Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf - PubMed (original) (raw)

Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf

Riva P Oliveira et al. Aging Cell. 2009 Sep.

Abstract

Studies in model organisms have identified regulatory processes that profoundly influence aging, many of which modulate resistance against environmental or metabolic stresses. In Caenorhabditis elegans, the transcription regulator SKN-1 is important for oxidative stress resistance and acts in multiple longevity pathways. SKN-1 is the ortholog of mammalian Nrf proteins, which induce Phase 2 detoxification genes in response to stress. Phase 2 enzymes defend against oxygen radicals and conjugate electrophiles that are produced by Phase 1 detoxification enzymes, which metabolize lipophilic compounds. Here, we have used expression profiling to identify genes and processes that are regulated by SKN-1 under normal and stress-response conditions. Under nonstressed conditions SKN-1 upregulates numerous genes involved in detoxification, cellular repair, and other functions, and downregulates a set of genes that reduce stress resistance and lifespan. Many of these genes appear to be direct SKN-1 targets, based upon presence of predicted SKN-binding sites in their promoters. The metalloid sodium arsenite induces skn-1-dependent activation of certain detoxification gene groups, including some that were not SKN-1-upregulated under normal conditions. An organic peroxide also triggers induction of a discrete Phase 2 gene set, but additionally stimulates a broad SKN-1-independent response. We conclude that under normal conditions SKN-1 has a wide range of functions in detoxification and other processes, including modulating mechanisms that reduce lifespan. In response to stress, SKN-1 and other regulators tailor transcription programs to meet the challenge at hand. Our findings reveal striking complexity in SKN-1 functions and the regulation of systemic detoxification defenses.

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Figures

Fig. 1. Identification of SKN-1-regulated genes

mRNA samples were generated under the indicated conditions, with skn-1(−) referring to skn-1 RNAi and skn-1(+) to RNAi control. Pairs of samples designated by arrows were compared on Agilent 44X oligonucleotide microarrays to identify genes that are regulated by SKN-1 under normal conditions (red arrow), and in response to treatment with Arsenite (As) (dark blue arrows), or tert-Butyl hydroperoxide (t-BOOH) (teal arrows). Genes that are regulated by SKN-1 under normal conditions were identified by both SAM and hierarchical clustering (red arrow). _skn-1_-dependent and –independent genes that respond to As or t-BOOH stress were identified by hierarchical clustering. While the As-induced response seemed to be entirely dependent upon skn-1, t-BOOH induced both _skn-1_-dependent and independent gene sets (see text). Some gene categories that we identified as being prominent in stress- and SKN-1-upregulated gene sets are listed in bold, with those that were overrepresented in only one or two sets indicated in italics.

Fig. 2. Genes that are up-regulated by SKN-1 under normal conditions

(A) Hierarchical clustering of gfp control (skn-1(+)) and skn-1(−) RNAi samples that were analyzed on microarrays (7 sample sets in total). Incubation conditions under which these samples were obtained are indicated. A representative subset of SKN-1-upregulated genes is shown. (B) Representation of functional group categories among the 233 SKN-1-upregulated genes that were identified by hierarchical clustering and SAM (Table S1). Genes were classified according to their molecular or biological function, based upon GO terms. The CUB/CUB-like group was classified by presence of these motifs (Blanc et al. 2007). The following broad categories were created by combining GO-terms: Detoxification/Stress response, Defense/Immunity, Signaling/Transcriptional regulation, and Protein folding/degradation. (C) Enrichment of SKN-1-binding motifs at SKN-1-upregulated genes. RSAT and Weeder Web were used to identify novel sequence motifs that are overrepresented in the predicted promoters of SKN-1-up-regulated genes, as defined by the 2 Kb or less of intergenic sequence upstream of each ORF. The consensus identified by Weeder Web is represented by WebLogo (Crooks et al. 2004). (D) Enrichment of functional gene categories among SKN-1-upregulated genes, compared to a set of Nrf2-upregulated genes. Highly represented GO terms are graphed for SKN-1-upregulated genes, and for Nrf2-dependent genes that were identified by expression profiling of primary cortical astrocytes from Nrf2 −/− and Nrf2 +/+ mice under non-stressed conditions (Lee et al. 2003a).

Fig. 3. Genes that are down-regulated by SKN-1 under normal conditions

(A) Hierarchical clustering (pictured) and SAM analysis of 7 sample sets identified a set of 63 SKN-1-downregulated genes (Table S2). (B) Representation of functional gene groups among the SKN-1-downregulated genes, analyzed as in Fig. 2B. (C) Enrichment of SKN-1 binding motifs in SKN-1-downregulated genes, analyzed as in Fig. 2C.

Fig. 4. SKN-1 regulation of overlapping gene groups under normal and Arsenite stress conditions

(A) Hierarchical clustering of genes that are differentially regulated in response to As treatment (4 sample sets, see Experimental Procedures). Genes were identified that are As-upregulated and _skn-1-_dependent, but none were identified that are As-upregulated and _skn-1-_independent. A subset of the genes identified by hierarchical clustering is shown. (B) Enrichment of SKN-1-binding motifs in SKN-1-downregulated genes, analyzed as in Fig. 2C. (C) Venn diagram showing overlap among genes that were upregulated by SKN-1 under normal and As stress conditions. (D) Comparison of SKN-1-upregulated genes identified under normal, As-treatment, and t-BOOH-treatment conditions, grouped by GO terms. Note that some GO terms are overrepresented among only one or two of these gene groups.

Fig. 5. SKN-1-dependent and -independent responses to an organoperoxide

(A) t-BOOH treatment affects regulation of _skn-1_-dependent and _skn-1_-independent gene programs. Hierarchical clustering identified genes that are up- or down-regulated in response to t-BOOH treatment, and unaffected by skn-1 RNAi (skn-1(−)) (SKN-1-independent genes). A subset of the genes identified from 3 sample sets by hierarchical clustering is shown, along with motifs that were identified as being overrepresented in their predicted upstream promoters (determined as in Fig. 2C). NGM corresponds to normal conditions (see Experimental Procedures). (B) Venn diagram of genes that were upregulated by SKN-1 under normal conditions and t-BOOH treatment. (C) Genes that were upregulated by t-BOOH treatment (Tables S8, S9), graphed as in Fig. 4D. GO terms that are overrepresented among t-BOOH-induced SKN-1-upregulated and SKN-1-independent genes are compared.

Fig. 6. SKN-1-regulated genes influence oxidative stress resistance and lifespan

(A) Many SKN-1-upregulated genes promote oxidative stress resistance. SKN-1-upregulated genes (Table S1) were knocked down by RNAi, then survival of young adults (8–9 hr) was assayed at the indicated times after introduction into 4 mM As. A representative experiment is shown in which 5 wells of 10 worms each were examined. Error bars indicate the SEM, and p values (Student’s t-test) indicate comparison to control RNAi. *p ≤ 0.0008; **p ≤ 0.008 (Student’s t-test). (B) Many SKN-1-downregulated genes reduce oxidative stress resistance. Resistance to As was analyzed after RNAi of the indicated genes (Table S2) as in (A). Other experiments and analyses of additional genes are described in (C) and Fig. S5. *p ≤ 0.0008; **p ≤ 0.008 (Student’s t-test). (C) Analysis of As resistance in young adults (2–6 hr). Experimental and control RNAi worms were placed in 5mM As, then the fraction surviving was counted 16, 24, and 40 hrs later. Results are presented as a graph from which we calculated the approximate fraction of animals in each set that were alive when 20% of the control animals were still surviving (black vertical line). A comparison of this fraction to control is plotted in Fig. S5. 6 samples of 10 worms each were examined for every condition. p-value of fraction alive compared to control at 20% control survival is < 0.05 for all genes shown (Student’s t-test performed across samples). Error bars = SEM. (D) Many SKN-1-downregulated genes decrease lifespan. A set of SKN-1-downregulated genes was analyzed for effects on longevity using a feeding RNAi longevity assay in RNAi-sensitive rrf-3(pk1426) worms at 20°C. Genes for which RNAi extended lifespan significantly in 3/3 trials (p<0.01, log-rank) are diagrammed, with data from a single trial shown (Table S11B, Experiment 2). Control is empty RNAi feeding vector L4440. Data and statistical analyses for all experiments and genes tested are provided in Table S11A-D.

Fig. 7. A model for SKN-1 functions under normal conditions

A positive feedback interaction with ins-7 and the DAF-2 pathway is featured. SKN-1 upregulates many genes that promote detoxification and stress resistance, and also downregulates genes that decrease stress resistance, lifespan, or both. Among the SKN-1-downregulated genes are both ins-7 and pdk-1 (not shown), each of which promotes DAF-2 pathway signaling (see text). The DAF-2 pathway in turn inhibits SKN-1 (Tullet et al. 2008).

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Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf - PubMed (original) (raw)