Transcriptional regulation of Caenorhabditis elegans FOXO/DAF-16 modulates lifespan

Abstract

Background: Insulin/IGF-1 signaling plays a central role in longevity across phylogeny. In C. elegans, the forkhead box O (FOXO) transcription factor, DAF-16, is the primary target of insulin/IGF-1 signaling, and multiple isoforms of DAF-16 (a, b, and d/f) modulate lifespan, metabolism, dauer formation, and stress resistance. Thus far, across phylogeny modulation of mammalian FOXOs and DAF-16 have focused on post-translational regulation with little focus on transcriptional regulation. In C. elegans, we have previously shown that DAF-16d/f cooperates with DAF-16a to promote longevity. In this study, we generated transgenic strains expressing near-endogenous levels of either daf-16a or daf-16d/f, and examined temporal expression of the isoforms to further define how these isoforms contribute to lifespan regulation.

Results: Here, we show that DAF-16a is sensitive both to changes in gene dosage and to alterations in the level of insulin/IGF-1 signaling. Interestingly, we find that as worms age, the intestinal expression of daf-16d/f but not daf-16a is dramatically upregulated at the level of transcription. Preventing this transcriptional upregulation shortens lifespan, indicating that transcriptional regulation of daf-16d/f promotes longevity. In an RNAi screen of transcriptional regulators, we identify elt-2 (GATA transcription factor) and swsn-1 (core subunit of SWI/SNF complex) as key modulators of daf-16d/f gene expression. ELT-2 and another GATA factor, ELT-4, promote longevity via both DAF-16a and DAF-16d/f while the components of SWI/SNF complex promote longevity specifically via DAF-16d/f.

Conclusions: Our findings indicate that transcriptional control of C. elegans FOXO/daf-16 is an essential regulatory event. Considering the conservation of FOXO across species, our findings identify a new layer of FOXO regulation as a potential determinant of mammalian longevity and age-related diseases such as cancer and diabetes.

Keywords: Aging; C. elegans; DAF-16/FOXO; Isoforms; Longevity; Transcription.

Figure 1

Structure of daf-16 isoforms. Coding regions are orange-filled boxes, and introns are lines. The SL1 trans-spliced region is indicated with a green line. The SL1 trans-spliced reads for the different isoforms are: daf-16d/f, 8 reads; daf-16d, 9 reads; daf-16a, 208 reads; daf-16b, 14 reads. See Additional file 1: Table S1 for ESTs associated with the different isoforms.

Figure 2

Effects of altering DAF-16a dosage on lifespan. (A) Expression levels of daf-16a mRNA in various daf-16(mgDf50); daf-2(e1370); daf-16a::gfp strains were compared to that of daf-2(e1370) worms. The graph is plotted on a log2 scale. Error bars represent standard deviation (S.D.) from two independent repeats. Statistical values are given in Additional file 1: Table S2. (B) Lifespan of daf-16(mgDf50) worms carrying various daf-16a::gfp transgenes as well as low-copy daf-16d/f::gfp^HT. Three high-copy daf-16a transgenic worms live longer than wild type. daf-16a::gfp^HT worms lived shorter than wild-type, while the lifespan of the daf-16d/f::gfp^HT strain is comparable to wild type. (C) Lifespan of daf-16(mgDf50); daf-2(e1370) worms carrying various daf-16::gfp transgenes. daf-16a::gfp^HT worms lived shorter than the daf-2(e1370) mutants. daf-16(mgDf50); daf-2(e1370); daf-16a::gfp^GR worms had the shortest lifespan among the high-copy daf-16a transgenic strains. daf-16d/f::gfp^HT transgene alone fully rescued the lifespan extension of daf-2(e1370) mutants. One lifespan experiment is shown from a total of three repeats; each with similar results. All lifespan data are shown in Additional file 1: Table S3.

Figure 3

DAF-16d/f and DAF-16a respond differently to changes in insulin/IGF-1 signaling. (A) Lifespan analysis of daf-16(mgDf50); daf-2(e1368) worms carrying daf-16a::gfp^HT or daf-16d/f::gfp^HT. One of three repeats is shown; each with similar results. All of the lifespan data are shown in Additional file 1: Table S4. (B) Dauer formation in daf-16(mgDf50); daf-2(e1370) or daf-16(mgDf50); daf-2(e1368) worms carrying daf-16a::gfp^HT or daf-16d/f::gfp^HT at 20°C. The daf-16(mgDf50); daf-2(e1368); daf-16d/f strain did not form dauers, while daf-16(mgDf50); daf-2(e1370); daf-16d/f strain formed a significant fraction of dauers. Dauer formation data represents one experiment with additional repeats showing similar results (Additional file 1: Figure S5). (C) Localization of DAF-16 isoforms in daf-2(e1368) and daf-2(e1370) mutant backgrounds. daf-16(mgDf50); daf-2(e1368) or daf-16(mgDf50); daf-2(e1368) worms carrying daf-16a::gfp^HT or daf-16d/f::gfp^HT, as well as the daf-2 mutants expressing endogenous daf-16 isoforms, were visualized after incubation at 20°C for 20 hours. Red arrows indicate nuclear DAF-16. (D) Quantification of the nuclear localization of the DAF-16 isoforms in daf-2(e1368) and daf-2(e1370) mutant backgrounds. Nuclear enrichment of DAF-16d/f is observed in daf-2(e1370) worms but not in daf-2(e1368) worms, while nuclear enrichment of DAF-16a is observed in both daf-2 alleles.

Figure 4

daf-16d/f is regulated at the level of transcription in both a temporal and spatial manner. (A,B) Quantitative RT-PCR analysis of endogenous daf-16a and daf-16d/f mRNA levels during larval development (L1-L4) and in the adult. In both wild-type and daf-2(e1370) mutants, the level of daf-16d/f mRNA significantly increases with age. Additional details are shown in Additional file 1: Figure S7. (C,D) Changes in DAF-16d/f and DAF-16a protein levels with age. Equal volumes of worms were used for each larval stage. Western blots with the worm lysates were probed with anti-α-DAF-16 antibody (top panel) and anti-α-tubulin (bottom panel). DAF-16d/f shows a dramatic increase in protein levels with age as compared to DAF-16a. (E) Quantification of the fluorescence intensity in Pdaf-16a::gfp and Pdaf-16d/f::gfp transgenic worms as they age The GFP intensity was quantified using ImageJ software and normalized to the L1 stage for each strain. (F) Transcriptional regulation of daf-16d/f determines longevity. daf-16(mgDf50); daf-2(e1370); daf-16d/f transgenic worms were treated with diluted daf-16 RNAi bacteria from the L4 stage to day 2 adulthood. All lifespan data are shown in Additional file 1: Table S5.

Figure 5

ELT-2 and SWSN-1 are required for daf-16 gene expression. (A)Pdaf-16d/f::gfp expression grown on control, elt-2 RNAi or swsn-1 RNAi bacteria. Left and middle panels worms grown on RNAi from hatching, right panel worms grown on RNAi from L4. Red arrows indicate the head region of the worms. (B) Close-up of worms shown in Panel A Left and middle columns with additional GFP RNAi control. Pdaf-16d/f::gfp expression in the head and anterior intestine of worms grown on control, gfp, elt-2, or swsn-1 RNAi bacteria from hatching. (C) Quantitative RT-PCR analysis of endogenous daf-16d/f in L4 larvae and Days 1, 2, and 5 adult daf-2(e1370) worms. For elt-2, worms were grown on RNAi bacteria from L4 stage and for swsn-1 were grown on RNAi bacteria from hatching. The expression of daf-16d/f is reduced by elt-2 or swsn-1 RNAi treatment. Additional details are shown in Additional file 1: Figure S13.

Figure 6

ELT-2, ELT-4, and SWI/SNF promote longevity by regulating daf-16 gene expression. (A-D) elt-2 and swsn-1 RNAi were started at the L4 stage. (E-H) elt-4 and swsn-2.1 RNAi were started at the L1 stage (hatching). Lifespan analyses of daf-2(e1370) (A, E), daf-16(mgDf50); daf-2(e1370) (B, F), daf-16(mgDf50); daf-2(e1370); daf-16a::gfp (C, G), and daf-16(mgDf50); daf-2(e1370); daf-16d/f::gfp (D, H). One of three repeats is shown; each with similar results. All lifespan data are summarized in Additional file 1: Table S6.

Figure 7

Model of the transcriptional regulation of daf-16. daf-16d/f plays a more prominent role than daf-16a in lifespan regulation. The thickness of the line represents the strength of regulation. ELT-2 and ELT-4 are shown as direct regulators of DAF-16. SWI/SNF is shown as the direct regulator of DAF-16d/f and not DAF-16a.