Distal enhancers play pivotal roles in development and disease yet remain one of the least understood regulatory elements. We used massively parallel reporter assays to perform functional comparisons of two leading enhancer models and find that gene-distal transcription start sites are robust predictors of active enhancers with higher resolution than histone modifications. We show that active enhancer units are precisely delineated by active transcription start sites, validate that these boundaries are sufficient for capturing enhancer function, and confirm that core promoter sequences are necessary for this activity. We assay adjacent enhancers and find that their joint activity is often driven by the stronger unit within the cluster. Finally, we validate these results through functional dissection of a distal enhancer cluster using CRISPR–Cas9 deletions. In summary, definition of high-resolution enhancer boundaries enables deconvolution of complex regulatory loci into modular units.

远端增强剂在发育和疾病中发挥关键作用，但仍然是最不为人知的调节元件之一。我们使用大规模平行报告基因分析对两个主要增强子模型进行功能比较，发现基因远端转录起始位点是活性增强子的可靠预测因子，其分辨率高于组蛋白修饰。我们表明，活性增强子单元由活性转录起始位点精确描绘，验证这些边界足以捕获增强子功能，并确认核心启动子序列对于该活动是必需的。我们分析了相邻的增强子，发现它们的联合活动通常是由集群中更强的单元驱动的。最后，我们通过使用 CRISPR-Cas9 缺失对远端增强子簇进行功能解剖来验证这些结果。总之，高分辨率增强子边界的定义能够将复杂的调控位点反卷积成模块化单元。