Characterization of hamster CYP1A1 gene: Inducible expression and negative regulation

Ikuko Sagami, Hideaki Kikuchi, Shuntaro Ikawa, Minro Watanabe

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

Abstract

A genomic clone encoding the hamster CYP1A1 gene was isolated from a hamster EMBL-3 genomic library and characterized. The CYP1A1 gene contained seven exons including the noncoding first exon as determined for CYP1A1 of other species. DNA sequence analysis up to -2307 bp of the CYP1A1 gene revealed the occurrence of five consensus xenobiotic responsive elements (XREs) and one basal transcription element (BTE) in addition to the canonical TATA box. For functional analysis, transfection experiments were performed in human hepatoma HepG2 cells with reporter gene constructs consisting of fragments with various lengths of the 5'-flanking region of the CYP1A1 gene and bacterial chloramphenicol acetyltransferase (CAT) gene. External deletion of the upstream region from the reporter gene resulted in a stepwise decrease of the CAT activity, suggesting that XREs were responsible for inducible expression of CYP1A1 gene by 3-methylcholanthrene (MC). A negative regulatory element (NRE) was also identified in the 5' -flanking region at -833 to -642. Removal of the NRE from the CYP1A1-CAT fusion gene resulted in about 3-fold increase of MC-inducible CAT activity. Using gel retardation assays with HepG2 nuclear extract, we demonstrated the presence of a specific protein which bound to the NRE fragment. Further competition analysis and methylation interference assays revealed that the nuclear protein bound to a 22-base fragment (from -688 to -709) of the NRE region, whose sequences were conserved among hamster, human, and rat CYP1A1 genes.

Original languageEnglish
Pages (from-to)801-810
Number of pages10
JournalJournal of biochemistry
Volume116
Issue number4
DOIs
Publication statusPublished - 1994 Oct

Keywords

  • CAT assay
  • Cytochrome P-4501A1
  • DNA-protein interaction
  • Negative regulatory element
  • Transcriptional regulation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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