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Conserved NF-κB signaling pathways shape immune responses in animals. In mammals, NF-κB activation patterns and downstream transcription vary with stimulus, cell type, and stochastic differences among identically treated cells. Whether animals without adaptive immunity exhibit similar heterogeneity or rely on distinct immune strategies remains unknown. We engineered Drosophila melanogaster S2* reporter cells as an immune-responsive model to monitor the dynamics of an NF-κB transcription factor, Relish, and downstream transcription in single, living cells. Following immune stimulation, Relish exhibits diverse nuclear localization dynamics that fall into distinct categories, with both the fraction of responsive cells and their activation speed rising with stimulus dose. Pre-stimulus features, including Relish nuclear fraction, 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. The number of NF-κB binding sites tunes transcriptional activity among immune enhancers. 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. 

Innate immune priming increases an organism’s survival of a second infection after an initial, non-lethal infection. We usedDrosophila melanogasterand an insect-derived strain ofEnterococcus faecalisto study transcriptional control of priming. In contrast to other pathogens, the enhanced survival in primed animals does not correlate with decreasedE.faecalisload. Further analysis shows that primed organisms tolerate, rather than resist infection. Using RNA-seq of immune tissues, we found many genes were upregulated in only primed flies, suggesting a distinct transcriptional program in response to initial and secondary infections. In contrast, few genes continuously express throughout the experiment or more efficiently re-activate upon reinfection. Priming experiments in immune deficient mutants revealed Imd is largely dispensable for responding to a single infection but needed to fully prime. Together, this indicates the fly’s innate immune response is plastic—differing in immune strategy, transcriptional program, and pathway use depending on infection history. 

Transcription is controlled by interactions of cis -acting DNA elements with diffusible trans -acting factors. Changes in cis or trans factors can drive expression divergence within and between species, and their relative prevalence can reveal the evolutionary history and pressures that drive expression variation. Previous work delineating the mode of expression divergence in animals has largely used whole-body expression measurements in one condition. Because cis -acting elements often drive expression in a subset of cell types or conditions, these measurements may not capture the complete contribution of cis -acting changes. Here, we quantify the mode of expression divergence in the Drosophila fat body, the primary immune organ, in several conditions, using two geographically distinct lines of D. melanogaster and their F1 hybrids. We measured expression in the absence of infection and in infections with Gram-negative S. marcescens or Gram-positive E. faecalis bacteria, which trigger the two primary signaling pathways in the Drosophila innate immune response. The mode of expression divergence strongly depends on the condition, with trans -acting effects dominating in response to Gram-negative infection and cis -acting effects dominating in Gram-positive and preinfection conditions. Expression divergence in several receptor proteins may underlie the infection-specific trans effects. Before infection, when the fat body has a metabolic role, there are many compensatory effects, changes in cis and trans that counteract each other to maintain expression levels. This work shows that within a single tissue, the mode of expression divergence varies between conditions and suggests that these differences reflect the diverse evolutionary histories of host–pathogen interactions. 

In all higher organisms, life begins with a single cell. During the early stages of development, this single cell grows and divides multiple times to develop into the many different kinds of cells that make up an organism. This is a highly regulated process during which cells receive instructions telling them what kind of cell to become. These instructions are relayed via genes, and a particular combination of activated genes determines the cell’s fate. Specific pieces of DNA, known as enhancers, act as switches that control when and where genes are active, while so-called shadow enhancers are found in groups and work together to turn on the same gene in a similar way. Shadow enhancers are often active during the early stages of life to direct the formation of specialized cells in different parts of the body. But so far, it has been unclear why it is beneficial to the divide the role of activating genes across several shadow enhancers rather than a single one. Here, Waymack et al. examined shadow enhancers around a gene called Kruppel in embryos of the fruit fly Drosophila melanogaster . Manipulating the shadow enhancers showed that they help to make gene activity more resistant to changes. Factors such as fluctuations in temperature have different effects on each shadow enhancer. Having several shadow enhancers working together ensures that, whatever happens, the right genes still get activated. For genes like Kruppel , which are key for healthy development, the ability to withstand unexpected changes is a valuable evolutionary benefit. The study of Waymack et al. reveals why shadow enhancers are involved in the regulation of many genes, which may help to better understand developmental defects. Many conditions caused by such defects are influenced by both genetics and the environment. Genetic illnesses can vary in severity, which may be related to the roles of shadow enhancers. As such, studying shadow enhancers could lead to new approaches for treating genetic diseases.