Transcriptional activation of cytochrome P450 CYP2C45 by drugs is mediated by the chicken xenobiotic receptor (CXR) interacting with a phenobarbital response enhancer unit

Cytochromes P450 (CYP)-2C enzymes fulfill an important role in xenobiotic metabolism and therefore have extensively been studied in rodents and humans. However, no CYP2C genes have been described in avian species to date. In this paper, we report the cloning, functional analysis, and regulation of chicken CYP2C45. The sequence shares up to 58% amino acid identity with CYP2Cs in other species. The overexpression of CYP2C45 in chicken hepatoma cells leghorn male hepatoma (LMH) led to increased scoparone metabolism. CYP2C45 regulation was studied in LMH cells at the mRNA level and in reporter gene assays using a construct containing 2.6 kb of its 5'-flanking region. Exposure of LMH cells to phenobarbital or metyrapone led to a 95- or 210-fold increase in CYP2C45 mRNA and a 140- or 290-fold increase in reporter gene expression, respectively. A phenobarbital response enhancer unit (PBRU) of 239 bp containing a DR-4 nuclear receptor binding site was identified within the 2.6-kb fragment. Site-specific mutation of the DR-4 revealed the requirement of this motif for CYP2C45 induction by drugs. The chicken xenobiotic receptor CXR interacted with the PBRU in electromobility shift and transactivation assays. Furthermore, the related nuclear receptors, mouse PXR and mouse CAR, transactivated this enhancer element, suggesting evolutionary conservation of nuclear receptor-DNA interactions in CYP2C induction.     

Genome-Wide Discovery of Drug-Dependent Human Liver Regulatory Elements

Inter-individual variation in gene regulatory elements is hypothesized to play a causative role in adverse drug reactions and reduced drug activity. However, relatively little is known about the location and function of drug-dependent elements. To uncover drug-associated elements in a genome-wide manner, we performed RNA-seq and ChIP-seq using antibodies against the pregnane X receptor (PXR) and three active regulatory marks (p300, H3K4me1, H3K27ac) on primary human hepatocytes treated with rifampin or vehicle control. Rifampin and PXR were chosen since they are part of the CYP3A4 pathway, which is known to account for the metabolism of more than 50% of all prescribed drugs. We selected 227 proximal promoters for genes with rifampin-dependent expression or nearby PXR/p300 occupancy sites and assayed their ability to induce luciferase in rifampin-treated HepG2 cells, finding only 10 (4.4%) that exhibited drug-dependent activity. As this result suggested a role for distal enhancer modules, we searched more broadly to identify 1,297 genomic regions bearing a conditional PXR occupancy as well as all three active regulatory marks. These regions are enriched near genes that function in the metabolism of xenobiotics, specifically members of the cytochrome P450 family. We performed enhancer assays in rifampin-treated HepG2 cells for 42 of these sequences as well as 7 sequences that overlap linkage-disequilibrium blocks defined by lead SNPs from pharmacogenomic GWAS studies, revealing 15/42 and 4/7 to be functional enhancers, respectively. A common African haplotype in one of these enhancers in the GSTA locus was found to exhibit potential rifampin hypersensitivity. Combined, our results further suggest that enhancers are the predominant targets of rifampin-induced PXR activation, provide a genome-wide catalog of PXR targets and serve as a model for the identification of drug-responsive regulatory elements.     

A duplicated HNF-3 binding site in the CYP2H2 promoter underlies the weak phenobarbital induction response

We are investigating induction of chicken cytochrome P450 genes by the sedative phenobarbital in chick embryo hepatocytes. The steady-state level of induced mRNA for the gene CYP2H1 is about 10-fold higher than that of a second gene, CYP2H2. Here, we show that a difference in drug-responsive enhancer activity does not underlie the differential response of these genes to phenobarbital since upstream enhancer regions are identical in these genes. The first 198 bp of CYP2H2 promoter sequence is identical to the CYP2H1 gene promoter, except that the functional HNF-3 binding site in the CYP2H1 promoter is replaced with a duplicated HNF-3 sequence in the CYP2H2 promoter. Transient expression analysis established that the promoter activity of the CYP2H2 gene was about ninefold lower than the CYP2H1 gene. Mutagenesis of either of the partially overlapping HNF-3 sites in the CYP2H2 gene substantially induced drug induction. Gel-shift analysis established that each of these HNF-3 sites bound HNF-3, most likely HNF-3beta. In-vitro footprint analysis demonstrated that all the identified sites in the CYP2H2 promoter bound protein except the duplicated HNF-3 region. However, protein binding was observed by in-vitro footprint analysis if either of the HNF-3 sites was mutated in the CYP2H2 promoter. Hence, duplication of the HNF-3 site in the CYP2H2 promoter does not allow binding of HNF-3 in the promoter context and may be predominantly, if not exclusively, responsible for the poor response of the CYP2H2 gene to phenobarbital.     

Effects of phenylarsine oxide on expression of heme oxygenase-1 reporter constructs in transiently transfected cultures of chick embryo liver cells

Heme oxygenase catalyzes the first and rate-controlling step of heme catabolism. Induction of heme oxygenase-1 can be caused by numerous factors, including heme, other metalloporphyrins, transition metal ions, heat shock, ultraviolet light, phorbol esters, sodium arsenite, and phenylarsine oxide (PAO). Induction of this enzyme may protect cells from oxidative damage. Using heme oxygenase-1 promoter/reporter gene constructs, we have previously reported that the sodium arsenite-mediated induction of heme oxygenase-1 in chick embryo liver cells and chicken hepatoma (LMH) cells involves an AP-1 element. We have now investigated whether the PAO-mediated induction of heme oxygenase-1 also involves an AP-1 element. Primary cultures of chick embryo liver cells were transiently transfected with heme oxygenase-1 promoter/reporter gene constructs, treated with PAO, and reporter gene activities were measured. We found that the PAO-mediated increase in reporter gene activity was dose- and time-dependent. This activity was decreased by prior treatment with N-acetylcysteine. Studies with mutated constructs showed that both an AP-1 element and a metal responsive element are involved in the PAO-mediated induction of the heme oxygenase-1 reporter construct. Electrophoretic mobility shift assays showed that nuclear proteins from PAO-treated cells had increased binding to an AP-1 probe, and that this increase was abrogated by N-acetylcysteine. These findings support the hypothesis that the PAO-mediated induction of heme oxygenase-1 is caused by activation of AP-1 and MRE/cMyc elements and may involve nuclear proteins whose states of phosphorylation determine binding to regulatory elements, and thus the level of expression of heme oxygenase-1.