Stabilization of DNA G-quadruplexes on the 5'-flanking region of CYP3A4 promotes transcription.

G-quadruplexes (G4s) are higher-order nucleic acid structures in genomic DNA that stack two or more G-quartets formed by Hoogsteen base pairing between four guanines. These structures play a critical regulatory role in transcription. We noticed potential G4-forming sequences in the 5'-flanking region of the gene encoding cytochrome P450 3A4 (CYP3A4), an enzyme that contributes most significantly to drug metabolism in humans. In the present study, we investigated the effects of these G4s on CYP3A4 expression. Using a qPCR stop assay and a chromatin immunoprecipitation (ChIP) assay with a G4-specific antibody, we found that three guanine-rich sequences in this region could indeed form a G4 structure. Treatment with the G4 stabilizer pyridostatin increased CYP3A4 expression in HepG2 cells. Formaldehyde-assisted isolation of regulatory elements and ChIP assays demonstrated that pyridostatin-mediated G4 stabilization relaxed the chromatin structure, enhancing the binding of the pregnane X receptor (PXR) to response elements of CYP3A4. Screening of an FDA-approved drug library using a qPCR stop assay identified digoxin as a G4 stabilizer of CYP3A4, which induces CYP3A4 expression, even though it is not a PXR ligand. These findings demonstrate that G4s on CYP3A4 can enhance transcription by relaxing the chromatin structure. Drugs currently in clinical use and new chemical entities capable of modulating G4 formation may cause drug-drug interactions with CYP3A4 substrates through a novel mechanism that alters CYP3A4 expression.