Abstract
The regulation of cytochrome P450 3A (CYP3A) enzymes is established in humans, but molecular mechanisms of its basal and xenobiotic-mediated regulation in cattle are still unknown. Here, ~10 kbp of the bovine CYP3A28 gene promoter were cloned and sequenced, and putative transcription factor binding sites were predicted. The CYP3A28 proximal promoter (PP; -284/+71 bp) contained DNA elements conserved among species. Co-transfection of bovine nuclear receptors (NRs) pregnane X and constitutive androstane receptor (bPXR and bCAR) with various CYP3A28 promoter constructs into hepatoma cell lines identified two main regions, the PP and the distal fragment F3 (-6899/-4937 bp), that were responsive to bPXR (both) and bCAR (F3 fragment only). Site-directed mutagenesis and deletion of NR motif ER6, hepatocyte nuclear factor 1 (HNF-1) and HNF-4 binding sites in the PP suggested either the involvement of ER6 element in bPXR-mediated activation or the cooperation between bPXR and liver-enriched transcription factors (LETFs) in PP transactivation. A putative DR5 element within the F3 fragment was involved in bCAR-mediated PP+F3 transactivation. Although DNA enrichment by anti-human NR antibodies was quite low, ChIP investigations in control and RU486-treated BFH12 cells, suggested that retinoid X receptor α (RXRα) bound to ER6 and DR5 motifs and its recruitment was enhanced by RU486 treatment. The DR5 element seemed to be recognized mainly by bCAR, while no clear-cut results were obtained for bPXR. Present results point to species-differences in CYP3A regulation and the complexity of bovine CYP3A28 regulatory elements, but further confirmatory studies are needed.
Highlights
The bovine CYP3A28 proximal promoter (PP) contained an everted repeat motif with a 6-nucleotide spacer (ER6)-type motif for pregnane X receptor (PXR) and constitutive androstane receptor (CAR) at -173/-156 bp, which aligned well with the known human CYP3A4 proER6 element (Fig 1)
This element was flanked by an hepatocyte nuclear factor (HNF)-3β site (5’) and two hepatocyte nuclear factor-4 (HNF-4) sites (3’)
nuclear receptor (NR)-mediated regulation of bovine CYP3A28 transcription was investigated through DNA re-sequencing, in silico prediction of NR/transcription factor (TF) binding sites, gene reporter assays of wild-type and mutated promoter fragments, induction studies in a bovine-derived hepatic cell line and chromatin immunoprecipitation (ChIP) assays
Summary
ReagentsThe established activators of human and mouse CAR and PXR have been reviewed [29,30,31]. All other chemicals used in the study are commercially available and of molecular biology grade.
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