Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, Virginia 22906
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ABSTRACT |
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Transcriptional
activity of the smooth muscle (SM) -actin gene is differentially
regulated in SM vs. non-SM cells. Contained within the rat SM
-actin
promoter are two MCAT motifs, binding sites for transcription enhancer
factor 1 (TEF-1) transcriptional factors implicated in the regulation
of many muscle-specific genes. Transfections of SM
-actin
promoter-CAT constructs containing wild-type or mutagenized MCAT
elements were performed to evaluate their functional significance.
Mutation of the MCAT elements resulted in increased transcriptional
activity in SM cells, whereas these mutations either had no effect or
decreased activity in L6 myotubes or endothelial cells. High-resolution
gel shift assays resolved several complexes of different mobilities
that were formed between MCAT oligonucleotides and nuclear extracts
from the different cell types, although no single band was unique to
SM. Western blot analysis of nuclear extracts with polyclonal
antibodies to conserved domains of the TEF-1 gene family revealed
multiple reactive bands, some that were similar and others that
differed between SM and non-SM. Supershift assays with a polyclonal
antibody to the TEF-related protein family demonstrated that TEF-1 or
TEF-1-related proteins were contained in the shifted complexes. Results
suggest that the MCAT elements may contribute to cell type-specific
regulation of the SM
-actin gene. However, it remains to be
determined whether the differential transcriptional activity of MCAT
elements in SM vs. non-SM is due to differences in expression of TEF-1
or TEF-1-related proteins or to unique (cell type specific)
combinatorial interactions of the MCAT elements with other
cis-elements and trans-factors.
vascular smooth muscle cells; transcription enhancer factor 1; transcriptional regulation
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INTRODUCTION |
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SMOOTH MUSCLE CELLS (SMC) in both atherosclerotic and myointimal lesions exhibit alterations in their differentiated state (52, 57). These changes include decreased expression of contractile proteins characteristic of SMC as well as altered growth responsiveness, changes in lipid metabolism, and increased production of extracellular matrix molecules. It is believed that such alterations in the differentiated state of the vascular SMC play a critical role in the progression of vascular disease. To understand the cellular and molecular regulation of differentiation, it is important to identify mechanisms that regulate the expression of genes that distinguish one cell type from another and are required for their differentiated function.
The process of differentiation requires the coordinate regulation of many sets of genes that enable the mature cell to perform its specialized functions (51, 69). Genes that are co-regulated often share cis-elements that are targets for common transcription factors. For example, studies of skeletal muscle development have led to the characterization of several important transcriptional regulatory factors, such as the MyoD family of helix-loop-helix factors and the myocyte-specific enhancer-binding factor-2 (MEF-2) family (for review, see Refs. 51, 69). It is likely that SMC differentiation is governed by an analogous system of transcriptional regulation by specific families of factors. However, as yet no smooth muscle (SM)-specific differentiation control factors have been identified.
Genes encoding the contractile proteins are candidates for studies of
SM-specific transcriptional regulation (reviewed in Ref. 52). In
particular, the SM myosin heavy chain, SM-22, h1-calponin, and SM
-actin
genes are appropriate for studies of SM-specific transcriptional
regulation, since they are products of single genes and are required
for contractile function of SMC. SM
-actin is the first known marker
of differentiated SMC to be expressed in the developing vasculature
(27), and it is the most abundant of the contractile proteins in mature
vascular SMC. Although it is transiently expressed in developing
skeletal and cardiac muscle (55, 70) and in myofibroblasts within
tumors (8) and healing wounds (12), it is exclusively expressed in SMC
in the normal adult animal (21, 70). Interestingly, SM
-actin
expression is reduced in SMC within human atherosclerotic lesions (20,
23, 66), although the mechanisms that mediate reduced expression are
presently unknown, including whether changes occur at the
transcriptional or posttranscriptional level.
Earlier studies of the chicken, human, mouse, and rat SM -actin gene
promoters have demonstrated that regulation is cell specific and
ultimately determined by a complicated orchestration of both positive
and negative signals from interactions of
cis-elements and
trans-factors (5, 6, 47, 50, 61). The
SM
-actin promoter contains a number of highly conserved
cis-elements. For example, we (61) and
others (38, 63, 68) have demonstrated that two highly conserved CArG
boxes within the 5'-flanking region of the SM
-actin gene are
required for tissue-specific transcription and that serum response
factor (SRF) or an SRF-like protein binds to these elements (61). The
SM
-actin promoter also contains two highly conserved MCAT elements
at
184 (designated MCAT-1) and at
320 (designated
MCAT-2), located in a region of the promoter, which, based on deletion
analysis, contains elements that have negative regulatory activity
within a SMC context (61). MCAT elements bind the transcription
enhancer factor 1 (TEF-1) family of transcription factors (16, 62, 71)
and have been implicated in the transcriptional activation of cardiac
and skeletal troponin T (29, 43-45), skeletal
-actin (37, 42),
-myosin heavy chain (18, 35, 36, 59, 60, 65),
-myosin heavy chain (24, 48), and the
-acetylcholine receptor (2). Several nonmuscle
promoters have also been shown to be regulated by MCAT elements, such
as the viral SV40 enhancer (13, 28, 71), HPV-16 E6 and E7 oncogenes
(30), and the human chorionic somatomammotropin (hCS) enhancer (31, 33,
67). MCAT-dependent regulation of gene expression has been shown to be
extremely complex, involving binding of multiple different TEF-1 and
TEF-1-related binding proteins (17). For example, there are four TEF-1
genes, including N-TEF-1, which
encodes at least eight different isoforms (17, 62; P. Simpson, personal
communication). In addition, MCAT-dependent regulation has been shown
to involve multiple TEF-1 cofactors as well as interaction with other
cis-regulatory elements, their binding
factors, and basal transcriptional regulatory machinery (17, 62).
Nothing is known regarding the contributions of MCAT elements to
transcriptional regulation in SMC. Strauch and colleagues (9, 63, 64)
provided evidence of functionality of the MCAT-1-containing region of
the murine SM -actin gene promoter in AKR-2B fibroblasts. Based on
promoter deletion studies, transcriptional activity in AKR-2B cells was
shown to be limited to a construct containing the first 191 bp of the
5'-flanking sequence. Their interpretation was that
transcriptional activity in AKR-2B fibroblasts was dependent on a
positive element between
150 and
191 (this region
includes MCAT-1) and removal of a negative element between
191
and
224 that suppressed promoter activity in AKR-2B and BC3H1
myoblasts (19, 63). Mutation of the MCAT-1 element in the context of the 191-bp construct decreased transcriptional activity in the AKR-2B
cell line but had no effect on transcriptional activity in pre- and
postconfluent myoblasts (9). Although these results demonstrated a
transcriptional regulatory role for the MCAT-1 element of the 191-bp
promoter construct in fibroblasts and myoblasts, studies did not
address the function of either MCAT-1 or MCAT-2 in SMC or the function
of MCAT-2 in non-SMC. The aims of the present study were
1) to determine whether the two MCAT
elements of the rat SM
-actin promoter are important in cell
type-specific transcriptional regulation and
2) to perform initial
characterization of the interactions of nuclear proteins with these
cis-elements.
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MATERIALS AND METHODS |
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Construction of mutant promoter-CAT expression plasmids.
Site-directed mutagenesis was performed using the Altered Sites II in
vitro mutagenesis system (Promega) as recommended by the
manufacturer. The oligonucleotides that were used to mutate the
-actin promoter sequence were as follows (underlined bases show
mutations): mutated MCAT-1 oligo,
5'-P-GGTCTCTTCCAC
CCTCTGCTCTGCTC-3'; mutated MCAT-2 oligo,
5'-P-AGGCATGGTTTG
TCAGAAGATGCC-3'. The mutants were subcloned into p371CAT, the pCAT-Basic plasmid (Promega) containing the first 371 bp of the rat SM
-actin promoter. The mutant clones were sequence verified by the Sanger dideoxy sequencing procedure (56) using a Sequenase kit (US Biochemical).
Cell culture.
SMC from rat thoracic aorta were isolated and cultured as previously
described (22). Rat aortic SMC used in the present studies were between
passages 13 and
30 and cultured under conditions that
we have previously shown to maximize expression of a variety of
SM-specific proteins including SM -actin (53), SM myosin heavy chain
(54), SM myosin light chain (49), and SM
-tropomyosin (26). Rat L6
skeletal myoblasts, originally isolated by Yaffe (72), were obtained
from the American Type Culture Collection and cultured as recommended.
L6 myoblasts were maintained in 10% FBS-containing medium. Myoblast
differentiation into myotubes was induced by reducing the FBS
concentration to 1% when cells reached confluence and by maintaining
cells for a minimum of 3 days. More than 70% of myoblasts were induced
to differentiate into myotubes by this procedure. Bovine aortic
endothelial cells (EC) were isolated (41) and cultured (5) as
previously described. AKR-2B mouse embryonic fibroblasts were the gift
of Dr. Harold Moses (Vanderbilt University, Nashville, TN) and were
cultured as previously described in McCoy's 5A medium (63).
DNA transfections.
Cells for transient transfection assays were plated into six-well
plates (Corning Glass, Corning NY) at a density of 2 × 104
cells/cm2 for SMC and for AKR-2B,
3 × 104
cells/cm2 for EC, and 4 × 104
cells/cm2 for L6 myoblasts and
myotubes. These densities were chosen so that the cells would be at
60-80% confluence at the time of transfection at 20-22 h
postplating. Transient transfection experiments consisted of
transfecting each promoter-CAT plasmid in triplicate using DOTAP
reagent according to the manufacturer's recommendations (Boehringer
Mannheim) or Transfectam (Promega) for EC as previously described (61):
specifically, 4 µg DNA plus 30 µl DOTAP/well for SMC, myoblasts,
and myotubes; 3 µg DNA plus 15 µl DOTAP for AKR-2B; and 2.5 µg
DNA plus 7.5 µl Transfectam for EC. After 48 h from the time of
transfection, the cells were washed twice in 4 ml of ice-cold PBS,
harvested by scraping in harvesting buffer of 40 mM Tris (pH 7.5), 1 mM
EDTA, and 150 mM NaCl, pelleted, resuspended in 100 µl 25 mM Tris (pH
7.5), and stored at 70°C.
Reporter gene assays.
Extracts from transfected cells were prepared by three freeze-thaw
cycles, followed by centrifugation to remove cellular debris. The
supernatant was heat inactivated at 65°C for 15 min and then pelleted, and a 55-µl aliquot was assayed for CAT activity by enzymatic butyrylation of tritiated chloramphenicol (DuPont-NEN) as
previously described (58). All CAT activity values were normalized to
the protein concentration of each cell lysate as measured by the
Bio-Rad microtiter plate assay. Early transfection studies were
performed by cotransfection with a -galactosidase (
-Gal) vector
to correct for changes in transfection efficiency. Because the
-Gal
measurements did not result in qualitative changes in the data and
viral-driven
-Gal genes could potentially compete for limited
trans-factors that regulate SM
-actin transcription (15), the cotransfections were discontinued. In
each experiment, a promoterless CAT construct, pCAT-Basic, was
transfected to serve as the baseline indicator of CAT activity; the
activity of the other constructs was expressed relative to pCAT-Basic
set equal to one, unless otherwise stated. In addition, an SV40
enhancer/promoter-CAT construct, pCAT-Control, served as a positive
control of transfection and CAT activity. All CAT activity values
represent at least three independent transfection assays with each
construct tested in triplicate per experiment and are expressed as
means ± SE. Data from replicate independent experiments for each
cell type were analyzed using a two-factor ANOVA with "experimental
group" differences as the main effect and the "experimental
day" as the interacting factor. The latter was necessary due to
experiment-to-experiment differences in the absolute levels of reporter
activity that are common in transient transfection experiments and
would result in a nonparametric distribution if experimental data were
simply pooled and analyzed by one-way ANOVA. Where appropriate, post hoc comparisons between experimental groups for a given cell type were
made using a Student-Newman-Keuls multicomparison test. Values of
P < 0. 05 were considered
statistically significant.
Preparation of nuclear extracts and electrophoretic mobility shift assays. Crude nuclear extracts (NE) were purified by the method of Dignam et al. (14) with the addition of 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µM 4-(2-aminoethyl)benzenesulfonyl fluoride (Sigma) to all solutions. The protein concentration of each NE was measured by the microtiter plate assay of Bio-Rad. The HeLa cell NE were purchased commercially (Santa Cruz Biotechnologies, Santa Cruz, CA).
Oligonucleotides were synthesized and HPLC purified by Operon Technologies (Alameda, CA). The 17-mer MCAT oligonucleotides consisted of the 7-bp motif plus the additional 5 bp immediately 5' and 3' to the motif. Complementary strands were end labeled separately with T4 polynucleotide kinase (Promega) and [Western analysis.
NE (15 µg) were boiled for 2 min, centrifuged 3 min to pellet debris,
and separated by SDS-PAGE on an 11% gel. After transfer to
nitrocellulose (Amersham), membranes were probed with polyclonal antisera to TEF-1 kindly provided by I. Farrance (Veterans Affairs Medical Center, San Fransisco, CA; Ref. 62), followed by a secondary antibody direct conjugate donkey -rabbit-peroxidase (Amersham) and
detected using enhanced chemiluminescence (Amersham) as described previously (17).
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RESULTS |
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SM -actin MCAT elements exhibited differential
activity in SMC vs. non-SMC.
The SM
-actin promoter contains two MCAT elements, designated MCAT-1
(
184 to
178) and MCAT-2 (
320 to
314), that
are highly conserved across mammalian species (61). To evaluate the
functional significance of the MCAT motifs in the transcriptional
regulation of the rat SM
-actin gene, mutations of either or both
elements were made in the context of the first 371 bp of the promoter
linked to a CAT reporter gene (Fig. 1). The
mutations tested were based on the work of Mar and Ordahl (44), who
showed that these mutations abolished activity of the cTNT promoter in
transfection assays and disrupted binding in footprinting assays. To
assess the transcriptional activity of the constructs, wild-type and
mutant promoter-CAT plasmids were transiently transfected into cultured
rat aortic SMC, which express high levels of the endogenous SM
-actin gene, and several non-SMC lines. The latter included
1) AKR-2B mouse embryonic
fibroblasts, which do not normally express the endogenous SM
-actin
gene but have been shown to express a promoter/reporter construct
containing 191 bp of the 5'-region of the SM
-actin promoter
in response to serum stimulation (63), and
2) L6 skeletal myoblasts, which
express SM
-actin at very low levels but show marked induction of
expression when induced to differentiate into myotubes (1), and bovine
aortic EC, which do not express the endogenous gene but do express the
p371CAT reporter construct at modest levels (61). Results of transient
transfections demonstrated that mutations of the MCAT elements had
different effects in different cell lines (Fig.
2). In SMC, the p371CAT wild-type construct
had approximately eightfold greater activity than a promoterless
control construct. Mutation of the MCAT-1 element resulted in a twofold increase in activity compared with the wild-type construct, whereas mutation of MCAT-2 led to a threefold increase (Fig. 2). Simultaneous mutation of both the MCAT-1 and -2 elements resulted in activity equivalent to that of the MCAT-1 mutation, rather than having an
additive effect. This suggests that at least part of the increase in
activity seen with the MCAT-2 mutation was dependent on having an
intact MCAT-1 element or that mutation of the MCAT-1 element resulted
in a state of promoter upregulation which was stable and could not be
altered by additional mutation of MCAT-2. Taken together, data
demonstrate that, in the context of the first 371 bp of the promoter,
the two MCAT elements function as repressors of transcription in SMC in
that mutation of either or both resulted in a two- to threefold
increase in activity compared with the wild-type promoter.
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MCAT elements bound nuclear factors from SMC and non-SMC.
To study the interaction of proteins with the MCAT elements of the SM
-actin promoter, EMSA were performed with NE from both SMC and
non-SMC. Double-stranded 17-mer oligonucleotide probes, containing the
7-bp MCAT motif plus 5 bp of the 5'- and 3'-flanking sequence, were end labeled with
32P. Wild-type MCAT probes formed
several shift bands with SMC NE (Fig.
3A,
lanes 2 and
9) that were abolished with addition
of excess cold wild-type oligonucleotides (lanes
3-5 and
10-12). However, mutant
oligonucleotide competitor DNA at a 250-fold molar excess failed to
compete for binding (lanes 6-7
and 13-14).
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NE from SMC and non-SMC lines contained multiple proteins in 50- to
60-kDa size range that cross-reacted with antibodies to TEF-1.
Because there were multiple and different complexes formed in the
high-resolution EMSA, we hypothesized that there might be multiple
TEF-1 or TEF-1-related proteins responsible for the formation of these
different complexes. To ascertain the presence of TEF-1 family members
in SMC and non-SMC, NE were subjected to SDS-PAGE, transferred to
nitrocellulose, and probed with TEF-1 antiserum (generous gift of I. Farrance). The antiserum was raised against pooled peptide sequences
derived from conserved regions of the TEF-1 gene family so that the
antiserum would have a broad specificity for diverse TEF-1 proteins
(17). Results of immunoblot analyses with this TEF-1 antibody showed
that NE from each of the cell lines tested contained proteins that were
antigenically related to TEF-1 and that there were differences in the
mobilities of the immunoreactive bands (Fig.
5). Of particular interest, a single distinct reactive band was seen with SMC NE that was not seen with NE
from any of the other cell types tested with the possible exception of
HeLa cells. These observations are provocative and provide a potential
basis to explain the differential functional activity of the SM
-actin MCAT elements in SMC vs. non-SMC (Fig. 2). However, whereas
results of these Western analyses suggest that different complements of
TEF-1-related proteins exist in SMC vs. non-SMC extracts, they do not
provide evidence that such proteins actually bind to the MCAT elements.
It is also possible that at least some of the differences in mobility
of TEF-1 immunoreactive proteins reflect species differences rather
than unique TEF-1-related proteins.
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SM -actin MCAT elements bound TEF-1 or TEF-1-related
proteins present in SMC and non-SMC NE.
To determine whether TEF-1 or TEF-1-related proteins were present in
the MCAT shift complexes, we attempted supershift analyses with the
same antibody employed in the Western analyses shown in Fig. 5. Despite
repeated attempts under many gel shift conditions and using various
antibody and NE concentrations, no effect on MCAT-1 or -2 shift
complexes was observed. However, this antibody is known to be of
relatively low affinity and does not work well in supershift analyses
unless the site affinity for TEF-1 is extremely high (I. Farrance and
P. Simpson, personal communication). As such, we repeated supershift
assays employing a polyclonal chicken IgY antibody raised against a
mouse GAL-4/TEF-1-related protein fusion protein (a generous gift of N. Shimizu). This antibody has previously been shown to react specifically
with recombinant TEF-1 proteins under the EMSA conditions employed in
the present studies (73). Addition of this antibody to gel shifts
resulted in formation of supershift complexes with each of the cell
extracts, and reduced but did not abolish the shift complexes. Binding
reactions lacking NE but including antisera did not contain any shifted complexes (data not shown). Control chicken IgY antibodies did not
affect the shift complexes (Fig. 6,
A and
B). These results suggest that at
least some of the MCAT binding factors are TEF-1 or TEF-1-related
proteins, thus implicating a possible role for this family of
transcription factors in control of cell type-specific expression of SM
-actin. However, results do not identify which, if any, of the known
TEF-1 or TEF-1-related protein isoforms are involved. In addition, our
observations that MCAT shift complexes were not abolished by inclusion
of high concentrations of antibody in the EMSA reaction leave open the
possibility that non-TEF-1 proteins and/or TEF-1 family members
that are not recognized by the GAL-4/TEF-1-related protein antibody may
be involved as well.
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DISCUSSION |
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It is well established that TEF-1 protein interactions with MCAT
elements are involved in and are sometimes required for the cell-specific transcriptional activation of many cardiac and skeletal muscle-specific genes in vitro (2, 18, 24, 29, 35, 37, 42-45, 48,
59, 60, 65). However, our report is the first to demonstrate a
transcriptional regulatory role for MCAT elements in SMC. Results of
the present studies demonstrated that the MCAT motifs were important
for the transcriptional activity of the SM -actin promoter/reporter
constructs and that these elements had surprisingly different effects
in SMC vs. non-SMC. In SMC, the MCAT elements functioned as repressors,
whereas, in L6 myotubes and EC, the MCAT elements acted as activators
(Fig. 2). To our knowledge, this is the first time that an MCAT element has been shown to serve as a negative regulatory element in the context
of a muscle-specific promoter, although there are several reports of
MCAT elements exerting repressor effects in nonmuscle promoters. For
example, Berger et al. (3) presented evidence indicating that TEF-1
functioned as a repressor of the SV40 late promoter. Jiang and
Eberhardt described repression by TEF-1/MCAT interactions of the hCS
gene enhancer (32) and heterologous RSV and TK promoters in BeWo
choriocarcinoma cells (33). However, in none of the preceding cases
have the molecular mechanisms responsible for MCAT repressor activity
been elucidated.
The mechanisms by which MCAT elements mediate the repression of SM
-actin transcription in SMC are also not known at this time.
Moreover, the importance of the MCAT elements in repressing SM
-actin transcription under physiological or pathological
circumstances is unclear. Unfortunately, studies in this area are
hampered by the lack of a model system in which the SM
-actin
promoter is repressed at the transcriptional level. There is extensive
evidence showing that SM
-actin expression can be repressed in a
highly selective manner by stimulation with platelet-derived growth
factor BB in cultured SMC (10, 11). However, decreases in expression of
the protein under these circumstances are mediated at the
posttranscriptional level through mRNA and protein destabilization, not
a change in transcription rate as measured by run-on analyses.
Likewise, whereas it is well established that expression of SM
-actin, as well as other SMC differentiation markers, is reduced
within intimal SMC in vivo in response to vascular injury or in
atherosclerotic lesions, it is unclear if these changes are mediated at
the transcriptional level (52). As such, there is no system currently
available with which to test for the repressor function of the MCAT
elements of the SM
-actin promoter.
Results of our EMSA analyses demonstrated that multiple and different
shift complexes were formed between MCAT oligonucleotide probes and NE
from SMC and non-SMC (Figs. 3 and 4). However, consistent with
extensive studies of the role of MCAT elements in multiple skeletal-
and cardiac-specific genes (17), our results provided no simple or
obvious explanation for the differential effects of MCAT-1 and -2 mutations in different cell types. For example, similar shift bands
were seen between SMC and L6 myotubes (Fig. 4,
A and
B, lanes
1 and 4), despite
the fact that MCAT mutations had opposite effects in these two cell
types, i.e., a two- to threefold increase in activity in SMC but a 60%
reduction in activity in L6 myotubes (Fig. 2). There are several
plausible mechanisms, which are not mutually exclusive, to explain
these observations. First, differences in functional activity may be
the result of binding of different TEF-1 family members or their splice
variants that are not resolvable in mobility shift assays. This is
certainly possible given the known complexity of the TEF-1 gene family
(17, 62) and results of Western analyses in the present studies
suggesting that SMC may express a unique TEF-1-related protein (Fig.
5). There is also direct precedence for this mode of MCAT-dependent regulation. For example, Stewart et al. (62) described the isolation of
two splice variants of chicken RTEF-1 and demonstrated that chimeric
proteins containing the activation domains of these isoforms possessed
differing abilities to transactivate GAL-4-dependent reporter
constructs. A second possibility is that the transcriptional activity
of TEF-1 proteins may be differentially regulated in different cell
types by posttranslational modifications. Farrance and Ordahl (17)
found that three species of TEF-1 proteins in chicken primary muscle
cultures were differentially phosphorylated and suggested that these
modifications may modulate the interaction of TEF-1 proteins with MCAT
elements or with cofactors to provide cell-specific transcriptional
activity. A third possiblity is that the observed differences in
binding properties and functional activities of the SM -actin MCAT
elements may be due to 1)
binding of TEF-1 cofactors, 2)
binding of factors other than TEF-1 proteins, and/or
3) combinatorial interactions with
other regulatory cis-elements within
the promoter and their binding factors. A recent report by Larkin et
al. (40) demonstrated that the sequences flanking the MCAT motifs of
the cardiac troponin T promoter directed the binding of TEF-1 cofactors
that conferred tissue-specific activity to promoter/reporter constructs
in cultured cells. Indeed, cofactors that are required for TEF-1
activity have been partially purified from HeLa cells (7, 48). Jiang
and Eberhardt (32) reported that COS and BeWo cells contain a protein
distinguishable from TEF-1, designated CSEF-1, that binds to the MCAT
elements of the hCS gene enhancer. Importantly, activation of the
skeletal
-actin promoter in response to
1-adrenergic stimulation
required not only the MCAT element, but also the CArG and Sp1 elements
(37). Furthermore, studies published by the Robbins laboratory (39) demonstrated that in vivo, combinatorial interactions between the MCAT,
C-rich, and
e3 elements were required for high level tissue-specific
activity of the
-myosin heavy chain gene promoter. Of interest, our
laboratory has previously demonstrated that transcriptional activity of
the SM
-actin gene in SMC is dependent on multiple cis-elements. This includes two highly
conserved CArG elements at
62 and
112 (61), a novel
transforming growth factor-
1 control element at
42 (25), and
at least one of two E-box elements at
214 and
252
respectively (34) (see Fig.
7). Although direct evidence is lacking, it is thus possible that the differential function
of MCAT elements in SMC is dependent on interaction with one or more of
these cis-elements in a manner
analogous to the MCAT elements found in a number of skeletal- and
cardiac-specific gene promoters (37, 39). However, in contrast to these
genes, a unique feature of the SM
-actin MCAT elements is that they act as cell type-specific repressor elements rather than activators. As
such, the repressor activity of the MCAT elements within a SMC context
may be due to interaction with one or more of the known positive
regulatory elements of the promoter, an as yet unidentified positive
regulatory element, or with the basal transcriptional machinery (or
some combination thereof). Thus elucidation of the mechanisms whereby
the SM
-actin MCAT elements act as repressors is likely to be
difficult and will require clear identification of the specific TEF-1
proteins and cofactors that bind to these control elements and how
these factors influence other control elements within the promoter.
|
In conclusion, we have demonstrated that the MCAT elements of the rat
SM -actin promoter bind TEF-1 or TEF-like proteins and contribute to
the differential regulation of transcription of this gene in different
cell types (61). The elements acted as repressors in SMC, whereas they
had either no effect or functioned as activators in non-SMC. Although
the precise mechanisms that govern this cell-specific regulation remain
to be elucidated, this report is the first to demonstrate functional
activity of the SM
-actin MCAT motifs within SMC. Moreover, it is
the first report, to our knowledge, to show a repressor activity of
MCAT elements in a muscle-specific promoter context. These studies underscore the utility of the SM
-actin promoter in dissecting the
variable and very complex interactions between MCAT elements and the
large family of TEF-1 and TEF-1-related proteins, although it remains
to be determined whether and by what mechanisms the MCAT elements
mediate transcriptional repression of this gene in vivo under
conditions in which expression of the SM
-actin gene is decreased at
the transcriptional level.
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ACKNOWLEDGEMENTS |
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We thank Dr. Iain Farrance for the gift of the rabbit TEF-1 antiserum and his very helpful comments and suggestions; Dr. Noriko Shimizu for sharing the chicken GAL-4/TEF-related protein antibodies; Drs. Alexandre Stewart and Paul Simpson for helpful discussions; Dr. Harold Moses for the AKR-2B cell line; and Andrea Tanner, Diane Raines, and Jennifer Clatterbuck for outstanding technical advice and assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants 5T32-HL-07824-19 (E. A. Swartz), F32-HL09648-01 (to A. D. Johnson), RO1-HL-38854 (to G. K. Owens), and PO1-HL-19242 (to G. K. Owens).
Address for reprint requests: G. K. Owens, Dept. of Molecular Physiology and Biological Physics, PO Box 10011, Charlottesville, VA 22906-0011.
Received 22 October 1997; accepted in final form 18 May 1998.
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