Resolving systematic errors in widely-used enhancer activity assays in human cells

The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that two previous observations relating to plasmid-transfection into human cells render such assays unreliable: (1) the function of the bacterial plasmid origin-of-replication (ORI) as conflicting core-promoter and (2) the activation of a type-I-interferon (IFN-I) response. These problems cause strongly confounding false-positives and -negatives in luciferase assays and STARR-seq screens. We overcome both problems by employing the ORI as core-promoter and by inhibiting two IFN-I-inducing kinases. This corrects luciferase assays and enables genome-wide STARR-seq screens in human cells. In HeLa-S3 cells, we uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells, and are key to the characterization of human enhancers.