Cloning and characterization of the 5′-flanking region of human cytokeratin 19 gene in human cholangiocarcinoma cell line

Background/Aims: The regulatory mechanism of cell-specific gene expression in cholangiocytes has not been sufficiently evaluated. In this study, we characterized the 5’-flanking region of the cytokeratin 19 gene expressed specifically in cholangiocytes. Methods: We cloned a 2952-bp fragment in the 5’-flanking region of cytokeratin 19 and evaluated the regulatory mechanism of gene expression in this region by assaying transient expression of reporter gene and DNA footprinting. Results: This segment of the 5’-flanking region of the human cytokeratin 19 gene shows an intense transcriptional activity in the cholangioma cell line KMBC, which was about 10 times its activity in the osteosarcoma cell line Saos-2, which does not express cytokeratin 19. From the results of reporter assays, important transcription regulatory elements are considered to be located in the segments from −2249 to −2050 bp and from −732 bp to the first ATG, and six protein-binding sites were detected in the segment from −732 bp to the first ATG by the DNA footprinting technique. Conclusions: Sp1 site, CCAAT box, and TATA box were present in the segment from −374 to the first ATG, and they are considered to constitute a cholangiocyte-specific promoter.