Structure and characterization of the 5'-flanking region of the mouse smooth muscle myosin heavy chain (SM1/2) gene

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 a...

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Bibliographic Details
Published in:Circulation research 1996-06, Vol.78 (6), p.978-989
Main Authors: WATANABE, M, SAKOMURA, Y, KURABAYASHI, M, MANABE, I, AIKAWA, M, KURO-O, M, SUZUKI, T, YAZAKI, Y, NAGAI, R
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Biological and medical sciences
Blood vessels and receptors
Cells, Cultured
Fundamental and applied biological sciences. Psychology
Mice
Molecular Sequence Data
Muscle, Smooth - chemistry
Mutagenesis, Site-Directed
Myosin Heavy Chains - genetics
Promoter Regions, Genetic
Rabbits
Sp1 Transcription Factor - physiology
Vertebrates: cardiovascular system
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Summary: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.
ISSN:0009-7330
1524-4571
DOI:10.1161/01.res.78.6.978