Down-regulation of NF-kappaB target genes by the AP-1 and STAT complex during the innate immune response in Drosophila.

The activation of several transcription factors is required for the elimination of infectious pathogens via the innate immune response. The transcription factors NF-kappaB, AP-1, and STAT play major roles in the synthesis of immune effector molecules during innate immune responses. However, the fact that these immune responses can have cytotoxic effects requires their tight regulation to achieve restricted and transient activation, and mis-regulation of the damping process has pathological consequences. Here we show that AP-1 and STAT are themselves the major inhibitors responsible for damping NF-kappaB-mediated transcriptional activation during the innate immune response in Drosophila. As the levels of dAP-1 and Stat92E increase due to continuous immune signaling, they play a repressive role by forming a repressosome complex with the Drosophila HMG protein, Dsp1. The dAP-1-, Stat92E-, and Dsp1-containing complexes replace Relish at the promoters of diverse immune effector genes by binding to evolutionarily conserved cis-elements, and they recruit histone deacetylase to inhibit transcription. Reduction by mutation of dAP-1, Stat92E, or Dsp1 results in hyperactivation of Relish target genes and reduces the viability of bacterially infected flies despite more efficient pathogen clearance. These defects are rescued by reducing the Relish copy number, thus confirming that mis-regulation of Relish, not inadequate activation of dAP-1, Stat92E, or Dsp1 target genes, is responsible for the reduced survival of the mutants. We conclude that an inhibitory effect of AP-1 and STAT on NF-kappaB is required for properly balanced immune responses and appears to be evolutionarily conserved.

Precocious expression of Drosophila Rbp9 inhibits ovarian germ cell proliferation.

We previously reported that Rbp9 is expressed in the germarium region as soon as germ cells complete mitosis and form 16 interconnected cystocytes. Mutation of Rbp9 caused over-proliferation of cystocytes and generated an ovarian tumor phenotype. This led us to conclude that Rbp9 either inhibits cell proliferation or is required for cell differentiation. To examine the role of Rbp9, we over-expressed it ectopically in germline stem cells and early stage cystocytes that had not yet formed 16 cell clusters. The egg chambers of the newly eclosed transgenic flies looked normal. However, on day 12, most of the developing egg chambers had disappeared and only the germarium region remained. Staining of the Rbp9 over-expressing transgenic ovaries with mitosis markers revealed almost no mitotic divisions in the transgenic germaria. Further examination of these cystocytes with HTS [hu-li tai shao protein], which labels the fusome structure, revealed that the cells in the germarium region consisted of stem cells and cystocytes that had not yet completed four mitotic divisions. These observations suggest that precocious expression of Rbp9 inhibits cystocyte proliferation. Therefore the precise onset of Rbp9 expression in germarium region 2a is critical for inhibiting their further proliferation and achieving their correct differentiation.

Expression of Rbp9 during mid-oogenesis induces apoptosis in egg chambers.

The Drosophila RNA binding protein RBP9 is expressed in both ovarian germline cells and neuronal cells of the adult central nervous system. Expression is limited to postmitotic cells in both cases. It has been suggested that Rbp9 is involved in the regulation of genes that function in cell proliferation or differentiation. We examined the effect of ectopic expression of Rbp9 in germline and somatic cells using the Gal4 system. Over-expression of Rbp9 in various tissues caused severe developmental defects. In particular, expression during mid-oogenesis using nos-Gal4:VP16 caused apoptosis in stage 10 egg chambers. A similar phenotype has been reported when one of the effector caspases of Drosophila, Dcp-1, is ectopically expressed during mid-oogenesis with the same Gal4 driver. Tight control of Rbp9 transcription seems to be critical for the proper development of Drosophila.