Heterogeneous NF-κB activation and enhancer features shape transcription in Drosophila immunity

reporter cells as an immune-responsive model to monitor the dynamics of an NF-κB transcription factor, Relish, and downstream transcription in single cells. After immune stimulation, Relish exhibits diverse nuclear localization dynamics, with both the fraction of responsive cells and the speed of Relish activation increasing with stimulus dose. Prestimulus features, including the amount of nuclear Relish under basal conditions, predict a cell's responsiveness to stimulation. Simultaneous measurement of Relish and downstream transcription revealed that the probability of transcriptional bursts from immune-responsive enhancers correlates with Relish nuclear fraction. Enhancers containing more κB binding sites have a higher likelihood of activating at the population level. Our study uncovers heterogeneity in NF-κB activation and target gene expression within Drosophila, illustrating how dynamic NF-κB behavior and enhancer architecture tune gene regulation.