Structure and Characterization of the 5′-Flanking Region of the Mouse Smooth Muscle Myosin Heavy Chain (SM1/2) Gene

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

Research output: Contribution to journalArticle

34 Scopus citations

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 SMl 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.

Original languageEnglish (US)
Pages (from-to)978-989
Number of pages12
JournalCirculation research
Volume78
Issue number6
DOIs
StatePublished - Jun 1996

Keywords

  • CACC
  • Myosin heavy chain
  • SM1/2
  • Smooth muscle
  • Sp1

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

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    Watanabe, M., Sakomura, Y., Kurabayashi, M., Manabe, I., Aikawa, M., Kuro-o, M., Suzuki, T., Yazaki, Y., & Nagai, R. (1996). Structure and Characterization of the 5′-Flanking Region of the Mouse Smooth Muscle Myosin Heavy Chain (SM1/2) Gene. Circulation research, 78(6), 978-989. https://doi.org/10.1161/01.RES.78.6.978