Identification of CCAAT enhancer binding protein α binding sites on the human α-fetoprotein gene

Development- and tissue-specific α-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/EBPα-binding sites in the promoter and enhancer regions. An electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis identified two and five C/EBPα-binding sites located in the promoter and enhancer regions, respectively. Chromatin immunoprecipitation analyses showed that C/EBPα 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/EBPα binding to each of the elements. These results indicate that C/EBPα regulates AFP gene expression through direct binding to multiple sites in the human AFP gene in cultured human cells.