Promoter-proximal gatekeepers restrict pleiotropic enhancer inputs to achieve tissue specificity

Developmental enhancers are central regulatory elements that can activate multiple genes, yet how they selectively regulate one gene over its neighbours remains unclear. Using the Drosophila twist E3 enhancer as a model, we show that this selectivity is encoded within promoter-proximal regions of target genes. We found that the E3 enhancer is pleiotropic, activating at least four functionally unrelated genes. Despite receiving the same enhancer input each target gene displays distinct and non-overlapping expression patterns. These differences cannot be attributed to context-dependent chromatin looping. Instead, we demonstrate that they arise from the action of each genes’ promoter-proximal sequence. These sequences act as “gatekeepers” that restrict enhancer input into precise tissue- and stage-specific transcription, thus functioning as active interpreters rather than passive recipients of enhancer signals. We propose that promoter-proximal gatekeepers provide a critical but under-appreciated layer of regulatory specificity within complex gene expression programs.