Differential integration of activation and repression signals in a multi-enhancer system

Transcription in the early Drosophila blastoderm is coordinated by the collective action of hundreds of enhancers. Many genes are controlled by so-called “shadow enhancers,” which provide resilience to environment or genetic insult, allowing the embryo to robustly generate a precise transcriptional pattern. Emerging evidence suggests that many shadow enhancer pairs do not drive identical expression patterns, however the biological significance of this remains unclear. In this study we characterize the shadow enhancer pair controlling the gene short gastrulation ( sog ). We removed either the intronic proximal enhancer or the upstream distal enhancer, and monitored sog transcriptional kinetics. Notably, each enhancer differs in sog spatial expression, timing of activation, and RNA Polymerase II loading rates. Additionally, modeling of individual enhancer activities demonstrates that these enhancers integrate activation and repression signals differently. While activation is due to the sum of the two enhancer activities, repression appears to depend on synergistic effects between enhancers. Finally, we examined the downstream signaling consequences resulting from the loss of either enhancer, and found changes in tissue patterning that are well explained by the differences in transcriptional kinetics measured. SUMMARY STATEMENT Non-intuitive shadow enhancer synergies are revealed by measuring transcriptional kinetics at the endogenous short gastrulation locus, giving rise to distinct patterning consequences in the dorsal ectoderm of Drosophila embryos. ### Competing Interest Statement The authors have declared no competing interest.