A 65-KDA PROTEIN MEDIATES THE POSITIVE ROLE OF HEME IN REGULATING THETRANSCRIPTION OF CYP2B1 B2 GENE IN RAT-LIVER/

Heme deficiency precipitated by CoCl2 administration to rats leads to a striking decrease in the inducibility of CYP2B1/B2 mRNA levels and i ts transcription by phenobarbitone (PB), besides decreasing the basal levels. Exogenous hemin administration counteracts the effects of CoCl 2 administration. The binding of nuclear proteins to labeled positive cis-acting element (-69 to -98 nucleotides) in the near 5'-upstream re gion of the gene is inhibited by CoCl2 administration to saline or PR- treated rats, as assessed in gel shift assays. Administration of exoge nous 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 a ffinity column chromatography. This 65-kDa protein manifests very litt le binding to the positive element, but in the presence of certain oth er nuclear proteins, shows a strong heme-responsive binding. The purif ied 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, manif esting heme response. It is concluded that the 65-kDa protein mediates the constitutive requirement of heme for the transcription of CYP2B1/ B2 gene.