© 1996 American Heart Association, Inc.
Articles |
Structure and Characterization of the 5'-Flanking Region of the Mouse Smooth Muscle Myosin Heavy Chain (SM1/2) Gene
Presented in part at Keyston Symposia, Molecular Biology of the Cardiovascular System, January 30, 1996.
From The Third Department of Internal Medicine, University of Tokyo (Japan).
Correspondence to Ryozo Nagai, MD, The Second Department of Internal Medicine, University of Gunma, 3-39-15, Syowa-machi, Maebashi-shi, Gunma-ken, 371, Japan. E-mail nagai@sb.gunma-u.ac.jp.
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Abstract We have previously shown that smooth muscle myosin heavy chain isoforms (SMs), including SM1, SM2, and SMemb, are differentially expressed during vascular development and in vascular lesions, such as atherosclerosis. The SM1/2 gene is expressed exclusively in smooth muscle cells and generates SM1 and SM2 mRNAs by alternative splicing. Whereas SM1 is constitutively expressed from early development, SM2 appears only after birth. In this study, we have isolated and characterized the 5'-flanking region of the mouse SM1/2 gene. Transient transfection assays using a series of promoter-luciferase chimeric constructs demonstrated that tandem elements of the CCTCCC sequence, located at -89 and -61 bp relative to the transcription start site, were essential for transcriptional activity of the SM1/2 gene in primary cultured rabbit aortic smooth muscle cells and smooth muscle cell lines derived from the rabbit aorta but not in non–smooth muscle cells. Gel mobility shift assays indicated that CCTCCC was a binding site for nuclear proteins prepared from smooth muscle cells. Double-stranded oligonucleotides containing either the CACC box or the Sp1 consensus sequence efficiently competed with the CCTCCC elements for binding the nuclear extracts. Site-specific mutations of CCTCCC elements resulted in a significant reduction of the promoter activity. Moreover, CCTCCC elements are evolutionary conserved between mouse and rabbit. In conclusion, the results of this study indicate an important role for the interaction of the CCTCCC sequence with Sp1 or related factors in activating transcription from the SM1/2 gene promoter.
Key Words: SM1/2 • smooth muscle • myosin heavy chain • Sp1 • CACC
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Introduction |
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Smooth muscle cells are the predominant cell type contributing to the formation of many vascular lesions characterized by a fibroproliferative response of the intima. SMCs increase in number in atherosclerotic lesions, and their rapid growth causes coronary artery restenosis after interventional therapy such as PTCA. A number of studies on morphological changes of the SMCs have shown that smooth muscles variously change their phenotype from the "synthetic type" to the "contractile type" according to the developmental and differential state.1 2 3 In normal adult arterial walls, contractile-type SMCs are observed, which have well-developed contractile apparatus, and their cytoplasms are packed with numerous filaments. On the other hand, fetal arteries or vascular lesions are characterized by synthetic-type SMCs, which are enriched with Golgi complexes, mitochondria, endoplasmic reticula, and free ribosomes. Phenotype of the SMCs reflects the expression of smooth muscle–specific genes, including cytodifferentiation-related contractile and cytoskeletal proteins, such as smooth muscle
-actin, h-caldesmon, meta-vinculin,4
SM22,5 calponin,6 SM1, and SM2. Among
those expressed specifically in SMCs, the promoter sequences of only a
few genes have been determined and characterized. Although the promoter
region of the smooth muscle -actin has been most vigorously
investigated, the questions as to the mechanisms of cell
type–specific expression remain to be addressed.7 8 9
Smooth muscle -actin is expressed at selected sites outside the
SMCs, such as skeletal and cardiac muscles in fetus,10 11
and continues to be expressed even in the atherosclerotic lesions where
SM1 and SM2 are no longer expressed.12 In contrast, SM1
and SM2 are the products of a single gene, SM1/2, which has been
demonstrated by us and others to be exquisitely regulated in relation
to smooth muscle development and differentiation.13 14 15
Thus, we have pursued the study of the mechanisms regulating SM1/2
expression to reveal the molecular mechanisms underlying the phenotypic
modulation of SMCs, since mapping and functional assessment of
cis elements required for cell-specific gene expression
have been a fruitful approach in the study of the differentiation
mechanisms of several distinct cell types, such as skeletal muscle,
cardiac muscle, hematopoietic cells, and adipocytes. We have previously shown that SMs are differentially expressed during normal vascular development and in vascular lesions12 13 16 and that these changes in gene expression precede smooth muscle phenotypic modulation in evaluation of the process of atherosclerosis and the formation of restenosis after PTCA.12 Smooth muscle myosin heavy chain has at least four types of isoforms, SM1 (204 kD), SM2 (200 kD), SMemb (NMMHC-B, 200 kD), and NMMHC-A (196 kD).13 14 15 16 17 18 19 Expression of SM1/2 is differentially regulated at the level of RNA processing during vascular development, whereby SM1 is constitutively expressed from early development, but SM2 appears after birth.13 Consequently, the mechanism of SM1/2 gene expression seems to be a key in the understanding of the development and differentiation of smooth muscles.
In order to determine the molecular mechanism of SM1/2 gene expression, we have isolated and characterized the 5'-flanking region of the mouse SM1/2 gene and found that tandem cis elements of CCTCCC sequence in the proximal upstream region are required for SM1/2 expression.
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Isolation of a Mouse Genomic DNA Clone (MF31), Including 5'-Flanking Region and First Exon
Methods for construction of a rabbit fetal aorta cDNA library and screening were previously described.16 A rabbit fetal aorta cDNA library was screened using
-32P–labeled
1571-nt cDNA fragments from the rabbit SM1/2 cDNA clone
containing a 3'-untranslated region, SMHC29, which we previously
isolated and reported.17 The rabbit cDNA library was
screened again with the most distal 5' end of the cDNA clone selected
in the former screening. A cDNA clone, 1H61, containing more upstream
nucleotides, was chosen and was 4 kbp long (between
-64 and +4 kbp from the ATG start codon). In order to obtain a
5'-upstream region of the mouse SM1/2 gene by homology to the rabbit
1H61 clone, the 
gt11 mouse uterus cDNA library (Stratagene) was
screened with a 350-bp EcoRI-HindIII subfragment
(between -64 and +286 bp from the ATG start codon) of the clone.
The mouse SM1/2 cDNA clone, MS62, was obtained. Sequencing and mapping
determined MS62 to be 
2 kbp in length and to contain 80 bp of the
5'-flanking sequences. The sequence between +107 and +400 bp of MS62
shares 88.4% identity at the nucleic acid level and 94.9% identity at
the amino acid level with rabbit SM1/2, demonstrating that MS62 was
definitely a part of the mouse SM1/2 gene. In order to obtain the
unknown 5'-flanking region of mouse SM1/2 gene, we used selective
amplification of the gene by cassette ligation–mediated
PCR.20 21 Mouse genomic DNA, which was extracted from
NIH3T3 cells by the GENOME DNA isolation kit (BIO101), was digested
with Bgl II. The products of digestion were ligated to
the double-stranded DNA cassette primer Sau3AI cassette (TAKARA).
The cassette-ligated fragment was amplified by PCR using the 23-mer
cassette primer C1 (TAKARA) and the 21-mer primer
(5'-ATTAGCAGGAGGACACCGGAT-3'), which is complementary to the
5'-flanking region between 25 and 45 bp of MS62. The products of
the first PCR were amplified again by the second PCR using the 23-mer
cassette primer C2 (TAKARA) and the 20-mer primer
(5'-AGCTCGGATCTGGCACTGAT-3'), which is complementary to the 5'-flanking
region between 1 and 20 nt of MS62. A more specific 400-bp fragment was
obtained. DNA sequence analysis of the fragment revealed that
it contained the 5'-untranslated sequence plus the 5'-flanking region
of the mouse SM1/2 gene. Finally, the FIXII mouse genomic library
(Stratagene) was screened with the PCR product and a clone, MF31,
which included the first exon, and the 5'-flanking region was
isolated.
Primer Extension Analysis
Total RNA was prepared from mouse aorta, uterus, and intestine
by the acid guanidinium thiocyanate–phenol chloroform
method.22 A primer, PEP-1 (5'-AGAGCTCGGATCTGGCACTG-3'),
was end-labeled with T4 kinase. The primer was coprecipitated with
20 µg each of total RNA and subsequently hybridized and extended with
reverse transcriptase (Superscript, Promega). After ethanol
precipitation, the products were electrophoresed on a 6% urea
polyacrylamide gel. A dideoxy sequencing reaction using the
same oligonucleotide primer was conducted, and samples
were electrophoresed in adjacent lanes to determine more precisely the
3' end of the extended species.
Subcloning and Sequencing
An insert from the phage clone MF31 was excised by digestion
with Not I and subcloned into pBluescript SK(-) (NN4).
For mapping and sequencing, several smaller genomic fragments were
subcloned, and the first exon of 1500 bp in length of the
5'-flanking region was sequenced by using the dideoxy–chain
termination method in denatured double-stranded plasmids.
RNase Protection Assay
Total RNA was prepared from C2/2 cells, an established cell line
derived from rabbit aortic SMCs23 by the methods described
previously.22 The 266-nt Pst I fragment from
the 3'-coding region and the 3'-untranslated region of the rabbit SM1/2
cDNA clone, SMHC29,17 was subcloned into pBluescript
SK(-). After linearizing plasmid DNA by digestion with
Not I, the RNA probe was synthesized with T7 RNA polymerase
and [-32P]UTP. The RNase protection assay was carried
out by modified procedures of the Riboprobe Gemini System II kit
(Promega) and Ribonuclease Protection Assay kit (Ambion). The probe and
protected fragments were analyzed on a denaturing urea–5%
polyacrylamide gel. This RNA probe was 340 nt long, including
the multicloning site of the vector, and able to distinguish SM1 and
SM2 mRNAs. One partially protected probe representing SM2
mRNA was 224 nt, and the other partially protected fragments
representing SM1 mRNA were 106 and 79 nt.
Promoter-Luciferase Vector Chimeric Constructs
The SM1/2 promoter–luciferase reporter genes were
constructed by cloning a Sac I–Sac I fragment
that corresponded to nucleotides +47 to -1226
relative to the transcription start site. This fragment was inserted
into PGVB (PicaGene, Nippon Gene) to yield the construct SCSC-PGV. The
EVSC-PGV construct was prepared by subcloning a blunted
EcoRV–Sma I fragment from NN4 into the
Sma I–Sma I site of SCSC-PGV (Fig 1A
). A series
of six recombinant plasmids of progressively shorter lengths were
constructed by subcloning the isolated restriction fragments from NN4
into the Sma I–Sac I site of the luciferase
expression vector PGVB. By using selective unique restriction sites
within the 5' region of the SM1/2 gene, deletions were constructed at
-576 bp (DEL1), -261 bp (DEL2), -188 bp (DEL3), and
-92 bp (DEL4) by digestion with BstXI,
PvuII, EcoO109I, and BamHI,
respectively. Deletion constructs (DEL5, -80 bp; DEL6, -72
bp) were also prepared using oligonucleotide primers
and PCR to amplify sequences that were ligated to PGVB. In order to
test the effects of the mutation of the CCTCCC sequence in the SM1/2
promoter, mutations were introduced into the promoter construct DEL1 by
an overlapping PCR method24 using synthesized
oligonucleotides, including mutations. Three mutated
plasmids, BM80, BM60, and BM-D, contained a cluster of substituted
nucleotides from CCC to AAA, which spanned from -86
to -84 bp, -58 to -56 bp, and both of the two,
respectively. Reporter constructs used to test the effects of the
CCTCCC sequence on the heterologous promoter were prepared by
amplifying the sequences between -104 and -44 bp using
upstream primers that had the Sac I site in their 5' ends
(primer 1, 5'-GCGGAGCTCAGGGAAGAGGACCT-3') and downstream primers that
had the Xho I site in their 3' ends (primer 2,
5'-CTACTCGAGAGTGGAAAG-3') to allow, upon their digestion, its cloning
into the corresponding restriction sites immediately upstream from a
truncated SV40 promoter–luciferase reporter construct (pGL3
promoter vector, Promega). The resulting plasmid was named BS-pro.
DM-pro, a mutated version of BS-pro, was constructed by an overlapping
PCR method24 using the oligonucleotides
containing mutations in the CCTCCC sequence along with either primer
1 or primer 2. The sequences of all constructs described were
confirmed by sequencing. As the positive control, we used the plasmid
PGVC (Nippon Gene) containing the SV40 promoter and enhancer. As the
negative control, the plasmid PGVB, which does not contain any promoter
or enhancer, was used.
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Cell Culture and DNA Transfection
Primary cultures of rabbit SMCs were prepared
enzymatically by a previously described method.25 26 27 Stock
cultures of rabbit C2/2 SMCs were obtained from Life Science Center,
Biochemical Research Lab, Asahi Chemical Industry. C2/2 cells were
cultured in DMEM (GIBCO) with 5% fetal calf serum. NIH3T3 cells were
cultured in high-glucose DMEM with 10% fetal calf serum. COS7
cells were cultured in DMEM with 10% fetal calf serum. Plasmids were
transfected cells during logarithmic phase growth (50% to 60%
confluent in serum-supplemented medium) in 60-mm tissue culture
plates. To monitor the differences in transfection efficiency, 5 µg
of chimeric constructs were cotransfected with the 2-µg construct,
pEFSA-LacZ, which contained the coding region of the
ß-galactosidase gene under the control of the human elongation
factor 1 promoter. Transient transfections of primary cultures, C2/2
cells, and COS7 cells were performed by the lipofectin (GIBCO) method
as described in the manufacturer's protocol.28 The dose
of lipofectin used for the transfection was 20 µL for primary culture
and 5 µL for other cells. Transient transfections of 3T3 cells were
performed by calcium phosphate precipitation (Stratagene) according to
the manufacturer's protocol.29
Luciferase Assay and ß-Galactosidase Activity Assay
Transfected primary cultures were harvested for extract
preparation 72 hours after transfection. The procedure was performed in
triplicate. Other cell cultures were harvested 48 hours after
transfection. Transfections were performed in duplicate with at least
two separate preparations of each plasmid. Cell extracts were prepared
by use of a luciferase assay kit (PicaGene system, Nippon Gene), and
levels of luciferase activity were measured by the Lumat LB9501
luminometer (Brethold). ß-Galactosidase assays were performed by
using o-nitrophenyl ß-D-galactopyranoside
(Sigma Chemical Co). The luciferase activity was normalized to
ß-galactosidase activity generated from cotransfected pEFSA-LacZ.
The relative luciferase activity was shown by the standardization of
positive controls to 1000.
Preparation of Nuclear Extracts and Gel Mobility Shift
Assays
Nuclear extracts were prepared from C2/2 cells as
described previously.30 In each gel mobility shift assay,
10 µg of nuclear extract was used. Annealed double-stranded
oligonucleotides synthesized by Nippon Bio-Service (Fig 4A) were -32P–labeled by a fill-in reaction of
Klenow fragments. The binding reactions were preincubated for 5 minutes
at room temperature in a total volume of 20 µL containing 10 mmol/L
Tris-HCl at pH 7.5, 50 mmol/L NaCl, 0.5 mmol/L dithiothreitol, 10%
glycerol, 0.05% NP-40, 2 µg of poly(dI-dC) as a nonspecific
competitor, and nuclear extracts. After addition of
1.0x104 cpm of labeled probe, the reactions were incubated
for an additional 20 minutes at room temperature and then
analyzed in 5% polyacrylamide nondenaturing gel in
low-ionic-strength buffer at 4°C. For the supershift assay
using anti-Sp1 polyclonal antibody (Santa Cruzu Biotechnology, Inc) and
anti–smooth muscle -actin antibody (DAKO), the mixture was
additionally incubated for 15 minutes at room temperature.
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Sequence Analysis of the SM1/2 Gene Promoter and 5'-Flanking Region
We have isolated a mouse genomic clone, MF31, through several steps from the rabbit SM1/2 cDNA clone, SMHC29.17 MF31 was
14 kbp long and included the first exon and 5'-flanking region of
the mouse SM1/2 gene promoter. Fig 1A
shows the
restriction map of MF31. To determine the size of the untranslated
exon, primer extension analysis was carried out by using total
RNA from mouse aorta, uterus, and small intestine (Fig 1B). In each
lane, a single signal was detected at the same position, -106 bp
from ATG, indicating that the mouse SM1/2 gene had a single
transcription start site in all of these tissues.
Consequently, the expression of the SM1/2 gene is not controlled
by selection of the transcription start sites. The first exon and the
second exon contained 90 and 16 bp of the noncoding region,
respectively. Therefore, the transcription start site was located in
the second exon, and the first intron was >10 kbp long.
The sequence of the 1526-bp 5'-flanking region and a part of the first
exon is shown in Fig 1C. The SM1/2 gene has a canonical TATA box at
-28 bp. The motifs of known cis elements were searched
by transcription factor databases. A GATA box at -43 bp, two CArG
boxes at -965 and -1297 bp, two CArG-like boxes at
-1085 and -1161 bp, and eight E boxes at -122,
-264, -342, -707, -1238, -1244,
-1442, and -1479 bp were identified. Other
muscle-relating transcription factor binding sites, such as M-CAT
and MEF-2 binding consensus sequences, are not detected in this
5'-flanking region.
Detection of SM1/2 Gene Expression in C2/2 Cells by RNase
Protection Assay
Physiological and microelectronic
studies have demonstrated that C2/2 cells, which were derived from the
rabbit aortic SMCs, conserve smooth muscle
characterization.23 In order to verify the expression of
SM1/2 gene in C2/2 cells, an RNase protection assay was performed. In
each lane of Fig 2, there are two protected bands that
are 106 and 79 nt long, representing SM1 mRNA. These
results indicate that at least SM1 mRNA is expressed in C2/2 cells in
both the logarithmic growth phase and confluent phases and justify the
use of C2/2 cells for analysis of transcription activity of the
SM1/2 gene.
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Promoter Activity of the SM1/2 Gene Promoter in C2/2 Cells,
Primary Cultures, and Other Non-SMCs
In order to assess the promoter activity of the 5'-flanking region
of the mouse SM1/2 gene, SM1/2 promoter–luciferase fusion reporter
plasmids were constructed. We transfected these constructs into C2/2
cells together with pEFSA-LacZ, which contained the coding region of
the ß-galactosidase gene under the control of the human
elongation factor 1 promoter and served as an internal control to
standardize the transfection efficiency. The results are shown in Fig 3A. The longest plasmid (EVSC, -3.4 kbp to +47 bp
from the transcription starting site) and the second longest plasmid
(SCSC, -1226 to +47 bp) had >50-fold higher transcription
activity compared with the promoterless plasmid (PGVB). Although
deletions from -1226 to -188 bp did not significantly
affect the promoter activity (SCSC, DEL1, DEL2, and DEL3), another
108-bp deletion (DEL5) reduced the expression level by 90%. Marked
reduction was seen by the deletion between -92 and -80 bp,
and the transcriptional activity of DEL5 was 25% compared with that of
DEL4. These results indicate that the region in or adjacent to the
sequence between -92 and -80 bp is important for full
promoter activity and may contain a positive cis element in
C2/2 cells. Because DEL6 (-72 bp) was approximately ninefold more
active than PGVB, there remain minimum cis elements
necessary for the transcription within the most proximal 72 bp. To
ascertain the significance of these regions, we performed the same sets
of assays using primary SMCs and other non-SMCs (Fig 3B). In primary
SMCs, promoter activity of each deletion construct was markedly higher
(>15-fold) than that in non-SMCs. It is also important to note that
deletion between -92 and -80 bp resulted in the significant
reduction in the promoter activity in primary cultures of SMCs but not
in non-SMCs. Although the promoter activity of the SM1/2 gene in the
C2/2 cells was 25% of that in primary cultures of SMCs, comparable
effects of the deletions between C2/2 and primary cultures of SMCs on
the promoter activity suggest that C2/2 cells are germane to the
present study.
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Gel Mobility Shift Assays With Tandem Elements of
CCTCCC
To identify the nuclear factors binding to the cis
elements delineated by the transient transfection assays, we performed
gel mobility shift assays. The double-stranded synthetic
oligonucleotide, SMS80, containing the
nucleotides between -103 and -68 bp of the
SM1/2 gene, was used as a probe (Fig 4A). As shown in
Fig 4B, SMS80 interacted with nuclear proteins prepared from C2/2 cells
and gave rise to two shifted complexes (complexes 1 and 2). Formation
of these complexes was completely abolished by an excess of unlabeled
SMS80. These results indicated that both complex 1 and 2
represent sequence-specific binding activity.
To determine their binding requirements further, substitution mutations
were introduced into SMS80. Four mutants were prepared, and each mutant
contained a clustered mutation that was a conversion of three
successive nucleotides in the sequences of SMS80 (-92
to -90 bp in BM1, -89 to -87 bp in BM2, -86 to
-84 bp in BM3, and -83 to -81 bp in BM4), as shown in
Fig 4A. Although both BM1 and BM4 could compete for the wild-type
oligonucleotide SMS80, neither BM2 nor BM3 abolished
the binding completely (Fig 4B). These results suggest that the
sequence CCTCCC, located between -89 and -84 bp, is
specifically bound to by the nuclear proteins from cultured SMCs.
Furthermore, as a candidate for a minimum cis element
necessary for the transcription within the most proximal 72 bp, we also
tested whether nuclear factors would also bind to the CCTCCC sequence
located at -61 bp. We prepared the synthetic
oligonucleotide SMS60, which contains the
nucleotides between -68 and -49 bp (Fig 4A). As
shown in Fig 4C, two shifted complexes were formed. SMS60 was proved to
be combined to the nuclear proteins of C2/2. These interactions were
inhibited by SMS80 as well as by SMS60, but not by SMS60M, which
contained a clustered mutation that was a conversion of three
successive nucleotides from -58 to -56 bp in
the sequences of SMS60 (Fig 4C). These results suggest that nuclear
factors that bind to CCTCCC elements at -89 and -61 bp are
related or identical.
To determine whether factors binding to CCTCCC sequence belong to the
family of known transcription factors, we used four double-stranded
oligonucleotides, which include consensus sequences for
a CACC box,31 which was originally identified as a
critical element for erythrocyte-specific expression of the
ß-globin gene, the Sp1 binding site,32 the CAT
box,33 and the M-CAT box34 (Fig 4A).
Unlabeled oligonucleotides carrying either a CACC
binding site or an Sp1 binding site could compete with both SMS60 and
SMS80 for binding to the nuclear protein from cultured SMCs (Fig 4B and 4C). These results suggest that despite some sequence divergence from
the consensus Sp1 or CACC binding proteins, factors binding to CCTCCC
sequence share binding specificities with Sp1 or CACC binding
proteins.
Using the commercially available polyclonal antibodies against Sp1, gel
mobility shift assays were performed to examine whether the complexes
formed with C2/2 nuclear extracts would actually contain Sp1. The
polyclonal antibody against Sp1 clearly supershifted the bindings over
either SMS80 or SMS60 probes as well as the bindings over the consensus
sequence for the Sp1 binding site (Fig 4E), whereas no effect on the
bindings was observed with the unrelated polyclonal antibody. These
results indicated that Sp1 is the constituent of the complexes formed
with the CCTCCC sequence in C2/2 nuclear extracts. In accordance with
these observations, we found that CCTCCC binding proteins are not
restricted to SMCs by showing the binding activities in the nuclear
extracts from COS7 cells, which are apparently non-SMCs (Fig 4D). As in
the case with C2/2 nuclear extracts, the bindings to CCTCCC sequence
were abolished by the consensus Sp1 binding site and were supershifted
by the addition of the Sp1 antibodies (Fig 4E). These results
ascertained the CCTCCC binding proteins to be present in both SMCs
and non-SMCs.
Effects of Site-Specific Mutations on SM1/2 Promoter
Activity
We next investigated whether the inhibition of protein binding to
the CCTCCC element actually reduces promoter activity. Mutations were
introduced into the promoter construct DEL1. Three mutated plasmids,
BM80, BM60, and BM-D, contained a cluster of substituted
nucleotides from CCC to AAA, which spanned from -86
to -84 bp, -58 to -56 bp, and both of the two,
respectively. Promoter activity of BM60, BM80, and BM-D was 46.9%,
49.3%, and 26.2%, respectively, compared with the activity of
wild-type DEL1 (Fig 5). From these results, we
conclude that the tandem CCTCCC elements, located at -89 and
-61 bp, are necessary for full promoter activity of the SM1/2
gene.
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Since GATA binding proteins have been implicated in the cell
type–specific expression, it may be worthwhile to examine the
importance of the GATA box located at -43 bp. A substitute
mutation at -41 bp from A to C was introduced into the promoter
construct DEL1 by using the PCR technique. Since the activity of this
mutant reporter gene was almost the same as that of the wild type, the
GATA box did not seem to play a role in controlling the promoter
activity of the SM1/2 gene (Fig 5).
Effects of the CCTCCC Sequence on the Heterologous
Promoter
In order to determine whether CCTCCC sequence can confer the
tissue-specific enhancement in the promoter activity on the
heterologous promoter, we linked 61 bp of the SM1/2 promoter region,
which contains two copies of CCTCCC sequence, to a truncated SV40
promoter and transfected them into SMCs and non-SMCs. To assess the
role of CCTCCC sequence specifically, mutations were introduced in
order to disrupt the nuclear factor binding. Promoter activities were
compared with the activity of the SV40 basal promoter–luciferase
construct (pGL3 promoter vector) in each cell type. As shown in Fig 6, insertion of the wild-type sequence had either no
effect or a non–sequence-specific effect on the luciferase
activity in every cell type tested. This result suggests that the
CCTCCC sequence alone is not sufficient for the tissue-specific
expression of the SM1/2 promoter and that the presence of the
additional elements is required for exerting its tissue-specific
role.
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Comparison With the Rabbit SM1/2 Gene Promoter
By sequence comparison of the SM1/2 gene promoter between mouse
and rabbit, we found two regions that contain several stretches of
conserved sequences (Fig 7). One region is located
between -120 and +40 bp and contains a canonical TATA box and
tandem elements of CCTCCC. Evolutionary conservation of the CCTCCC
sequence between mouse and rabbit supports our hypothesis that these
elements play a role in regulating the expression of the SM1/2 gene.
There does not exist a GATA box in the rabbit promoter. However, given
that GATA binding proteins constitute a multigene family whose members
are tissue-restricted in activity and regulate a variety of genes
within those cell types,35 36 further studies may be
necessary to affirm its function. The other conserved region, which is
located between -1500 and -1000 bp from the transcription
start site (Fig 7B), contains several conserved elements, including two
CArG-like boxes and one CArG box, which were originally emphasized as
regulatory elements in the promoter region of the smooth muscle
-actin gene.7 In the mouse promoter, there were
eight E-box motifs (CANNTG), which were reported to be necessary for
the activation of the smooth muscle -actin gene8
but were not conserved in the rabbit promoter. In our experiment, none
of these elements acted as an activator or as a suppresser.
A previous study on the rabbit SM1/2 gene promoter has shown that a
promoter fragment 2266 bp upstream from the transcription start site
has the highest reporter activity in cultured rat aortic SMCs and
implies that the MEF2-like sequence located at -1540 may play a
role.37 In spite of careful inquiry, we could not find an
MEF2-like element in the mouse SM1/2 promoter comparable to the one in
the rabbit promoter. The reason for such an apparent discrepancy of the
regulatory mechanism of the SM1/2 gene between mouse and rabbit is not
clear.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SM1/2 gene has recently been proved to provide an excellent model in the study of the molecular mechanisms of smooth muscle–specific expression, since we and others showed its exclusive expression in SMCs.12 13 15 In the present study, we have characterized the mouse SM1/2 gene promoter and determined the sequences required for its expression in cultured SMCs by 5' deletion and site-specific mutation analyses. The 6-bp sequence CCTCCC, located at -89 and -61 bp, serves as a binding site of nuclear factors, which share the binding specificity with either Sp1 or CACC-box binding proteins. Moreover, the sequence CCTCCC is conserved between mouse and rabbit. Thus, we propose that interaction of the CCTCCC sequence with its binding proteins is required for the activation of the SM1/2 gene.
Our gel-shift assays using the unlabeled competitors showed that the CACC box efficiently competes for the binding of nuclear factors to the CCTCCC sequence, suggesting that the CCTCCC sequence is bound by CACC-box binding proteins. There are some precedents to show that CACC binding proteins and their cognate binding sites play a role in controlling cell-specific gene expression in particular cell types. The best example is the importance of the CACC box in the regulation of ß-globin gene expression. Mutation of the CACC box in the promoter of the ß-globin gene causes a part of ß-thalassemia, resulting in the significant reduction of erythrocyte-specific expression of the ß-globin gene.38 Miller and Bieker39 have isolated an erythroid-specific Krüppel factor (EKLF), containing proline-rich domains and three zinc-finger structures that bear some similarities with those in the Krüppel family of transcription factors, as a protein binding to the CACC-box of ß-globin. The knockout of EKLF leads to the selective loss of the ß-globin gene expression and results in death due to severe ß-thalassemia during fetal liver erythropoiesis.40 A second example suggesting the potential role of the CACC box in cell type–specific expression was offered by the studies of Parmacek et al31 and Bassel-Duby et al,41 who showed that the CACC box may function as an important sequence element for muscle-specific expression of troponin C and myoglobin genes. Subsequent characterization of the CACC-box binding proteins revealed the 40-kD protein, which is ubiquitously expressed in many tissues. However, it seems unlikely that CACC-box binding protein described by Williams et al41 is identical to CCTCCC binding proteins, since the mobility on the EMSA gel of shifted complexes formed by CCTCCC probe is almost equal to that of Sp1, whose molecular weight is much larger than 40 kD. Last, the nuclear factor binding to the CACC box in the T-cell receptor gene has been identified; it is referred to as htß.42 htß contains a negatively charged region and four zinc-finger domains like Sp1 and is present in a variety of cell types. Thus, it is commonly noticed that the Sp1 binding sequence is bound by diverse transcription factors, many of which belong to the zinc-finger protein family of transcription factors. In this regard, it is tempting to speculate that the factors with zinc-finger domains distinct from Sp1 participate in the regulation of the SM1/2 gene expression through binding to the CCTCCC sequence.
Our data indicating that the CCTCCC element is important for the SM1/2
genes corroborate the recent findings that the promoter region of the
smooth muscle–specific SM22 gene and smooth muscle
-actin gene contain the CACC box and GGGAGG, respectively, yet
the functional role of these sequences in each gene remains to be
examined.5 43 44 In the case of the rabbit SM1/2 gene, the
region between -1.5 and -1.0 kbp has been shown to play a
role in cell type–specific gene expression. Although the
SMC-specific expression of the SM1/2 gene is markedly diminished when
5'-flanking sequences are deleted to -1.0 kbp in the rabbit gene,
our data indicate that deletion of the equivalent region minimally
affects the expression of the mouse SM1/2 gene. In view of the
conservation of the essential mechanisms across the species, an
apparent discrepancy of the regulatory mechanisms between rabbit and
mouse was somewhat surprising. Whether such an
inconsistency is due to the species difference of the
genes or transfected cells or is due to the difference of the position
of the 3' end of the promoter within the constructs is uncertain.
On the basis of the observation that the CCTCCC binding proteins are
present in the nuclear extracts from both C2/2 and COS7 cells and
that the Sp1 antibodies supershifted the complexes, it is probable that
Sp1 itself plays a role in regulating the SM1/2 promoter. Although we
have not yet vigorously tested whether ubiquitously expressed Sp1 plays
a role in regulating the tissue-specific expression of the SM1/2
gene in vivo, we can envisage several explanations supporting this
hypothesis. First, Sp1 may function as a cofactor that forms a
functional complex with tissue-specific transcription factors.
Studies involving the muscle-specific expression of cardiac
-actin and skeletal troponin I genes provide precedent for this
mode of function of Sp1. It has been formally established that Sp1,
SRF, and MyoD are required for the muscle-specific expression of
the cardiac -actin gene.45 Likewise, interaction
between Sp1 and MyoD is necessary for the muscle-specific
expression in both troponin I46 and acetylcholine receptor
delta genes.47 In this regard, it is relevant to speculate
that there exists a potential binding site downstream from the CCTCCC
sequence for cell type–specific factors. In fact, we found that
CCTCCC sequence alone is not sufficient to confer smooth
muscle–specific expression, whereas these sequences increase the
transcription from the SM1/2 promoter more efficiently in SMCs than in
non-SMCs. These observations are consistent with the notion
that Sp1 may function as a component of the tissue-specific
transcription factor complex formed over the proximal promoter region
of the SM1/2 gene. Given that the minimal promoter region spanning
nucleotides -72 to +47 is highly conserved between
mouse and rabbit, there may exist another important sequence required
for the tissue-specific expression of the SM1/2 gene in this
region. Second, interaction of Sp1 with other transcription factors may
be regulated in a cell type–specific manner. It has been shown
that the retinoblastoma gene product differentially regulates
several genes, including c-fos and transforming growth
factor-ß1 genes,48 depending on the cell types in which
the retinoblastoma gene is expressed. Experiments using the
Sp1-deficient Drosophila cells along with the Sp1 expression
vectors may help us to determine whether Sp1 activates the
SM1/2 gene through the CCTCCC sequence.
In conclusion, the present study raises the possibility that the interaction between the CCTCCC sequence and its cognitive binding proteins, Sp1 or related transcription factors, regulates the expression of the SM1/2 gene. Our observation that the CCTCCC sequence is unable to confer tissue-specific characteristics to the heterologous promoter may indicate that additional elements are required for the tissue-specific expression of this gene. Having noticed that there remain other important cis elements, we may further examine the trans-acting DNA binding proteins and their mutual associations. Further studies may contribute to the control of atherosclerosis and restenosis after PTCA.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We gratefully acknowledge Yasuharu Sasaki for providing us with the cultured SMC line, C2/2, and Hyo-Soo Kim and Kenji Nakahara for helping with the RNase protection assay. We also thank Yukari Nakajima, Midori Ogawa, and Masako Nakamura for their excellent technical assistance.
Received January 4, 1996; accepted February 16, 1996.
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References |
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