(Received for publication, October 13, 1994; and in revised form, December 19, 1994)
From the
To identify potential regulators of smooth muscle cell (SMC)
differentiation, we studied the molecular mechanisms that control the
tissue-specific transcriptional expression of SM -actin, the most
abundant protein in fully differentiated SMCs. A construct containing
the region from -1 to -125 of the promoter (p125CAT) had
high transcriptional activity in SMCs (57-fold > promoterless) and
endothelial cells (ECs) (18-fold) but not in skeletal myoblasts or
myotubes. Mutation of either of two highly conserved
CC(AT-rich)
GG (CArG) motifs at -62 and -112
abolished the activity of p125CAT in SMCs but had no effect in ECs. In
contrast, high transcriptional activity in skeletal myotubes, which
also express SM
-actin, required at least 271 base pairs of the
promoter (-1 to
-271). Constructs containing 547 base
pairs or more of the promoter were transcriptionally active in SMCs and
skeletal myotubes but had no activity in skeletal myoblasts or ECs,
cell types that do not express SM
-actin. Electrophoretic mobility
shift assays provided evidence for binding of a unique serum response
factor-containing complex of factors to the CArG box elements in SMCs.
Results indicate that: 1) transcriptional expression of SM
-actin
in SMCs requires the interaction of the CArG boxes with SMC
nucleoprotein(s); 2) expression of SM
-actin in skeletal myotubes
requires different cis-elements and trans-factors
than in SMCs; and 3) negative-acting cis-elements are
important in restricting transcription in cells that do not express SM
-actin.
Atherosclerosis, which accounts for a large proportion of the
morbidity and mortality associated with cardiovascular
disease(1, 2) , is characterized by the abnormal
growth and phenotypic modulation of vascular smooth muscle cells
(SMCs)()(3) . Intimal SMCs within atherosclerotic
lesions of man, as well as experimental animals, show diminished
expression of a number of proteins that are characteristic of fully
differentiated SMCs, including SM
-actin and SM myosin heavy
chain(4, 5) . As such, an understanding of the
mechanisms that control SMC differentiation is likely to be important
for understanding atherogenesis.
A key to understanding SMC differentiation is to identify factors that regulate the coordinate expression of cell-specific genes that distinguish one cell type from another. Studies of skeletal muscle development have lead to the characterization of a number of ``master control'' genes that regulate skeletal muscle differentiation or lineage determination, including myoD and related members of the helix-loop-helix family(6, 7) . These factors have been shown to activate the expression of genes characteristic of mature skeletal muscle cells by functioning as transcription factors that bind to a specific DNA-binding motif, referred to as an E-Box, that is found in the 5`-flanking regions of a number of skeletal muscle-specific genes (7) . More recently, Tontonoz et al.(8) identified a helix- loop-helix transcription factor that appears to regulate differentiation-specific gene expression in adipocytes. Thus, it is likely that the expression of at least some SM-specific genes and the molecular mechanisms involved in SMC differentiation rely on analogous transcriptional regulatory systems, although no SM-specific regulatory/transcription factors have as yet been characterized.
The principle function of mature, fully
differentiated SMCs is contraction; therefore, genes encoding proteins
involved in SM contraction, such as -actin, myosin heavy chain,
myosin light chain, caldesmon, vinculin, calponin, and metavinculin,
are candidates to study transcriptional regulatory systems in SMC
differentiation. The SM isoforms of myosin light chain, caldesmon,
vinculin, and metavinculin are produced by alternative splicing of gene
transcripts expressed in a variety of cell
types(9, 10, 11, 12, 13) .
Thus, these genes are not likely to be suitable models to study
SM-specific transcriptional regulatory systems. The SM myosin heavy
chain gene shows a high degree of SM-specific expression(14) ;
however, sequence information on this promoter has not been reported.
Currently, one of the best genes to begin to characterize
transcriptional cis-elements and trans-factors
involved in control of SMC differentiation is SM
-actin, which is
the most abundant of the SM contractile proteins in mature vascular
SMC(15) . Although SM
-actin is transiently expressed in
cardiac (16, 17) and skeletal (17) muscle
during development and in myofibroblasts within tumors and healing
wounds (18) , it is exclusively expressed in SMCs and
SM-related cells in the normal adult
animal(19, 20, 21) . Additionally, the
expression of SM
-actin is modulated in proliferating SMCs found
within human atherosclerotic
lesions(4, 22, 23) .
Studies of the
chicken, mouse, and human SM -actin genes have been performed
previously and have demonstrated the importance of cell context in the
characterization of promoter elements that function in transcriptional
regulation(24, 25, 26, 27) . Studies
of the chicken SM
-actin gene demonstrated that the first 122 bp
of the promoter were sufficient to confer a moderate to high level of
transcriptional activity in chicken SMCs, skeletal myoblasts, and
fibroblasts and that the addition of a 29-bp promoter segment (to
-151) restricted expression in fibroblasts and abolished
expression in skeletal myoblasts. However, this same segment induced a
doubling of activity in SMCs(24, 27) . Studies
utilizing the mouse SM
-actin promoter transfected into mouse
AKR-2B embryonic fibroblasts and subconfluent mouse skeletal
myoblast-like BC3H1 cells demonstrated that the region between
-224 and -192 abolished expression of the first 191 bp of
the promoter in both cell types. However, site-directed mutagenesis
studies showed that the negative-acting cis-element within the
-244 to -192 region that abolished expression in BC3H1
cells was different from the negative element in
fibroblasts(25) . These studies, therefore, demonstrated that
the regulatory elements responsible for transcriptional expression of
the SM
-actin gene are highly dependent upon the cell type in
which the promoter is studied.
The SM -actin promoter contains
a number of highly conserved putative regulatory elements. One such
element is the CArG box, which has the general sequence motif
CC(A/T-rich)
GG. The 5`-flanking regions of both the
skeletal and cardiac
-actin genes contain conserved CArG box
elements that have been shown to direct developmental and
tissue-specific expression of these
genes(28, 29, 30, 31) , and CArG
elements have also been implicated in the regulation of the myosin
light chain(32, 33) , muscle creatine
kinase(34) , and dystrophin genes(35) . Additionally, a
CArG box motif is the core component of the serum response element
(SRE), a DNA element that is required for the transient transcriptional
response of many nonmuscle-specific immediate-early response genes,
such as c-fos and
actin, upon serum or growth factor
stimulation(36, 37) . Although previous studies have
demonstrated that the SM
actin CArG elements can function in
serum-inducible expression of the gene in
fibroblasts(38, 39) , the expression of the endogenous
SM
-actin gene is not serum-inducible in SMCs, even though
-actin expression is serum-stimulated under the same
conditions(40) . Indeed, although previous studies have shown
that promoter regions containing the CArG boxes were transcriptionally
active in SMCs(26, 27) , no direct evidence for a
regulatory role of the SM
-actin CArG elements in SMCs has been
reported.
The goals of the current study were to identify cis-acting DNA elements within the rat SM -actin promoter
that govern SM-specific transcriptional expression, to determine
whether the CArG motifs play an important role in the regulation of SM
-actin expression in SMCs, and to determine whether there is
evidence for the binding of SM nuclear proteins to these regulatory
sites. Although not a goal of the present study, the long term
objective is to determine if these SM nuclear proteins act as
transcription factors that not only regulate the transcription of SM
-actin but also function as global regulators of other SM-specific
genes; i.e. function as master regulatory factors. The results
of these studies provide evidence that 1) the cell-specific expression
of SM
-actin is due to a complex combination of both negative and
positive acting cis-elements found within the first 547 bp of
the promoter; 2) the CArG box elements, along with additional 5`- or
3`-flanking regions, are important in the tissue-specific positive
activation of SM
-actin; and 3) the SM-specific trans-factors that bind the CArG boxes include serum response
factor (SRF) or an SRF-like protein. These studies also support the
utility of the SM
-actin promoter in understanding the molecular
regulation of SMC differentiation and also underscore the potential
usefulness of this promoter in expressing bioactive molecules in a
SMspecific manner.
Site-directed CArG mutations were generated using the Altered Sites in vitro mutagenesis system (Promega) according to the manufacturer's recommendations. The mutated promoter fragments were then PCR amplified, subcloned into pCAT-Basic, and sequence verified as described above.
All promoter-CAT plasmid DNAs used for transient transfections were prepared using an alkaline lysis procedure (42) followed by banding on two successive ethidium bromide-cesium chloride gradients. Each plasmid preparation was examined by electrophoresis on 1% agarose gels, and preparations were judged acceptable if >50% of the DNA was supercoiled and if the relative amount of supercoiled to nicked plasmid DNA was approximately the same for all constructs used in the same set of transfections. Each promoter construct is designated by a ``p'' followed by a number that indicates the 5`-most promoter sequence relative to the transcription start site. The 3` end of each promoter fragment ends at position +20 within the first exon.
Cells were seeded for transient transfection assays into
6-well plates (Corning Glass; Corning, NY) at a density of 2
10
cells/cm
for SMCs and L6 skeletal myoblasts,
3
10
cells/cm
for BAECs, and 4
10
cells/cm
for RAECs. These densities were
chosen so that the cells would be at 60-80% confluency at the
time of transfection (30 h after plating). Transient transfection
experiments consisted of transfecting each promoter-CAT plasmid in
triplicate using Transfectam reagent according to the
manufacturer's recommendations (Promega). Briefly, for each well
to be transfected, 5 µg of a plasmid construct (in 10 µl of 10
mM Tris (pH 8.0), 1 mM EDTA) was incubated for 10 min
in a tissue culture-sterile microcentrifuge tube with 10 µl of
Transfectam reagent and 380 µl of transfection media. During the
incubation, each well was washed twice with 3 ml of transfection medium
to remove residual serum, and after the second media wash was removed,
250 µl of fresh transfection medium was added to each well. After
incubation, 400 µl of DNA/Transfectam/media solutions were added to
each well. The transfection media consisted of the following: a 1:1
formulation of Dulbecco's modified Eagle's medium and
Ham's F-12 medium (Life Technologies, Inc.) for SMCs,
Dulbecco's modified Eagle's medium for L6 skeletal
myoblasts and myotubes, MCDB 105 for RAECs, and Waymouth's basal
medium (Life Technologies, Inc.) for BAECs. Each of the transfection
mediums also contained 0.68 mML-glutamine, 100
units/ml penicillin, and 100 µg/ml streptomycin. At 14 h
post-transfection, the cells were changed to the cell-specific culture
medium described above. In the case of skeletal myotubes,
culture/differentiation medium was the same as for the skeletal
myoblasts except that only 1% FBS was included. After 48 h in culture
medium, the cells had reached confluency and were harvested by scraping
in harvesting buffer (30 mM Tris, 6 mM EDTA, 150
mM NaCl (pH 7.5)), pelleted, resuspended in 100 µl of cold
100 mM Tris (pH 7.5), and stored at -70 °C.
Cell
extracts were prepared by four cycles of freezing in a dry ice/ethanol
bath, thawing, and vigorous vortexing followed by centrifugation to
remove cellular debris. A 60-µl aliquot of each cell extract was
heat-inactivated, and 55 µl of each heat-inactivated extract was
transferred to a fresh microfuge tube and was assayed for CAT activity
by enzymatic butyrylation of tritiated chloramphenicol (DuPont NEN) as
described previously(49) . All CAT activity values were
normalized to the protein concentration of each cell lysate as measured
by the Bradford assay (50) using Bio-Rad's protein assay
reagent (Bio-Rad). Early transfection experiments utilized a
-galactosidase plasmid as a co-transfection partner to measure
transfection efficiency. However, the
-galactosidase activity
measurements did not result in qualitative changes in the data nor did
they affect experimental variability. Since the co-transfections
conferred no advantage in the reduction of experimental error and had
the potential of competing for limited trans-factors that
regulate SM
-actin transcription, the
-galactosidase
co-transfections were discontinued. In each experiment, the
promoterless pCAT-Basic construct was also transfected into triplicate
wells to serve as the base line indicator of CAT activity, and the
activity of each promoter construct is expressed relative to the
promoterless construct set to one. Additionally, a SV40
enhancer/promoter-CAT construct (Promega) served as a positive control
of transfection and CAT activity. All CAT activity values represent at
least three independent transfection experiments with each construct
tested in triplicate per experiment. Relative CAT activity data are
expressed as the means ± standard error computed from the
results obtained from each set of transfection experiments. One-way
analysis of variance followed by the Newman-Keuls' multiple range
test were used for data analysis. Values of p < 0.05 were
considered statistically significant.
The 95-bp promoter segments were
generated by PCR amplification using wild-type or CArG box-mutated
promoter-CAT plasmids as the templates and PCR primers containing EcoRI restriction sites. After the reactions were chloroform
extracted, the PCR products were EtOH precipitated, and EcoRI
restriction endonuclease (Promega) was digested and gel-purified in a
1% agarose gel using the Magic PCR prep DNA purification system
(Promega). The EcoRI 5`-overhangs were filled in using Klenow
enzyme (Boehringer-Mannheim), [-
P]dATP
(6000 Ci/mmol) (DuPont NEN), and unlabeled dCTP, dGTP, and dTTP
(Promega). The unincorporated nucleotides were removed using NucTrap
Push columns (Stratagene) as recommended by the manufacturer. After
fill-in labeling, the 95-bp fragments included nucleotide positions
-137 to -43.
CArG oligonucleotides for EMSA contained 5
bp 5` and 3` of each CArG element (20 bp total). Mutant CArG oligos
involved changing both the flanking CCs and GGs of the CArG motif
(CC[AT]GG) to AAs. Both oligonucleotides of a
double-stranded duplex were end-labeled separately with T4
polynucleotide kinase (Promega) and [
-
P]ATP
(6000 Ci/mmol) (DuPont NEN) and annealed together. Unincorporated
nucleotides were removed as described above.
EMSA were performed
with 20 µl of binding reaction that contained 0.24 ng of
P-labeled 95-bp fragment or
30 pg of
P-labeled annealed oligonucleotides, 5 or 10 µg of
crude nuclear extract (in 5 µl of Dignam buffer D(51) ), or
2.5 µl of in vitro synthesized SRF, 35 mM Tris
(pH 7.5), 5 mM HEPES, 200 mM KCl, 1.125 mM dithiothreitol, 1.05 mM EDTA, 0.25 µg of poly(dI-dC),
10% glycerol, and competitor oligonucleotides where indicated. All
binding reactions were incubated for 30 min at room temperature. When
affinity-purified anti-SRF antibodies (generous gifts from Ravi P.
Misra and Michael E. Greenberg; Harvard Medical School; Boston, MA)
were included in the EMSA, they were also incubated in binding
reactions for 30 min as described above except without the radiolabeled
DNA. The radiolabeled DNA was subsequently added and incubated for an
additional 30 min. All binding reactions were then loaded on 5%
polyacrylamide gels (30:1 acrylamide/bis-acrylamide (Bio-Rad)), which
had been prerun at 170 V for 30 min and electrophoresed at 170 V in 0.5
TBE (45 mM Tris borate, 1 mM EDTA). The gels
were then dried and exposed to Kodak X-Omat LS film at -70 °C
in the presence of an intensifying screen. The binding reactions for UV
cross-linking experiments were performed in a similar manner except
that 7.5 µg of SMC nuclear extract or 4 µl of in vitro synthesized SRF were used per 20-µl reaction. Following
electrophoresis in 5% polyacrylamide gels, the gels were UV irradiated
at a wavelength of 254 nm and a distance of 6 cm for 30 min at 4 °C
and exposed to film at 4 °C overnight. The bands corresponding to
the protein-DNA complexes of interest were excised from the gels,
placed in 50 µl of sample buffer (50 mM Tris (pH 6.8), 1%
SDS, 25 mM dithiothreitol, 10% glycerol, and 0.001% bromphenol
blue tracking dye), and electrophoresed on one-dimensional
SDS-polyacrylamide gels as described previously(15) . The gels
were then dried and autoradiographed as described above.
Figure 1:
A, structures of the rat SM
-actin promoter-CAT reporter gene constructs. Promoter sequences
were PCR amplified and ligated into a plasmid containing a promoterless
CAT gene. The 5`-most nucleotide position is indicated above the diagram of each chimera. The relative positions of putative cis-acting regulatory regions are also indicated. B,
expression of SM
-actin promoter-CAT constructs in adult rat
aortic smooth muscle cells. Subconfluent cultures were transiently
transfected with the plasmid constructs pictured in A. CAT
activity was expressed relative to the base-line CAT activity of a
promoterless-CAT construct set to one. An SV40 enhancer/promoter-CAT
construct served as a positive control for transfection and CAT
activity. The data represent six independent experiments, each
experiment performed with triplicate samples per plasmid
construct.
To identify cis-elements
responsible for cell-type specific regulation of SM -actin, the
truncated promoter-CAT constructs were also transfected into non-SMC
lines. These cell lines included aortic endothelial cells, which do not
express endogenous SM
-actin based on both Northern analysis as
well as evaluation of SM
-actin protein expression by
two-dimensional gel electrophoresis and immunostaining with SM
actin-specific antibody (data not shown), and L6 skeletal
myoblasts, which also do not express SM
-actin but which stimulate
endogenous SM
-actin expression when induced to differentiate into
myotubes ((53) and data not shown). In skeletal myoblasts, the shorter
promoter constructs (p125CAT to p371CAT) only modestly activated the
transcription of the CAT reporter gene (up to 6-fold above
promoterless-CAT), while the longer promoter constructs (p547CAT to
pPromCAT) failed to elicit any increase in CAT expression over that of
the promoterless-CAT construct (Fig. 2A). These data
suggest that the promoter contains an element(s) within the region
between -547 and -371 that represses transcriptional
expression of SM
-actin in skeletal myoblasts. This contrasts with
results seen in SMCs in which there was a prominent CAT activity
produced by the shorter promoter constructs as well as by p547CAT to
pPromCAT.
Figure 2:
Expression of SM -actin promoter-CAT
constructs in rat L6 skeletal myoblasts and myotubes (A) and
rat and bovine aortic endothelial cells (B). Subconfluent
cultures were transiently transfected with the plasmid constructs
pictured in Fig. 1A. CAT activity was expressed
relative to the base-line CAT activity of a promoterless-CAT construct
set to one. An SV40 enhancer/promoter-CAT construct served as a
positive control for transfection and CAT activity. The skeletal
myoblast, myotube, and RAEC data represent three independent
experiments, and the BAEC data represent five independent experiments,
each experiment performed with triplicate samples/plasmid
construct.
When the truncated promoter-CAT constructs were
transiently transfected into skeletal myoblasts that were then induced
to differentiate into myotubes (Fig. 2A), the results
again differed from that seen in SMCs in that the shortest promoter
constructs (p125CAT to p208CAT) moderately activated transcription (up
to 9-fold), similar to the level of activity seen in skeletal
myoblasts. However, upon addition of the region between -271 and
-208 (p271CAT), which contains two putative E-Box motifs, CAT
expression increased 18-fold compared with promoterless-CAT in skeletal
myotubes but had no affect in skeletal myoblasts or SMCs. Additionally,
the longest promoter-CAT constructs retained transcriptional activity
(up to 20-fold above promoterless-CAT) in skeletal myotubes, similar to
that seen in SMCs. The differential expression of the full-length
promoter (pPromCAT) between SMCs, skeletal myoblasts, and skeletal
myotubes correlated with endogenous SM -actin mRNA levels in these
cells in that SMCs and skeletal myotubes expressed a high level of SM
-actin mRNA, as determined by Northern analysis, while skeletal
myoblasts did not (data not shown).
Cell-type specific expression of
the truncated promoter-CAT constructs was further tested in RAECs,
another cell line that does not express its endogenous SM -actin
gene. Surprisingly, p125CAT exhibited high CAT activity (up to 18-fold
above promoterless-CAT). This was subject to negative regulation with
addition of upstream promoter sequences (Fig. 2B).
However, in contrast to the activity observed in SMCs, the longer
promoter-CAT constructs (p547CAT to pPromCAT) had no activity above the
promoterless-CAT level in RAECs. Studies were repeated in BAECs, which
also did not express their endogenous SM
-actin gene at the mRNA
or protein level (data not shown). As shown in Fig. 2B,
the general pattern of promoter activities in BAECs was the same as
seen in RAECs. The similarity between RAECs and BAECs suggests that the
mechanisms and trans-factors that regulate SM
-actin
expression in RAECs and BAECs are likely to be the same. Since RAECs
were more difficult to transfect and more costly to grow in culture
than BAECs, subsequent studies requiring endothelial cells utilized
BAECs.
The transfection results in endothelial cells demonstrated
that there are negative-acting cis-elements within the
promoter regions -547 to -371, -208 to -271,
and -125 to -155 that repress expression of SM -actin
in these cells. These results are similar to those obtained in skeletal
myoblasts in which the larger promoter constructs were not
transcriptionally active. In contrast, in SMCs and skeletal myotubes,
which transcribe their endogenous SM
-actin gene, there appeared
to be differential regulation of the gene since the first 125 bp of the
promoter were sufficient to express a very high level of
transcriptional activity in SMCs but not in skeletal myotubes. Taken
together, these data demonstrate that the first 547 bp of the promoter
are sufficient to confer tissue-specific expression of the SM
-actin gene in these cultured cell types.
Figure 3:
Smooth muscle -actin promoter
sequence. This figure compares the 5`-flanking regions of rat (27) , mouse(25) , human(84) , and chicken (85) SM
-actin genes. Nucleotides conserved with the rat
sequence are indicated by dashedlines. Spaces indicate gaps introduced in the sequence to optimize the
alignment. Numbers in the right margin refer to the
position of the last nucleotide in each line relative to the
transcriptional start site. Putative regulatory sequences that are
conserved between at least two of the depicted species are boxed. This figure was previously published by Blank et
al.(27) except for the nucleotide corrections in the rat
sequence in the region between -161 and
-159.
Figure 4:
A, CArG mutations introduced into the rat
SM -actin promoter-CAT reporter constructs. Site-directed
mutations were introduced using Altered Sites in vitro mutagenesis system. The relative positions of mutated CArG box
elements are also indicated. B, effects of CArG mutations in
SM
-actin p125CAT constructs transfected into rat aortic SMCs and
endothelial cells. Subconfluent cultures were transiently transfected
with CArG mutant constructs pictured in Fig. 4A. CAT
activity was expressed relative to the base-line CAT activity of a
promoterless-CAT construct set to one. The SMC data represent four
independent experiments, and the EC represent three independent
experiments, each experiment performed with triplicate samples/plasmid
construct. Similar results were obtained with mutation of the CArG
motifs in the p271CAT construct (data not
shown).
To determine whether mutation of CArG-A or CArG-B would also abolish
the transcriptional activity induced by the full-length promoter, the
same site-specific CArG mutations were created in the pPromCAT
construct. This resulted in approximately a 50% reduction in the CAT
activity in SMCs (Fig. 5), demonstrating that while both CArG-A
and CArG-B were essential for p125CAT to be transcriptionally active in
SMCs, there is likely an additional upstream cis-element(s)
required for full activity of the full-length promoter. This additional
positive-acting cis-element(s) may be upstream of -699
of the promoter since activity of pPromCAT had significantly greater
activity (p < 0.05) than p699CAT (Fig. 1B).
Consistent with this, in studies with the human SM -actin gene
transiently transfected into rat SMCs, Nakano et al.(26) found suggestive evidence for a positive-acting
upstream regulatory element in the region between -891 and
-597 of the human promoter.
Figure 5:
Effects of CArG mutations in SM
-actin pPromCAT constructs transfected into SMCs and skeletal
myotubes. Subconfluent cultures were transiently transfected with
pPromCAT or pPromCAT constructs mutated in CArG-A, CArG-B, or both. CAT
activity was expressed relative to the base-line CAT activity of a
promoterless-CAT construct set to one. The data for each cell line
represents three independent experiments, each experiment performed
with triplicate samples/plasmid construct.
The pPromCAT/CArG mutant
constructs were also studied in skeletal myotubes, which express their
endogenous SM actin gene. Results demonstrated that mutation of
either CArG box reduced transcriptional activity of pPromCAT in
myotubes by approximately 50% (Fig. 5). Therefore, the CArG
boxes appear to be involved in the transcription of SM
-actin in
both SMCs and skeletal myotubes. However, the mode of regulation via
the CArG box elements is different in the two cell-types. Whereas the
first 125 bp of the promoter were sufficient for a high level of
transcription in SMCs (Fig. 1B) and the activity was
completely dependent upon the CArG boxes in SMCs (Fig. 4B), the same 125-bp promoter segment induced
only modest activity in skeletal myotubes (Fig. 2A).
This implies that transcriptional activation via the CArG elements
requires an interaction with sequence elements upstream of -125
in skeletal myotubes but not in SMCs. The upstream element(s) required
for expression in skeletal myotubes may reside between -271 and
-208, which contains two putative E-Boxes, since addition of this
region was necessary for high levels of transcriptional activation (Fig. 2A). Therefore, although the CArG box elements
functioned in the transcriptional regulation of SM
actin in both
SMCs and skeletal myotubes, there was a qualitative difference in the
regulation of transcription via the CArG box elements in these
cell-types, and this difference is likely to be due to a complex array
of cell-specific cis-elements and trans-factors.
Figure 6:
A, structure of rat SM -actin
promoter segments used in electrophoretic mobility shift assays.
Oligonucleotide primers with engineered EcoRI restriction
sites were used to PCR amplify the 95-bp promoter sequences for
subsequent restriction and Klenow enzyme fill-in with
[
-
P]dATP. The 20-bp CArG box
oligonucleotides were end-labeled with
[
-
P]ATP and T4 polynucleotide kinase and
annealed to form double-stranded duplexes. The 5`- and 3`-most
nucleotide positions are indicated above the diagram of each
promoter segment. The relative positions of mutated CArG box elements
are also indicated. B, binding of SMC nuclear proteins to the
wild-type 95-bp SM
-actin promoter segment. The radiolabeled
5`-flanking DNA segment from -137 to -43 (*WT
), which contained wild-type CArG-A
and CArG-B sequences, was incubated with 5 µg of SMC nuclear
extract. Competition reactions were performed with 20-bp
double-stranded oligonucleotide duplexes of the CArG-A element (wtA), CArG-B element (wtB), or their mutations (mA and mB, respectively). Competitor
oligonucleotides were added at a 50-400-fold molar excess
relative to the radiolabeled DNA. The positions of the three principle
nucleoprotein/DNA complexes (labeled 1-3) are indicated
by the arrows.
Incubation of the
wild-type 95-bp fragment (*WT) with SMC NE resulted in the
formation of three shift bands as well as a fourth band below band 3
with some SMC NE preparations (Fig. 6B, lane1). Addition of unlabeled 20-bp double-stranded
oligonucleotide duplexes homologous to CArG-A (wtA) or CArG-B (wtB) sequences competed for binding of nuclear factors and
inhibited the formation of bands 1 and 2 (Fig. 6B, lanes2-6 and 9-13,
respectively). In contrast, mutant double-stranded CArG-A and CArG-B
oligonucleotide duplexes (mA and mB, respectively)
failed to compete (lanes7-8 and 14-15). This demonstrates that SMC nuclear proteins are
binding the CArG-A and CArG-B elements leading to shift bands 1 and 2.
This contrasts with band 3 (and 4) that appeared to represent binding
to other areas of the 95-bp segment since these bands were not competed
for by CArG box oligonucleotides.
As a further test of CArG box interaction with SMC nuclear proteins, the 95-bp DNA segment containing mutations of CArG-A and/or CArG-B were used in EMSA. Mutation of CArG-A preferentially decreased formation of shift band 1 relative to shift band 2, and competition with double-stranded CArG-A and CArG-B oligonucleotides inhibited formation of shift band 1 but not band 2 (Fig. 7A). In a similar manner, mutation of CArG-B by itself or of CArG-A and CArG-B together nearly abolished shift band 1 formation, and competition with double-stranded CArG-A and CArG-B oligonucleotides had no additional affect on any of the shift bands (Fig. 7B). For reasons that are not clear, CArG oligonucleotide competitors did not compete for formation of band 2 when using the 95-bp DNA segments containing mutations of CArG-A or -B alone or together, although they did compete for this band using the wild-type 95-bp DNA segment (Fig. 6B).
Figure 7:
Binding of SMC nuclear proteins to the
95-bp SM -actin promoter segment containing mutations of CArG-A (A) or mutations of CArG-B or CArG-AB (B).
Radiolabeled 5`-flanking DNA segment from -137 to -43 that
contained a mutated CArG-A (*mA
), a
mutated CArG-B (*mB
), or both (*mAB
) was incubated with 5 µg of
SMC nuclear extract. The wild-type 95-bp segment (*WT
) was also included in these
experiment as a comparison (lanes1). Competition
reactions were performed with 20-bp double-stranded oligonucleotide
duplexes of the CArG-A element (wtA), CArG-B element (wtB), or their mutations (mA and mB,
respectively). Competitor oligonucleotides were added at a
50-400-fold molar excess relative to the radiolabeled DNA. The
positions of the three principle nucleoprotein/DNA complexes (labeled 1-3) are indicated by the arrows.
EMSA
experiments using P-labeled 20-bp double-stranded CArG-A
and CArG-B oligonucleotide duplexes (Fig. 6A) were also
performed to determine whether these short CArG fragments are
sufficient to bind nuclear factors and whether the factors that bind
the different CArG boxes are equivalent. Incubation of the wild-type
CArG-B oligonucleotide duplex (*wtB
) with SMC NE produced
two slowly migrating shift bands, labeled A and B, that were both
specifically competed away with increasing amounts of CArG-A and CArG-B
competitor oligonucleotides (Fig. 8, lanes2-4 and 7-9; also see Fig. 10A, lane3) but not with mutant
competitors (Fig. 8, lanes5-6 and 10-11). Shift bands A and B were also formed with the
wild-type CArG-A oligonucleotide duplex (*wtA
) (see Fig. 10A, lane1), although their
intensities were less. These were also inhibited by CArG
oligonucleotide competitors (data not shown). In contrast, shift bands
A and B did not form when the mutated CArG-A and CArG-B oligonucleotide
duplexes (*mA
and *mB
) were used (see Fig. 10A, lanes2 and 4),
further demonstrating the CArG specificity of bands A and B. Reaction
of the CArG oligonucleotides with SMC NE also resulted in the formation
of two faster migrating shift bands/complexes (labeled C and D).
However, these shift bands/complexes appeared to represent nonspecific
binding since mutation of CArG-A or CArG-B (*mA
and
*mB
) did not abolish formation of these shift bands (see Fig. 10A) and competitions with unlabeled CArG
oligonucleotides did not effectively decrease their formation (Fig. 8). Thus, these results confirm that SMC nuclear proteins
can bind specifically to both CArG box elements, consistent with EMSA
using the 95-bp segments. Additionally, the results shown in Fig. 6B and Fig. 8also revealed that lower
concentrations of CArG-B competitor were needed to inhibit the
formation of CArG-specific shift bands, suggesting that CArG-B has a
greater affinity for the CArG binding factor(s) than does CArG-A. The
differences in shift band intensities between *wtA
and
*wtB
were not due to a difference in the specific
activities of those oligonucleotides since the difference in shift band
intensities were observed even when the CArG-A oligonucleotide duplex
had a higher specific activity.
Figure 8:
Binding of SMC nuclear proteins to the
wild-type CArG-B. Radiolabeled wild-type 20-bp double-stranded CArG-B (*wtB) oligonucleotide duplex was
incubated with 5 µg of SMC nuclear extract. Competition reactions
were performed with unlabeled 20-bp double-stranded oligonucleotide
duplexes of the CArG-A element (wtA), CArG-B element (wtB), or their mutations (mA and mB,
respectively). Competitor oligonucleotides were added at a
25-400-fold molar excess relative to the radiolabeled DNA. The
positions of four nucleoprotein/DNA complexes (labeled A-D) are indicated by the arrows.
Figure 10:
A, binding of SMC and EC nuclear proteins
to the wild-type and mutated 20-bp CArG-A and CArG-B elements and to
the 22-bp SRE CArG element. Radiolabeled wild-type and mutated 20-bp
double-stranded CArG-A (*wtA and *mA
, respectively), CArG-B (*wtB
and *mB
, respectively), and the 22-bp SRE
CArG (*SRE) oligonucleotide duplexes were incubated with 10
µg (for CArG-A duplexes) or 5 µg (for CArG-B and SRE duplexes)
of SMC or EC nuclear extracts. The positions of four nucleoprotein-DNA
complexes (labeled A-D) are indicated by the arrows. B, binding of nuclear proteins from skeletal
myoblast and myotube cell lines to the wild-type 95-bp SM
-actin
promoter segment. The radiolabeled wild-type 5`-flanking DNA segment
from -137 to -43 (*WT
) was
incubated with 5 µg of rat SMC NE (lane1), rat
L6 skeletal myoblast NE (lane2), or rat L6 skeletal
myotube NE (lane3). The arrows labeled 1 and 2 indicate the CArG-specific SMC nucleoprotein
complexes. The unlabeled arrows identify the positions of the
nucleoprotein complexes formed with L6 skeletal cell line nuclear
extracts. C, comparison of SMC nuclear proteins and in
vitro synthesized serum response factor bound to the wild-type
95-bp SM
-actin promoter segment and the wild-type 20-bp CArG-B
element. Lane1, radiolabeled wild-type 95-bp
promoter segment (*WT
) reacted with 5
µg of SMC nuclear extract; lane2, radiolabeled
wild-type CArG-B oligonucleotide duplex (*wtB
) with 5 µg of SMC nuclear
extract; lane3, *WT
incubated with 2.5 µl of SRF in vitro synthesized with rabbit reticulocyte lysate; lane4, *WT
incubated with 2.5
µl of unprogrammed (UP) rabbit reticulocyte lysate; lane5, *wtB
with 2.5
µl of SRF lysate; and lane6, same as lane1. This gel was run twice as long as the previous gels in
order to optimize comparisons of shift band positions. The three
principle SMC nucleoprotein complexes that form with *WT
are indicated by the arrows labeled 1-3 (compare Fig. 6B).
Three of the principle SMC nucleoprotein complexes that form with *wtB
are indicated by the arrows labeled A-C (compare Fig. 8B).
Initial studies utilized an
affinity-purified polyclonal antibody raised against the amino terminus
of SRF (referred to as R1119, a generous gift from Ravi P. Misra and
Michael E. Greenberg; Harvard Medical School; Boston, MA). This
antibody was previously shown to react with SRF from both murine and
human cell lines, as well as with in vitro synthesized human
SRF(64) . In EMSA, the antibody reacted with the SMC nuclear
factors that form CArG-specific shift bands 1 and 2 with *WT and resulted in at least one supershifted band of slower
electrophoretic mobility (Fig. 9A). The anti-SRF
antibody did not, however, react with nuclear factors in
non-CArG-specific shift band 3. In a similar manner, the migration of
CArG-specific shift bands A and B formed with *wtB
was
also altered when reacted with the same antibody, while bands C and D
were unaffected. In contrast, addition of pre-immune serum or non-SRF
antibodies did not alter shift patterns (data not shown). A second
affinity purified antibody against the amino terminus of SRF (R1122)
and immune serum raised against SRF's carboxyl terminus (R1443;
also gifts from R.P. Misra and M. E. Greenberg) yielded similar results
to those with SRF R1119 antibody (data not shown). Furthermore, UV
cross-linking assays demonstrated that the size of the SM CArG
box-binding protein binding to the CArG-B element was approximately 67
kDa (data not shown), similar to the known size of SRF(52) .
This strongly suggests that SRF, or a protein related to SRF in size
and antigenicity, is at least one of the SMC nuclear factors binding
the SM
-actin CArG elements.
Figure 9:
A, binding of the wild-type 95-bp SM
-actin promoter segment and the wild-type 20-bp CArG-B element to
SMC nuclear proteins that had been reacted with an antibody raised
against the amino terminus of serum response factor. The leftside of the figure shows the results with wild-type
5`-flanking DNA segment from -137 to -43 (*WT
). The arrows labeled 1-3 indicate the positions of the three principle
nucleoprotein/DNA complexes that formed when *WT
was reacted with SMC nuclear extract by itself (lane1). The rightside of the figure shows
the results with the wild-type CArG-B oligonucleotide duplex (*wtB
). The arrows labeled A and B indicate the positions of the two
CArG-specific nucleoprotein/DNA complexes that formed when *wtB
was reacted with SMC nuclear
extract by itself (lane7). The radiolabeled DNAs
were incubated with 5 µg of SMC nuclear extract and various
dilutions of affinity-purified polyclonal antibodies (referred to as
R1119) raised against SRF. The top, unlabeled arrows identify the supershifted nucleoprotein-DNA complexes that formed
upon reaction with anti-SRF antibody. B, binding of in
vitro synthesized serum response factor to the wild-type 20-bp
CArG-B element. Radiolabeled wild-type CArG-B oligonucleotide duplex (*wtB
) was incubated with 2.5 µl of
SRF in vitro synthesized with rabbit reticulocyte lysate.
Competition reactions were performed with unlabeled 20-bp
double-stranded oligonucleotide duplexes of the CArG-A element (wtA), CArG-B element (wtB), or their mutations (mA and mB, respectively). Competitor
oligonucleotides were added at a 100-400-fold molar excess
relative to the radiolabeled DNA. *wtB
incubated with 5 µg of SMC nuclear extract was also
included in this experiment as a comparison (lane1),
and the positions of the two CArG-specific SMC nucleoprotein/DNA
complexes (labeled A and B) are indicated by the arrows. Other comparison lanes included *wtB
incubated with 2.5 µl of
unprogrammed (UP) rabbit reticulocyte lysate (lane13), mutated CArG-B oligonucleotide duplex (*mB
) incubated with 2.5 µl of SRF
lysate (lane14), and wild-type CArG-A
oligonucleotide duplex (*mA
) incubated
with 2.5 µl of SRF lysate (lane15).
To further investigate the
possible involvement of SRF in binding the SM -actin CArG box
elements, the binding characteristics of in vitro synthesized
SRF were compared with the SMC nuclear proteins. As shown in Fig. 9B, reacting *wtB
with in vitro synthesized SRF resulted in shift bands with similar
electrophoretic mobilities and CArG box-specific competition properties
as SMC NE bands A, B, and C (compare with Fig. 8and 10A).
As with CArG-specific shift bands A and B with the SMC NE, the two
slowest migrating SRF shift bands also appeared to be CArG box-specific
for the following reasons. First, competition reactions with the
wild-type CArG-B oligonucleotides inhibited the formation of the two
top bands (Fig. 9B, lanes8-10),
while the mutant CArG-B oligonucleotides did not (lanes11 and 12). Second, unprogrammed lysate, which was reacted
with *wtB
(lane13), and SRF lysate,
which was reacted with the mutated CArG-B oligonucleotides
(*mB
) (lane14), did not result in
formation of shift band B and formed a greatly diminished shift band A.
These results provide additional evidence that SRF is the factor in SMC
NE that contributes to shift bands A and B. In contrast, the wild-type
CArG-A oligonucleotides were not effective competitors (lanes3-7) nor did *wtA
efficiently bind SRF
lysate to form bands A or B (lane15). This is also
consistent with earlier results demonstrating that CArG-B had a greater
affinity for the SMC NE CArG-binding factor(s) than did CArG-A (Fig. 6B, 8, and 10A).
Fig. 10A also demonstrates the results of reacting SMC nuclear extract with
a radiolabeled 22-bp double-stranded oligonucleotide duplex conforming
to SRF's optimum CArG box binding site (*SRE; lane5)(65) . This resulted in shift bands similar to
those formed with labeled SM -actin CArG-B (*wtB
; lane3), providing additional evidence that the SM
-actin CArG box elements are binding SRF, or SRF-like factor, in
SMC nuclear extract.
To further characterize whether the same CArG-specific nuclear
factors bind the CArG-B oligonucleotide duplex and the 95-bp DNA
segment, the electrophoretic mobilities of the CArG-specific shift
bands formed with the two DNA segments were compared. In order to
optimize comparisons between bands, these gels were run twice as long
as previous shifts (see ``Materials and Methods''). Results
demonstrated that reaction of the *WT probe with SMC NE
resulted in a band with a slower electrophoretic mobility (Fig. 10C, lanes1 and 6, band1) than was evident with any of the other
combinations tested. This included *wtB
probe + SMC
NE (Fig. 10C, lane2), *WT
probe + in vitro synthesized SRF (lane3), or *wtB
+ in vitro synthesized SRF (lane5). The difference in
mobility of band 1 was not due to a difference in DNA fragment size
since we found no difference in shift bands between *WT
and *wtB
when reacted with in vitro synthesized SRF. Results suggest either 1) that the wild-type
95-bp promoter fragment is able to interact with an SRF-like protein
that is larger than SRF, while the 20-bp double-stranded CArG
oligonucleotides contain only enough sequence information to bind SRF
or 2) that the complexes formed with the 95-bp fragment and the 20-bp
double-stranded oligonucleotides both contain SRF but that the 95-bp
promoter fragment also contains DNA sequence information required to
bind an SRF accessory protein(s), and the binding of the accessory
protein(s) results in the decreased electrophoretic mobility.
The goals of the present study were to identify cis-elements and trans-factors that govern
transcriptional expression of SM -actin in vascular SMCs. Results
demonstrated that tissue-specific expression was dependent upon both
negative- and positive-acting cis-elements, which include the
CArG box elements, and upon SM trans-factors or complexes of
factors that bound over the CArG boxes. Suppression of SM
-actin
promoter-CAT reporter gene constructs in cells that do not express
their endogenous SM
-actin gene was dependent upon negative-acting cis-elements that repressed the activity of shorter promoter
constructs. For example, in skeletal myoblasts, addition of the
promoter region between -547 and -371 abolished the
transcriptional activity of the shorter constructs. Similarly, in
endothelial cells, negative-acting cis-elements within three
regions of the promoter, including the -547 and -371
region, abolished the high transcriptional activity of the p125CAT
construct. Previous reports with the mouse SM
-actin promoter also
described a negatively acting cis-element, whose 5`-boundary
was at -195, that was responsible for suppressing the
transcriptional activity of a shorter promoter-reporter gene construct
in fibroblasts and subconfluent (undifferentiated) BC3H1 skeletal-like
myoblasts, which do not express their endogenous SM
-actin
gene(66) . Additionally, a 29-bp region (-151 to
-123) of the chicken SM
-actin promoter significantly
reduced expression of the p122CAT ``core'' promoter in
chicken fibroblasts and abolished expression in chicken skeletal
myoblasts(24) . As of yet, the human SM
-actin 5`-flanking
region has not been studied sufficiently to determine whether it also
contains a promoter region that turns off gene transcription. However,
CArG box-binding factor-A has been shown to suppress transcription of
the human SM
-actin gene in mouse C2 skeletal myoblasts. The
expression of CArG box-binding factor-A was also shown to decrease as
the myoblasts were induced to form myotubes (67) , consistent
with the induction of SM
-actin during differentiation in this
cell line(53) .
High transcriptional activity of SM
-actin in SMCs required both CArG boxes and one or more additional
upstream DNA elements. The CArG elements were also shown to bind SMC
nuclear factors and to function in the complex regulation of
tissue-specific expression. While mutation of either CArG by itself in
the context of the 125-bp promoter abolished transcriptional expression
in SMCs, it had no effect in endothelial cells. Only when both CArG
boxes were mutated together was there any reduction in transcription,
but this double CArG-mutated promoter still retained at least half of
the activity seen with the wild-type sequence. In contrast to the high
activity of the rat 125-bp promoter, previous studies in our laboratory
utilizing the chicken 122-bp SM
-actin promoter, which contains
CArG boxes that are 100% conserved with the rat promoter but with
extensive divergence outside the CArG elements (Fig. 3), was not
active in endothelial cells(27) . Taken together, these
findings suggest that the transcriptional activity of p125CAT in
endothelial cells is elicted by nonconserved sequences outside the CArG
motifs. Although the SM
-actin CArG boxes may have some function
in endothelial cells, there is clearly a qualitative difference in
transcriptional activation via the CArG box elements in SMCs versus endothelial cells.
Skeletal muscle cells also showed a
qualitative difference in the transcriptional activation of SM
-actin via the CArG box elements. Although mature skeletal muscle
cells do not express SM
-actin in vivo, the gene is
transiently expressed in immature skeletal cells during development in vivo(17) and constitutively expressed in cultured
skeletal muscle cells upon differentiation into myotubes(53) .
Mutating either CArG-A or CArG-B in the context of the full-length
promoter resulted in about a 50% reduction of CAT activity in both SMCs
and skeletal myotubes. However, the CArG elements were sufficient to
induce a high level of transcriptional activity in the context of the
p125CAT construct in SMCs but not in skeletal myotubes. In contrast to
SMCs, activity of the CArG boxes in skeletal myotubes was dependent
upon inclusion of the promoter region between -271 and
-208. Interestingly, this region contains two E-Boxes at
-252 and -214. E-Box elements bind to myoD and
other members of the helix-loop-helix family and have been shown to be
required for CArG-dependent transcriptional expression of a number of
muscle-specific genes in skeletal muscle cells, including cardiac
-actin(68, 69, 70) . The hypothesis that
the differential regulation of SM
-actin in SMCs versus skeletal myotubes involves the CArG elements is also supported by
results of EMSA demonstrating that the SMC and skeletal myotube shift
bands are distinct in the context of a 95-bp promoter segment
containing two highly conserved CArG elements but not in the context of
the CArG elements alone. Therefore, CArG boxes may function in the
positive transcriptional activation of SM
-actin within a variety
of cultured cell lines that express their endogenous gene, but the
mechanisms of differential transcriptional regulation are likely to
involve cell-specific CArG box-binding proteins and/or protein
complexes that involve more than one DNA element.
To our knowledge,
this is the first report that provides direct evidence that the
CArG-box binding activity in SMCs includes SRF or an SRF-like factor.
The identification of SRF as one of the putative SMC transcription
factors was demonstrated by 1) supershifting the SMC CArG-specific
shift bands with anti-SRF antibodies, 2) determining that the SMC CArG
box-binding factor was similar in size to SRF, 3) establishing that the
shift bands formed with in vitro synthesized SRF had the same
mobilities and CArG-specific competition characteristics as the shift
bands formed with SMC nucleoproteins whether the proteins were reacted
with the oligonucleotide duplexes of CArG-A, CArG-B, or SRF's
optimum binding site (SRE), and 4) ascertaining that the relative
binding affinity of in vitro synthesized SRF for the CArG-A
and CArG-B oligonucleotide duplexes was the same as the relative
binding affinity of SMC nuclear factors for the same oligonucleotides.
Although these results do not prove that SRF is involved in the
transcriptional activation of SM -actin in SMCs, we also
demonstrated that CArG box mutations that abolished transcription also
inhibited in vitro binding of both SRF and the SMC CArG
box-binding factor. Results are similar to those obtained in previous
studies of the human (62) and Xenopus(54) cardiac
-actin genes that demonstrated that SRF
was likely to be the factor that regulates these genes.
This report
provides evidence that SRF functions as a part of the tissue-specific
transcriptional machinery of SM -actin. However, SRF was
originally identified as the factor that binds to the SRE of the
c-fos proto-oncogene(52, 55) , a member of a
family of genes termed immediate-early response genes, many of which
are thought to be of cardinal importance in governing cell growth and
differentiation in nonmuscle (71, 72, 73) as
well as in muscle cells(74, 75) . At the core of the
c-fos SRE is a CArG motif, which has been shown to be the
primary element required for SRF binding(55) . CArG box motifs
have also been found in the SREs of a variety of other immediate-early
response genes, and there is evidence for involvement of SRF in their
regulation as well(36) . Although previous studies in our
laboratory have shown that the transcription of nonmuscle
-actin
is rapidly and transiently stimulated in SMCs when exposed to
serum-containing medium, the transcription of the endogenous SM
-actin in SMCs was not altered under the same
conditions(40) . Similar results have been reported for SM
-actin in fibroblast cells(38) , as well as for the
skeletal and cardiac
-actins(62, 76) . Additional
studies in our laboratory demonstrated that transcription of the
truncated SM
actin promoter-CAT constructs in SMCs was not
affected by serum stimulation. (
)Therefore, very similar
SRE/CArG promoter elements appear to be involved in both the
nontissue-specific transcription of a variety of nonmuscle-specific
genes inducible by growth factors as well as in the constitutive
expression of a number of muscle-specific genes in a tissuespecific
manner. Several models, which are not mutually exclusive, could be
postulated to explain this phenomenon.
First, we cannot rule out the
possibility that the CArG box-binding factor that activates SM
-actin in SMCs is similar but not identical to SRF. The SMC factor
might be completely distinct from SRF or only one monomer subunit of
the SRF dimer need be different to form a tissue-specific unit. Similar
to helix-loop-helix factors(77) , dimerization among
heterologous SRF-like subunits could produce a variety of heterodimers
from a limited number of protein monomers, thereby expanding the
regulatory potential. In this report, we demonstrated a difference in
mobility between SMC nucleoprotein complexes and in vitro synthesized SRF complexes formed with the 95-bp promoter fragment,
while the same complexes formed with the 20-bp CArG-containing
oligonucleotides did not show this difference (Fig. 10C). This is consistent with a SMC nuclear
factor distinct from SRF interacting with the SM
-actin CArG
elements, but only in the context of sequences contained within the
95-bp promoter fragment. This model is also consistent with studies by
Walsh and co-workers(57) , which demonstrated that the chicken
skeletal
-actin CArG elements bound a chicken skeletal muscle
nuclear factor distinct from SRF in EMSA. Alternatively, if SRF is the
factor that activates SM
-actin expression, a second model might
involve tissue- or stimulus-specific, post-translational modifications
of SRF that would dictate its activation properties. SRF is a
phosphoprotein(62, 78) , and it is possible that
distinct signaling pathways function to activate SRF under various
conditions or in different cell-types. Alternatively, variations at the
gene level, such as methylation patterns or permissive versus nonpermissive chromatin configurations, might only allow SRF to
form an active complex over muscle CArG elements under basal
conditions, while the interaction of SRF over immediate-early gene CArG
elements might require a growth stimulus. A third possibility to
explain SRF's involvement in both muscle specificity and serum
inducibility is that SRF might bind in conjunction with a diverse group
of accessory factors that are themselves responsible for the tissue-
and stimulus-specific activities. The data in Fig. 10C are consistent with this model in that the unique slower mobility
shift band seen with the 95-bp fragment reacted with SMC nuclear
proteins may represent the binding of an accessory factor that is
present only in SMC nuclear extract and whose binding is dependent on
CArG flanking sequences not found in the 20-bp CArG oligonucleotides.
Indeed, studies of c-fos regulation have shown that SRF binds
an accessory protein to form a ternary complex over the
SRE(79) , and it has been suggested that the accessory factor
functions to integrate different signal transduction pathways at the
SRE(79, 80) . In the case of the SRF accessory
proteins p62/TCF, Elk-1, and SAP-1, a motif known as the Ets
domain-binding element is located on the 5` side of the CArG box in the
c-fos SRE and is required for binding these accessory
proteins(79, 81, 82) . SM
-actin CArG
elements are not flanked by Ets domain-binding motifs; however, a yet
uncharacterized DNA element may be located in the evolutionarily
conserved regions that flank the 5` or 3` sides of CArG-A and/or
CArG-B. The presence of CArG accessory factors specific to SMCs would
also explain the difference in electrophoretic mobilities between shift
bands formed with SMC versus skeletal myoblast/myotube NEs (Fig. 10B) and explain the differential expression of
the promoter-CAT constructs in the various cell lines.
Consistent
with all of the above models of SRF activity is the hypothesis that
either the internal CArG nucleotides, the sequences that immediately
flank the CArG elements, and/or cis-elements some distance
from the CArG boxes are essential in determining the nature of the
complexes that bind the CArG elements. This is true for
helix-loop-helix proteins, which have distinct binding site preferences
that depend on variable nucleotides within and around the E-box motif.
Although the internal nucleotides are extremely variable from one CArG
to the next, the internal sequence of any particular CArG element is
usually highly conserved, and there is evidence that modification of
the A/T core has a pronounced affect on transcriptional
activity(83) . Additionally, the two 10-bp CArG elements in the
SM -actin gene are each located within larger 16-bp domains that
are 100% conserved between all species in which the promoter has been
cloned, and a variable amount of flanking sequence is conserved for
other gene CArG elements as well. Observations in the present studies
suggest that the flanking and/or internal CArG sequences may also be
important. First, as noted above, the 95-bp fragment bound a larger
protein or complex of proteins in SMC nuclear extract that the 20-bp
CArG oligonucleotides were not able to bind, suggesting that the
flanking sequences outside the 5 bp surrounding the CArG elements are
important in factor binding. Second, the affinities of SMC CArG
box-binding factors and in vitro synthesized SRF for the
CArG-B oligonucleotide duplex was consistently greater than for the
CArG-A duplexes. Since these oligonucleotide duplexes appeared to bind
the same factors, as judged by the electrophoretic mobilities of the
shift bands, their differences in binding affinities were likely the
result of differences in either the internal and/or flanking CArG
nucleotides. Functional specificity may also be dictated by
interactions with cis-elements that are located distantly.
This should not be surprising since the control of gene expression
often involves the interaction of multiple regulatory elements.
Consistent with this hypothesis is the evidence that the CArG elements
within the 125-bp promoter are not sufficient to activate transcription
in skeletal myotubes; however, the CArGs acquire the ability to
activate transcription with the addition of an upstream promoter
element(s).
In summary, this report provides evidence that the
tissue-specific expression of SM -actin is dependent upon a
complex combination of both positive- and negative-acting cis-elements, that both promoter CArG boxes are required for
tissue-specific positive activation in cultured SMCs, and that the
SM-specific factors or complexes of factors that bind over the CArG
boxes include SRF, an SRF-like protein, and/or SRF accessory proteins.
Since all of the ``master'' regulatory proteins characterized
in skeletal muscle are transcription factors, it is interesting to
speculate that the SM-specific transcription factor(s) that activates
SM
-actin via the CArG elements may also serve in the
transcriptional regulation of other SM-specific genes, thereby playing
a more general role in the control of differentiation in SMCs.
Alternatively, the SM-specific CArG box transcription factor(s) may not
be a master regulator itself, but its expression may in turn be
controlled by an upstream SM-specific regulatory protein.