Signaling and transcriptional regulation of antimicrobial peptide genes in Drosophila melanogaster

Sammanfattning: Insects rely solely on innate immune reactions for protection against infect-ing microbes in their environment. In Drosophila, one major defense mechanism is the production of a battery of antimicrobial peptides (AMPs). The expression of AMPs is primarily regulated at the level of transcription and constitutes both constitutive expression in a tissue-specific manner and inducible systemic expression in response to infection. The aim of my thesis has been to investigate the regulation of AMP gene expression at different levels. I have studied a novel cis-regulatory element, Region 1 (R1) found in the proximal promoter of all Cecropin genes in Drosophila melanogaster, as well as in other species of Drosophila. We found that the R1 element was important for the expression of CecropinA1 (CecA1) both in vitro and in vivo. A signaling-dependent R1-binding activity (RBA) was identified in nuclear extracts from Drosophila cells and flies. The molecular nature of the RBA, has despite considerable effort, not yet been identified. I also have studied the role of the JNK pathway in transcriptional regulation of AMP genes. The role of the JNK pathway in the regulation of AMP genes has long been elusive, however, in this study we showed that the pathway is directly involved in the expression of AMP genes. Analysis of cells mutant for JNK pathway components showed severely reduced AMP gene expression. Fur-thermore, over-expression of a JNK pathway-inhibitor also inhibited AMP gene expression. Lastly, I have studied transcription factors that have not previously been implicated in transcriptional regulation of AMP genes. In a yeast screen, three members of the POU family of transcription factors were identified as regulators of CecA1. Two of them, Drifter (Dfr) and POU do-main protein 1 (Pdm1) were further characterized. We showed that Dfr was able to promote AMP gene expression in the absence of infection, suggest-ing it to play a role in constitutive expression of AMP genes. Indeed, down-regulation of Dfr expression using RNAi severely reduced the constitutive expression of AMP genes in the male ejaculatory duct. We also identified an enhancer element important for Dfr-mediated expression of CecA1. Pdm1, on the other hand, was shown to be important for the systemic expression of AMP genes. In Pdm1 mutant flies, several AMP genes are systemically expressed even in the absence of infection, suggesting that Pdm1 works as a repressor of those genes. However, at least on AMP gene, AttacinA (AttA) requires Pdm1 for its expression, suggesting that Pdm1 works as an activator for this gene. Upon infection, Pdm1 was rapidly degraded, but, regenerated shortly after infection. We propose that the degradation of Pdm1 is important for the activation of the Pdm1-repressed genes and that regeneration sup-ports the expression of AttA.