NEAT1_2 and DAZAP1, Paraspeckle Components, Interact with PXR to Negatively Regulate CYP3A4 Induction.

Human pregnane X receptor (PXR) is a major nuclear receptor that upregulates the expression of drug-metabolizing enzymes such as CYP3A4. In our recent study, it was revealed that PXR interacts with DAZ-associated protein 1 (DAZAP1), which is an essential component of the paraspeckle, a membraneless nuclear body, and the interaction was disassociated by rifampicin, a ligand of PXR. The purpose of this study was to clarify the roles of paraspeckles in PXR-mediated transcriptional regulation. Immunoprecipitation assays using PXR-overexpressing HepG2 (ShP51) cells revealed that PXR interacts with not only DAZAP1 but also NEAT1_2, a long noncoding RNA included in the paraspeckle, and that the interaction between PXR and NEAT1_2 was disassociated by rifampicin. These results suggest that PXR is trapped in paraspeckles and that the activation of PXR by its ligands facilitates its disassociation from paraspeckles. Induction of CYP3A4 by rifampicin was significantly enhanced by the knockdown of NEAT1_2 or DAZAP1 in ShP51 cells and their parental HepG2 cells. A luciferase assay using a plasmid containing the PXR response elements of CYP3A4 revealed that the increased CYP3A4 induction by siNEAT1_2 or siDAZAP1 was due to the increased transactivation by PXR. These results suggest that paraspeckles play a role in trapping nuclear PXR in the absence of the ligand to negatively regulate transactivation of its downstream gene. Collectively, this is the first study to demonstrate that the paraspeckle components NEAT1_2 and DAZAP1 negatively regulate CYP3A4 induction by PXR. SIGNIFICANCE STATEMENT: This study revealed that PXR interacts with paraspeckle components NEAT1_2 and DAZAP1 to suppress CYP3A4 induction by PXR, and the interaction is dissociated by PXR ligands. This finding provides a novel concept that paraspeckles formed by liquid-liquid phase separation potentially affect drug metabolism via negative regulation of PXR function.

Adenosine N6-methylation upregulates the expression of human CYP2B6 by altering the chromatin status.

N6-Methyladenosine (m6A) modification is the most prevalent RNA modification in mammals. We have recently demonstrated that inhibition of m6A modification by 3-deazaadenosine results in an increase in the expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, and CYP2C8 in human liver-derived cells. In the present study, we aimed to clarify the mechanism of m6A-mediated regulation of CYP2B6 expression. RNA immunoprecipitation using an anti-m6A antibody revealed that CYP2B6 mRNA in human liver and hepatocarcinoma-derived HepaRG cells was m6A-modified around the stop codon. In contrast to the treatment with 3-deazaadenosine, double knockdown of methyltransferase like (METTL) 3 and METTL14 (METTL3/14) resulted in a decrease in the levels of CYP2B6 mRNA in Huh-7 and HepaRG cells and a decrease in bupropion hydroxylase activity, a marker activity of CYP2B6, in HepaRG cells. The stability of CYP2B6 mRNA was not influenced by siMETTL3/14. Reporter assays using the plasmids containing the last exon or 5'-flanking region of CYP2B6 indicated that reporter activities were not influenced by knockdown of METTL3/14. The expression levels of the constitutive androstane receptor, pregnane X receptor, and retinoid X receptor, which are the nuclear receptors regulating the transcription of CYP2B6, were not influenced by siMETTL3/14. The chromatin immunoprecipitation and formaldehyde-assisted enrichment of regulatory elements assays revealed that H3K9me2, a repressive histone marker, was enriched in the vicinity of the upstream region of CYP2B6, and knockdown of METTL3/14 induced the condensation of the chromatin structure in this region. In conclusion, we demonstrated that METTL3/14 upregulated CYP2B6 expression by altering the chromatin status.

The N6-methyladenosine modification posttranscriptionally regulates hepatic UGT2B7 expression.

UDP-glucuronosyltransferases (UGTs) are enzymes catalyzing the glucuronidation of various endogenous and exogenous compounds. In this study, we examined the possibility that N6-methyladenosine (m6A) modification affects hepatic UGT expression. Treatment of HepaRG cells with 3-deazaadenosine, an inhibitor of RNA methylation, significantly increased UGT1A1, UGT1A3, UGT1A4, UGT1A9, UGT2B7, UGT2B10, and UGT2B15 mRNA levels (1.3- to 2.6-fold). Among them, we focused on UGT2B7 because it most highly contributes to glucuronidation of clinically used drugs. Methylated RNA immunoprecipitation assays revealed that UGT2B7 mRNA in HepaRG cells and human livers is subjected to m6A modification mainly at the 5' untranslated region (UTR) and secondarily at the 3'UTR. UGT2B7 mRNA and protein levels in Huh-7 cells were significantly increased by double knockdown of methyltransferase-like 3 (METTL3) and METTL14, whereas those were decreased by knockdown of fat mass and obesity-associated protein (FTO) or alkB homolog 5, RNA demethylase (ALKBH5), suggesting that m6A modification downregulates UGT2B7 expression. By experiments using actinomycin D, an inhibitor of transcription, it was demonstrated that ALKBH5-mediated demethylation would attenuate UGT2B7 mRNA degradation, whereas METTL3/METTL14 or FTO-mediated m6A modification would alter the transactivity of UGT2B7. Luciferase assays revealed that the promoter region at -118 to -106 has a key role in the decrease in transactivity of UGT2B7 by FTO knockdown. We found that hepatocyte nuclear factor 4α (HNF4α) expression was significantly decreased by knockdown of FTO, indicating that this would be the underlying mechanism of the decreased transactivity of UGT2B7 by knockdown of FTO. Interestingly, treatment with entacapone, which is used for the treatment of Parkinson's disease and is an inhibitor of FTO, decreased HNF4α and UGT2B7 expression. In conclusion, this study clarified that RNA methylation posttranscriptionally controls hepatic UGT2B7 expression.