Identification of cis-elements of the human endothelin-A receptor gene and inhibition of the gene expression by the decoy strategy

J. Yamashita, T. Yoshimasa, H. Arai, J. Hiraoka, K. Takaya, Y. Miyamoto, Y. Ogawa, H. Itoh, K. Nakao

研究成果: ジャーナルへの寄稿記事

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Previously, we succeeded in molecular cloning of the cDNA and the gene for human endothelin-A receptor (ET-AR). In the present study, we define cis- elements in the 5'-flanking region of the ET-AR gene. Deletion analyses were performed in A7r5 cells, rat vascular smooth muscle cell line, and Chinese hamster ovary cells using ET-AR promoter-luciferase gene constructs including 5 kilobases of the 5'-flanking region. These analyses demonstrated the existence of one negative regulatory element (-2.0 kilobases to -857 bases) and two positive regulatory elements (-137 to -53 and -53 to +251). Gel mobility shift assay revealed a nuclear protein binding to the region (-104 to -78) (R1). DNase I footprinting analysis showed a footprint spanning from -91 to -83 whose sequence is CCCCACCTT (ETA-P1). When a plasmid including R1 fragments (R1 decoy) was co-transfected into A7r5 cells with ET-AR (-137 to +251)-luciferase gene construct, it significantly reduced the luciferase activity in a dose-dependent manner. Moreover, R1 decoy down-regulated the endogenous ET-AR mRNA in A7r5 cells by a maximum of 75%. Thus, we identified cis-elements that regulate basal transcriptional activity of the ET-AR gene and proved the feasibility to suppress the expression of the ET-AR gene by the DNA decoy strategy using the positive regulatory element we identified.

元の言語英語
ページ(範囲)15993-15999
ページ数7
ジャーナルJournal of Biological Chemistry
273
発行部数26
DOI
出版物ステータス出版済み - 6 26 1998
外部発表Yes

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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