Steroid signaling is required for longevity induced by decreased mTOR signaling.

a, Schematic overview of the endogenous dafachronic acid biosynthesis pathway in C. elegans. In the first step, the Rieske oxygenase DAF-36/Neverland serves as a cholesterol 7-desaturase, catalyzing the oxidation of cholesterol to 7-dehydrocholesterol (Wollam, Magomedova et al. 2011). An unknown enzyme catalyzes the reduction of the Δ-5 bond to lathosterol. Short-chain dehydrogenase DHS-16 carries out the oxidation of the 3-hydroxy to 3-keto derivative to yield lathosterone (Wollam, Magner et al. 2012). In the final step, cytochrome P450 DAF-9/CYP27A1 catalyzes the oxidation of the sterol sidechain to the carboxylic acid moiety (Motola, Cummins et al. 2006), resulting in the formation of Δ7-DA. Δ7-DA functions as a ligand for the nuclear hormone receptor DAF-12/FXR to promote development. Unliganded DAF-12 binds to its corepressor DIN-1s inducing dauer diapause. b-f, Representative life spans comparing wild type (N2), raga-1(ok701), and steroid mutants. b) daf-36(k114) and raga-1;daf-36, c) daf-9(rh50) and raga-1;daf-9, d) daf-12(rh61rh411) and raga-1;daf-12, e) din-1(dh127) and raga-1;din-1 f) nhr-8(hd117) and raga-1;nhr-8. Statistical analysis for life spans was performed using the log-rank (Mantel–Cox) test. ns, non-significant (p>0.05), **** (p<0.001). Data and statistics are summarized in Supplementary Table 1. g, Representative life spans comparing N2 and daf-12(rh61rh411) on let-363/mTOR and control (luc) RNAi. Eggs were cultivated on regular NGM plates until day 1 of adulthood, then worms were transferred to the respective RNAi plates. Statistical analysis for life spans was performed using the log-rank (Mantel-Cox) test. **** (p<0.005). Data and statistics are summarized in Supplementary Table 1. h-i, Life span upon 0.5 µM Δ7-DA supplementation or vehicle (EtOH) control egg-on, comparing h) raga-1(ok701) and raga-1;daf-9(rh50), and i) raga-1(ok701) and raga-1;daf-12. Statistical analysis for life spans was performed using the log-rank (Mantel–Cox) test. ns (p>0.05), *** (p<0.005), n=2. Data and statistics are summarized in Supplementary Table 1. j, Relative endogenous concentrations of cholesterol, lathosterol, and Δ7-dafachronic acid (DA) from N2 and raga-1(ok701) day 1 adults. DA concentrations were normalized to PQN, n=4. Mean ± SD. Statistical analysis was performed using the Student’s t-test. ns (p>0.05), * (p<0.01)

daf-12 modulates the raga-1 mutant transcriptome.

a, Principal component analysis of wild type (N2), raga-1(ok701), daf-12(rh61rh411), and raga-1;daf-12 mutant transcriptomes in day 1 adults. b, Volcano plot of differentially expressed genes comparing raga-1;daf-12/raga-1(ok701). Black dots mark significantly changed genes (Adj.p<0.05), grey dots mark non-significantly changed genes (Adj.p>0.05), dhs-26 is highlighted as a red dot. Blue area highlights 970 significantly down-regulated genes; the red area highlights 1314 significantly up-regulated genes. c, KEGG pathway enrichment (FDR<0.05) of significantly (Adj.p<0.05) down-regulated (blue) and up-regulated genes (red) comparing raga-1;daf-12/raga-1(ok701). The gene list of KEGG enrichments is contained in Supplementary Table 3. d, Heatmap of log2(FC) of the top 15 down-regulated (blue) and top 15 up-regulated genes (red) comparing raga-1;daf-12/raga-1(ok701). *** (Adj.p<0.005). e, Z-score heatmap of genes involved in steroid signaling and mTOR signaling comparing N2, raga-1(ok701) and raga-1;daf-12. Comparing raga-1/N2, * (Adj.p<0.05), ** (Adj.p<0.01), *** (Adj.p<0.005). Comparing raga-1;daf-12/raga-1(ok701), ## (Adj.p<0.01), ### (Adj.p<0.005).

Functional genomic screen reveals that DAF-12 regulated gene dhs-26 affects raga-1 life span.

a, Pearson correlation analysis of the Log2(FC) of all genes significantly changed in expression comparing raga-1;daf-12/raga-1(ok701) (Y axis) and raga-1(ok701)/N2 (X axis). 536 genes are significantly down-regulated in raga-1;daf-12/raga-1(ok701) and up-regulated in raga-1(ok701)/N2 (brown). dhs-26 gene is indicated by the red dot. 383 genes are significantly up-regulated in raga-1;daf-12/raga-1(ok701) and raga-1(ok701)/N2 (yellow). 86 genes are significantly down-regulated in raga-1;daf-12/raga-1(ok701) and raga-1(ok701)/N2 (blue). 56 genes are significantly up-regulated in raga-1;daf-12/raga-1(ok701) and down-regulated in raga-1(ok701)/N2 (white). b, The 536 candidate genes shown were filtered using Log2(FC)>0.5, reads/million>10, availability of the respective RNAi clone, presence of gene orthologues in human, mice or D. melanogaster, resulting in 185 candidate genes. Schematic overview of the functional genomic screen. 185 genes were individually knocked down by RNAi in raga-1 mutants to screen for influence on adult life span. c, Bar graph displaying the relative mean life span change (%) of raga-1 mutants fed RNAi bacteria of candidate genes. Mean life span was assessed by feeding worms with RNAi bacteria egg-on and scoring for survival every two to three days. raga-1(ok701) and raga-1;daf-12 grown on luciferase (luc) RNAi were included as reference controls. Statistical analysis was performed using the log-rank (Mantel-Cox) test, n=1. d, Heat map displaying significant life span change (p<0.001) induced by RNAi clones on raga-1 life span displayed in c.

raga-1 mutants require the highly conserved dehydrogenase DHS-26/DHRS1 for life span extension.

a, Multiple sequence amino acid alignment of DHS-26 with orthologs from other species using constraint-based multiple sequence alignment tool (COBALT). Red=identical amino acids, grey=non-identical. Above, the NAD(P)-binding Rossman fold domain is shown. Below, the Rossman motif (yellow) and the catalytic center (red) are highlighted. b, Sequence alignment of the Rossman motif (TGxxxGxG, yellow), and the catalytic center (YxxxK, red) of different organisms. c, Structural alignment of the AlphaFold 3-predicted model of human DHRS1 (PDB Q96LJ7, grey) with the AlphaFold 3-predicted model of C. elegans DHS-26 (PDB Q23612, turquoise). The Rossman motif (yellow) and catalytic center (red) are highlighted. r.m.s.d. = 2.748 Å. d, Representative life span comparing wild type (N2) and raga-1(ok701) on dhs-26/DHRS1 and control (luc) RNAi treatment egg-on. Statistical analysis was performed using the log-rank (Mantel-Cox) test. * (p<0.05), **** (p<0.001), n=3. Data and statistics are summarized in supplementary Table 1. e, Schematic of the genomic dhs-26 locus and structure of the dhs-26(syb8968) deletion mutant (X: 3,360,722-3,362,493). Triangle indicates the endogenous insertion site of flag::Scarlet tag at the C-terminus. f, Representative life span analysis of N2, dhs-26(syb8968), raga-1(ok701), and raga-1;dhs-26 mutants. Statistical analysis was performed using the log-rank (Mantel-Cox) test. **** (p<0.001), n=4. All data and statistics are summarized in supplementary Table 1. g, dhs-26 mRNA levels measured by RNA sequencing in the indicated mutant genotypes. Statistical analysis was performed using One-way ANOVA, ** (p<0.01), **** (p<0.001), n=4. h, Immunoblots with lysates from 500 day 1 N2 and raga-1(ok701) worms with endogenously tagged DHS-26::FLAG::Scarlet, probed with the indicated antibodies. DHS-26::FLAG::Scarlet fusion protein is expected at 73 kDa. n=4. i, Quantification of immunoblots displaying fold change of FLAG-band intensities normalized to β-Actin loading control and N2. Statistical analysis was performed using Student’s t-test, * (p<0.05), n=4.

DHS-26/DHRS1 regulates steroid signaling and is required for life span extension induced by reduced mTOR

a, Representative images of DHS-26::FLAG::Scarlet (DHS-26::Scarlet) expression in the canal associated neurons (CAN) of day 1 adults, lateral aspect midbody. Worms were supplemented with 100 nM Δ7-DA or vehicle control (EtOH) egg-on. Scale bar, 5 µm. b, Representative quantification of DHS-26::Scarlet fluorescence intensity from a. Fluorescence intensity of each region of interest (ROI) was calculated by subtracting the background fluorescence of each image from the ROI. Statistical analysis was performed using One-way ANOVA, **** (p<0.001), n=3. c, Representative images of DHS-26::Scarlet expression in the CAN of day 1 N2 adults, lateral aspect midbody. Worms were cultured on regular NGM plates containing 5 µg/mL cholesterol (Chol) or on NGM plates without cholesterol supplementation (No Chol) egg-on. n=5, Scale bar, 5 µm. d, Quantification of DHS-26::Scarlet fluorescence intensity from c. Fluorescence intensity of each region of interest (ROI) was calculated by subtracting the background fluorescence of each image from the ROI. Statistical analysis was performed using paired t-test, * (p<0.005), n=5. e, Representative life span of raga-1(ok701) and raga-1;dhs-26 animals supplemented with 0.5 µM Δ7-DA or EtOH control. Statistical analysis was performed using the log-rank (Mantel-Cox) test. ns (p>0.05), **** (p<0.001), n=3. Data and statistics are summarized in supplementary Table 1. f, Representative images of mir-84p::GFP expression in seam cell V2 of animals (highlighted) at larval stages L4.3-L4.5 (Mok, Sternberg et al. 2015). Larval stage was assessed by vulval morphology. Differential interference contrast (DIC) and fluorescence images were taken at midbody for mir-84p::GFP, and head for TTX-3::RFP of the same worm. Scale bar, 10 µm. g, Representative quantification of mir-84p::GFP expression in seam cell V2 of raga-1(ok701) and raga-1;dhs-26 animals. Fluorescence intensity of each region of interest (ROI) was calculated by subtracting the background fluorescence of each image from the ROI, and was additionally normalized to TTX-3::RFP expressed in AIY interneurons. Statistical analysis was performed using Student’s t-test, **** (p<0.001), n=3. h, Relative concentrations of Δ7-dafachronic acid (DA) and its precursors cholesterol and lathosterol extracted from raga-1(ok701) and raga-1;dhs-26 day 1 adults. Measured concentrations were normalized to protein levels. Statistical analysis was performed using Student’s t-test, * (p<0.05), ** (p<0.01), *** (p<0.005), n=4. i, Working model. mTOR inhibits DA biosynthesis. Reduced mTOR activity elevates DA levels. Increased DA binding to the nuclear hormone receptor DAF-12 drives the expression of DAF-12 target genes such as dhs-26, which positively regulates DA production. Activation of DAF-12 and DHS-26 additionally induce downstream processes linked to longevity. j, KEGG pathway enrichment (FDR<0.05) of significantly (Adj.p<0.05) downregulated (blue) genes comparing raga-1;dhs-26/raga-1.The gene list of KEGG enrichments is listed in supplemenrary Table 3. k, KEGG pathway enrichment (FDR<0.05) of significantly (Adj.p<0.05) upregulated genes (red) comparing raga-1;dhs-26/raga-1(ok701). The gene list of KEGG enrichments is listed in supplementary Table 3. l, Dhrs1 mRNA levels measured by RNA sequencing in the brain and liver of 15 day old female TK2KI animals treated with rapamycin or vehicle control (EtOH) by Siegmund et al. (Siegmund, Yang et al. 2017). Rapamycin and EtOH were administered in drinking water. During pregnancy the administered rapamycin concentration was 8 µg/ml, and 40 µg/ml after delivery. Untreated control litters were given water with equivalent concentrations (0.1%) of vehicle alone (100% ethanol). Statistical analysis was performed using Student’s t-test, ns (p>0.05), * (p<0.05), n=3. m, DHRS1 mRNA levels measured by RNA sequencing in the liver of male C57BL/6 control and Fxr−/− mice by Peng et al. (Peng, Piekos et al. 2017). Animals were sacrificed at day −2 (gestational day 17.5), day 1 (exactly 24 hours after birth), and days 5, 20, 25, and 60 (collected at approximately 9:00 AM). Statistical analysis was performed using 2-Way ANOVA of paired mean difference, **** (p<0.0001), n=3.

Steroid signaling does not affect mTOR activity.

a, Schematic of TORC1 induced inhibition of HLH-30/TFEB by phosphorylation. Phosphorylation of HLH-30/TFEB inhibits its nuclear translocation and target gene expression. Cellular localization of HLH-30/TFEB was visualized and quantified using the expression of a hlh-30::Neongreen (HLH-30::NG) reporter in the seam cells. Representative images of N2 animals with cytosolic, and nuclear localization in seam cells, outlined and marked with arrow heads. Scale bar 20 µm. b, Quantification of HLH-30::NG cellular localization in seam cells at day 1 of adulthood. Statistical analysis was performed using One-way ANOVA, ns (p>0.05), ** (p<0.01), *** (p<0.005), n=4. c-d, Representative immunoblots with lysates from day 1 worms probed with the indicated antibodies. P-AMPK targets AMPKα phosphorylated at position Thr172. n=3. e-g, Quantification of immunoblots displaying P-AMPK intensities normalized to the loading controls β-Actin, or α-Tubulin. Statistical analysis was performed using One-way ANOVA, ns (p>0.05), * (p<0.01), n=3.

raga-1 mutation induces transcriptomic changes but does not regulate expression of steroid genes.

a, Volcano plot of differentially expressed genes identified between raga-1/N2. Grey dots mark non-significantly changed genes, black dots mark significantly changed genes (Adj.p<0.05). Yellow area highlights 4578 genes significantly up-regulated, the white area shows 4319 genes that are significantly down-regulated. b, KEGG pathway enrichment (FDR<0.05) of significantly (Adj.p<0.05) downregulated genes (blue) comparing raga-1(ok701)/N2. The gene list of KEGG enrichments is listed in supplementary table 3. c, KEGG pathway enrichment (FDR<0.05) of significantly (Adj.p<0.05) upregulated genes (red) comparing raga-1(ok701)/N2. The gene list of KEGG enrichments is listed in supplementary table 3. d, Representative images of DAF-36::GFP expression at day 1 of adulthood comparing N2 and raga-1(ok701). Differential interference contrast (DIC) and fluorescence images were taken of the animals’ full body. Scale bar 200 µm. e, Quantification of DAF-36::GFP expression at L2, L3, L4 and day 1 of adulthood. The developmental stage of every imaged animal was assessed by vulval morphology. f, Representative images of DHS-16::GFP expression at L4 stage. DIC and fluorescence images were taken of the animals’ full body. Scale bar 200 µm. g, Quantification of DHS-16::GFP expression at L4 stage. The developmental stage of every imaged animal was assessed by vulval morphology. h, Representative images of DAF-9::GFP expression at day 1 of adulthood. DIC and fluorescence images were taken of the animals’ full body. Scale bar 200 µm. i, Quantification of DAF-9::GFP expression at L2, L3, L4 and day 1 of adulthood. The developmental stage of every imaged animal was assessed by vulval morphology. j, Representative images of DAF-12::GFP expression at day 1 of adulthood. DIC and fluorescence images were taken of the animals’ head. Scale bar 50 µm. k, Quantification of DAF-12::GFP expression at L2, L3, L4 and day 1 of adulthood. The developmental stage of every imaged animal was assessed by vulval morphology. For e, g, i, and k, for samples with n=3 statistical analysis was performed using Student’s t-test, ns (p>0.05), * (p<0.05). Bars represent mean±SD, L2 (n=2), L3 (n=3), L4 (n=3) and day 1 of adulthood (n=3).

Characterization of DHS-26 conservation and dhs-26 mutation.

a, Triangle heatmap showing percent identity between aligned DHS-26/DHRS1 proteins of different organisms using ClustalW. b, Phylogenetic analysis of C. elegans dhs-26 using WormFlux. Sequence labels indicate taxonomy or model organism, organism name, and gene name, respectively. c, Representative body size quantified by measuring worm area comparing N2 and dhs-26(syb8968). Statistical analysis was performed using Student’s t-test, ns (p>0.05), n=3. d, Representative brood size measurement of individual N2 and dhs-26(syb8968) animals over a time course of 3 consecutive days starting at day 1 of adulthood. Statistical analysis was performed using Student’s t-test, ns (p>0.05), n=3. e, Quantification of the mean of total progeny per worm measured in (c) comparing N2 and dhs-26(syb8968). Bars represent mean±SD, statistical analysis was performed using Student’s t-test, ns (p>0.05), n=3. f, RT-qPCR quantification of dhs-26 mRNA levels in wild type (N2), daf-12(rh61rh411), raga-1(ok701), and raga-1;daf-12. Statistical analysis was performed using One-way ANOVA, * (p<0.005), n=4. Primer sequences are listed in table 4. g, Representative microscopy images of DHS-26::FLAG::Scarlet (DHS-26::Scarlet) and DAF-12::GFP::FLAG of day 1 adult worms. Expression of DHS-26::FLAG::Scarlet in the head region requires supplementation of 100 nM Δ7-DA to be visible. Expression of DHS-26::FLAG::Scarlet and DAF-12::GFP::FLAG in canal associated neurons (CAN) was visible under regular growth conditions. Fluorescence and DIC images of animals’ head and mid-section. Scale bar 20 µm for the head, and 5 µm for CAN. h, Representative brightfield images of raga-1;dhs-26 worms showing regular crawling (no omega phenotype) and curled body morphology (omega phenotype). Scale bar represents 200 µm. i, Quantification of omega phenotype penetrance comparing raga-1(ok701), raga-1;dhs-26, and raga-1;dhs-26::FLAG::Scarlet. Bars represent mean±SD, statistical analysis was performed using Student’s t-test, ns (p>0.05), * (p<0.05), n=2.

DHS-26 does not function as a canonical DA biosynthetic enzyme in wild type animals.

a, Schematic of the positive regulatory arm of Δ7-DA-bound DAF-12 resulting in the transcriptional activation of its target gene mir-84. Representative images of the mir-84p::GFP expression pattern in seam cell V2 at larval stages L4.3-L4.5 (Mok, Sternberg et al. 2015). The larval stage of every imaged animal was assessed by vulval morphology. Differential interference contrast (DIC) and fluorescence images were taken of the midbody for mir-84p::GFP, and head for TTX-3::RFP. Scale bar represents 50 µm. b, Representative images of mir-84p::GFP in seam cell V2 of animals at larval stages L4.3-L4.5 (Mok, Sternberg et al. 2015). The larval stage of every imaged animal was assessed by vulval morphology. DIC and fluorescence images were taken of the animals’ midbody for mir-84p::GFP, and head for TTX-3::RFP. Scale bar represents 10 µm. c, Representative quantification of mir-84p::GFP expression in seam cell V2 of N2, dhs-26(syb8968), and daf-9(rh50) animals. Fluorescence intensity of each region of interest (ROI) was calculated by subtracting the background fluorescence of each image from the ROI. mir-84p::GFP was normalized to TTX-3::RFP expression in AIY interneurons of the same animal. Statistical analysis was performed using One-way ANOVA, ns (p>0.05), **** (p<0.001), n=4. d, Relative concentrations of Δ7-dafachronic acid (DA) and its precursors cholesterol and lathosterol extracted from wild type (N2) and dhs-26(syb8968) day 1 adults. Measured concentrations were normalized to protein levels. Statistical analysis was performed using Student’s t-test, ns (p>0.05), n=4. e, Representative measurement of pharyngeal pumping rates of day 1 adults measured in 30 s. Bars represent mean±SD, statistical analysis was performed using Student’s t-test, ns (p>0.05), n=3. f-i, Quantification of dauer phenotype penetrance 48h after egglay, assessing mutation in dhs-26 on the following pathways: insulin signaling at 22.5°C (e, n=9), TGFβ signaling at 25°C (f, n=6), and steroid signaling in daf-36(k114) (g, n=3) and daf-9(rh50) (h, n=3) at 27°C. Bars represent mean±SD, statistical analysis was performed using One-way ANOVA, ns (p>0.05), * (p<0.05), **** (p<0.0001). j-k, Quantification of dhs-26(syb8968) effects on distal tip cell migration defects in L4 animals grown on cholesterol-free plates egg-on in steroid signaling mutants daf-36(k114) (i) and daf-9(rh50) (j). Bars represent mean±SD, statistical analysis was performed using One-way ANOVA, ns (p>0.05), n=3.

Proteomics changes induced by loss of dhs-26 overlap with changes induced by mutation of daf-12.

a, PCA plot of single worm proteomics data comparing wild type (N2), daf-12(rh61rh411), dhs-26(syb8968), raga-1(ok701), raga-1;daf-12, and raga-1;dhs-26 in day 1 adults. b, Volcano plot of differentially expressed genes comparing raga-1;dhs-26/raga-1(ok701). Black dots mark significantly changed genes (Adj.p<0.05), grey dots mark non-significantly changed genes (Adj.p>0.05). Blue area highlights 515 significantly down-regulated genes, the red area highlights 1093 significantly up-regulated genes. c, Volcano plot of differentially expressed genes comparing raga-1;daf-12/raga-1(ok701). Black dots mark significantly changed genes (Adj.p<0.05), grey dots mark non-significantly changed genes (Adj.p>0.05). Blue area highlights 102 significantly down-regulated genes, the red area highlights 112 significantly up-regulated genes. d, Venn diagram overlapping significantly regulated proteins comparing raga-1;dhs-26/raga-1(ok701) and raga-1;daf-12/raga-1(ok701), resulting in the identification of 93 shared genes. e, KEGG pathway enrichment (FDR<0.05) of the 93 shared genes shown in f. Gene list and the respective ortholog of the enriched pathways is displayed. The gene list of KEGG enrichments is additionally listed in supplementary Table 3.