Large-scale analysis of the integration of enhancer-enhancer signals by promoters

Genes are often regulated by multiple enhancers. It is poorly understood how the individual enhancer activities are combined to control promoter activity. Anecdotal evidence has shown that enhancers can combine sub-additively, additively, synergistically, or redundantly. However, it is not clear which of these modes are more frequent in mammalian genomes. Here, we systematically tested how pairs of enhancers activate promoters using a three-way combinatorial reporter assay in mouse cells. By assaying about 69,000 enhancer-enhancer-promoter combinations we found that enhancer pairs generally combine near-additively. This behaviour was conserved across seven developmental promoters tested. Surprisingly, these promoters scale the enhancer signals approximately following a power-law, but the exponent of this response varies between promoters. A housekeeping promoter showed an overall different response to enhancer pairs, and a smaller dynamic range. Thus, our data indicate that enhancers mostly act additively, but promoters transform their collective effect non-linearly. Per EEP library 1 iPCR sample was prepared and sequenced paired end. The Klf2 library iPCR was sequenced twice, first on a MiSeq and then on a NovaSeq 6000. 1 pDNA sample was prepared per EEP library. For cDNA samples 3 Biological replicates were performed for each EEP library. All pDNA and cDNA samples were sequenced twice on a NovaSeq 6000