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 such assays are rendered unreliable by two previously reported phenomena relating to plasmid transfection into human cells: (i) the bacterial plasmid origin of replication (ORI) functions as a conflicting core promoter and (ii) a type I interferon (IFN-I) response is activated. These cause 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, enabling 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.

鉴定人类基因组中的转录增强子是生物学的主要目标。增强子通常通过染色质标记进行预测，但它们的功能主要通过基于质粒的报告分析来评估。在这里，我们表明，由于先前报道的两种与质粒转染人类细胞有关的现象，此类测定变得不可靠：（i）细菌质粒复制起点（ORI）充当冲突的核心启动子和（ii）I型干扰素（ IFN-I) 反应被激活。这些在荧光素酶测定和 STARR-seq 筛选中导致混淆的假阳性和阴性。我们通过使用 ORI 作为核心启动子并通过抑制两种 IFN-I 诱导激酶来克服这两个问题，从而在人类细胞中实现全基因组 STARR-seq 筛选。在 HeLa-S3 细胞中，我们发现了强增强子、IFN-I 诱导的增强子、和增强子在染色质水平内源性沉默。我们的研究结果适用于哺乳动物细胞中的所有附加型增强子活性测定，并且是表征人类增强子的关键。