Analysis of long-lived C. elegans daf-2 mutants using serial analysis of gene expression

Abstract

We have identified longevity-associated genes in a long-lived Caenorhabditis elegans daf-2 (insulin/IGF receptor) mutant using serial analysis of gene expression (SAGE), a method that efficiently quantifies large numbers of mRNA transcripts by sequencing short tags. Reduction of daf-2 signaling in these mutant worms leads to a doubling in mean lifespan. We prepared C. elegans SAGE libraries from 1, 6, and 10-d-old adult daf-2 and from 1 and 6-d-old control adults. Differences in gene expression between daf-2 libraries representing different ages and between daf-2 versus control libraries identified not only single genes, but whole gene families that were differentially regulated. These gene families are part of major metabolic pathways including lipid, protein, and energy metabolism, stress response, and cell structure. Similar expression patterns of closely related family members emphasize the importance of these genes in aging-related processes. Global analysis of metabolism-associated genes showed hypometabolic features in mid-life daf-2 mutants that diminish with advanced age. Comparison of our results to recent microarray studies highlights sets of overlapping genes that are highly conserved throughout evolution and thus represent strong candidate genes that control aging and longevity.

Figure 1.

Survival curves for double mutant daf-2(m41); fer-15(b26ts) (DR1993) and infertile control fer-15(b26ts) (DR1830) adults at 25.5°C. The double mutant lived significantly longer than control strains (P < 0.0001, log rank test). The two arrowheads at the top of the graph correspond to days 6 and 10 of the study, the times at which 100% and 95 ± 3% of the control and daf-2 populations were alive, respectively. Wild type (N2), circles; fer-15, inverted triangles; fer-15; daf-2, diamonds. Filled and open symbols represent data from two independent trials. The mean lifespan (±SE) of wild type was 14.3 ± 0.5 d (n = 222); fer-15, 14.9 ± 0.1d(n = 232); and fer-15; daf-2, 27.3 ± 1.7d(n = 235). Maximum lifespans for the strains were 20 d, 22 d, and 41 ± 1 d, respectively.

Figure 2.

Expression analysis of daf-2 SAGE libraries. Gene expression was analyzed in daf-2 worms at days 1, 6, and 10 of adulthood. Tags that met our selection criteria were grouped according to biological function. Mean fold differences of tag abundance were calculated between day 1 and day 10 and are shown in (A). The number of genes within a given group or gene family is noted. (B) The expression profile of four hsp-16-like genes in daf-2 and control worms. Expression levels of these genes drastically increase (∼60-fold) over daf-2 lifespan but are only marginally expressed in controls. Genes that are plotted together are closely related and their SAGE tags are indistinguishable; these tag numbers represent the sum of SAGE tags for both genes.

Figure 3.

Expression analysis of 6-d-old daf-2 and control worms. Genes matching our selection criteria were classified according to biological function. Mean fold differences for these genes are shown. The numbers of genes constituting these groups are indicated. Genes associated with major metabolic pathways are expressed at lower levels in daf-2 mutants.

Figure 4.

Expression analysis of 10-d-old daf-2 and 6-d-old control worms. Genes matching our selection criteria were classified based on biological function. Mean fold differences for all genes within a group are shown. The numbers of genes constituting these groups are indicated. Differences in metabolic activities seen in daf-2 on day 6 are noticeably reversed.

Figure 5.

Global expression analysis of metabolism-associated genes in daf-2 and control worms. Genes associated with the GO term “metabolism” were analyzed. We compared expression profiles of the 900 most abundant genes in 6-d-old daf-2 vs. 6-d-old control worms (A) and 10-d-old daf-2 vs. 6-d-old control worms (B). The insets in both graphs show the section between tag #50 and #300 at a greater resolution. (A) At day 6, metabolism-associated tags in the control library are more abundant than in the daf-2 library (3.5-fold). (B) Comparison of daf-2 mutants (day 10) and controls (day 6) revealed a less distinct differential expression profile (1.6-fold lower expression of metabolism-associated tags in daf-2).

Figure 6.

Global gene expression differences of metabolism-associated genes in daf-2 and control worms. The graph schematically illustrates metabolic activity in mutant and control worms. Fold differences of all differentially expressed metabolism-associated genes between SAGE libraries are indicated. Arrowheads point to the SAGE library with the higher metabolic activity in a given comparison. Generally, metabolism-associated transcripts were more abundant in controls than in daf-2, and metabolic activity also increased with age.