(Received for publication, November 18, 1994)
From the
The muscle-specific -enolase gene is expressed in
proliferating adult myoblasts as well as in differentiated myotubes.
Through deletion-transfection analysis, we identified a 79-base pair
enhancer from the
-enolase gene that leads to high level
expression of a reporter gene in myoblasts, but not in fibroblasts.
Following myoblast differentiation into myotubes, the activity of the
enhancer declined, indicating that
-enolase gene expression in
myotubes is mediated by other regulators, possibly the myogenic
helix-loop-helix family of transcription factors. Electrophoretic
mobility shift assays indicated that proteins present in myoblast
nuclear extracts specifically bind to the 3` half of the 79-base pair
enhancer. This region contains an ets DNA-binding motif which
is required not only for high level activity in myoblasts, but also for
repressing activity in fibroblasts. Furthermore, the
-enolase
myoblast-specific enhancer shows limited similarity to the
myoblast-specific enhancer associated with the human desmin gene,
suggesting that gene expression in adult myoblasts may be coordinately
regulated.
In adult muscle, activation of myoblasts, called satellite
cells, is one mechanism for maintaining skeletal muscle mass. Although
quiescent in normal muscle, satellite cells proliferate and
differentiate in response to muscle damage or degeneration, thereby
regenerating muscle fibers. We reported previously that the
muscle-specific -enolase gene is expressed in proliferating
myoblasts of adult, but not fetal human muscle(1) . The
appearance of
-enolase mRNA in myoblasts prior to birth may be
indicative of the emergence of adult satellite cells in humans,
although they arise somewhat later during development than the
population of myoblasts resistant to the phorbol ester
12-O-tetradecanoylphorbol-13-acetate reported previously to
represent the emergence of satellite cells(2) .
-Enolase
expressing myoblasts emerge at a comparable stage to the distinct
population of adult myoblasts that appear during mid to late fetal
development in avian embryos(3, 4) . In cell culture,
adult myoblasts differ from fetal myoblasts in responsiveness to
12-O-tetradecanoylphorbol-13-acetate, acetylcholine, and
platelet-derived growth factor(5, 6, 7) , and
in myosin heavy chain expression following
differentiation(8, 9) .
Although fetal and adult
myoblasts can be distinguished based on -enolase mRNA
accumulation, this difference disappears following differentiation so
that myotubes derived from both cell types accumulate comparable levels
of
-enolase transcript(1, 10) . Thus, not only do
the regulatory mechanisms that control
-enolase gene expression
change with developmental stage, but also with the differentiated state
of the cell. It is likely that the helix-loop-helix (HLH) (
)family of myogenic regulators (MyoD, myogenin, myf-5, and
MRF4) are involved in controlling
-enolase gene expression
following differentiation (for review, see (11) and (12) ). Transcripts encoding
enolase are detectable in the
somites (from which skeletal myoblasts are derived) of 8.75-day
postcoitum mouse embryos shortly after the first myogenic HLH factors
are detected(13) . Moreover, the HLH myogenic regulators have
been shown to activate muscle-specific gene expression, including
-enolase(14) , when expressed in non-muscle cells. In
adult muscle fibers in vivo,
-enolase gene expression is
also linked to the contractile and metabolic properties of muscle
fibers.
-Enolase transcripts accumulate to high levels in fast
twitch relative to slow twitch muscle(1) . Alteration of the
exogenous stimuli which control fiber type such as innervation and
hormone levels results in changes in
-enolase
expression(15, 16) .
The myogenic HLH regulators
are not likely to control enolase gene expression in proliferating
myoblasts. Although present in myoblasts, the myogenic HLH proteins
appear inactive as transcription factors for muscle-specific
genes(17) . Growth factors necessary to maintain myoblasts in a
proliferative, undifferentiated state, such as basic fibroblast growth
factor and transforming growth factor
, appear to inhibit the
activity of the myogenic HLH proteins(18, 19) .
Consistent with this data,
-enolase mRNA accumulation was
unchanged in myoblasts in which the expression of the entire family of
HLH myogenic regulators had been abolished(1) . It is likely
that the transcription factor(s) that control myoblast-specific gene
expression are activated by growth factors, perhaps in a manner
analogous to activation of serum response factor that controls
expression of many cellular immediate-early genes(20) .
In
the present study, we identified a cis-regulatory DNA sequence
that controls expression of the -enolase gene specifically in
adult myoblasts. As this enhancer did not activate transcription
following differentiation, distinct regulatory factors control
-enolase gene expression in proliferating myoblasts and
differentiated myotubes. Site-directed mutagenesis indicated that an ets motif within the enhancer is required for transcriptional
activation in myoblasts and repression in fibroblasts, suggesting that
Ets domain proteins activate or repress transcription of the
-enolase gene depending on the cellular context.
Figure 5:
The ets motif within the
-enolase myoblast-specific enhancer shows limited similarity to a
region within the myoblast-specific enhancer from the human desmin
gene. The region within the desmin myoblast-specific enhancer important
for activity (34) is shown compared with the
-enolase
regulatory region. The consensus ets motif (32) is
also shown. The bracket indicates the region protected from
DNase digestion by myotube nuclear extracts during footprint analysis
of the desmin enhancer (Mb, (34) ). The sequences of the two
oligonucleotides (wild type and mutant) used as competitors in EMSA are
shown. Bold letters indicate the region that corresponds to
-583 to -563 from the
-enolase gene. Additional bases
were added to facilitate cloning. Mutated bases are boxed.
Figure 4:
An ets motif within the 79bp
myoblast-specific enhancer (-628 to -549) is important for
activity. Schematic representation of constructs pJC13-pJC16 in A, and pJC3 and pJC17-pJC19 in B is shown on
the left and the relative luciferase activity produced by each
following transient transfection into C2C12 myoblasts and 10T1/2
fibroblasts is shown on the right. Hatched boxes represent regions of -enolase 5`-flanking DNA, and the thin line represents the SV40 minimal promoter, cloned
upstream of the luciferase reporter gene (LUC). The ets motif is represented by the stippled box in B.
The double point mutation (GA
CT) within the ets motif
of pJC18 is indicated. Numbers denote position relative to the
start site of transcription of the
-enolase gene. Relative
luciferase values from five separate experiments were
averaged.
Subconfluent cells were
transfected on either 60- or 100-mm dishes with 6 or 10 µg of
luciferase plasmids, respectively, using a modification of the calcium
phosphate precipitation method described by Sternberg et
al.(25) . The DNA precipitate was allowed to stand on the
cells overnight, at which time the cells were washed twice in
phosphate-buffered saline and given fresh media. For transient
transfections, a vector expressing the lacZ gene under the
control of the Rous sarcoma virus long terminal repeat was included as
an internal control for transfection efficiency(26) .
Approximately 48 h following addition of DNA, total protein content in
cell extracts was determined (Bio-Rad Protein Assay) and luciferase
assays were performed using the Promega luciferase assay kit and a
TD-20e luminometer (Turner Designs), normalizing values to
-galactosidase activity, quantitated using the Galacto-Light kit
(TROPIX, Inc.).
For stable transfections, the pSV2neo vector was included at [1/10] the concentration of the test plasmid. Following overnight incubation with DNA, cells were washed and fresh media was added. After allowing the cells to recover for an additional day, cells were split 1:2 and selected in 400 µg of active G418 (Life Technologies, Inc.)/ml. As colonies began to appear, several hundred were pooled and the cells were split to maintain myoblasts at low density in high serum. Luciferase assays were performed on myoblasts and on myotubes following 3 days of myoblast differentiation as described above.
Figure 1:
Identification of -enolase
5`-flanking DNA that promotes high level luciferase activity in
myoblasts. Schematic representation of constructs (pJC1, pJC3, pJC5,
pJC7, pJC10) is shown on the left, and the relative luciferase
activity produced by each following transient transfection into C2C12
myoblasts and 10T1/2 fibroblasts is shown on the right.
Fragments containing different regions of
-enolase 5`-flanking DNA (hatched boxes) were generated by PCR and cloned upstream of
the luciferase reporter gene (LUC). The thin line in
pJC10 represents the SV40 minimal promoter. Numbers denote
position relative to the start site of transcription of the
-enolase gene. Relative luciferase values from five separate
experiments were averaged.
To determine if the region of DNA between -628 and -362 will enhance transcription in association with a heterologous promoter, this region was amplified by PCR and cloned into the pGL2 vector containing the luciferase reporter gene downstream of the SV40 minimal promoter (pJC10, Fig. 1). Following transient transfection, pJC10 reproducibly demonstrated significantly higher activity in myoblasts than fibroblasts, suggesting that a myoblast-specific enhancer is present within this 266-bp DNA fragment.
Figure 2:
The -enolase enhancer located between
-628 and -362 is not active following differentiation of
myoblasts into myotubes. Schematic representation of constructs (pJC3, pJC310, pJC311, pJC312) is shown on the left and the relative luciferase activity produced by each following
stable transfection into C2C12 myoblasts is shown on the right. The thin line in pJC10-pJC12 represents
the SV40 minimal promoter. pJC3 and 10 were as described in the legend
to Fig. 1. pJC11 contains a 225-bp fragment of
DNA, and
pJC12 contains the 500-bp myosin light chain 1/3 enhancer. Myoblasts
were cotransfected with the luciferase reporter constructs together
with pSV2neo and selected with G418. Stable clones were pooled and
assayed for luciferase activity while growing in high serum (myoblasts)
or following differentiation for 3 days in low serum
(myotubes).
Figure 3:
The nucleotide sequence of the 266-bp DNA
element that promotes high level myoblast-specific expression of the
luciferase reporter gene. Potential binding sites for known
transcription factor families are
indicated(29, 30, 31, 32, 33) . Numbers indicate position relative to the start site of
transcription of the -enolase gene. This region was divided at the
position indicated by the arrow(-549) into two smaller
fragments (79 and 187 bp), and each was tested for activity as
described below.
The 79-bp enhancer contains
consensus binding sites for GATA and Ets domain transcription factors.
In addition, the ets motif within the enhancer showed some
homology (10 of 14 bp) with a region within the myoblast-specific
enhancer from the human desmin gene shown to be important for activity ((34) ; Fig. 5). We examined the role of the ets motif by mutating 2 base pairs within the GGA core consensus from
GA to CT within pJC3 to generate pJC18. Similar to pJC17, this
construct was nearly 15-fold less active in myoblasts than pJC3 (Fig. 4B), suggesting that an Ets domain protein may be
involved in controlling high level myoblast-specific expression of the
-enolase gene. However, a construct with three copies of the ets motif upstream of the SV40 minimal promoter (pJC19) was
inactive in myoblasts, indicating that this site alone is not
sufficient for myoblast-specific activity (Fig. 4B).
The ets motif also appeared important for inhibiting activity
in fibroblasts as pJC18 containing the mutated ets motif was
consistently more active than pJC3 in 10T1/2 cells (Fig. 4B). In fact, pJC17 and 18 were more active in
10T1/2 cells than in C2C12 myoblasts. Thus, the ets motif
appears to be involved in controlling tissue-specific expression by
enhancing promoter activity in myoblasts and repressing activity in
fibroblasts.
Figure 6:
The 79-bp myoblast-specific enhancer binds
to proteins present in myoblast nuclear extracts. EMSA were performed
using P-labeled 79-bp DNA (-628 to -362) and
nuclear extracts derived from C2C12 myoblasts (lanes
2-4). Two complexes were formed (A and B)
which could be specifically competed with unlabeled 79-bp DNA (lane
3) but not with a 25-bp DNA containing an Sp1 binding site (lane 4). A 100-fold molar excess of each competitor was
added. Lane 1 shows the migration of labeled fragment in the
absence of extract.
Figure 7:
Localization of protein binding within the
79-bp DNA. EMSA were performed using P-labeled 79-bp DNA
(-628 to -362) and nuclear extracts derived from C2C12
myoblasts (lanes 2-11). Increasing concentrations (1-,
10-, and 100-fold molar excess) of three different unlabeled DNAs were
used as competitors: the 79-bp DNA and two overlapping subfragments of
the 79-bp DNA, 40-bp (-628 to -588) and 58 bp (-607
to -549) in length. Whereas the 40-bp DNA did not compete (lanes 6-8), the 79-bp fragment (lanes
3-5), and the 58-bp fragment (lanes 9-11)
competed efficiently for protein binding in complexes A and B. Lane
1 shows the migration of labeled fragment in the absence of
extract.
Figure 8:
Protein binding to the myoblast-specific
enhancer requires an ets motif. EMSA were performed using P-labeled 58-bp DNA (-607 to -549) and nuclear
extracts derived from C2C12 myoblasts (lanes 2-8).
Complexes A and B were formed and were specifically competed with
unlabeled 58-bp DNA (lane 3) but not with the 40-bp DNA
(-628 to -588, lane 4) or a 25-bp DNA containing
an Sp1 binding site (lane 5). Complex A was specifically
competed with the wild type 25-bp DNA from -583 to -563 (wt, lane 7; also see Fig. 5), whereas a mutated DNA,
also shown in Fig. 5, failed to compete for protein binding (lane 8). A 100-fold molar excess of each competitor was
added. Lane 1 shows the migration of labeled fragment in the
absence of extract.
-Enolase belongs to a relatively small group of
muscle-specific gene products expressed in proliferating myoblasts, as
well as differentiated myotubes. Regulatory mechanisms that control
gene expression in myoblasts have not been defined, but appear to
change with the age of the muscle donor, so that myoblasts from
embryonic, fetal, and adult muscle have distinct phenotypes. As a first
step to isolating regulators of the adult myoblast phenotype, we
identified an enhancer responsible for controlling the adult
myoblast-specific expression of the human
-enolase gene.
-Enolase mRNA is undetectable in fetal myoblasts which instead
accumulate the ubiquitous isoform
-enolase(1) . The switch
to the muscle-specific isoform occurs prior to birth so that myoblasts
derived from postnatal muscle tissues accumulate predominantly
-enolase. Previous analysis of the DNA sequence flanking the human
-enolase gene (21, 35) revealed the presence of
potential binding sites for several general (Sp1, AP1, and 2) and
muscle-specific transcription factors (E box for myogenic HLH factors,
CArG box, MEF2 site, and M-CAT
site(11, 27, 36, 37) . The latter
sites have been shown to be involved in controlling the expression of
genes induced during differentiation, such as myosin light chain 1/3
(MLC1/3). The MLC1/3 enhancer contains two E boxes and a MEF2 binding
site (38) and promotes low level expression in myoblasts and
high level expression in myotubes(24) . Our analysis showed
that a myoblast-specific enhancer is present between -628 and
-549 upstream of the transcription start site of the
-enolase gene and that the activity of this enhancer dropped
dramatically following removal of serum which induces differentiation
of myoblasts into myotubes, in marked contrast to the MLC1/3 enhancer.
An ets motif within the enhancer was important for
myoblast-specific activity and was bound by proteins present in
myoblast nuclear extract. Ets domain proteins comprise a family of
transcription factors that bind to purine-rich DNA sequences with a GGA
core consensus(39) . They have been shown to be involved in
regulating gene expression and controlling growth in a variety of
biological systems(32) . The subfamily of Ets domain proteins,
including Elk-1 and SAP-1, which bind DNA autonomously and as part of
the serum response factor ternary complex, are activated by
phosphorylation following growth factor stimulation(20) . Our
data suggest that growth factor stimulation may also be required for
activation of Ets-dependent
-enolase gene expression in myoblasts.
In addition to acting as an enhancer in myoblasts, the 79-bp DNA
fragment we identified appeared to mediate repression of the
-enolase gene in fibroblasts. Deletion of the 79-bp enhancer or
mutation of the ets motif resulted in elevated transcriptional
activity in fibroblasts relative to the parental construct. In
fibroblasts, it is possible that an Ets domain protein bound to DNA
acts to repress transcription. Net, another member of the Elk/SAP Ets
subfamily has negative effects on transcription and Ras expression
switches Net activity to positive(40) . It is possible that the
same Ets domain protein is acting on the enhancer in myoblasts and
fibroblasts, but that activity is modified in response to different
signal transduction pathways in the two cell types.
The ets motif alone did not display enhancer function in myoblasts,
suggesting that additional cis elements within the enhancer
are involved in regulating -enolase gene expression. We found that
protein complexes formed on the 79-bp DNA outside the ets motif, supporting the idea that multiple DNA-binding proteins must
interact for enhancer activity. A common feature of Ets domain proteins
is that they interact with other proteins to form multisubunit
complexes(20, 32) . Some Ets domain proteins can bind
to DNA autonomously, as is the case for Ets1 and the TCR
enhancer,
but transcriptional activation requires a second non-Ets
protein(41) . We have, as yet, been unable to identify
additional cis-regulatory elements within the 79-bp DNA (data
not shown). Sequences outside the enhancer also appear important for
-enolase gene expression. Several potential Sp1 sites are located
downstream of the enhancer and Sp1 may act alone or cooperatively with
the Ets domain protein to produce maximal transcriptional activity. Sp1
sites present within the enhancer of the
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene have been
proposed to regulate the expression of that gene in
myoblasts(42) . Furthermore, as the myoblast-specific enhancer
was most effective in association with its endogenous promoter, the
-enolase initiation complex may play an important role in
regulating expression. It is significant that the
-enolase
promoter (-131 to +62) demonstrated considerable
myoblast-specific activity. Several studies have suggested that
relatively short promoter regions can generate tissue-specific
transcription. For example, 133 bp of 5`-flanking DNA from the myogenin gene promotes appropriate temporal and spatial
expression of a reporter gene in transgenic animals(43) .
Finally,
-enolase gene expression may also be influenced by a
negative regulatory element located upstream of the myoblast-specific
enhancer between -917 and -628. The vimentin gene, which is
expressed in many cell types, including myoblasts, is down-regulated
during myogenesis (44) . The silencer located upstream of the
promoter (45, 46, 47) may participate in this
process. Further characterization of the putative negative regulatory
region associated with the
-enolase gene is required to determine
what, if any, role it plays in regulating its expression.
The
regulators that bind to the -enolase myoblast-specific enhancer
may also control the expression of other muscle-specific gene products.
Like
-enolase, carbonic anhydrase III, the integral membrane
protein H36 (
7 integrin), the intermediate filament desmin, and
MyoD are muscle-specific gene products expressed in proliferating
myoblasts (48, 49, 50, 51) . The
genes encoding the latter two proteins have been cloned and enhancers
active in myoblasts have been identified(52, 53) . No
significant homology was found between the
-enolase and MyoD
enhancers. The desmin gene is regulated by separate myotube- and
myoblast-specific enhancers that are adjacent to one another between
-973 and -693 relative to the transcription start
site(34) . The ets motif within the
-enolase
enhancer shares limited homology with the Mb sequence within the desmin
myoblast-specific enhancer, shown to be important for activity. The
enhancers associated with the human desmin and
-enolase genes must
be examined in greater detail to determine whether they are recognized
by similar regulatory factors.