A 65-kDa protein mediates the positive role of Heme in regulating the transcription of CYP2B1/B2 gene in rat liver

Abstract

Heme deficiency precipitated by CoCl₂ administration to rats leads to a striking decrease in the inducibility of CYP2B1/B2 mRNA levels and its transcription by phenobarbitone (PB), besides decreasing the basal levels. Exogenous hemin administration counteracts the effects of CoCl₂ administration. The binding of nuclear proteins to labeled positive cis-acting element (-69 to -98 nucleotides) in the near 5'-upstream region of the gene is inhibited by CoCl₂ administration to saline or PB-treated rats, as assessed in gel shift assays. Administration of exogenous hemin to the animal or addition in vitro to the extracts is able to overcome the effects of CoCl₂ treatment. The protein mediating this effect has been purified from CoCl₂ administered nuclear extracts by heparin-agarose, positive element oligonucleotide affinity, and heme affinity column chromatography. This 65-kDa protein manifests very little binding to the positive element, but in the presence of certain other nuclear proteins, shows a strong heme-responsive binding. The purified protein binds heme. It is also able to stimulate transcription of a minigene construct of the CYP2B1/B2 gene containing -179 nucleotides of the 5'-upstream region and the I exon in a cell-free system, manifesting heme response. It is concluded that the 65-kDa protein mediates the constitutive requirement of heme for the transcription of CYP2B1/B2 gene.