Promoter function and the role of cytokines in the transcriptional regulation of rabbit CYP2E1 and CYP2E2.
Rabbit CYP2E1 and CYP2E2 show considerable similarity in the 5' flanking region, but a 32-base-pair element (32-BPE) that is repeated in 2E1 is present only as a single inexact copy in 2E2. In the present investigation, footprinting disclosed two specific binding sites for liver nuclear proteins, and the DNase I sensitivity profiles of the two genes were found to be different. Several positive and negative regulatory elements were identified by transfection with a series of constructs of upstream CYP2E sequences fused to the luciferase gene. Both genes have an HNF-1 consensus motif with one nucleotide mismatch, which affects binding affinity and promoter activity. Investigation of DNA-protein interactions revealed that Sp1 and NFkappaB bind exclusively to the 32-BPE of 2E1 and 2E2, respectively, suggesting a possible regulatory role for the 32-BPE. Interleukin-1alpha (IL-1alpha) gave rise to a 2.5-fold increase in the promoter activity of 2E1 in HepG2 cells, and the IL-1alpha-mediated induction of reporter gene expression was almost completely prevented when the 32-BPE was deleted. Increased DNA binding and Sp1 protein content as a result of IL-1alpha treatment, as well as cotransfection experiments with pPacSp1, suggest that Sp1 is a transcription activator for the induction of 2E1 by IL-1alpha in HepG2 cells.
Peng
H M
HM
Department of Biological Chemistry, Medical School, The University of Michigan, Ann Arbor 48109, USA. hwpeng@umich.edu
Coon
M J
MJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D015151:Immunoblotting; D007375:Interleukin-1 / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D016207:Cytokines; D007375:Interleukin-1; D016328:NF-kappa B; D016329:Sp1 Transcription Factor; D003577:Cytochrome P-450 Enzyme System; D008156:Luciferases; D019392:Cytochrome P-450 CYP2E1; D003850:Deoxyribonuclease I