Chromobox 4 interacts with pregnane X receptor to repress CYP3A4 expression through polycomb repressive complex 1/2-mediated epigenetic regulation.

Polycomb repressive complexes (PRCs) 1 and 2 are epigenetic regulators that suppress gene transcription through histone H3 lysine K27 trimethylation. This modification, catalyzed by enhancer of zeste homolog 2 (EZH2) within PRC2, is recognized by chromobox (CBX) proteins within PRC1, leading to chromatin condensation and transcriptional repression. Although EZH2 and CBX inhibitors have been developed for cancer therapy, the role of PRC1/2 inhibition in the regulation of drug metabolism-related genes remains unclear. In this study, we investigated the regulatory mechanisms of CYP3A4 expression by PRC1/2. Treatment with GSK126, an EZH2 inhibitor, significantly increased CYP3A4 mRNA and protein levels in HepaSH cells and ShP51 cells. Among CBX family members, CBX4 knockdown increased CYP3A4 mRNA levels, whereas its overexpression suppressed them in ShP51 cells. Consistently, CBX4 knockdown enhanced rifampicin-induced CYP3A4 expression, whereas its overexpression attenuated this induction. A coimmunoprecipitation assay revealed that CBX4 interacts with pregnane X receptor (PXR) regardless of rifampicin treatment. Chromatin immunoprecipitation analysis demonstrated that both CBX4 and PXR bind to the promoter and enhancer regions of CYP3A4, and that CBX4 knockdown increases PXR binding. Formaldehyde-assisted isolation of regulatory elements assays showed that CBX4 knockdown relaxed chromatin structure at the CYP3A4 locus, which is further enhanced by rifampicin. Collectively, these findings indicate that CBX4 interacts with PXR and likely represses CYP3A4 transcription through recognition of EZH2-mediated trimethylation of lysine 27 on histone H3 and chromatin condensation, highlighting a potential risk of drug-drug interactions associated with CBX4 inhibition. SIGNIFICANCE STATEMENT: This study provides, to our knowledge, the first evidence of epigenetic mechanisms underlying CYP3A4 suppression mediated by PRC1/2. The findings demonstrate that CBX4 interacts with PXR and binds to the CYP3A4 regulatory regions, where it recognizes enhancer of zeste homolog 2-mediated trimethylation of lysine 27 on histone H3 and represses CYP3A4 transcription via chromatin condensation. Given the increasing clinical interest in CBX inhibitors for cancer therapy, these findings highlight a potential risk of drug-drug interactions associated with CBX4 inhibition.