Structural and functional characterization of liver cell-specific activityof the human sodium/taurocholate cotransporter

Bile salts are rapidly removed from the circulation by the liver-specific s odium/taurocholate cotransporter (SLC10A1), To understand factors controlli ng its liver-specific expression, we isolated human SLC10A1 from a YAC chro mosomal clone. SLC10A1 spans similar to 23 kb distributed over five exons, The major transcription start site is at 299 bp, and a minor start site is at 395 bp from the translational start site. A 1.2-kb portion of the 5' fla nking region was sequenced and shown. to contain a number of liver-enriched elements, but no TATA box. Using secreted alkaline phosphatase reporter co nstructs liver-specific expression was examined. Transient transfection dem onstrated that SLC10A1 promoter expression was selectively expressed eightf old in FAO and rat hepatocytes, while deletion mutants demonstrated liver-s pecific expression in a region extending from -5 to +198 bp, which containe d putative sites for C/EBP and HNF3, Mutations of the C/EBP site resulted i n loss of 77% of transcriptional activity. Cotransfection of C/EBP, but not other putative liver-enriched binding factors, increased SLC10A1 promoter activity. Electrophoretic mobility shift assays demonstrated specific prote in-DNA interactions that involved C/EBP alpha and beta, These studies demon strate that the TATA-less human SLC10A1 promoter exhibits liver-specific ac tivity and its regulatory elements contain binding sites for C/EBP, which c ontributes specifically to its transcriptional regulation, (C) 2000 Academi c Press.