TI - dFOXO upregulates transcription of the d4E-BP gene . AB - We have shown previously that Drosophila embryonic Kc167 cells respond to insulin stimulation with upregulated activities of dPKB and dS6K [53 , 54] . We performed mRNA profiling experiments using the Affymetrix GeneChip system to measure on a genome -wide scale the transcriptional changes induced by insulin in these cells . On the basis of the currently held model that FOXO transcription factors are transcriptional activators that are negatively regulated by insulin , we expected potential dFOXO target genes to be repressed in Kc167 cells upon insulin stimulation . Figure 6a shows a selection of dFOXO target gene candidates that are transcriptionally downregulated by a factor of two or more upon insulin stimulation and whose promoter regions contain one or more conserved forkhead-response elements ( FHREs ) with the consensus sequence (G/A) TAAACAA [55] . Three of these candidate gene products are each involved in one of two biological processes known to be negatively regulated by insulin , namely gluconeogenesis ( PEPCK ) and lipid catabolism ( CPTI and long-chain-fatty-acid-CoA-ligase ) . The remaining candidates are involved in stress responses ( cytochrome P450 enzymes ) , DNA repair ( DNA polymerase iota ) , transcription and translation control ( d4E-BP and CDK8 ) , and cell -cycle control ( centaurin gamma and CG3799 ) . Several of the insulin-repressed genes have been reported to be transcriptionally induced in Drosophila larvae under conditions of complete starvation ( d4E-BP and PEPCK ) or sugar-only diet ( CPTI and long-chain-fatty-acid-CoA-ligase ) [41,56] . We chose d4E-BP for further investigation , because it has previously been reported to be insulin-regulated at the level of protein PHOSphorylation , but not at the level of gene expression [57] . The d4E-BP gene encodes a translational repressor and was initially identified as the immune-compromised Thor mutant in a genetic screen for genes involved in the innate immune response to bacterial infection [58,59] . Figure 6b shows the presence of several FHREs in the genomic region around the d4E-BP locus . The d4E-BP protein is negatively regulated by insulin through LY294002 - and rapamycin-sensitive PHOSphorylation [57] , suggesting involvement of the Dp110 and dTOR signaling pathways . PHOSphorylation of d4E-BP leads to the dissociation of d4E-BP from its binding partner , the translation initiation factor deIF4E , which then participates in the formation of a functional initiation complex . Positive transcriptional regulation of d4E-BP by dFOXO , which corresponds to negative transcriptional regulation by insulin , would be a complementary mechanism of regulation . We then investigated whether overexpression of endogenous dFOXO could induce transcriptional upregulation of the d4E-BP gene . On the basis of our overexpression results , we chose the Dp110DN-dFOXO coexpression to efficiently activate dFOXO . Eye imaginal discs from Dp110DN-expressing third instar larvae display a low level of basal d4E-BP transcription throughout the disc , which is not induced by the driver construct alone ( Figure 6d ) . Coexpression of dFOXO elicited a dramatic upregulation of d4E-BP transcription posterior to the morphogenetic furrow ( Figure 6e ) . Consistent with this observation , we were able to induce expression of the d4E-BP enhancer trap line Thor1 with human FOXO3a-TM ( Figure 6f-h ) . It remained unclear , however , whether regulation of d4E-BP expression by dFOXO is of physiological relevance . It has been previously reported that overexpression of d4E-BP partially suppresses the dPKB overexpression phenotype [57] , but as ectopic expression experiments have to be interpreted with some caution , we assessed whether loss of d4E-BP function suppresses the cell -number reduction in insulin-signaling mutants as does loss of dFOXO function . We generated double-mutant flies for dPKB and d4E-BP and observed that the Thor1 mutation slightly but significantly suppressed the reduced cell -number phenotype in a dose -dependent manner . The Thor1 mutation itself had no effect on ommatidial number compared to wild-type flies ( data not shown ) , so we can rule out additive effects of d4E-BP and dPKB . These observations strongly argue that under conditions of reduced insulin-signaling activity the dFOXO -dependent reduction in cell number is in part mediated by the transcriptional upregulation of its target d4E-BP . Microarray studies in both mammalian [23] and Drosophila [52] cells imply that FOXO transcription factors exert their physiological functions by modulating expression of large sets of target genes .