C/EBPalpha is a regulator of the UDP glucuronosyltransferase UGT2B1 gene.
The rat UDP glucuronosyltransferase, UGT2B1, is expressed in the liver where it glucuronidates steroids, environmental toxins, and carcinogens. A region between -88 and -111 base pairs upstream from the UGT2B1 gene transcription start site contains a CCAAT enhancer binding protein (C/EBP)-like element and was previously shown by Dnase I footprint analysis to bind to proteins in both rat liver and human hepatoma (HepG2) cell nuclear extracts. In this study, the importance of this region in the regulation of the UGT2B1 gene was assessed by functional and DNA binding assays. Varying lengths of the UGT2B1 gene promoter, with and without the C/EBP-like element, were fused to the chloramphenicol acetyltransferase reporter gene and transfected into HepG2 cells. Transcriptional activity of the UGT2B1 promoter construct containing the C/EBP-like element was strongly elevated in the presence of a cotransfected C/EBPalpha expression vector. In contrast, no change was observed when an expression vector encoding C/EBPbeta was cotransfected with the UGT2B1 promoter constructs. Introduction of point mutations into the C/EBP-like element prevented any C/EBPalpha-mediated increase in chloramphenicol acetyltransferase activity. Gel shift analyses demonstrated that the C/EBP-like element binds a complex of nuclear proteins present in both HepG2 cells and rat liver. The presence of C/EBPalpha in this complex was confirmed by supershift analysis with antiserum to this factor. These data strongly suggest that the liver-enriched factor C/EBPalpha binds to, and activates, the UGT2B1 gene promoter. The importance of C/EBPalpha in the regulation of the homologous mouse UGT2B1 gene was also assessed in vivo. Transcripts homologous to UGT2B1 were detected in the livers of mice containing intact c/ebpalpha and c/ebpbeta genes and in mice containing a homozygous null mutation in the c/ebpbeta gene. In contrast, these transcripts were not detected in mice with a disrupted hepatic c/ebpalpha gene. These data extend the findings with the rat UGT2B1 gene promoter and establish that C/EBPalpha, but not C/EBPbeta, is an essential transcriptional regulator of the homologous UGT2B1 gene in the mouse.
Hansen
A J
AJ
Department of Clinical Pharmacology, Flinders University School of Medicine, Flinders Medical Centre, Bedford Park, South Australia, 5042, Australia.
Lee
Y H
YH
Sterneck
E
E
Gonzalez
F J
FJ
Mackenzie
P I
PI
D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic; D014453:Glucuronosyltransferase / Q000235:genetics*; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D014407:Tumor Cells, Cultured
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014453:Glucuronosyltransferase