A genome-wide survey reveals that a diverse array of enhancers coordinates the Drosophila innate immune response

To defend against microbes, animals regulate a complex immune response. The Drosophila innate immune system deploys a large transcriptional induction of signaling proteins, antimicrobial effectors, and other critical immune factors. This transcriptional response is encoded in enhancers, cis-regulatory sequences that modulate gene expression by binding transcription factors (TFs). Although enhancers and transcription factor binding sites (TFBSs) have been identified for several immune responsive genes in Drosophila, most enhancers that regulate immune-induced genes are unknown. By identifying enhancers, we can understand how their composition controls expression and contributes to infection outcome. We employ self-transcribing active regulatory-region sequencing (STARR-seq) in a hemocyte-like cell line to identify immune-specific enhancers across the D. melanogaster genome and perform ATAC-seq in hemocytes extracted from adult flies to assess the chromatin state of these enhancers before and after immune stimulus. We identify hundreds of enhancers responsive to IMD stimulation, one of the two primary immune signaling pathways in Drosophila As expected, immune enhancers are enriched for motifs of Relish, an NF-kB factor, and Kay/Jra, a bZip heterodimer pair, involved in the Imd and JNK pathways respectively, compared with enhancers active in unstimulated cells. However, when grouping enhancers by their target gene's expression timing or functional role or by the enhancers' chromatin accessibility pre- or post-stimulus, different groups of TFBS motifs are enriched, suggesting distinct regulatory logic for different parts of the immune response. Identification and characterization of the diverse array of enhancers that regulate the innate immune response expand our understanding of how animals fight infections.