Regulation of cytochrome P-450b/e gene expression by a heme- and phenobarbitone-modulated transcription factor.

Abstract

The cloned DNA fragment of the cytochrome P-450b/e gene containing the upstream region from position -179 through part of the first exon is faithfully transcribed in freeze-thawed rat liver nuclei. Phenobarbitone treatment of the animal strikingly increases this transcription, and the increase is blocked by cycloheximide (protein synthesis inhibitor) or CoCl2 (heme biosynthetic inhibitor) treatment of animals. This picture correlates very well with the reported cytochrome P-450b/e mRNA levels in vivo and run-on transcription rates in vitro under these conditions. The upstream region (from position -179) was assessed for protein binding with nuclear extracts by nitrocellulose filter binding, gel retardation, DNase I treatment ("footprinting"), and Western blot analysis. Phenobarbitone treatment dramatically increases protein binding to the upstream region, an increase once again blocked by cycloheximide or CoCl2 treatments. Addition of heme in vitro to heme-deficient nuclei and nuclear extracts restores the induced levels of transcription and protein binding to the upstream fragment, respectively. Thus, drugmediated synthesis and heme-modulated binding of a transcription factor(s) appear involved in the transcriptional activation of the cytochrome P-450b/e genes, and an 85-kDa protein may be a major factor in this regard.

Heme-Reversible Impairment of CYP2B1/2 Induction in Heme-Depleted Rat Hepatocytes in Primary Culture: Translational Control by a Hepatic α-Subunit of the Eukaryotic Initiation Factor Kinase?

“…Yet, even this profound heme depletion had no effect on PB-mediated transcriptional activation of rat CYP2B1 and CYP2B2 genes. Our studies, which used a model of an even more severe hepatic heme pool depletion and highly sensitive quantitative PCR analyses that could distinguish between the highly homologous CYP2B1 and CYP2B2 genes, support previous conclusions that heme is not required for PB-mediated transcriptional activation of either P450 (Srivastava et al, 1989;Sinclair et al, 1990;Jover et al, 2000), as proposed previously (Dwarki et al, 1987;Rangarajan and Padmanaban, 1989).…”

“…Just one group to date has advocated a positive role for heme in CYP2B transcriptional activation (Dwarki et al, 1987;Rangarajan and Padmanaban, 1989). Two other research teams having examined this possibility concluded that heme is not involved in CYP2B transcriptional activation (Hamilton et al, 1988;Srivastava et al, 1989;Sinclair et al, 1990).…”

A 65-kDa Protein Mediates the Positive Role of Heme in Regulating the Transcription of CYP2B1/B2 Gene in Rat Liver

“…Run-on Transcription-This was carried out by the procedure of Guertin et al (19) and has been described (3,4). Briefly, nuclei were incubated with 200 Ci of [␣-…”

“…Studies in this laboratory have led to the identification of a positive cis-acting element (Ϫ69 to Ϫ98 nt) and a negative cis-acting element (Ϫ126 to Ϫ160 nt) in the near 5Ј-upstream region of the CYP2B1/B2 gene (3)(4)(5). The positive element includes the 17-base pair PB-responsive consensus element, referred to as Barbie Box, identified first by Fulco and co-workers (6 -8) in Bacillus megaterium, rat, mice, and other organisms of barbiturate inducible cytochrome P-450 and other proteins.…”

Molecular Mechanism Governing Heme Signaling in Yeast: a Higher-Order Complex Mediates Heme Regulation of the Transcriptional Activator HAP1

“…Heme also directly regulates various molecular and cellular processes for systems that use or sense oxygen. For example, heme stimulates the expression of globin chains in erythroid cells and cytochrome P-450 in hepatic cells (6,9,36), activates protein synthesis through the heme-regulated inhibitor kinase (7), and regulates the transport of numerous enzymes and the assembly of hemoglobin and cytochrome complexes (24,31). The roles of heme are largely conserved from yeast to humans, and recent evidence indicates that heme might regulate diverse processes through similar mechanisms (24,48).…”