CRISPR-Cas9-Mutated Pregnane X Receptor (pxr) Retains Pregnenolone-induced Expression of cyp3a65 in Zebrafish (Danio rerio) Larvae

Abstract

Pregnane X receptor (PXR; NR1I2) is a nuclear receptor that regulates transcriptional responses to drug or xenobiotic exposure, including induction of CYP3A transcription, in many vertebrate species. PXR is activated by a wide range of ligands that differ across species, making functional studies on its role in the chemical defensome most relevant when approached in a species-specific manner. Knockout studies in mammals have shown a requirement for PXR in ligand-dependent activation of CYP3A expression or reporter gene activity. Morpholino knockdown of Pxr in zebrafish indicated a similar requirement. Here, we report on the generation of 2 zebrafish lines each carrying a heritable deletion in the pxr coding region, predicted to result in loss of a functional gene product. To our surprise, 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. Thus, zebrafish carrying pxr alleles with deletions in either the DNA binding or the ligand-binding domains did not yield a loss-of-function phenotype, suggesting that a compensatory mechanism is responsible for cyp3a65 induction. Alternative possibilities are that Pxr is not required for the induction of selected genes, or that truncated yet functional mutant Pxr is sufficient for the downstream transcriptional effects. It is crucial that we develop a better understanding for the role of Pxr in this important biomedical test species. This study highlights the potential for compensatory mechanisms to avoid deleterious effects arising from gene mutations.

Genotype to Phenotype: CRISPR Gene Editing Reveals Genetic Compensation as a Mechanism for Phenotypic Disjunction of Morphants and Mutants

“…Several examples of genetic compensation have been recently documented. Salanga et al reported notable discrepancies between knockdown and knockout studies of zebrafish pregnane X receptor ( pxr; nr112 ), a nuclear receptor and zinc finger transcription factor involved in transcriptional responses to xenobiotic exposure with conserved functions across various species [ 36 , 80 ]. Among other functions, Pxr, upon ligand binding, induces the expression of the xenobiotic-metabolizing enzyme cytochrome P450 3A (Cyp3a).…”

“…The sequence of the expressed transcript from the exon 7, 8 mutant allele revealed the direct splicing of exons 6–9. Notably, PTCs were identified in coding strand DNA; unexpectedly, both mutants retained the wild-type-like activation of cyp3a65 in response to pxr ligand, pregnenolone suggesting the expression of functional gene product [ 36 ]. Although this study did not investigate the possibility of compensatory mechanisms of pxr , this pathway should be investigated for genetic compensation considering the conserved evolution of pxr across species.…”

Vitamin K in Vertebrates’ Reproduction: Further Puzzling Pieces of Evidence from Teleost Fish Species

“…As a NR, PXR modulates the transcription of genes showing specific response elements (REs) on their promoter regions to which it binds through their DNA binding domain (DBD), once it is activated by known ligands [ 71 ]. In contrast to the DBD, its ligand binding domain (LBD) exhibited an evolutionary low degree of conservation [ 71 ] and thus, PXR ligands might differ across species [ 71 , 72 ]. PXR signaling cascade regulations seem to be further puzzling, as PXR subcellular localization and transcriptional activity are also dependent on multiple post-translational modifications, including phosphorylation, acetylation, SUMOylation, and ubiquitination [ 73 ].…”

Orphan cytochrome P450 20A1 CRISPR/Cas 9 mutants and neurobehavioral phenotypes in zebrafish

“…See Supplementary Table 1 for primer sequences and cycling conditions. sgRNA templates were prepared as described in [16–18] [44]. Briefly, a universal reverse primer was combined with a forward primer containing a 5’ T7 polymerase binding site, a gene-specific target sequence, and approximately 20 nucleotides of a 3’ sequence complementary to the universal reverser primer in a 100 μl reaction at a 500 nM final concentration for each primer, dNTPs at a 200 μM final concentration, Q5 reaction buffer at a 1x final concentration, and 2U of Q5.…”