Over 700 direct transcriptional targets of the dFOXO transcription factor are identified in the adult fruit fly. dFOXO-bound genes are conserved between worm and fly, but dFOXO is not the sole mediator of the transcriptional response to changes in insulin signalling in the fly.
More than 700 direct transcriptional targets of dFOXO were determined in the adult Drosophila female, in the wild-type or an insulin-signalling mutant.dFOXO has an important role in the wild-type fly and is important for transcription of numerous signalling components including TOR and Sos.While dFOXO is an important effector of the insulin signalling pathway, it is required for only a portion of the transcriptional changes that occur in response to alterations in the pathway in the fly, and in many cases indirectly.There is strong evolutionary conservation of dFOXO-bound genes between the worm and the fly, specifically enriched in regulatory genes.
Forkhead Box-O (FoxO) transcription factors are crucial players in numerous cellular and organismal processes including metabolism, stress protection, cellular differentiation, cell-cycle arrest, apoptosis and lifespan. FoxOs are regulated by a number of signalling pathways, including negative regulation by insulin/insulin-like growth factor signalling (IIS) (Partridge and Bruning, 2008; Salih and Brunet, 2008). The fruit fly Drosophila melanogaster has a single FoxO orthologue—dFOXO. dFOXO is capable of extending fly lifespan, as well as being required for lifespan extension in response to downregulation of IIS (Giannakou et al, 2004; Hwangbo et al, 2004; Slack et al, 2011). To further our understanding of dFOXO biology, we uncover over 700 direct dFOXO targets in the adult female fly, both in the wild-type fly and in a mutant with reduced IIS activity.
dFOXO is directly required for transcription of several components of IIS and interacting pathways, such as the gene encoding the target of rapamycin (TOR) kinase, in the wild-type fly. Indeed, the removal of dFOXO results in reduced signal through these pathways. The genomic locations occupied by dFOXO in adults are different from those observed by others in larvae or cultured cells (Puig et al, 2003; Teleman et al, 2008), indicating that dFOXO binding is influenced by developmental stage and/or cell type. These locations remain unchanged upon activation of dFOXO by stresses or reduced IIS in the adult, but the binding of dFOXO to the same sites is increased. Genetically induced reduction in IIS results in activation/repression of a greater number of direct dFOXO targets than observed in the wild-type fly.
To determine the relationship between dFOXO and IIS in the adult fly, we identify the part of the IIS transcriptional response that is controlled by dFOXO, both directly and indirectly. We observe that aspects of the transcriptional response to changes in IIS can take place in the absence of dFOXO, indicating that other transcriptional regulators must be involved. This is different to the situation in the worm Caenorhabditis elegans where the worm FoxO, encoded by the daf-16 gene, is required for all the effects of a reduction in IIS (Kenyon et al, 1993; Gems et al, 1998; Murphy et al, 2003). On the other hand, the existence of dFOXO-independent effects is in accordance with genetic experiments in the fly where lifespan extension and xenobiotic resistance caused by a reduction in IIS are dependent on dFOXO, while lowered fecundity and body size, delayed development and resistance to paraquat are not (Slack et al, 2011). Promoter analyses revealed GATA and other forkhead factors as candidate mediators of the dFOXO-independent effects in the fly.
Despite the different topology of the transcriptomic response to IIS changes in the two organisms, there is genome-wide evolutionary conservation of dFOXO targets between the fly and the worm (Figure 9), enriched for a second tier of regulators including the dHR96/daf-12 nuclear hormone receptor.
FoxO transcription factors, inhibited by insulin/insulin-like growth factor signalling (IIS), are crucial players in numerous organismal processes including lifespan. Using genomic tools, we uncover over 700 direct dFOXO targets in adult female Drosophila. dFOXO is directly required for transcription of several IIS components and interacting pathways, such as TOR, in the wild-type fly. The genomic locations occupied by dFOXO in adults are different from those observed in larvae or cultured cells. These locations remain unchanged upon activation by stresses or reduced IIS, but the binding is increased and additional targets activated upon genetic reduction in IIS. We identify the part of the IIS transcriptional response directly controlled by dFOXO and the indirect effects and show that parts of the transcriptional response to IIS reduction do not require dfoxo. Promoter analyses revealed GATA and other forkhead factors as candidate mediators of the indirect and dfoxo-independent effects. We demonstrate genome-wide evolutionary conservation of dFOXO targets between the fly and the worm Caenorhabditis elegans, enriched for a second tier of regulators including the dHR96/daf-12 nuclear hormone receptor.