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Role of the AP‐1 binding site in regulating PKCε promoter activity in the fetal heart
Author(s) -
Zhang Haitao,
Zhang Lubo
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1413
Subject(s) - microbiology and biotechnology , binding site , chromatin immunoprecipitation , promoter , electrophoretic mobility shift assay , biology , protein kinase c , oligonucleotide , mutant , transcription factor , dna , gene , gene expression , kinase , biochemistry
To determine whether the AP‐1 binding site is involved in regulating PKCε promoter activity, the activities of 5′ deletion mutants of a 2000‐bp PKCε promoter region were evaluated in the rat embryonic ventricular myocyte cell line H9C2. The levels of basal full‐length promoter activity obtained with pPKCε2k‐Luc was at least 15‐fold greater than the vector alone in a high glucose medium. Basal promoter activity fell by 42% with a deletion from −2000 to −398, which contains the AP‐1 binding site, and it fell 92% with a deletion from −2000 to −361, which lacks the AP‐1 binding site. Electrophoretic mobility shift assays with oligonucleotide probes encompassing the putative AP‐1 site showed a major DNA‐protein complex using nuclear extracts from fetal rat hearts. Super‐shift analyses showed that c‐Jun, but not c‐Fos or Fra‐1, antibody caused a super‐shifting of the AP‐1 DNA‐protein complex. In addition, chromatin immunoprecipitation assays demonstrated a binding of AP‐1/c‐Jun to the AP‐1 binding site in the promoter region of the PKCε gene in intact chromatin in vivo in the fetal heart. The results indicate a strong stimulatory role of AP‐1 (c‐Jun homodimer) binding in PKCε promoter activity in the fetal heart. (Supported in part by NIH grant HL82779).