A review of Genes that Act Downstream of the DAF-16 FOXO Transcription Factor to Influence the Life Span of C. Elegans

Summary

In the work we have described here (Murphy et al. 2003), an unbiased micro-array analysis was used to identify transcriptional targets of the DAF-2/DAF-16 pathway, and the identified genes were tested for their individual contributions to longevity. Because we applied an unbiased method of gene expression profiling of a combination of multiple mutant alleles as well as a time course of RNAi treatment, resulting in data from over 70 arrays, we were able to significantly reduce the number of false positives. We found not only the very few previously identified DAF-16 targets but also many novel targets, and the previously described DBE and a new motif were overrepresented in the promoters of the genes. The diversity of the core set of downstream targets suggests that the coordination of expression through DAF-16 is critical in the regulation of longevity.

DAF-16, a FOXO-family transcription factor, influences the rate of aging of C. elegans in response to insulin/IGF-1 signaling. Using DNA microarray analysis, we found that DAF-16 affects the expression of a set of genes during early adulthood, the time at which this pathway is known to influence aging, and we have shown that many of these genes influence the aging process (Murphy et al. 2003). We also identified a DNA motif, in addition to the canonical DAF-16 motif, that is overrepresented in the promoters of the DAF-16-regulated genes. The insulin/IGF-1 pathway functions cell non-autonomously to regulate life span, and our findings suggest that it signals other cells, at least in part, by feedback regulation of two insulin/IGF-1 homologs.

Our findings suggest that the insulin/IGF-1 pathway ultimately exerts its effect on life span by up-regulating a wide variety of genes, including cellular stress response and anti-microbial genes, fat and steroid hormone synthesis genes, and many genes of unknown function, and by down-regulating specific life-shortening genes. Because the genes seem to act in a cumulative manner to affect life span, this study demonstrates the power of functional microarray analysis for dissecting complex regulatory systems.

C. elegans has proven to be an excellent model system for the study of aging. In addition to its general utility in genetic approaches (Brenner 1974), the worm also displays distinct phenotypes of aging during its short life span, allowing researchers to study long-and short-lived mutants (Johnson 1990; Kenyon et al. 1993; Linet al. 1997; Lakowski and Hekimi 1996; Ewbank et al. 1997; Dillin et al. 2002a) as well as treatments that affect longevity (Lakowski and Hekimi 1998; Melov 2002). Two recent studies (Herndon et al. 2002; Garigan et al. 2002) carefully described the stochastic progression of aging in worms, which is marked by general tissue deterioration and reduction of motility. Age-related changes include sarcopenia, distortion of the cuticle, collapse and bacterial packing of the pharynx, distortion of gonadal nuclei, and the accumulation of fat in droplets in the head (Herndon et al. 2002; Garigan et al. 2002). Many of these phenotypes are reminiscent of human aging, and the fact that the genetic pathways known to affect life span in worms are highly conserved (Kenyon 2001; Guarente and Kenyon 2000) suggests that what we glean through studies of C. elegans aging will shed light on the mechanisms of aging regulation in humans.

Among the genetic mechanisms known to affect aging in C. elegans, the DAF-2/Insulin-IGF-like receptor (IIR) pathway (Kenyon et al. 1993) has perhaps the most dramatic effects and is one of the best studied. daf-2 mutants are not only long-lived, with a life span of two to three times that of wild type worms, but the mutants are also extremely healthy and active much later than wild type (Kenyon et al. 1993). Many of the components of this signaling pathway have been cloned and characterized and include a P13-kinase (age-1; Morris et al. 1996), a PTEN phosphatase (daf-18; Ogg and Ruvkun 1998), and additional kinases (akt-1, akt2, pdk-1, and sgk-1; Paradis et al. 1999; Paradis and Ruvkun 1998; Hertwick et al. 2004). The activation of DAF-2/IIR activates this kinase cascade, culminating in the phosphorylation and nuclear exclusion of the DAF-16/FOXO transcription factor (Lin et al. 1997; Ogg et al. 1997; Lee et al. 2001). The activity of DAF-16/FOXO is required for all of the known phenotypes of daf-2 mutants, including its extended longevity (Kenyon et al. 1993). However, the downstream targets of this transcription factor were largely unknown before the work we will describe here. Additionally, the only mechanism that had been hypothesized to function downstream of DAF-16/FOXO to extend life span involved the mediation of reactive oxygen species (Honda and Honda 1999). This paper will describe the work that we have done to discover these genes and test their roles in longevity. (For additional details and supplementary data, please see Murphy et al. 2003.)