DNA-binding-independent mechanisms of metabolic regulation by the Drosophila FOXO transcription factor

Forkhead box-O (FOXO) transcription factors are evolutionarily conserved regulators of several biological processes, including development, stress responses, metabolism and ageing. As downstream effectors of nutrient-dependent cell signalling pathways, including insulin/IGF signalling, they integrate signals from multiple stimuli to orchestrate appropriate transcriptional responses to changes in the nutritional environment. Traditionally, FOXO-dependent responses have been attributed to target gene regulation through direct interactions with regulatory regions by DNA-binding via the conserved Forkhead (FH) domain. However, emerging evidence suggests that FOXO proteins may also influence gene expression through DNA-binding-independent mechanisms. However, differences in transcriptional outputs between DNA-binding dependent and independent FOXO functions have yet to be explored. Here, we have used genomic engineering of the endogenous Drosophila foxo locus to disrupt the DNA-binding activity of the single fly FOXO orthologue, allowing us to dissect the in vivo contributions of canonical and non-canonical dFOXO functions. We show that while DNA-binding is essential for several dFOXO-mediated phenotypes including female fecundity, lifespan, and resistance to oxidative and xenobiotic stress, other traits such as adult body size and survival during starvation remain intact. Notably, DNA-binding-deficient dFOXO flies exhibit defective lipid mobilisation under starvation, implicating a DNA-binding-independent role for dFOXO in metabolic regulation. Differential gene expression analysis during starvation in these mutants revealed key transcriptional changes in genes encoding metabolic regulators as well as regulators of transcription and chromatin structure. Together, these findings reveal distinct modes of dFOXO transcriptional regulation that depend on its direct association with DNA.