Sex‐specific regulation of aging in Caenorhabditis elegans

Summary A fascinating aspect of sexual dimorphism in various animal species is that the two sexes differ substantially in lifespan. In humans, for example, women's life expectancy exceeds that of men by 3–7 years. Whether this trait can be attributed to dissimilar lifestyles or genetic (regulatory) factors remains to be elucidated. Herein, we demonstrate that in the nematode Caenorhabditis elegans, the significantly longer lifespan of hermaphrodites—which are essentially females capable of sperm production—over males is established by TRA‐1, the terminal effector of the sex‐determination pathway. This transcription factor directly controls the expression of daf‐16/FOXO, which functions as a major target of insulin/IGF‐1 signaling (IIS) and key modulator of aging across diverse animal phyla. TRA‐1 extends hermaphrodite lifespan through promoting daf‐16 activity. Furthermore, TRA‐1 also influences reproductive growth in a DAF‐16‐dependent manner. Thus, the sex‐determination machinery is an important regulator of IIS in this organism. These findings provide a mechanistic insight into how longevity and development are specified unequally in the two genders. As TRA‐1 is orthologous to mammalian GLI (glioma‐associated) proteins, a similar sex‐specific mechanism may also operate in humans to determine lifespan.


| INTRODUCTION
A remarkable phenomenon in aging biology is that the two genders display significantly different lifespans in divergent, sexually dimorphic animal species. For example, in flies, mice, and humans, females have a tendency to live longer than males (in human populations, the lifespan advantage of women over men can achieve up to 7-8 years; Blagosklonny, 2010;Eskes & Haanen, 2007;Gems, 2014;La Croix et al., 1997;Lints, Bourgois, Delalieux, Stoll & Lints, 1983;Tower, 2006;Tower & Arbeitman, 2009;Vina, Borr as, Gambini, Sastre & Pallard o, 2005). In these species, the heterogametic sex (XY) is male. In contrast, in species where the heterogametic sex (ZW) is female (e.g., in most bird species), males tend to live longer than females. Moreover, genetic and environmental factors that influence lifespan often have a larger effect in one sex than the other (Partridge, Gems & Withers, 2005). The question whether sex-specific differences in lifespan are determined by genetic regulatory mechanisms or are merely the by-products of different lifestyles (e.g., males are generally more predisposed than females to engage in fights) remains a great challenge for science, one with significant medical and social implications (Blagosklonny, 2010).
The nematode Caenorhabditis elegans develops as either a male having only one sex chromosome (XO) in the somatic cells or a hermaphrodite with two sex chromosomes (XX) in the somatic cells (the self-fertile hermaphrodite is essentially a female that produces sperm for a brief period before oogenesis; Zarkower, 2006). Gametes with no sex chromosome are generated as a consequence of chromosome nondisjunction during meiosis which is a rather rare event; in wildtype populations, males present only at low (~0.3%) frequency.
At adult stages, a TRA-1A variant, TRA-1 100 , which is a C-terminally truncated form of TRA-1A isoform, accumulates at much higher levels in hermaphrodites than in males, and this sex-specific difference appears to result from the proteolytic degradation of TRA-1 100 in males (Schvarzstein & Spence, 2006;Starostina et al., 2007).
In mixed C. elegans populations containing both sexes, hermaphrodites significantly outlive males (Gems & Riddle, 1996;Johnson & Hutchinson, 1993;Johnson & Wood, 1982). Lifespan advantage in hermaphrodites disappears when animals are grown individually or unable to physically interact with each other: the lifespan of solitary or paralyzed males is nearly 30% longer than that of isolated or grouped hermaphrodites (Gems & Riddle, 2000). In the absence of hermaphrodites, however, males frequently leave the area of food source (Escherichia coli bacteria) to find a mating partner-this phenomenon is called mate-searching behavior (Lipton, Kleemann, Ghosh, Lints & Emmons, 2004)-and males leaving the bacterial layer are subjected to calorie restriction or intermittent/prolonged starvation. Both conditions are known to extend lifespan significantly in various animal species (Koubova & Guarente, 2003). It is worth noting that severely paralyzed nematodes that were previously placed onto the bacterial layer consume bacterial cells nearby their body, thereby also becoming starved by time. Another study on mixed C. elegans populations showed recently that males shorten the lifespan of hermaphrodites via secreted pheromones (Maures et al., 2014). In the experimental design, the analysis applied a nearly equal number of males and hermaphrodites were assayed on each test plate (200-200 hermaphro-dites and males/plate). Under these circumstances, hermaphrodites did not display a longevity advantage over males, rather the two genders lived almost identical long. The 1:1 sex ratio and relatively high population density however are quite far from that observed in nature.
So, free-living hermaphrodite animals are likely not to be exposed to such high doses of male substances, and their lifespan is probably less significantly affected by the opposite sex. In addition, both mating and male pheromone, although through distinct mechanisms, shorten lifespan in males (Shi, Runnels & Murphy, 2017). The former factor also limits lifespan in hermaphrodites (Shi & Murphy, 2014). Thus, many aspects of sexual interaction strongly affect the lifespan of both sexes in this organism.
The use of mutations in key sex-determination genes revealed that the presence of two X chromosomes restricts hermaphrodite lifespan (Hartman & Ishii, 2007). Expression data of autosomal and X chromosome-linked genes suggested that the level of dosage compensation (this mechanism equalizes the expression of X chromosome-linked genes between the two sexes) declines as the hermaphrodite animal ages. Age-related decrease in dosage compensation may limit lifespan in XX animals. Together, the aging process is determined unequally in the two C. elegans sexes, and lifespan regulation occurs in a complex way that involves different environmental, behavioral, and genetic factors, including the dosage compensation machinery.
In this work, we aimed to culture nematodes under conditions that approximate to those found in their natural environments (relatively low population density and male scarcity). Under these settings, hermaphrodites lived significantly longer than males. We also found that increased longevity in hermaphrodite animals depends on the nematode sex-determination pathway and that this regulatory gene cascade influences the activity of the Forkhead-like transcription factor DAF-16 (dauer formation defective), the effector of insulin/IGF-1 (insulin-like growth factor 1) signaling (IIS). Thus, IIS, which regulates aging across divergent animal phyla, is adjusted unevenly between the two genders in this organism and perhaps in other animal species. 2 | RESULTS 2.1 | Hermaphrodites live longer than males in mixed C. elegans populations with hermaphrodite abundance We measured the lifespan of nematodes that were maintained in groups: 60-70 hermaphrodites and five males were placed on each test plate (this sex ratio approaches to those found in natural C. elegans populations in which males are present at low frequency; Brenner, 1974). In good accordance with previous data (Johnson & Wood, 1982;Johnson & Hutchinson, 1993), under these circumstances-such population density and sex ratio enable both individual and social behavioral patterns including response to crowding/dauer pheromone, mating, and being in hiding-hermaphrodites lived about 2 days longer than males at 25°C (Figure 1a,a'). This is a nearly 20% longevity difference in favor of hermaphrodites. Similar results were obtained when animals were grown on media lacking the DNA synthesis inhibitor FUdR (5-fluoro-2-deoxyuridine; Figure S1), which is generally used in C. elegans lifespan assays to confer sterility to the treated animals and known to affect longevity in the wild-type at higher temperatures (Angeli et al., 2013). The tendency of hermaphrodites to live longer than males was also evident when populations were maintained at 20°C ( Figure S2). Thus, the longer lifespan of hermaphrodites over males in mixed populations with hermaphrodite abundance appears to be established largely independently of several environmental conditions, raising the potential involvement of genetic (regulatory) factors in determining sex differences in lifespan.
2.2 | In nematodes defective for insulin/IGF-1 signaling, the longevity advantage of hermaphrodites over males depends on DAF-16/FOXO activity Next, we aimed to explore the regulatory mechanisms underlying the hermaphrodite bias in longevity. The IIS pathway plays a pivotal role in the control of C. elegans aging (Kenyon, 2010;Lin, Dorman, Rodan & Kenyon, 1997;Ogg et al., 1997). Upon ligand binding, the insulin/IGF-1 plasma membrane receptor DAF-2 (constitutive dauer formation) activates a cascade of downstream cytoplasmic kinases, which eventually inhibits the FOXO-like transcription factor DAF-16 (Ogg et al., 1997;Figure 1B). When IIS is lowered, DAF-16 effectively translocates into the nucleus to dictate the expression of target genes required for lifespan extension, stress resistance, and dauer larval formation (dauer is an alternative, nonaging developmental diapause triggered by starvation, crowding, and high temperatures in the wild-type; Fielenbach & Antebi, 2008;Vellai et al., 2003). We found that in long-lived daf-2(-) loss-of-function mutant strains maintained at temperatures permitting reproductive growth, hermaphrodites also tend to live longer than males (Figure 1c,c' and Figure S3). Note that a previous study reported a male longevity advantage when animals defective for DAF-2 were fed killed E. coli bacteria as a food, showing that males are more susceptible to live E. coli toxicity (Gems & Riddle, 2000). daf-16 deficiency however suppressed the longer lifespan hermaphrodites exhibit over males in daf-2(-) mutant genetic backgrounds (Figure 1c,c' and Figure S3).
These data indicate that DAF-16 mediates increased hermaphrodite longevity in this sensitized IIS-defective genetic background (in wellfed wild-type animals, daf-16 is largely repressed by IIS).
2.3 | The longevity advantage of hermaphrodites over males depends on TRA-1 activity How is daf-16 controlled differently in hermaphrodites and males?
To test whether the nematode sex-determination cascade (Figure 1d) is implicated in the sex-specific regulation of aging, we monitored the lifespan of mutant animals with decreased or elevated TRA-1 activity. According to these results, tra-1(-) mutations transforming animals with XX (hermaphrodite) karyotype into males significantly reduced lifespan (Figure 1e,e'). Consistent with this finding, a tra-1 gain-of-function (gf) mutation, e1575, increased lifespan by F I G U R E 1 The terminal sex-determining factor TRA-1 promotes hermaphrodite longevity by enhancing daf-16 activity. (a, a') When maintained in groups containing both sexes, and with a great majority of hermaphrodite animals, wild-type hermaphrodites (red curve) live significantly longer than males (blue curve). (b) The insulin/IGF-1 signaling pathway in C. elegans. DAF-2: insulin/insulin-like growth factor receptor 1; AGE-1: type I phosphatidylinositol-3-kinase; PDK-1: phosphoinositide-dependent kinase; AKT-1, -2: AKT/PKB-AKT8 virus protooncogene/protein kinase B; DAF-16: FOXO-like transcription factor. (c, c') In daf-2(-) mutant background, the longer lifespan of hermaphrodites over males depends on daf-16 activity. Animals were maintained at 20°C until they developed into the L4 larval stage and then transferred at 25°C. (d) The C. elegans sex-determination cascade. TRA-1, the terminal effector of the cascade, is essentially active only in hermaphrodites but not in males. X: sex chromosome; A: autosome. (e, e') Inactivation of tra-1 decreases while inactivation of fem-3 (which corresponds to hyperactive tra-1) increases lifespan. A tra-1 gain-of-function (gf) mutation, e1575gf, promotes longevity. A tra-3(lf) mutation was used to maintain tra-1(e1575gf) mutant animals that are essentially females. (f-g') Both mutational inactivation (f and f') and RNA interference-mediated depletion (g, g') of daf-16 suppress lifespan extension in fem-3(-) mutant (i.e., tra-1 hyperactive) animals. (h-i') Mutational inactivation (h, h') and depletion (i, i') of daf-16 suppress the longer lifespan of tra-1(e1575gf) mutants. On panels (b, c, and e-i), Kaplan-Meier lifespan curves (log-rank tests) while on panels (b', c', e'-i'), the corresponding mean survival data (independent samples t tests with Bonferroni correction) are shown. On the latter, *p < .05, **p < .001, ***p < .0001; NS: not significant. On panels (a'-i'), bars represent AESEM. Statistics and data are included in Table S1. On panels (g, g' and i, i'), "ev" denotes empty vector (animals were fed with bacteria expressing the empty vector only). tra-3(lf) indicates the loss-of-function allele tra-3(e1767). On panels (a and d), arrows indicate activations, bars represent inhibitory regulatory interactions. On panels (e-g'), wild-type corresponds to wild-type hermaphrodites. On panels (a, c and e-i), animals were maintained at 25°C approximately 30% (animals with hyperactive TRA-1 function are feminized; Figure 1e . We conclude that TRA-1 promotes longevity in hermaphrodites by enhancing daf-16 activity. In other words, TRA-1 strengthens the function of daf-16 in aging control, explaining why hermaphrodites live significantly longer than males in populations containing both sexes. At first sight, these results were somewhat unexpected as TRA-1 had previously been known as a transcriptional repressor rather than an activator (Berkseth et al., 2013;Chen & Ellis, 2000;Conradt & Horvitz, 1999;Hargitai et al., 2009;Mason et al., 2008;Schwartz & Horvitz, 2007;Szab o et al., 2009;Yi et al., 2000). In case of mammalian GLI proteins, however, both activation and inhibition functions were observed (Hui & Angers, 2011).  (Kwon, Narasimhan, Yen & Tissenbaum, 2010). Performing a sequence analysis, we have identified two conserved TRA-1-binding sites (Conradt & Horvitz, 1999) in the daf-16 locus. The sites were also found in the orthologous genomic regions of C. briggsae, a closely related Caenorhabditis species ( Figure 2a).

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These data, together with results provided by a previous chromatin immunoprecipitation assay followed by deep sequencing (ChIP-seq; Berkseth et al., 2013), raised the possibility of a direct regulatory interaction between TRA-1 and daf-16 isoforms involved in aging control. One of these consensus TRA-1-binding sites is located at 3kilobase (kb) upstream of the daf-16d/f isoforms, while the other is located within the first exon of the daf-16a isoform (exonic sequences often serve as binding elements for transcriptions factors; Stergachis et al., 2013). The ChIP-seq analysis provided by Berkseth and colleagues also identified two potential TRA-1-binding sites in the daf-16 coding region (Berkseth et al., 2013) which are however slightly diverged from the canonical one (Conradt & Horvitz, 1999;Hargitai et al., 2009) and the daf-16-specific TRA-1-binding sites identified by our present study (Figure 2a). To assess the functionality of these potential TRA-1-binding sites, we generated two transgenic strains expressing isoform-specific gfp-(green fluorescent protein) tagged daf-16 reporter constructs, daf-16d/f::gfp and daf- Red triangles indicate conserved TRA-1-binding sites (the consensus sequence is shown on the top; red letters indicate identical nucleotides; green nucleotides indicate conserved nucleotides; N denotes arbitrary nucleotides). The structures of daf-16d/f::gfp and daf-16a::gfp reporters are also shown. Blue letters indicate difference from the consensus sequence, and gray letters indicate nucleotides that are missing in the mutated constructs. (b) daf-16d/f::gfp expression is decreased in XO males and in tra-1 (-) mutant XX males and increased in fem-3(-) mutant backgrounds (top row). mut daf-16d/f::gfp (a mutated derivative of the wild-type construct, lacking several nucleotides in the predicted TRA-1 binding site) expression is independent of fem-3 and tra-1 activities (bottom row). All fluorescence pictures were taken with the same exposure time (500 ms) and magnification (1009). (c) High-resolution confocal microscopy images of daf-16d/f expression in a wild-type (control, left) vs. a tra-1(-) mutant (right) animal. Fluorescence pictures were taken with the same exposure setting. (d, e) Quantification of the relative expression intensity of daf-16d/f::gfp (d) and mut daf-16d/f::gfp (e) reporters. daf-16d/f::gfp expression is increased in fem-3(-) mutant animals but decreased in XO males and in tra-1(-) mutant XX animals. Interestingly, the hypomorphic tra-1(e1488) mutation has a stronger effect on reporter expression than the genetic null allele tra-1(e1099) does which may be due to maternal effect (tra-1 null mutants can be maintained as heterozygous animals) (d). In contrast, expression levels of mut daf-16d/f::gfp is largely independent of TRA-1 activity (e). (f, g) Expression intensity of daf-16a::gfp (f) and mut daf-16a::gfp (g) reporters at the late L1 larval stage when dauer development is initiated. Expression of mut daf-16a appears to be independent of TRA-1. In panels (d-g), *p < .05; **p < .01, ***p < .001, independent samples t tests; bars represent AESEM; "NS" denotes not significant. Statistics and data are included in Table S2. (h, i) Dauer development in daf-2(-) mutants is increased by FEM-3 deficiency and decreased by TRA-1 deficiency. The percentage of dauer larvae is determined at 20°C (h) and 23°C (i). **p < .01, ***p < .001; chi-squared tests with Bonferroni correction. Statistics and data are included in Table S3 HOTZI ET AL.
| 5 of 15 demonstrated that intracellular levels of daf-16d/f::gfp expression are markedly attenuated by TRA-1 deficiency (Figure 2c). Sex differences in daf-16d/f::gfp expression remained on in later developmental stages and throughout adulthood (Figures S4 and S5, and Tables   S6 and S7). Together, we suggest that TRA-1 enhances the transcriptional activity of daf-16d/f presumably through the predicted binding site.
daf-16a::gfp was mainly expressed in neuronal and hypodermal cells in the head and tail body regions ( Figure S6). Its expression intensity was significantly decreased in tra-1(-), but enhanced in fem-3(-), mutant worms at the L1/2 larval stages when developmental decision between normal reproductive growth and dauer larva formation occurs (Figure 2f and Figure S6). A binding site mutant version of the reporter, mut daf-16a::gfp (Figure 2a), also displayed decreased expression levels, as compared with the corresponding wild-type reporter ( Figure 2g and Figure S6). Moreover, at these larval stages, mut daf-16a::gfp expression appeared to be largely independent of TRA-1 activity. Thus, the expression of daf-16a is also enhanced by TRA-1.
Interestingly, after the L1/2 larval stages when the reproductive growth vs. dauer development decision is already determined, daf-16a::gfp expression appeared to be no longer activated by TRA-1 ( Figure S7 and Table S8). At the L4 larval and young/aged adult stages, daf-16a was expressed at similar or even higher levels in XO males and tra-1(-) mutant XX animals than in XX hermaphrodites.
Thermosensitive (ts) daf-2(-) ts mutant animals enter into the dauer larval stage at the restrictive temperature (25°C). The manifestation of the Daf-constitutive phenotype in daf-2(-) ts mutants requires DAF-16 activity; daf-2(-) ts ; daf-16(-) double-mutant animals grow as reproductive adults even at 25°C. daf-16a isoform is known to promote dauer development but has no effect on lifespan (Kwon et al., 2010). We found that dauer larval formation in daf-2(-) ts mutants is increased in fem-3(-) and inhibited in tra-1(-) mutant backgrounds at temperatures (20-23°C) where only a portion of the population develops as dauer larvae (Figure 2h,i). These data imply that TRA-1 also modulates IIS by enhancing daf-16a activity in controlling the decision between reproductive growth and dauer larval development in hermaphrodites.
2.5 | A functional daf-16d/f transgene promotes longevity more effectively and is expressed at higher levels in hermaphrodites than in males daf-16 encodes several isoforms, among which daf-16d/f, together with daf-16b, were identified as the main transcripts that regulate nematode lifespan (Kwon et al., 2010). We crossed an integrated, full-length (translational fusion) daf-16d/f::gfp reporter transgene, lpIs14 (Kwon et al., 2010), into a daf-16(-) mutant background, and found that it is capable of rescuing normal lifespan in both hermaphrodites and males (Figure 3a-b'). However, the lifespan extending effect of lpIs14 transgene was more evident in hermaphrodite animals than in males. Under conditions of 10:1 hermaphrodite:male population ratio, daf-16(-) mutant hermaphrodites transgenic for lpIs14 lived even longer than wild-type hermaphrodites (p < .0001; see Figure 3a,a'), whereas the lifespan of daf-16(-); daf-16d/f (lpIs14) males did not exceed that of the wild-type (Figure 3b,b'). When males were maintained in single-sex groups, daf-16(-) mutants transgenic for Ipls14 lived only a slightly longer than wild-type males (p < .05; Figure S8). Consistent with these data, lpIs14 was expressed at higher levels in hermaphrodites, as compared with males, in an otherwise wild-type background (Figure 3c,c' and Table S2). Interestingly, the expression was also obvious in the nucleus of intestinal cells in hermaphrodites but not in males. Hence, the expression of daf-16d/f, two daf-16 isoforms that control the rate at which cells age, is influenced by the sex of the animal in favor of hermaphrodites. This can explain why hermaphrodites live longer than males in populations containing both genders, and with hermaphrodite excess. An unbiased TRA-1-specific ChIP-seq study performed at four different developmental time points also identified TRA-1 binding to daf-16 (Berkseth et al., 2013), but the binding sites the authors determined in the daf-16 locus are not the same as those we identified in this study. Here, we carried out ChIP-qPCR (more sensitive and powerful than ChIP-seq) on ultrasound fragmented chromatin to 500-bp-long fragments, prepared from mixed-stage animals. We used a positive (xol-1) and a negative (daf-11) control regions, two different TRA-1-specific antibodies, and IgG as a negative control HOTZI ET AL. | 7 of 15 ( Figure 4). In the ChIP-seq analysis, 184 TRA-1-binding sites were identified, which are fewer than typical for site-specific transcription factors. Indeed, several previously described TRA-1 target genes such as egl-1, ceh-30, and lin-39 (Conradt & Horvitz, 1999;Schwartz & Horvitz, 2007;Szab o et al., 2009)

remained unidentified by this
ChIP-seq analysis (Berkseth et al., 2013). However, we know that a direct evidence for the functionality of the TRA-1-binding site we determined in the daf-16 locus would be the elimination of the binding site by CRISPR/Cas technology which would block the regulatory interaction between TRA-1 and daf-16d/f and a isoforms in vivo.

| DISCUSSION
Caenorhabditis elegans is a tractable model system to study the molecular mechanisms underlying sex-specific differences in various biological processes and anatomical features. In this work, we explored a novel regulatory interaction that determines lifespan and reproductive growth unequally between hermaphrodite and male animals. First, we observed that both wild-type and IIS-deficient daf-2(-) mutant hermaphrodites live significantly longer than the corresponding males (Figure 1 and Figures S1-S3). Sex differences in longevity in daf-2(-) mutants disappeared in daf-16(-) mutant genetic backgrounds (Figure 1 and Figure S3). Thus, the sex-specific regulation of nematode lifespan depends on DAF-16 activity. Next, we showed that the master sex-determining factor TRA-1 promotes the transcriptional activity of certain daf-16 isoforms, d/f and a.
TRA-1 and these daf-16 isoforms hence act in the same genetic pathway to modulate lifespan or development. As DAF-16 functions as the main target of IIS in the regulation of lifespan and development, TRA-1, and thereby the sex-determination machinery, is an important modulator of this signaling system ( Figure 5). This implies that IIS is adjusted in a sex-specific way, leading to significant sex differences in the activity of several biological processes. Indeed, the expression of daf-16d/f playing an important role in longevity control (Kwon et al., 2010) is elevated by TRA-1 in hermaphrodites but not in males (Figures 2-4). Depending on population density and the ambient temperature, daf-16a controls the decision between reproductive growth and dauer larva development (Figures 2 and 4). TRA-1 also increases the expression of this daf-16 isoform in hermaphrodite animals. These regulatory interactions elucidate the hermaphrodite bias toward a longer lifespan (this study) and increased dauer larval formation (Vellai, McCulloch, Gems & Kov acs, 2006). Similarly, a marked sex-specific difference was previously observed in C. elegans learning capacity, a trait that also relies on IIS (Vellai et al., 2006). It would be relevant to examine whether the TRA-1-daf-16 regulatory axis is involved in the control of associative learning. In case of positive results, one could provide an explanation for the F I G U R E 3 A functional, full-length daf-16d/f::gfp transgene (lpIs14) extends lifespan more effectively in hermaphrodites than in males. (a) Lifespan curve of wild-type hermaphrodites, daf-16(-) mutant hermaphrodites, and daf-16(-) mutant hermaphrodites transgenic for lpIs14. lpIs14 denotes an integrated, full-length daf-16d/f::gfp transgene (Kwon et al., 2010). It rescues normal lifespan in animals defective for DAF-16. (a') The corresponding mean lifespan data. (b) Lifespan curve of wild-type males, daf-16(-) mutant males and daf-16(-) mutant males transgenic for lpIs14. (b') The mean lifespan data. lpIs14 promotes longevity more significantly in daf-16(-) mutant hermaphrodites (a, a') than in daf-16(-) mutant males (b, b'). In panels (a' and b'), NS indicates not significant, *** indicates p < .001, log-rank and independent samples t test. (c) Expression of lpIs14 in young hermaphrodites and males. (c') Relative expression levels of lpIs14 in hermaphrodites vs. males (the expression is higher in the former). Bars represent AESEM, *** indicates p < .001, independent samples t test. For statistics and data, see Tables S1 and S2 tendency of hermaphrodites to perform an associative learning paradigm more effectively. In nematodes, lipid metabolism and stress resistance are also influenced by DAF-2 and DAF-16 (Ashrafi et al., 2003;Scott, Avidan & Crowder, 2002). Through enhancing the activity of certain daf-16 isoforms, TRA-1 may also strengthen these biological processes in hermaphrodite animals.
Until recently, DAF-16/FOXO was known to be regulated pre-  (Figure 2 and 4). This raises the relevant question of whether increasing merely daf-16 transcription is sufficient for lifespan extension. In animals transgenic for a functional daf-16 reporter construct, lpIs14 (Kwon et al., 2010), lifespan was shown to increase proportionately with the copy number of the transgene (Bansal et al., 2014). Furthermore, the chromatin remodeler SWI/SNF complex was demonstrated to extend lifespan through controlling daf-16 transcription. These data revealed that elevating daf-16 transcript levels is indeed capable of extending lifespan significantly.
In the nematode sex-determination cascade, FEM-1 inhibits TRA-1 (Figure 1d), which is orthologous to human GLI proteins (Robbins, Fei & Riobo, 2012;Zarkower & Hodgkin, 1992) acting as downstream effectors of Hh signaling. Accumulating evidence indicates that Hh signaling plays a pivotal role in mammalian sexual differentiation (Franco & Yao, 2012) and that the mammalian Fem1b protein, an orthologue of FEM-1, also suppresses the transcriptional activity F I G U R E 4 The daf-16 isoforms d/f and a are direct targets of the sex-determining factor TRA-1. (a-c) ChIP (chromatin immunoprecipitation) data showing TRA-1 binding to target sequences. (a) TRA-1 binds to a xol-1-specific genomic fragment containing the conserved binding site (positive control), but not to daf-11-specific one (inner negative control). (b) TRA-1 binds to a daf-16d/f isoform-specific DNA fragment that contains the binding sequence (Figure 2a) in vivo. (c) TRA-1 binds to a daf-16a-specific genomic fragment with the conserved binding site (Figure 2a). On panels (a-c), "Vellai lab": TRA-1 antibody generated by our laboratory (see Figures S9 and S10); "com": commercially available TRA-1 antibody; Ab: antibody; mouse IgG: negative control. Bars represent AESEM *p < .05; **p < .01; ***p < .001. Independent samples t tests with Bonferroni correction. Statistics and data are included in Table S4. (d) daf-16d/f transcript levels are significantly higher in wild-type hermaphrodites than in males. (e) tra-1(gf) mutation increases daf-16d/f transcript levels, as compared with controls. (f, g) At the late L1 (f) and dauer (g) larval stages, the expression of daf-16a is decreased in tra-1(-) but increased in fem-3(-) mutant backgrounds. In panels (d-g), quantitative RT-PCR data are shown; bars represent mean AE SD, *p < .05, **p < .01; ***p < .001, Pair Wise Fixed Reallocation Randomization test. Statistics and data are included in Table S5 of GLI1 (Gilder, Chen, Jackson, Jiang & Maher, 2013). In addition, Hh signaling regulates whole-body energy metabolism via activating the Akt/FOXO pathway (Zhang, Cheng, Wang, Leung & Mak, 2017) and also influences IIS activity in certain developmental events (Lipinski et al., 2005). This is particularly interesting as in mammals, IIS is implicated in the sex-specific regulation of tissue differentiation (Lam, Shah & Brosens, 2012;Pitetti et al., 2013). These data prompted us to perform an in silico analysis of the human genome for the presence of conserved GLI binding sites in the FOXO3 locus ( Figures S11 and S12). In mammals, FOXO proteins are encoded by four genes, FOXO1, 3, 4, and 6, and allelic variations of FOXO3 have been correlated with longevity in numerous human populations (Martins, Lithgow & Link, 2016). By performing a locus-specific sequence analysis, we uncovered conserved GLI binding sites in the regulatory regions of human FOXO genes, especially FOXO3 (Figures S11 and S12). A potential consensus site found in the first intronic sequence of FOXO3 showed a strong conservation across the orthologous regions of mammalian, in particular primate, genomes. In addition, accumulating evidence indicates that Hh signaling plays a fundamental role in mammalian sexual differentiation (Franco & Yao, 2012;Wang et al., 2013). Based on this evolutionary conservation and gender-specific activity of Hh signaling, we speculate that a similar regulatory interaction between Hh signaling and FOXO-like transcription factors may also operate in humans to determine lifespan unequally in the two genders. Although nematodes lack certain components of the canonical Hh signaling pathway and primary sexdetermination in mammals is strictly chromosomal (i.e., depends on the presence of chromosome Y), genetic interaction between functionally conserved proteins and genes (TRA-1/GLI and daf-16/ FOXO3) may affect lifespan in a sex-specific manner across divergent animal taxa. Together, these data raise the possibility that molecular interactions between TRA-1/GLI and DAF-16/FOXO proteins are evolutionarily conserved from worms to humans. It is possible that in mammals, the presence of chromosome Y somehow determines the activity of Hh signaling in a sex-specific manner, which in turn influences IIS through a GLI-FOXO regulatory interaction.
In mammals, IIS controls various cellular, physiological, and developmental functions, including apoptosis, aging, metabolism, systemic body growth, self-renewal of stem cells, and behavior. Many of such functions manifest in a sex-specific manner. For example, in humans, the control of behavior, sensory information transmission, learning, and memory processing all depend on IIS and display a marked sex bias: women tend to behave less aggressively, have a better sense of smell, and learn skills faster than men (Londorsf, Eberly & Pusey, 2004). Uncovering the regulatory role of the C. elegans sex-determining protein TRA-1 in daf-16 activity may help to understand better how IIS affects diverse biological processes unequally between women and men.

| Strains and genetics
Nematodes were maintained and propagated on Nematode Growth Medium-(NGM) containing plates and fed with Escherichia coli OP50 bacteria. The following C. elegans strains were used in this study: Bristol (N2) as wild-type.

| Lifespan assays
Lifespan assays were carried out at 25°C. daf-2(-) ts mutant animals were maintained at 20°C until the L4 larval stage, then transferred at 25°C (otherwise indicated), and scored for mean lifespan. For synchronization, 20-30 gravid, well-fed adults were transferred to a new agar plate containing NGM seeded with E. coli OP50 bacteria to lay embryos for 4-5 hr and then removed. Alternatively, embryos were prepared by NaOH-hypochlorite treatment. Approximately 60-70 F1 young (nongravid) adults were transferred to new NGM plates supplemented with 300-400 mg/ml FUdR (5-fluoro-2 0 -deoxyuridine, Sigma; t = 0). Sterile F1 adults were then assayed. Animals that climbed up the wall of plastic dishes or exhibited a protruded vulva phenotype were excluded from the analysis. Animals were considered dead when they stopped pharyngeal pumping and responding to touching. In hermaphrodite vs. male lifespan assays, approximately 50-60 hermaphrodites and five to six males were maintained on each plate. SPSS 17 software was used to calculate mean lifespan and perform statistical analysis. p values for comparing Kaplan-Meier survival curves between two groups were determined using log-rank (Mantel-Cox) tests, and p values for comparing mean lifespans were determined using independent samples t tests with Bonferroni correction. Escherichia coli HT115(DE3) RNA interference (RNAi)-feeding bacteria were grown overnight in LB medium containing 50 lg/ml ampicillin and 6.25 lg/ml tetracycline in final concentration. L4/ young-stage adults were transferred to plates containing 300-400 mg/ml FUdR, 50 lg/ml ampicillin, 6.25 lg/ml tetracycline, and 0.4 mM IPTG in final concentration. Strains were grown for two generations on RNAi bacteria before assaying for lifespan. After FUdR treatment (about 24-48 hr), animals were transferred to novel RNAi plates. Empty vector-containing bacteria were used as controls.

| Dauer formation assay
L1-stage larvae were synchronized by isolating eggs from gravid adults. 100-200 embryos were pipetted onto NGM plates seeded with E. coli OP50 bacteria and kept for 60-72 hr at appropriate temperatures (20-23°C). Animals were well-fed and maintained at a relatively low population density to avoid dauer formation response triggered by environmental cues. The number of dauer larvae and L4-stage larvae/adults was determined visually, and scoring was con- acid-long polypeptide (translated from the 14 th exon of tra-1), which was subsequently used as epitope to generate rabbit polyclonal TRA-1 antibody. The antigen was expressed in QIAexpress system (Qiagen), and 6xHis tag was used for the purification after dialysis.
The raised antibody specifically labels a 175-kDa protein (Figures S9 and S10).