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INTRODUCTION |
Expression of the rat
SkM11 sodium channel isoform
is restricted almost exclusively to skeletal muscle; following a rapid
post-natal increase in mRNA and protein levels, SkM1 becomes the
predominant voltage-dependent sodium channel expressed in
adult skeletal muscle (1, 2). The spatial distribution of the channel
within a myofiber is also tightly regulated, with the highest
density of channel protein found within the folds of the neuromuscular
junction, and lower levels throughout the sarcolemma and T-tubular
membrane (3-5). Given the multiple levels at which channel expression is regulated, complex interactions of transcription factors probably govern SkM1 transcription.
We have previously characterized several cis-regulatory elements that
control expression of this gene in a primary muscle culture system (6).
We found that both positive and negative mechanisms combine to modulate
expression, and that two E-boxes play pivotal roles in this process.
One E-box, located at
31/
26 within the promoter, works with other
elements to orchestrate positive regulation of the gene, while a second
E-box, located at
90/
85 within a larger upstream repressor region,
confers muscle-specific expression on the basal promoter that otherwise lacks cell-type specific function.
One of the unresolved issues from our earlier work was the mechanism by
which the upstream repressor region achieved muscle-specific function.
Transcription factors that bind to this region are present in all cell
types examined, and transfer of either the entire repressor or its
various sub-components to a heterologous rat brain type II sodium
channel (RBII) promoter repressed expression in muscle cells as well as
non-muscle cells. We postulated that the native SkM1 promoter
influenced the ability of the upstream repressor region to act
selectively in non-muscle cells, perhaps through the E-box within the
SkM1 promoter.
Several muscle-specific genes, including those for troponin I, desmin,
and the acetylcholine receptor (AChR)
,
,
,
, and
subunits, contain E-boxes within their promoter regions that are
involved in regulating positive gene expression. These E-boxes function
in part through their interaction with myogenic basic helix-loop-helix
(bHLH) proteins (7-14). Although positive regulation through the E-box
is common to all these genes, the interplay between the bHLH factors
and other transcription factors is more variable, and in some cases has
not been completely resolved. The E-box within the promoter of the
desmin gene coordinates positive regulation through interaction with a
distal enhancer that contains a second E-box and an MEF2 binding site
(7), while the E-boxes of the
AChR subunit promoter interact with
an M-CAT sequence adjacent to it (10). The AChR
subunit and SkM1
5'-flanking sequences have substantial sequence and functional
similarities (6, 12, 13). However, the E-box within the
subunit
promoter controls both positive and negative regulation of that gene,
while these functions are split between two E-boxes within the SkM1 sequence (6, 13).
In this report we demonstrate that the SkM1 promoter E-box influences
the ability of the upstream repressor region to function, and that the
binding of bHLH factors to the promoter E-box releases repression
exerted by this element in the muscle lineage. Furthermore, comparison
of repressor function in different muscle cell types and at different
developmental stages reveals specificity in the ability of particular
myogenic bHLH factors to effect this release of repression.
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EXPERIMENTAL PROCEDURES |
Generation of Reporter Gene Constructs--
The
174,
135,
95, and
85 promoter E-box mutations were created from previously
characterized full-length (
2800/+249) promoter E-box mutations
contained in pCAT-Basic (Promega; 6). Briefly, PCR was performed using
either the c/g or tcc/gaa mutant
2800/+254 as a template. The
5'-primer contained 20 base pairs of SkM1 sequence starting at the
designated point and a restriction site (either HindIII or
PstI) for cloning purposes. The 3'-primer was complementary
to +56 to +78 of the SkM1 sequence. The PCR products were digested with
HindIII and SacI (
135,
95,
85) or
PstI and SacI (
174) and cloned into the same
sites of the corresponding wild-type 5'-deletion mutant. Mutants
generated by PCR were sequenced (Sequenase; U. S. Biochemical Corp.).
All other mutations used in this report were created and characterized previously (6).
Cell Culture and Transient Expression Assays--
Culture and
transfection of primary muscle cells was carried out as reported
previously (2, 6). The C2C12 cell line was maintained in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) containing 10%
fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin (Life Technologies, Inc.). To initiate and maintain
differentiated C2C12 cells, 2% horse serum replaced the fetal bovine
serum. LipofectAMINE and OptiMEM (Life Technologies, Inc.) were used to
transfect C2C12 cells, according to manufacturer directions. To allow
comparison between calcium phosphate-transfected primary cultures and
LipofectAMINE-transfected C2C12 cells, a constant molar ratio of test
DNA (4-6.4 µg) to pCAT-C (0.8 µg) was maintained. Normalization
and quantitation of CAT assays were carried out as reported previously
using the pCAT-Control (Promega) vector expressing the gene for
chloramphenicol acetyltransferase driven by the SV-40 promoter and
enhancer as a positive control, and the pCAT-Basic vector as a negative
control (6).
Cells that were both transfected with reporter gene constructs and
infected with the CMV-myogenin internal ribosome entry sequence (IRES)
-galactosidase adenovirus were transfected first for 3-4 h, then
infected with 2 × 1010 particles of the adenovirus
and maintained overnight (16-18 h). Control cells were fed medium
without adenovirus. Cells were either switched to differentiation
medium or maintained in medium containing 10% fetal bovine serum for
an additional 28-30 h prior to harvest. Cells that were not
transfected with reporter gene constructs were infected with the
CMV-myogenin IRES
-galactosidase adenovirus according to the same
paradigm and harvested for nuclear extracts or membrane proteins.
Gel-shift Assays--
Gel-shift assays and supershift assays
were carried out as previously reported (6) with the following
modifications and additions. Gel-shift assays for the repressor probes
were carried out at 4 °C rather than room temperature. The
antibodies used to supershift the various bHLH factors were obtained
from Santa Cruz (E2A and myf-6), PharMingen (myogenin), and Novocastra (MyoD).
Preparation of Protein Fractions and Western
Blotting--
Membrane fractions containing sodium channel protein or
nuclear extracts containing transcription factors were prepared as reported previously (6, 15). Gel electrophoresis and Western blotting
were carried out as reported using the Western Star kit (Tropix; Ref.
15). The primary antibodies used to detect the bHLH factors were the
same as those used in the supershifts. To remove particulate matter and
reduced background staining, the myf-6 antibody was treated as follows.
The antibody was diluted 1:50 in 10% heat-inactivated horse serum in
phosphate-buffered saline and incubated with 60 mg of porcine liver
extract (Sigma) for 1 h a 4 °C. The extract and particulate
matter were removed by centrifugation at 100,000 × g
for 2 h. The final antibody solution was diluted 1:250 in 10%
horse serum, 0.4% I-block (Tropix), and 0.1% Tween in
phosphate-buffered saline. Final dilutions for the MyoD antibody was
1:250, and the myogenin antibody 1:500. The monoclonal antibody (L/D3)
used to detect the sodium channel has been extensively characterized
and is specific for the SkM1 isoform of sodium channel (16, 17).
Generation of Replication-deficient CMV-myogenin IRES
-Galactosidase Adenovirus--
The dl327 adenovirus and pAd-Link
vector used to create the CMV-myogenin IRES
-galactosidase
adenovirus were obtained from the Vector Core of the University of
Pennsylvania (18, 19). 293 cells (American Type Cell Culture) were
grown in Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
The polylinker in the pCI expression vector (Promega) was altered to
contain restriction sites for the enzymes EcoRI,
StyI, NotI, SnaBI, and
BclI, and an EcoRI to StyI fragment
containing myogenin was inserted. A NotI to BamHI
fragment containing an IRES in frame with the
-galactosidase gene,
obtained from pLIGns (20), was inserted between the NotI and
BclI site of CMV-myogenin to yield CMV-myogenin IRES
-galactosidase. This construct was digested to completion with
BglII, and a partial digest carried out with ClaI
to yield a fragment extending from the CMV promoter to the SV40 poly(A)
site of the expression vector. This fragment was cloned into the
BglII and ClaI sites of pAd-Link and prepared for
recombination by linearizing with NheI.
The dl327 adenovirus was grown in 293 cells and prepared for
recombination by digestion with ClaI as reported (19). The initial transfection was carried out as reported previously (19), but
the standard agar overlay procedure was replaced by plaque-purification using 96-well plates. Serial dilutions of the transfected cells were
combined with 1 × 106 293 cells in a 20-ml total
volume and dispensed into 96-well plates using 100 µl/well. The
plates were maintained for 6 days, then fed with 100 µl of medium.
After another 5-6 days, plaques were observed by eye. Dilutions
resulting in more than 20 plaques/plate were discarded. A total of 30 plaques were screened by Southern blot, and of these, 9 were positive.
Plaque purification was carried out in the same manner, using serial
dilutions ranging between 10
6 and 10
10.
Eleven plaques were screened in a Southern blot, and all were positive.
One of these was expanded for large scale production according to
published methods (19).
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RESULTS |
Arrangement and Activity of Cis-regulatory Elements--
We
previously characterized several cis-regulatory elements that control
expression of the SkM1 gene (6, Fig. 1).
For most of the experiments reported here, we focused on four major
functional elements between
174 and +49. Our previous results are
summarized as follows. The promoter E-box at
31/
26
directs positive modulation of the gene through an interaction with
elements elsewhere in the SkM1 genomic sequence. Myogenic bHLH proteins
play a role in this interaction. The activity of the
85/
57
positive element is largely muscle-specific and confers 7-fold
higher expression levels on the promoter, although its activity is
masked in cultured muscle cells by the repressor E-box
immediately upstream at
90/
85. DNase footprinting of the
transcription factors that bind the upstream repressor
region or repressor have shown that this element extends upstream to approximately
135, and functional studies described below substantiate an independent function for these upstream repressor sequences. A 3'-positive
element that includes part of the 5'-untranslated region and part
of the first intron lies between +50/+254; this element increases
expression levels 10-fold in muscle cells.

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Fig. 1.
Cis-regulatory elements that control SkM1
gene expression. The locations of previously characterized
cis-regulatory elements are indicated, with elements that modulate
positive expression shown in black and those that modulate
negative expression shown in white (6). An upstream
repressor region or repressor contains both upstream repressor
sequences and a repressor E-box. These elements repress SkM1 expression
in non-muscle cells in conjunction with the native SkM1 promoter. The
85/ 57 positive element contains within it two smaller motifs at
83/ 78 and 64/ 59 that are required for the binding of the
cognate transcription factor; the full element is shown as two
connected boxes containing plus signs, indicating the two motifs. The
promoter, which by itself is broadly expressed in many cell types,
contains within it a promoter E-box that binds the myogenic bHLH
factors. In the context of the full-length 2800/+254 SkM1 regulatory
sequence, but not the promoter region itself, mutations in the promoter
E-box severely reduce muscle-specific expression, indicating that the
promoter E-box works with other elements outside the promoter to
control positive modulation of the gene. The 3'-positive element
located downstream of the transcription initiation sites encompasses
part of the 5'-untranslated region and part of the first intron. This
element confers 10-fold higher levels of reporter gene expression in
muscle cells.
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Myogenic bHLH Proteins Bind the Wild-type SkM1 Promoter E-box but
Not the Promoter E-box Mutants--
The SkM1 promoter E-box binds
multiple proteins in both muscle and non-muscle cells, but the myogenic
bHLH proteins MyoD, myogenin, and MRF4 were present only in muscle
cells, as indicated by supershift assays with the appropriate
antibodies (Fig. 2, A-E).
There was heterogeneity in the expression of these myogenic factors in
muscle at different stages of development and in different muscle cell
types (13). For example, MyoD, but not myogenin, was expressed in C2C12
myoblasts (panel C), while both MyoD and myogenin
were expressed in C2C12 myotubes (panel D).
Likewise, gel-shifts obtained with nuclear extracts prepared from
primary muscle cells after 4 days or 7 days in culture differed in the number of transcriptional complexes formed with the wild-type SkM1
E-box probe (panels A and B). MyoD and
myogenin were present at both time points, while MRF4 was detected only
on day 7.

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Fig. 2.
Myogenic bHLH proteins bind the wild-type
SkM1 promoter E-box but not the promoter E-box mutants. The
wild-type SkM1 promoter E-box region (cagCAGCTGtcc), a c/g mutant
promoter E-box region (cagGAGCTGtcc), and a tcc/gaa mutant
promoter E-box region (cagCAGCTGgaa) were used in gel-shift
assays with nuclear extracts prepared from the following cell types:
A, day 4 primary muscle cells; B, day 7 primary
muscle cells; C, C2C12 myoblasts; D, C2C12 day 2 myotubes; E, PC12 cells. For all panels, antibodies against
the following factors were used for supershift assays: 1, no
antibody; 2, MyoD; 3, myogenin; 4,
MRF4; 5, E2A. The supershifts are indicated by
asterisks. The SkM1 promoter E-box bound multiple proteins
in both muscle and non-muscle cells, but the myogenic bHLH proteins
were bound only in muscle cells, as indicated by the
asterisks. The two mutations in the promoter E-box either
abolished (c/g mutant), or markedly reduced (tcc/gaa mutant) the
ability of this region to bind myogenic bHLH factors. The c/g mutant
also severely reduced the binding of non-bHLH proteins, while the
tcc/gaa mutant retained binding for these additional factors. The
tcc/gaa mutant also bound an additional factor not observed with the
wild-type probe; this new complex co-migrated with the MyoD gel-shift
but was not supershifted by the MyoD antibody. In panel
F, a direct comparison was made between the supershifts of
the MyoD and E2A antibodies in muscle cells at different stages of
development. Two alternative supershifted states of MyoD existed, with
the higher complex predominating in myoblasts, and the lower in
myotubes. An antibody to the MyoD dimerization partner, E2A,
demonstrates that E2A was involved only in the higher complex. The E2A
antibody did not supershift the complex corresponding to myogenin. The
nuclear extracts were prepared from the cell type indicated beneath
each set of lanes.
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We showed previously that two mutants in the promoter E-box either
abolished (c/g mutant) or markedly reduced (tcc/gaa mutant) the ability
of this region to orchestrate positive interactions with other elements
in the SkM1 gene (6). We tested these mutants directly in gel-shift
assays to determine the effect of each mutation on transcription factor
binding (Fig. 2). The c/g mutation abolished the binding of most
transcription factors, including the myogenic bHLH factors in all cells
types (Fig. 2). Functional data published previously (6) and additional
data presented below indicate that this mutant is a true null mutation.
The results obtained for the tcc/gaa promoter mutation are more
complex. This mutation greatly reduced the binding of the myogenic bHLH
proteins, as indicated by the loss of supershifts generated by the
antibodies to the factors, although some residual MyoD binding was
observed in myoblasts and day 4 primary cultures (Fig. 2). The binding
of non-bHLH protein complexes that interact with the wild-type promoter
region was not affected by the tcc/gaa mutation. In the experiments
that follow, these binding characteristics helped to differentiate
between the functional impact of the myogenic bHLH proteins and the
action of other transcription factors that bind the promoter E-box in
primary muscle cultures. In contrast, the c/g mutation affected the
binding of all factors in these nuclear extracts.
The tcc/gaa probe also binds a new factor not observed with the
wild-type probe (Fig. 2, A-D). Although this additional
complex co-migrated closely with the wild-type band supershifted by the MyoD antibody, this mutant complex did not contain MyoD. However, functional assays reported below indicate that the effects of the
tcc/gaa mutation on gene expression reflect loss of function, rather
than the gain of function generated by the binding of this new factor.
Although the primary purpose of these experiments was to determine
whether or not the myogenic bHLH proteins could bind the promoter E-box
mutants, we also noted unexpected complexities in the MyoD supershift
patterns. Comparison of the size of the supershift generated by the
MyoD antibody in C2C12 myoblast and myotubes extracts, or day 4 and day
7 primary culture extracts, revealed that two alternative supershifted
states of MyoD existed, with the higher complex predominating in
myoblasts, and the lower in myotubes (Fig. 2F). An antibody
to the MyoD dimerization partner, E2A, further underscored the
difference between the MyoD complexes in the two developmental states
(Fig. 2F). In C2C12 myoblasts and day 4 primary cultures,
similar discrete supershifts were observed with both the E2A and MyoD
antibodies, while in more mature myotubes, the E2A supershift was
similar in location and intensity only to the higher MyoD complex,
suggesting that E2A was involved in the higher complex, but not the
lower. The E2A antibody did not supershift the complex corresponding to
myogenin, again suggesting that myogenin does not dimerize with E2A.
Overall, our data suggested that the dimerization partners of the
myogenic bHLH proteins vary depending on the state of differentiation, although we did not pursue this point further.
The Transcription Factors That Bind the
85/
75 Positive Element,
Repressor E-box, and the Upstream Repressor Sequences Are Expressed in
All Cell Types--
The
85/
57 positive element binds a single
complex in all cell types examined, including PC12 and muscle cells
(Fig. 3A). This factor was
displaced by the wild-type competitor (Fig. 3A, lane 2) but not a mutant competitor that altered
two short motifs at
83/
78 and
64/
59, represented by the
connected boxes in Fig. 1 (Fig. 3A,
lane 3). Although this factor is present in all cell types, the
85/
57 positive element exhibited greater activity in differentiated muscle cells, as published previously and shown below
(6).

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Fig. 3.
Identification of the transcription factors
that bind to other SkM1 elements. For both A and
B, the cell types from which the nuclear extracts were
prepared is shown beneath the lanes. A, the 85/ 57
positive element bound a transcription factor expressed in all cell
types; this factor was displaced by the wild-type competitor, but not
the mutant. 1, no competitor; 2, wild-type competitor
(GAAGATTGGCCCAGTCCTCAGGTTTCACT); 3, mutant
competitor (GAGCTAGCGCCCAGTCCTCAGCCCGGGCT). B,
the repressor region bound multiple factors in all cell types. For all
cell types, the following probes were used: 1, 135/ 82
probe containing both upstream repressor sequences and repressor E-box;
2, 135/ 95 probe containing only upstream repressor
sequences; 3, 93/ 82 probe containing only repressor
E-box. The highest complex associated more uniquely with the repressor
E-box and was bound to the probes containing the repressor E-box, while
the middle complex associated more uniquely with the upstream repressor
sequences and was bound to the probes containing these sequences. A
lower band associated with both elements, although the intensity of the
gel-shift for all factors was greatest on the probe containing both
components of the upstream repressor region.
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The upstream repressor region is comprised of two components, a
repressor E-box and the upstream repressor sequences. Previously, we
identified a broad gel-shift that exhibited an extensive footprint covering sequences between
135 and
82 (6). Gel-shift assays carried
out at low temperatures revealed an additional factor that associates
more uniquely with the repressor E-box. Both the
135/
82 probe that
included the repressor E-box and the repressor E-box probe alone
associated with a sharp gel-shift that runs as the highest complex,
while the shorter
135/
95 probe that excluded the repressor E-box
did not bind this factor. The broad middle gel-shift was found with the
probes that include the upstream repressor sequences, while the lowest
gel-shift band appeared with all three probes. The longest probe,
containing both components of the upstream repressor region, generated
the most intense gel-shift, suggesting that these factors stabilize
each other on the DNA. The repressor-binding transcription factors were
found in all cell types.
The Promoter Determines the Cell Type in Which the SkM1 Repressor
Functions--
The
174/+49 SkM1 sequence with the wild-type promoter
E-box or the
174/+49 sequence with either the c/g or tcc/gaa mutant promoter E-box were examined in transient expression assays both in
primary muscle cells, which express the SkM1 gene, and PC12 cells,
which express the RBII gene (21, 22). As reported for other cell types
previously (6), the upstream repressor region functioned in the
non-muscle PC12 cell line, but allowed expression in muscle cells.
However, mutations in the promoter E-box that interfered with binding
of the myogenic bHLH factors caused the repressor to function in muscle
cells to the same extent it did in the non-muscle cell line. These same
mutations did not further reduce expression in PC12 cells. These data
suggest that the "default" setting of the upstream repressor region
is to function except when the promoter E-box binds bHLH factors.
Transfer of the upstream repressor region to the heterologous RBII
sodium channel promoter reduced expression of that promoter in muscle
cells, while permitting expression in PC12 cells (Fig. 4, bottom panel).
The repressor binding-proteins were clearly present in PC12 cells (Fig.
3B), and the repressor was able to function in conjunction
with the native SkM1 promoter in these cells (Fig. 4, top
panel), indicating that the repressor-binding proteins were
functionally active in PC12 cells. Together, these data indicate that
the promoter, and specifically the promoter E-box, determines the cell
type in which the repressor functions.

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Fig. 4.
The promoter determines the cell type in
which repression occurs. In the top panel,
the 174/+49 SkM1 sequence with the wild-type promoter E-box or the
174/+49 sequence with either the c/g or tcc/gaa mutant promoter E-box
were inserted into a vector containing the reporter gene for CAT. These
constructions were assayed in transient expression assays in primary
muscle cells, which express the SkM1 gene, or PC12 cells, which express
the RBII gene. Both cell types were transfected with the pCAT-Control
(pCAT-C) plasmid as a positive control. In conjunction with its native
promoter, the repressor functioned in the non-muscle PC12 cell line,
while it allowed expression in muscle cells. Mutations that disrupt the
ability of the promoter E-box to bind the myogenic factors caused the
repressor to function in primary muscle cells, but did not further
reduce gene expression in the negative PC12 cell line. In the
lower panel, the 174/ 50 sequence containing
the entire upstream repressor region was transferred onto the
heterologous RBII promoter, which also contains an E-box, and analyzed
in both cell types. This switch in promoters caused the repressor to
function in primary muscle cells rather than PC12 cells. The activity
of the RBII promoter without added sequences is also shown in both cell
types.
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Expression of SkM1 Sodium Channel Protein and Myogenic Factors as a
Function of Development in C2C12 Cells--
We have previously used
primary muscle cultures to study the expression of SkM1 mRNA levels
and the function of the SkM1 cis-regulatory elements because this
system relates most closely to the in vivo setting (1, 2,
6). However, primary muscle cultures express detectable levels of SkM1
mRNA and protein at the earliest times measured in culture, even
before myotubes form (2).2 We
therefore examined C2C12 cells as an alternative to primary muscle
cultures since C2C12 cells can be maintained in culture as myoblasts,
yet reliably form myotubes when culture conditions are altered. We
first determined the levels of SkM1 sodium channel protein expressed in
C2C12 cells at various stages of development (Fig.
5). The SkM1 protein was not detected in
myoblasts. Upon differentiation to form myotubes, the level of SkM1
protein gradually increased; highest levels were attained only at days
5 and 7, several days after myotubes had formed. Thus, C2C12 cells
differed from primary muscle cultures in that SkM1 protein was not
expressed in undifferentiated cells, and the level of expressed protein in myotubes continued to increase with maturation in culture.

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Fig. 5.
The initiation of myogenin expression
precedes the onset of SkM1 expression in C2C12 cells. A membrane
protein fraction or a nuclear extract protein fraction was isolated
from C2C12 myoblasts (MB) or myotubes at the indicated day
following application of differentiation medium (D1= day 1, etc.). As positive and negative controls, the same fractions were
isolated from day 7 primary muscle cells (D7, PM)
or NIH 3T3 cells. After SDS-polyacrylamide gel electrophoresis,
membrane proteins were analyzed for the sodium channel using a
monoclonal antibody (L/D3) to the SkM1 sodium channel, or
nuclear extract proteins were analyzed for the myogenic transcription
factors using antibodies against the individual factors.
MGN, myogenin.
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We then examined the appearance of the myogenic factors MyoD, myogenin,
and MRF4 (Fig. 5). MyoD was present at the highest levels in myoblasts
and decreased following differentiation. Myogenin was not present in
myoblasts, but appeared within a day following transfer to
differentiation medium, prior to the rise in the sodium channel protein
level. Myogenin peaked at day 2, and expression decreased at later
times. We were unable to detect MRF4 by Western blot in C2C12 cells,
although MRF4 could be detected by day 4 in C2C12 myotubes using the
more sensitive supershift assay (data not shown). All three myogenic
factors were present at higher levels in day 7 primary muscle cultures
than day 7 C2C12 myotubes. Since the temporal pattern of channel
protein and myogenic factor expression was most clearly defined in
C2C12 cells, we carried out further functional studies of the SkM1
cis-regulatory elements in C2C12 myoblasts, day 7 C2C12 myotubes, and
day 7 primary muscle cultures.
The Upstream Repressor Sequences Play a Key Role in the Interaction
with the SkM1 Promoter E-box--
To determine which element(s)
interacted with the promoter E-box, we sequentially added upstream
elements to the wild-type or promoter mutants. The promoter alone was
expressed at high levels in both myoblasts and myotubes (Fig.
6A), consistent with our
previous observations that the promoter is broadly expressed in both
positive and negative cell types (6). The c/g and tcc/gaa mutations
reduced promoter function slightly in primary muscle cells, with less
effect in C2C12 myoblasts and myotubes. These data indicate that the
promoter E-box does not play an important role in function of the basal
promoter and that the promoter E-box by itself does not confer specific
expression in differentiated muscle cells.

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Fig. 6.
Mutations in the promoter E-box that disrupt
binding of the myogenic factors interfere with the muscle-specific
activity of upstream cis-regulatory elements, with the most prominent
interaction taking place between the upstream repressor sequences and
the promoter E-box. The diagram at the top of each graph
schematically depicts the elements that were inserted into the
pCAT-Basic vector for the set of assays shown in that graph;
arrows indicate the position of the c/g and tcc/gaa
mutations in the promoter E-box that disrupt binding of the myogenic
bHLH factors. The following abbreviations are used to denote the
elements: PEB, promoter E-box; REB,repressor
E-box; URS, upstream repressor sequences. The full positive
element is denoted by the connected black
boxes containing plus signs. Transient
expression assays were carried out in the indicated cell types, and CAT
activity was normalized relative to the control plasmid (pCAT-C). The
designation on the abscissa indicates whether the wild-type,
c/g, or tcc/gaa promoter E-box mutation was used. In panel
A, the promoter was expressed broadly in all cell types, and
the promoter E-box mutations had little effect on expression levels. In
panel B, the 85/ 57 positive element increased
expression to the greatest extent in differentiated muscle cells;
mutations in the promoter E-box partially counteracted this increase.
In panel C, the repressor E-box reduced
expression in all cell types, but greater levels of expression remained
in differentiated muscle cells, especially primary muscle cells.
Although the tcc/gaa mutation did not alter the behavior of this
combination of elements, the c/g mutation reduced the expression in
differentiated cells to the same level as seen in myoblasts. In
panel D, the inclusion of the upstream repressor
sequences reduced expression in both myoblasts and myotubes, but the
greatest degree of muscle-specific expression was observed with the
inclusion of this element, with primary muscle cells exhibiting 10-fold
higher expression levels than C2C12 myoblasts. Both the c/g and tcc/gaa
mutants reduced level of expression in primary muscle cells to nearly
the same level as in myoblasts.
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Addition of the
85/
57 element contributed to positive regulation in
differentiated myotubes (Fig. 6, A and B; note
difference in scale). Although this positive element increased the
level of gene expression in both myoblasts and myotubes, consistent with the presence of a ubiquitous transcription factor, the
augmentation produced in differentiated muscle cells was 4-6-fold
greater than in myoblasts (Fig. 2B). Both mutations in the
promoter E-box significantly reduced this differentiation-specific
activity (Fig. 6B), indicating that at least part of the
activity was derived through an interaction with the promoter
E-box.
Addition of the repressor E-box to the combined promoter and positive
element reduced expression in all cells, but to a greater degree in
C2C12 myoblasts and myotubes (10-fold) than primary muscle cells
(7.5-fold). The level of expression observed in myoblasts approached
that reported previously for the negative NIH 3T3 cell line (6), and
neither promoter mutation further reduced expression levels in
myoblasts. In differentiated cells, the tcc/gaa mutation did not
significantly alter repressor activity, while the c/g promoter mutation
reduced expression in both C2C12 and primary muscle myotubes to nearly
the same level as myoblasts, suggesting that the promoter E-box binding
proteins that interact with the repressor E-box are the non-bHLH factors.
The greatest effect of the promoter E-box mutations was observed in
constructs that included the upstream repressor sequences (Fig.
6D). Addition of these sequences further reduced expression in both myoblasts and myotubes, but the incremental decrease was much
less in myotubes, particularly primary muscle myotubes. Promoter mutations had little effect on the residual expression in myoblasts, but both the c/g and tcc/gaa mutations virtually eliminated expression above background in myotubes, producing a 90% reduction in primary culture myotubes. Our data suggest that the upstream repressor sequences play an important role in the interaction with the promoter E-box, and that mutations in the promoter E-box allow the combined components of the upstream repressor region to function to the same
degree in differentiated muscle cells as in non-muscle cells. The
combination of all four elements produces the highest degree of
developmental specificity of SkM1 expression.
Myogenin Releases Repression of the SkM1 Upstream Repressor Region
in C2C12 Myoblasts--
To determine if the interaction between the
promoter E-box and the upstream repressor sequences was mediated by
particular bHLH proteins, we forced expression of myogenin in C2C12
myoblasts using a recombinant adenovirus, and tested the functional
impact of this transcription factor on either the wild-type
174/+49 sequence, or the corresponding c/g or tcc/gaa promoter E-box mutants. The production of myogenin in the infected cells was verified by
Western blot (Fig. 7). In the absence of
myogenin, myoblasts did not express either the wild-type or mutant
174/+49 sequences at levels above background. Introduction of
myogenin resulted in expression of the wild-type sequence in both
myoblasts and 1 day myotubes at levels 9-fold higher than background
(Fig. 7), but the mutations in the promoter E-box interfered with the
ability of myogenin to potentiate this increase. The overall level of myogenin-driven expression attained with the
174/+49 construct in
these cells was comparable to that observed in primary muscle cells
(Figs. 6D and 7).

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Fig. 7.
Forced expression of myogenin in C2C12
myoblasts disrupts activity of the upstream repressor and initiates
transcription of the endogenous SkM1 gene. The wild-type or mutant
174/+49 SkM1 flanking sequences were assayed in transient expression
assays in either control cells or cells infected with an adenovirus
expressing myogenin. Following transfection and infection, cells were
either maintained in 10% fetal bovine serum (FBS) or
switched to 2% horse serum (HS) for 28 h prior to
harvesting. Forced expression of myogenin increased expression of the
wild-type 174/+49 sequence, while the mutations in the promoter E-box
blocked this increase either completely (c/g) or partially (tcc/gaa).
MGN, myogenin. Inset, the level of myogenin
protein in the nuclear extract fraction and the level of SkM1 sodium
channel protein in the membrane protein fraction were assayed by
Western blotting. Control C2C12 myoblasts did not express either
myogenin or sodium channel protein, while treatment with myogenin
adenovirus induced expression of both proteins. Treatment with horse
serum for 28 h (day 1 myotubes) also induced expression of both
proteins, but treatment with myogenin adenovirus further increased the
level of both myogenin and sodium channel.
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Myogenin Is Sufficient to Initiate Expression of the Endogenous
Sodium Channel Gene in C2C12 Myoblasts--
Although our data indicate
that myogenin releases repression exerted by the upstream repressor
region in the small segment of SkM1 flanking sequence used in our
functional assays, there are additional elements that control
expression of the endogenous gene. The ability of myogenin to initiate
expression of the endogenous sodium channel gene was therefore assessed
by directly measuring sodium channel protein levels in the same
experimental paradigm used for the functional assays. In control
myoblasts, no SkM1 protein product was detected, while the forced
expression of myogenin was sufficient to up-regulate SkM1 protein
levels to an extent comparable to control day 1 C2C12 myotubes (Fig. 7,
inset).
Later Phases of Sodium Channel Up-regulation Correlate with the
Activity of a 3'-Positive Element--
Although the initiation of SkM1
transcription correlates with the appearance of myogenin in C2C12
myotubes, both MyoD and myogenin levels are relatively low at later
times in culture when the highest levels of SkM1 protein are detected,
and only low levels of MRF4 are found in these cells, as determined by
Western blot (Fig. 5). These observations suggest that additional
factors must act to maintain transcription of the SkM1 gene at later
times. Since we previously demonstrated the contribution of a
3'-positive element to positive regulation of the SkM1 gene (6), we
compared the activity of SkM1 constructs with and without the
3'-positive element during late myotube development in C2C12 cells
(Fig. 8). Although the enhanced
expression produced by this positive element in C2C12 cells was less
than in primary muscle cultures, the magnitude of the effect did
correlate with developmental up-regulation of SkM1 protein expression
in C2C12 cells.

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Fig. 8.
The late increase in SkM1 protein level in
differentiating cultures correlates with the activity of an additional
3'-positive element. The activity of the entire SkM1 5'-flanking
sequence ( 2800/+49) either with or without the additional 3'-positive
element (+50/+254) was assayed as a function of development in C2C12
cells at days 4, 5, and 7, corresponding to the time at which the late
increase in sodium channel protein levels occurs. As a positive
control, activity was also analyzed in day 7 primary muscle cells. The
3'-positive element produced enhanced expression in C2C12 cells most
prominently at the latest time in culture, and even higher levels of
expression were observed in primary muscle cultures, where this element
increased expression 10-fold.
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DISCUSSION |
We have shown previously that multiple cis-regulatory elements
control expression of the SkM1 sodium channel gene in primary muscle
cells, and two E-boxes located within several larger elements play a
dominant role (6). Although we had demonstrated that the promoter E-box
interacts with elements outside the promoter to control positive
regulation of the gene, we had not determined with what element(s) it
interacted or how this positive regulation was achieved. Our earlier
experiments also indicated that the native SkM1 promoter controls the
cell type in which the upstream repressor region can function, but the
mechanism underlying the promoter/repressor interaction was unclear. In
this report, we have focused most of our effort on understanding the
interactions between four major elements located between
174 and +49
in order to systematically approach these unresolved issues.
The promoter E-box influences the ability of upstream repressor region
to act in differentiated muscle. As we have shown previously and again
in this report, the promoter itself is expressed broadly in many cell
types, even though the promoter E-box is the one element characterized
to date that binds cell-type specific factors, the myogenic bHLH
proteins. Mutations in the promoter E-box reduce expression of the
basal promoter in all cell types to a relatively small degree,
indicating both that the promoter E-box is not important to the
function of the basal promoter and that the promoter E-box cannot
confer specificity by itself. It is the addition of upstream elements
that increase expression in differentiated muscle relative to
undifferentiated muscle, even though the upstream elements bind
ubiquitously expressed transcription factors. Mutations that disrupt
the ability of the promoter E-box to bind either the myogenic bHLH
factors or non-bHLH factors interfere with the specificity conferred by
these upstream elements, indicating that factors bound at the promoter
E-box interact with those bound at upstream elements. The interaction
of these factors potentiate higher levels of expression in
differentiated muscle relative to myoblasts, especially in primary
muscle cells where the amount of the myogenin is highest.
Two different mutations in the promoter E-box were used to help
characterize the interaction between the promoter E-box and upstream
elements. One mutation (c/g) abolishes the binding of both myogenic
bHLH proteins and non-bHLH proteins, while a second (tcc/gaa) severely
reduces binding of the bHLH proteins but does not diminish binding of
the non-bHLH transcription factors. These mutants distinguish the
impact of the bHLH factors from that of the other proteins that bind to
the promoter.
The interaction between the promoter E-box and the
85/
57 positive
element is a supportive one in that factors bound to the promoter E-box
"aid" the action of the transcription factor that binds the
positive element. Since both promoter mutations affect this
interaction, it appears that the myogenic bHLH proteins are involved.
The upstream repressor region is comprised of two individual
components, the repressor E-box and the upstream repressor sequences, that have different relationships with the promoter E-box. For both of
these components, factors bound to the promoter E-box inhibit the function of the repressor specifically in
differentiated muscle cells, resulting in the retention of positive
expression in primary muscle cells, and to a lesser extent in C2C12
myotubes. The factors responsible for the promoter E-box/repressor
E-box interaction appear to be the non-bHLH factors, since the c/g
mutation, but not the tcc/gaa mutation, affect the interaction. Both
promoter E-box mutations allow full repression by the upstream
repressor sequences, demonstrating that myogenic bHLH are responsible
for this release of repression.
Experiments that we carried out previously, in which various
subcomponents of the upstream repressor region were transferred to the
heterologous RBII promoter, demonstrated that the repressor E-box was
both necessary and sufficient for negative regulation (6). Although the
data presented in this paper do not allow us to conclude if the
upstream repressor sequences can function without the repressor E-box
in the
174/+49 sequence, it is clear that the upstream repressor
sequences play a key and distinct role in interacting with the bHLH proteins.
The promoter determines in what cell type gene expression is allowed.
Transfer of the SkM1 repressor to the RBII promoter results in
repression in primary muscle cells, while expression is allowed in PC12
cells, which express the RBII gene (21, 22). One prediction from these
data is that the E-box within the RBII promoter will bind bHLH proteins
specific to neuronal cells and that these factors might act in PC12
cells to inhibit the action of the SkM1 repressor much as the myogenic
factors do in conjunction with the wild-type SkM1 promoter in myotubes.
Indeed, bHLH factors have been found in PC12 cells, and increased
levels of specific neuronal bHLH factors have been shown to correlate
with increased expression of the RBII gene in this cell type when
treated with nerve growth factor, suggesting neuronal bHLH proteins
play an important role in the regulation of the RBII gene (21, 23). Other neuronal genes expressed in PC12 cells are regulated by interactions between E-boxes and separate regulatory sequences, and
this interaction is mediated in part through bHLH proteins (24, 25). We
anticipate that there may be parallel mechanisms controlling expression
of the SkM1 and RBII genes, with regulation by bHLH factors acting as a
common theme.
Not all bHLH proteins that bind the promoter E-box are equivalent in
their ability to release the repression exerted by the upstream
sequences. A complex program initiates development in muscle, with
different myogenic factors expressed at different times. C2C12
myoblasts, which do not contain detectable levels of SkM1 protein,
express only MyoD partnered with E2A. Upon differentiation, myogenin is
rapidly up-regulated. Initiation of SkM1 gene expression takes place at
this time. This correlation led us to hypothesize that myogenin plays
an important role in the initiation of SkM1 expression.
Direct introduction of myogenin in combination with the entire
174/+49 sequence into C2C12 myoblasts and day 1 myotubes released repression, leading to expression levels comparable to those observed for this same construct in primary muscle cells. Mutations in the
promoter E-box either abolish (c/g) or greatly reduce (tcc/gaa) the
release of repression that myogenin can confer, confirming that
myogenin binding at the promoter E-box directly affects negative regulation. Myogenin also activates expression of the endogenous SkM1
gene. However, unlike its action on the short regulatory sequences, the
effect of myogenin on the endogenous gene is potentiated by culture
conditions that induce differentiation, suggesting that myogenin may
initiate expression of the endogenous SkM1 gene through multiple mechanisms.
Levels of SkM1 increase at later times in C2C12 differentiation,
suggesting later action of another factor, particularly since the
levels of the bHLH factors themselves decrease. We previously demonstrated that the 3'-positive element plays a major role in tissue-specific SkM1 expression (6), and the activity of this element
correlates with the late increase in the endogenous SkM1 protein in
C2C12 cells, although it never confers the level of activity in C2C12
myotubes that it does in primary muscle cells. Part of the activity of
the 3'-positive element is derived through a specific interaction with
MRF4, since forced expression of MRF4 in C2C12 cells increases the
activity of the 3'-positive element to the same level observed in
primary muscle cells.2 The absence of high levels of SkM1
gene expression in the constructs that lack the 3'-positive element
indicates its important role, particularly for the maintenance of
expression in later stages of differentiation.
Although our initial analysis presents the relationship between the
promoter E-box-binding proteins and other transcription factors as a
one-on-one interaction, this is certainly an oversimplification of an
association that is probably far more complex, with changes occurring
in the entire transcription initiation complex to switch it from an
"inactive" to and "active" state. The myogenic bHLH proteins
and perhaps factors that are still unknown may independently contribute
to the assembly of the transcription initiation complex. Our data
suggest that only specific myogenic bHLH proteins can function in
conjunction with the other SkM1 factors, and it may be that the bHLH
proteins confer the muscle-specific action to the complex. However, the
myogenic bHLH factors cannot act alone. Our future work will be
directed toward identifying all of the factors involved and
understanding the interplay between them.