Abstract
Phenobarbital-responsive DNA elements were identified in the 5'-flanking region of the chicken CYP2H1 gene by in reporter gene assays in a chicken hepatoma cell line (leghorn male hepatoma (LMH)). A 264-base pair (bp) enhancer sequence (phenobarbital-responsive unit (PBRU)) responded to phenobarbital and a variety of phenobarbital-type inducers. Analysis of putative transcription factor binding sites within the 264-bp element revealed a nuclear receptor half-site repeat (DR-4) neighboring a putative nuclear factor-1 site. This motif resembles phenobarbital response elements in the flanking regions of three phenobarbital-inducible genes, rat CYP2B2, mouse Cyp2b10, and human CYP2B6. Activation of the 264-bp element was eliminated after site-directed mutagenesis of the DR-4 hexamer half-sites. Evidence for evolutionary conservation of this recognition site was indicated by activation in LMH cells of a mouse Cyp2b10 phenobarbital-responsive enhancer by the same spectrum of inducers that activate the CYP2H1 264-bp PBRU. Inhibition of this activation by okadaic acid may explain the reported inhibitory effects on induction of CYP2B1/2 and Cyp2b10 by this phosphatase inhibitor. We show that this inhibition occurs directly on the 264-bp PBRU, whereas the proximal promoter of CYP2H1 is induced by okadaic acid in reporter gene assays. These experiments exploit the unique phenobarbital inducibility of the hepatoma-derived cell line LMH.
Highlights
The cytochrome P-450 (CYP)1 gene superfamily encodes proteins responsible for the metabolism of numerous xenobiotic and endobiotic substrates [1]
A major breakthrough has been the recent observation in several laboratories that orphan nuclear receptors are involved in CYP regulation. These findings emerged from the discovery and analysis of well defined PBresponsive elements in the flanking region of rat CYP2B2 and CYP3A1 [13,14,15,16], mouse Cyp2b10 [17, 18], and human CYP2B6 and CYP3A4 [19, 20]
The results revealed two major inducible regions, a 1376-bp fragment located at the 5Ј end of the 5.9-kb flanking region (–5913/– 4537) that was induced about 5-fold and a 264-bp fragment located at bp –1657/–1393 mediating a 6 –7
Summary
The cytochrome P-450 (CYP) gene superfamily encodes proteins responsible for the metabolism of numerous xenobiotic and endobiotic substrates [1]. Phenobarbital (PB) induces predominantly enzymes from the CYP2B, -2C, and -3A subfamilies [2, 5, 7]. These CYPs, as well as more than 50 other genes, are affected by PB and a number of structurally unrelated compounds classified as PB-like inducers [8]. The 5Ј-flanking region of CYP2H1 has been analyzed by Hahn et al [25], and a 4.8-kb PB-responsive enhancer fragment (–5898 to –1057) was identified Within this large DNA region, a small enhancer element of 240 bp was isolated [26]. A limiting factor in these studies was that the 240-bp fragment mediated only a small response to PB in primary hepatocyte cultures when compared with activation of transcription in vivo [26]
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