Structural and functional characterization of liver cell-specific activity of the human sodium/taurocholate cotransporter.
Bile salts are rapidly removed from the circulation by the liver-specific sodium/taurocholate cotransporter (SLC10A1). To understand factors controlling its liver-specific expression, we isolated human SLC10A1 from a YAC chromosomal clone. SLC10A1 spans approximately 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' flanking region was sequenced and shown to contain a number of liver-enriched elements, but no TATA box. Using secreted alkaline phosphatase reporter constructs liver-specific expression was examined. Transient transfection demonstrated that SLC10A1 promoter expression was selectively expressed eightfold in FAO and rat hepatocytes, while deletion mutants demonstrated liver-specific expression in a region extending from -5 to +198 bp, which contained putative sites for C/EBP and HNF3. Mutations of the C/EBP site resulted in 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 protein-DNA interactions that involved C/EBPalpha and beta. These studies demonstrate that the TATA-less human SLC10A1 promoter exhibits liver-specific activity and its regulatory elements contain binding sites for C/EBP, which contributes specifically to its transcriptional regulation.
Shiao
T
T
Department of Medicine, University of Colorado Health Sciences Center and Denver Veterans Affairs Medical Center, 4200 East Ninth Avenue, Denver, Colorado 80262, USA.
Iwahashi
M
M
Fortune
J
J
Quattrochi
L
L
Bowman
S
S
Wick
M
M
Qadri
I
I
Simon
F R
FR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D004285:Dogs; D005786:Gene Expression Regulation; D051541:Hepatocyte Nuclear Factor 3-alpha; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000502:physiology*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009928:Organ Specificity; D029363:Organic Anion Transporters, Sodium-Dependent; D011401:Promoter Regions, Genetic; D051381:Rats; D017422:Sequence Analysis, DNA; D027981:Symporters; D014157:Transcription Factors; D014158:Transcription, Genetic
D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C496499:FOXA1 protein, human; C496501:Foxa1 protein, rat; D051541:Hepatocyte Nuclear Factor 3-alpha; D009687:Nuclear Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; D014157:Transcription Factors; C071908:sodium-bile acid cotransporter