Overexpression nub-RB and Pdm2-RB. Drosophila melanogaster

The intricate regulation of gut homeostasis is dependent on a fine-tuned regulation of the host defense to avoid detrimental immune activation. We have previously applied Drosophila to show that mutants of the short isoform of the POU transcription factor Pdm1/nubbin (nub) have a constitutively active immune response, altered gut microbiota and shortened lifespan. Here, we studied the role of Nub-PB, the larger isoform derived from the nub gene. Both Nub protein isoforms contain the C-terminal POU and Homeo domains whereas Nub-PB encompasses a longer N-terminal end. Microarray analysis revealed that targeted misexpression of Nub-PB in immunocompetent organs triggers a strong and broad expression of immune genes in a comparable manner to Nub-PD mutants. The seemingly antagonistic functions of the two isoforms were confirmed by co-overexpression of both forms, which resulted in normal expression levels of antimicrobial peptide (AMP) genes. Cell line experiments demonstrated a synergistic activation of AMPs by the combined transfection with Nub-PB and NF-kB/Relish. In agreement with this finding, Relish was indispensable for Nub-PB-driven diptericin expression, suggesting that Nub-PB acts as a Relish co-activator. Moreover, Nub-PB triggered strong expression of gut-specific drosomycin-like genes in a JAK/STAT pathway-dependent manner. Using RNA interference, we found that Nub-PB influences antimicrobial peptide expression in gut but not fat body tissues. Moreover, Nub-PB expression levels in gut enterocytes correlated with the gut bacterial load as well as host lifespan, suggesting a profound role in host immunity. Surprisingly, Nub-PB-overexpression caused a hypersensitive infection phenotype as flies succumbed within 24 h to orally administered Erwinia carotovora carotovora 15. Nub-PB thus appears to be a central activator of gut immunity that requires tight control, executed at least in part, by isoform antagonism to maintain immune homeostasis. Overall design: Overexpression of nub-RB and Pdm2-RB, was carried out in flies of the genotype w1118;UAS-nub-RB/c564-Gal4 and w1118;UAS-Pdm2-RB/c564-Gal4 respectively, and compared with flies carrying the c564-Gal4 driver but no UAS target gene (w1118;c564-Gal4/+), called w1118 in the data. Flies were reared in mixed gender populations and kept at 25°C for moderate overexpression. Female flies were used at 10 days of age, dissected to separate guts (“gut”) and the rest (flies without guts and heads). Total RNA was extracted followed by DNAse treatment, RNA purification, labeling and hybridization.