Enhancer control of promoter activity and variability via frequency modulation of clustered transcriptional bursts

Gene expression in mammalian cells is controlled by enhancers that are often dispersed across large cis -regulatory landscapes around a promoter. Yet how enhancers determine transcription of their target genes, and how this depends on their relative position inside a cis -regulatory landscape remains unclear. Here we use live-cell imaging to track the activity of a promoter under the control of the same enhancer, but inserted at different positions across a simplified regulatory landscape with minimal complexity. Combined with mathematical modeling, this reveals that RNA production from the promoter occurs in clusters of transcriptional bursts, with enhancer position controlling the frequency at which such clusters appear. This results in bursts being more frequent and occurring more uniformly across cells when the enhancer is genomically close to the promoter than when it is located at large genomic distance. Mathematical modeling further indicates that the enhancer modulates the promoter’s ability to transition from its basal transcriptional state to a regime where clusters of bursts become more frequent. Our results challenge existing models of mammalian promoter operation, and reveal that enhancer position within a cis -regulatory landscape critically controls the timing and variability of transcriptional output in single cells.