Interaction of CArG Elements and a GC-rich Repressor Element in Transcriptional Regulation of the Smooth Muscle Myosin Heavy Chain Gene in Vascular Smooth Muscle Cells
1997; Elsevier BV; Volume: 272; Issue: 47 Linguagem: Inglês
10.1074/jbc.272.47.29842
ISSN1083-351X
AutoresCort S. Madsen, Christopher P. Regan, Gary K. Owens,
Tópico(s)RNA modifications and cancer
ResumoWe have previously shown that maximal expression of the rat smooth muscle myosin heavy chain (SM-MHC) gene in cultured rat aortic smooth muscle cells (SMCs) required the presence of a highly conserved domain (nucleotides −1321 and −1095) that contained two positive-acting serum response factor (SRF) binding elements (CArG boxes 1 and 2) and a negative-acting GC-rich element that was recognized by Sp1 (Madsen, C. S., Hershey, J. C., Hautmann, M. B., White, S. L., and Owens, G. K. (1997)J. Biol. Chem. 272, 6332–6340). In this study, to better understand the functional role of these three ciselements, we created a series of SM-MHC reporter-gene constructs in which each element was mutated either alone or in combination with each other and tested them for activity in transient transfection assays using primary cultured rat aortic SMCs. Results demonstrated that the most proximal SRF binding element (CArG-box1) was active in the absence of CArG-box2, but only upon removal of the GC-rich repressor. In contrast, regardless of sequence context, CArG-box2 was active only when CArG-box1 was present. We further demonstrated using electrophoretic mobility shift assays that Sp1 binding to the GC-rich repressor element did not prevent SRF binding to the adjacent CArG-box2. Thus, unlike other proteins reported to inhibit SRF activity, the repressor activity associated with the GC-rich element does not appear to function through direct inhibition of SRF binding. As a first step toward understanding the importance of these elementsin vivo, we performed in vivo footprinting on the intact rat aorta. We demonstrated that both CArG boxes and the GC-rich element were bound by protein within the animal. Additionally, using the rat carotid injury model we showed that Sp1 protein was significantly increased in SMCs located within the myointimal lesion, suggesting that increased expression of this putative repressor factor may contribute to the decreased SM MHC expression within SMCs found in myointimal lesions. We have previously shown that maximal expression of the rat smooth muscle myosin heavy chain (SM-MHC) gene in cultured rat aortic smooth muscle cells (SMCs) required the presence of a highly conserved domain (nucleotides −1321 and −1095) that contained two positive-acting serum response factor (SRF) binding elements (CArG boxes 1 and 2) and a negative-acting GC-rich element that was recognized by Sp1 (Madsen, C. S., Hershey, J. C., Hautmann, M. B., White, S. L., and Owens, G. K. (1997)J. Biol. Chem. 272, 6332–6340). In this study, to better understand the functional role of these three ciselements, we created a series of SM-MHC reporter-gene constructs in which each element was mutated either alone or in combination with each other and tested them for activity in transient transfection assays using primary cultured rat aortic SMCs. Results demonstrated that the most proximal SRF binding element (CArG-box1) was active in the absence of CArG-box2, but only upon removal of the GC-rich repressor. In contrast, regardless of sequence context, CArG-box2 was active only when CArG-box1 was present. We further demonstrated using electrophoretic mobility shift assays that Sp1 binding to the GC-rich repressor element did not prevent SRF binding to the adjacent CArG-box2. Thus, unlike other proteins reported to inhibit SRF activity, the repressor activity associated with the GC-rich element does not appear to function through direct inhibition of SRF binding. As a first step toward understanding the importance of these elementsin vivo, we performed in vivo footprinting on the intact rat aorta. We demonstrated that both CArG boxes and the GC-rich element were bound by protein within the animal. Additionally, using the rat carotid injury model we showed that Sp1 protein was significantly increased in SMCs located within the myointimal lesion, suggesting that increased expression of this putative repressor factor may contribute to the decreased SM MHC expression within SMCs found in myointimal lesions. Vascular lesions resulting from either atherosclerotic disease or restenosis are characterized by the significant presence of SMCs 1The abbreviations used are: SMC, smooth muscle cell; SM-MHC, smooth muscle myocin heavy chain; SRF, serum response factor; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; PBS, phosphate-buffered saline; DMS, dimethyl sulfate; TE, Tris-EDTA buffer; LMPCR, ligation-mediated polymerase chain reaction; PCR, polymerase chain reaction; bp, base pair(s). that are phenotypically distinct from fully differentiated medial SMCs (2Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9990) Google Scholar, 3Kocher O. Gabbiani G. Hum. Pathol. 1986; 17: 875-880Crossref PubMed Scopus (124) Google Scholar, 4Kocher O. Gabbiani F. Gabbiani G. Reidy M.A. Cokay M.S. Peters H. Huttner I. Lab. Invest. 1991; 65: 459-470PubMed Google Scholar). Major modifications include decreased expression of smooth muscle isoforms of contractile proteins, increased matrix production, altered growth regulatory properties, altred lipid metabolism, and decreased contractility (5Owens G.K. Physiol. Rev. 1995; 75: 487-517Crossref PubMed Scopus (1397) Google Scholar). The process by which SMCs undergo such changes is referred to as "phenotypic modulation" (6Chamley-Campbell J.H. Campbell G.R. Atherosclerosis. 1981; 40: 347-357Abstract Full Text PDF PubMed Scopus (258) Google Scholar). Importantly, these alterations in SMC protein expression patterns cannot simply be viewed as a consequence of vascular disease, but rather are likely to contribute to its progression. The identification of the molecular mechanisms that regulate protein expression in the normal SMC should provide for a better understanding of how these processes are altered in phenotypically modified SMCs. SM-MHC represents one of the best studied markers of the SMC (5Owens G.K. Physiol. Rev. 1995; 75: 487-517Crossref PubMed Scopus (1397) Google Scholar). Four SM-MHC isoforms (SM-1A, SM-1B, SM-2A, and SM-2B) have been identified (7Nagai R. Kuro-o M. Babij P. Periasamy M. J. Biol. Chem. 1989; 264: 9734-9737Abstract Full Text PDF PubMed Google Scholar, 8White S. Martin A.F. Periasamy M. Am. J. Physiol. 1993; 264: C1252-C1258Crossref PubMed Google Scholar, 9Kelley C.A. Adelstein R.S. Can. J. Physiol. Pharmacol. 1994; 72: 1351-1360Crossref PubMed Scopus (16) Google Scholar), all of which are derived from alternative splicing of a single gene product (10Babij P. Periasamy M. J. Mol. Biol. 1989; 210: 673-679Crossref PubMed Scopus (154) Google Scholar), that is exclusively expressed in SMCs (11Miano J. Cserjesi P. Ligon K. Periasamy M. Olson E.N. Circ. Res. 1994; 75: 803-812Crossref PubMed Scopus (320) Google Scholar). Expression of the SM-MHC gene has been shown to be regulated in a manner that exquisitely "marks" the differentiated state of the SMC both during normal vascular development and in vascular disease. In humans and rabbit, SM-1 is expressed during early fetal development with SM-2 appearing only after birth in what is thought to be the fully differentiated or mature SMC (12Aikawa M. Sivam P.N. Kuro Kimura K. Nakahara K. Takewaki S. Ueda M. Yamaguchi H. Yazaki Y. Periasamy M. Circ. Res. 1993; 73: 1000-1012Crossref PubMed Scopus (308) Google Scholar, 13Frid M.G. Printesva O.Y. Chiavegato A. Faggin E. Scatena M. Koteliansky V.E. Pauletto P. Glukhova M.A. Sartore S. J. Vasc. Res. 1993; 30: 279-292Crossref PubMed Scopus (71) Google Scholar, 14Kuro-o M. Nagai R. Tsuchimochi H. Katoh H. Yazaki Y. Ohkubo A. Takaku F. J. Biol. Chem. 1989; 264: 18272-18275Abstract Full Text PDF PubMed Google Scholar). Moreover, several studies have shown that SM-MHC expression is significantly decreased in vascular lesions (12Aikawa M. Sivam P.N. Kuro Kimura K. Nakahara K. Takewaki S. Ueda M. Yamaguchi H. Yazaki Y. Periasamy M. Circ. Res. 1993; 73: 1000-1012Crossref PubMed Scopus (308) Google Scholar, 15Holifield B. Helgason T. Jemelka S. Taylor A. Navran S. Allen J. Seidel C. J. Clin. Invest. 1996; 97: 814-825Crossref PubMed Scopus (100) Google Scholar), and the loss of this marker protein has been used to evaluate the differentiated state of the SMC as related to vascular disease progression (16Aikawa M. Kim H.S. Kuro-o M. Manabe I. Watanabe M. Yamaguchi H. Yazaki Y. Nagai R. Ann. N. Y. Acad. Sci. 1995; 748: 578-585Crossref PubMed Scopus (21) Google Scholar, 17Suzuki J. Aikawa M. Isobe M. Sekiguchi M. Yazaki Y. Nagai R. Transplant. Proc. 1995; 27: 578PubMed Google Scholar). Thus, SM-MHC represents an excellent gene for delineating the transcriptional regulatory mechanisms that define the differentiated state of the SMC. The 5′-flanking regions of SM-MHC genes cloned from rat (18White S.L. Low R.B. J. Biol. Chem. 1996; 271: 15008-15017Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), rabbit (19Katoh Y. Loukianov E. Kopras E. Zilberman A. Periasamy M. J. Biol. Chem. 1994; 269: 30538-30545Abstract Full Text PDF PubMed Google Scholar), and mouse (20Wantanabe M. Sakomura Y. Kurabayashi M. Manabe I. Aikawa M. Kuro-o M. Suzuki T. Yazaki Y. Nagai R. Circ. Res. 1996; 78: 978-989Crossref PubMed Scopus (34) Google Scholar), all contain a highly conserved 227-bp domain located between nucleotides −1321 and −1095 in rat (1Madsen C.S. Hershey J.C. Hautmann M.B. White S.L. Owens G.K. J. Biol. Chem. 1997; 272: 6332-6340Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In a recent study, we demonstrated that this domain was required for maximal SM-MHC promoter activity in vascular SMCs, and that present within this domain were two positive-acting CArG boxes that were specifically recognized by SRF, and a negative-acting GC-rich element that was recognized by Sp1 (1Madsen C.S. Hershey J.C. Hautmann M.B. White S.L. Owens G.K. J. Biol. Chem. 1997; 272: 6332-6340Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In a separate investigation of the rabbit SM-MHC promoter, transient transfection studies and analysis of protein-DNA interactions revealed that an approximately 100-bp segment of the conserved domain, located just upstream of CArG-box2, contained multiple enhancer elements each interacting with unidentified factors, two of which were thought to be SM-specific (21Kallmeier R.C. Somasundaram C. Babij P. J. Biol. Chem. 1995; 270: 30949-30957Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). An additional study of rat SM-MHC promoter activity in a variety of cell types, revealed that the region spanning nucleotides −1317 to −1249 was important for the restriction of gene expression to SMCs (18White S.L. Low R.B. J. Biol. Chem. 1996; 271: 15008-15017Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Thus, this very complex and highly active domain appears to contain a multitude of cis elements that interact with multiple factors, of which only a few have been putatively identified. In addition to this conserved 227-bp domain, two tandemly oriented CCTCCC elements, located proximal to the transcriptional start site, have been identified and also shown to be important for SM-MHC expression (20Wantanabe M. Sakomura Y. Kurabayashi M. Manabe I. Aikawa M. Kuro-o M. Suzuki T. Yazaki Y. Nagai R. Circ. Res. 1996; 78: 978-989Crossref PubMed Scopus (34) Google Scholar). The CArG box motif has been demonstrated to be important for expression of multiple SM-specific genes including SM-MHC (1Madsen C.S. Hershey J.C. Hautmann M.B. White S.L. Owens G.K. J. Biol. Chem. 1997; 272: 6332-6340Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), SM α-actin (22Shimizu R.T. Blank R.S. Jervis R. Lawrenz-Smith S.C. Owens G.K. J. Biol. Chem. 1995; 270: 7631-7643Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), SM-22α (23Kim S. Ip H.S. Lu M.M. Clendenin C. Parmacek M.S. Mol. Cell. Biol. 1997; 17: 2266-2278Crossref PubMed Scopus (190) Google Scholar), and telokin (24Herring B.P. Smith A.F. Am. J. Physiol. 1997; 41: C1394-C1404Crossref Google Scholar). Thus, a better understanding of CArG-dependent regulation for any one of these genes could lead to the identification of an important common transcriptional mechanism. In the present study, we generated a series of combinatorial mutants to better understand how CArG boxes 1 and 2 functioned within the SM-MHC promoter in vascular SMCs and how a GC-rich element, which is located between the two CArGs and which is specifically recognized by Sp1, influenced their activity. These data revealed that, upon removal of the GC-rich repressor, CArG-box1 could sustain activity in the absence of CArG-box2. In view of the fact that both CArGs in the SM α-actin promoter were absolutely required for activity (22Shimizu R.T. Blank R.S. Jervis R. Lawrenz-Smith S.C. Owens G.K. J. Biol. Chem. 1995; 270: 7631-7643Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), this finding suggests that activation through the CArG motif may involve different gene-specific mechanisms. Furthermore, unlike other factors shown to inhibit SRF activity, gel-shift analysis of Sp1 and SRF binding to the juxtaposed GC-rich repressor and CArG-box2 elements, revealed that the mechanism of repression was not likely to be mediated through direct inhibition of SRF binding. To assess their likely importance for SM-MHC expression in the fully differentiated vascular SMC, we performed in vivo footprinting on the intact rat aorta to demonstrate that protein-DNA interaction occurred at both CArG boxes and the GC-rich repressor element. Finally, we found that Sp1 presence was increased in myointimal SMCs in a rat carotid injury model, suggesting that the GC-rich repressor element may contribute to reduced SM-MHC protein expression in dedifferentiated myointimal SMCs. The cloning, determination of the +1 start site, and sequencing of the rat SM-MHC promoter have been previously described (1Madsen C.S. Hershey J.C. Hautmann M.B. White S.L. Owens G.K. J. Biol. Chem. 1997; 272: 6332-6340Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 18White S.L. Low R.B. J. Biol. Chem. 1996; 271: 15008-15017Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The construction of pCAT-1346, pCAT-1182, and pCAT-1102 promoter-chloramphenicol reporter plasmids containing progressive deletions of rat SM-MHC 5′-flanking DNA was described previously (1Madsen C.S. Hershey J.C. Hautmann M.B. White S.L. Owens G.K. J. Biol. Chem. 1997; 272: 6332-6340Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Site-directed mutagenesis of the pCAT-1346 construct was performed using the Ex-site mutagenesis kit according to the manufacturer's instructions (Stratagene). The integrity of each mutated construct was determined by dideoxy sequencing (25Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52678) Google Scholar). SMCs from rat thoracic aorta were isolated and cultured under conditions that result in retention of expression of multiple SMC differentiation marker proteins including SM1 (26Geisterfer A.A. Peach M.J. Owens G.K. Circ. Res. 1988; 62: 749-756Crossref PubMed Scopus (1011) Google Scholar). SMCs (passage 10–22) were seeded for transient transfection assays into six-well plates at a density of 2 × 104. These densities were chosen so that the cells would be at 70–80% confluence at the time of transfection (24 h after plating). Transfections of the CAT-reporter gene constructs, subsequent growth conditions of the SMCs, and determination of CAT activities, were all done as described previously (1Madsen C.S. Hershey J.C. Hautmann M.B. White S.L. Owens G.K. J. Biol. Chem. 1997; 272: 6332-6340Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The promoterless-pCAT construct was transfected into triplicate wells to serve as the base-line indicator of CAT activity, and the activity of each promoter construct is expressed relative to the promoterless construct set to one. Additionally, a SV40 promoter-CAT construct with enhancer (Promega) served as a positive control of transfection and CAT activity. All SMC CAT activity values represent at least three independent experiments with each construct tested in triplicate per experiment. Relative CAT activity data are expressed as the means ± S.D. computed from the results obtained from each set of transfection experiments. One-way analysis of variance followed by the Newman-Keuls's multiple range test were used for data analysis. Values of p < 0.05 were considered statistically significant. Recombinant SRF was generated using anin vitro transcription and translation kit (Stratagene) as described previously (27Hautmann M.B. Madsen C.S. Owens G.K. J. Biol. Chem. 1997; 272: 10948-10956Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Oligonucleotide affinity-column purified Sp1 was obtained from a commercial source (Santa Cruz). The oligonucleotides used for EMSAs were purchased commercially (Operon Technologies, Inc.) and included the following: C2 oligo, 5′-cctggcctttttgggttgtt-3′; GC oligo, 5′-ggttgtttcccgcccaggcc-3′, and C2 + GC oligo, 5′-cctggcctttttgggttgttcccgcccaggcc-3′. EMSAs were performed with 20 μl of binding reaction that included ∼30 pg of32P-end-labeled annealed oligonucleotides, SRF and Sp1 as indicated, and 0.25 μg of poly(dI-dC) in 1 × binding buffer (12 mm HEPES (pH 7.9), 100 mm KCl, 5 mmMgCl2, 4 mm Tris-HCl (pH 7.5), 0.6 mm EDTA, 0.6 mm dithiothreitol, and 10% glycerol). Following a 20-min incubation at room temperature, the samples were subjected to electrophoreses on a 5% polyacrylamide gel, which had been pre-run at 170 V for 1 h. Electrophoresis was performed at 170 V in 0.5 × 0.45 mm Tris borate and 1 mm EDTA. Gels were dried and exposed to film for 24–48 h. The anti-Sp1 and anti-SRF antibodies used for EMSA supershift experiments were all purchased commercially (Santa Cruz). EMSA binding reactions were set up as described above and incubated for 20 min; 2 μl of the antibody were added to the mixture; and the reaction was incubated for another 15 min at room temperature and then loaded onto the gel for electrophoresis. The thoracic aortas from eight adult Sprague-Dawley rats were dissected individually from each animal and immediately placed in 5 ml of 37 °C phosphate-buffered saline (PBS) containing 0.5% dimethyl sulfate (DMS). The aortas were incubated for 2 min with constant gentle shaking and rinsed three times in 50 ml of ice-cold PBS containing 50 mm Tris-HCl (pH 8.0). Aortas were stored in this same solution until all dissections were completed. The adventitial and endothelial layers of the aortae were mechanically removed to minimize contribution of non-SMC DNA in the subsequent polymerase chain reactions. The aortae were then cut into small pieces, pooled, and placed in a lysis solution containing 50 mm NaCl, 50 mm Tris-HCl (pH 8.0), 25 mm EDTA (pH 8.0), 0.5% sodium dodecyl sulfate, 0.3 mg of proteinase K per ml. Following an overnight room temperature incubation, chromosomal DNA was isolated by extraction with phenol (pH 8.0) (twice), phenol-chloroform-isoamyl alcohol (twice), and chloroform (once) with gently mixing by inversion. After adjusting the NaCl concentration to 0.2 m, the DNA was precipitated with the addition of 2.5 volumes of ethanol, and the precipitate was transferred to 0.5 ml of Tris-EDTA buffer (TE) in a new tube, redissolved, and precipitated again with ethanol. The precipitate was dissolved in 0.4 ml of TE, and the concentration was measured by optical density at 260 nm. For the in vitro methylated controls, 100 μg of Sprague-Dawley rat kidney DNA was extensively deproteinized as described above with phenol and chloroform, modified with 0.5% DMS, and precipitated with ice-cold DMS stop solution. A plasmid containing the SM-MHC promoter (pCAT-1346) was also subjected to DMS treatment and used as an additional in vitro control and also for sequence verification. An approximate equivalent amount ofin vivo methylated DNA was also precipitated with DMS stop solution. The samples were dissolved in 180 μl of H2O, adjusted to a concentration of 10% piperidine, and incubated at 90 °C for 30 min. The reaction products were cooled on ice and precipitated with the addition of 0.2 mm sodium acetate (pH 7.0) and ethanol. After two more rounds of ethanol precipitation, the samples were dried overnight, precipitated again, and resuspended in TE to 0.4 mg/ml. The ligation-mediated polymerase chain reaction (LMPCR) performed on the DMS-treated DNA samples was as described previously (28Mueller P.R. Wold B. Science. 1989; 246: 780-786Crossref PubMed Scopus (798) Google Scholar), with minor exceptions. First strand synthesis and subsequent PCRs were performed with Taq polymerase using the reaction buffer supplied by the manufacturer (Perkin-Elmer Corp.). Following the first strand synthesis step and prior to the ligation reaction, samples were extracted with phenol-chloroform and precipitated with ethanol. This added extraction step seemed to increase ligation efficiency. Three sets of oligonucleotide primers (nine total) were utilized to analyze the CArG-box2-, GC-rich-, and CArG-box1-containing regions of the 227-bp conserved element. These were as follows: MHCP1, 5′-gaatcccttttctctaagga-3′; MHCP2, 5′-ttctctaaggacgcaggatctgggt-3′; MHCP3, 5′-aggatctgggtggctgcagggagcgagg-3′; MHCP4, 5′-aggcagagttggcctcatg-3′; MHCP5, 5′-caggccgggggacgcctcgctc-3′; MHCP6, 5′-gacgcctcgctccccgcgcacggac-3′; MHCP7, 5′-agggaggaccagctcagga-3′; MHCP8, 5′-ctcaggacctcgagggtccgtgcg-3′; MHCP9, 5′-ctcgagggtccgtgcgcggggagcga-3′. To evaluate the potential for reaction variability, multiple LMPCRs were performed on the in vivo aortic, in vitrocontrol kidney, and SM-MHC plasmid DNAs. All samples were subjected to electrophoresis on 6% sequencing gels along with an additional32P-end-labeled molecular weight ladder (Life Technologies, Inc.) and exposed to film. We observed that band intensities were highly consistent between individual LMPCRs, and that the in vitro control kidney DNA and the SM-MHC plasmid DNA both yielded essentially identical patterns of methylation reactivity (not shown). Quantitation and identification of protected or hypermethylated guanine residues was as described previously (29Ghivizzani S.C. Madsen C.S. Hauswirth W.W. J. Biol. Chem. 1993; 268: 8675-8682Abstract Full Text PDF PubMed Google Scholar). Rat carotid arteries injured by balloon catheterization were prepared by Zivic-Miller Laboratories. Fourteen days after injury the left injured and the right uninjured carotid vessels were perfusion fixed with 4% paraformaldehyde and processed for routine histology. Six-mm sections were cut and placed on ProBond slides (Fisher). Paraffin-embedded sections were deparaffinized with xylene (three changes, 10 min each) and rehydrated through a graded ethanol series of 100, 95, and 70% (two changes each at 3 min per change). Endogenous peroxidase activity was blocked by treating with 0.3% H2O2 in methanol for 30 min at room temperature. Blocking of nonspecific binding was accomplished with a 1-h incubation in 2.0% goat serum and 3% bovine serum albumin in PBS. The slides were incubated overnight in the primary anti-Sp1 antibody (Santa Cruz) diluted 1:1000 in PBS. Binding of the primary antibody was detected with a biotinylated goat anti-rabbit antibody that was subsequently detected with an avidin-biotin complex with horseradish peroxidase (Vectostain Elite ABC kit, Vector Laboratories) using diaminobenzinide tetrahydrochloride as the peroxidase substrate. The SM-MHC, SM-22α, and SM α-actin promoters all contain a set of paired CArG boxes each of which has been demonstrated to specifically bind SRF (1Madsen C.S. Hershey J.C. Hautmann M.B. White S.L. Owens G.K. J. Biol. Chem. 1997; 272: 6332-6340Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 22Shimizu R.T. Blank R.S. Jervis R. Lawrenz-Smith S.C. Owens G.K. J. Biol. Chem. 1995; 270: 7631-7643Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 23Kim S. Ip H.S. Lu M.M. Clendenin C. Parmacek M.S. Mol. Cell. Biol. 1997; 17: 2266-2278Crossref PubMed Scopus (190) Google Scholar). For all three promoters, both CArG elements were shown to be required for maximal transcriptional activity in cultured vascular SMCs. However, in contrast to the SM-MHC and SM α-actin promoters where mutation of either CArG element resulted in complete abrogation of all CArG-dependent activity, the most proximal CArG box in the SM-22α promoter was capable of sustaining activity on its own, albeit at a reduced level. Thus, although the transcriptional mechanisms that utilize this dual-CArG motif are likely to have several features in common, other gene-specific differences may also exist. To better understand CArG-dependent regulation of the SM-MHC gene and to gain insight regarding the role of the GC-rich repressor element, we analyzed the activity of these elements in a variety of sequence contexts. From our previous deletion analysis (1Madsen C.S. Hershey J.C. Hautmann M.B. White S.L. Owens G.K. J. Biol. Chem. 1997; 272: 6332-6340Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), we determined that a SM-MHC reporter-gene construct containing nucleotides −1346 to +88 (pCAT-1346), that included the entire 227-bp conserved domain, was sufficient for maximal transcriptional activity in vascular SMCs. Using this pCAT-1346 construct as a template, both CArG boxes and the GC-rich repressor element were mutated either alone or in combination with each other, and tested for alterations of transcriptional activity in transient transfection assays using cultured vascular SMCs (Fig. 1). For comparative purposes wild-type deletion constructs pCAT-1102, pCAT-1182, and pCAT-1346 were also included in the analysis. For ease of reference we refer to activity levels at or below 20-fold over promoterless pCAT (the level of pCAT-1102 construct which does not contain the 227-bp domain) as minimal. The activity levels of pCAT-CArG1Mut, pCAT-CArG2MutA, and pCAT-GC-richMutA were previously described (1Madsen C.S. Hershey J.C. Hautmann M.B. White S.L. Owens G.K. J. Biol. Chem. 1997; 272: 6332-6340Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), however, because CArG-box2 (CCTTTTTGGG) does not conform to the classic CArG consensus site (CC(A/T)6GG) (30Wynne J. Treisman R. Nucleic Acids Res. 1992; 20: 3297-3303Crossref PubMed Scopus (76) Google Scholar), we generated a different mutation within this element (pCAT-CArG2MutB) that was previously shown to be important for SRF binding (22Shimizu R.T. Blank R.S. Jervis R. Lawrenz-Smith S.C. Owens G.K. J. Biol. Chem. 1995; 270: 7631-7643Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 31Walsh K. Mol. Cell. Biol. 1989; 9: 2191-2201Crossref PubMed Scopus (80) Google Scholar). Also for verification purposes, we created an additional mutant construct that targeted the GC-rich repressor element (pCAT-GCrichMutB) at nucleotide positions known to be required for Sp1 binding (32Courey A.J. Tjian R. McKnight S.L. Yamamoto K.R. Transcriptional Regulation. Cold Spring Harbor Laboratory Press, Plainview, NY1992: 743-769Google Scholar). As shown in Fig. 1 the activity levels of the pCAT-CArG2MutA and pCAT-CArG2MutB constructs were approximately equal (20- and 17-fold over promoterless pCAT, respectively). This similar decrease in activity levels suggests that both mutations equally inhibited binding of the same positive acting factor, most likely SRF. Likewise, the similar increases in activity levels observed for pCAT-GCrichMutA (87-fold over promoterless pCAT) and pCAT-GCrichMutB (94-fold over promoterless pCAT), indicated the abrogation of binding of a common negative-acting factor, potentially Sp1 or a related family member. Based on the juxtapositioning of the GC-rich repressor element and CArG-box2, we originally hypothesized that the primary mechanism of repression was likely to be mediated through factor binding to the GC-rich sequence leading to inhibition of SRF binding to CArG-box2. However, the combinatorial mutation data strongly suggests that the presence of the GC-rich element can also negatively regulate CArG-box1 activity. As shown in Fig. 1, the activity of pCAT-1182 which contains CArG-box1 was greater than that of pCAT-1102 (34- versus20-fold over promoterless pCAT). This finding suggested that CArG-box1 contained within the pCAT-1182 construct was capable of enhancing transcription above the 20-fold minimal level in the absence of CArG-box2. However, when CArG-box2 was mutated (pCAT-CArG2MutA and -MutB) within the context of the pCAT-1346 construct, transcriptional activity decreased to the 20-fold level of pCAT-1102 and not the expected 34-fold level observed for the pCAT-1182 construct, even though CArG-box1 remained intact. These data suggested that a repressor element located between nucleotides −1182 and −1346 negatively influenced CArG-box1 activity. To test if the previously identified GC-rich repressor element was responsible for the putative decrease in CArG-box1 activity, we generated a construct in which both CArG-box2 and the GC-rich repressor element were mutated (pCAT-CArG2/GC-richMut). Mutation of CArG-box2 along with the GC-rich element allowed us to rule out the possibility that any noted increases in activity could be due to alleviation of repressor effects on CArG-box2 activity. The activity level of this construct was determined to be 35-fold over that of promoterless-pCAT, essentially identical to the 34-fold activity level of the pCAT-1182 construct. Activity returned to a minimal level when CArG-box1 was mutated along with the CArG-box2 and GC-rich elements (pCAT-CArG1/CArG2/GC-richMut). These data strongly suggest that the GC-rich element, located greater
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