Identification of CCAAT enhancer binding protein alpha binding sites on the human alpha-fetoprotein gene.
Development- and tissue-specific alpha-fetoprotein (AFP) gene expression is controlled by various transcription factors including hepatocyte nuclear factors (HNFs), and a number of cis-acting elements. We recently identified multiple CCAAT/enhancer binding protein (C/EBP) binding sites in the enhancer of the human AFP gene. In this study, we have identified and functionally characterized seven C/EBPalpha-binding sites in the promoter and enhancer regions. An electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis identified two and five C/EBPalpha-binding sites located in the promoter and enhancer regions, respectively. Chromatin immunoprecipitation analyses showed that C/EBPalpha binds both enhancer and promoter regions of the AFP gene in human AFP-producing hepatoma and stomach cancer cells, but not in non-AFP-producing cells. Reporter transfection assays showed that transcription was stimulated by C/EBPalpha binding to each of the elements. These results indicate that C/EBPalpha regulates AFP gene expression through direct binding to multiple sites in the human AFP gene in cultured human cells.
Li
Hong Mei
HM
Department of Biochemistry, Hokkaido Graduate School of Medicine, Sapporo, Japan.
Ikeda
Hiromi
H
Nakabayashi
Hidekazu
H
Nishi
Shinzo
S
Sakai
Masaharu
M
D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D047369:Chromatin Immunoprecipitation; D018983:DNA Footprinting; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D006801:Humans; D011401:Promoter Regions, Genetic; D000509:alpha-Fetoproteins / Q000235:genetics*
D022762:CCAAT-Enhancer-Binding Proteins; C515848:CEBPA protein, human; D000509:alpha-Fetoproteins