The search for DAF-16/FOXO transcriptional targets: Approaches and discoveries
Introduction
The Caenorhabditis elegans FOXO transcription factor, DAF-16, is controlled by the activity of the DAF-2 insulin receptor, repressing DAF-16 activity through phosphorylation and cytoplasmic retention (Lin et al., 2001, Henderson and Johnson, 2001). In the absence of DAF-2/insulin receptor signaling, DAF-16/FOXO moves into the nucleus and regulates transcription of its targets. The identification of these target genes has been the focus of much attention because they may mediate the various phenotypes of daf-2 mutants, including longevity determination (Kenyon et al., 1993), fat metabolism (Lee et al., 2003, Ashrafi et al., 2003), and formation of the diapause state known as dauer (Riddle and Albert, 1997). The genes making up the DAF-16-regulated transcriptome are likely to carry out the biochemical activities necessary for these phenotypes. The roles that DAF-16/FOXO performs in C. elegans may be broadly conserved, as its homologs in flies (dFOXO) and mammalian cells (FOXO3a, FOXO1) are also critical for lifespan (Hwangbo et al., 2004) and cell survival (Brunet et al., 2004). Elimination of the insulin receptor specifically from adipose tissue (the Fat Insulin Receptor KnockOut, or FIRKO, mouse) results in increased lifespan (Bluher et al., 2003), highlighting the importance of the longevity-determining genes regulated by FOXOs in mammals. The identification of FOXO target genes in all of these organisms will help unravel the mechanisms underlying longevity and cell survival. Once the roles of individual genes are elucidated, some may serve as targets for clinical intervention, both for normal aging and for age-related disease.
Section snippets
Approaches used to identify DAF-16 targets
Of all the FOXO transcription factors, most is known about the targets of the C. elegans homolog, DAF-16. Identifying DAF-16 targets through classical genetics is challenging: an EMS screen for suppressors of the dauer-constitutive phenotype of daf-2 in the presence of a daf-16 transgene yielded many alleles of daf-18 (the PTEN phosphatase upstream of daf-16) rather than genes downstream of daf-16 (Lin et al., 2001). This is perhaps less surprising in light of later evidence that the functions
Why might the DAF-16 target sets vary?
It is fair to ask at this point how much overlap exists between the target sets produced by these studies, and if there are differences, what are the sources of this variation? In fact, many of the significantly changed genes have been identified in multiple studies (Murphy et al., 2003, McElwee et al., 2003, McElwee et al., 2004, Halaschek-Wiener et al., 2005), so it is likely that most of the approaches have resulted in valuable data. However, both biological factors and technical
DAF-16 target genes: clues to underlying biological mechanisms
Now that several approaches have identified DAF-16 targets, we can begin to think about how they contribute to daf-16-mediated phenotypes. Many of the DAF-16 targets revealed by genome-wide approaches were expected, as they had already been studied in candidate approaches. Additionally, many are shared with the dauer transcriptome (McElwee et al., 2004, Halaschek-Wiener et al., 2005, Wang and Kim, 2003); this is not surprising, given that daf-2 mutants form dauers constitutively at high
Conclusions
What has been learned by amassing this collection of genes regulated by daf-2 and daf-16? While many of the targets confirm previous hypotheses (protection from a variety of stresses, overlap with dauer genes), many of the biological functions of FOXO/DAF-16 targets remain undiscovered. The vast majority of the genes have not yet been tested for their contributions to daf-2 phenotypes; until those genes are analyzed, it seems premature to claim that the mechanisms by which insulin receptor
Acknowledgements
The author thanks Zemer Gitai, Wendy Shaw, Shijing Luo, and Max Jan for useful comments. Due to space limitations, many relevant studies could not be addressed in this review, and the author apologizes for these omissions.
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