Department of Biology, Williams College, Williamstown, Massachusetts 01267
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ABSTRACT |
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The myosin heavy chain (MHC) IIB gene is preferentially expressed in fast-twitch muscles of the hindlimb, such as the tibialis anterior (TA). The molecular mechanism(s) for this preferential expression are unknown. The goals of the current study were 1) to determine whether the cloned region of the MHC IIB promoter contains the necessary cis-acting element(s) to drive fiber-type-specific expression of this gene in vivo, 2) to determine which region within the promoter is responsible for fiber-type-specific expression, and 3) to determine whether transcription off of the cloned region of the MHC IIB promoter accurately mimics endogenous gene expression in a muscle undergoing a fiber-type transition. To accomplish these goals, a 2.6-kilobase fragment of the promoter-enhancer region of the MHC IIB gene was cloned upstream of the firefly luciferase reporter gene and coinjected with pRL-cytomegalovirus (CMV) (CMV promoter driving the renilla luciferase reporter) into the TA and the slow soleus muscle. Firefly luciferase activity relative to renilla luciferase activity within the TA was 35-fold greater than within the soleus. Deletional analysis demonstrated that only the proximal 295 base pairs (pGL3IIB0.3) were required to maintain this muscle-fiber-type specificity. Reporter gene expression of pGL3IIB0.3 construct was significantly upregulated twofold in unweighted soleus muscles compared with normal soleus muscles. Thus the region within the proximal 295 base pairs of the MHC IIB gene contains at least one element that can drive fiber-type-specific expression of a reporter gene.
muscle; transcription; somatic gene transfer; luciferase; fiber type
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INTRODUCTION |
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SKELETAL MUSCLE FIBERS are heterogeneous with respect to contractile properties. Skeletal muscle fiber diversity is attributable to differential expression of isoforms of contractile proteins, including myosin isoforms and several thin filament proteins. Differential expression of myosin isoforms can dictate contractile properties such as maximal velocity of shortening and force-velocity characteristics (6). Myosin molecules are hexameric; they consist of two myosin heavy chain (MHC) proteins and two pairs of myosin light chain proteins. Differentiated mammalian muscle fibers have been shown to be capable of expressing at least four isoforms of MHC: slow type I, intermediate type IIA, fast type IIX, and fast type IIB (reviewed in Refs. 20 and 25). Distinct genes encode each of these MHC isoforms.
Muscle fibers that express different MHC isoforms are adapted for different motor tasks. Motor units used in short-duration, high-power-output activity, such as sprinting and power lifting, express primarily the IIB and IIX MHC isoforms (25). Fast motor units used for endurance activity, i.e., the deep red regions of most muscles, abundantly express the IIA MHC isoform (25). Fibers that express the type I MHC are abundant in muscle groups adapted for sustained periods of tonic contractile activity, such as anti-gravity function or joint stabilization. Expression of this isoform predominates in the soleus and vastus intermedius muscles, two slow-twitch muscles of the hindlimb.
There is an extensive body of evidence characterizing the adaptive
changes in MHC phenotype in response to various types of mechanical and
hormonal environments (reviewed in Refs. 20 and 25). Of critical
importance to the current study, muscle unweighting, induced either by
hindlimb suspension (12) or spaceflight (14), causes a slow- to
fast-type fiber transition whereby the MHC IIB is expressed de novo in
the soleus muscle. Transcriptional processes have been shown to
regulate in part expression of some members of the MHC gene family,
including the MHC IIB gene (10), cardiac -MHC (2, 27, 33), and
cardiac
-MHC (also the type I MHC in skeletal muscle; see Refs. 2,
27, and 33). However, the underlying mechanisms, i.e., signaling
pathways and transcription factors, that determine diversity and
adaptability among skeletal muscle fibers remain to be elucidated. The
lack of a cell culture system that can accurately mimic physiological
phenomena such as hindlimb unweighting has impeded our understanding of
some of the molecular mechanisms that dictate maintenance and
transition of muscle fiber type. The purpose of this study was to
delineate the critical cis-acting
region(s) within the MHC IIB promoter that drive fiber-type-specific
expression of this gene in vivo. To accomplish this goal, somatic gene
transfer was used. Numerous groups have shown that plasmid DNA can be
injected directly into muscle for adequate expression of reporter genes
(1, 5, 7-9, 17, 19, 23, 35, 36, 38). In the current study, the
murine IIB promoter was linked to a firefly luciferase reporter gene.
It is presented here that only the proximal 295 base pairs (bp) of the
MHC IIB promoter are required for both
1) muscle fiber-type-specific expression of reporter gene activity and
2) appropriate upregulation of
reporter gene activity within the unweighted soleus muscle.
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MATERIALS AND METHODS |
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Cloning and generation of deletion constructs for the murine IIB
promoter.
Polymerase chain reaction (PCR) primers were designed to anneal to the
sequenced region of the MHC IIB promoter (30). Using high-fidelity long
PCR (Boehringer Mannheim, Indianapolis, IN) from mouse genomic DNA, the
PCR product generated was cloned upstream of the firefly luciferase
gene (pGL3basic, Promega, Madison, WI) and termed pGL3IIB2.6. This
construct contains 2,560 bp of sequence upstream of the transcription
start site (TSS) and 13 bp downstream of the TSS. Four deletion
constructs were made by using pGL3IIB2.6 as a template. pGL3IIB0.3 and
pGL3IIB0.06 are constructs that contain 295 and 63 bp, respectively, of
sequence upstream of the TSS. To generate these constructs, pGL3IIB2.6
was digested with Asp718, which lies
upstream of the most 5'-sequence (part of the pGL3 vector), and
either EcoR I (pGL3IIB0.3) or
Pvu II (pGL3IIB0.06). The DNA was
recircularized with blunt ligation after the ends were polished with
Klenow. pGL3IIB0.88 was generated by restriction of pGL3IIB2.6 with
Pvu II, which cleaves the promoter
region at positions
63 and
943 upstream of the TSS. The
product, missing the internal 880 bp, was recircularized with blunt
ligation. The pGL3IIB1.3 construct was generated using PCR. The
5'-primer was designed to anneal from position
1282 to
position
1259 upstream of the TSS. The PCR product was cloned
upstream of the firefly luciferase reporter gene in the
Xho I site of the multiple cloning region.
Preparation of DNA for injections. Plasmid DNA for injections was prepared from large-scale growth of bacteria harboring the plasmid of interest. The plasmid DNA was purified using Qiagen columns, resuspended in sterile phosphate-buffered saline, quantitated using 260-nm OD spectrophotometric measurements, and stored at a concentration of 4 mg/ml.
DNA injections. Plasmid DNA for the experimental construct (pGL3IIB2.6 or derivative) was combined with pRL-CMV plasmid DNA. pRL-CMV is a CMV-driven renilla luciferase reporter gene (Promega). The final concentration of each construct was 2 mg/ml. The DNA solution was pipetted into an insulin-syringe fitted with a 28-gauge needle. Animals were anesthetized using a cocktail [(in mg/kg body wt) 25 ketamine, 1 acepromazine, and 5 xylazine], and the hindlimbs were shaved. Fifty microliters of the DNA solution (100 µg of each construct) was directly injected into tibialis anterior (TA) muscle. The insertion point of the needle was ~1 cm on the proximal side of the ankle joint. The injection tract ran parallel with the muscle fibers. To inject the soleus muscle, an incision was made on the lateral aspect of the hindlimb. The distal one third of the soleus was isolated for visualization. Twenty-five microliters of the DNA solution (50 µg of each construct) were injected into the soleus muscle. The hindlimb was stitched using 5-0 suture, and the animal was returned to its cage.
Determination of reporter gene activity.
One week after the injections, the animals were killed, and the
injected muscles were quickly removed, trimmed of all connective tissue, weighed, and frozen in liquid nitrogen. Muscles were
homogenized in 2 ml of a buffer containing 100 mM
tris(hydroxymethyl)aminomethane, pH 7.8, 1 mM EDTA, 1 mM
dithiothreitol, 2 µg/ml aprotinin, 5 µg/ml leupeptin, 170 µg/ml
phenylmethylsulfonyl fluoride, and 0.7 µg/ml pepstatin. The
homogenate was centrifuged at 4°C for 15 min at 10,000 g. The supernatant was stored at
80°C until analysis. The protein extracts were brought to
room temperature, and 20 µl of these extracts were used to perform
the luciferase assays. To measure reporter gene activity, the
dual-luciferase reporter assay (Promega) was used. This assay measures
and distinguishes luciferase activity from the firefly luciferase
protein and the renilla luciferase protein. Light from the chemical
reactions was integrated over 10 s using a Turner Designs luminometer.
All activities reported were standardized to a reading of 50% on the luminometer.
Immunoprecipitation and Western blot analysis of firefly luciferase. Firefly luciferase was immunoprecipitated from muscle extracts using a commercially available polyclonal antibody directed against firefly luciferase (Promega) and protein G Sepharose (Boehringer Mannheim) using the manufacturer's recommendations. After immunoprecipitation using 5 µl of the antibody, the eluted sample was separated on a 10% sodium dodecyl sulfate gel, transferred to a membrane (Bio-Rad, Richmond, CA), and incubated with the luciferase antibody. A chemiluminescence kit (New England Biolabs, Boston, MA) was used for detection of firefly luciferase on the membrane. As a control, 1 ng of purified firefly luciferase (Promega) was also loaded on the gel.
Hindlimb suspension.
Sprague-Dawley rats were assigned to one of two groups:
1) normal control
(n = 11) and
2) hindlimb suspension
(n = 11). For hindlimb suspension, the
basic protocol of Diffee et al. (12) was used. Five animals in each
group were suspended for 3 wk. At that time, the animals were killed
and the soleus muscles were removed, weighed, quickly frozen in liquid
nitrogen, and stored at 80°C until analysis. The other six
animals in each group were suspended for 2 wk. At that time, the soleus
muscles from both normal and hindlimb suspension animals were injected
with 50 µg of both pGL3IIB0.3 and pRL-CMV. Care was taken to prevent
any weight bearing in the hindlimb suspension animals during
preparation and recovery from the injection surgery as well as at the
time of death. After one additional week of suspension of the hindlimb suspension group, the animals were killed and the soleus muscles were
removed, weighed, and stored as above. Luciferase assays were performed
on muscle extracts as described in Determination of
reporter gene activity.
MHC mRNA analysis.
To extract total RNA, muscles were homogenized in 4 M guanidinium
thiocyanate, 20 mM sodium acetate, and 100 mM -mercaptoethanol. Homogenates were extracted in phenol-chloroform and precipitated with
isopropanol. RNA pellets were incubated in 4 M LiCl for 30 min on ice
and resuspended in formamide. Electrophoresis and transfer of RNA were
performed as described previously (26). Blots were probed with an oligo
probe specific for the IIB MHC mRNA (28), stripped, and subsequently
probed with an oligo probe specific for 28S rRNA.
Statistics. Data for the variables studied are reported as means ± SE. Statistically significant differences were determined using standard t-tests (hindlimb suspension) or Tukey's test (deletional analysis) after an analysis of variance. The 0.05 level of confidence was accepted for statistical significance.
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RESULTS |
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Verification of the use of a second normalizing gene construct. One inherent property of injections of plasmid DNA into rat muscle is the variability in uptake of that DNA between muscles and the resultant variability in reporter gene activity (35, 38). A crucial first experiment was performed to determine whether the variability in DNA uptake and reporter gene expression could be normalized using a second construct that is constitutively active. Figure 1 shows that when two promoters, CMV-driven firefly luciferase (pGL3-CMV) and CMV-driven renilla luciferase (pRL-CMV) were co-injected into the rat TA muscle, the relative expression of the reporter genes was the same across a 10-fold difference in activity. Firefly luciferase activity strongly correlated (r2 = 0.89) with renilla luciferase activity (Fig. 1).
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Deletion analysis of the MHC IIB promoter in the TA muscle.
Using the known sequence, the promoter of the MHC IIB gene was
amplified with PCR and cloned upstream of the firefly luciferase gene
(pGL3IIB2.6). This construct contains 2,560 bp upstream of the TSS of
the MHC IIB gene. Four separate deletion mutants of the MHC IIB
promoter linked to the firefly luciferase were made (Fig.
2). These contain 1,282 bp upstream
of the TSS (pGL3IIB1.3), 295 bp upstream of the TSS
(pGL3IIB0.3), 63 bp upstream of the TSS (pGL3IIB0.06), and an
internal deletion of 880 bp within the context of pGL3IIB2.6
(pGL3IIB0.88: between
63 and
943). These constructs
were co-injected with pRL-CMV into the TA muscle, a fast-twitch
dorsi-flexor muscle that expresses the IIB gene. One week after
injections, the activities of firefly and renilla luciferases within
muscle homogenates were determined (Table
1). Renilla luciferase expression derived
from the pRL-CMV construct was relatively constant between the
different sets of injections. When injecting 2.6 kb of the MHC IIB
promoter linked to a firefly luciferase reporter gene (pGL3IIB2.6) into
the rat TA, firefly luciferase activity was ~1/20 the activity of the
strong CMV-driven renilla luciferase (Table 1). When pGL3IIB1.3 or
pGL3IIB0.3 was injected into the TA, similar levels of firefly
luciferase activity were observed. However, when pGL3IIB0.06 was
injected, activity of the firefly luciferase gene significantly dropped
30-fold (Table 1). Similar results were obtained when 880 bases of the
promoter (pGL3IIB
0.88) were deleted within the context of the 2.6-kb
promoter. The decrease in activities from these two promoters was
likely not a result of lack of uptake of DNA by the muscle; both sets of injections showed renilla luciferase activity equivalent to that of
the other sets of injections (Table 1). Firefly luciferase activity in
muscles injected with either pGL3IIB0.06 and pGL3IIB
0.88 was
slightly above background, as determined in pGL3basic controls (Table
1). This suggests that a crucial region for high level of expression in
the rat TA of the MHC IIB gene lies between bases
295 and
63.
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Slow- vs. fast-twitch muscle expression of the IIB MHC promoter.
To determine if any or all of these MHC IIB promoter constructs contain
the necessary cis-acting element(s)
required for muscle fiber-type-specific expression of the reporter
gene, each construct was injected into the soleus muscle, a slow-twitch
muscle of the hindlimb that does not normally express the MHC IIB gene.
When 50 µg of pGL3IIB2.6 was co-injected with 50 µg of pRL-CMV into the soleus muscle, firefly luciferase activity was significantly less
than in the TA (Fig. 3). This was also true
for pGL3IIB1.3 and pGL3IIB0.3. The firefly luciferase activity derived
from both pGL3IIB0.06 and pGL3IIB0.88 was similarly very low in the
TA and the soleus. Because it is possible that the soleus muscle may
not express the firefly luciferase gene well, it was critical to inject
a different promoter to drive the firefly luciferase. As a
positive control, a skeletal
-actin promoter driving firefly luciferase (HSA2000, a kind gift from Dr. Robert Wade) was co-injected with pRL-CMV into the soleus and TA muscles. Unlike the MHC IIB gene,
the endogenous skeletal
-actin is expressed equally in both fast-
and slow-twitch muscle. The firefly luciferase activity from this
reporter gene construct was not significantly different between the
soleus and TA muscles (Fig. 3, P > 0.05). The lack of expression of the pGL3IIB-derived constructs in the
slow-twitch soleus muscle was thus likely due to the
fiber-type-specific expression pattern of this gene and suggests that
at least one cis-acting element(s)
required for fiber-type-specific expression of this promoter lies
downstream of position
295.
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Induction of activity of the IIB promoter with hindlimb suspension. The lack of weight bearing in the soleus of a rat undergoing hindlimb suspension induces an increase in MHC IIB mRNA expression in the soleus muscle (Fig. 5A and Refs. 12 and 14). Animals were placed into two groups: normal and hindlimb suspended. After 3 wk of suspension, soleus muscle weight to body weight ratios significantly declined (0.417 ± 0.017 vs. 0.233 ± 0.012 mg/g, P < 0.05). IIB mRNA was not detected in any of the control soleus muscles but was expressed in the unweighted soleus muscles at a level ~10% of that in the TA (Fig. 5, A and B). In animals not used for RNA analysis, pGL3IIB0.3 and pRL-CMV were co-injected into the soleus muscles of normal and hindlimb-suspended rats after 2 wk of suspension, and the hindlimb-suspended rats were suspended for one additional week. Renilla luciferase activity did not vary between the two groups (normal, 7466 ± 2168; hindlimb suspended, 10421 ± 2471, P > 0.05). As Fig. 5B shows, hindlimb suspension induced a significant twofold increase in firefly luciferase activity in the soleus muscle.
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DISCUSSION |
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Major findings. The primary findings of this study were 1) demonstration that only 295 bp of the proximal upstream regulatory region of the MHC IIB gene were required for accurate reproduction of endogenous gene expression in vivo and 2) that same region contained at least one element that was responsive to unweighting in the soleus muscle.
Use of DNA injection in vivo for the study of gene regulation.
Recently, the use of somatic gene transfer into muscle has been shown
to be a viable technology for the study of transcriptional regulation
of gene expression in muscle (1, 5, 7-9, 17, 19, 23, 35, 36, 38).
Initial studies by Wolff et. al. (38) demonstrated an inherent
variability of reporter gene activity after injection into muscle. It
was also shown that a second vector co-injected could correct for that
variability (36, 38), a finding confirmed here (Fig. 1). On analysis of
the data presented in Table 1, it is apparent that the variability of
the firefly luciferase activity was substantial, with standard errors
ranging from one-third to one-half of the mean value. This was also
true for renilla luciferase activities. After normalization of the firefly luciferase activity to the renilla luciferase activities, it
became quite apparent that the variance in firefly luciferase activity
from any given promoter was a function of the efficiency of gene
transfer. Thus somatic gene transfer appears to be a feasible tool for
probing transcriptional control in vivo. In fact, other investigators
using DNA injections have demonstrated an element of the skeletal
-actin gene responsive to muscle overload (7, 8), a
slow-muscle-specific element within the troponin I slow promoter (9), a
pressure overload response element within the c-fos promoter (1),
cardiac expression of the
-MHC gene (36), differential expression of
the creatine kinase M gene in cardiac and skeletal muscle (35), and
thyroid responsiveness of the IIB MHC gene (23) and the
-MHC gene
(5, 17, 19).
Genetic control of fiber-type-specific expression of IIB
reporter genes.
Three constructs tested here, pGL3IIB2.6, pGL3IIB1.3, and pGL3IIB0.3,
all generated luciferase activities well above background, as
determined by injections of the parent vector, pGL3basic (Table 1 and
Fig. 3). As Fig. 3 shows, this IIB promoter elicited greater activity
than a previously tested skeletal -actin promoter (9), although some
of this difference may be attributable to different parent vectors
(pGL3 vs. pGL2). No significant decrease in promoter activity in the TA
was detected between pGL3IIB2.6 and pGL3IIB0.3, suggesting that the
regulatory elements that drive high-level expression in the TA are
likely located downstream of
295 (the most 5'-end of
pGL3IIB0.3). The pGL3IIB0.3 construct, as well as the larger
constructs, displayed fiber-type specificity. Expression in the soleus
muscle, which does not express the IIB mRNA or protein at detectable
levels (Fig. 5A), was only one-tenth
of the level in the TA (Fig. 3). Thus the region upstream of the most
5'-end of the pGL3IIB0.06 must contain at least one element
required for high levels of reporter gene activity and may contain an
element that drives expression specifically in IIB-expressing fibers. Certainly there may be elements upstream of the 5'-end of the pGL3IIB0.3 promoter that may be able to direct high levels of muscle
fiber-type-specific expression of reporter genes. However, the data
obtained in the current study suggest that the region between
295 and
63 of the IIB promoter is necessary for muscle fiber-type-specific expression of reporter genes.
Genetic control of fiber-type adaptation.
When exposed to various loading conditions, skeletal muscle has the
ability to alter gene expression (20, 25). The MHC IIB gene is
exquisitely regulated by weight bearing in the soleus muscle. The
normal rat soleus does not express detectable levels of the MHC IIB
mRNA by Northern blot analysis, whereas after 3 wk of hindlimb
suspension, the unweighted soleus muscle expresses the MHC IIB mRNA
(Fig. 5). Although this mRNA is de novo expressed, the level of
expression in the unweighted soleus of the MHC IIB mRNA was much less
than in the normal TA muscle (Fig. 5). Firefly luciferase expression
off of the injected pGL3IIB0.3 reporter construct was also upregulated
significantly in the unweighted soleus muscle above that of the normal
soleus muscle and was less than that of the normal TA (Fig. 5). Thus
the region downstream of 295 of the MHC IIB promoter contains at
least one element that was responsive to unweighting in the soleus
muscle. The cis-acting element(s)
within the 295-bp region responsible for load responsiveness and
fiber-type-specific expression of the IIB gene remain to be determined.
It has been recently shown that only 293 bp of the mouse
-MHC
promoter is required for appropriate upregulation in the functionally
overloaded plantaris muscle (32) and downregulation in the unweighted
soleus muscle (18). Interestingly, those elements of the
-MHC
promoter shown to be important for high levels of expression in cell
culture (31) are not required for either the upregulation (32) or
downregulation (18) of expression from this promoter. Differences in
analysis of cis-acting elements within
the rat
-MHC promoter have also been found between cell culture and
whole animal studies (5). Thus the present data suggest that the use of
somatic gene transfer to identify important cis-acting elements for
fiber-type-specific expression of the MHC IIB gene appears to be both
feasible and the appropriate approach.
Elements within the MHC IIB gene.
Because only the proximal 295 bp are required for fiber-type-specific
activity, the region downstream of 295 likely contains at least
one cis-acting element that drives
fiber-type-specific expression. Within this region are numerous
candidate elements that may bind transcription factors in a
fiber-type-specific manner. The MHC IIB gene has a typical TATA box
located
27 relative to the TSS which cannot be exchanged for
another TATA box, implicating the importance of the context with which
the TATA box resides (11). There is also a CArG box, found in numerous
genes expressed in a muscle-specific fashion (35), located at
109 to
88. The MHC IIB gene also has three A-T-rich
regions within the proximal 220 bp of the TSS which may bind members of
the myocyte enhancer factor 2 family (4, 13, 39). An E-box
(centered around
63) which serves as an element for
transcription factors of the basic helix-loop-helix family, including
the four identified myogenic proteins (MyoD, MRF-4, Myf-5, myogenin)
also lies within the proximal 295 bp. Interestingly, MyoD and myogenin
have been shown to preferentially accumulate in fast and slow adult
muscles, respectively (15, 16, 37). The A-T elements, CArG box, and the
E-box are missing from both pGL3IIB0.06 and pGL3IIB
0.88. Both of
these reporter gene constructs demonstrate low activity in all muscle
(Fig. 3) and in cell culture (data not shown). It remains to be
determined which, if any, of these elements is required for the
high-level activity in skeletal muscle, and further, high-level
activity in fast-twitch muscle.
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ACKNOWLEDGEMENTS |
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Special thanks are given to Matt Wheeler and Melissa Patterson.
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FOOTNOTES |
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This work was supported in part by National Science Foundation Grant IBN-9723351.
Address reprint requests to S. J. Swoap.
Received 24 July 1997; accepted in final form 1 December 1997.
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