Simultaneous measurement of intrinsic promoter and enhancer potential reveals principles of functional duality and regulatory reciprocity

Abstract

Accumulating evidence indicates that both promoters and enhancers are capable of exerting promoter and enhancer functions; however, the relationship between these two activities within individual elements and the determinants of this dual functionality remain poorly understood. We developed a massively parallel dual reporter assay that enables simultaneous assessment of the intrinsic promoter and enhancer potential exerted by the same sequence. Parallel quantification for thousands of elements reveals that canonical human promoters and enhancers can act as both promoters and enhancers under the same contexts, and that promoter activity may be necessary but not sufficient for enhancer function. Perturbations to element transcription factor binding motifs lead to disruptions in both activities, implicating a shared syntax for the two regulatory functions. Combinations of elements with different minimal promoters reveal reciprocal activity modulation, which, together with a strong correlation between promoter and enhancer functions, imply a bidirectional feedback loop to sustain environments of high transcriptional activity. Finally, we validate this reciprocity and correlation in situ using CRISPR activation at the human {beta}-globin locus. Our approach reveals that the functional convergence between promoters and enhancers arises from a shared regulatory logic and sequence syntax, advancing a unified model for regulatory element biology.