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

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.


daf-16d/f is the most abundant daf-16 isoform in adult worms
We find that daf-16 is regulated at the level of transcription as worms age. To systematically analyze the comparative level of each daf-16 isoform in the early adult stage, we designed isoform-specific primers as well as primers covering 3' sequences shared by each isoform (Table S7). Isoform-specific primers were specific to their respective target (data not shown). Since it is possible that the different daf-16 primer sets could show different amplification efficiency, we first measured the daf-16 mRNA level of daf-16 isoform transgenic worms using isoform-specific primers as well as isoform-common primers ( Figure S8A, B). Since these worms express the isoform specific transgene, the Ct values should be the same between the common and isoform specific values. However, we found that the isoform-specific primers and isoform-common primers produce different Ct values, Ct iso vs Ct com respectively, and this was most apparent for daf-16b ( Figure S8-S9). Therefore, to directly compare the levels between the different daf-16 isoforms, we calculated the ΔCt by subtracting the Ct com value from the Ct iso value (S10C). This is the ΔCt calibration (ΔCt S8 ) shown in Figure S9. Importantly, for each daf-16 isoform, the two biological repeats provided similar ΔCt calibration (ΔCt S8 ) (Ct iso -Ct com ) values ( Figure S8C).
To compare the level of each daf-16 isoform in day 1 adult daf-2(e1370) mutants, we first measured Ct value for each daf-16 isoform using isoform-specific primers ( Figure S9A); Ct iso daf-16a , Ct iso daf-16b , Ct iso daf-16d/f . Then, using the common primers, we measured the Ct value to obtain the Ct com daf-16 total . The ΔCt for each isoform was determined by Ct isoform -Ct endogenous control ( Figure S9).
Then to determine the ΔΔCt, we took the ΔCt isoform -ΔCt calibrator(from S8) as shown in Figure S9. To determine the relative abundance of each daf-16 isoform in day 1 adult daf-2 mutants, we calculated 2 -ΔΔCt ( Figure S9). Importantly, the sum of the relative levels of each daf-16 isoform was around 1 ( Figure S9B). Last, in two independent sample repeats, daf-16d/f was found to comprise more than 80 % of total daf-16, suggesting DAF-16d/f is the major isoform in early adult worms ( Figure S9C). Figure S1. Comparative DAF-16 protein levels in different daf-16a transgenic worm strains.

Supplementary Figures:
Equal amounts of lysates prepared from approximately 100 worms were loaded onto 10%SDS PAGE gel, and immunoblotted with anti-α-DAF-16 and anti-α-tubulin. See Methods for additional details.

Figure S2. Effect of dosage of transgene on the spatial distribution of DAF-16a
Nuclear/cytosolic distribution of DAF-16a in four daf-16(mgDf50); daf-2(e1370); daf-16a transgenic worms grown at 15°C. The nuclear/cytosolic ratio of DAF-16a::GFP was measured using Image J software. The mean ratio is plotted and the error bars represent the Standard Deviation. Two independent repeats are shown.

Figure S15: Lack of an effect of FuDR on lifespan.
The strains show similar lifespan curves without FuDR. A concentration of 400uM FuDR was used.
Supplementary Tables: Table S1-Analysis of the ESTs that correspond to daf-16        Figure 2B and 2C.