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Nature. Author manuscript; available in PMC 2011 August 17.
Published in final edited form as:
PMCID: PMC3098900
NIHMSID: NIHMS292497

Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB

Abstract

Activating AMPK or inactivating calcineurin slows ageing in Caenorhabditis elegans1,2 and both have been implicated as therapeutic targets for age-related pathology in mammals35. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs)6 are a family of cofactors involved in diverse physiological processes including energy homeostasis79, cancer10 and endoplasmic reticulum stress11. Here we show that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. We demonstrate that CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity.

AMPK and calcineurin antagonistically regulate CRTCs in mammals8 to modulate energy homeostasis and endoplasmic reticulum (ER) stress9,11. We therefore hypothesized that CRTCs may be critical longevity targets of AMPK and calcineurin, promoting their effects on lifespan through transcriptional regulation. We found a single C. elegans CRTC by homology search (Y20F4.2, now re-named CRTC-1) (Fig. 1a and Supplementary Fig. 1). crtc-1 was expressed throughout the intestine of the worm, as well as in head and tail neurons (Fig. 1b), overlapping the expression pattern of the calcineurin catalytic subunit, tax-6 (ref. 2) and the AMPK catalytic subunit, aak-2 (Supplementary Fig. 2a). Strikingly, inhibition of crtc-1 via RNA interference (RNAi) (Supplementary Fig. 2b) extended wild-type median lifespan by up to 53% (Fig. 1c), comparable to the effects of tax-6 RNAi or AAK-2 activation (Fig. 1c, d). Given the significant role of CRTC-1 in longevity, we investigated whether AMPK and calcineurin modulate ageing through CRTC-1.

Figure 1
CRTC-1 regulates longevity

In mammals, activated AMPK blocks the transcriptional function of CRTC2 by restricting it to the cytosol9. We therefore tested the effect of starvation and heat stress, two conditions known to activate AMPK in C. elegans1, on CRTC-1 cellular localization. A transgenic strain expressing CRTC-1::RFP revealed that CRTC-1 was present throughout the nucleus and cytosol under basal conditions (Fig. 1e and Supplementary Fig. 2b). Starvation and heat stress both induced CRTC-1::RFP translocation to the cytosol and nuclear exclusion in intestinal cells (Fig. 1f–h and Supplementary Fig. 3), illustrating that environmental stimuli that activate AMPK inactivate CRTC-1.

Next we investigated whether direct activation of AMPK rendered CRTC-1 cytosolic. Mammalian AMPKα catalytic subunits are activated by phosphorylation of threonine 172 in their activation loop. Mutation of this residue to aspartic acid or alanine results in a constitutively active or kinase-dead AMPK, respectively12. Expression of CRTC-1::RFP with the equivalent activated AAK-2 mutation (amino acids (aa) 1–321, T181D)::GFP, caused nuclear exclusion of CRTC-1 under fed conditions at 20 °C (Fig. 2a). In contrast, coexpression of kinase-dead AAK-2 (aa 1–321, T181A)::GFP and CRTC-1::RFP did not induce nuclear exclusion (Fig. 2a), demonstrating that catalytic activation of AAK-2 by threonine 181 phosphorylation is required for AAK-2-dependent CRTC-1 nuclear exclusion.

Figure 2
CRTC-1 is a target of AAK-2 and TAX-6

Treatment with tax-6 RNAi caused similar nuclear exclusion of CRTC-1::RFP under fed conditions (Fig. 2b). In addition, tricaine, a class of anaesthetic known to increase calcium flux13, induced tax-6-dependent nuclear localization of CRTC-1::RFP (Supplementary Fig. 4a). Suggesting CRTC-1 is a direct calcineurin target, CRTC-1::RFP containing site-specific mutations in the calcineurin binding site did not translocate to the nucleus in response to tricaine and was retained in the cytosol (Supplementary Fig. 4b).

Similar to mammalian CRTCs8, cytosolic retention of CRTC-1 required 14-3-3 proteins, as simultaneous RNAi knockdown of the two C. elegans 14-3-3 proteins ftt-1 (also known as par-5) and ftt-2 (ref. 14) via RNAi resulted in CRTC-1::RFP accumulation within the nucleus (Fig. 2c) and blocked CRTC-1::RFP cytosolic sequestering after heat stress (Supplementary Fig. 5). In addition, we determined that AMPK directly phosphorylates CRTC-1 at conserved 14-3-3-binding sites. Incubation of CRTC-1 with purified AMPK and AMP in an in vitro kinase assay resulted in phosphorylation of CRTC-1, as detected by anti-phospho-Ser 14-3-3 binding motif antibody (Fig. 2d).

Collectively, these data illustrate that in response to pro-longevity perturbations to AMPK and calcineurin, CRTC-1 becomes phosphorylated, cytosolically sequestered and inactivated.

To determine if the lifespan effects of AMPK and calcineurin are due to the inactivation of CRTC-1, we first examined if crtc-1 RNAi-mediated longevity was epistatic to tax-6. Although crtc-1 RNAi increased the lifespan of wild-type worms, it had no additive effect on the extended lifespan of tax-6 mutants (Fig. 3a), in which CRTC-1 is already rendered cytosolic and inactive (Supplementary Fig. 4a), suggesting that these lifespan mediators function in a linear pathway.

Figure 3
Calcineurin and AMPK regulate lifespan through phosphorylation of CRTC-1

To examine if CRTC-1 was a direct longevity target of AMPK and calcineurin, we inhibited phosphorylation of CRTC-1 at two conserved AMPK/calcineurin sites, S76 and S179, both of which reside within 14-3-3-binding motifs (Supplementary Fig. 1). Previous studies show that 14-3-3 proteins commonly bind tandem phosphorylated sites within a protein, resulting in significantly increased affinity over solitary sites due to cooperative binding15,16. Compound mutation of serines 76 and 179 to alanines in CRTC-1 rendered it constitutively nuclear and refractory to tax-6 deactivation or aak-2 activation (Fig. 3b, c and Supplementary Fig. 6).

Notably, although tax-6 RNAi robustly extended the lifespan of C. elegans expressing wild-type CRTC-1::RFP, which translocated to the cytoplasm freely when calcineurin was not present (Fig. 3d), it had no effect on worms expressing constitutively nuclear CRTC-1 (S76A, S179A) (Fig. 3d). Post-translational modification of CRTC-1 is therefore critical for the effects of calcineurin on longevity. This longevity suppression was not due to general sickness as there was no significant difference between the lifespan of wild-type worms and those expressing CRTC-1 (S76A, S179A) (Supplementary Fig. 7a). Knocking down tax-6 specifically during adulthood again increased wild-type lifespan but had no effect on the CRTC-1 (S76A, S179A) mutant (Supplementary Fig. 7 b–d), indicating that the CRTC-1-dependent effects of tax-6 on lifespan are not solely acting during development.

We used the CRTC-1 (S76A, S179A) mutant to ask whether the extended lifespan of activated AMPK was also mediated by CRTC-1. Expression of CRTC-1 (S76A, S179A) fully suppressed the lifespan extension seen in AAK-2-overexpressing worms (Fig. 3e). This demonstrates that CRTC-1 is both a critical and direct target of aak-2-mediated longevity, and indicates that AMPK and calcineurin function upstream of a shared longevity pathway that signals through CRTC-1.

To understand the downstream effectors of CRTC-1, we examined CRH-1, the single C. elegans orthologue of the cyclic AMP response element binding (CREB) transcription factor family17. Mammalian CREBs (CREB, CREM and ATF1) associate with CRTCs to activate transcription and are involved in diverse processes including memory, immunity, DNA repair, energy homeostasis, fat storage and ER stress18,19. crh-1 is expressed throughout the worm, in overlapping tissues to crtc-1 (Supplementary Fig. 8a). Co-immunoprecipitation of Flag::CRTC-1 and HA::CRH-1 demonstrated that these proteins interact in vivo (Fig. 4a and Supplementary Fig. 8b). The role of CRTC-1 in CRH-1 transcriptional activation was assessed by the CREB reporter construct pCRE::GFP, which was significantly repressed by RNAi against crh-1, crtc-1 and tax-6 (Fig. 4b, c).

Figure 4
CREB activity regulates lifespan

If the lifespan extension seen by activating AMPK or deactivating calcineurin functions through CRTC-1 to inactivate CREB, inactivating crh-1 directly should increase longevity. Indeed, RNAi of crh-1 increased the lifespan of both wild-type and RNAi-sensitive rrf-3 (pk1426) mutants (Fig. 4d and Supplementary Fig. 9). Furthermore, lifespan extension by crtc-1 RNAi was not seen in crh-1 (nn3315) null mutants (Fig. 4e), indicating that the longevity effects of inactivating crtc-1 are mediated by crh-1.

We examined the effects of AMPK and calcineurin on CREB-regulated genes by comparing whole-genome gene expression of activated aak-2, tax-6 null and crh-1 null mutant animals to wild-type worms (Supplementary Table 1). Despite the many distinct roles of AMPK and calcineurin, we found that long-lived worms with activated aak-2 or deactivated tax-6 had transcriptional profiles significantly similar to crh-1 null animals (Fig. 4f, g and Supplementary Fig. 10). The directionality of the transcriptional changes induced by activated aak-2 and inactivated tax-6 was also remarkably similar to crh-1 nulls, with the majority of genes (150 or 67.5%) affected by all mutants exhibiting shared patterns of expression (Fig. 4f and Supplementary Fig. 11). Further, differentially expressed genes across all groups were highly enriched for cAMP regulatory elements (CRE) and the presence of a TATA box in their upstream promoter region (Fig. 4h and Supplementary Fig. 12a), two signatures of highly inducible CREB targets. Interestingly, and in contrast to CREB function in mammals, gene expression analysis revealed that CRH-1 may function as a bifunctional transcriptional regulator, as both upregulated and downregulated genes in crh-1 null animals were enriched for CREs (Supplementary Fig. 12b).

In mammals, AMPK and CREB are involved in energy homeostasis, particularly in response to starvation. Surprisingly, differentially expressed genes in aak-2-overexpressing, tax-6 null mutant and crh-1 null mutant animals were not markedly enriched for genes related to metabolism. Rather, there was strong upregulation of genes involved in ER stress, with 55% of known activated in blocked unfolded protein response family members (ABU)20, upregulated by all mutants (P = 1.7× 10−8, Fisher’s exact test; Fig. 4f, Supplementary Fig. 10 and Supplementary Table 2). abu genes are induced in response to ER stress when the unfolded protein response pathway (UPR) is blocked and are therefore thought to act in parallel to the UPR to maintain protein homeostasis20. abu genes are required for innate immunity21 and, notably, are activated by resveratrol and critical for its effects on longevity in C. elegans22. Furthermore, overexpression of abu family members increases lifespan in the worm22. It will be interesting to determine the potential role of ER stress in lifespan extension via AMPK–calcineurin–CRTC-1 signalling and whether CRTC-1 has a role in resveratrol-mediated lifespan extension.

Our data indicate that CRTC-1 is the critical direct longevity target of both AMPK and calcineurin in C. elegans and identify a new role for CRTCs and CREB in modulating longevity. They also represent the first analysis of the transcriptional profiles of long-lived activated AMPK and deactivated calcineurin organisms and suggest the primary longevity-associated role of these perturbations is the modulation of CRTC-1 and CRH-1 transcriptional activity. Notably, both the FOXO transcription factor daf-16 (ref. 23) and genes involved in autophagy24 have also been implicated in AMPK and calcineurin longevity, respectively. Further work to determine precisely where the AMPK–calcineurin–CRTC-1 pathway converges with FOXO and autophagy will be enlightening. It will also be interesting to determine if CRTC-1 mediates downstream effects of kinases other than AMPK. In mammals, CRTCs are regulated by multiple CAMKL kinase family members8,9,25 (Supplementary Table 3), and we saw additive effects of AMPK and related kinases on the localization of CRTC-1, in particular the MAP/microtubule affinity-regulating kinase (MARK) par-1, indicating that this kinase may also regulate CRTC-1 in vivo (Supplementary Fig. 13c, d). At present, however, AMPK is the only CAMKL kinase shown to be a positive regulator of longevity.

Collectively, these data identify CRTC-1 as a central node linking the upstream lifespan modifiers AMPK and calcineurin to CREB activity via a shared signal-transduction pathway, and demonstrate that post-translational modification of CRTC-1 is required for their effects on longevity (Supplementary Fig. 14). Complementing the pro-longevity effects of inhibiting CRTC function in C. elegans, reducing components of the CRTC/CREB pathway has recently been shown to confer health benefits to mice9,19,2628. Given the evolutionary conservation of this pathway from C. elegans to mammals29 it will be fascinating to determine the role of CRTCs both as mammalian ageing modulators and as potential drug targets for patients with metabolic disorders and cancer.

METHODS SUMMARY

A detailed description of all experimental methods including C. elegans strains (Supplementary Table 4), growth, imaging, lifespan analysis and RNAi application is provided in Methods. None of the RNAi treatments used affected feeding rates (Supplementary Fig. 15). Transgenic strains were generated via microinjection into the gonad of adult hermaphrodites using standard techniques. Integrated transgenic lines were generated using gamma irradiation and outcrossed to wild-type at least four times. All lifespans were conducted at 20 °C with deaths scored and live worms transferred to new plates every 1–2 days, see Supplementary Table 5 for statistical analysis and replicate data. JMP 8/Graphpad Prism 5 and R/Bioconductor software were used for all statistical analyses.

METHODS

Lifespan studies

All lifespan experiments were performed on standard 6-cm nematode growth media plates30 supplemented with 100μg ml−1 carbenicillin at 20 °C. Plates were removed from 4 °C storage 2 days before seeding with 100μl of Escherichia coli HT115 containing either empty vector or RNAi inducing plasmids. RNAi for a particular gene can be readily achieved in the worm by feeding C. elegans E. coli (HT115) that express double-stranded RNA of the gene of interest31. Bacterial cultures were grown overnight at 37 °C in the presence of both carbenicillin (100μg ml−1) and tetracycline (10μg ml−1) before seeding onto NGM plates. Once seeded, bacterial lawns were grown at room temperature (25 °C) for 48 h. RNAi was induced with 100μl IPTG (100 mM) 2 h before worms were added to plates. To age-synchronize worms, five gravid adults (24 h post larval stage four, L4) were placed on plates with the appropriate control or RNAi bacteria and allowed to lay eggs for 5 h before being removed. These eggs were then cultured to adulthood (72 h post egg lay at 20 °C) before being moved to fresh plates at a density of 10 worms per plate, 10 plates per treatment. Age = 0 was defined as the day adults were moved to 10 worms a plate. Worms were moved to fresh plates every 1–2 days until day 14, after which only those on mould-contaminated plates were transferred. Worms were censored at the first sign of any bacterial contamination. Death was scored by gentle agitation with a worm pick and confirmed with no response after three attempts at both the head and tail. Death was scored every 1–2 days throughout.

RNAi constructs

RNAi tax-6 and crh-1 (1) (Supplementary Fig. 9) constructs used were taken from the Vidal RNAi library. The ctrc-1 (1) (Supplementary Fig. 9) RNAi plasmid was made by cloning full-length crtc-1 cDNA between the two inverted T7 promoters in the pAD12 RNAi plasmid and transformed into HT115 cells. Remaining RNAi constructs used in Supplementary Fig. 9 are in the L4440 plasmid. Primers against cDNA were as follows. CRTC-1 (2): 5′-GGATCACCGGTTCAGATGCAT, 3′-AGGTGCACCTTCAGCATTTGT. CRTC-1 (3): 5′-GAGCTCCAAGGACATCGAAGTCG, 3′-CTCGAGTGGATGCATTGGAACCATACC. CRH-1 (2): 5′-GAGCTCATGGCCACAATGGCGAG, 3′-CTCGAGCTTATCCGCCGTTTCTA.

DAPI staining

Worms were washed in 1 ml M9 (ref. 30) in a watch glass (1–3 times, until bacteria is removed). M9 was almost completely removed before 100–200 μl DAPI (200 ng ml−1 in ethanol) was added. DAPI treatment was incubated in darkness for 20 min or until the ethanol had evaporated. One millilitre of M9 was then added to rehydrate for 1 h (up to 5 h at room temperature or overnight at 4 °C). One drop (~3 μl) of ProLong mounting medium was placed on a slide before transferring the stained worm. A cover slip was added and sealed with nail polish. Fluorescence was examined at 358 nm.

Heat stress and starvation assays

Effects of heat and starvation on CRTC-1::RFP was measured by placing C. elegans expressing crtc-1::RFP onto either OP50-seeded NGM plates at 33 °C overnight or into 9-well plates containing M9 media at 20 °C. Controls were C. elegans fed OP50 at 20 °C.

Microscopy

All microscopy was performed using 0.1 mg ml−1 tetramisole hydrochloride in M9 as an anaesthetic, which pilot experiments revealed had no effect on CRTC-1 localization. Except for tricaine time-course experiments, worms were in 5 μl anaesthetic mounted on 2% agarose pads on glass slides under glass cover slips. All photographs were taken using a Zeiss Axiovert microscope and AxioCam. Pictures in Figs 2a and and3b3b used ApoTome optical sectioning. For the tricaine experiments (Supplementary Fig. 4a, b), L4 worms were placed in wells of a 96-well plate in 100 μl of tetramisole/M9 with or without tricaine (2 mg ml−1). Pictures were taken during the time-course through the 96-well plate.

Kinase redundancy assays

Worms were subjected to RNAi for CAMKL kinases from hatch. Twenty-four hours post-L4 worms were then picked into M9 with tricaine (2 mg ml−1) in wells of a 9-well plate. Worms were left on a rotational shaker at 20 °C for 2 h. Using a glass pipette32 worms were then placed onto NG plates seeded with E. coli (OP50). When tricaine solution had evaporated (approx 20 min), worms were picked onto fresh OP50 plates, 5 worms per plate. Localization of CRTC-1::RFP was then scored as ‘all nuclear’ (all intestinal cells showed only punctate nuclear CRTC-1), ‘some cells nuclear’ (intestinal cells showed mix of punctate nuclear CRTC-1 and cytosolic CRTC-1) and ‘cytosolic’ (CRTC-1 was dispersed evenly throughout nucleus and cytosol in all intestinal cells). Time = 0 was defined as when worms were moved to fresh OP50 plates.

Transgenic strain construction

Expression constructs were based on pPD95.77 from the Fire laboratory C. elegans vector kit. RFP in the manuscript refers to tdTOMATO, which replaced the GFP in pPD95.77. Transgenic strains were generated via microinjection into the gonad of adult hermaphrodites using standard techniques. Integrated transgenic lines were generated using gamma irradiation and out-crossed to wild-type at least four times.

Calcineurin-binding mutant

QuikChange mutagenesis was used to mutate residues within the conserved calcineurin-binding site in CRTC-1. This resulted in changing the amino acid sequence (aa 423–428) as follows. Wild type: EALDIPKLTITNAEGA; calcineurin-binding mutant: EALDIAKATAANAEGA.

Single-worm PCR for genotyping

Single-worm lysis buffer (SWLB): 30 mM Tris pH 8.0, 8 mM EDTA, 100 mM NaCl, 0.7% NP-40, 0.7% Tween-20. Proteinase K was added to a final concentration of 100μg ml−1 just before use. To prepare the DNA template, one worm was added to a PCR tube containing 5μl SWLB supplemented with Proteinase K and incubated for 60 min at 60 °C. Proteinase K was then heat-inactivated at 95 °C for 15 min before the reaction was cooled to 4 °C. To setup the PCR reaction, 5μl of the worm lysate was used as template.

Expression of C. elegans proteins in 293T cells

Full-length C. elegans crtc-1 and crh-1 cDNA was cloned using gateway recombination into mammalian expression plasmid pcDNA3 containing in-frame 5′ Flag or HA tag respectively. 293T cells were transfected using lipofectamine 2000 (Invitrogen) following manufacture’s guidelines. Primer information is available on request.

293T cells immunoprecipitation

293T cells were washed 1× with PBS and resuspended in 500 l PLB supplemented with protease inhibitor cocktail (Roche). Cells were incubated for 10 min at 4 °C then sonicated to disrupt nuclei. Lysate was centrifuged at 10,000g for 30 min at 4 °C then precleared with Protein A/G PLUS-Agarose (Santa Cruz). Cleared lysate was incubated with anti-Flag M2 affinity gel (Sigma) overnight at 4 °C. For control anti-Flag M2 affinity gel was blocked with 50 μg Flag peptide for 1 h at 4 °C then lysate was added and incubation continued overnight at 4 °C Immune complexes were collected by centrifugation and washed extensively. Complexes were eluted with Flag peptide then resolved by SDS–PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed with anti-Flag M2-Peroxidase (Sigma) and anti-HA-Peroxidase (Roche).

C. elegans immunoprecipitation

Approximately 25,000 N2 and AGD 744 animals were treated with 2 mg ml−1 Tricaine for 2 hours then, flash-frozen, then ground to fine powder using a mortar and pestle on dry ice. Powder was collected in 400 μl cell lysis buffer (Cell Signaling) supplemented with protease inhibitor cocktail (Roche) and sonicated to disrupt nuclei. Lysate was centrifuged at 16,000g for 30 min at 4 °C then precleared with Protein A/G PLUS-Agarose (Santa Cruz). Cleared lysate was incubated with EZview Red anti-Flag M2 affinity gel (Sigma) for 2 h at 4 °C. Immune complexes were collected by centrifugation and washed extensively. Complexes were eluted with 3× Flag peptide then resolved by SDS–PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed with anti-Flag M2-Peroxidase (Sigma) and anti-HA (Abcam).

In vitro kinase assay

Recombinant GST::CRTC-1 and GST proteins (2 μg) were incubated with 100 mU purified AMPK (Millipore) in kinase reaction buffer (2.5 mM Tris, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, 150 μM AMP, 125 μM ATP) for 30 min at 30 °C. Samples were resolved by SDS–PAGE then transferred to nitrocellulose membrane. Phosphorylation was determined by immunoblotting with phospho-Ser 14-3-3-binding motif antibody (Cell Signaling).

Microarray procedure

Egg preparations were made using standard bleaching techniques and larval stage one (L1) worms were synchronized by hatching eggs overnight in M9 buffer. Seven hundred and fifty L1 larvae were seeded per 10 cm NG plate seeded with OP50. Worms were harvested for RNA extraction when they reached L4 larval stage by snap-freezing in liquid nitrogen in TRIzol. All L1 larvae were seeded at the same time and samples frozen at different times to account for variation in development time between groups. Three thousand worms were used for each sample and three biological replicates done for each experimental group: N2, AGD383, RB1667 and crh-1 (nn3315). Biological samples were prepared on separate days, RNA preparations were carried out at the same time (12 h before array) using TRIzol/chloroform extraction and then run through an RNeasy cleanup column. Arrays were done on Affymetrix C. elegans Genome Array from the same batch.

Microarray data analysis

Raw expression data files were obtained for three replicates each of L4 crh-1 (nn3315), tax-6 (ok2065) and aak-2 (aa 1–321) mutants and three N2 control replicates with the Affymetrix C. elegans Genome Array. All microarray analysis was performed with Bioconductor33. Standard data quality validation as suggested by Affymetrix34 was carried out with the ‘simpleaffy’ package, followed by ‘affyPLM’, which identified no problematic chips. The raw data were preprocessed according to the GC-RMA method35 (implemented in ‘gcrma’), which performs probe-sequence-based background adjustment, quantile normalization, and utilizes a robust multi-chip average to summarize information into single expression measurements for each probeset (Supplementary Table 1). Before statistical testing, the data were submitted to a non-specific filter (via the package ‘genefilter’) that removed probesets with an expression interquartile range smaller than 0.5. To identify genes that were significantly differentially expressed between each mutant and the control, linear modelling and empirical Bayes analysis was performed using the ‘limma’ package36. Limma computes an empirical Bayes adjustment for the t-test (moderated t-statistic), which is more robust than the standard two-sample t-test comparisons. To correct for multiple testing, Benjamin and Hochberg’s method to control for false discovery rate was used37. Genes with an adjusted P value of 0.05 or smaller and a fold-change in expression larger than twofold were considered differentially expressed (Supplementary Table 2).

cAMP response element (CRE) identification

We gathered intergenic upstream sequences (up to 5 kb, from WS198) for differentially expressed genes and used MATCH38 to search against the TRANSFAC39 CRE matrix (M00039), an experimentally derived matrix based on 29 human CREB1-binding sequences40. In the MATCH search we used the ‘minFP’ score cutoff, which aims to minimize false positives. In addition, we estimated the number of such sites that can be found by chance (background) by using the same procedure to search the upstream sequences of ten similarly sized samples of C. elegans genes that were not affected by any of the mutants. We used the same procedure to search against the TRANSFAC TATA matrix (M00252)41.

Supplementary Material

Supplemental Excel Files

Supplemental Info

Table S1

Table S2

Table S3

Acknowledgments

W.M. is funded by the George E. Hewitt Foundation for Medical Research, the American Federation for Aging Research and the Glenn Foundation for Medical Research. R.J.S. is funded by National Institutes of Health (NIH) R01 DK080425 and P01 CA120964. A.P.C.R. and G.M. are funded by NIH R01 HG004164, AG031097 and CA14195. A.D. is supported by NIH R01 DK070696 and AG027463. We thank the Caenorhabditis Genetics Center, the National Bioresource Project for the Nematode and Mark Alkema for providing worm strains. We are grateful to M. Raices and M. D’Angelo for critical analysis of the manuscript, DAPI images and the NUP-160::GFP construct. We also thank members of the A.D. laboratory and M. Hansen for comments on the manuscript and discussion and K. Butler for technical assistance in the early stages of this project.

Footnotes

Author Contributions W.M., I.M., M.M., R.J.S. and A.D. designed the experiments. W.M. and I.M. performed the experiments. A.P.C.R analysed the microarray data and performed the promoter analysis and W.M. analysed and performed statistical analysis on all other data. The manuscript was written by W.M. and edited by I.M., A.P.C.R., G.M., R.J.S. and A.D. All authors discussed the results and commented on the manuscript.

Author Information Data have been deposited at GEO under accession number GSE25513. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/nature.

Supplementary Information is linked to the online version of the paper at www.nature.com/nature.

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