Abstract
The CYP3A4 enzyme is the most abundant drug-metabolizing enzyme in the liver, metabolizing ~50% of commonly used medications. CYP3A4 displays large interperson variability in expression and enzyme activity with unknown causes. This study aims to identify cis-acting regulatory elements controlling the transcription of CYP3A4, using chromatin conformation capture (4C and 3C assays), chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR), clustered regularly interspaced short palindromic repeats (CRISPR)-mediated deletions of genomic regions and reporter gene assays in primary culture human hepatocytes and hepatic cell lines. 4C assays identified four regions (R1-R4) interacting with the CYP3A4 promoter, one of which overlaps with the previously identified upstream enhancers CLEM4/XREM (R2) while the other three are novel. ChIP-qPCR, reporter gene assays and CRISPR-mediated deletion experiments indicate regulatory roles for both R2 and R4. Interestingly, the deletion of R4 increased CYP3A4 while decreasing CYP3A43 expression, possibly due to competitive domain-domain interactions within the CYP3A cluster, supported by deletion of R4 increasing interaction between the CYP3A4 promoter and R2. We also identified a single nucleotide polymorphism rs62471956 within R4, with the variant allele A having increased transcriptional activity in a reporter gene assay. The rs62471956 A allele is associated with higher CYP3A43 expression and lower CYP3A4 expression in a cohort of 136 liver samples, further supporting the opposing effects of R4 on CYP3A4 and CYP3A43. rs62471956 is in complete linkage disequilibrium with CYP3A4*22, potentially contributing to reduced expression of CYP3A4*22. These results validate previously identified enhancers (CLEM4 and XREM) of CYP3A4 and demonstrate additional regulatory mechanisms underlying CYP3A4 transcriptional control via competitive domain-domain interactions within the CYP3A cluster.