(Received for publication, July 25, 1996, and in revised form, December 3, 1996)
From the Department of Molecular Physiology and
Biological Physics, University of Virginia, Charlottesville, Virginia
22908 and the § Department of Molecular Physiology and
Biophysics, University of Vermont, Burlington, Vermont 05405
To identify cis- and
trans-acting factors that regulate smooth muscle-specific
gene expression, we studied the smooth muscle myosin heavy chain gene,
a rigorous marker of differentiated smooth muscle. A comparison of
smooth muscle myosin heavy chain promoter sequences from multiple
species revealed the presence of a highly conserved 227-base pair
domain (nucleotides 1321 to
1095 in rat). Results of a deletion
analysis of a 4.3-kilobase pair segment of the rat promoter
(nucleotides
4220 to +88) demonstrated that this domain was necessary
for maximal transcriptional activity in smooth muscle cells. Gel-shift
analysis and site-directed mutagenesis demonstrated that one true CArG
and another CArG-like element contained within this domain were both
recognized by the serum response factor and were both required for the
positive activity attributable to this domain. Additional studies
demonstrated that mutation of a GC-rich sequence within the 227-base
pair conserved domain resulted in a nearly 100% increase in
transcriptional activity. Gel-shift analysis showed that this GC-rich
repressor element was recognized by both Sp1 and Sp3. These data
demonstrate that transcriptional control of the smooth muscle myosin
heavy chain gene is highly complex, involving both negative and
positive regulatory elements, including CArG sequences found in the
promoters of multiple smooth muscle differentiation marker genes.
Intimal migration and proliferation of vascular smooth muscle cells (SMCs)1 are known to play an integral role in development of atherosclerotic disease (1, 2), and numerous factors have been identified that have growth promoting and/or chemotactic activity for SMCs (2). An additional feature of SMCs within atherosclerotic lesions is that cells exhibit marked differences in morphology and protein expression patterns as compared with normal medial SMCs (3-6), a process referred to as "phenotypic modulation" (7). This is characterized by decreased expression of proteins that are characteristic of differentiated SMCs, including the SM isoforms of contractile proteins, as well as altered growth regulatory properties, lipid metabolism, matrix production, and decreased contractility (reviewed in Ref. 8). Of particular significance, many of these phenotypic alterations in intimal SMCs cannot simply be viewed as a consequence of atherosclerotic disease, but rather are likely to play a major role in its development and/or progression.
Although the SMC exhibits a high degree of plasticity and, unlike
skeletal and cardiac myocytes, does not undergo terminal differentiation, it is a very specialized cell, whose differentiated function is dependent upon the coordinate regulation of a large number
of cell type-specific/selective products, including contractile proteins, receptors, signal transduction molecules, and ion channels (8). Contractile proteins that are highly abundant and specific to the
SMC represent obvious candidates for studying molecular control of SMC
differentiation. Consequently, it is not unexpected that the best
studied SM-specific marker is smooth muscle -actin, which first
appears in the putative SMCs that envelope the dorsal aortae (9).
Although it is transiently expressed during development in other
mesodermally derived tissues (10) and in fibroblasts during wound
repair (11), smooth muscle
-actin is normally expressed only in SMCs
and SMC-like cells in the adult (12). A second well characterized
SM-specific contractile protein is smooth muscle myosin heavy chain
(SM-MHC). The SMC contains four isoforms of SM-MHC (SM-1A, SM-1B,
SM-2A, SM-2B) (13-15), which are all derived from a single gene via
alternative splicing (16). In situ hybridization and RNase
protection analysis of SM-MHC gene expression during mouse
embryogenesis revealed that SM-MHC mRNAs were present only in
smooth muscle-containing tissues; no transcripts were detected in the
developing brain, heart, or skeletal muscle (17). Immunohistochemical
studies that discriminated between the SM-1 and SM-2 isoforms revealed
that, in both rabbit and human aortae, SM-MHC protein was specific to
the SMC and that SM-1 predominated early during embryonic development,
with SM-2 appearing postnatally (18, 19). These studies indicate that, similar to smooth muscle
-actin, SM-MHC represents a highly rigorous SMC marker and a good candidate gene for discerning transcriptional mechanisms important for maintenance of the differentiated SMC phenotype.
The promoters of the rat, mouse, and rabbit SM-MHC genes have been
recently cloned and partially characterized. A reporter construct
containing 2266 bp of the 5-flanking region of the rabbit SM-MHC gene
was shown to be highly active in cultured rat aortic SMCs and only
minimally active in other cell types (20). In a separate study on the
rabbit SM-MHC promoter, Kallmeier et al. (21) identified a
107-bp fragment (nucleotides
1332 to
1225) that enhanced
transcription in what appeared to be a highly SMC-specific manner.
Analysis of the rat SM-MHC promoter in multiple cell types indicated
that a 68-bp domain (nucleotides
1249 to
1317) was important for
restriction of expression to the SMC (22). Constructs longer than
nucleotide
1317 were found to be significantly active only in the
SMC. Another recent report on mouse SM-MHC showed that, similar to rat
and rabbit, 1500 bp of the mouse SM-MHC promoter was strongly active
only in cultured SMCs (23). In all of the above studies, several
potential cis-elements, including multiple E boxes, GC
boxes, and CArG or CArG-like boxes, were identified based on sequence
similarities to known cis-elements. Some of these potential
elements were also present in regions with functional activity.
However, only in the mouse promoter study by Watanabe et al.
(23) were any potential cis-elements specifically mutated
and tested for function. They demonstrated by site-directed mutagenesis
that two CCTCCC boxes located proximal to the TATA box were bound by
Sp1 and functioned as positive-acting cis-elements.
In this study, we utilized ~4.2 kilobase pairs of the rat SM-MHC gene
5-flanking region and created a series of mutated CAT fusion
constructs to identify functionally important cis-elements. We specifically targeted those sequences that exhibited homology to
known cis-elements and that were also conserved between
species. A sequence alignment of the rat, mouse, and rabbit SM-MHC
promoters revealed a highly conserved 227-bp domain that spanned
nucleotides
1321 to
1095 in rat. Deletion analysis showed that this
domain was required for maximal promoter activity in SMCs.
Site-directed mutagenesis was utilized to further demonstrate that two
of the three CArG or CArG-like boxes contained within the 227-bp domain functioned as positive-acting cis-elements recognized by the
serum response factor (SRF) or an SRF-like protein. A GC-rich sequence located within this domain was determined to function as a
negative-acting cis-element recognized by members of the Sp1
family. The fact that CArG motifs are required for expression of both
the SM-MHC gene (this study) and the SM
-actin gene (24) suggests
that this motif may be important for the coordinate regulation of
SM-specific genes during SMC differentiation.
The cloning, determination of the +1 start site, and
nucleotide sequence (nucleotides 1699 to +121) of the rat SM-MHC
promoter have been previously reported (22). In this study, we ligated a 4308-bp BglII fragment, spanning nucleotides
4220 to
+88, into a partially filled-in SalI site of the pCAT-Basic
reporter vector (Promega). This clone (pCAT
4220) was sequenced by the
dideoxy method of Sanger et al. (25) using either an
automated sequencer (Applied Biosystems Inc.) or a Sequenase kit
(U. S. Biochemical Corp.). The sequence from nucleotides
4220 to
2400 was generated from analysis of only a single strand, whereas
both strands were sequenced from nucleotides
2400 to +88. Serial
deletion constructs were generated from the pCAT
4220 clone using
exonuclease III and the protocol provided by the manufacturer
(Stratagene). Deletion clones that targeted specific sites were
generated using Taq polymerase and a thermal cycler
(Perkin-Elmer). Oligonucleotide primers were purchased from a
commercial source (Operon Technologies, Inc.) and contained
HindIII and XbaI linkers such that PCR products could be directionally cloned into the pCAT-Basic vector (Promega). Site-directed mutagenesis of the pCAT
1346 construct was performed using the Ex-site mutagenesis kit according to the manufacturer's instructions (Stratagene). The orientation and integrity of the mutated constructs were determined by dideoxy sequencing
(25).
The nucleotide sequences of
the rabbit (nucleotides 2266 to +1) (20), mouse (nucleotides
1500
to +1) (23), and rat (nucleotides
4220 to +1) SM-MHC promoters were
analyzed with computer assistance using sequence similarity programs
(Genetics Computer Group, Madison, WI).
SMCs from rat thoracic aorta were isolated and cultured as
described previously (26). Rat L6 skeletal myoblasts were cultured as
described previously (24) and fused into myotubes using the protocol of
Yaffe (27). SMCs (passages 10-22) and L6 myoblasts were seeded for
transient transfection assays into 6-well plates at a density of 2 × 104. These densities were chosen so that the cells would
be 70-80% confluent at the time of transfection (24 h after plating).
Transfections of the CAT reporter gene constructs, subsequent growth
conditions of the SMCs and L6 myoblasts, and preparation of the cell
extracts for measurement of CAT activity were all performed as
described previously (24), with only slight modification, namely, the amount of plasmid DNA added per well was 4 µg, and
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (7.5 µl/µg of DNA; Boehringer Mannheim) was utilized as the transfection reagent. All CAT activity values were normalized to
the protein concentration of each cell lysate as measured by the
Bradford assay (28). Early transfection experiments included a
-galactosidase plasmid as a cotransfection partner for measurement of transfection efficiency. However, the
-galactosidase activity measurements did not result in qualitative changes in the data, nor did
they affect experimental variability. Since the cotransfections conferred no advantage in the reduction of experimental error and had
the potential of competing for limited trans-acting factors that regulate SM-MHC transcription, the
-galactosidase
cotransfections were discontinued. In each experiment, the promoterless
pCAT 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 1. Additionally, an SV40 promoter-CAT construct with enhancer
(Promega) served as a positive control of transfection and CAT
activity. All SMC CAT activity values represent at least three
independent experiments, with each construct tested in triplicate per
experiment. L6 myoblast and myotube CAT activity values represent two
independent experiments. Relative CAT activity data are expressed as
the means ± S.D. computed from the results obtained from each set
of transfection experiments. One-way analysis of variance followed by
the Newman-Keuls's multiple range test were used for data analysis.
Values of p < 0.05 were considered statistically
significant.
Nuclear extracts were prepared from rat aortic SMCs grown to confluency under the same media conditions utilized for the transfection assays. The actual preparative method used was essentially that of Dignam et al. (29); protease inhibitors were added as described previously (24).
The oligonucleotides used for EMSAs were purchased commercially (Operon
Technologies, Inc.) and included the following: CArG3, 5-cgggaccatatttagtcagg-3
; CArG2, 5
-cctggcctttttgggttgtt-3
; CArG1,
5
-gacttccttttatggcctga-3
; GC-rich, 5
-ggttgtttcccgcccaggcc-3; CArG2Mut, 5
cctggatcctttgggttgtt-3
; GC-richMut,
5
-ggttgtttcccggatccaggcc-3
; Sp1, 5
-gttcgccccgccccgatcgaat-3
; and
SRE, 5
-ggatgtccatattaggacatct-3
. EMSAs were performed with 20 µl of
binding reaction that included ~30 pg of 32P-end-labeled
annealed oligonucleotides, 10 µg of nuclear extract (unless otherwise
indicated), and 0.25 µg of poly(dI-dC) in 1 × binding buffer
(12 mM HEPES (pH 7.9), 100 mM KCl (50 mM for GC-rich oligonucleotide EMSA), 5 mM
MgCl2, 4 mM Tris-HCl (pH 7.5), 0.6 mM EDTA, 0.6 mM dithiothreitol, and 10%
glycerol). Following a 20-min incubation at room temperature, the
samples were subjected to electrophoresis on a 5% polyacrylamide gel,
which had been pre-run at 170 V for 1 h. Electrophoresis was
performed at 170 V in 0.5 × 45 mM Tris borate and 1 mM EDTA. Gels were dried and exposed to film for
24-72 h.
The Sp1, Sp3, SRF, and CTF/NF1 antibodies used for EMSA supershift experiments were all purchased commercially (Santa Cruz). EMSA binding reactions were set up as described above and incubated for 20 min; 2 µl of the antibody was added to the mixture; and the reaction was incubated for another 15 min at room temperature and then loaded onto the gel for electrophoresis.
DNase I FootprintingBinding conditions were as
described for EMSAs, except that 40 µg of SMC nuclear extract
was used per reaction. DNase I digestions and electrophoresis of the
samples were performed as described previously (30). Location of the
footprinted regions was determined by including on the gel a dideoxy
sequence marker ladder, and a Maxam-Gilbert G-specific ladder of the
labeled fragment was also generated to verify the integrity of the
probe (data not shown). The plasmid construct pCAT1346 was utilized
as template DNA for PCR-generated footprint probes. The
oligonucleotides utilized in the PCRs were 5
-ccataacgctccatgggct-3
(nucleotides
1346 to
1328) and 5
-gcatggagagtgggaggga-3
(nucleotides
1089 to
1071). The
1346/
1328 oligonucleotide
primer used in the PCRs was labeled at the 5
-end with T4
polynucleotide kinase (Life Technologies, Inc.) and
[
-32P]ATP (DuPont NEN) to generate an asymmetrically
labeled double-stranded probe. Probes were purified on a 5%
nondenaturing polyacrylamide gel.
The rat SM-MHC
promoter has been recently cloned and sequenced from bp 1699 to +121
(22). To assist in the identification of potential
cis-elements, we sequenced an additional 2500 bp of the rat
SM-MHC promoter (up to nucleotide
4220) and compared the rat promoter
sequence with the published sequences of rabbit (
2266 bp) (20) and
mouse (
1500 bp) (23). We identified only two domains that shared
obvious sequence similarities among all three species (Fig.
1A). The distal (relative to the +1 start site) conserved domain was located in the rat promoter between nucleotides
1321 and
1095 (Fig. 1B). Within this 227-bp
domain, we identified five known cis-elements, all of which
were totally conserved between the three species. These elements
included two CArG-like boxes and one true CArG box (hereafter referred
to as CArG boxes 1, 2, and 3, in order of proximal to distal), a
GC-rich element, and a CTF/NF1 site. These three elements represent
potential binding sites for SRF (31), Sp1 (32), and CTF/NF1 (33), respectively. It should be noted that the centrally located nucleotides of the 13-bp CTF/NF1 site (TGGC(N)5GCCA) contain sequence
differences between the three species; however, these residues are not
considered to be important for factor binding (34). The second
conserved domain encompassed the TATA box and extended to nucleotide
128 in rat. Contained within this domain were two conserved CCTCCC elements that were previously shown in transient transfection studies
to function as positive-acting cis-elements in the mouse SM-MHC promoter (23).
Deletion Analysis of the Rat SM-MHC Promoter Revealed That the Conserved 227-bp Domain Is Required for Maximal Activity
To
identify functional elements contained within the rat SM-MHC promoter,
a 4308-bp BglII fragment (bp 4220 to +88) was cloned into
a pCAT reporter vector. A series of deletion constructs were created by
treating the pCAT
4220 construct with exonuclease III and, when
targeting specific regions of interest, by PCR (see "Materials and
Methods"). Transient transfection of these constructs into cultured
rat aortic SMCs revealed that pCAT
1346, which contained both of the
conserved domains, was the most active construct, with an activity
level ~48-fold over promoterless pCAT (Fig. 2). The
addition of more promoter 5
-flanking DNA reduced transcriptional activity to 29-fold (pCAT
3443) and 24-fold (pCAT
4220) over
promoterless pCAT. This observed decrease in activity suggests that
negative regulatory elements exist upstream of nucleotide
1346. The
pCAT
1182 construct, which is missing CArG boxes 3 and 2 and the
GC-rich element, yielded an activity 34-fold over promoterless pCAT.
When the entire 227-bp conserved domain was deleted (pCAT
1102),
transcriptional activity further decreased to ~17-fold over
promoterless pCAT. The activity of the pCAT
562 construct was found to
be approximately equal (14-fold over promoterless pCAT) to that of the
pCAT
1102 construct. The notable difference in activity between
pCAT
1346 and pCAT
1102 suggests that a positive-acting
element(s) resides within the 227-bp conserved domain. The incremental
gain of activity noted for the pCAT
1182 and pCAT
1346
constructs indicates that more than one positive-acting
cis-element is present within this region.
Transcriptional activity of the SM-MHC promoter has been measured in
several other non-SMC types, including L8 skeletal myoblasts (22),
NIH3T3 mouse fibroblasts (23), and C2C12 mouse
myoblasts (20). For comparative purposes, the deletion constructs were tested for activity in L6 myoblasts and myotubes (Fig.
3). Transient transfection analysis of L6 myotubes
revealed that all constructs were only minimally active relative to
SMCs, ranging from 8 to 14-fold activity over promoterless pCAT.
Similar results were seen in L6 myoblasts, except that the range in
activity (4-7-fold over promoterless pCAT) was about half that of
myotubes. In contrast to the SMC data, the addition of the 227-bp
domain failed to enhance transcription in either L6 myoblasts or
myotubes, suggesting that the activity of this domain is regulated in a
cell type-specific manner.
Multiple Sites of Protein-DNA Interaction Are Present within the 227-bp Conserved Domain
To identify potential
cis-elements within the 227-bp domain, DNase I footprint
experiments were performed using nuclear extracts from cultured rat
aortic SMCs. Using a probe that extended from nucleotides 1346 to
1071, we identified three domains of protein binding (Fig.
4). These same three domains were also identified in a
DNase I analysis of the antisense strand (data not shown). A summation
of the footprint data shows that footprint 3 extended from nucleotides
1234 to
1206 and encompassed CArG box 2 and an adjacent GC-rich
element. Footprint 2 extended from nucleotides
1146 to
1121 and
encompassed the CTF/NF1 site. Footprint 1 extended from nucleotides
1115 to
1098 and encompassed CArG box 1. The identification of
multiple sites of protein-DNA interaction is consistent with the
promoter deletion data that indicated the presence of multiple
regulatory elements within this region.
CArG Boxes 1 and 2 Are Positive-acting cis-Elements, and the GC-rich Sequence Functions as a Negative-acting cis-Element
To
determine if the sites of protein-DNA interaction described in Fig. 4
represented functional elements, we mutated each of the four known
cis-elements contained within footprints 1-3. CArG boxes 1 and 2, the GC-rich element, and the CTF/NF1 site were all mutated
within the context of the most active construct (pCAT1346) and tested
for functional activity. Although CArG box 3 did not display any
protein binding activity, we mutated this element anyhow due to its
similarity to other SRF recognition sites and sequence conservation
among all three species. Transient transfection of the mutated
constructs into rat aortic SMCs revealed that the SM-MHC promoter was
regulated by both positive- and negative-acting cis-elements. As shown in Fig. 5A,
CArG box 3 was totally deleted, and CArG boxes 1 and 2 were mutated at
the conserved 5
-CC doublet. The presence of these two cytosine
residues has been previously shown to be necessary for SRF binding and
functional activity (24). Transfection analysis of the three CArG boxes
showed that deletion of CArG box 3 had virtually no effect on
functional activity relative to wild-type pCAT
1346 (Fig.
5B). However, mutations of CArG boxes 1 and 2 resulted in an
~50% decrease in transcriptional activity (20- and 23-fold over
promoterless pCAT, respectively) when compared with wild-type
pCAT
1346 (48-fold over promoterless pCAT). The reduced activity of
pCAT-CArG1Mut and pCAT-CArG2Mut suggests that a major portion of the
positive activity of the 227-bp conserved domain (the difference in
activity between pCAT
1102 and pCAT
1346) is dependent on the
presence of CArG boxes 1 and 2.
The sequence identified as the GC-rich element contains a core Sp1 site
(CCCGCCC); thus, the central nucleotides of this sequence were
specifically mutated such that any putative Sp1 binding would be
abrogated (32). Alteration of this element resulted in a marked
increase in transcriptional activity (Fig. 5B). The actual level of activity (94-fold over promoterless pCAT) was approximately double that of wild-type pCAT1346 and nearly equal to that of the
powerful SV40 viral promoter (pSV40-CAT). This result suggests that the
GC-rich element located at nucleotide
1215 functions as a
negative-acting cis-element. The CTF/NF1 site in pCAT-NF1Mut was completely deleted due to previous reports indicating that this
factor is capable of binding to either half of its palindromic recognition sequence (TGGC(N)5GCCA) (35). The activity of
pCAT-NF1Mut was determined to be slightly higher than that of
pCAT
1346 (52- versus 47-fold over promoterless pCAT).
However, these differences were not statistically significant, and
further analyses of this element were not performed.
The five pCAT1346 mutated constructs were transfected into L6
myoblasts and myotubes to test whether any of the sequence mutations
affected activity in different cell types. Relative to wild-type
pCAT
1346, none of the mutated cis-elements resulted in any
significant gain or loss of transcriptional activity (Fig. 5C). Of particular interest, the GC-rich mutant
(pCAT-GC-richMut), which was almost twice as active as wild-type
pCAT
1346 in SMCs, was actually slightly less active than the wild
type in L6 myoblasts or myotubes. These data suggest that the positive
and negative elements contained within the 227-bp conserved domain
function in a cell type-specific manner.
EMSA and antibody supershift analysis were
utilized to determine if the three CArG boxes identified in Fig. 1
functioned as SRF recognition sites. Individual oligonucleotides
containing CArG boxes 1-3, as well as 5 bp of 5- and 3
- flanking
sequence, were labeled, incubated with 10 µg of SMC nuclear extracts,
and analyzed by EMSA (Fig. 6A). Duplicate
reactions that included an SRF antibody were also electrophoresed on
the same gel (Fig. 6A, right panel). For
comparison, we included a similar sized oligonucleotide that contained
the well characterized SRF-binding site present in the c-fos
promoter, the SRE (31). Similar to a previous study on SRF binding to
the SM
-actin CArG boxes (24), we identified two closely migrating
protein-DNA complexes with the CArG1 probe. The mobilities of these two
bands were supershifted upon SRF antibody addition. Two bands were also
identified with the SRE probe that comigrated alongside the CArG1·SRF
complexes and supershifted with SRF antibody addition. These data
demonstrate that the protein-CArG1 complexes contain SRF or an SRF-like
factor. No protein-DNA complexes whose mobilities supershifted upon
addition of SRF antibody were detected with either the CArG box 3 or
box 2 probes.
Transfection data of pCAT-CArG3Mut indicated that CArG box 3 was
nonfunctional; as such, we did not further pursue experimentation on
protein binding to this site. However, we did perform further EMSAs on
CArG box 2. By increasing the amount of SMC nuclear extract from 10 to
30 µg in the gel-shift reaction, we were able to detect a band that
comigrated with the SRE·SRF complex and also supershifted upon SRF
antibody addition (Fig. 6B). We have previously demonstrated that mutation of the 5-CC doublet of the CArG box completely abrogated
SRF binding (24). However, given the amount of extract needed for
detection of the CArG2·SRF (or SRF-like) complex, we wanted to verify
that, under these conditions, SRF did not bind to the mutated CArG2
oligonucleotide. A CArG2Mut oligonucleotide that duplicated mutations
created in the pCAT-CArG2Mut construct was synthesized and tested for
SRF binding. As shown in Fig. 6C (right lane),
the CArG2Mut probe failed to generate a band that comigrated with the
SRE·SRF complex, even when 30 µg of SMC extract was used. In
conjunction with the transfection data, these protein binding studies
suggest that SRF interaction with CArG boxes 1 and 2 functions to
activate transcription of the SM-MHC gene. Although the binding
affinities for the three CArG boxes were not determined, the data
presented in Fig. 6 suggest that the binding affinity of SRF, or an
SRF-like protein, is much greater for CArG1 than for CArG2.
Due to the considerable sequence similarities
between the negative-acting GC-rich element located within the 227-bp
conserved domain and the published Sp1-binding site, we wanted to
determine if this element functioned as a Sp1 recognition site. Using a labeled oligonucleotide that encompassed the SM-MHC GC-rich element, EMSAs were performed in the presence of different unlabeled competitors (Fig. 7). The GC-rich probe formed three predominant
protein-DNA complexes (bands 1-3) in the presence of SMC nuclear
extract. Bands 1 and 2 exhibited very similar mobilities and migrated
through the gel slower than did band 3. This particular migration
pattern of protein-DNA complexes was similar to the pattern previously described for Sp1 and Sp3 binding; using antibody supershift analysis and a Sp1 probe, Dennig et al. (36) demonstrated that band 1 contained Sp1 protein and that bands 2 and 3 represented Sp3-probe complexes. It should be noted that with longer electrophoresis times,
we were able to better resolve bands 1 and 2 (data not shown). Further
analysis of Fig. 7 shows that the consensus Sp1 oligonucleotide
competed very efficiently for factor binding, even more so than did the
wild-type GC-rich competitor. The fact that all bands disappeared
equally with addition of the Sp1 competitor oligonucleotide is
consistent with Sp1 and Sp3 binding since both factors bind to the same
element with equal affinities (37). Nucleotide alterations in the
GC-richMut oligonucleotide were created to duplicate mutations made in
the pCAT-GC-richMut construct. No competition was evident for the
GC-richMut oligonucleotide even at the highest concentration utilized
(250 M excess). Antibody supershift analysis were employed
to determine if the bands that were competed away with the Sp1
consensus element truly represented Sp1- and Sp3-containing complexes
(Fig. 8). Similar to the results found in the
aforementioned study, the addition of Sp1 antibody resulted in the
disappearance of band 1 and the formation of supershifted complexes.
Likewise, the addition of Sp3 antibody resulted in the disappearance of
bands 2 and 3 and the formation of supershifted complexes. The data
from Figs. 7 and 8 demonstrate that the GC-rich element located within
the 227-bp conserved domain functions as a recognition element for both
Sp1 and Sp3. Furthermore, the lack of competition with the GC-richMut
oligonucleotide suggests that any potential Sp1/Sp3 binding at this
site would have been abolished in the highly active pCAT-GC-richMut
construct.
The goal of this study was to identify cis-elements and
trans-acting factors important for transcriptional
regulation of the SM-MHC gene. By comparing the 5-flanking sequences
of the rat, mouse, and rabbit SM-MHC genes, we identified two domains
that were conserved among all three species. The most proximal domain encompassed the TATA box and contained two conserved CCTCCC elements. This region was previously studied in mouse, and the CCTCCC elements were shown to be necessary for the basal promoter activity ascribed to
this domain (23). Therefore, we focused our studies on the distally
conserved domain located between nucleotides
1095 and
1321 in rat.
From deletion analysis, we determined that inclusion of this 227-bp
domain was necessary for maximal expression of the SM-MHC gene in
cultured rat aortic SMCs. We further demonstrated that several of the
known cis-elements present within this domain were important
for SM-MHC expression, both as positive and negative regulators.
The presence of this 227-bp domain failed to enhance transcription in
either rat L6 myoblasts or myotubes. This result suggests that the
positive effects attributable to this region are potentiated in a cell
type-specific manner. This observation is in agreement with an earlier
SM-MHC promoter mapping study by Kallmeier et al. (21). They
demonstrated that a rabbit SM-MHC construct containing 1332 bp of
5-flanking DNA (including the 227-bp conserved region) was highly
active in primary cultures of rabbit aortic SMCs (6-fold over the basal
activity level of the
112 promoter construct). This
1332 construct
was only minimally active (basal level activity) in five other non-SMC
types. However, a complete review of the literature indicates that
there exist several discrepancies with respect to certain SM-MHC
deletion constructs and their activity levels in both SMC and non-SMC
types. Katoh et al. (20) analyzed the rabbit promoter in
primary cultures of rat aortic SMCs and found that
509 and
1392
deletion constructs were both at ~25% relative CAT activity when
compared with the
2266 construct (100% CAT activity), their most
active construct. All of the constructs tested were only minimally
active in other non-SMCs. In contrast, White and Low (22) analyzed the
rat SM-MHC promoter in primary cultures of rat aortic SMCs and reported
that a
1249 deletion construct was the most active construct,
exhibiting twice the level of activity of the next most active
construct,
602 (26- versus 12-fold activity over
promoterless plasmid). More important, they found that with the
addition of 68 bp of more 5
-flanking sequence (the
1327 construct),
transcriptional activity decreased to one-half that of the
602
construct. A significant decrease in activity was also noted when the
1249 and
1327 constructs were compared in rat L8 myoblasts. The
noted high level of activity (150-fold over promoterless plasmid) of
the
1249 construct in L8 myoblasts and the subsequent drop in
activity (barely detectable levels) with the addition of 68 bp of
5
-flanking sequence led the authors to conclude that this domain was
likely to contain a repressor element(s) necessary for cell
type-specific expression. In another study, Watanabe et al.
(23) reported that a mouse
188 deletion SM-MHC promoter construct,
when tested in primary cultures of rabbit aortic SMCs, was the most
active construct. The addition of up to 3 kilobase pairs of more
5
-flanking sequence only led to a reduction in transcriptional
activity. No increase in activity was observed with inclusion of CArG
boxes 1 and 2.
It is difficult to reconcile the discrepancies in the data presented in
the above studies, not only with respect to our data, but also with
respect to each other. Particularly perplexing is the result in mouse,
where maximal activity in SMCs was achieved with a 188 construct. One
possible explanation for many of the observed differences between these
studies may be differences in the phenotypic state of the SMC in which
the activity of the SM-MHC promoter was assessed. It is well documented
that SMCs undergo rapid phenotypic modulation when placed in culture
(38). In particular, there is an immediate and significant decrease in
expression of the SM-MHC gene (39). However, we have demonstrated that
rat aortic SMCs used in our studies re-express the SM-MHC gene upon
reaching confluency (39). Western blot analysis of the SMCs utilized in
this study showed that, although there was a marked decrease in SM-2
protein, the SM-1 isoform was present in confluent cells up to passage
22, the last passage analyzed.2 Moreover,
we have shown by protein electrophoretic analysis, Northern analysis,
and nuclear run-on analysis (39, 40)3 that
the endogenous SM-MHC gene is actively transcribed in our SMC cultures
under the exact transfection conditions used in the present study. In
all but one of the studies cited above (22), it was not made clear if
the endogenous SM-MHC gene was determined to be active at the time when
the SM-MHC promoter-reporter gene fusion constructs were analyzed.
Based on our observations, it is possible that differences in
confluency at the time of transfection and harvest and differences
related to culture methodology could account for the noted differences
in expression. Additionally, inherent phenotypic differences between
cultured rat and rabbit SMCs may represent another possible
explanation. Despite several attempts, we have been unable to culture
rabbit aortic SMCs such that they continue to express their endogenous
SM-MHC gene, even at early passages. Potential effects due to
phenotypic differences become even more plausible in view of the
observation of White and Low (22) that certain SM-MHC deletion
constructs were differentially expressed in SMCs isolated from either
rat aortic or tracheal tissues. Finally, sequence differences
(resulting from either construct design or species divergence) in the
deletion constructs being compared could certainly explain some of the
differences in expression. However, it is doubtful that this
explanation could account for the mouse data, where the addition of
CArG boxes 1 and 2 or any other 5
-flanking sequence failed to enhance
transcription above the level of the
188 construct. Clearly,
discerning the many factors likely to be involved in the expression of
the SM-MHC gene in cultured SMCs will require further careful study in
which each of the potential mitigating factors is analyzed.
This study demonstrated through site-directed mutagenesis that SM-MHC
CArG boxes 1 and 2 functioned as positive-acting
cis-elements. We further demonstrated that both CArGs, in
the presence of aortic SMC nuclear extract, formed protein-DNA
complexes that contained a factor antigenetically related to SRF. These
data provide evidence that CArG-SRF interaction within the vascular SMC
contributes significantly to the expression of the SM-MHC gene. Our
laboratory previously demonstrated by site-directed mutagenesis that
two CArG boxes located at nucleotides 112 and
62 in the rat SM
-actin promoter were absolutely required for transcriptional
activity in cultured SMCs (24). Li et al. (41) further
demonstrated that deletion of a region containing two CArG boxes in the
mouse SM22
gene (another highly specific SMC marker) resulted in a significant drop in transcriptional activity in cultured SMCs. Thus,
similar to several cardiac- and skeletal muscle-specific genes
(42-44), CArG-SRF interaction appears to serve as a common transcriptional activation mechanism for multiple SM-specific genes.
It was somewhat surprising that mutation of the highly conserved CTF/NF1 site present within the 227-bp domain did not result in any significant alteration in activity. However, several studies indicated that, at least in some circumstances, CTF/NF1 functions to regulate transcription via a mechanism that is likely to involve higher order chromatin structures (45, 46). Such an activity would likely be missed in a transient transfection reporter gene analysis where "naked" plasmid DNA is utilized (35).
Another important regulatory element identified in this study is the
GC-rich repressor that was recognized by both Sp1 and Sp3. Multiple
mechanisms can be envisioned whereby Sp1 and/or Sp3 binding could
inhibit transcription. The fact that the GC-rich element is positioned
adjacent to CArG box 2 suggests that it may function as a negative
regulator by preventing SRF binding to the CArG2 element in a manner
similar to YY1-SRF interaction in the cardiac -actin gene (47).
Also, in contrast to Sp1, a number of recent promoter studies indicate
that binding of Sp3 appears to negatively regulate transcription
(48-50). It will be of interest in the future to investigate how the
elements identified in this study, as well as their potential binding
factors, are involved in the altered expression of the SM-MHC gene in
phenotypically modified SMCs found in atherosclerotic lesions in man
(51).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U83321[GenBank].
We gratefully acknowledge the expert technical assistance of Diane Raines and Andrea Tanner.