Guttman, Y.; Krupkin, B.; Ahituv, N.

Massively parallel reporter assays (MPRAs) enable high-throughput functional testing of thousands of cis-regulatory elements (CREs). However, conventional MPRAs typically use short CRE fragments combined with minimal promoters, which limits their ability to capture native regulatory interactions. Here, we present a modified MPRA framework that assays CREs together with their target promoter. We apply this approach to CYP3A4, a major pharmacogene with extensive interindividual expression variability. We assayed 1,214 variants across six CYP3A4 regulatory regions, integrating variants from global populations, cancer genomes, and archaic humans. Most variants exerted modest to no effects on regulatory activity, consistent with strong functional constraint at the CYP3A4 locus. However, we identified a subset of variants with significant regulatory impact, including variants that could be associated with altered drug response and cancer severity. Together, these results provide a comprehensive functional map of CYP3A4 regulatory variation and establish a generalizable MPRA strategy for studying enhancer-promoter interactions.