CRISPR-Cas9 mutated pregnane x receptor (pxr) retains pregnenolone-induced expression of cytochrome p450 family 3, subfamily A, polypeptide 65 (cyp3a65) in zebrafish (Danio rerio) larvae

Abstract

AbstractPregnane x receptor (PXR) is a nuclear receptor that regulates transcriptional responses to drug or xenobiotic exposure in many vertebrate species. One key response is the induction of cytochrome P450 3A (CYP3A) transcription. PXR is a promiscuous receptor activated by a wide range of ligands that can differ across species, making functional studies on its role in the chemical defensome, most relevant when approached in a species-specific manner. Genetic knockout studies in mammals have shown a requirement for PXR in ligand-dependent activation of CYP3A expression or reporter gene activity. Transient knockdown in zebrafish revealed a similar requirement, however it is not known what the effect of a genetic knockout would be in the zebrafish model. Here, we report on generation of two zebrafish lines each carrying a genetic deletion in the pxr coding region, predicted to result in loss of a functional gene product. To our surprise zebrafish larvae, homozygous for either of the pxr mutant alleles, retain their ability to induce cyp3a65 mRNA expression following exposure to the established zebrafish Pxr ligand, pregnenolone (PN). Thus, zebrafish carrying pxr alleles with sizable deletions in either the DNA binding or the ligand binding domains do not yield a loss-of-function phenotype, suggesting that a compensatory mechanism is responsible for cyp3a65 induction. Two alternative possibilities are that Pxr is not required for the effect or that truncated yet functional mutant Pxr is sufficient for the effect.