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DC1Supplementary Materials forA Steroid Receptor–MicroRNA Switch Regulates Life Span in Responseto Signals from the GonadYidong Shen, Joshua Wollam, Daniel Magner, Oezlem Karalay, Adam Antebi**To whom correspondence should be addressed. E-mail: antebi@age.mpg.dePublished 14 December 2012, Science 338, 1472 (2012)DOI: 10.1126/science.1228967This PDF file includes:Materials and MethodsFigs. S1 to S8Tables S1 to S3Full Reference List

Supplemental MaterialsMaterials and MethodsC. elegans strains and cultureNematodes were grown with standard techniques at 20ºC unless otherwise noted (23). Allstrains with glp-1(e2141) were maintained at 15ºC and grown at 25ºC to inducegermlineless phenotype. NGM plates with ethanol or Δ4-dafachronic acid were preparedas reported (4). daf-16(mu86);muIs109(Pdaf-16::daf-16::gfp; Podr-1::rfp), pha1(e2123);arEx1273(lin-14::gfp), and nhr-80(tm1011);glp-1(e2141) were kindlyprovided by Dr. Kenyon (UCSF), Dr. Greenwald (Columbia U.) and Dr. Aguilaniu (ENSde Lyon) respectively. Strains used are listed in Table S2.TransgenesPlasmids, akt-1p::DR::akt-1u, akt-1p::DR::unc-1u, pdk-1p::DR::pdk-1u, pdk1p::DR::unc-1u, lin-14p::DR::lin-15u, and lin-14p::DR::unc-1u were injected into N2 togenerate dhEx765, dhEx768, dhEx772, dhEx776, dhEx779, and dhEx781, respectively.For dhEx842, N2 was injected with mir-241p::mir-241, mir-84p::mir-84, and a cotransformation marker, myo-2::mCherry.RNA interferenceRNAi experiments were performed as described (24). For RNAi of cco-1, daf-2 and daf16, synchronized eggs were seeded on HT115 plates expressing corresponding dsRNA.For RNAi of akt-1, lin-14, and other heterochronic genes, synchronized larvae were

grown on OP50 plates until late L4 and transferred to corresponding HT115 plates forRNA interference.Lifespan assaysAblation of germline precursor cells by laser microsurgery and adult lifespan analysiswere performed as previously reported (25). Worms undergoing internal hatching,bursting vulva, or crawling off the plates were censored. Statistical analysis wasperformed with the Mantel-Cox Log Rank method.GC/MS/MS analysisWorms were synchronized by bleaching and grown until day 1 of adulthood for analysis.Lipids were extracted from worms via the Bligh and Dyer method before derivitizing forGC-MS analysis (26). 7-dehydrocholesterol and dafachronic acid (DA) levels wereanalyzed by GC/MS/MS on a 7000A Triple Quadrupole GC/MS instrument (AgilentTechnologies) equipped with an ESI source and an HP5-ms column. Whole-worm lipidextracts were spiked with cholesterol-d7 and 5β-cholanic acid as internal standards andthen derivitized with either Fluka III (Sigma) or trimethylsilyldiazomethane (Sigma) for7-dehydrocholesterol or DA analysis, respectively. Compounds were analyzed in MRMmode using the following transitions: cholesterol-d7 (m/z 465.4Æ360.3), 7dehydrocholesterol (m/z 350.2Æ195.0), 5β-cholanic acid (m/z 374.3Æ264.0) and Δ7-DA(m/z 428.3Æ229). The results were from three independent experiments.

Plasmid constructionTo generate pDR, pMyo-LD17, a gift from Dr. Lo, was modified by first removing themyo-2 promoter and L-HDAg gene within, then replacing the original unc-54 3’UTRwith unc-1 3’UTR. The Dual Reporter (DR) module in pDR consists of gfp::icr::rfp asreported (27). DR reporters were subsequently generated from pDR as follows:akt-1p::DR::akt-1u and akt-1p::DR::unc-1uThe akt-1 promoter of 3.2 kb was amplified from genomic DNA by PCR and inserted infront of the DR module. For akt-1p::DR::akt-1u, a 444 bp akt-1 3’UTR was amplifiedfrom genomic DNA and cloned after the DR module.pdk-1p::DR::pdk-1u and pdk-1p::DR::unc-1uThe pdk-1 promoter of 2.9 kb was amplified from genomic by PCR and inserted in frontof the DR module. For pdk-1p::DR::pdk-1u, pdk-1 3’UTR of 492 bp was amplified fromgenomic DNA and cloned after the DR module.lin-14p::DR::lin-14u and lin-14p::DR::unc-1uThe lin-14 promoter (5.2 kb) was amplified from genomic DNA by PCR and clonedbefore the DR module. For lin-14p::DR::lin-14u, a 1.5 kb lin-14 3’UTR was amplifiedfrom genomic DNA and cloned after the DR module.To generate mir-241p::mir-241 and mir-84p::mir84, a 2.0 kb mir-241 promoter, a 2.8 kbmir-84 promoter, and the microRNA stem-loops together with 200 bp of flankingsequence were PCR amplified from genomic DNA. PCR fragments were subsequentlycloned into L3781 (Fire vector kit 1997).

For luciferase assays, 3’UTRs of interest were amplified from genomic DNA by PCR andcloned after the luciferase gene in pMIR-REPORT Luciferase (Ambion). For plasmidsexpressing mature microRNAs, the stem-loop of tested microRNA and 200 bp offlanking sequence were amplified from genomic DNA by PCR and cloned into the3’UTR of GFP in pEGFP-C1.Cell culture and transfectionHEK293T cells were maintained in DMEM medium supplemented with 10% fetal bovineserum (Gibco). Transfections were performed with FuGENE HD (Roche) according tothe manufacturer’s instruction.Luciferase assayPlasmids expressing microRNAs were co-transfected into HEK293T cells with fireflyand renilla luciferase reporters. Luciferase activities were measured with Dual-LuciferaseReporter Assay System (Promega) 24 h post transfection. The 3’UTR of interest wasfused with firefly luciferase and renilla luciferase serves as an internal control. At leastthree independent experiments were performed.qRT-PCRSynchronized worms were grown until day 1 of adulthood unless otherwise noted.Afterwards, worms were collected in TRIzol (Invitrogen) and frozen in liquid nitrogen.For mRNA, total RNA was prepared by RNeasy Mini kit (QIAGEN) and cDNA wassubsequently generated by Superscript III First Strand Synthesis System with randomhexamers (Invitrogen). For microRNA, miRNeasy Mini kit (QIAGEN) and TaqManMicroRNA Reverse Transcription kit (Applied Biosystems) were used for total RNA and

cDNA preparation respectively. qRT-PCR was performed with Power SYBR Greenmaster mix (Applied Biosystems) on a 7900HT Fast Real-Time PCR System (AppliedBiosystems). Three or four technical replicates were performed in each reaction. ama-1and Sno-RNA U18 were used as internal controls for mRNA and microRNA qRT-PCRrespectively. The results were from at least three biological replicates. Primer sequencesare listed in Table S3.Analysis of fluorescence intensityFluorescence intensity was measured by COPAS Biosort (Union Biometrica), or frommicroscopic pictures by Photoshop (Adobe) or ImageJ (http://rsbweb.nih.gov/ij/) asindicated. For microscopic pictures, background signal was substracted as reported (28).Fluorescence microscopy was performed on a Carl Zeiss Axio Imager Z1. For analysis ofmir-84p::GFP and mir-241p::GFP, images of indicated tissues were calibrated withInSpeck Green Microscopy Image Intensity Calibration Beads (Molecular Probes).Statistical analysisResults are presented as mean SD unless noted otherwise. Statistical tests wereperformed with either One-way ANOVA with Tukey test (ANOVA) or Student’s t-test(t-test) as indicated using GraphPad Prism (GraphPad software).

Supplementary Figures and TablesFig. S1. Loss of the germline alters the expression of hormone synthetic genes.A. qRT-PCR results of the indicated genes in WT and glp-1 worms in Day 1 adults. ttest, * P 0.05, *** P 0.001, ns: non-significant.B and C. The upregulation of daf-36 expression in germlineless animals is not regulatedby DAF-16/FOXO (B) or DAF-12 (C) in Day 1 adults. ANOVA: * P 0.05, ns: nonsignificant.D. At day 1 adulthood, daf-36::gfp expression is elevated in the absence of germline.3403 dhEx320 and 3141 glp-1;dhEx320 worms were analyzed by COPAS Biosort infive independent experiments. t-test, *** P 0.001.

Fig. S2. Analysis of microRNA expression patterns in germlineless animals.A. qRT-PCR results showing mir-48, mir-228, and let-7 levels at indicated time points.L3, L4: 3rd and 4th larval stage. D1: day 1 adult, D4: day 4 adult, and D10: day 10adult. Error bars: SEM.B-D. let-7 (B), mir-48 (C) and mir-228 (D) are not regulated by DA/DAF-12 signaling.Worms with indicated genotypes were grown on plates with ethanol (EtOH) or Δ4-

dafachronic acid (DA) until young adulthood without bearing eggs. ANOVA: vs glp1 EtOH, * P 0.05, ** P 0.01, ns: non-significant.E. Quantification of mir-84p::GFP and mir-241p::GFP intensity in WT and glp-1 wormsat day 1 of adulthood. For mir-84p::GFP, 31 WT and 33 glp-1 worms were examined.For mir-241p::GFP, 44 WT and 49 glp-1 worms were examined. The results werefrom three independent experiments. t-test: * P 0.05, ** P 0.01.F. The upregulation of mir-48, mir-84, and mir-241 in germlineless animals isindependent of nhr-80. Error bars: SEM. ANOVA, ns: non-significant.Fig. S3. mir-84 and mir-241 specifically regulate gonadal longevity.A. Gonadal longevity is significantly reduced in mir-84;mir-241;glp-1 worms.

B. The transgene dhEx842 (mir-84 mir-241) restores gonadal longevity in mir-84;mir241;glp-1 worms.C. Overexpressing mir-84 and mir-241 rescues the oxidative stress resistance of mir84;mir-241;glp-1 worms. ANOVA: ** P 0.01, *** P 0.001, ns: non-significantFig. S4. mir-84 and mir-241 regulate DAF-16/FOXO activity in gonadal longevity.A and B. Nuclear localization of DAF-16::GFP is impaired in mir-84;mir-241;glp-1compared to glp-1 animals.C. Overall DAF-16::GFP level in intestinal cells is not affected by mutating mir-84 andmir-241. DAF-16::GFP intensity was measured from 80 anterior intestinal cells inthree independent experiments. t-test: *** P 0.001, ns: non-significant.

Fig. S5. mir-84 and mir-241 regulate DAF-16/FOXO transcriptional activity in gonadallongevity.A. qRT-PCR results showing the indicated genes regulated by both daf-12 and daf-16upon germline removal.

B. Mutation of mir-84 and mir-241 reduces expression of the indicated genes ingermlineless animals.C. Mutation of mir-84 and mir-241 reduces sod-3::gfp expression in germlinelessanimals.D. daf-16RNAi does not further decrease the expression of indicated genes in mir84;mir-241;glp-1 compared to glp-1 animals.Bars: SEM. ANOVA: * P 0.05, ** P 0.01, *** P 0.001, ns: non-significant.Fig. S6. The effect of mir-84 and mir-241 on the transcriptional output of other majorregulators in gonadal longevity.A. fard-1 is regulated by daf-12 but not daf-16 upon germline removal.

B and C. Upon germline ablation, mutation of mir-84 and mir-241 does not affect theexpression of fard-1 (B) but mildly reduces the level of cdr-6 (C).D and E. mir-84 and mir-241 do not influence the expression of lgg-1 (D), unc-51 (E), orfat-6 (F). Bars: SEM.ANOVA: * P 0.05, ** P 0.01, ns: non-significant.

Fig. S7. akt-1 and lin-14 are targets of mir-84 and mir-241.A and B. mir-84 and mir-241 target the 3’UTRs of akt-1 and lin-14. HEK293T cellswere transfected as indicated to co-express luciferase reporter with the 3’UTR of

interest and corresponding microRNAs. ANOVA: vs Vector, *** P 0.001, ns: nonsignificant.C. A diagram showing the working mechanism of the DR reporter. A DR reporter, basedon the operon CEOP5428, expresses two fluorescent proteins simultaneously.Whereas the expression of GFP is controlled only by the promoter, RFP is under theregulation of both the promoter and the fused 3’UTR. When the 3’UTR of interest istargeted by microRNA, the GFP expression remains unchanged while the RFP levelwill be reduced. Therefore, a decreased RFP/GFP ratio indicates the interactionbetween the microRNA and the 3’UTR.D and E. Upon germline ablation, lin-14::gfp is downregulated in intestinal nuclei of lateL4 worms. 61 intestinal nuclei from 21 mock ablated worms, and 91 intestinal nucleifrom 27 ablated worms were scored. Arrow heads: intestinal nucleus. t-test: ***P 0.001.F. sod-3 expression is restored in mir-84;mir-241;glp-1 animals upon akt-1 or lin-14RNAi treatment. ANOVA: ** P 0.01, ns: non-significant.G and H. pdk-1-3’UTR-DR construct is upregulated in mir-84;mir-241 mutantscompared to WT.I. glp-1 mutants do not show downregulation of the pdk-1-3’UTR-DR construct.