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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

A conserved nuclear receptor consensus sequence (DR-4) mediates transcriptional activation of the chicken CYP2H1 gene by phenobarbital in a hepatoma cell line.

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.[1]


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