Abstract:

Canonical models suggest that regulatory enhancers function independently of genomic distance, position, and orientation to activate the target gene. Indeed, a wide range of genomic distances and relative enhancer positions exist in endogenous contexts. However, systematic analysis of how specific enhancer-promoter configurations affect transcriptional dynamics has been limited by technical constraints. Here, we employ single-cell MS2/MCP-based live imaging in Drosophila embryos to systematically analyze transcriptional dynamics driven by varying enhancer-promoter configurations. Our analyses reveal that while linear enhancer-promoter distance moderately tunes transcriptional output, downstream enhancer positioning relative to the promoter reduces mRNA output by 70%. Each configuration modulates distinct transcriptional parameters: linear distance governs initiation kinetics, while relative enhancer positioning dictates transcriptional stability. Although the downstream enhancer positioning destabilized the enhancer-promoter interactions, transcription factor hub properties remained comparable between two configurations, revealing a clear dissociation between TF hub dynamics and E-P configuration-dependent transcriptional activity. These effects are consistent across varied enhancer and reporter sequences, revealing configuration-dependent cis-regulatory element arrangement as an intrinsic mechanism for transcriptional fine-tuning. This work challenges the paradigm of configuration-independent enhancer function and establishes a framework to dissect the interplay between genome architecture and trans-acting factors.