Functional characterization of the bovine conglutinin promoter: presence of a novel element for transcriptional regulation of a C-type mammalian lectin containing a collagen-like domain.
Bovine conglutinin is a Ca2+-dependent serum lectin that is specific for N-acetylglucosamine and a member of the collectin (collagen-like lectin) family. Here we report the identification of the cis-acting elements involved in regulating expression of the conglutinin gene. The 5'-flanking region of the conglutinin gene was cloned and sequenced by gene walking using vector (cassette)-ligation mediated PCR. A genomic fragment encompassing -741 to +50 bp had significant promoter activity when linked to the luciferase reporter gene and transfected into the human hepatoma cell line HepG2. Transfection analysis using a series of luciferase vector/5'-stepwise deletion mutants of the promoter constructs indicated that the sequence of 7 base pairs at around -180 bp from the transcription initiation site was necessary for the full expression of the conglutinin gene. The site-directed mutagenesis in the AP-1 (Activator Protein-1) sequence, immediately downstream of the positively controlling cis-element at around -180 bp, resulted in a marked loss of the promoter activity. The novel positively controlling cis-element and the AP-1 sequence regulated synergistically the expression of the conglutinin gene. Gel retardation assay and DNase I footprint analysis demonstrated the presence of the nuclear proteins that bind to these two cis-elements.
Kawasaki
N
N
College of Medical Technology, Kyoto University, Sakyo-ku, Kyoto, Kyoto, 606-8507, Japan.nkawasak@kuhp.kyoto-u.ac.jp
Satonaka
M
M
Imagawa
M
M
Naito
H
H
Kawasaki
T
T
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002417:Cattle; D037222:Collectins; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D005786:Gene Expression Regulation; D006801:Humans; D037102:Lectins / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012712:Serum Globulins / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D037222:Collectins; D037102:Lectins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D012712:Serum Globulins; D018808:Transcription Factor AP-1; C024014:conglutinin; D008156:Luciferases; D003850:Deoxyribonuclease I