The DAF-16 FoxO forkhead transcription factor regulates lifespan in response to changes in the insulin/insulin-like growth factor signalling (IIS) pathway. Microarray studies in long-lived
daf-2 insulin/IGF receptor mutants have implied a role for DAF-16 in many biological processes (e.g. somatic maintenance) that regulate ageing 1,2. However, it is unknown if DAF-16 directly regulates these processes. We therefore set out to identify direct targets of DAF-16, to try to bridge the gap between a single transcription factor and the many downstream genes and processes it regulates. DAF-16 binding in vivo was surveyed using an approach not previously tried in C. elegans: DNA adenine methyltransferase (Dam) identification3. We expressed a chimeric DAF-16::Dam protein in C. elegans, and treated them with
daf-2 RNAi to activate DAF-16. Dam then acts to methylate adenosines in GATC sequences near its binding site. Methylated DNA was then isolated and hybridized to whole genome tiling arrays. We found an enrichment of DAF-16 binding to promoter regions and in genes containing DAF-16 binding elements (DBEs), including genes previous identified using chromatin immmunoprecipitation (ChIP) 4. In total, we identified 907 genes with DAF-16::Dam methylation peaks and interestingly these were significantly enriched for genes up-regulated in
daf-2 mutants (p = 1.4e-11), but not for down-regulated genes. To create a list of higher confidence DAF-16 targets for further study (e.g. ChIP-PCR, longevity assays), we chose 65 genes that were both DAF-16 bound and up-regulated in long-lived
daf-2 mutants. These include several genes where the mammalian orthologs are FoxO regulated (e.g. PEPCK). Against expectation, somatic maintenance genes (e.g. detoxification enzymes, chaperonins) are not over-represented among these 65 genes and the data suggests that DAF-16 does not directly regulate these processes. Instead, we find genes linked to sugar homeostasis and IIS itself (e.g.
ist-1 ,
akt-1 and
akt-2), and regulators of stress resistance, including
skn-1,
sek-1 and AMP kinase. This implies that DAF-16 may act as a master regulator of other regulatory pathways, acting as a central node within several positive feedback loops. 1. McElwee et al. J. Biol. Chem. 279, 44533 (2004). 2. Murphy et al. Nature 424, 277 (2003). 3. van Steensel, Henikoff, Nat Biotechnol 18, 424 (2000). 4. Oh et al., Nat Genet 38, 251 (2006).