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Longevity Genes Revealed by Integrative Analysis of Isoform-Specific daf-16/FoxO Mutants of Caenorhabditis elegans

FoxO transcription factors promote longevity across taxa. How they do so is poorly understood. In the nematode Caenorhabditis elegans, the A- and F-isoforms of the FoxO transcription factor DAF-16 extend life span in the context of reduced DAF-2 insulin-like growth factor receptor (IGFR) signaling. To elucidate the mechanistic basis for DAF-16/FoxO-dependent life span extension, we performed an integrative analysis of isoform-specific daf-16/FoxO mutants. In contrast to previous studies suggesting that DAF-16F plays a more prominent role in life span control than DAF-16A, isoform-specific daf-16/FoxO mutant phenotypes and whole transcriptome profiling revealed a predominant role for DAF-16A over DAF-16F in life span control, stress resistance, and target gene regulation. Integration of these datasets enabled the prioritization of a subset of 92 DAF-16/FoxO target genes for functional interrogation. Among 29 genes tested, two DAF-16A-specific target genes significantly influenced longevity. A loss-of-function mutation in the conserved gene gst-20, which is induced by DAF-16A, reduced life span extension in the context of daf-2/IGFR RNAi without influencing longevity in animals subjected to control RNAi. Therefore, gst-20 promotes DAF-16/FoxO-dependent longevity. Conversely, a loss-of-function mutation in srr-4, a gene encoding a seven-transmembrane-domain receptor family member that is repressed by DAF-16A, extended life span in control animals, indicating that DAF-16/FoxO may extend life span at least in part by reducing srr-4 expression. Our discovery of new longevity genes underscores the efficacy of our integrative strategy while providing a general framework for identifying specific downstream gene regulatory events that contribute substantially to transcription factor functions. As FoxO transcription factors have conserved functions in promoting longevity and may be dysregulated in aging-related diseases, these findings promise to illuminate fundamental principles underlying aging in animals.

FoxO3 polymorphisms are associated with extreme longevity in multiple independent cohorts of centenarians (Lunetta et al. 2007;Willcox et al. 2008;Anselmi et al. 2009;Flachsbart et al. 2009;Li et al. 2009;Pawlikowska et al. 2009). The evolutionary conservation of FoxO functions in promoting longevity suggests that understanding the molecular basis for FoxO transcription factor action will illuminate fundamental mechanisms that govern aging.
The well-established role of FoxO transcription factors as targets of insulin-like signaling first came to light from studies in Caenorhabditis elegans, where the insulin/insulin-like growth factor receptor (IGFR) ortholog DAF-2 promotes reproductive development and limits adult life span by inhibiting the FoxO transcription factor DAF-16 via a conserved phosphoinositide 3-kinase (PI3K)/Akt pathway-dependent mechanism. Engagement of DAF-2/IGFR by agonist insulin-like ligands activates the PI3K/Akt pathway, resulting in Akt-dependent phosphorylation of three DAF-16/FoxO amino acid residues that lie within conserved RxRxxS/T motifs. Phosphorylated DAF-16/ FoxO is subsequently exported from the nucleus and sequestered in the cytoplasm. When DAF-2/IGFR pathway activity is reduced, unphosphorylated DAF-16/FoxO translocates to the nucleus, where it regulates the expression of numerous genes, including those that control metabolism, immunity, and detoxification (Murphy and Hu 2013). The inhibition of FoxO by insulin-like signaling is evolutionarily conserved, as reduction of FoxO activity ameliorates biological phenotypes associated with reduced insulin-like signaling in flies (Junger et al. 2003;Slack et al. 2011;Yamamoto and Tatar 2011) and mice Nakae et al. 2002;Dong et al. 2008;Cheng et al. 2009).
DAF-16/FoxO also promotes life span extension in animals lacking a germline (Hsin and Kenyon 1999). Although DAF-2/ IGFR and the germline both control DAF-16/FoxO activity by regulating its subcellular localization (Henderson and Johnson 2001;Lee et al. 2001;Lin et al. 2001), they may do so through distinct mechanisms, as the molecular requirements for DAF-16/FoxO regulation by DAF-2/IGFR signaling and the germline differ (Berman and Kenyon 2006;Ghazi et al. 2009).
Work from multiple groups has identified hundreds of genes that are regulated by DAF-16/FoxO in the context of reduced DAF-2/IGFR signaling (Lee et al. 2003;McElwee et al. 2003;Murphy et al. 2003). Our understanding of the functional significance of DAF-16/FoxO target genes in mediating DAF-16/FoxO-dependent longevity is based primarily on a study in which nearly 60 DAF-16/FoxO target genes were tested for roles in DAF-16/FoxO-dependent life span extension using RNAi-based assays. This revealed that most single gene RNAi knockdowns have relatively small effects on longevity, leading to the conclusion that DAF-16/FoxO promotes longevity through the cumulative regulation of hundreds of genes (Murphy et al. 2003).
The roles of most individual DAF-16/FoxO-dependent gene regulatory events in life span control have not been assessed experimentally. This may be due in large part to the abundance of genes regulated by DAF-16/FoxO. Efforts to prioritize DAF-16/FoxO target genes based on direct binding of DAF-16/ FoxO to promoters succeeded in identifying aakg-4 as a gene that is directly induced by DAF-16/FoxO and required for DAF-16/FoxO-dependent life span extension (Schuster et al. 2010;Tullet et al. 2014). Therefore, strategies to prioritize subsets of DAF-16/FoxO target genes can lead to the discovery of new longevity genes by permitting detailed functional analysis of a relatively small number of genes.
The daf-16 genomic locus encodes three groups of transcripts (a, b, and d/f/h) that are transcribed from distinct promoters (Lin et al. 1997;Ogg et al. 1997;Kwon et al. 2010). The encoded proteins possess phosphorylation sites conserved in humans and other species (Supporting Information, Figure S1, A and B). daf-16d, f, and h are transcribed from the same promoter but have distinct 59 ends and translational start sites ( Figure S1C; Figure S2; WormBase, www.wormbase.org) (Kwon et al. 2010;Murphy and Hu 2013). For clarity, we refer to the d/f/h group of transcripts and polypeptides collectively as daf-16f and DAF-16F, respectively. In animals with diminished DAF-2/IGFR signaling, mutations that reduce daf-16a and f but not daf-16b activity shorten life span to the same extent as daf-16 null mutations (Lee et al. 2001). Furthermore, overexpression of DAF-16B under the control of its endogenous promoter does not extend the life span of daf-16/FoxO null mutants (Lee et al. 2001;Kwon et al. 2010). These data implicate DAF-16A and DAF-16F as the critical targets of DAF-2/IGFR in life span control. Analysis of functional isoform-specific DAF-16::GFP reporters demonstrates that DAF-16A and DAF-16F have overlapping spatiotemporal expression patterns and are both expressed in multiple tissues (Kwon et al. 2010). Notably, although the set of genes collectively regulated by DAF-16/ FoxO is well defined, the roles of specific DAF-16/FoxO isoforms in gene regulation are obscure.
In light of the differential influence of DAF-16/FoxO isoforms on longevity (Kwon et al. 2010), we hypothesized that detailed characterization of isoform-specific daf-16/FoxO mutants would illuminate the molecular basis for DAF-16/FoxO action in life span control by enabling the prioritization of a subset of DAF-16/FoxO target genes for further investigation.

C. elegans strains and maintenance
Strains used in this study are listed in Table S17; Table S20. Animals were maintained at 15°on nematode growth media (NGM) plates seeded with Escherichia coli OP50. Double mutants were constructed using standard genetic techniques. Genotypes were confirmed by PCR amplification to detect restriction fragment length or PCR polymorphisms. Percival I-36NL incubators (Percival Scientific, Perry, IA) were used for maintenance, dauer arrest assays, and life span assays.

RNA isolation
Animals were washed twice in M9 buffer. Total RNA was isolated using TRIzol reagent (Invitrogen) and purified using an RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.
Quantitative real-time reverse-transcriptase PCR cDNA was synthesized using a SuperScript III Reverse Transcriptase kit and random hexamers (Invitrogen, Carlsbad, CA). Real-time PCR was then performed in triplicate using Power SYBR Green PCR master mix (Applied Biosystems, Warrington, UK) and a Mastercycler ep realplex thermal cycler (Eppendorf North America, Westbury, NY). A total of 10 ng of cDNAwas used as a template in 15 ml reaction volume. Primers were selected initially using GETPrime (Gubelmann et al. 2011) and Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and subsequently validated by melt curve analysis and agarose gel electrophoresis. Primer sequences are listed in Table S21. Relative expression levels and technical error were determined by the DD2C t method (Nolan et al. 2006). Gene expression levels were normalized to actin (act-1), and the ratio of expression relative to act-1 was then compared to the same ratio in N2 Bristol wild type. Statistical analysis was performed in Graph-Pad Prism (GraphPad Software, La Jolla, CA) using the paired ratio t-test.

Rapid amplification of complementary DNA (cDNA) ends
Total RNA was isolated from young adult animals. daf-16a and daf-16f cDNA was prepared using a 59 rapid amplificiation of cDNA ends (RACE) system version 2.0 (Invitrogen). First-strand cDNA was synthesized using a daf-16a/f genespecific primer and SuperScript II. The original mRNA template was degraded by RNase H and RNase T1. After purification, a homopolymeric tail was added using terminal deoxynucleotidyl transferase to the 39 end of cDNA. Standard PCR was performed using Taq DNA polymerase, a nested daf-16a or daf-16f-specific primer, and the abridged anchor primer complementary to the homopolymeric tail. After visualization of products on a standard agarose gel, the reaction mix was cloned into pCR4-TOPO vector using a TOPO TA Cloning kit (Invitrogen) and transformed into chemically competent E. coli DH5a. Clones were selected on LB plates containing 50 mg/ml ampicillin, and plasmids were analyzed by Sanger sequencing. Numbers of clones analyzed for each strain are indicated in Table S22.

Dauer arrest assays
Dauer assays were performed at 25°as previously described (Hu et al. 2006). Briefly, animals were synchronized in a 4-hr egglay at 15°and grown at 25°on NGM plates. Animals were scored when wild-type animals were gravid adults and daf-2 animals had arrested as dauers (60-72 h after egglay). Statistical significance was assessed using a two-tailed, unpaired t-test with Welch's correction.

Life span assays
Life span assays were performed as previously described (Chen et al. 2013a), with minor modifications. Animals derived from a synchronized 4-hr egglay were grown at 15°u ntil the L4 larval stage and then shifted to 20°. Plates harboring any males were discarded. Animals were grown for an additional 20-24 hr to day 1 of adulthood and then placed on life span plates containing 25 mg/ml 5-fluoro-29-deoxyuridine (FUDR) (Sigma-Aldrich, St. Louis) to prevent progeny growth. glp-1 mutant animals were raised for 48 hr at the restrictive temperature (25°) to ablate the germline and then shifted to 20°. Statistical significance was assessed using the standard chi-square-based log-rank test in GraphPad Prism.

RNAi
RNAi clones were identical to previously published isoformspecific and pan-daf-16 RNAi clones (Kwon et al. 2010). Feeding RNAi was performed using standard procedures (Kamath et al. 2001). All RNAi NGM plates contained 5 mM IPTG and 25 mg/ml carbenicillin. NGM plates were seeded with an overnight culture of E. coli HT115 with either control L4440 vector or RNAi plasmid. For RNAi life span assays, HT115 was concentrated 53. Plasmids from E. coli clones were sequenced for every experimental replicate to confirm their identity.

MosI-mediated single copy insertion
Using the full build service platform at Knudra Transgenics, the plasmids pNU164 and pNU191 were constructed by inserting daf-16 cargo into MosI-mediated single copy insertion (MosSCI) vector backbones. For pNU164, the daf-16a cDNA was PCR amplified from yk13f11/yk1006c10 plasmid, and the full-length daf-16 39 UTR was PCR amplified from wildtype genomic DNA. These two were ligated by PCR-mediated overlap extension (Heckman and Pease 2007) and cloned into a pCFJ151 plasmid. A hybrid segment composed of the 39 portion of the cDNA and the 39 UTR was amplified from this plasmid, while daf-16a promoter segments and partial daf-16a genomic sequence was PCR amplified from wild-type genomic DNA. All parts were then inserted into the multiple cloning site (MCS) of pCFJ151 via Gibson ligation. For pNU191, the promoter and unique daf-16f exons were PCR amplified from wild-type genomic DNA. The common daf-16a/f coding segment and the end of the coding region through the 39 UTR were amplified from pNU164. All parts were then inserted into the MCS of pCFJ151 via Gibson ligation. All coding regions of plasmids were verified by Sanger sequencing. For production of transgenic animals, plasmids were used in a MosSCI injection mix and used to create single copy insertions at the ttTi5605 locus as per published methods (Frokjaer-Jensen et al. 2008).

Stress-resistance assays
For all stress assays, animals derived from a synchronized 4-hr egglay were grown at 15°until the late L3 larval stage and then shifted to 20°and grown for an additional 12 hr until the L4 larval stage. Plates harboring any males were discarded.
For oxidative stress, animals were transferred to plates containing 7.5 mM tert-butyl hydroperoxide (t-BOOH) (Sigma-Aldrich) and scored approximately three times per day for survival. For ultraviolet (UV) stress, animals were transferred to plates lacking bacteria and irradiated with 1200 J/m 2 UV-C using a Stratalinker 2400 UV crosslinker (Stratagene). UV-treated animals were then transferred to seeded plates containing 25 mg/ml FUDR and scored daily for survival. For thermotolerance, animals were transferred to seeded plates containing 25 mg/ml FUDR, grown for an additional 12 hr at 20°, shifted to 33°, and scored approximately four times per day for survival. For all assays, animals that dessicated on the side of plates were censored. Statistical significance was assessed in GraphPad Prism using the standard chi-square-based log-rank test.
Whole transcriptome profiling (RNA-Sequencing) Animals were grown as described for life span assays. After picking a subset of the population for life span assays, the remaining animals were harvested for isolation of total RNA. The Agilent TapeStation was used to assess RNA quality. Samples with RNA integrity numbers (RINs) of eight or greater were prepped using the Illumina TruSeq mRNA Sample Prep v2 kit (catalog nos. RS-122-2001 andRS-122-2002). Messenger RNA (mRNA) was isolated from 0.1 to 3 mg of total RNA by polyA + purification, fragmented, and copied into first-strand cDNA using reverse transcriptase and random primers. The 39 cDNA ends were then adenylated and adapters were ligated. One of the adapters contained a six-nucleotide barcode to enable multiplexing of samples. Products were purified and enriched by PCR to create the final cDNA library. Libraries were checked for quality and quantity by Agilent TapeStation and quantitative PCR (qPCR) using a library quantification kit for Illumina sequencing platforms (catalog no. KK4835, Kapa Biosystems, Wilmington, MA). Clonal clusters were generated using cBot (Illumina, San Diego). Quadriplexed samples were sequenced using the HiSequation 2000 system (Illumina) with a 100-cycle paired-end run in high output mode using version 3 reagents according to manufacturer's protocols.

RNA-seq analysis
Individual read files for each sample were concatenated into a single .fastq file. Raw reads data for each sample were checked using FastQC (version 0.10.0, Babraham Bioinformatics, Cambridge, United Kingdom; http://www.bioinformatics. bbsrc.ac.uk/projects/fastqc/) to identify features potentially indicative of quality issues (e.g., low-quality scores, overrepresented sequences, and inappropriate GC content). We used the Tuxedo Suite (Langmead et al. 2009;Trapnell et al. 2009Trapnell et al. , 2013 for alignment, differential expression analysis, and postanalysis diagnostics. Briefly, reads were aligned to the reference genome (University of California Santa Cruz, UCSC ce10; http://genome.ucsc.edu/) using TopHat (version 2.0.9) ) and Bowtie (version 2.1.0.0) (Langmead 2010). Alignments were performed using default parameter settings, with the exception of "-b2-very-sensitive" and "-no-coverage-search." Additionally, the "max_intron_length" parameter was set to 25 kb to minimize false positive splice junction discovery. A second round of quality control was then performed using FastQC to ensure that only high-quality data were analyzed further. Cufflinks/CuffDiff (version 2.1.1) (Trapnell et al. 2013) was used for quantification of expression and differential expression analysis, using UCSC ce10.fa as the reference genome and UCSC ce10.gtf as the reference transcriptome (http://genome.ucsc.edu/). For this analysis, we used parameter settings "-multi-read-correct" to adjust expression calculations for reads that map to more than one locus, as well as "-compatible-hits-norm" and "-upper-quartile -norm" for normalization of expression values. We generated diagnostic plots using the CummeRbund package (Trapnell et al. 2012) as a quality check on normalized data.
For functional enrichment testing, we identified enriched Gene Ontology (GO) biological process terms (Ashburner et al. 2000) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (Kanehisa and Goto 2000) using LRpath, a logistic regression method that overcomes limitations associated with the use of arbitrary significance cutoff values by taking into account the distribution of significance levels of all profiled genes (Sartor et al. 2009). Enriched GO terms and KEGG pathways were defined as those having a false discovery rate of ,0.01. We used a directional LRpath test to distinguish between up-regulated and down-regulated functions. Overlapping and redundant GO terms were clustered semantically with REVIGO (Supek et al. 2011). We employed a semantic similarity (SimRel) cutoff of 0.7 to define representative terms. LRpath and REVIGO outputs are shown in Table S15.
Classes of DAF-16A/F targets were defined by the following criteria: DAF-16A-specific, I A . 0.8 and I F , 0.2; DAF-16F-specific, I F . 0.8 and I A , 0.2; and redundantly regulated, I A , 0.2 and I F , 0.2. Genes not binned into these three categories were partitioned into one of the following three groups: shared A-dominant (shared A . F), I A /I F . 2; shared F-dominant (shared F . A), I F /I A . 2; or equally shared (shared A = F), 0.5 # I A /I F # 2.

RNAi-defective assays
Assays were performed essentially as described (Billi et al. 2012). L1 larvae were picked onto RNAi plates (5 mM IPTG, 25 mg/ml carbenicillin) seeded with an overnight culture of E. coli HT115 containing a pos-1 RNAi plasmid, which causes embryonic lethality (Tabara et al. 1999). rde-4(ne301) was used as an RNAi-defective (rde) control (Tabara et al. 2002). Animals were grown for 84 hr at 20°to allow for development and egglaying and then removed from the plate. Eggs were counted immediately, live progeny were counted 48 hr later, and percentage of nonviable eggs was calculated.

Data availability
Strains are available upon request. Whole transcriptome profiling data are available at GEO with the accession number: GSE72426.

Molecular characterization of isoform-specific daf-16/FoxO mutants
To isolate daf-16a-and daf-16f-specific mutant alleles, we screened for mutants with deletions in the unique N-terminal exons of each isoform (Gengyo-Ando and Mitani 2000). Two independent alleles, tm5030 and tm5032, are deletions in the daf-16a-specific exon ( Figure 1). The 59 RACE analysis shows that these mutations result in frameshifts and predicted early translation termination ( Figure 1B, Figure S3, Figure S4, and Figure S5). As expected, both tm5030 and tm5032 significantly reduced daf-16a transcript levels as measured by quantitative real-time reverse transcriptase PCR (qPCR; Figure S6 A and B; Table S1), consistent with nonsense-mediated decay secondary to premature translation termination. Neither mutation influenced the integrity or quantity of daf-16b or daf-16f transcripts ( Figure S6, C and D; Table S1). tm6659 is a deletion that spans all four daf-16f-specific exons, including all three putative transcriptional and translational start sites ( Figure 1A; Figure S1C) (Kwon et al. 2010;Murphy and Hu 2013). daf-16f transcripts were undetectable in animals harboring tm6659 by 59 RACE and qPCR ( Figure S3; Figure S6D; Table S1), and neither daf-16a nor daf-16b transcripts were affected ( Figure S6 A-C; Table S1). Total daf-16 levels are sharply reduced in tm6659 mutants (Figure S6 E and F; Table  S1). Importantly, our data show no compensatory increase in daf-16f transcripts in either daf-16a(tm5030) or daf-16a (tm5032) ( Figure S6D) and no increase in daf-16a transcripts in daf-16f(tm6659) ( Figure S6, A and B).
DAF-16A promotes dauer arrest in animals with reduced DAF-2/IGFR signaling In response to adverse environmental conditions, C. elegans larvae undergo developmental arrest in an alternative larval stage known as dauer (Riddle 1988). daf-2/IGFR and daf-16/ FoxO mutants were first isolated in genetic screens for animals with dauer-constitutive (Daf-c) and dauer-defective (Daf-d) phenotypes, respectively (Riddle et al. 1981). daf-2/IGFR mutants constitutively arrest as dauers at 25°in a daf-16/ FoxO-dependent manner (Riddle et al. 1981;Vowels and Thomas 1992;Gottlieb and Ruvkun 1994). To examine the roles of distinct DAF-16/FoxO isoforms in dauer regulation, we determined the influence of isoformspecific daf-16/FoxO mutations on dauer-constitutive phenotypes in two daf-2/IGFR mutant backgrounds: e1368, a missense mutation in the DAF-2/IGFR extracellular ligand-binding domain, and e1370, a missense mutation in the cytoplasmic tyrosine kinase domain (Kimura et al. 1997). e1370 may be a stronger loss-of-function allele than e1368, as it causes a more penetrant dauer-constitutive phenotype and extends life span to a greater extent than e1368 (Figure 2 and Figure 3) (Gems et al. 1998).
daf-16a no. 1 and no. 2 mutations partially reduced mean life spans of both daf-2(e1368) and daf-2(e1370) mutants, whereas daf-16a/f mutation decreased mean life spans to the same extent as daf-16 null mutation (Figure 3, A and C;  Table S6). These results indicate that DAF-16A is partially required for the longevity of daf-2/IGFR mutants. In contrast, daf-16f mutation did not reproducibly influence life span in either daf-2/IGFR mutant background (Figure 3, B and D; Table S5; Table S6). This finding was unexpected in light of previous studies implicating DAF-16F in life span control by the DAF-2/IGFR pathway (Kwon et al. 2010;Bansal et al. 2014). Taken with our finding that the tm6659 mutation likely fully eliminates daf-16f activity (Figure 1, A and B; Figure S1C; Figure S6D), our data are consistent with a hierarchical model of DAF-16/FoxO isoform function in promoting longevity in the context of reduced DAF-2/IGFR signaling. DAF-16A is necessary for full life span extension. However, DAF-16F is dispensable for longevity, as DAF-16A is sufficient to fully extend life span even when daf-16f is mutated.
Given that daf-16a/f mutation shortens life span in animals with reduced DAF-2/IGFR activity to a much greater extent than daf-16a mutation alone (Figure 3, A and C; Table S5;  Table S6), we tested the possibility that DAF-16F is required for life span extension in the absence of DAF-16A by performing isoform-specific daf-16/FoxO RNAi (Kwon et al. 2010). As previously shown, inactivation of either daf-16a or daf-16f alone by RNAi had a modest effect on the longevity of daf-2/IGFR mutant animals compared to RNAi of all daf-16/FoxO isoforms ("pan-daf-16 RNAi"; Figure S7A; Table S7) (Kwon et al. 2010). However, daf-16f RNAi shortened the mean life span of daf-16a;daf-2 double mutant animals to nearly the same extent as pan-daf-16 RNAi ( Figure 3E; Table S7). Therefore, although DAF-16F is dispensable for longevity in the presence of DAF-16A, it is required for life span extension in animals lacking DAF-16A. The life span shortening effect of daf-16a RNAi in daf-16a;daf-2 double mutants may be a consequence of off-target RNAi effects (Ma et al. 2006).
We also wished to determine the extent to which longevity in daf-16f;daf-2 double mutants requires DAF-16A. To address this question, we performed isoform-specific daf-16/ FoxO RNAi on daf-16f;daf-2 double mutants. daf-16a RNAi shortened the mean life span of daf-16f;daf-2 double mutant animals by nearly the same amount as pan-daf-16 RNAi (Figure 3F; Table S7). In contrast, daf-16f RNAi had a negligible effect on daf-16f;daf-2 mean life span. Therefore, when DAF-16F is absent, DAF-16A is likely the sole FoxO isoform that promotes longevity. In aggregate, our results indicate that DAF-16A is the primary isoform that controls C. elegans aging in the context of reduced DAF-2/IGFR signaling. DAF-16F is not required for longevity when DAF-16A is present, but it promotes long life in the absence of DAF-16A.
Rescue of mutant phenotypes with single-copy isoform-specific DAF-16/FoxO transgenes Our data on DAF-16/FoxO isoform function are in agreement with a previous study implicating both DAF-16A and DAF-16F in dauer regulation in the daf-2(e1370) mutant background (Kwon et al. 2010). However, our results contradict the contention that DAF-16F plays a more prominent role in life span control than DAF-16A (Kwon et al. 2010;Bansal et al. 2014). This discrepancy may be a consequence of distinct experimental strategies used to assess the function of DAF-16/FoxO isoforms in life span control. We used isoformspecific deletion mutants in which other daf-16/FoxO isoforms remained under the control of endogenous regulatory elements and continued to be expressed at physiological levels ( Figure S6). In contrast, Kwon et al. (2010) based their analysis on strains harboring both a daf-16/FoxO null mutation and transgenes overexpressing cDNAs encoding individual daf-16/FoxO isoforms.
In an effort to reconcile our results with those of Kwon et al. (2010), we generated integrated transgenic strains expressing either daf-16a or daf-16f under the control of their native promoters ( Figure S8). These transgenes were modeled after those reported by Kwon et al. (2010). To minimize potentially confounding influences pertaining to overexpression and/or differences in genomic integration sites, we constructed these strains using MosSCI (Frokjaer-Jensen et al. 2008). We crossed these single-copy transgenes into daf-16a/f and daf-16a mutant backgrounds to assess their relative activity in promoting longevity and dauer arrest.
Promoter-swap experiments show that daf-16a or daf-16f transgenes driven by the daf-16f promoter rescue longevity to a greater extent than the same transgenes driven by the daf-16a promoter (Kwon et al. 2010). Intriguingly, WormBase (www.wormbase.org) annotations document the existence of daf-16a transcripts that originate from the daf-16f promoter. We verified this in two ways. First, we used PCR amplification and sequencing to identify splice junctions unique to these transcripts ( Figure S11, A and B). Furthermore, we identified sequence reads in whole transcriptome profiling data that correspond to such transcripts ( Figure S11B). Therefore, daf-16a is transcribed from two promoters, one of which is the same promoter that drives expression of daf-16f. As primary transcripts originating from the daf-16f promoter contain all daf-16a exons ( Figure 1A), alternative splicing of such pre-mRNAs may generate mature daf-16a transcripts. Taken together with the relative importance of the daf-16f promoter in extending life span (Kwon et al. 2010), this finding could explain why our single-copy daf-16a transgene ( Figure S8) has less activity than the endogenous daf-16a locus ( Figure 3G; Figure S9, A-C; Figure S10, A-D).
To test the activity of our single-copy daf-16f transgene, we constructed daf-16a/f;daf-2(e1368) and daf-16a/f;daf-2(e1370) animals harboring the daf-16f transgene. The life spans of these animals were comparable to the life spans of daf-16a;daf-2 double mutant animals ( Figure 3H; Figure  S9D; Table S8). Since daf-16f activity is the major contributor to longevity in daf-16a;daf-2 double mutants ( Figure 3E), the activity of daf-16f encoded by this single-copy transgene is comparable to the activity of the endogenous daf-16f locus. Dauer arrest phenotypes of these animals also mirrored those of daf-16a;daf-2 double mutants (Figure S10, E and F). As previously observed in strains harboring multicopy daf-16 transgenes (Kwon et al. 2010), animals with the daf-16f transgene lived much longer than those with the daf-16a transgene (Figure 3, G and H; Table S8). Notably, the daf-16f transgene significantly increased both the life span and the penetrance of the dauer-constitutive phenotype of daf-16a;daf-2(e1370) double mutant animals that contain intact endogenous daf-16f ( Figure 3H; Figure S10E). Therefore, the longevity and dauer-constitutive phenotypes of daf-2 mutant animals are highly sensitive to daf-16f gene dosage. Figure 2 daf-16a-specific mutations suppress dauer arrest in daf-2/IGFR mutants. (A) The dauer-constitutive phenotype of daf-2(e1368) animals is fully suppressed by daf-16a/f mutation and both daf-16a mutations but is unaffected by daf-16f mutation. (B) The dauerconstitutive phenotype of daf-2(e1370) animals is fully suppressed by daf-16a/f mutation, partially suppressed by both daf-16a mutations, and unaffected by daf-16f mutation. Mean and standard deviation for at least three biological replicates are presented. Statistics and raw data are presented in Table S2; Table S3; Table S4.
Collectively, our results with single-copy isoform-specific daf-16/FoxO transgenes, while consistent with published results using multicopy transgenes (Kwon et al. 2010;Bansal et al. 2014), strongly suggest that the previously reported difference in longevity-promoting activity of multicopy daf-16a-and daf-16f-specific transgenes (Kwon et al. 2010;Bansal et al. 2014) is due to both overexpression of daf-16f as well as the failure of the daf-16a-specific transgene to recapitulate endogenous daf-16a activity.

DAF-16A promotes longevity in animals lacking a germline
Although DAF-16/FoxO is also required for longevity in animals lacking a germline (Hsin and Kenyon 1999), life span extension caused by germline ablation requires molecules such as KRI-1 and TCER-1 that are not necessary for longevity in daf-2/IGFR mutants (Berman and Kenyon 2006;Ghazi et al. 2009). These observations suggest that DAF-2/IGFR and the germline could control life span by coupling to distinct DAF-16/FoxO isoforms. To test this hypothesis, we determined the effect of isoform-specific daf-16/FoxO mutations on life span in animals harboring a temperature-sensitive glp-1 mutation that develops without a germline when grown at 25°(Arantes-Oliveira et al. 2002). As we observed in animals with reduced DAF-2/IGFR activity, the life spans of germline-ablated animals were modestly reduced by daf-16a mutation ( Figure 4A; Table S9). daf-16f mutation did not influence the life span of germline-ablated animals in a consistent manner ( Figure 4B; Table S9).
Collectively, these data show that DAF-16A is also the main FoxO isoform that promotes longevity in animals lacking a germline, while DAF-16F is not required for life span extension in this context. Furthermore, they demonstrate that the distinct molecular requirements for life span extension in daf-2/IGFR mutants and germline-ablated animals (Berman and Kenyon 2006;Ghazi et al. 2009) cannot be explained by the coupling of these upstream pathways to disparate DAF-16/FoxO isoform outputs.
DAF-16A promotes stress resistance in animals with reduced DAF-2/IGFR signaling As DAF-16/FoxO is required for the resistance of daf-2/IGFR mutants to various environmental insults (Murakami and Johnson 1996;Honda and Honda 1999;McColl et al. 2010), we determined the impact of daf-16a-and daf-16fspecific mutations on the sensitivity of daf-2(e1370) mutants to heat, oxidative stress, and UV radiation. The effect of these mutations on stress tolerance was similar in each condition. As expected, the daf-2/IGFR mutant was significantly more resistant to heat, oxidative stress, and UV radiation ( Figure  S12; Table S12) than wild-type animals. daf-16a/f mutation completely abolished the resistance of the daf-2/IGFR mutant to heat and oxidative stress and strongly suppressed its resistance to UV radiation. In all three contexts, daf-16a mutation partially suppressed stress resistance of the daf-2/IGFR mutant, whereas daf-16f mutation did not influence its ability to withstand insult. These results mirror the impact of isoform-specific mutations on life span (Figure 3; Figure 4).

DAF-16/FoxO target gene regulation by DAF-16A and DAF-16F
To illuminate the mechanistic basis for DAF-16/FoxO isoform-specific functions in life span control, we performed whole transcriptome profiling of young adult daf-2(e1370) mutant animals in the context of wild-type daf-16/FoxO and isoform-specific daf-16/FoxO mutant alleles. A subset of animals from each experimental replicate was subjected to life span assays to confirm that all mutants from which RNA was isolated had the expected life span phenotypes. Identification of genes that were differentially expressed in wild type and daf-2(e1370) and differentially expressed in the opposite direction in both daf-16(null);daf-2 and daf-16a/f;daf-2 double mutants compared to daf-2 mutants defined a set of 399 genes that are targets of DAF-16A and/or DAF-16F ( Figure  5; Table S13; henceforth referred to as "DAF-16A/F target genes"). We validated our profiling results by measuring transcript levels corresponding to 15 genes that emerged from this analysis using qPCR. DAF-16/FoxO-dependent reg-ulation was confirmed for all 15 of these genes ( Figure 6; Figure S13; Table S14).
To estimate the influence of specific DAF-16/FoxO isoforms on aspects of whole organism physiology, we performed gene set enrichment analysis of DAF-16A/F target genes using GO biological process terms (Ashburner et al. 2000) and KEGG pathways (Kanehisa and Goto 2000) ( Figure S14; Table  S15). GO analysis revealed specific up-regulation of ribosome biogenesis genes and specific down-regulation of replication and cell death genes in the daf-16f mutant. Immune response genes were up-regulated in daf-16a mutants and downregulated in the daf-16f mutant ( Figure S14A; Table S15). KEGG analysis also unveiled enrichment of genes involved in ribosome biogenesis in the daf-16f mutant while showing reduction of genes involved in glycolysis and gluconeogenesis in daf-16a mutants. Genes involved in cysteine and methionine metabolism were depleted in the daf-16f mutant but enriched in daf-16a mutants ( Figure S14B; Table S15).
To gain insight into the relative magnitude of DAF-16Aand DAF-16F-specific contributions to the regulation of individual DAF-16A/F target genes, we compared the effect of either daf-16a or daf-16f mutation on DAF-2/IGFR-dependent gene regulation to the effect of daf-16a/f mutation on DAF-2/IGFR-dependent gene regulation, thus calculating an "A-index" (I A ) and "F-index" (I F ) for each DAF-16A/F target gene (see Materials and Methods). Hypothetically, an idealized DAF-16A-specific target gene would have I A = 1.0 and I F = 0, whereas a DAF-16F-specific target gene would have I A = 0 and I F = 1.0 ( Figure 5A). I A and I F calculated using FPKM values from whole transcriptome profiling correlated well with I A and I F derived from qPCR data for 15 genes tested ( Figure 6; Figure S13; Table S13; Table S16).
We first generated a scatterplot of I A and I F for the entire set of 399 DAF-16A/F target genes ( Figure 5B; Figure S15). This depiction indicates that I A . I F for most genes, suggesting that DAF-16A plays a larger role in gene regulation than DAF-16F. To further explore this question, we plotted I A and I F of the entire set of DAF-16A/F target genes from lowest to highest I A ( Figure 5C) and I F ( Figure 5D). This allowed us to visualize the magnitude of isoform-specific regulation for both DAF-16A and DAF-16F across the entire set of target genes while potentially revealing relationships between the isoforms in target gene regulation. These illustrations confirm that for most target genes, DAF-16A has a greater impact on expression than DAF-16F. Furthermore, they reveal no obvious global relationship between the degree of regulation of any single gene by one isoform and the impact of the other isoform on its expression.

Categorization of DAF-16A/F target genes
The life span phenotypes of daf-16a/f, daf-16a, and daf-16f mutants ( Figure 3) suggested that sorting of DAF-16A/F target genes into categories based on their regulation by DAF-16A and/or DAF-16F might help to prioritize subsets of DAF-16/FoxO genes that are likely to contribute significantly to longevity. Therefore, we placed each DAF-16A/F target gene into one of four categories based on the impact of each isoform on expression: DAF-16A-specific (I A . 0.8 and I F , 0.2), DAF-16F-specific (I F . 0.8 and I A , 0.2), redundant (I A and I F both , 0.2), and shared (all others). We further subdivided genes in the "shared" category into three subgroups: genes for which DAF-16A plays a greater role than DAF-16F in regulation (shared A . F; I A /I F . 2.0), genes for which DAF-16F has a greater impact on regulation than DAF-16A (shared F . A; I F /I A . 2.0), and genes that are regulated by both isoforms (shared A = F; 0.5 # I A /I F # 2.0) (Figure 5, E and F).
Two DAF-16A-specific target genes play important roles in life span control Our main goal at the outset of this line of inquiry was to prioritize a tractable subset of DAF-16/FoxO target genes for functional analysis. Based on the life span phenotypes of isoform-specific daf-16/FoxO mutants (Figure 3; Figure 4), Figure 4 Effects of daf-16aand daf-16fspecific mutations and RNAi on life span in animals lacking a germline. (A and B) Effects of daf-16a (A) and daf-16f (B) mutations on life spans of germline-ablated glp-1(e2141) animals. (C and D) daf-16f is required for daf-16a;glp-1 longevity and vice versa. Survival curves are presented for (C) daf-16a;glp-1 and (D) daf-16f;glp-1 mutant animals upon exposure to isoform-specific daf-16 RNAi. Figure S7B shows control glp-1(e2141) survival when treated with isoform-specific daf-16 RNAi. See text for details. Statistics and raw data are presented in Table S9; Table S10.
we surmised that the subgroups of 57 DAF-16A-specific and 35 redundant target genes had a high probability of containing genes that would impact longevity significantly. DAF-16Aspecific genes likely contribute to the life span differential between daf-16a;daf-2 and daf-2 mutants, whereas redundantly regulated genes might be expected to account for the difference in the life spans of daf-16a/f;daf-2 and daf-16a; daf-2 double mutants (Figure 3, A and C).
To test this hypothesis, we obtained strains readily available to the community that harbored probable strong loss-of-function mutations in twenty DAF-16A-specific, two DAF-16F-specific, and nine redundant target genes (Table S17). Life spans were assayed after exposure to either control bacteria or bacteria engineered to synthesize double-stranded RNA corresponding to the daf-2 gene (daf-2 RNAi). In control experiments, life span extension induced by daf-2 RNAi was completely abrogated in daf-16 null mutants and partially reduced in daf-16a-specific mutants (Figure 7, A and B).
Among 19 strains harboring mutations in genes induced by DAF-16/FoxO, daf-2-RNAi-induced life span extension was significantly less than that observed in wild-type controls in a strain harboring a mutation in gst-20 ( Figure 7C; Table S18). This strain was not RNAi defective (Rde; Figure S16), indicating that the reduced life span extension upon exposure to daf-2 RNAi is unlikely to be a consequence of a background mutation that reduces the efficiency of daf-2 inactivation by RNAi. Furthermore, examination of the mutational load in this strain, which was generated as part of the Million Mutation Project (Thompson et al. 2013), revealed no obvious strong loss-of-function mutations in genes known to influence aging or RNAi (Table S19).
To verify this result, we constructed a gst-20;daf-2(e1368) double mutant and compared its life span to that of gst-20 and daf-2 single mutant siblings. gst-20 mutation did not significantly shorten life span in animals with wild-type daf-2 (Figure 7, D and E; Table S18), indicating that this mutation does not cause general frailty or sickness. Intriguingly, gst-20 mutation significantly  Figure S15. I A and I F for all genes are listed in Table S13. (C and D) Plots of I A and I F for all DAF-16A/F target genes from lowest to highest I A (C) or I F (D). Solid lines correspond to indices of 0 and 1. Three genes with indices .2.2 or , 21.2 were omitted for presentation purposes. Gene rankings are listed in reduced life span extension caused by daf-2 mutation when animals were fed E. coli HT115 ( Figure 7D; Table S18) but did not influence the life span of daf-2(e1368) mutants when they were fed E. coli OP50 ( Figure 7E; Table S18).
gst-20 encodes a glutathione-S-transferase family member homologous to human hematopoietic prostaglandin D synthase, an enzyme that catalyzes the glutathione-dependent isomerization of prostaglandin H2 to prostaglandin D2 (Kanaoka and Urade 2003). We hypothesize that the induction of gst-20 by DAF-16A ( Figure S13A; Table S14) contributes to DAF-16/FoxO-dependent life span extension in a manner dependent upon bacterial food source.
Among 12 strains with mutations in genes repressed by DAF-16/FoxO, one strain containing a mutation in srr-4 lived significantly longer than wild-type animals exposed to control RNAi. This strain also did not contain strong loss-of-function mutations in genes that affect aging (Table S19). Life span assays with outcrossed srr-4 mutant animals and wild-type siblings verified that srr-4 mutation extends life span ( Figure 7F; Table S18). As observed for gst-20 mutation, we found that the impact of srr-4 mutation on life span was also dependent upon bacterial food source; srr-4 mutation extended life span significantly when animals were fed E. coli HT115 ( Figure 7F; Table S18) but had less of an effect on longevity when animals were fed E. coli OP50 ( Figure 7G; Table S18). srr-4 encodes a putative seven-transmembrane G-protein-coupled receptor that is repressed by DAF-16A ( Figure S13B; Table S14). The SRR-4 protein is conserved in other Caenorhabditis species and is predicted to contain a DUF267 (domain of unknown function) domain. We postulate that DAF-16A may promote longevity in part by reducing srr-4 expression.

Discussion
Over 20 yr after the initial demonstration that DAF-16/FoxO promotes longevity (Kenyon et al. 1993), the question of Asterisks indicate statistically significant changes (P , 0.05 by paired ratio t-test). I A and I F were calculated using mean expression values measured by qPCR, and correlate with indices calculated using RNA-seq measurements (Table S16). Statistics and data are summarized in Table S14. which DAF-16/FoxO target genes are important for life span extension remains unanswered. Efforts to address this important issue have been hindered by the prodigious number of genes that are regulated by DAF-16/FoxO. Most of the hundreds of DAF-16/FoxO target genes that are regulated by DAF-2/IGFR signaling (Lee et al. 2003;McElwee et al. 2003;Murphy et al. 2003) have not been interrogated for roles in life span control.
Approaches to prioritize subsets of DAF-16/FoxO target genes for detailed investigation have succeeded in identifying DAF-16/FoxO target genes with previously unappreciated functions in promoting longevity (Schuster et al. 2010;Tullet et al. 2014). We have combined phenotypic analysis and whole transcriptome profiling of isoform-specific daf-16/ FoxO mutants to define a subset of 92 DAF-16/FoxO target genes (composed of 57 DAF-16A-specific and 35 redundant target genes; Figure 5, E and F; Table S13) that we have prioritized for further study. We hypothesize that this subset may be enriched in genes likely to play important roles in life span control; regulation of DAF-16A-specific genes may account for the difference in life span between daf-2 mutants and daf-16a;daf-2 double mutants (Figure 3, A and C), and regulation of redundant genes may contribute to life span differences between daf-16a/f;daf-2 double mutants and both daf-16a;daf-2 (Figure 3, A and C) and daf-16f;daf-2 double mutants (Figure 3, B and D).
Our initial phenotypic analysis of 29 strains harboring strong loss-of-function mutations in genes from this subset has revealed potentially important functions for two DAF-16/ FoxO target genes, gst-20 and srr-4, in controlling life span. Neither of these genes had previously been implicated in DAF-16/FoxO-dependent longevity. Our discovery of these new longevity genes underscores the efficacy of our integrative strategy in identifying DAF-16/FoxO target genes that  Table S18.
are likely to influence life span. We anticipate that functional analysis of the remaining 63 genes within this prioritized subset of DAF-16/FoxO targets will reveal further insights into the mechanistic basis for life span extension by DAF-16/FoxO. As most transcription factors regulate the expression of hundreds of genes, our strategy may be generally applicable to the study of target genes and their relative importance in mediating transcription factor function.
Notably, our life span assays revealed diet-dependent effects of gst-20 and srr-4 on longevity. Mutations in both genes influenced life span when animals were fed E. coli HT115 but did not significantly affect longevity when animals were fed E. coli OP50 (Figure 7; Table S18). Given that the main RNAi library used in the C. elegans field was constructed using E. coli HT115 , which is distinct from the standard OP50 strain used in C. elegans studies, our findings underscore the importance of verifying RNAi-based life span assays in the context of E. coli OP50.
Although we have focused our efforts on genes within the DAF-16A-specific and redundant categories of DAF-16/FoxO targets ( Figure 5E; Table S13), genes in other categories may also influence life span significantly. Indeed, aakg-4, which encodes an atypical AMP-activated protein kinase gamma isoform that promotes longevity in the context of reduced DAF-2/IGFR activity (Chen et al. 2013b;Tullet et al. 2014), emerged from our analysis in the shared A . F category, which was the largest subset of DAF-16/FoxO targets ( Figure  5E; Table S13). We are exploring complementary integrative approaches to define other subsets of DAF-16/FoxO target genes that may direct our attention to specific genes within the larger subsets generated in this study.
Our analysis of isoform-specific daf-16/FoxO mutants indicates that DAF-16A is the major FoxO isoform that controls dauer arrest (Figure 2), longevity (Figure 3; Figure 4), and stress resistance ( Figure S12). In the presence of physiologic levels of DAF-16A, DAF-16F is dispensable for dauer regulation, life span control, and stress resistance. In the absence of DAF-16A, DAF-16F promotes dauer arrest and longevity, as demonstrated by the incomplete suppression of dauerconstitutive and life span extension phenotypes by daf-16a mutation ( Figure 2B; Figure 3, A and C; Figure 4A) and the influence of daf-16f RNAi on life span in daf-2/IGFR mutant animals and germline-ablated animals that lack daf-16a ( Figure 3E; Figure 4C).
These conclusions are at odds with previous suggestions that DAF-16F is the major isoform that promotes longevity in the context of reduced DAF-2/IGFR signaling (Kwon et al. 2010;Bansal et al. 2014). As this interpretation of the primacy of DAF-16F in life span control was based in large part on life span phenotypes of transgenes overexpressing either daf-16a or daf-16f (Kwon et al. 2010;Bansal et al. 2014), we conducted our own investigations of single-copy daf-16a and daf-16f transgenes driven by the same promoters used by Kwon et al. (2010) (Figure S8). Our results (Figure 3, G and H; Figure S9; Figure S10) strongly suggest that the difference in the longevity-promoting activities of daf-16a and daf-16f transgenes that they observed is a consequence of both the sensitivity of the daf-16f transgene to dosage ( Figure  3H; Figure S10E) as well as the failure of the daf-16a transgene to fully recapitulate the activity of the endogenous daf-16a locus ( Figure 3G; Figure S9A; Figure S10, A-D), rather than greater longevity-promoting activity per se of daf-16f compared to daf-16a.
The expression profiling experiments presented here are the first to define the relative contributions of endogenous DAF-16/FoxO isoforms to the regulation of DAF-16/FoxO target genes. They reveal a dominant role for DAF-16A relative to DAF-16F in regulating gene expression in young adult animals ( Figure 5; Table S13) that is commensurate with the relative influence of DAF-16A and DAF-16F on adult life span (Figure 3; Figure 4). These results further support the conclusion that DAF-16A is the major FoxO isoform that promotes longevity in C. elegans.
Based on our results, we propose a hierarchical model of DAF-16/FoxO isoform function in life span control ( Figure  7H). DAF-2/IGFR inhibits both DAF-16A and DAF-16F (Kwon et al. 2010). In the context of reduced DAF-2/IGFR signaling, both DAF-16A and DAF-16F contribute to longevity and the regulation of DAF-16/FoxO target genes. In the absence of DAF-16A, the altered expression of genes regulated mainly by DAF-16A shortens life span. When DAF-16F is inactive, genes regulated primarily by DAF-16F are misregulated, but this does not influence life span. When neither DAF-16A nor DAF-16F is present, DAF-2/IGFR mutation does not promote longevity due to the misregulation of both DAF-16A-specific target genes as well as genes that are regulated by both DAF-16A and DAF-16F.
The conserved role of IGFR signaling and FoxO transcription factors in life span control was first discovered in C. elegans (Friedman and Johnson 1988a,b;Johnson 1990;Kenyon et al. 1993). Decades later, how FoxO transcription factors extend life span remains poorly understood. Here we report a novel approach to delineate the contributions of specific FoxO target genes to FoxO-dependent longevity. IGFR signaling is now known to influence aging in mammals (Bluher et al. 2003;Holzenberger et al. 2003) and possibly humans (Suh et al. 2008;Tazearslan et al. 2011), and the prolongevity function of FoxO that is conserved in invertebrates (Kenyon et al. 1993;Giannakou et al. 2004;Hwangbo et al. 2004;Slack et al. 2011;Yamamoto and Tatar 2011) and mice (Shimokawa et al. 2015) is likely relevant to human aging and aging-related diseases. By directing focus to a subset of target genes, our strategy will facilitate the elucidation of the molecular basis for life span extension by FoxO transcription factors. Further investigation of DAF-16/FoxO target gene function in C. elegans promises to illuminate phyletically general functions of FoxO transcription factors in controlling aging. tance with whole transcriptome profiling, the UM Bioinformatics Core and Mallory Freeberg for help with analysis of transcriptome profiling data, and John Kim and Colin Delaney for comments on the manuscript. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health (NIH) Office of Research Infrastructure Programs (P40 OD010440). This work was supported by the Biology of Aging training grant T32AG000114, awarded to the UM Geriatrics Center by the National Institute on Aging (A.T.-Y.C.), the Functional Assessment Core of the UM Nathan Shock Center of Excellence in the Basic Biology of Aging, research scholar grant DDC-119640 from the American Cancer Society (P.J.H.), and R01 grant AG041177 from the NIH (P.J.H.).    No daf-16h transcripts were detected by 5' RACE.

Figure S3 Overview of strategy for characterizing daf-16/FoxO transcripts in isoform-specific mutants.
Spliced transcripts are shown, and exons are separated by black lines. Note that daf-16a(tm5032) disrupts a 5' splice site and therefore intronic sequence is included.
For 5' RACE, a semi-synchronized population of young adults was harvested for RNA. RNA was purified and analyzed as described in Methods. The A/F-RACE primer was used for first-strand cDNA synthesis. PCR amplification was performed using either the A30-RACE primer, A32-RACE primer, or the F-RACE primer, in combination with the Abridged Anchor Primer from the Invitrogen RACE kit. For daf-16(tm5032), a different primer A32-RACE was used. RACE sequences are presented in Figures S2 and S4.
qPCR results are presented in Fig. S6 and Table S1.

Figure S4 (continued)
Additional legend-specific to daf-16a(tm5032) lower-case: intronic sequence introduced by tm5032 mutation tm5032 #1 vs. #2: Two products detected by cDNA sequencing. #1 is the major product, formed by cryptic splice site activation. * denotes cryptic splice site. -16a(tm5030) and daf-16a(tm5032) genomic sequence (nt 6064-6837    Legend | = identity with wild-type sequence Underline: start of Forkhead domain Bold: RxRxxT AKT family phosphorylation motif  = phosphothreonine * = early stop Blue: amino acids included in the protein products of R13H8.1c but not R13H8.1b due to inclusion of 6 nucleotides that do not affect the reading frame (see figure S4). In wild-type, the three indicated amino acids are present in R13H8.1c, but are replaced by a single glutamic acid residue in R13H8.1b. In daf-16a(tm5030), the three indicated amino acids are present in R13H8.1c, but are replaced by a single lysine residue in R13H8.1b.

Figure S6 qPCR measurements of daf-16/FoxO transcripts in isoform-specific mutants.
Mean values are presented from three biological replicates, error bars represent standard deviation and asterisks indicate samples that display statistically significant changes (p < 0.05 by paired ratio ttest). Data and statistics are summarized in Table S1. Note that pan-daf-16 #1 primers anneal within the deletion of the daf-16(mu86) null allele, and pan-daf-16 #2 primers anneal outside of the deletion. Likewise, tm5030-specific primers anneal within the tm5032 deletion and vice versa. See Fig. S3 for a visual representation. daf-16a and daf-16f transgenes are both integrated on chromosome II at the ttTi5605 locus. Promoter 1 is 3kb while Promoter 2 is 4kb. The complete 1.7kb 3'UTR was incorporated. Note that a C. briggsae unc-119 sequence is incorporated 5' upstream of both transgenes for strain generation and selection. The daf-16a transgene does not rescue daf-16a mutant dauer arrest in the daf-2(e1368) background. (E-F) daf-16f-rescued daf-16a/f;daf-2 animals exhibit the same phenotypes as daf-16a;daf-2, suggesting that the daf-16f single-copy transgene has the same activity as daf-16f expressed from the genomic locus. Note: the daf-16a;daf-2(e1368) in D and F are the same, as they were performed in the same biological replicates, and are shown twice for relevant comparisons. See Table S11 for data and statistics. For all panels, the average and SD of three biological replicates are plotted. Note that RNA-seq quantification suggests the "long" daf-16a transcript driven by the daf-16f promoter is found at 200X lower concentration than daf-16a and 500X lower than daf-16f, strongly suggesting that this transcript is trans-spliced to yield the canonical daf-16a transcript. Thus, the upstream sequence constitutes outrons. (B) cDNA sequence amplified from wild-type RNA. Bolded sequence indicates sequence confirmed by RNA-seq reads. Green indicates daf-16a outrons that correspond to daf-16f exons. Orange corresponds to the daf-16a 5'UTR.
Figure S12 Thermotolerance, oxidative stress resistance, and UV stress resistance of daf-16 isoform-specific mutants in the daf-2(e1370) background. Survival data for animals exposed to 33°C (A), 7.5mM t-BOOH (B), or 1200 J/m 2 UV (C) are shown. See Methods for details. For thermotolerance and oxidative stress assays, patterns and absolute values of survival were very similar for all 3 replicates, and therefore combined data for all replicates are presented. For UV stress resistance, one representative trial is shown, but note that daf-16f mutation reduced UV resistance in 1 of 3 trials. See Table S12 for data and statistics. Fig. 6. Mean and standard deviation are plotted for three biological replicates of young adults. Asterisks denote statistically significant changes compared to daf-2(e1370) control (p < 0.05, paired ratio ttest). Data and statistics are presented in Table S14.  Figure S17 Comparison of effects of two daf-16a alleles on expression of DAF-16A/F target genes. Log 2 fold-change (FC) is shown for daf-2(e1370) vs. daf-16a(tm5030);daf-2 on the X-axis, and log 2 FC is shown for daf-2(e1370) vs. daf-16a(tm5032);daf-2 on the Y-axis.   Fig. 2. Column statistics are calculated from multiple replicates listed in Tables S3 and S4. *** p-value cannot be calculated because SD = 0, but effectively p < 0.0001 ^ p-value cannot be calculated because SD = 0, but effectively p = 1 # daf-16(tm5030);daf-2(e1370) and daf-2(tm5032);daf-2(e1370) non-dauer larvae developed into sterile adults after an additional 48 hours at 25°C     Table S4 daf-2(e1370) dauer arrest raw data. Column statistics are calculated from measurements from three plates per genotype for each replicate. *** p-value cannot be calculated because SD = 0, but effectively p < 0.0001 ^ p-value cannot be calculated because SD = 0, but effectively p = 1 # daf-16(tm5030);daf-2(e1370) and daf-2(tm5032);daf-2(e1370) non-dauer larvae developed into sterile adults after an additional 48 hours at 25°C       S14 qPCR data and statistics for Fig. 6 and Fig. S13. Mean and standard error of the mean for each cohort is calculated based on triplicate measurements. Mean and standard deviation overall is calculated based on means of three biological replicates.           -16(tm5032), a different primer A32-RACE was used, indicated by asterisks. See Figure S3 for details. All indicated RACE clones were consistent with sequences presented in Figures S2 and S4. Note that clones without SL1 trans-spliced leaders (w/o SL1) were partial fragments of the sequences.