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Circulation Research. 1996;78:978-989

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(Circulation Research. 1996;78:978-989.)
© 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.

Masafumi Watanabe, Yasunari Sakomura, Masahiko Kurabayashi, Ichiro Manabe, Masanori Aikawa, Makoto Kuro-o, Toru Suzuki, Yoshio Yazaki, Ryozo Nagai

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.


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {alpha}-actin, h-caldesmon, meta-vinculin,4 SM22{alpha},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 {alpha}-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 {alpha}-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.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {alpha}-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 {lambda}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 {approx}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 {lambda}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 {approx}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 [{alpha}-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 1ADown). 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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  		Figure 1. Structure and nucleotide sequence of the mouse SM1/2 gene. A, Schematic diagram of the promoter region of the mouse SM1/2 gene. Restriction map of the clone MF31 is shown. Open box indicates the first exon. Two lines, designated as EVSC and SCSC, indicate the regions that were ligated upstream from the luciferase gene. Restriction enzyme sites are indicated as follows: N, Not I; E, EcoRV; S, Sac I; and Sm, Sma I. B, Mapping of the transcription start site by primer extension. Lanes C, T, A, and G contained sequencing reactions primed with the same antisense oligonucleotides used for mapping and were electrophoresed in parallel. The arrowhead indicates the extension products. The arrow indicates the position where the transcription starts. C, The nucleotide sequence of the promoter region of the mouse SM1/2 gene. Numbers appearing in the left margin refer to the first nucleotide listed on that line. Numbers indicate the position of the nucleotide relative to the transcription start site, which is referred to as +1. The portion of the sequences in the first exon is contained within the rectangle at the bottom.

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{alpha} 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 4ADown) were {alpha}-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 {alpha}-actin antibody (DAKO), the mixture was additionally incubated for 15 minutes at room temperature.





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  		Figure 4. Gel mobility shift assays. A, The oligonucleotides used in the gel mobility shift assays are listed. The oligonucleotide SMS80 and SMS60 contains the nucleotide sequence of the SM1/2 gene spanning from -103 to -68 bp and from -68 to -49 bp relative to the transcription start site, respectively. Mutations, indicated by underlining, were introduced in or adjacent to the CCTCCC sequence to assess their respective effects on the binding activity. The CCTCCC sequences, which are consensus binding sites for transcription factors Sp1, CACC, CAT, and M-CAT, are outlined. C.S. indicates consensus sequence. B, The double-stranded oligonucleotide SMS80 was labeled by end-filling using Klenow in the presence of [{alpha}-32P]dCTP and was incubated with C2/2 cell nuclear extracts in the absence (-) or presence (+) of a 1000-fold molar excess of each unlabeled competitor, as indicated. The sequence-specific complexes (complexes 1 and 2) and the free probe are indicated by arrows. C, The double-stranded oligonucleotide SMS60 was end-labeled with [{alpha}-32P]dCTP and was incubated with C2/2 cell nuclear extracts in the absence (-) or presence (+) of a 1000-fold molar excess of each unlabeled competitor, as indicated. D, The double-stranded oligonucleotide SMS80 was end-labeled with [{alpha}-32P]dCTP and was incubated with COS7 cell nuclear extracts in the absence (-) or presence of (+) of a 1000-fold molar excess of each unlabeled competitor, as indicated. E, End-labeled, double-stranded oligonucleotides containing the Sp1 consensus sequence, SMS60, or SMS80 were labeled with [{alpha}-32P]dCTP and were incubated with C2/2 cell and COS7 cell nuclear extracts in the presence of antibody specific for either Sp1 (S) or {alpha}-actin (A).


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 {approx}14 kbp long and included the first exon and 5'-flanking region of the mouse SM1/2 gene promoter. Fig 1AUp 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 1BUp). 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 1CUp. 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 2Down, 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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  		Figure 2. RNase protection assays. SM1/2 mRNA levels were determined in a ribonuclease protection assay in C2/2 cells of either the log phase or the confluent phase. Probe is indicated by the arrowhead. The protected fragments, which are indicated by arrows, corresponding to the SM1 mRNA, were {approx}106 and 79 bp. The SM2 mRNA, whose estimated size is 224 bp, was not detected.

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{alpha} promoter and served as an internal control to standardize the transfection efficiency. The results are shown in Fig 3ADown. 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 3BDown). 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 {approx}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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  		Figure 3. The 5'-deletion analysis of the mouse SM1/2 promoter. A, The 5'-flanking sequence spanning from -3.4 kbp to +47 bp containing promoter sequences and a part of the first exon of the mouse SM1/2 gene was fused to the luciferase reporter gene in the PGVB vector (construct EVSC). Construct SCSC was generated by inserting the Sac I–Sac I restriction fragment spanning from -1226 to +47 bp into the Sac I site in the PGVB vector. The constructs DEL1, DEL2, DEL3, DEL4, DEL5, and DEL6 were generated from construct SCSC by deletions from its 5' end. Each construct was transiently transfected into C2/2 cells. The construct PGVC, which contains the promoter and enhancer of SV40, was assayed in parallel as a positive control for transfection. Promoter activities were estimated by luciferase values normalized to ß-galactosidase and are shown here by the relative values to the activity of the plasmid PGVC scaled to 1000. Transfections were performed in duplicate with at least two separate preparations of each plasmid. Values are mean± SEM. B, The constructs EVSC, SCSC, DEL1, DEL4, and DEL5 were transiently transfected into the primary cultures of rabbit aortic SMCs, NIH3T3 mouse fibroblasts, and COS7 monkey kidney epithelial cells. The construct PGVC was assayed in parallel as a positive control for transfection. Promoter activities were estimated by luciferase values normalized to ß-galactosidase and are shown here by the relative values to the activity of the plasmid PGVC scaled to 1000. Values correspond to the average of either two or three experiments.

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 4AUp). As shown in Fig 4BUp, 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 4AUp. Although both BM1 and BM4 could compete for the wild-type oligonucleotide SMS80, neither BM2 nor BM3 abolished the binding completely (Fig 4BUp). 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 4AUp). As shown in Fig 4CUp, 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 4CUp). 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 4AUp). 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 4BUp and 4CUp). 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 4EUp), 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 4DUp). 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 4EUp). 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 5Down). 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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  		Figure 5. Mutation of promoter elements of the SM1/2 gene. Mutations were introduced into the two CCTCCC boxes individually or in combination or into the consensus sequence for the GATA binding site. The DEL1 construct and the mutant constructs were transiently transfected into the C2/2 cells. The PGVC construct, which contains the promoter and enhancer of SV40, was assayed in parallel as a positive control for transfection. Promoter activities were estimated by luciferase values normalized to ß-galactosidase and are shown here by the relative values to the activity of the plasmid PGVC scaled to 1000. Transfections were performed in duplicate with at least two separate preparations of each plasmid. Values are mean±SEM.

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 5Up).

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 6Down, 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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  		Figure 6. The CCTCCC sequence on the heterologous promoter. BS-pro was constructed by inserting an SM1/2 promoter region between -44 and -104 bp immediately upstream from the truncated SV40 promoter–luciferase construct. Mutations of the CCTCCC sequence within the plasmid BS-pro yielded the plasmid DM-pro. These constructs were transiently transfected into the primary cultures of rabbit aortic SMCs, C2/2 cells, and COS7 cells. The construct pGL3 promoter vector was assayed in parallel as a control for transfection. Promoter activities were estimated by luciferase values normalized to ß-galactosidase and are shown here by the relative values to the activity of the pGL3 promoter vector scaled to 100. Transfections were performed in duplicate with at least two separate preparations of each plasmid. Values are mean±SEM.

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 7Down). 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 7BDown), 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 {alpha}-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 {alpha}-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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  		Figure 7. Sequence comparison of the SM1/2 promoter between rabbit and mouse. Nucleotides identical to rabbit SM1/2 are indicated by the asterisks, and gaps introduced to optimize the alignment are denoted by dashes. Two promoter regions, proximal (A) and distal (B) portions of the promoter, with conserved sequences between mouse and rabbit are shown. Numbers appearing in the left margin refer to the first nucleotide listed on that line. Numbers indicate the position of the nucleotide relative to the transcription start site, which is referred to as +1.


*    Discussion
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 *Discussion
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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{alpha} gene and smooth muscle {alpha}-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 {alpha}-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 {alpha}-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.


*    Selected Abbreviations and Acronyms
 
PCR = polymerase chain reaction
PTCA = percutaneous transluminal coronary angioplasty
SM = smooth muscle myosin heavy chain isoform
SMC = smooth muscle cell


*    Acknowledgments
 
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.


*    References
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  1. Chamley CJ, Campbell GR. What controls smooth muscle phenotype? Atherosclerosis. 1981;40:347-357. [Medline] [Order article via Infotrieve]
  2. Chamley CJ, Campbell GR, Ross R. Phenotype-dependent response of cultured aortic smooth muscle to serum mitogens. J Cell Biol. 1981;89:379-383. [Abstract/Free Full Text]
  3. Chamley CJ, Campbell GR, Ross R. The smooth muscle cell in culture. Physiol Rev. 1979;59:1-61. Review. [Free Full Text]
  4. Glukhova MA, Kabakov AE, Frid MG, Ornatsky OI, Belkin AM, Mukhin DN, Orekhov AN, Koteliansky VE, Smirnov VN. Modulation of human aorta smooth muscle cell phenotype: a study of muscle-specific variants of vinculin, caldesmon, and actin expression. Proc Natl Acad Sci U S A. 1988;85:9542-9546. [Abstract/Free Full Text]
  5. Solway J, Seltzer J, Samaha FF, Kim S, Alger LE, Niu Q, Morrisey EE, Ip HS, Parmacek MS. Structure and expression of a smooth muscle cell-specific gene, SM22{alpha}. J Biol Chem. 1995;270:13460-13469. [Abstract/Free Full Text]
  6. Frid MG, Shekhonin BV, Koteliansky VE, Glukhova MA. Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and calponin. Dev Biol. 1992;153:185-193. [Medline] [Order article via Infotrieve]
  7. Min BH, Foster DN, Strauch AR. The 5'-flanking region of the mouse vascular smooth muscle alpha-actin gene contains evolutionarily conserved sequence motifs within a functional promoter. J Biol Chem. 1990;265:16667-16675. [Abstract/Free Full Text]
  8. Foster DN, Min B, Foster LK, Stoflet ES, Sun S, Getz MJ, Strauch AR. Positive and negative cis-acting regulatory elements mediate expression of the mouse vascular smooth muscle alpha-actin gene. J Biol Chem. 1992;267:11995-12003. [Abstract/Free Full Text]
  9. Blank RS, McQuinn TC, Yin KC, Thompson MM, Takeyasu K, Schwartz RJ, Owens GK. Elements of the smooth muscle alpha-actin promoter required in cis for transcriptional activation in smooth muscle: evidence for cell type-specific regulation. J Biol Chem. 1992;267:984-989. [Abstract/Free Full Text]
  10. Ruzicka DL, Schwartz RJ. Sequential activation of alpha-actin genes during avian cardiogenesis: vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation. J Cell Biol. 1988;107:2575-2586. [Abstract/Free Full Text]
  11. Woodcock MJ, Mitchell JJ, Low RB, Kieny M, Sengel P, Rubbia L, Skalli O, Jackson B, Gabbiani G. Alpha-smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles. Differentiation. 1988;39:161-166. [Medline] [Order article via Infotrieve]
  12. Aikawa M, Sivam PN, Kuro-o M, Kimura K, Nakahara K, Takewaki S, Ueda M, Yamaguchi H, Yazaki Y, Periasamy M, Nagai R. Human smooth muscle myosin heavy chain isoforms as molecular markers for vascular development and atherosclerosis. Circ Res. 1993;73:1000-1012. [Abstract/Free Full Text]
  13. Kuro-o M, Nagai R, Tsuchimochi H, Katoh H, Yazaki Y, Ohkubo A, Takaku F. Developmentally regulated expression of vascular smooth muscle myosin heavy chain isoforms. J Biol Chem. 1989;264:18272-18275. [Abstract/Free Full Text]
  14. Nagai R, Kuro-o M, Babij P, Periasamy M. Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis. J Biol Chem. 1989;264:9734-9737. [Abstract/Free Full Text]
  15. Babij P, Periasamy M. Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing. J Mol Biol. 1989;210:673-679. [Medline] [Order article via Infotrieve]
  16. Kuro-o M, Nagai R, Nakahara K, Katoh H, Tsai RC, Tsuchimochi H, Yazaki Y, Ohkubo A, Takaku F. cDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in arteriosclerosis. J Biol Chem. 1991;266:3768-3773. [Abstract/Free Full Text]
  17. Nagai R, Larson DM, Periasamy M. Characterization of a mammalian smooth muscle myosin heavy chain cDNA clone and its expression in various smooth muscle types. Proc Natl Acad Sci U S A. 1988;85:1047-1051. [Abstract/Free Full Text]
  18. Kawamoto S, Adelstein RS. Chicken nonmuscle myosin heavy chains: differential expression of two mRNAs and evidence for two different polypeptides. J Cell Biol. 1991;112:915-924. [Abstract/Free Full Text]
  19. Katsuragawa Y, Yanagisawa M, Inoue A, Masaki T. Two distinct nonmuscle myosin-heavy-chain mRNAs are differentially expressed in various chicken tissues: identification of a novel gene family of vertebrate non-sarcomeric myosin heavy chains. Eur J Biochem. 1989;184:611-616. [Medline] [Order article via Infotrieve]
  20. Compton J. Nucleic acid sequence-based amplification. Nature. 1991;350:91-92. [Medline] [Order article via Infotrieve]
  21. Isegawa Y, Sheng J, Sokawa Y, Yamanishi K, Nakagomi O, Ueda S. Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): application to sequencing 6.5 kb genome segment of hantavirus strain B-1. Mol Cell Probes. 1992;6:467-475. [Medline] [Order article via Infotrieve]
  22. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]
  23. Sasaki Y, Uchida T, Sasaki Y. A variant derived from rabbit aortic smooth muscle: phenotype modulation and restoration of smooth muscle characteristics in cells in culture. J Biochem. 1989;106:1009-1018. [Abstract/Free Full Text]
  24. Higuchi R, Krummel B, Saiki RK. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988;16:7351-7367. [Abstract/Free Full Text]
  25. Campbell GR, Campbell JH. Smooth muscle phenotypic changes in arterial wall homeostasis: implications for the pathogenesis of atherosclerosis. Exp Mol Pathol. 1985;42:139-162. Review. [Medline] [Order article via Infotrieve]
  26. Campbell JH, Reardon MF, Campbell GR, Nestel PJ. Metabolism of atherogenic lipoproteins by smooth muscle cells of different phenotype in culture. Arteriosclerosis. 1985;5:318-328. [Abstract/Free Full Text]
  27. Campbell JH, Campbell GR. Endothelial cell influences on vascular smooth muscle phenotype. Annu Rev Physiol. 1986;48:295-306. Review. [Medline] [Order article via Infotrieve]
  28. Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987;84:7413-7417. [Abstract/Free Full Text]
  29. Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987;7:2745-2752. [Abstract/Free Full Text]
  30. Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983;11:1475-1489. [Abstract/Free Full Text]
  31. Parmacek MS, Ip HS, Jung F, Shen T, Martin JF, Vora AJ, Olson EN, Leiden JM. A novel myogenic regulatory circuit controls slow/cardiac troponin C gene transcription in skeletal muscle. Mol Cell Biol. 1994;14:1870-1885. [Abstract/Free Full Text]
  32. Briggs MR, Kadonaga JT, Bell SP, Tjian R. Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science. 1986;234:47-52. [Abstract/Free Full Text]
  33. Dorn A, Bollekens J, Staub A, Benoist C, Mathis D. A multiplicity of CCAAT box-binding proteins. Cell. 1987;50:863-872. [Medline] [Order article via Infotrieve]
  34. Mar JH, Ordahl CP. M-CAT binding factor, a novel trans-acting factor governing muscle-specific transcription. Mol Cell Biol. 1990;10:4271-4283. [Abstract/Free Full Text]
  35. Orkin SH. GATA-binding transcription factors in hematopoietic cells. Blood. 1992;80:575-581. Review. [Free Full Text]
  36. Laverriere AC, MacNeill C, Mueller C, Poelmann RE, Burch JB, Evans T. GATA-4/5/6, a subfamily of three transcription factors transcribed in developing heart and gut. J Biol Chem. 1994;269:23177-23184. [Abstract/Free Full Text]
  37. Katoh Y, Loukianov E, Kopras E, Zilberman A, Periasamy M. Identification of functional promoter elements in the rabbit smooth muscle myosin heavy chain gene. J Biol Chem. 1994;269:30538-30545. [Abstract/Free Full Text]
  38. Kulozik AE, Bellan KA, Bail S, Kohne E, Kleihauer E. Thalassemia intermedia: moderate reduction of beta globin gene transcriptional activity by a novel mutation of the proximal CACCC promoter element. Blood. 1991;77:2054-2058. [Abstract/Free Full Text]
  39. Miller IJ, Bieker JJ. A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Kruppel family of nuclear proteins. Mol Cell Biol. 1993;13:2776-2786. [Abstract/Free Full Text]
  40. Perkins AC, Sharpe AH, Orkins SH. Lethal ß-thalassaemia in mice lacking the erythroid CACCC-transcription factor EKLF. Nature. 1995;375:318-322. [Medline] [Order article via Infotrieve]
  41. Bassel-Duby R, Hernandez MD, Gonzalez MA, Krueger JK, Williams RS. A 40-kilodalton protein binds specifically to an upstream sequence element essential for muscle-specific transcription of the human myoglobin promoter. Mol Cell Biol. 1992;12:5024-5032. [Abstract/Free Full Text]
  42. Wang Y, Kobori JA, Hood L. The ht beta gene encodes a novel CACCC box-binding protein that regulates T-cell receptor gene expression. Mol Cell Biol. 1993;13:5691-5701. [Abstract/Free Full Text]
  43. Reddy S, Ozgur K, Lu M, Chang W, Mohan SR, Kumar CC, Ruley HE. Structure of the human smooth muscle alpha-actin gene: analysis of a cDNA and 5' upstream region. J Biol Chem. 1990;265:1683-1687. [Abstract/Free Full Text]
  44. Carroll SL, Bergsma DJ, Schwartz RJ. Structure and complete nucleotide sequence of the chicken alpha-smooth muscle (aortic) actin gene: an actin gene which produces multiple messenger RNAs. J Biol Chem. 1986;261:8965-8976. [Abstract/Free Full Text]
  45. Sartorelli V, Webster KA, Kedes L. Muscle-specific expression of the cardiac alpha-actin gene requires MyoD1, CArG-box binding factor, and Sp1. Genes Dev. 1990;4:1811-1822. [Abstract/Free Full Text]
  46. Lin H, Yutzey KE, Konieczny SF. Muscle-specific expression of the troponin I gene requires interactions between helix-loop-helix muscle regulatory factors and ubiquitous transcription factors. Mol Cell Biol. 1991;11:267-280. [Abstract/Free Full Text]
  47. Simon AM, Burden SJ. An E box mediates activation and repression of the acetylcholine receptor delta-subunit gene during myogenesis. Mol Cell Biol. 1993;13:5133-5140. [Abstract/Free Full Text]
  48. Kim SJ, Lee HD, Robbins PD, Busam K, Sporn MB, Roberts AB. Regulation of transforming growth factor beta 1 gene expression by the product of the retinoblastoma-susceptibility gene. Proc Natl Acad Sci U S A. 1991;88:3052-3056.[Abstract/Free Full Text]



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