Cis-acting regulatory elements regulating CYP3A4 transcription in human liver.

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 CYP3A422, potentially contributing to reduced expression of CYP3A422. 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.