Received for publication, February 8, 2001, and in revised form, February 23, 2001
Skeletal myofibers of vertebrates acquire
specialized metabolic and physiological properties as a consequence of
developmental cues in the embryo and different patterns of contractile
activity in the adult. The myoglobin gene is regulated stringently in
muscle fibers, such that high myoglobin expression is observed in
mitochondria-rich, oxidative myofibers (Types I and IIa) compared with
glycolytic fibers (Type IIb). Using germ-line transgenesis and somatic
cell gene transfer methods, we defined discrete regions of the murine and human genes encoding myoglobin that are sufficient to confer muscle- and fiber type-specific expression to reporter genes. Mutational analysis confirms the importance of A/T-rich, MEF2-binding motifs in myoglobin gene regulation, as suggested by previous studies
using different experimental approaches. In addition, we demonstrated a
previously unsuspected role for an intragenic E-box motif as a negative
regulatory element contributing to the tightly regulated variation in
myoglobin gene expression among particular myofiber subtypes.
 |
INTRODUCTION |
Skeletal myofibers of vertebrates acquire specialized metabolic
and physiological properties as a consequence of developmental cues in
the embryo and different patterns of motor neuron activity in the adult
(1-3). Among different myofiber subtypes, myoglobin is expressed
selectively in mitochondria-rich, oxidative fibers that express Type I
or Type IIa myosin. In contrast, fast glycolytic fibers that express
Type IIb myosin are virtually devoid of myoglobin. Differential
expression of myoglobin in particular subtypes of myofibers is not
apparent during embryonic or fetal life but is established within the
early post-natal period (4). In adult mammals, myoglobin expression is
modulated by environmental stimuli including chronic hypoxia, endurance
exercise training, and most potently by sustained changes in the
pattern of motor nerve activation (5-8).
Several molecular signaling mechanisms have been found to participate
in the control of myofiber specialization (2, 3, 9). Recently, we and
others have presented evidence that the protein phosphatase
calcineurin, in conjunction with calmodulin-dependent protein kinases, act upon NFAT and MEF2 proteins to transduce signals
generated by changes in intracellular calcium so as to regulate
transcriptional activity of oxidative muscle genes such as myoglobin
(10-13). These signaling pathways have been demonstrated clearly in
cultured skeletal myotubes, but their relevance to gene regulation
within the muscles of intact animals remains uncertain. In particular,
no previous studies have examined the function of specific
transcriptional control elements of the myoglobin gene in skeletal
muscles of intact animals. Here we show that sequences contained within
the proximal 5' flanking regions of either the murine or human
myoglobin genes are sufficient to confer muscle- and fiber
type-specific expression to a reporter gene in transgenic mice.
Analysis of expression patterns resulting from mutated forms of the
myoglobin promoter confirms the importance of A/T-rich, MEF2-binding
motifs in this process, as suggested by previous research using
different experimental approaches (14, 15). In addition, such studies
demonstrate a previously unsuspected role for an intragenic E-box motif
as a negative regulatory element contributing to the tightly regulated
variation in myoglobin gene expression among different myofiber subtypes.
 |
EXPERIMENTAL PROCEDURES |
Indirect Immunofluorescence--
Hind limb muscles
(gastrocnemius, soleus, and plantaris) were harvested from 6-week-old
mice, immersion-fixed in 4% paraformaldehyde overnight at 4 °C,
cryoprotected with 10% sucrose in phosphate-buffered saline,
and frozen in isopentane at
70 °C. Frozen sections (8 µm) were
hydrated in phosphate-buffered saline and immunostained for myoglobin
expression as previously described (4).
Transgenic Mice--
A
21-kb1 mouse genomic fragment
containing the complete myoglobin gene with all 3 exons and 2 introns
was isolated by screening a mouse genomic P1 library and was subcloned
as separate fragments into either pBluescript (Stratagene) or pGEM
(Promega) vectors. PCR-based mutagenesis was used to insert a
hemagglutinin (HA) epitope tag between the second and third codons with
no other disruptions of the genomic structure of the myoglobin gene.
The insertion generated an Asp718 restriction site at the
beginning of the HA tag to facilitate genotyping. The 21-kb myoglobin
transgene (Fig. 2A) carrying an HA tag was reassembled by
multiple steps of subcloning and verified by restriction enzyme mapping
and Southern blot analysis. DNA fragments containing either the
complete myoglobin transgene (HA-Mb) (Fig. 3A) or truncated
forms thereof (HA-Mb3'
and HA-Mb5'
) (Fig. 3A) were
gel-purified and introduced by microinjection into fertilized oocytes
of C57Bl6/C3H mice. The resulting transgenic offspring were identified
by Southern blot analysis of genomic DNA and used to establish
transgenic lines.
The proximal promoter region of the mouse myoglobin gene (
357 to +55)
was isolated by PCR using the P1 clone containing the myoglobin
gene as template with a forward primer 5'-AGCAAGATGCCTGTGCCCAA-3' and a
reverse primer 5'-GGAAGATCTGGTGGCTTCTAAAGAGGAC-3'. The resulting 430-base pair PCR product was subcloned into PCR II TA vector (Invitrogen), verified by sequence analysis. This myoglobin promoter region was cloned subsequently into the luciferase reporter plasmid pGL3 (Promega) at BglII and Asp718 sites to
construct the myoglobin-luciferase reporter plasmid Mb357-Luc.
PCR-based site-directed mutagenesis was then performed using the
following primers: Mb
E-box 1, forward (5'-GCTCCCACAATGGAGCTCGCCCCAAAATAGC-3') and
reverse (5'-GCTATTTTGGGGCGAGCTCCATTGTGGGAGC-3') (SacI); Mb
E-box 3, forward
(5'-GTGGCCTCAAATCTGCAGTGAGAGCCAGCCC-3') and
reverse
(5'-GGGCTGGCTCTCACTGCAGATTTGAGGCCAC-3')
(PstI); Mb
A/T, forward
(5'-GGCACTTGCCCCAAGCTAGCTGCCCATGTG-3') and
reverse (5'-CACATGGGCAGCTAGCTTGGGGCAAGTGCC-3')
(NheI).
These mutations disrupt putative binding sites for myogenic
regulatory proteins. Mutations were identified by novel restriction sites generated at the site of mutagenesis and confirmed by sequence analysis. The wild type or mutant DNA fragments were gel-purified and
introduced by microinjection into fertilized oocytes of C57Bl6/C3H mice. The resulting transgenic offspring were identified by Southern blot analysis of genomic DNA and used for founder analysis.
Immunoblot Analysis--
Tissues were harvested from either wild
type or transgenic mice, disrupted in lysis buffer (1×
phosphate-buffered saline, 1% Triton X-100, 20% glycerol, 10 mM EDTA, and protease inhibitor mixture (Roche
Molecular Biochemicals)), homogenized with a Teflon pestle, and cleared
by centrifugation. Proteins (10 µg) were separated by 12%
SDS-polyacrylamide gel electrophoresis and immunoblotted with
anti-myoglobin antibody (Dako) using the ECL detection system (Amersham Pharmacia Biotech). The intensity of bands was quantified by
PhosphorImager/Storm860 analysis (Molecular Dynamics) using ImageQuant software.
Regeneration Model--
Muscle degeneration/regeneration was
induced in Wistar rats as previously described by Schiaffino and
colleagues (16) using intramuscular injections of bupivacaine. Plasmid
DNA (50 µg) containing segments of the human myoglobin gene promoter
(19) was introduced into the soleus or extensor digitorum longus (EDL)
muscle 3 days after injury. Muscles were harvested 10 days after
bupivacaine injection and assayed for reporter activity.
Luciferase Assay--
Tissues were harvested from transgenic
mice at 7-10 weeks of age and assayed for expression of luciferase as
previously described (17).
 |
RESULTS |
Stringent Regulation of Myoglobin Protein Expression in Different
Myofiber Subtypes--
Indirect immunofluorescence analysis of the
hind limb muscles of the mouse illustrates the marked differences in
expression of myoglobin among different myofibers (Fig.
1). A high level expression of myoglobin
was detected in >90% of myofibers in the soleus muscle (Fig.
1B) as expected from previous analyses (4, 18). In the
gastrocnemius muscle, the percentage of fibers expressing myoglobin
decreases from >75% in the deep portion (Fig. 1B) to ~50% in the intermediate region (Fig. 1C) and to <10%
in the superficial region (Fig. 1D).

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Fig. 1.
Stringent regulation of myoglobin gene
expression in different myofiber subtypes. Myoglobin protein
expression in skeletal muscle of young adult mice (6 weeks old) was
detected by indirect immunofluorescence staining of a cross-section
from hind limb muscles. A, low magnification view oriented
with the deep portion on the left (adjacent to the tibia)
and the superficial portion on the right. High magnification
images are indicated as insets. B, the deep
portion of the hind limb muscles including the soleus (left)
and plantaris (right) muscles. C, intermediate
portion of the gastrocnemius muscles. D, superficial portion
of the gastrocnemius muscle. Each scale bar = 50 µm.
|
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Transcriptional Control of the Murine Myoglobin Gene Assessed in
Transgenic Mice--
In the first phase of this analysis, a 21-kb
genomic DNA region encompassing all 3 exons and intervening sequences
of the murine myoglobin gene, as well as 5 kb of 5' flanking DNA and 8.5 kb of 3' flanking DNA, was cloned and modified to serve a reporter
function by the insertion of an HA epitope tag into Exon 1 (Fig.
2A). The rationale for this
strategy was to preserve the native genomic organization as much as
possible. In addition, this approach permits direct assessment of
transgene expression in comparison to endogenous myoglobin gene
expression at the protein level within the same muscle extracts (Fig.
2B).

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Fig. 2.
Schematic diagram and expression pattern of
HA-tagged myoglobin transgene. A, a 21-kb mouse genomic
fragment contains the intact myoglobin gene with all 3 exons
(open boxes for untranslated regions and solid
boxes for coding regions) and 2 introns (solid lines between
boxes), a 5' flanking region (5 kb) and a 3' flanking region (8.5 kb). PCR-based mutagenesis was used to insert an HA tag
(oval with arrowhead) between the second
and the third codon. B, immunoblot of tissue extracts from a
transgenic mouse carrying the HA-tagged myoglobin transgene, HA-Mb,
using anti-myoglobin antibodies. The HA-tagged form of myoglobin
(HA-Mb) migrates more slowly in the gel than the endogenous
myoglobin (Mb) protein. Data from ImageQuant analysis of
PhosphorImager scanned immunoblots are presented as a percentage of the
endogenous myoglobin in the heart (from two founder lines
n = 4 transgenic animals). C, immunoblot
analysis of the developmental expression of HA-tagged myoglobin and
endogenous myoglobin in cardiac and skeletal muscles. Data from
ImageQuant analysis of PhosphorImager scanned blots are presented as a
percentage of the endogenous myoglobin expression in hearts of adult
mice (from one founder line n = 4 transgenic
animals).
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|
In transgenic mice established with the 21-kb myoglobin genomic
fragment, the transgene product (HA-Mb) was expressed in the tissues of
adult animals in a pattern that faithfully recapitulated that of the
endogenous myoglobin gene (Fig. 2B). Data from a different transgenic line carrying the same transgene within a different chromosomal insertion site gave the same result (not shown). There was
no apparent relationship between copy number of integrated transgenes
(estimated to be 3 and 21 in these two transgenic lines) and the
pattern of expression of HA-myoglobin. In addition, the developmental
timing of the transgene expression mirrored that of the endogenous gene
in the cardiac ventricles and atria and in the skeletal muscles of the
hind limb; however, the level of expression of HA-myoglobin protein
compared with endogenous myoglobin protein was less in adult skeletal
muscle (Fig. 2C).
The parent 21-kb transgene construction (HA-Mb-WT) was modified to
eliminate large segments of the 5' or 3' flanking regions. As
illustrated schematically in Fig.
3A, a construct designated as
HA-Mb
3' removed all but 0.5 kb from the 3' flanking region, whereas
a construct designated as HA-Mb
5' retained only 0.4 kb of the 5'
promoter regions. Transgenic mice carrying these truncated versions of
the myoglobin transgene expressed less HA-Mb as a fraction of
endogenous myoglobin (HA-Mb-WT; Fig. 3B, upper
panels) than mice carrying the HA-Mb transgene. However, the
normal pattern of expression among specific muscle groups
(e.g. soleus
white vastus lateralis) was maintained
even when most of the 3' and 5' flanking regions were deleted from the
transgene construction (Fig. 3B, lower panels).

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Fig. 3.
Schematic diagram and expression pattern of
HA-tagged myoglobin transgene constructs. A, the
full-length (21 kb) HA-tagged myoglobin transgene, HA-Mb, is presented
as in Fig. 2A. The 5' deletion
(HA-Mb 5') was constructed by removing a 4.5-kb
5' fragment using restriction enzymes BglII and
HindIII. The 3' deletion (HA-Mb 3')
was constructed by removing an 8.0-kb 3' fragment using restriction
enzymes BamHI and HindIII. B,
expression pattern of endogenous myoglobin or the myoglobin transgene
in soleus (SOL) and white vastus lateralis (WV)
muscles of transgenic mice. Data are presented relative to endogenous
myoglobin expression in the soleus muscle (top panel) or
relative to expression in the soleus muscle of either endogenous
myoglobin (Mb) or the transgene product (HA-Mb)
(bottom panel). The numbers of animals examined
were: HA-Mb-WT, n = 7; HA-Mb 3', n = 3; and HA-Mb 5', n = 6.
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Transcriptional Activity of the Human Myoglobin Gene Promoter in
Regenerating Muscles of the Rat--
Previous studies of the human
myoglobin gene promoter in cultured cells (14, 18) and following
somatic cell gene transfer into the hearts of intact rats (19)
suggested that important transcriptional regulatory elements reside in
the proximal 5' flanking region. Here we determined that a segment of
the human myoglobin gene extending from
373 to +7 base pairs relative
to the transcriptional initiation site is sufficient to direct
muscle-specific expression of a luciferase reporter gene in a pattern
that mirrors that of the endogenous myoglobin gene. Using a model
system characterized extensively by Schiaffino and colleagues (16),
plasmid DNA was introduced into the hind limb muscles of adult rats
that were undergoing regeneration following chemical injury. Human
myoglobin gene promoter fragments linking either 2 or 0.4 kb of 5'
flanking sequences to a luciferase reporter gene were expressed more
abundantly in the soleus (SOL) than the EDL muscles,
reflecting the pattern of the endogenous myoglobin gene (Fig.
4). Absolute levels of luciferase
expression were greater with the larger promoter fragment, pMb2kb-Luc,
but the fiber type-specific pattern was maintained with both the 2-kb
(pMb2kb-Luc) or the
373 to +7 promoter
(pMb380-Luc) fragments (Fig. 4, right
panel).

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Fig. 4.
Human myoglobin promoter after somatic cell
gene transfer. Luciferase activity was measured in regenerating
rat soleus and EDL muscles 7 days after intramuscular injection of the
human myoglobin promoter-reporter plasmids pMb2kb-Luc or pMb380-Luc or
of the cytomegalovirus promoter-reporter plasmid CMV-Luc. The
data were normalized to RSV-CAT activity and expressed as
absolute luciferase activity (left panel) or relative to
expression in soleus muscle (right panel). The
numbers of animals examined following injection of each
plasmid were: pMb2kb-Luc plasmid in soleus (SOL)
(n = 8) and in EDL (n = 10); pMb380-Luc
in soleus (n = 10) and in EDL (n = 7);
CMV-Luc in soleus (n = 4) and EDL (n = 4). RLU, relative light units.
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Specific Nucleotide Sequence Elements Required for Correct
Patterning of Murine Myoglobin Promoter Activity--
A proximal
promoter fragment of the murine myoglobin gene (
357 to +55) was
linked to a luciferase reporter gene, and this DNA construct
(Mb357-Luc) was used to generate transgenic mice. The sequence of this
genomic fragment is highly conserved among myoglobin genes of different
mammalian species (Fig. 5A).
As illustrated in Fig. 5B, the parent Mb357-Luc reporter
gene construct was modified by nucleotide substitutions to disrupt
specific nucleotide sequence motifs within the myoglobin promoter
(Mb
A/T-Luc, Mb
E-box1-Luc, and
Mb
E-box3-Luc). These sites were chosen for
mutational analysis on the basis of their conservation in myoglobin
genes of other mammalian species (Fig. 5A) and because of
previous data from cell culture models (14, 15, 19).

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Fig. 5.
Sequence elements controlling transcriptional
activity of the myoglobin gene promoter. A, DNA
sequences from mouse (mMb), human (hMb), rat
(rMb), and seal (sMb) myoglobin promoters
obtained from GenBankTM (accession numbers X04405,
X00371, AF278533, and V00471, respectively) were aligned by DNAstar.
Putative regulatory elements are indicated as: E-box1,
E-box2, and E-box3, for E-box motifs (CANNTG);
A/T for the AT-rich, MEF2 binding site; and TATA
for TATA box. The underlined letters below the boxed
sequence show nucleotide substitutions generated by site-directed
mutagenesis. The numbers designate the nucleotide positions
relative to the transcription start site of the mouse myoglobin gene.
B, schematic representation of myoglobin-luciferase reporter
gene constructs used in mutational analysis. The murine myoglobin 5'
promoter region from 357 to +55 was fused to the firefly luciferase
gene (Mb357-Luc). PCR-based site-directed mutagenesis
disrupted E-box1 (Mb E-box1- Luc), E-box3 (Mb E-box3-Luc), or the
A/T element (Mb A/T-Luc). C,
luciferase activities (corrected for protein concentration) in the
soleus (SOL) and white vastus lateralis (WV)
muscles from founder transgenic mice are expressed as absolute values
(upper panel in log scale), the means of absolute values
(middle panel), or the means of paired comparisons relative
to expression in the soleus muscle (lower panel). The
numbers of founder animals used was: for Mb357-Luc, n = 10; for Mb357 Ebox1-Luc, n = 5; for
Mb357 Ebox3-Luc, n = 8; and for Mb357 A/T-Luc,
n = 5. RLU, relative light units.
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The
357 to +55 segment of the murine myoglobin gene was sufficient to
direct high levels of luciferase expression in myoglobin-rich soleus
muscles as compared with myoglobin-deficient white vastus lateralis
muscles in each of 10 independent transgenic founder mice carrying this
transgene (Fig. 5C). In mice, the soleus muscle consists
almost entirely of Type I and Type IIa fibers (20) that express
endogenous myoglobin (Fig. 1). In contrast, more than 80% of fibers
within the white vastus lateralis muscle are Type IIb glycolytic fibers
within which the endogenous myoglobin gene is transcriptionally
inactive. Mb357-Luc was active selectively in muscle tissues, as
evidenced by the high ratio of luciferase activity in heart and soleus
skeletal muscle versus liver or lung (>20:1) from these
animals (not shown). This pattern was consistent in 10 independent
founder mice representing different chromosomal insertion sites.
Disruption of an A/T-rich, MEF2-binding motif markedly reduced the
expression of luciferase in the soleus muscles of transgenic mice
compared with the native myoglobin sequence
(Mb357
A/T-Luc versus Mb357-Luc; Fig.
5C). This result supports the hypothesis that MEF2 proteins
serve as important regulators of fiber type-specific gene expression
(13).
Disruption of an E-box motif adjacent to the A/T-rich, MEF2 binding
element in the construct Mb
E-box1-Luc had no discernable effect in
altering the pattern of transcriptional activity driven by the
380 to +15 myoglobin genomic fragment in transgenic mice (Fig.
5C). We observed in previous studies of the human myoglobin gene promoter in cultured skeletal myotubes that neither of the two
conserved E-boxes flanking the MEF2 binding site (E-box1 and E-box2 in
Fig. 5) was required for full activity of the myoglobin promoter
(14).
No previous studies had explored the function of a third conserved
E-box motif (E-box3 in Fig. 5A) that we noted to be present within the first exon of myoglobin genes from humans, mice, and rats.
Our current analysis of murine myoglobin gene promoter function in
transgenic mice indicates that this intragenic E-box motif located
downstream of the transcriptional start site (+5 to +10) within the 5'
untranslated region of exon 1 has an important function. In animals
carrying the Mb
E-box3-Luc transgene, luciferase expression was
increased in white vastus muscles compared with the activity of the
parent pMb357-Luc transgene (Fig. 5C). The Mb
E-box3-Luc construct was expressed in soleus muscle to a level equal to or greater
than that of the native myoglobin promoter sequence (Mb357-Luc). However, the ratio of expression in soleus versus white
vastus lateralis was reduced from ~10:1 to 2:1 (Fig. 5C).
The E-box3 mutation did not result in ectopic transcriptional activity
in non-muscle tissues, since the ratio of expression in soleus muscle relative to lung or liver was unaltered (data not shown).
 |
DISCUSSION |
Transcription factors and signaling events required to promote
myogenic commitment and differentiation have been defined in considerable detail (9, 21). By contrast, the molecular mechanisms by
which skeletal myofibers assume one of several highly specialized phenotypes are much less well understood. New findings presented in
this study extend the understanding of transcriptional control elements
within the myoglobin gene, which serves as a representative of a large
set of genes that establish the oxidative (Types I and IIa) myofiber
phenotypes. We observed that transcriptional control elements
sufficient to recapitulate the pattern of expression of the endogenous
myoglobin gene in adult mice are contained within a 21-kb mouse genomic
fragment encompassing the myoglobin gene. More detailed analyses
indicated that the correct pattern of transcriptional regulation of the
myoglobin gene (striated muscles
non-muscle tissues and
soleus > white vastus lateralis muscle) requires only a small
segment of the proximal 5' flanking region extending from nucleotides
357 to +55 relative to the transcriptional start site. Mutations
within two protein binding motifs within this region, an A/T-rich, MEF2
binding sequence and an intragenic E-box, disrupt the proper function
of this regulatory region. Proteins binding at the A/T-rich element
(including but not necessarily limited to MEF2) are required to support
high levels of transcription in myofiber subtypes of the soleus muscle
(Types I and IIa) that are rich in myoglobin. In contrast, proteins
binding at the intragenic E-box motif appear to repress transcription
selectively in myofiber subtypes that are predominant in white vastus
lateralis muscles (primarily Type IIb).
During the embryonic development of birds or fish, myogenic precursor
cells are directed to a fast or slow fiber fate by cues that include
sonic hedgehog signaling (22-25). However, the relevance of the
hedgehog pathway to the determination and maintenance of specialized
phenotypes in innervated adult myofibers has not been addressed. We
have proposed a model in which the dominant influence of the sustained,
tonic patterns of motor neuron activity that establish the slow (Type
I) or oxidative (Type I and IIa) myofiber phenotypes is directed to the
relevant target genes, at least in part, by MEF2 and NFAT proteins
under the post-translational regulation of calcineurin, CaMK
(calmodulin-dependent protein kinase), and possibly other
calcium-regulated signaling pathways (9, 12, 13, 26). This viewpoint
has been supported by data from other laboratories (10, 11), but a
central role for this mechanism has been questioned by others (27, 28). As we have discussed elsewhere (9), our model should not be interpreted
as excluding important roles for other transcription factors in
addition to MEF2 and NFAT in the mechanisms of fiber type-specific gene
regulation. To the contrary, compelling data implicate Ras (29), MusTRD
(30), SIX (31), and nuclear receptor (32) proteins in regulation of
certain genes that are expressed selectively in particular myofiber
subtypes. However, binding sites for MusTRD, SIX, and nuclear receptor
proteins are not evident within the control region of the myoglobin
gene that we have shown to be sufficient for soleus-specific
transcription. The results from the present analysis add support to the
notion that MEF2 transcription factors and other proteins that form
complexes at the myoglobin A/T-rich element are important to the
stringently regulated expression of myoglobin among different myofiber
subtypes (13).
Other new observations presented here are of particular interest in
light of previous data that suggest a role for bHLH proteins in fiber
type-specific gene regulation. Hughes and colleagues (33) noted an
increased ratio of myogenin to MyoD in soleus versus EDL
muscles of rats; they generated transgenic mice to overexpress myogenin
in fast, glycolytic fibers under the control of the myosin light chain
1/3 promoter/enhancer. Expression of this transgene increased the
abundance of oxidative fibers within the EDL, although it did not
promote increased expression of Type I myosin (34). These data
implicate bHLH proteins in the specialization of fast fibers into
oxidative and glycolytic subtypes, an issue pertinent to myoglobin gene
regulation. Our findings with respect to the myoglobin E-box3 element
provide the first evidence that bHLH proteins may participate directly
in fiber type-specific transcription of the myoglobin gene. The
identity of the repressor factor or factors that recognize this
particular E-box motif remains to be determined. It is possible that,
like MEF2 (35, 36), bHLH proteins of the MyoD family can form complexes
with co-repressors to negatively regulate gene expression in certain
cell backgrounds or in the absence of appropriate activating stimuli
that unmask their transcriptional activation domains. On the other
hand, other E-box binding proteins are known to function as
transcriptional repressors (37-42) and may serve as cognate factors
that recognize the myoglobin E-box3 element.
Stringent regulation of myoglobin gene expression among different
myofiber subtypes (Fig. 1) seems likely to demand both positive and
negative control mechanisms. This study provides evidence for such dual
regulation, opening new opportunities to enhance the fundamental
understanding of the molecular basis of myofiber specialization.
We thank Dr. Kathy Graves, Brian
Mercer, Caroline Humphries, Ling Lin, April Hawkins, and John Shelton
for excellent technical assistance. We are grateful to Hai Wu for
useful discussions.
Published, JBC Papers in Press, February 26, 2001, DOI 10.1074/jbc.M101251200
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