Longevity Genes Revealed by Integrative Analysis of Isoform-Specific daf-16/FoxO Mutants of Caenorhabditis elegans

Abstract

FoxO transcription factors promote longevity across taxa. How they do so is poorly understood. In the nematode Caenorhabditis elegans, the A- and F-isoforms of the FoxO transcription factor DAF-16 extend life span in the context of reduced DAF-2 insulin-like growth factor receptor (IGFR) signaling. To elucidate the mechanistic basis for DAF-16/FoxO-dependent life span extension, we performed an integrative analysis of isoform-specific daf-16/FoxO mutants. In contrast to previous studies suggesting that DAF-16F plays a more prominent role in life span control than DAF-16A, isoform-specific daf-16/FoxO mutant phenotypes and whole transcriptome profiling revealed a predominant role for DAF-16A over DAF-16F in life span control, stress resistance, and target gene regulation. Integration of these datasets enabled the prioritization of a subset of 92 DAF-16/FoxO target genes for functional interrogation. Among 29 genes tested, two DAF-16A-specific target genes significantly influenced longevity. A loss-of-function mutation in the conserved gene gst-20, which is induced by DAF-16A, reduced life span extension in the context of daf-2/IGFR RNAi without influencing longevity in animals subjected to control RNAi. Therefore, gst-20 promotes DAF-16/FoxO-dependent longevity. Conversely, a loss-of-function mutation in srr-4, a gene encoding a seven-transmembrane-domain receptor family member that is repressed by DAF-16A, extended life span in control animals, indicating that DAF-16/FoxO may extend life span at least in part by reducing srr-4 expression. Our discovery of new longevity genes underscores the efficacy of our integrative strategy while providing a general framework for identifying specific downstream gene regulatory events that contribute substantially to transcription factor functions. As FoxO transcription factors have conserved functions in promoting longevity and may be dysregulated in aging-related diseases, these findings promise to illuminate fundamental principles underlying aging in animals.

Keywords: C. elegans; FoxO transcription factors; aging; insulin-like growth factor signaling; longevity.

Figure 1

daf-16/FoxO isoforms and isoform-specific mutations. (A) daf-16/FoxO genomic structure with isoform-specific mutations. Colors indicate unique N-terminal exons (green and orange) and Forkhead domains (yellow) that correspond to DAF-16/FoxO protein domains in Figure S1A. (B) Summary of isoform-specific mutant alleles. See text for details.

Figure 2

daf-16a-specific mutations suppress dauer arrest in daf-2/IGFR mutants. (A) The dauer-constitutive phenotype of daf-2(e1368) animals is fully suppressed by daf-16a/f mutation and both daf-16a mutations but is unaffected by daf-16f mutation. (B) The dauer-constitutive phenotype of daf-2(e1370) animals is fully suppressed by daf-16a/f mutation, partially suppressed by both daf-16a mutations, and unaffected by daf-16f mutation. Mean and standard deviation for at least three biological replicates are presented. Statistics and raw data are presented in Table S2; Table S3; Table S4.

Figure 3

Effects of daf-16a- and daf-16f-specific mutations, RNAi, and single-copy transgenic rescues on daf-2/IGFR life span. (A–D) Effects of daf-16a (A and C) and daf-16f (B and D) mutations on life spans of daf-2(e1368) (A and B) and daf-2(e1370) (C and D). (E and F) daf-16f is required for daf-16a;daf-2 longevity and vice versa. Survival curves are presented for (E) daf-16a;daf-2(e1370) and (F) daf-16f;daf-2(e1370) mutant animals upon exposure to isoform-specific daf-16 RNAi. Figure S7A shows control daf-2(e1370) survival when treated with isoform-specific daf-16 RNAi. (G) Effect of a single-copy daf-16a transgene on daf-16a/f;daf-2(e1370) life span. (H) Effect of a single-copy daf-16f transgene on daf-16a/f;daf-2(e1370) and daf-16a;daf-2(e1370) life span. See text for details. Statistics and raw data are presented in Table S5; Table S6; Table S7; Table S8.

Figure 4

Effects of daf-16a- and daf-16f-specific mutations and RNAi on life span in animals lacking a germline. (A and B) Effects of daf-16a (A) and daf-16f (B) mutations on life spans of germline-ablated glp-1(e2141) animals. (C and D) daf-16f is required for daf-16a;glp-1 longevity and vice versa. Survival curves are presented for (C) daf-16a;glp-1 and (D) daf-16f;glp-1 mutant animals upon exposure to isoform-specific daf-16 RNAi. Figure S7B shows control glp-1(e2141) survival when treated with isoform-specific daf-16 RNAi. See text for details. Statistics and raw data are presented in Table S9; Table S10.

Figure 5

DAF-16A and DAF-16F target genes identified by whole transcriptome profiling. (A) Depiction of the A-index (I_A) for three hypothetical target genes with I_A = 0, 0.5, and 1.0. Idealized expression profiles in daf-2(e1370), daf-16a/f;daf-2, and daf-16a;daf-2 are shown for all three genes. (B) Scatterplot comparing I_A and I_F for DAF-16A/F target genes. Dashed lines indicate I_A and I_F = 0 or 1. Only genes with indices from −0.2 to 1.2 are shown; a scatterplot with a wider range of indices is shown in Figure S15. I_A and I_F for all genes are listed in Table S13. (C and D) Plots of I_A and I_F for all DAF-16A/F target genes from lowest to highest I_A (C) or I_F (D). Solid lines correspond to indices of 0 and 1. Three genes with indices >2.2 or < −1.2 were omitted for presentation purposes. Gene rankings are listed in Table S13. (E) Tree diagram summarizing the categorization system for DAF-16A and DAF-16F target genes. See text and Materials and Methods for details. (F) Scatterplot from B, with individual genes color coded according to categories depicted in E. Dashed lines indicate I_A or I_F values of 0.2 or 0.8, corresponding to the cutoffs used to define redundantly regulated, A-specific, and F-specific targets.

Figure 6

qPCR validation of target gene regulation by DAF-16A and DAF-16F. (A–F) Expression of six DAF-16A/F target genes quantified by qPCR using RNA isolated from day 1 young adult animals. Values represent the mean from three biological replicates. Error bars represent standard deviation. Asterisks indicate statistically significant changes (P < 0.05 by paired ratio t-test). I_A and I_F were calculated using mean expression values measured by qPCR, and correlate with indices calculated using RNA-seq measurements (Table S16). Statistics and data are summarized in Table S14.

Figure 7

DAF-16A-specific targets influence life span. daf-16 (A) and daf-16a (B) are required for longevity induced by daf-2/IGFR RNAi. (C) The DAF-16A up-regulated target gene gst-20 is required for full life span extension induced by daf-2/IGFR RNAi. gst-20 promotes longevity in daf-2(e1368) mutants grown on E. coli HT115 (D) but not in animals grown on E. coli OP50 (E). The DAF-16A down-regulated target gene srr-4 limits longevity in animals grown on HT115 (F) but does not influence life span in animals grown on OP50 (G). (H) Model of life span control and gene regulation by DAF-16A and DAF-16F. See text for details. Life span statistics and data are presented in Table S18.