MULTIPLE FACTORS REGULATE THE RAT-LIVER BASOLATERAL SODIUM-DEPENDENT BILE-ACID COTRANSPORTER GENE PROMOTER

The hepatic uptake of bile acids from the portal circulation is primar ily dependent upon a sodium-dependent basolateral membrane transporter , In order to bean to investigate the factors controlling rat liver so dium-dependent bile acid cotransporter (ntcp) gene expression, we isol ated approximate to 30 kilobase pairs of rat genomic DNA in three over lapping lambda phage clones, The rat ntcp gene is distributed over 16. 5 kilobase pairs as five exons, Primer extension analysis revealed two closely spaced transcription initiation sites, 27 and 41 nucleotides downstream of a TATA sequence, Regulation of transcription was investi gated first by transfection of primary rat hepatocytes by a series of 5'-deleted rat ntcp promoter-driven luciferase constructs (from approx imate to -6 kilobase pairs to -59 base pairs of upstream sequences, te rminating at nucleotide +47), identifying a minimal promoter element: nucleotide -158 to +47, This minimal promoter was active in transfecte d HepG2, but inactive in NIH3T3, Caco-2, and Madin-Darby canine kidney cells, indicating that the determinants of hepatocyte-specific expres sion reside within this region, The individual elements within the min imal promoter were investigated via transfection of HepG2 cells by a s eries of 20 mutant plasmids, each containing a 10-base pair sequential block mutation, Eight mutant constructs profoundly suppressed promote r activity; encompassing sequences from -66 to +4 nt, and +15 to +24 n ucleotides, while no other 10-base pair mutation significantly interfe red with minimal promoter activity, Deoxyribonuclease I footprint anal ysis of the minimal promoter revealed three bound regions; -92 to -74 (footprint C), -50 to -37 (footprint B), and -17 to +12 (footprint A), Gel mobility shift assays provided evidence for hepatocyte nuclear fa ctor 1 binding within footprint A and a liver-enriched factor(s) that binds within a novel palindrome in footprint B, These studies indicate that three elements direct the basal and tissue-restricted expression of the rat ntcp promoter; a TATA element, the liver-enriched transcri ption factor hepatocyte nuclear factor 1, and an unknown liver-enriche d factor that binds within a novel palindrome in footprint B.