Regulation of the hepatic CYP 2E1 gene during chronic alcohol exposure: lack of an ethanol response element in the proximal 5'-flanking sequence.
Chronic exposure to ethanol is known to cause a dramatic increase in the level of CYP 2E1 apoprotein. More recently it has been demonstrated that under certain conditions the mRNA encoding cytochrome P450 2E1(CYP 2E1) is inducible; however, the mechanisms by which these increases occur are not well understood. In the current study, DNase I footprinting assays performed on the first kilobase of the CYP 2E1 5'-flanking sequences resulted in the identification of 13 sequence-specific protected regions using rat liver nuclear extracts isolated from either control or ethanol-treated animals. No differences were observed in the DNase I footprint patterns produced by the two different nuclear extracts. In addition, analysis by electrophoretic mobility shift assays (EMSA) revealed that with one exception, there were no differences in the level of binding complexes between the two extracts. However, EMSA analysis with an oligonucleotide to one footprint site (designated Site C) revealed that in nuclear extracts isolated from ethanol-treated animals there was a 2.9-fold increase in this binding complex when compared to control nuclear extracts. This site was previously shown to contain an HNF-1alpha binding site, and here we demonstrate that bacterially expressed HNF-1alpha in footprint assays bind Site C sequences and that HNF-1alpha transactivates the CYP 2E1 promoter in co-transfection experiments with HNF-1alpha expression plasmid and plasmids containing CYP 2E1 promoter sequences coupled to the chloramphenicol acetyl transferase gene. Furthermore, in contrast to the increase observed by EMSA in Site C binding, no increase was detected in the CYP 2E1 transcriptional rate supported by nuclear extracts from ethanol-treated animals over controls using in vitro transcription assays, suggesting that the increase by ethanol in CYP 2E1 transcription is not mediated through the HNF-1alpha site.
McGehee
R E
RE
University of Arkansas for Medical Sciences, Arkansas Children's Hospital Research Institute, Department of Pediatrics, Little Rock 72205, USA.
Ronis
M J
MJ
Badger
T M
TM
D000818:Animals; D001665:Binding Sites; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics*; D004268:DNA-Binding Proteins; D000431:Ethanol / Q000008:administration & dosage* / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D008099:Liver / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; C496489:Hnf1a protein, rat; D009687:Nuclear Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D000431:Ethanol; D019392:Cytochrome P-450 CYP2E1