to a nitrocellulose membrane as described above for Western
analysis. The membrane was incubated for 10 h at 4 °C in a
blocking solution composed of EMSA binding reaction buffer (minus
glycerol) containing 5% (w/v) nonfat milk. DNA-protein interaction
occurred during incubation of the membrane in a blocking solution
containing 0.25% nonfat milk and labeled d
NRE-S probe (2 × 106 cpm/ml) for 10 h at 4 °C. Following
hybridization, the membrane was washed three times for 5 min at room
temperature in a solution consisting of 50 mM Tris-HCl, pH
7.9, 30 mM KCl, 1 mM MgCl2, 0.5 mM EDTA, and 0.5 mM DTT, air-dried, and exposed
to film overnight.
Statistical Analysis--
All statistical analyses were
performed using the StatView SE + Graphics program (Version 4.1; Abacus
Concepts, Inc.). Student's t tests were used to assess
differences between group means. All data are reported as means ± S.E. The lowest significance level accepted was p < 0.05. Given the large number of transgenic mice required for mRNA
analysis (5 mice/n), we restricted our analysis to include
three independent measurements (n = 3) following the 2-week NWB period, which precluded a statistical analysis.
 |
RESULTS |
Changes in Morphology and Phenotype following NWB Activity or
MOV--
NWB activity imposed by hind limb suspension results in
muscle weight loss (atrophy) and a slow to fast myofiber transition accompanied by decreased
MyHC expression (18). There were no significant differences measured in the initial body weight of mice
representing either the control (n = 46) or NWB
(n = 46) groups (Table
II). However, 2 weeks of NWB activity
resulted in a significant decrease in body weight (NWB;
11.7%) when
compared with body weight values of mice representing the control group (Table II). Importantly, the initial and final body weights of mice
comprising the control group did not differ significantly over the
2-week NWB period, indicating that body weight loss for the NWB group
was the result of NWB activity induced by hind limb suspension and not
the inhibition of normal growth. In agreement with our previous study
(18), 2 weeks of NWB activity resulted in a significant decrease in
both absolute (mg) (
30.7%) and normalized (
19.2%) weights of the
NWB-S muscle as compared with CS muscle weight (Table II). To assess
whether NWB decreased endogenous
MyHC expression in the NWB-S,
Northern hybridization, SDS-polyacrylamide gel electrophoresis analysis
of MyHC content, and myosin ATPase histochemistry were performed. In
agreement with our previous findings, Northern hybridization showed a
38% decrease in endogenous
MyHC mRNA in the NWB-S following a
2-week period of NWB activity (Fig. 2) (18). The NWB-induced decrease
in endogenous
MyHC expression was also seen at the protein level as
assessed by myosin ATPase histochemistry and myosin heavy chain
separation (data not shown; see Ref. 18).
To evaluate the effect of MOV on muscle enlargement (hypertrophy),
normalized plantaris weight (mg/g of body weight) was obtained. Over
the 8-week experimental period, no significant differences were
measured in body weights between adult transgenic mice representing sham-operated control (n = 17) and MOV
(n = 17) groups (Table III). As expected, 8 weeks of MOV
resulted in a significant increase (92%) in normalized (mg/g of body
weight) MOV-P weight as compared with CP values (Table III). As
reported previously, the hypertrophic growth of the MOV-P was
accompanied by a concomitant increase in
MyHC mRNA and protein
expression (data not shown; see Ref. 17).
Transgene
293 Expression Pattern and Regulation in Response to
NWB Activity and MOV--
Our previous work revealed that an
NWB-responsive element(s) resides within a 600-bp region located
immediately 5' to the
MyHC gene transcription start site (Fig.
1) (18). To further delimit the location
of this element(s), we have generated multiple independent transgenic
mouse lines for each of two different transgenes (
293 and
350)
(Fig. 1). Since adult-stage
MyHC gene expression is primarily
restricted to slow type I muscle fibers (Fig. 1, schematic
diagram), we first examined the levels of CAT specific activity (pmol/µg of protein/min) in protein extracts obtained from
muscles containing different proportions of type I fibers, as well as
nonmuscle tissue. As shown in Fig. 3A, transgene
293 was
not expressed in the adult heart but was expressed in a muscle-specific manner in accordance to type I fiber composition (soleus > gastrocnemius > plantaris). Collectively, these results show that
transgene
293 expression qualitatively mimics the normal adult-stage
expression pattern of the endogenous
MyHC gene.

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Fig. 1.
Response of various
MyHC transgenes to NWB and MOV activity.
Transgenes consist of either 293 bp ( 293) or 350 bp ( 350) of
human MyHC 5'-flanking sequence and 120 bp of 5'-untranslated region
fused to the CAT reporter gene. Transgene 600 contains 600 bp of
mouse MyHC 5'-flanking sequence and 1600 bp of 5'-untranslated
region linked to the CAT reporter gene. Relative location of the highly
conserved proximal promoter cis-elements (MCAT, C-rich,
e3, and NRE) comprising the MyHC control region are as
designated. +, moderate to high levels of expression; , barely
detectable to no expression; , increased expression; decreased
expression. Data are compiled from Refs. 17 and 18 and data
herein.
|
|
We have previously shown that NWB activity decreases
MyHC expression
in the slow twitch soleus muscle (18). To determine if an NWB
element(s) resides within transgene
293, CAT specific activity was
measured in CS and NWB-S muscle following a 2-week period of NWB
activity. Surprisingly, CAT specific activity measured in NWB-S extract
revealed an unexpected increase (1.2-3.7-fold) as compared with CS
extract (Fig. 3B, Table IV).
To examine whether these results were due to post-transcriptional
regulation, we analyzed transgene
293 expression at the level of
mRNA accumulation (Fig. 2). In
contrast to the 38% decrease in endogenous
MyHC mRNA levels
measured after 2 weeks of NWB activity, exogenous CAT mRNA levels
measured for transgenic line
293:L7 increased, confirming our CAT
assay results (Figs. 2 and 3B;
Table IV).
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Table IV
Response of 293 transgene CAT specific activity to NWB and MOV
Values are means ± S.E. CAT specific activity (pmol/µg of
protein/min) was measured by incubation of protein extracts with 20 mM acetyl-CoA and 14C-chloramphenicol (0.2 µCi/0.35 µM) in 250 mM Tris-HCl, pH 7.8, at
37 °C. Incubation of muscle protein extracts representing each
transgenic line was performed as follows: MOV study using transgene
293 (line 293:L4, 5 µg/h; 293:L5, 3 µg/h; 293:L6, 30 µg/21 h; 293:L7 30 µg/17 h; 293:L96, 7.5 µg/30 min;
293:L99, 7.5 µg/30 min); NWB study using transgene 293
( 293:L4, 6 µg/h; 293:L5, 3 µg/h; 293:L6, 30 µg/17 h;
293:L7, 10 µg/17 h; 293:L96, 7.5 µg/15 min; 293:L99, 2 µg/30 min). Note: CP and MOV-P CAT specific activities for lines 4-6
were previously reported (Ref. 17 and references within) and are shown
for ease of comparison.
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Fig. 2.
Northern blot analysis of NWB soleus muscle
from transgenic mice harboring transgene 293
line 7 ( 293:L7). Total RNA isolated from
CS and NWB-S (6 µg) muscle pooled from 3-5 animals was fractionated
on a 1.5% agarose denaturing gel. Intensity of the hybridization
signal was quantitated using a PhosphorImager and normalized to 18 S
(24) values to account for loading differences between lanes.
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Fig. 3.
A, representative CAT assay
demonstrating the expression pattern of the 293:L96 transgene in
various adult tissues. Protein extracts (7.5 µg) prepared from
control muscle (soleus (CS), gastrocnemius (CG), plantaris (CP), and
heart (H)) and nonmuscle (lung (Lu), liver
(Li), and spleen (Sp)) tissues were incubated for
15 min at 37 °C. Extracts obtained from nontransgenic
(Ntg) mouse soleus muscle served as a negative control, and
purified CAT protein (70 ng) was used as a positive control.
B, representative CAT assay showing transgene 293:L96
expression following 2 weeks of NWB activity or 8 weeks of MOV. Protein
extracts (7.5 µg) were incubated for 15 min (CS and NWB-S) or 30 min
(CP and MOV-P) at 37 °C. Ntg CS was used as negative control, and 70 ng of purified CAT protein was used as a positive control. Note the
unexpected increase in CAT specific activity of NWB-S sample in
comparison with CS. The increase in CAT specific activity in response
to NWB activity was observed in all 293 transgenic lines tested (see
Table IV).
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In contrast to NWB activity, MOV induces endogenous
MyHC gene
expression in the fast twitch plantaris muscle of rodents (17) (Fig. 1,
schematic diagram). To determine if transgene
293 responds to MOV, the plantaris muscles of mice representing each
of six independent lines were overloaded and assayed for CAT specific activity 8 weeks later. Expression assays showed that CAT specific activity measured in MOV-P muscle extracts was 3-16-fold higher than
that measured in sham-operated CP muscle extracts (Fig. 3B, Table IV), which agrees with our previous findings for this transgene (17). Taken together, these data provide convincing evidence that NWB
and MOV element(s) are segregated to different regions within the
MyHC-proximal promoter.
Transgene
350 Expression Pattern and Regulation in Response to
NWB Activity and MOV--
In striking contrast to transgene
293,
under control conditions no detectable levels of CAT specific activity
could be measured in muscle or nonmuscle tissues of mice representing
each of 11 independent transgenic lines harboring transgene
350.
Since transgene
293 was up-regulated by NWB activity, we tested
transgene
350 expression following 2 weeks of NWB activity; however,
as under basal conditions, detectable levels of transgene
350
expression could not be measured in the NWB-S muscle. Furthermore, 8 weeks of MOV did not result in the induction of transgene
350
expression in the MOV-P muscle. These results could be accounted for if
transgene
350 integrated into an inactive chromosomal site. However,
due to the large number (11) of
350 lines tested for CAT expression, it is highly improbable that chromosomal position effects can account
solely for the lack of transgene expression. Therefore, we interpret
these in vivo results to implicate the
NRE (
332 to
300) as a negative element responsible for the transcriptional silencing of transgene
350 and suggest that the
NRE may also play
a role in NWB-induced decreases in
MyHC gene transcription.
Enriched DNA-Protein Interaction at
NRE--
Our transgenic
results point to a correlative link between the
NRE and the
transcriptional repression of transgene
350. Therefore, to
investigate the protein binding properties of the
NRE under control
and NWB conditions, we performed direct and competitive electrophoretic
mobility shift assays (EMSAs) using nuclear extract isolated from CS
and NWB-S muscle (Fig. 4, A
and B). Incubation of a double-stranded
32P-labeled
NRE probe (DS,
332 to
300; Table I) with
NWB-S nuclear extracts resulted in a barely detectable DNA-protein
complex that was not discernible when CS nuclear extracts were used
(Fig. 4A, lanes 1-3). Previous
studies have shown that
NRE interacts with proteins (e.g.
CNBP and YB-1) that bind double- or single-stranded nucleic acid (9,
10); therefore, we next examined DNA-protein interactions using various
single-stranded forms of the
NRE. Interestingly, when an
32P-labeled
NRE sense strand (
NRE-S, -332 to -300)
was incubated with NWB-S nuclear extracts, a binding complex was formed
that was highly enriched in comparison with that formed when CS nuclear extracts were used (Fig. 4A, lanes
4-6). In contrast, a binding complex was not formed when
32P-
NRE antisense (
NRE-AS,
332 to
300) or
32P-
NRE sense-proximal (p
NRE-S,
317 to
300)
strands were incubated with either CS or NWB-S nuclear extracts (Fig.
4A, lanes 7-12). However, a highly
enriched binding complex was detected when a 32P-labeled
NRE sense-distal strand (d
NRE-S,
332 to
311) was incubated
with NWB-S nuclear extracts (Fig. 4A, lanes
13-15). The formation of this binding complex was judged to
be sequence-specific as determined by competition with a 100-fold molar
excess of unlabeled wild type d
NRE-S (Fig. 4B,
lane 1 versus lane
2) and the lack of competition seen when either a 100-fold
molar excess of unlabeled mutant d
NRE-S probe (d
NRE-Smt1 and
d
NRE-Smt2; Table I) or nonspecific DNA
(sense strand of
MyHC C-rich element) are used in competition EMSAs
(Fig. 4B, lane 1 versus
lanes 3-5). These data demonstrate that highly
enriched, sequence-specific DNA binding activity exists in NWB-S
nuclear extracts that preferentially bind the d
NRE-S.

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Fig. 4.
A, EMSA of NRE-protein interaction. A
DNA-protein complex (SC, specific complex) was delimited to
the distal portion (lanes 13 and 14) of the
NRE sense strand. , no extract. B, competition EMSA.
100-fold molar excess of cold mutated d NRE-S probe (lanes
3 and 4) or nonspecific probe (lane 5) was
added to the binding reaction. C, two-dimensional UV
cross-linking of DNA-protein complex. Three bands with apparent
molecular masses of 50, 52, and 112 kDa were identified. D,
shift-Southwestern of DNA-protein complex. Two bands were detected (50 and 52 kDa) that corresponded to those identified by UV
cross-linking.
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Table V
Sequence alignment of human, rabbit, mouse, and rat NRE
NRE sequences were aligned using the BestFit program of the GCG
Wisconsin package. Boldface base pairs indicate a mismatch in
comparison with the human MyHC sequence. The italicized sequences
represent the distal NRE.
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Biochemical Analysis of DNA-binding Factor(s) Interacting at
d
NRE--
As an initial characterization of the factor(s) in NWB-S
nuclear extracts that bind to d
NRE-S, we performed two-dimensional UV cross-linking analysis. The d
NRE-S probe was incubated with NWB-S
nuclear extract, and the binding complex was separated from unbound
probe by EMSA. Subsequently, the EMSA gel was exposed to UV light (312 nm) for 30 min, and the enriched binding complex was excised from the
gel, electroeluted, and resolved on a 12% (w/v) SDS-polyacrylamide
gel. As shown in Fig. 4C, two-dimensional UV cross-linking
analysis detected three bands with apparent molecular masses of
116, 52, and 50 kDa. To further study the protein components of this
binding complex, we performed shift Southwestern blot analysis
essentially as described by Demczuk et al. (29). Following electroblotting of the enriched NWB-S binding complex, the protein was
eluted from the nitrocellulose membrane and subjected to Southwestern analysis using a 32P-labeled d
NRE-S probe. Two bands
were detected that correspond to the 52- and 50-kDa proteins detected
by UV cross-linking analysis (Fig. 4, C and D).
It is not clear why the 116-kDa band was not observed in shift
Southwestern analysis, but one possibility is that this band represents
a multimer of the binding complex formed during exposure to UV light.
These results indicate that the enriched binding complex formed at the
d
NRE-S site is composed of at least two distinct proteins whose
identities are presently unknown.
Expression Pattern of CNBP and YB-1 in CS and NWB-S
Muscle--
Previous investigations have shown that the zinc
finger-binding factor CNBP and the Y-box binding protein YB-1 bind the
NRE (9, 10). By Northern blot analysis, we show that both CNBP and
YB-1 are expressed in CS and NWB-S muscle; however, only YB-1 mRNA
levels decreased (
28%) following 2 weeks of NWB activity (Fig.
5A). To determine the
subcellular location of CNBP and YB-1, CS and NWB-S nuclear and
cytoplasmic extract was fractionated by SDS-polyacrylamide gel
electrophoresis and analyzed by Western blot (Fig. 5, B and
C). Western analysis using anti-CNBP serum revealed that
CNBP was detected in HeLa nuclear extracts but not in CS or NWB-S
nuclear extract (Fig. 5B, lanes 1-3),
whereas it was detected in CS and NWB-S cytoplasmic extracts (Fig.
5B, lanes 5 and 6). Western
analysis using a YB-1 polyclonal antibody detected a protein of
approximately 50 kDa in CS and NWB-S cytoplasmic extracts that had a
migration pattern similar to that of partially purified recombinant
YB-1 protein (Fig. 5C, lane 1 versus lanes 4 and 5). A
50-kDa protein was also detected in CS nuclear extracts that was barely
detectable in NWB-S nuclear extracts (Fig. 5C, lanes 1 versus 2-5).
Collectively, these data provide evidence that CNBP and YB-1 are
expressed in adult skeletal muscle; however, their absence in NWB-S
nuclear extracts supports the notion that they are not a component of
the binding activity in NWB-S nuclear extracts.

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Fig. 5.
A, Northern blot analysis of CNBP and
YB-1 expression within NWB soleus muscle from transgenic mice harboring
transgene 293 line 7 ( 293:L7). Total RNA was isolated from CS and
NWB-S (6 µg) muscle pooled from 3-5 animals, fractionated on a 1.5%
agarose denaturing gel, and quantitated for hybridization intensity as
described in the legend to Fig. 2. B, CNBP protein in
skeletal muscle and HeLa cell nuclear and cytoplasmic extracts. Western
blot analysis is shown of 50 µg of HeLa cell (lanes
1 and 4), CS (lanes 2 and
5), and NWB-S (lanes 3 and
6) nuclear (N.E.) or cytoplasmic extracts
(C.E.) using a human polyclonal CNBP antibody. Note the
absence of CNBP protein in NWB-S nuclear extracts. C, YB-1
protein in skeletal muscle nuclear and cytoplasmic extracts. Western
blot analysis is shown of 50 µg of NWB-S (lanes
2 and 4) and CS (lanes 3 and 5) nuclear or cytoplasmic extracts (N.E. and
C.E., respectively) using a YB-1 polyclonal antibody.
Partially purified rat YB-1 protein served as a positive control
(lane 1). Note the barely detectable levels of
YB-1 protein in NWB-S nuclear extracts.
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Is CNBP a Component of the Enriched d
NRE Binding
Complex?--
To directly establish whether CNBP is a component of the
highly enriched binding activity identified in NWB-S nuclear extracts, competition and supershift EMSAs were performed (Fig.
6). The addition of a 100-fold molar
excess of unlabeled 3-hydroxy-3-methylglutaryl CoA probe (previously
shown to bind CNBP (30)) to the binding reaction did not interfere with
complex formation, whereas a 100-fold molar excess of unlabeled
d
NRE-S probe did (Fig. 6, lanes 1-3). Furthermore, the addition of either preimmune serum or anti-CNBP serum
to the binding reaction neither supershifted nor inhibited binding
complex formation when NWB-S nuclear extracts were used (Fig. 6,
lanes 4 and 5). Since CNBP is a zinc
finger protein, we tested whether the addition of the metal ion
chelator 1,10-phenanthroline would inhibit binding complex formation.
Interestingly, when 4 mM of either 1,10-phenanthroline
(1,10-Phen) or the non-metal-chelating analog
1,7-phenanthroline (1,7-Phen) was added to the binding reaction, complex formation was not altered (Fig. 6, lanes
1, 6, and 7).

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Fig. 6.
EMSA analysis for
d NRE-S binding to NWB-S and HeLa cell nuclear
extracts. 32P-labeled d NRE-S probe and nuclear
extracts from NWB-S (1 µg) (lanes 1-7) and
HeLa cells (250 ng) (lanes 8-15) were incubated
for 20 min at room temperature in the absence (lanes
1 and 8) or presence of 100-fold molar excess of
unlabeled d NRE-S (lanes 2 and 9) or
3-hydroxy-3-methylglutaryl CoA probe (lanes 3 and
10). Thirty minutes prior to the addition of the d NRE-S
probe, preimmune serum (PI) (lanes 4 and 11), anti-CNBP antibody (lanes 5 and 12), 4 mM 1,10-phenanthroline
(1,10-Phen; lanes 6 and
13), 4 mM 1,7-phenanthroline
(1,7-Phen; lanes 7 and 14),
or 4 mM 1,10-phenanthroline and 0.5 mM
ZnCl2 (lane 15) were incubated with
nuclear extract at 4 °C. Binding complexes were resolved as
described under "Experimental Procedures."
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To further investigate whether CNBP interacts with the d
NRE-S probe,
EMSAs were performed using HeLa cell nuclear extracts, since these
extracts have been shown to be a rich source of CNBP (31). When
using HeLa nuclear extracts, EMSAs revealed a binding complex
with a higher mobility than that formed when using NWB-S nuclear
extracts (lane 1 versus
lane 8). The addition of a 100-fold molar excess
of unlabeled d
NRE-S and 3-hydroxy-3-methylglutaryl CoA probes to the
binding reaction as competitor led to either a complete or near
complete inhibition of 32P-labeled d
NRE-S binding to
HeLa cell nuclear extracts, respectively (Fig. 6, lanes
9 and 10). In contrast to the d
NRE-S-protein
complex formed with NWB-S nuclear extract, the addition of anti-CNBP
serum to the HeLa binding reaction resulted in a supershifted binding complex, whereas the addition of preimmune serum had no effect (Fig. 6,
lane 11 versus lane
12). Furthermore, 1,10-phenanthroline, but not
1,7-phenanthroline, inhibited the formation of a binding complex that
could be reconstituted by the addition of 0.5 mM ZnCl2 to the binding reaction (Fig. 6, compare
lanes 13-15). The results from our EMSAs provide
compelling evidence that CNBP can bind the
NRE-S probe but is not a
component of the specific binding complex formed when using NWB-S
nuclear extracts.
Is YB-1 a Component of the Enriched d
NRE-S Binding
Complex?--
YB-1 has been shown to bind the rabbit
MyHC
suppressor element (
311 to
294) (10) that has an 85% sequence
identity to the d
NRE-S probe (Table V). Accordingly, we performed
EMSAs to determine if YB-1 is a component of the NWB-S binding complex. Incubation of the 32P-labeled d
NRE-S probe with NWB-S
nuclear extract resulted in a binding complex that was competed away by
the addition of a 100-fold molar excess of unlabeled d
NRE-S probe
(Fig. 7, lane 1 versus lane 2). Although the addition
of excess unlabeled HF-1 probe (containing the HF-1a element previously
shown to bind YB-1 (14)) competed away 32P-labeled
d
NRE-S binding to NWB-S nuclear extracts (Fig. 7, lane 1 versus lane 3), it was
not as effective as the unlabeled d
NRE-S probe (Fig. 7,
lane 1 versus lane
2). Preincubation of NWB-S nuclear extracts with either
preimmune serum or a polyclonal YB-3 antibody neither abolished
32P-labeled d
NRE-S binding nor supershifted the specific
binding complex (Fig. 7, lanes 4 and
5). To determine whether YB-1 could interact with the human
MyHC d
NRE-S sequence, bacterially expressed recombinant YB-1
protein was used in EMSAs. In contrast to NWB-S nuclear extracts,
incubation of partially purified bacterially expressed recombinant YB-1
protein with the 32P-labeled d
NRE-S probe resulted in
the formation of two distinct binding complexes, one with an
electrophoretic mobility equivalent to the complex formed with NWB-S
nuclear extracts (Fig. 7, lane 6). The addition
of a 100-fold molar excess of unlabeled d
NRE-S or HF-1 probe nearly
abolished the formation of both YB-1 binding complexes (Fig. 7,
lanes 7 and 8). Preincubation of
recombinant YB-1 with preimmune serum did not interfere with YB-1
32P-labeled d
NRE-S complex formation; however,
preincubation with YB-3 polyclonal antibody (YB-3 protein has 89%
overall amino acid identity to YB-1) supershifted the YB-1
32P-labeled d
NRE-S binding complex with the highest
mobility (SC), indicating that the other binding complex was
due to nonspecific (NS) binding (Fig. 7, lanes
7-10). Collectively, these experiments show that
recombinant YB-1 protein can bind the human
MyHC d
NRE-S probe.
However, the lack of an anti-YB-3 supershifted complex when using NWB-S
nuclear extracts strongly suggests that YB-1 is not a component of the
specific DNA-protein complex formed between 32P-labeled
d
NRE-S and NWB-S nuclear extracts.

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Fig. 7.
EMSA analysis for
d NRE-S binding to NWB-S nuclear extracts and
recombinant YB-1. A 32P-labeled d NRE-S probe was
incubated with either NWB-S nuclear extract (1 µg) or partially
purified rat YB-1 protein (100 ng). The specific DNA-protein complex
(SC) was examined by competition with a 100-fold molar
excess of unlabeled d NRE-S probe (lanes 2 and
7) or the sense strand of HF-1 oligonucleotide
(lanes 3 and 8). Preimmune serum
(PI) (lanes 4 and 9) and
polyclonal YB-3 antibody (lanes 5 and
10) were incubated for 30 min at 4 °C prior to the
addition of the d NRE-S probe. The YB-3 antibody produced a
supershift (SS) of the lower YB-1 complex (lane
10). NS, nonspecific DNA-protein complex.
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DISCUSSION |
One mechanism by which adult-stage skeletal muscle adapts to new
functional demands is by modulating transcriptional activation and
repression of select muscle genes. Because the
MyHC gene is
regulated in a muscle type-specific fashion throughout development and
in response to a broad range of physiological stimuli, it represents an
excellent model system in which to study the mechanisms underlying
differential gene transcription. Our approach used to gain insight into
the mechanistic basis underlying
MyHC plasticity is to study its
regulation in response to altered neuromuscular activity induced by
altered mechanical loads. For example, under conditions of decreased
mechanical loading imposed by NWB we have previously demonstrated a
40-50% decrease in the basal expression level of transgenes composed
of either 5600 or 600 bp of
MyHC 5'-flanking DNA in the slow twitch
soleus muscle of adult transgenic mice (18). As concerns the regulation
of
MyHC expression in response to increased MOV, we have shown that
a transgene composed of 293 bp of
MyHC 5'-flanking DNA (
293) was
minimally sufficient to induce expression in both the fast twitch
plantaris and slow twitch soleus muscles (17). In an effort to extend
these findings, this study was undertaken to investigate whether
distinct DNA element(s) regulate the antithetic expression pattern of
the
MyHC gene in response to MOV and NWB activity. For this purpose,
transgenic mice harboring transgenes composed of different lengths of
MyHC 5'-flanking sequence were generated, and their expression was analyzed in response to MOV and NWB activity. Our in vivo
findings herein represent the first clear presentation of evidence that distinct
MyHC promoter sequence(s) mediate
MyHC expression in response to MOV and NWB activity and that these element(s) are segregated to different regions within the
MyHC proximal promoter.
In Vivo Determination of Segregated MOV and NWB Regulatory
Elements--
We initiated these studies by establishing the basal
pattern of transgene
293 and
350 expression within muscle and
nonmuscle tissues of adult mice and how these patterns change following either NWB activity or MOV. Although transgene
293 is not expressed at detectable levels in single fibers, measurements of CAT specific activity using whole tissue extracts qualitatively suggests that transgene
293 expression faithfully mirrored endogenous
MyHC expression, since its expression was not detected in adult ventricular muscles or nonmuscle tissues (Fig. 3, A and B;
Table IV). Furthermore, following an 8-week period of MOV, the
expression of transgene
293 was induced in the predominantly fast
twitch plantaris muscle at levels that were 3-16-fold higher than
control levels, clearly indicating that this transgene harbored
MOV-responsive element(s) (Fig. 3B, Table IV). In striking
contrast to the measured decrease in endogenous
MyHC mRNA (Fig.
2) and protein (data not shown (18)) expression following NWB activity,
transgene
293 expression was not down-regulated, but instead, its
expression level was unexpectedly increased by 1.2-3.7-fold above
expression levels measured in control soleus muscle (Fig. 3, Table IV).
At present, the precise molecular mechanism underlying transgene
293
up-regulation by NWB activity is unclear; however, it is clear that
sequences required for NWB regulation are missing. Importantly, this
surprising result was obtained for each of six independent transgenic
lines examined, and each line carries different relative transgene copy numbers; therefore, it is highly unlikely that transgene copy number or
chromosomal position effects can account for these results (Table IV).
Because transgene
293 responds to MOV, but not NWB activity, in a
manner similar to the endogenous
MyHC gene, the regulatory elements
required for MOV and NWB regulation must be segregated, thus
eliminating the possibility that a single MOV/NWB DNA element exists
for the
MyHC gene.
To locate regulatory element(s) involved in
NWB-dependent regulation of the
MyHC gene, we examined a
transgene containing 350 bp of human
MyHC 5'-flanking DNA (transgene
350) that harbored the
NRE at its 5'-terminal end. In contrast to
the unexpected increase in transgene
293 expression following NWB,
analysis of these transgenic lines revealed that expression had been
completely abolished in control, NWB soleus, and MOV-P muscle. This
result was unanticipated because two previous reports have demonstrated that rat
MyHC reporter constructs of comparable size and harboring the entire
NRE were expressed in permanent myogenic cell lines as
well as following direct injection into rabbit ventricular muscle and
rat control and pressure overloaded ventricular muscle (8, 32). A
number of possibilities exist that may explain the disparity between
our transgenic results and those of the aforementioned studies. First,
it is possible that minor sequence differences between the rat and
human
MyHC proximal promoter regions (>85% identity) used in these
studies can account for the observed differences in expression.
However, this possibility was eliminated when nine transgenic lines
carrying a rat-derived construct (
333;
333 to +34 rat
MyHC/luciferase reporter) that harbored the entire
NRE also did
not express.3 Second, it is
possible that transgenes
350 and
333 integrated into an inactive
chromosomal site; however, due to the large number of
350 (eleven)
and
333 (nine) transgenic lines tested for expression, it is
unlikely that chromosomal position effects can account solely for the
lack of transgene expression. An alternate and more likely explanation
for previous results is that when cell culture systems or direct DNA
injection was used, transgene
350 and
333 remained episomal,
whereas in our study these transgenes were exposed to an additional
level of regulation imposed by chromatin architecture or nuclear
microenvironment, and perhaps in this context the 5'-terminal location
of the
NRE conferred a dominant negative effect on transgene transcription. In fact, the
NRE has been reported to act in a positional manner in that its 5' location more effectively suppressed expression of both heterologous and
MyHC promoter/reporter
constructs (8). Similarly, the expression of a vascular smooth muscle
-actin reporter construct was shown to be repressed when a negative regulatory element was placed 5' and adjacent to an enhancer element containing an MCAT sequence (33). It is noteworthy that in the latter
experiments, the positioning of a negative element located at the
5'-terminal end and adjacent to a MCAT element is identical to the
linear arrangement of elements within transgene
350 used in this study.
CNBP Is Not a Component of the Highly Enriched d
NRE-S Binding
Complex--
As a first step toward identifying the protein(s) that
bind to the d
NRE-S binding site, and to determine if CNBP was a
component of the binding complex, we performed EMSA using nuclear
extracts isolated from control and NWB soleus muscle. At present, the
cellular function of CNBP has not been defined; however, multiple lines of evidence implicate CNBP as a regulator of both transcription and
translation. Convincing evidence that CNBP is involved in translational
control has recently been provided by Pellizzoni et al.
(34), who show that CNBP binds a highly conserved target sequence
within the 5'-untranslated region of ribosomal protein mRNAs. It is
thought that this type of interaction modulates the efficiency of
ribosomal protein mRNA translation and thus ribosome production.
Further support for a role for CNBP as a translational regulator comes
from the studies with mouse liver cells indicating that CNBP was
exclusively found in the cytoplasmic compartment (35). On the other
hand, the notion that CNBP functions as a transcription factor is based
on its documented binding to promoter elements of target genes that
have high sequence similarities to the 3-hydroxy-3-methylglutaryl CoA
reductase SRE (GTGC/GGGTG), and its ability to regulate different
promoter/reporter gene constructs in diverse cell contexts (30). For
example, a recent study has demonstrated that both CNBP
and CNBP
bind the
MyHC gene
NRE and that overexpression of CNBP
in
primary cardiocytes led to decreased
MyHC reporter gene expression
(9).
In this study, using EMSAs we have identified d
NRE-S binding
activity that is sequence-specific and highly enriched only in NWB-S
nuclear extracts (Fig. 4, A and B). While CNBP
has been shown to bind the
NRE and regulate the expression of
MyHC reporter gene constructs in cultured cardiac cells (9), our
experiments confirm that CNBP can bind the
NRE; however, we provide
multiple lines of evidence that CNBP is not a regulator of
MyHC
expression during NWB activity. First, an antibody that recognizes CNBP
detected its presence only in cytoplasmic extracts where it was more
abundant in NWB-S than control muscles (Fig. 5B). Second, in
mobility shift assays, the binding complex formed between d
NRE-S and
NWB-S nuclear extracts had a low mobility, and the complex was neither
abolished by the presence of the metal ion chelator 1,10-phenanthroline nor supershifted by antibody recognizing CNBP. In striking contrast, the binding complex formed with HeLa nuclear extracts, a known source
of CNBP, had a high mobility, was abolished by the metal ion chelator
1,10-phenanthroline, was subsequently reconstituted by the addition of
ZnCl2, and was supershifted by antibody recognizing CNBP
(Fig. 6). Third, by UV cross-linking and shift Southwestern analyses,
single-stranded binding proteins with greater molecular mass (50 and 52 kDa) than the 19-kDa CNBP were identified (Fig. 4, C and
D). An additional important finding that further
characterizes the d
NRE-S binding activity found in NWB-S nuclear
extracts is that metal ions are not required for binding to the
d
NRE-S site.
YB-1 Is Not a Component of the Highly Enriched d
NRE-S Binding
Complex--
Like CNBP, various Y-box binding factors have been shown
to regulate gene expression at the level of translation by binding to
mRNA, while other members interact with single and double-stranded DNA to regulate gene transcription (11). Transcriptional control by
Y-box proteins generally occurs by binding to regulatory elements containing a consensus inverted CCAAT box sequence located within the
transcriptional control region of a gene. Accumulating evidence has
implicated YB-1 as a general regulator of transcription and as playing
a role in directing tissue-specific expression in a variety of cell
contexts including muscle (11). For example, in vitro
expression assays have shown that a highly conserved 27-bp region (
87
to
60), composed of a Y-box and an adjacent upstream MCAT element,
was sufficient to confer muscle specific expression on the
c-fos basal promoter (12). Also, in vitro overexpression assays showed that two Y-box-binding factors, MY1 and
MY1a, led to decreased expression of the nicotinic acetylcholine receptor
-subunit promoter/reporter constructs only when a 47-bp (
47 to
1) activity-dependent (innervation) enhancer
element was present and that both Y-box factors bound specifically to the sense strand of this enhancer element (13). In addition to skeletal
muscle, Y-box binding factors have also been implicated in directing
cardiac specific expression. Specifically, YB-1 and an associated
factor have been shown to bind to the HF-1a site, which contains an
inverted CCAAT-like element and acts in conjunction with an adjacent
HF-1b/MEF2 site to confer ventricular specific expression to MLC-2v
reporter constructs and transgenes (14). Finally, independent
experimental approaches have shown that the ubiquitously expressed
Y-box binding factor YB-1 interacts with the
MyHC gene
NRE
(10).
Due to the relevance of recent findings demonstrating the involvement
of YB-1 in directing cardiac and skeletal muscle-specific gene
expression, we explored the possibility that YB-1 may be a component of
the NWB-S single-stranded binding activity identified in our mobility
shift assays. However, despite previous observations, our experiments
provide evidence that eliminates YB-1 as a component of the enriched
d
NRE-S binding activity in NWB-S extracts. Most compelling is that
in supershift EMSAs, a polyclonal YB-3 antibody did not abolish the
formation of a binding complex when using NWB-S nuclear extracts,
whereas it effectively supershifted the specific complex formed when
using partially purified YB-1 protein (Fig. 7, lane
6 versus lane 10).
Additionally, in competition EMSAs, the use of mutant forms of the
d
NRE-S site revealed the sequence-specific nature of the
single-stranded binding activity in NWB nuclear extracts, since mutant
forms did not compete with wild type d
NRE-S for complex formation
(Fig. 4B). These results were further confirmed when the
MLC2v HF-1 binding site was found to be qualitatively a less effective
competitor of complex formation between d
NRE-S and NWB-S nuclear
extracts, despite the high degree of sequence similarity (>85%) to
the d
NRE-S site (Fig. 7, lanes 1-3). In
addition, partially purified recombinant YB-1 protein bound to the
NRE antisense strand (data not shown), whereas this strand was not
bound by NWB-S nuclear extracts (Fig. 4A). Furthermore, an
antibody that recognizes YB-1 detected its presence in cytoplasmic extracts obtained from both NWB-S and CS muscles, where its abundance did not appear to differ qualitatively. Although YB-1 protein was
present in CS nuclear extracts, albeit at lower levels than in
cytoplasmic extracts, it was barely detectable in NWB-S nuclear extracts, effectively eliminating it as a potential regulator of gene
transcription (Fig. 5C). The low abundance of detectable nuclear localized YB-1 protein is partially corroborated by our Northern hybridization, which showed a 28% decrease in YB-1-specific mRNA following 2 weeks of NWB activity (Fig. 5A).
The in vivo identification of DNA regulatory element(s) and
their corresponding binding protein(s) that regulate
MyHC gene expression in response to MOV and NWB activity is important for a
better understanding of skeletal muscle adaptation to altered neuromuscular activity and fiber type-specific expression. This study
provides the first persuasive in vivo evidence that the regulatory element(s) directing
MyHC gene expression in response to
the diverse physiological stimuli of MOV and NWB activity segregate to
distinct regions within the
MyHC gene-proximal promoter. Although the exact mechanism underlying increased expression of transgene
293
in response to NWB activity is unclear at present, our findings suggest
that the
NRE may be involved in NWB-dependent decreases in
MyHC gene expression. This conclusion is based on our transgenic analysis showing that a 57-bp region harboring the
NRE added to the
5'-terminal end of transgene
293 results in a transcriptionally repressed transgene (
350) (Fig. 1). This conclusion was further supported by results indicating that enriched binding activity at the
d
NRE-S site was only seen under NWB conditions in EMSAs. While the
exact components of the d
NRE-S binding complex remain to be
identified, UV cross-linking and shift Southwestern analyses showed
that two proteins of approximately 50 and 52 kDa comprised the
d
NRE-S binding activity in NWB-S nuclear extracts. The
identification of these components induced by NWB activity will not be
trivial, since these pursuits will require extensive and careful
investigation incorporating intact animal models to ensure the
preservation of physiologically relevant regulatory pathways.
Nevertheless, our results provide critical insights essential for
determining how muscle cells transduce changing mechanical loads into
changes in transcription levels of specific genes. The elucidation of these mechanisms will probably lead to the development of
countermeasures aimed at preventing muscle wasting accompanying various
disease states and zero gravity exposure.
 |
ACKNOWLEDGEMENTS |
We thank P. Kingsley for the CNBP
cDNA, D. Levens for the CNBP antibody, P. Umeda for the YB-1
cDNA and protein, W. Reynolds for the Xenopus YB-3
antibody, and K. Chien for the YB-1 antibody. We also thank M. Hannink
and G. Weisman for critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants R01 AR41464 (to R. W. T.) and F32 AR08412 (to J. J. M.).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.
To whom correspondence should be addressed: Dept. of
Veterinary Biomedical Sciences and Dept. of Biochemistry, University of
Missouri, 1600 E. Rollins Ave., W112 VET Medicine Bldg., Columbia, MO
65211. Tel.: 573-882-6253; Fax: 573-884-6890; E-mail:
tsikar{at}missouri.edu.
2
J. McCarthy and R. W. Tsika, manuscript in preparation.
3
D. Vyas and R. Tsika, manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
MyHC, myosin heavy
chain;
MyHC,
-myosin heavy chain;
NRE,
MyHC negative
regulatory element;
d
NRE, distal region of
NRE;
d
NRE-S, distal
region of the
NRE sense strand;
CNBP, cellular nucleic acid-binding
protein;
YB-1 and -3, Y-box-binding factor 1 and 3, respectively;
MOV, mechanical overload;
NWB, non-weight-bearing;
bp, base pair(s);
CS, control-soleus;
NWB-S, NWB-soleus;
CP, control-plantaris;
MOV-P, MOV-plantaris;
CAT, chloramphenicol acetyltransferase;
DTT, dithiothreitol;
EMSA, electrophoretic mobility shift assay.
 |
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