Figures and data in miR-125-chinmo pathway regulates dietary restriction-dependent enhancement of lifespan in Drosophila

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11 figures, 1 table and 5 additional files

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Figure 1 with 2 supplements

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*let-7* and *miR-125* are required for DR-dependent enhancement of lifespan.

(A) Schematic of the *let-7-Complex* (*let-7-C*) rescuing transgenes. The *Drosophila let-7-Complex* (*let-7-C)* locus is located on chromosome two and encodes a 2435 nucleotide long primary transcript that is processed into three evolutionary conserved miRNAs: *miR-100*, *let-7* and *miR-125*. The *let-7-C* rescuing transgene includes a 17983 base-pair genomic fragment containing the *let-7-C* locus. The *ΔmiR-100*, *Δlet-7*, and *ΔmiR-125* are derivatives of the *let-7-C* transgenes that lack the mature *miR-100*, *let-7*, and *miR-125* sequences, respectively. The *let-7-C* hypomorph (*let-7-C^hyp*) encodes the *let-7-Complex* cDNA driven by a 3.3 kb promoter fragment in a transheterozygous *let-7-C* null mutant background (*let-7-C^KO2/let-7-C^GKI*). The *let-7-C^hyp/Rescue* line encodes a single copy of the *let-7-C^hyp* and a single copy of the *let-7-C* wild-type transgene. The *let-7-C^hyp/Δlet-7-C* line encodes a single copy of the *let-7-C^hyp* and a single copy of the ~18 kb *let-7-C* that lacks the mature *miR-100*, *let-7* and *miR-125* sequences. The *let-7-C^null* rescue line encodes a single copy of the ~18 kb *let-7-C* transgene in a *let-7-C* null mutant. (**B-D**) Quantitative RT-PCR of *miR-100* (B), *let-7* (C) and *miR-125* (D) in wild-type (*w¹¹¹⁸*) flies that were fed *Ad libitum* (AL) (pink pattern and pink solid bar) or DR diet (blue pattern and blue solid bar) for 7 days or 20 days. The levels of *miR-100*, *let-7* and *miR-125* increase upon 20d exposure to DR, while a significant increase in *miR-125* is detected upon 7d of DR. Expression levels were normalized to *SnoR442*. Values are mean ± SD, n = 6. (**E-G**) *Let-7-C ^hyp/Δlet-7-C* flies express significantly reduced levels of *miR-100*, *let-7,* and *miR-125* as compared to *Let-7-C ^hyp* rescue flies that were fed AL or DR diets for 7 days. For expression analysis of the rescue and mutant lines that were fed AL or DR diet for 20 days, please refer to Figure 1—figure supplement 2B-D. (H) The *let-7-C^hyp/Rescue* flies show a significant increase in lifespan upon dietary restriction ( blue line; n = 97) as compared to *let-7-C^hyp/Rescue* flies that were fed an ‘*ad libitum*’ diet ( red line; n = 95). (I) The *let-7-C^hyp* mutants (AL, red line; n = 111 and DR, blue line; n = 72) display a reduced DR-dependent increase in lifespan compared to the rescue line. (**J-M**) Compared to the *let-7-C^null* rescue flies (J) and *ΔmiR-100* (K) flies, *Δlet-7* (L) and *ΔmiR-125* (M) flies displayed a significantly dampened DR-dependent lifespan extension upon DR (blue line) when compared to flies that were fed an AL diet (red line). For statistical comparison of survival curves, p values and Χ² values were calculated with log rank test. Genotypes of strains used in this figure (**B-D**) *w¹¹¹⁸*; (E-G, H) *let-7-C^null; let-7-C^hyp/Rescue: w¹¹¹⁸; let-7-C^GKI/let-7-C^KO2, P{neoFRT}40A; P{w+, let-7-Cp^3.3kb::cDNA} / {v+, let-7-C}attP2*; (**E-G, I**) *let-7-C^hyp//Δlet-7-C: w¹¹¹⁸; let-7-C^GKI/let-7-C^KO2, P{neoFRT}40A; P{w+, let-7-Cp^3.3kb::cDNA} / {v+, let-7-C ^{Δlet-7-C miRNAs}}attP2;* (J) *let-7-C^null; Rescue/+: w¹¹¹⁸; let-7-C^GKI/let-7-C^KO2, P{neoFRT}40A; {v+, let-7-C} attP2 /+;* (K) *let-7-C^null; ΔmiR-100/+: w¹¹¹⁸; let-7-C^GKI/let-7-C^KO2, P{neoFRT}40A; {v+, let-7-C ΔmiR-100} attP2 / +;* (L) *let-7-C^null; Δlet-7: w¹¹¹⁸; let-7-C^GKI/let-7-C^KO2, P{neoFRT}40A; {v+, let-7-C ^Δlet-7} attP2 / +;* (M) *let-7-C^null; ΔmiR-125: w¹¹¹⁸; let-7-C^GKI/let-7-C^KO2, P{neoFRT}40A; {v+, let-7-C ^ΔmiR-125} attP2 / +*.

Figure 1—source data 1 Lifespan analysis of let-7-Complex^hyp, let-7-Complex^hyp rescue, let-7-Complex^null rescue, ΔmiR-100, Δlet-7, and ΔmiR-125 mutant lines.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig1-data1-v2.docx
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Figure 1—source data 2 Survival proportions for strains used in Figure 1.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig1-data2-v2.xlsx
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Figure 1—figure supplement 1

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Scheme for generation of experimental strains used in Figures 1 and 2.

This analysis compared flies that were generated using a scheme that ensured that they had identical genetic backgrounds. Flies that were analyzed (F14) were trans-heterozygous for two different *let-7-C* null alleles (dark pink and yellow), ensuring that phenotypes were not due to recessive mutations on either *let-7-C* mutant chromosome. In addition, third chromosomes that contained differing rescuing transgenes (light pink) were derived in parallel from the same population of flies. Finally, all flies had a common X-chromosome (green), derived from an isogenized stock. (S1-1) All rescuing transgenes, including the wildtype rescuing transgene as well as *let-7* and *miR-125* deleted versions, were injected into embryos from the same population of BL#25710. Resulting progeny were backcrossed twice to BL#32261 in order to select and balance *vermillion+* transformants (F1 and F2). Single transformants were subsequently backcrossed to an isogenized version of BL#3703 three times (F3-F5) in order to make balanced stocks with isogenized X chromosomes (F6). (S1-2) Stocks with differing rescuing transgenes were crossed to the same population of a stock that contained the *let-7-C^KO2* chromosome, an isogenized X chromosome, and two 3rd chromosome balancers. The *let-7-C^KO2* stock used in F7 was generated in a similar fashion as the rescuing transgenes stocks, by backcrossing three times to an isogenized version of BL#3703. Resulting stocks (F8) had common X (green), 2nd (yellow) and 3rd (light pink) chromosomes and were used in F13 to generate the experimental strains. (S1-3) A second *let-7-C* allele, *let-7-C^GKI*, was prepared by outcrossing twice to an isogenized stock, and then crossed to an isogenized stock containing a T (2:3) Cyo-TM6b compound chromosome. The *let-7-C* allele was selected based on mini-white, and the T (2:3) Cyo-TM6b balancer was selected based on the dominant Humoral marker. The resulting stock with a fixed second and third chromosome was amplified and used as the source for all virgins in the crosses that yielded the flies for analysis. (S1-4). Flies for analysis were generated by crossing virgins of the stock generated in F12 with males of stocks generated in F8 that harbored differing rescuing transgenes. For generation of the *let-7-C^hyp* rescue and mutant line the *let-7-C^hyp* transgenic was combined with *let-7-C^KO2* stock in the step S1-3. The remaining steps were similar to the scheme described.

Figure 1—figure supplement 2

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Lifespan analysis controls and expression analysis of strains used in Figure 1.

(A) *w¹¹¹*⁸ flies show a significant increase in lifespan upon DR (blue line; n = 98; median lifespan = 58) as compared to *w¹¹¹*⁸ flies that were fed an ‘*ad libitum*’ diet (red line; n = 99; median lifespan = 28). (**B–D**) Quantitative RT-PCR of *miR-100* (B), *let-7* (C) and *miR-125* (D) in *let-7-C ^hyp/Δlet-7-C* and *let-7-C^hyp/Rescue* flies that were fed AL or DR diet for 20 days. *Let-7-C ^hyp/Δlet-7-C* flies express dramatically reduced levels of *miR-100*, *let-7,* and *miR-125* as compared to *Let-7-C ^hyp* rescue flies that are fed AL or DR diets for 7 days. Expression levels were normalized to *SnoR442*. Values are mean ± SD, n = 6. (**E–M**) Lifespan analysis to examine the effect of genetic background of strains used in Figure 1. Crosses were established between *w¹¹¹⁸* flies, and the background strains and the progeny were sorted into two groups (AL and DR). (E) *Let-7-C^KO2/+; +/+* flies live significantly longer on DR diet (AL, red line, n = 110, median lifespan = 22; DR, blue line, n = 113, median lifespan = 34). (F) *Let-7-C^GKI/+; +/+* flies live significantly longer on DR diet (AL, red line, n = 106, median lifespan = 44; DR, blue line, n = 112, median lifespan = 70). (G) *Let-7-C^KO2/+; let-7-C^hyp/+* flies live significantly longer on DR diet (AL, red line, n = 139, median lifespan = 28; DR, blue line, n = 160, median lifespan = 40). (H) *Let-7-C^GKI/+; let-7-C/+* flies live significantly longer on DR diet (AL, red line, n = 102, median lifespan = 36; DR, blue line, n = 100, median lifespan = 48). (I) *Let-7-C^GKI/+; ΔmiR-100/+* flies live significantly longer on DR diet (AL, red line, n = 103, median lifespan = 12; DR, blue line, n = 79, median lifespan = 50). (J) *Let-7-C^GKI/+; Δlet-7/+* flies live significantly longer on DR diet (AL, red line, n = 83, median lifespan = 20; DR, blue line, n = 96, median lifespan = 43). (K) *Let-7-C^GKI/+; ΔmiR-125/+* flies live significantly longer on DR diet (AL, red line, n = 91, median lifespan = 32; DR, blue line, n = 100, median lifespan = 46). (M) *Let-7-C^GKI/+; Δlet-7-C/+* flies live significantly longer on DR diet (AL, red line, n = 96, median lifespan = 30; DR, blue line, n = 117, median lifespan = 36). For statistical comparison of survival curves, p values and Χ² values were calculated with log rank test. Genotypes of strains used in this figure (A) *w¹¹¹⁸*; (**B-D**) *Let-7-C^null; let-7-C^hyp/Rescue: w¹¹¹⁸; let-7-C^GKI/let-7-C^KO2, P{neoFRT}40A; P{w+, let-7-Cp^3.3kb::cDNA} / {v+, let-7-C}attP2*; (**B-D**) *Let-7-C^hyp//Δlet-7-C: w¹¹¹⁸; let-7-C^GKI/let-7-C^KO2, P{neoFRT}40A; P{w+, let-7-Cp^3.3kb::cDNA} / {v+, let-7-C ^{Δlet-7-C miRNAs}}attP2;* (E) *Let-7-C^KO2/+; +/+: w¹¹¹⁸; let-7-C^KO2, P{neoFRT}40A/+; +/+;* (F) *let-7-C^KO2/+; +/+: w¹¹¹⁸; let-7-C^GKI/+; +/+;* (G) *Let-7-C^KO2/+; let-7-C^hyp/+: w¹¹¹⁸; let-7-C^KO2, P{neoFRT}40A/+; P{w+, let-7-Cp^3.3kb::cDNA}/+;* (H) *let-7-C^GKI/+; let-7-C/+: w¹¹¹⁸; let-7-C^GKI/+; {v+, let-7-C}attP2/+;* (I) *let-7-C^GKI/+; ΔmiR-100/+: w¹¹¹⁸; let-7-C^GKI/+; {v+, let-7-C ΔmiR-100} attP2 /+;* (J) *Let-7-C^GKI/+; Δlet-7/+: w¹¹¹⁸; let-7-C^GKI/+;{v+, let-7-C ^Δlet-7} attP2 /+;* (K) *Let-7-C^GKI/+; ΔmiR-125/+: w¹¹¹⁸; let-7-C^GKI/+; {v+, let-7-C ^ΔmiR-125} attP2 / +*; (L) *let-7-C^GKI/+; Δlet-7-C/+: w¹¹¹⁸; let-7-C^GKI/+;{v+, let-7-C ^Δlet-7-C} attP2 /+*.

Figure 1—figure supplement 2—source data 1 Lifespan analysis of let-7-Complex^hyp, let-7-Complex^hyp rescue, let-7-Complex^null rescue, ΔmiR-100, Δlet-7, and ΔmiR-125 mutant lines.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig1-figsupp2-data1-v2.docx
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Figure 1—figure supplement 2—source data 2 Survival proportions for strains used in Figure 1.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig1-figsupp2-data2-v2.xlsx
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Figure 2 with 1 supplement

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Reducing *chinmo (*Δ*miR-125*, *chinmo^RNAi and* Δ*miR-125*, *chinmo¹)* levels in *miR-125* mutants suppresses the loss of DR-mediated increase in lifespan extension and decrease in triglyceride levels.

(A) Schematic representing post-transcriptional repression of *chinmo* mRNA by *let-7* and *miR-125* adapted from our previously published study (Chawla et al., 2016). *chinmo* mRNA 3’untranslated region (3’UTR) encodes multiple binding sites for *let-7* and *miR-125* miRNAs and is differentially regulated by *let-7* and *miR-125. Let-7* is the predominant repressor of *chinmo* during development and *miR-125* silences *chinmo* in the adult stage. (B) Loss of *miR-125* leads to derepression of *chinmo* in the adult brain and a concomitant increase in Chinmo signal in the adult fat tissue (Compare top two left panels with top two right panels). Confocal images of dissected tissues (brain and fat body) from adult *let-7-C ^null* rescue and *miR-125* mutant flies that were fed *ad libitum* (AL) or nutrient restricted (DR) diet for 5 days. (**C, D**) Rescue, UAS *chinmo^RNAi* flies and *ΔmiR-125, UAS chinmo^RNAi* flies show a 25% increase in median lifespan upon DR ( blue line) (Rescue, UAS *chinmo^RNAi*, χ² = 37.35; *ΔmiR-125, UAS chinmo^RNAi*, χ² = 57.71). (**E-F**) *Rescue, chinmo¹* flies display a 20% increase in median lifespan upon DR (blue line) and *ΔmiR-125, chinmo¹* flies display a 41% increase in DR-dependent increase in median lifespan (compare red and blue curves) (*Rescue, chinmo¹* χ² = 29.63; *ΔmiR-125, chinmo¹* χ² = 102.5). For statistical comparison of survival curves, p values and χ² were calculated with log rank test. (G) Quantitation of triglyceride (TAG) stored levels in Rescue line, *ΔmiR-125* line, Rescue, *UAS chinmo^RNAi* line, ΔmiR-125, UAS chinmo^RNAi line, *Rescue, chinmo¹* line and *ΔmiR-125, chinmo¹* line, AL (Red bars) and DR (Blue bars) fed 20-day-old flies. The bars represent mean ± SD, n = 6, p value was calculated with unpaired t-test with Welch’s correction. p Values are indicated in the graph. The TAG values were normalized to protein levels. The *ΔmiR-125* flies display significantly lower stored triglycerides compared to the genetically identical rescue line in both AL and DR diets. Reducing *chinmo* levels by RNAi or by reducing a copy of *chinmo* led to an increase in TAG levels upon DR. Consistent with the survival analysis, the increase in TAG levels in *ΔmiR-125, chinmo¹* flies TAG was similar to the increase in rescue flies (p value=0.6343), indicating that reducing a copy of *chinmo* was sufficient to rescue the TAG levels and the rescue was better than what was obtained with knockdown of *chinmo* by RNAi in cells that express *miR-125*. (**H, I**) Fat bodies/abdomens of female flies were dissected and stained for the content and diameter of lipid droplet (LD) (red are lipid droplets stained with Nile red and blue is Dapi). Scale bar, 25 μm. (I) Quantitation of lipid droplet (LD) diameter in (H). Quantitation of 15 largest LDs in five samples per condition. Error bars represent mean ± SD and p values are indicated in the figure. The reduction in lipid droplet size in *ΔmiR-125* mutants that are fed an AL and DR diet is rescued by reducing the dosage of *chinmo* in *ΔmiR-125, chinmo¹* and not by *chinmo^RNAi*. Genotypes of strains used in this figure: (**B, G-I**) *let-7-C^null; Rescue/+: w¹¹¹⁸; let-7-C^GKI/let-7-C^KO2, P{neoFRT}40A; {v+, let-7-C} attP2 /+;* (**B, G-I**) *let-7-C^null; ΔmiR-125: w¹¹¹⁸; let-7-C^GKI/let-7-C^KO2, P{neoFRT}40A; {v+, let-7-C ^ΔmiR-125} attP2 / +*; (**C, G-I**) *let-C^GKI/KO2; Rescue, chinmo^RNAi: w¹¹¹⁸; let-7-C^GKI/let-7-C^KO2, P{neoFRT}40A; {v+, let-7-C} attP2/P{w+, UAS-chinmo^{RNAi 148}}VK00033*; (**D, G-I**) *let-C^GKI/KO2; ΔmiR-125, chinmo^RNAi: w¹¹¹⁸; let-7-C^GKI/let-7-C^KO2, P{neoFRT}40A; {v+, let-7-C ^ΔmiR-125} attP2/P{w+, UAS-chinmo^{RNAi 148}}VK00033*; (**E, G-I**) *let-7-C^GKI/chinmo¹let-7-C^KO2; Rescue/+: w¹¹¹⁸; let-7-C^GKI/chinmo¹, let-7-C^KO2, P{neoFRT}40A; {v+, let-7-C} attP2 /+*; (**F, G-I**) *let-7-C^GKI/chinmo¹,let-7-C^KO2; ΔmiR-125/+: w¹¹¹⁸; let-7-C^GKI/chinmo¹, let-7-C^KO2, P{neoFRT}40A; {v+, let-7-C ^ΔmiR-125} attP2 / +.*

Figure 2—source data 1 Lifespan analysis of rescue, chinmo^RNAi and ΔmiR-125, chinmo^RNAi strains.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig2-data1-v2.docx
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Figure 2—source data 2 Lifespan analysis of rescue, chinmo¹ and ΔmiR-125, chinmo¹ strains.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig2-data2-v2.docx
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Figure 2—source data 3 Survival proportions for strains used in Figure 2.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig2-data3-v2.xlsx
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Figure 2—figure supplement 1

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Effect of genetic background and/or RU-486 in *UAS chinmo^RNAi/+* and *chinmo¹/+* strains used for analysis in Figure 2.

(A) *UAS chinmo^RNAi/+* flies show a significant increase in lifespan upon DR (DR+, blue dotted line; n = 103; median lifespan = 64 d; DR-, blue solid line; n = 96; median lifespan = 56 d) as compared to *UAS chinmo^RNAi/+* flies that were fed an ‘*ad libitum*’ diet (AL+, red dotted line; n = 99; median lifespan = 32 d; AL-, red solid line; n = 108; median lifespan = 32 d). RU-486 does not significantly change the lifespan of *UAS chinmo^RNAi/+* flies in AL or DR (p values and , χ² values are indicated in the figure panel). (B) *chinmo¹, let-7-C^KO2/+* flies show a significant increase in lifespan upon DR (DR, blue line; n = 90; median lifespan = 58) as compared to *chinmo¹, let-7-C^KO2/+* flies that were fed and AL diet (red line; n = 104; median lifespan = 38 d). For statistical comparison of survival curves, p values and χ² were calculated with log rank test. Genotypes of strains used in this figure: (A) *UAS chinmo^RNAi/+: w¹¹¹⁸; +/+; P {w+, UAS-chinmo^{RNAi 148}} VK00033/+;* (B) chinmo¹, let-7-C^KO2/+: w¹¹¹⁸; chinmo¹, let-7-C^KO2, P{neoFRT}40A/+; +/+.

Figure 2—figure supplement 1—source data 1 Lifespan analysis of flies encoding a single copy of the UAS chinmo^RNAi transgene and a strain that is heterozygous for let-7-C and chinmo.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig2-figsupp1-data1-v2.docx
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Figure 2—figure supplement 1—source data 2 Survival proportions for strains used in Figure 2—figure supplement 1.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig2-figsupp1-data2-v2.xlsx
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Figure 3 with 1 supplement

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Chinmo is nutrient-dependent downstream effector of the DR-pathway.

(A) Chinmo protein levels increase in adult fat tissue of *w¹¹¹⁸* flies that were fed a DR diet for 10 days. Confocal images of dissected fat body from adult *w¹¹¹⁸* flies that were fed an AL or DR diet for 10 days. Immunostaining was performed with anti-Chinmo, anti-La (nuclear protein), and Dapi. Scale bar, 10 μm. (**B, C**) Quantitation of Chinmo protein levels in adult fat tissue nuclear and peripheral (non-nuclear) regions as determined by measuring pixel intensity in 10 cells in five samples per condition. Error bars represent mean ± SD and p values were calculated by unpaired t test with Welch’s correction. (D) DR induces expression of *chinmo* in *w¹¹¹⁸* flies. RT-PCR quantitation of *chinmo* mRNA in head and decapitated body tissue (-Head) of *w¹¹¹⁸* flies that were fed an AL or DR diet for 10 days. (E) DR increases circulating levels of *chinmo* mRNA in *w¹¹¹⁸* flies. RT-PCR quantitation of *chinmo* mRNA in the hemolymph of *w¹¹¹⁸* flies that were fed an AL or DR diet for 10 days. (**F-J**) Reducing *chinmo* levels in the adult fat body increases lifespan and enhances TAG levels under AL conditions. (F) A transgene expressing a short hairpin to knockdown *chinmo* was expressed in the adult fat body using the steroid (RU-486) inducible gene switch Gal4 driver. Quantitative RT-PCR of *chinmo* from abdominal fat tissue of *FB GS >UAS chinmo^RNAi* flies in presence of RU-486 (bars with red and blue pattern) or in absence of RU-486 (bars with solid red and blue color) in flies that were fed Ad libitum (AL) (red) or DR diet (blue) for 10 days. Expression levels were normalized to *Actin*. Values are mean ± SD, n = 6. p Values are indicated in the graph and were calculated by unpaired t test with Welch’s correction. (G) Knockdown of *chinmo* in the adult fat tissue resulted in a 16% increase in the median lifespan of flies that were fed an AL diet (compare red solid line with the red dotted line) (Figure 3—source data 1A–B has details of experimental repeat). For statistical comparison of survival curves, p values and χ² were calculated with log rank test. (H) Quantitation of triglyceride (TAG) stored levels in AL-RU-486 (Solid red bars), AL + RU-486 (Red pattern bars), DR-RU-486 (Solid blue bars) and DR +RU-486 (Blue pattern bars) fed 20-day-old *FB GS >UAS chinmo^RNAi* flies. The bars represent mean ± SD, n = 5, p value was calculated with two tailed t-test. Significance levels: ***p<0.001. (I) Fat bodies of *FB GS >UAS chinmo^RNAi* female flies were dissected and stained for the content and diameter of lipid droplet (LD) (red are lipid droplets stained with Nile red and blue is Dapi). Scale bar, 25 μm. (J) Quantitation of lipid droplet (LD) diameter in (I). Quantitation of 15 largest LDs in five samples per condition. Error bars represent mean ± SD and p values are indicated in the graph and were calculated by using unpaired t test with Welch’s correction. Genotypes of strains used in this figure: (**A-E**) *w¹¹¹⁸*; (**F-J**) *FB GS/+; UAS chinmo^RNAi/+; w¹¹¹⁸; P{w[+mW.hs]=Switch1}106/+; P{w+, UAS-chinmo^{RNAi 148}}VK00033/+*.

Figure 3—source data 1 Lifespan analysis of FBGS >UAS chinmo^RNAi strain.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig3-data1-v2.docx
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Figure 3—source data 2 Survival proportions of lifespan curves of FBGS >UAS chinmo^RNAi strain.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig3-data2-v2.xlsx
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Figure 3—figure supplement 1

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Nutrient-dependent regulation of Chinmo in the adult fat tissue.

(A) Chinmo protein levels increase in adult fat tissue of *w¹¹¹⁸* flies that were fed a DR diet for 20 days. Confocal images of dissected fat body from adult *w¹¹¹⁸* flies that were fed an AL or DR diet for 20 days. Immunostaining was performed with Rat anti-Chinmo, Guinea pig anti-Chinmo, and Dapi. Scale bar, 10 μm. (**B, C**) Quantitation of Chinmo protein levels detected by the rat anti-Chinmo antibody in adult fat tissue nuclear and peripheral (non-nuclear) regions as determined by measuring pixel intensity in 10 cells in five samples per condition. Error bars represent mean ± SD and p values were calculated by unpaired t test with Welch’s correction. (**D, E**) Quantitation of Chinmo protein levels detected by the Guinea pig anti-Chinmo antibody in adult fat tissue nuclear and peripheral (non-nuclear) regions as determined by measuring pixel intensity in 10 cells in five samples per condition. Error bars represent mean ± SD and p values were calculated by unpaired t test with Welch’s correction. (F) DR induces expression of *chinmo* more significantly in adult fat tissue compared to adult head tissue. RT-PCR quantitation of *chinmo* mRNA in head and dissected fat tissue of *w¹¹¹⁸* flies that were fed an AL or DR diet for 20 days. (G) In contrast, DR induces *miR-125* levels more significantly in adult head tissue as compared to fat tissue. (H) Chinmo protein is not detectable in *w¹¹¹⁸* adult brains due to post-transcriptional silencing by *miR-125*. Confocal images of dissected brain from adult *w¹¹¹⁸* flies that were fed an AL or DR diet for 20 days. Immunostaining was performed with Rat anti-Chinmo, mouse anti-Dacshund and Dapi. Scale bar, 25 μm. (I) Schematic showing the differential tissue-specific regulation of *chinmo* in adult brain and fat tissue. *miR-125* silences *chinmo* in the adult brain. DR induces *miR-125* and *chinmo* levels in the adult brain and consequentially, *chinmo* continues to be post-transcriptionally repressed in this tissue under DR conditions. In the fat tissue, DR leads to an increase in *chinmo* mRNA and protein levels. Genotype of strain used in this figure: (**A-H**) *w¹¹¹⁸*.

Figure 4 with 2 supplements

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Export of Chinmo from the nucleus is dependent on deacetylation of Chinmo by dSir2.

(A) Chinmo redistributes to the cytoplasm upon cell starvation. Kc167 cells were transfected with Tubulin Gal4 and UAS-GFP::Chinmo constructs and redistribution of Chinmo in fed (*upper panel*) and starved (*lower panel*) cells was examined by immunostaining with GFP antibody (Chinmo), Fmr1 (cytoplasmic marker) and Dapi (blue). Chinmo is predominantly localized to the nucleus in the fed state and redistributes to the cytoplasm in starved Kc167 cells. (**B, C**) Localization of GFP::Chinmo in cells overexpressing Flag dSir2 under fed/control (Ctrl) conditions. Kc167 cells were transfected with Tubulin Gal4 and UAS-Chinmo:: GFP and dSir2 pAFW constructs and redistribution of Chinmo was examined by immunostaining cells with GFP (Chinmo), Flag (dSir2), and Dapi. Overexpression of dSir2 led to redistribution of Chinmo to the cytoplasm under fed conditions. (C) Bar graph showing quantitation of the percentage of cells in which Chinmo::GFP was localized to the nucleus in cells transfected with either empty vector or dSir2 expressing plasmid. A total of 100 cells were quantitated in a minimum of three independent experiments. (**D, E**) Acetylation of Flag-Chinmo in fed (Ctrl) and Starvation (Stv) conditions. Acetylation of Flag-Chinmo was detected by immunoprecipitation with a Flag antibody followed by western blot analysis with an anti-acetyl-lysine antibody. Three bands of Chinmo were detected in the Flag immunoprecipitates reproducibly with the top-most band corresponding to the expected size of the full-length protein. (E) Bar graph showing the fraction of acetylated lysine in full-length Flag-Chinmo (indicated by * in panel D) under Ctrl and Stv conditions, determined by three independent immunoprecipitation experiments. (F) Co-immunoprecipitation of Myc-dSir2 with Flag-Chinmo under control and starvation conditions. Flag antibody was used for immunoprecipitation of Flag-Chinmo followed by western blot analysis with Myc antibody to detect dSir2 in the immunoprecipitates. (G) Schematic representing the nutrient-dependent Chinmo relocalization by dSir2. (**H-L**) K47, K55, and K348 are the main acetylation sites of Chinmo. The GPS-PAIL online prediction tool was used to identify potential Lysine residues in Chinmo that were acetylated. For table with all predicted acetylation Lysine residues please refer to Figure 4—figure supplement 1A. Kc167 cells were transfected with Tubulin Gal4 and GFP-tagged Chinmo wild type or each of the mutants in which the lysine (K) residues were mutated to Glutamine (Q) and the nuclear-cytoplasmic distribution of the tagged Chinmo proteins was examined under fed and starvation conditions by immunostaining with GFP(Chinmo) and Dapi. Scale bars represent 5 µm. (**H-I**) Majority of the wild-type GFP: : Chinmo protein redistributes to the cytoplasm upon starvation. (**H, J-L**) K47Q, K55Q double mutant, K348Q single mutant and K47Q, K55Q, K348Q triple mutant predominantly localize to the nucleus even upon starvation. (**M-P**) The K47Q, K55Q double mutant and K348Q single mutant show significant redistribution of Chinmo in the cytoplasm upon overexpression of dSir2. However, combining the three mutations in K47Q, K55Q, K348Q triple mutant abrogates the cytoplasmic export of Chinmo upon dSir2 overexpression. (Q) Schematic indicating the three lysine residues that are required for dSir2-mediated redistribution of Chinmo to the cytoplasm. The statistical results in all the bar graphs (**C, E, I-P**) are shown as mean ± SD (n = 3). p Values are calculated using unpaired t-test with Welch’s correction and indicated in each of the graphs.

Figure 4—source data 1 Uncropped western blots with relevant bands labeled for Figure 4D–F.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig4-data1-v2.docx
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Figure 4—source data 2 Original image files of the unedited for western blots in Figure 4D.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig4-data2-v2.zip
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Figure 4—source data 3 Original image files of the unedited for western blots in Figure 4E.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig4-data3-v2.zip
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Figure 4—source data 4 Original image files of the unedited for western blots in Figure 4F.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig4-data4-v2.zip
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Figure 4—source data 5 Morphology and fluorescence microscopy of Kc167 cells used in Figure 4.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig4-data5-v2.docx
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Figure 4—figure supplement 1

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Evaluation of predicted acetylated lysine residues in Chinmo.

(A) Table indicating potential Lysine residues in Chinmo that are sites for acetylation based on the GPS-PAIL online prediction tool. GPS-PAIL was run with all stringency options and there were very few sites obtained at the stringent (0) or medium (2) cut-offs. All the Lysine residues that were predicted at low cut-off are represented in (A). (B) Schematic representation of the Chinmo protein with predicted Lysines that may undergo Acetylation/deacetylation-mediated regulation. (C) Western blot analysis to confirm expression of GFP-tagged Chinmo in Kc167 cells transfected with Tubulin Gal4 plasmid and either empty vector (pUASTattB) in fed conditions (Ctrl) or UAS GFP Chinmo in fed (Ctrl) and starvation conditions (Stv). (D) All Lysine (K) residues that were predicted to be acetylated were mutated to Glutamine (Q). The UAS-GFP Chinmo mutant constructs were transfected into Kc167 cells together with Tubulin GAL4 and the nucleo-cytoplasmic distribution of the protein was examined after immunostaining of fixed cells exposed to fed (Ctrl) or 10 hr starvation. (D) Majority of single mutants analyzed (K27Q, K47Q, K55Q, K114Q, K288Q, K358Q, K414Q, K506Q, K515Q, and K569Q) redistributed to the cytoplasm upon 10 hr starvation. K348Q and K47QK55Q double mutant and K47QK55QK348Q triple mutant predominantly localized to the nucleus even upon starvation (Figure 4H–L). Scale bars represent 5 µm.

Figure 4—figure supplement 1—source data 1 Uncropped western blots with relevant bands labeled for Figure 4—figure supplement 1C.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig4-figsupp1-data1-v2.docx
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Figure 4—figure supplement 1—source data 2 Original image files of the unedited for western blots in Figure 4—figure supplement 1C.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig4-figsupp1-data2-v2.zip
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Figure 4—figure supplement 2

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Chinmo protein and mRNA are upregulated in adult and larval fat tissue upon starvation.

(A) Chinmo protein levels increase in adult fat tissue of *w¹¹¹⁸* flies that were exposed to starvation for 2 days. Confocal images of dissected fat body from adult *w¹¹¹⁸* flies that were exposed to normal diet or starvation for 2 days. Immunostaining was performed with anti-Chinmo, anti-Woc (nuclear protein) and Dapi. Scale bar, 10 μm. (**B, C**) Quantitation of Chinmo protein levels in adult fat tissue nuclear and peripheral (non-nuclear) regions as determined by measuring pixel intensity in 10 cells in five samples per condition. Chinmo levels increase in the nuclear and periphery regions upon starvation. Error bars represent mean ± SD and p values were calculated by unpaired t test with Welch’s correction. (D) Chinmo protein levels increase in larval fat tissue upon 16 hr of starvation. Confocal images of dissected fat body from *w¹¹¹⁸* 2nd instar larvae that were exposed to normal diet or starvation for 16 hr. (**E, F**) Quantitation of Chinmo protein levels in larval fat tissue nuclear and peripheral (non-nuclear) regions as determined by measuring pixel intensity in 10 cells in five samples per condition. Chinmo levels increase in the nuclear but not periphery regions upon starvation. Error bars represent mean ± SD and p values were calculated by unpaired t test with Welch’s correction. (**G-H**) *Chinmo* mRNA levels increase upon starvation. RT-PCR quantitation of fold change in *chinmo* mRNA levels in *w¹¹¹⁸* 2^nd instar larvae (G), adult whole flies (H) that were fed a Ctrl/normal food (Red bars) or exposed to starvation (Blue bars) for 2 days. (I) The DR-dependent increase in *chinmo* is higher in peripheral tissues (decapitated body tissue) as compared to the head tissue. RT-PCR quantitation of *chinmo* mRNA in head and decapitated body tissue of *w¹¹¹⁸* flies that were fed a normal diet or exposed to starvation for 2 days. Genotypes of strains used in this figure: (**A-I**) *w¹¹¹⁸*.

Figure 5 with 1 supplement

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Chinmo co-fractionates with exosomal proteins upon nutrient deprivation.

(A) Schematic of the 3X Flag-Chinmo protein indicating the BTB and Zinc finger domains. (B) Western blot analysis to confirm expression of Flag-tagged Chinmo in Kc167 cells transfected with Tubulin Gal4 plasmid and either empty vector (pUASTattB) or UAS Flag Chinmo. (**C-N**) Kc167 cells were transfected with Tubulin Gal4 and Flag Chinmo and distribution of Chinmo was examined in fixed cells after immunostaining with Flag and Dapi. Chinmo is predominantly localized to the nucleus in the fed state (**C-E**) and redistributes to cytoplasm when Kc167 cells are starved for 8 hr (**F-H**). (**I-N**) Chinmo distribution in Kc167 starved cells that are shifted to medium (CCM3) for 4 hr (**I-K**) and 16 hr (**L-N**). Chinmo translocates to the nucleus when cells are shifted from starvation to nutrient-rich conditions for 16 hr. Confocal images were acquired at ×100 magnification. Scale bars represent 8 μm. (O) Western blot analysis of cell lysate, concentrated culture medium and purified exosomes from Kc167 cells. Kc167 cells were transfected with Tubulin Gal4 and Flag Chinmo and 72 hr post-transfection cells were divided into flasks with either medium (CCM3) or 2 mg/ml glucose (PBS) for 8 hr. The medium from both conditions was collected and concentrated using Amicon centrifugal columns. Exosomes were purified from concentrated medium by gel exclusion chromatography. The exosomal fraction, concentrated medium and, cell lysate were analyzed by western blotting with Flag (Flag Chinmo), Csp 1 (exosome marker), and Syntaxin 1A (exosome marker) antibodies. Flag Chinmo was detected in exosomes and concentrated medium of Kc167 cells that were starved for 8 hr (Asterisk symbol). (P) Schematic representing dynamic redistribution of Chinmo in Kc167 cells exposed to starvation for 8 hr followed by a shift to medium for 16 hr.

Figure 5—source data 1 Uncropped western blots with relevant bands labeled for Figure 5B and O.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig5-data1-v2.docx
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Figure 5—source data 2 Original image files of the unedited western blots in Figure 5B and O.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig5-data2-v2.zip
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Figure 5—figure supplement 1

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Elution of vesicles using gel filtration columns.

(A) Elution profile with 100 µl of concentrated Kc167 cell culture medium loaded. OD600nm (blue line) and OD280nm (red line) was measured in each of the collected fractions. The elution of vesicles peaked at 200 µl after void volume (1000 µl) when 100 µl were collected. The protein fraction eluted in much later fractions (~1000 µl) after the void volume. (B) Western blot analysis of fractions collected (A) with antibodies to exosomal proteins cysteine string protein (CSP), syntaxin 1A (Syx1A) and hepatocyte growth factor regulated tyrosine kinase substrate (Hrs).

Figure 5—figure supplement 1—source data 1 Uncropped western blots with relevant bands labeled for Figure 5—figure supplement 1B.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig5-figsupp1-data1-v2.docx
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Figure 5—figure supplement 1—source data 2 Original image files of the unedited western blots in Figure 5—figure supplement 1B.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig5-figsupp1-data2-v2.zip
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Figure 6 with 3 supplements

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Ectopic expression of *chinmo* in adult neurons and fat body mimics *miR-125* mutant DR phenotype.

*UAS chinmo* was expressed in adult neurons and adult fat tissue using the steroid (RU-486) inducible gene switch Gal4 drivers. (A) Female flies that were fed an RU-486 supplemented diet for 5 days displayed increased levels of Chinmo in neuronal cells, as detected by Chinmo. Woc (nuclear marker) and Dapi staining of dissected adult fly brains. (**B, D**) *3X ElavGS >UAS chinmo* (B) or *3X ElavGS >UAS Flag chinmo* (D) flies that were not fed RU-486 show a significant increase in lifespan upon DR (DR, blue line) as compared to *3X ELavGS >UAS chinmo* (B) or *3X ElavGS >UAS Flag chinmo* (D) flies that were fed an *ad libitum* (AL) diet (red line)(3X *ElavGS >UAS chinmo*: AL-, n = 98; median lifespan = 32 d; DR-, n = 99; median lifespan = 42; *3X ElavGS >UAS Flag-chinmo*: AL-, n = 107; median lifespan = 44 d; DR-, n = 99; median lifespan = 70 d). RU-486 fed *3X ElavGS >UAS chinmo* flies or *3X ElavGS >UAS Flag-chinmo* display a significantly dampened DR-dependent increase in lifespan (blue and red dotted lines) (*3X ElavGS >UAS chinmo*: AL+, n = 100; median lifespan = 10 d; DR+, n = 95; median lifespan = 16 d; *3X ElavGS >UAS Flag-chinmo:* AL+, n = 137; median lifespan = 18 d; DR+, n = 100; median lifespan = 26 d). (**C, E**) *FB GS >UAS chinmo* or *FB GS >UAS Flag-chinmo* flies that were fed RU-486 displayed a stronger DR phenotype relative to *3X ElavGS >UAS Chinmo* or *3X ElavGS >UAS Flag chinmo* (blue and red dotted lines)(*FB GS >UAS chinmo:* AL-, n = 186; median lifespan = 26 d; DR-, n = 187; median lifespan = 52 d; AL+, n = 213; median lifespan = 16 d; DR+, n = 209; median lifespan = 16 d; *FB GS >UAS Flag-chinmo:* AL-, n = 96; median lifespan = 42; DR-, n = 91; median lifespan = 54; AL+, n = 83; median lifespan = 14 d; DR+, n = 73; median lifespan = 14 d) (Compare p values and Χ² in panels B/D with C/E) compared to the flies that were fed the Ethanol (blue and red solid lines). For statistical comparison of survival curves, p values and Χ² were calculated with log rank test. (**F, G**) Quantitation of triglyceride (TAG) stored levels in AL-RU-486 (Solid red bars), AL + RU-486 (Red dotted bars), DR-RU-486 (Solid blue bars) and DR +RU-486 (Dotted blue bars) fed 20d old (F) *3XElavGS > UAS chinmo* or (G) *3XElavGS > UAS Flag-chinmo* flies. Increasing Chinmo levels in adult neurons is sufficient to lead to a significant reduction in the systemic triglyceride levels under both AL and DR conditions. This decrease mimics the decrease in TAG levels observed in *miR-125* mutants. The bars represent mean ± SD, n = 5, p value was calculated with unpaired two tailed t-test with Welch’s correction. (**H, I**) Fat bodies/abdomens of female flies were dissected and stained for the content and diameter of lipid droplet (LD) (red are lipid droplets stained with Nile red and blue is Dapi). For representative confocal images, please refer to Figure 6—figure supplement 1H–I. Quantitation of lipid droplet (LD) diameter in *3XElavGS > UAS chinmo* (H) and *3XElavGS > UAS Flag-chinmo* (I). A significant reduction is seen in lipid droplet size in the abdominal fat tissue when Chinmo is expressed in adult neurons. Quantitation of 15 largest LDs in five samples per condition. Error bars represent mean ± SD. (**J–K**) *3X ElavGS >UAS chinmo* flies express increased levels of *chinmo* mRNA in both head and fat tissue. (**L–M**) *FBGS >UAS chinmo* flies predominantly express chinmo mRNA in the adult fat tissue. (**J–M**) Quantitative RT-PCR of RNA extracted from dissected tissues (head and fat body) of *3XElavGS > UAS chinmo* (**J, K**) and *FBGS >UAS chinmo* (**L, M**) flies that were fed an AL (Red and pink bars) and DR (Dark Blue and Light blue bars) under uninduced (Red and Dark blue bars) and steroid induced (Pink and Light blue bar) conditions. Expression levels were normalized to *Actin5c*. Values are mean ± SD, n = 6. p Values were calculated using unpaired t test with Welch’s correction. Genotypes of strains used in this figure: (**A, B, F, H, J-M**) *3X ElavGS >UAS chinmo: P{elav-Switch.O}GS −1A / +; P{elav-Switch.O}GS-3A, P{elav-Switch.O} GSG301/P{w+, UAS-chin::SV40}/+;* (**D, G,I**) *3X ElavGS >UAS Flag chinmo: P{elav-Switch.O}GS −1A / +; P{elav-Switch.O}GS-3A,P{elav-Switch.O} GSG301/P{w+, UAS Flag-chin::SV40} attP2 / +;* (C) *FBGS >UAS chinmo: w¹¹¹⁸; P {w [+mW.hs]=Switch1}106/+; P{w+, UAS-chin::SV40}/+;* (E) *FBGS >UAS Flag-chinmo: w¹¹¹⁸; P{w[+mW.hs]=Switch1} 106/+; P{w+, UAS Flag-chin::SV40} attP2 / +.*

Figure 6—source data 1 Lifespan analysis of strains used in Figure 6.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig6-data1-v2.docx
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Figure 6—source data 2 Survival proportions of lifespan curves of strains used in Figure 6B–E.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig6-data2-v2.xlsx
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Figure 6—figure supplement 1

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Lifespan and molecular analysis of strains used in Figure 6.

(**A–C**) Effect of diet and RU-486 on lifespan of *3X ElavGS X w¹¹¹⁸* (A), *UAS chinmo X w¹¹¹⁸* (B) and *UAS Flag chinmo X w¹¹¹⁸* (C) flies. (A) Crosses were established between *3X ElavGS* and *w¹¹¹⁸* and the progeny was sorted into four groups (AL-, DR-, AL+ and DR+). Median lifespan in: AL- is 33 days; AL + is 40 days: DR- is 65 days and DR +is 61 days. (B) Crosses were established between *UAS chinmo* and *w¹¹¹⁸* and the progeny was sorted into four groups (AL-, DR-, AL+ and DR+). Median lifespan in: AL- is 24 days; AL + is 20 days: DR- is 34 days and DR +is 38 days. (C) Crosses were established between *UAS Flag chinmo* and *w¹¹¹⁸* and the progeny was sorted into four groups (AL-, DR-, AL+ and DR+). Median lifespan in: AL- is 26 days; AL + is 30 days: DR- is 38 days and DR +is 38 days. For statistical comparison of survival curves, p values and Χ² were calculated with log rank test. (D) Over expression of chinmo in the adult fat tissue reduces TAG levels. *UAS chinmo* was expressed in adult fat body using the drug (RU-486)-inducible gene switch Gal4 driver. Female flies that were fed an RU-486 supplemented diet for 5 days displayed increased levels of Chinmo in fat body cells, as detected by Chinmo, Woc (nuclear marker), and Dapi staining of dissected fat body. (E) Quantitation of stored triglyceride (TAG) levels in -RU-486 and +RU-486 fed 20d old *FBGS >UAS chinmo* flies. (F) Fat bodies/abdomens of female flies were dissected and stained for the content and diameter of lipid droplet (LD) (red are lipid droplets stained with Nile red and blue is Dapi). Scale bar, 25 μm. (G) Quantitation of lipid droplet (LD) diameter in (F). Quantitation of 15 largest LDs in five samples per condition. Error bars represent mean ± SD. (**H, I**) Representative nile red images of fat bodies of (H) *ElavGS >UAS chinmo* and (I) *ElavGS >UAS Flag chinmo* adult female flies stained with Nile red and Dapi. (J) Schematic displaying systemic reduction in TAG levels by ectopic expression of Chinmo using *ElavGS* or *FBGS* Gal4 drivers. Genotypes of strains used in this figure: (A) *ElavGS/+; ElavGS, ElavGS/+: P{elav-Switch.O}GS −1A / +; P{elav-Switch.O}GS-3A, P{elav-Switch.O} GSG301/+;* (B) *+/+; UAS chinmo/+: w¹¹¹⁸; +/+; P{w+, UAS-chin::SV40}/+*; (C)*+/+; UAS Flag chinmo/+: w¹¹¹⁸; +/+; P{w+, UAS-Flag chin::SV40} attP2 /+*; (**D-G**) *FBGS >UAS chinmo: w¹¹¹⁸; P{w[+mW.hs]=Switch1}106/+; P{w+, UAS-chin::SV40}/+*; (H) *3XElavGS > UAS chinmo: P{elav-Switch.O}GS-1A/ +; P{elav-Switch.O}GS-3A, P{elav-Switch.O} GSG301/P{w+, UAS-chin::SV40}/+*; (I) *3XElavGS > UAS Flag chinmo: P{elav-Switch.O}GS −1A / +; P{elav-Switch.O}GS-3A,P{elav-Switch.O} GSG301/P{w+, UAS-Flag chin::SV40} attP2 / +.*

Figure 6—figure supplement 1—source data 1 Lifespan analysis of strains used in Figure 6—figure supplement 1A–C.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig6-figsupp1-data1-v2.docx
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Figure 6—figure supplement 2

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Non-autonomous expression of Chinmo in abdominal fat tissue of *3XElavGS > UAS Chinmo* flies.

(**A–H and A’–H’**) To verify specificity of the gene switch GAL4 drivers, *UAS GFP* was expressed in a steroid-inducible manner with *3X Elav-GS* (**A-H**) or *FB-GS* (**A’-H’**). Confocal images of dissected *Drosophila* adult brain and fat body from fruit flies that were fed an AL diet (**A, B, E, F, A’, B’, E’, F’**) or DR diet (**C, D, G, H, C’, D’, G’, H’**) under uninduced (**A-D, A’-D’**) and steroid-induced (**E-H, E’-H’**) conditions. *3x ElavGS >UAS GFP* flies that were fed RU-486 for 5 days displayed increased levels of Chinmo in the brain and neurons innervating the adult fat tissue as detected by GFP staining of dissected brains and fat body (**E, F, G, H**) compared to flies that were not fed RU-486 (**A, B, C, D**). In contrast, *3x ElavGS >UAS chinmo* flies that were fed RU-486 for 5 days displayed increased levels of Chinmo in the adult fat tissue (**N, P**), in addition to the adult brain (**M, O**) compared to flies that were not fed RU-486 (**I-L**) *FB GS >UAS GFP* and *FB GS >UAS chinmo* flies that were fed RU-486 for 5 days displayed increased levels of GFP (**F’, H’**) and Chinmo in the fat tissue (**N’, P’**), respectively. Scale bar, 50 μm. Genotypes of strains used in this figure: (**A-H**) *3x ElavGS >UAS GFP: P{elav-Switch.O}GS-1A/P{y[+t7.7] w[+mC]=10XUAS-IVS-myr::GFP}attP40; P{elav-Switch.O}GS-3A, P{elav-Switch.O}GSG301 /+;* (**I-P**) *3x ElavGS >UAS chinmo: P{elav-Switch.O}GS-1A / +; P{elav-Switch.O}GS-3A,P{elav-Switch.O}GSG301/P{w+,UAS-chin::SV40}/+;* (**A’-H’**) *FBGS >UASGFP:w¹¹¹⁸; P{w[+mW.hs]=Switch1} 106/P{y[+t7.7] w[+mC]=10XUAS-IVS-myr::GFP}attP40; +/+*: (**I’-P’**) *FB GS >UAS chinmo: w¹¹¹⁸; P {w [+mW.hs]=Switch1}106/+; P{w+, UAS-chin::SV40}/+*.

Figure 6—figure supplement 3

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Non-autonomous expression of Chinmo in abdominal fat tissue of *3XElavGS > UAS Flag chinmo* flies.

(A) Confocal images of dissected *Drosophila* adult brain from fruit flies that were fed an AL diet (*top two panels*) or DR (*bottom two panels*) diet under uninduced (AL-, DR-) and steroid-induced (AL+, DR+) conditions. *3x ElavGS >UAS Flag chinmo* flies that were fed RU-486 for 10 days displayed increased levels of Chinmo in the brain tissue as detected by anti-flag and anti-chinmo staining of dissected brains compared to flies that were not fed RU-486. (**B, C**) *3x ElavGS >UAS chinmo* flies that were fed RU-486 for 10 days also displayed increased levels of Chinmo in the adult fat tissue (AL+, DR+), in addition to the adult brain compared to flies that were not fed RU-486 (AL-, DR-). Scale bar, 25 μm. (**D, E**) Quantitation of Chinmo protein levels as detected by staining with anti-Flag and anti-chinmo antibody in adult fat tissue nuclei as determined by measuring pixel intensity in 10 cells in five samples per condition. Error bars represent mean ± SD and p values were calculated by unpaired t test with Welch’s correction. Genotypes of strains used in this figure: (**A-E**) *3x ElavGS >UAS Flag chinmo: P{elav-Switch.O}GS −1A / +; P{elav-Switch.O}GS-3A, P{elav-Switch.O} GSG301/P{w+, UAS-Flag chin::SV40} attP2/+.*

Figure 7 with 3 supplements

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Ectopic expression of *chinmo* in adult neurons represses transcription of genes involved in fat metabolic processes.

(A) Quantitation of *chinmo* mRNA levels in RNA extracted from *3X ElavGS >UAS chinmo* flies that were fed a solvent (light blue bars) or RU-486 (pink bars) diet for 5 and 10 days. (**B, C**) Quantitation of *chinmo* mRNA levels in RNA extracted from head tissue (B) and decapitated body tissue (C) of *3X ElavGS >UAS chinmo* flies that were fed ethanol (blue bar) or RU-486 (pink bar) diet for 10 days. Expression levels were normalized to *Actin5c*. Values are mean ± SD, n = 3. (D) Venn diagram of 1175 proteins identified across the two groups (-RU-486 and +RU-486) and 91% were common between the two groups. (E) Volcano plot illustrating significantly differentially abundant proteins. Forty proteins were found to be differentially expressed by using a cutoff on p value ≤ 0.05 and log₂FC ≥ 1 (7 upregulated) and ≤−1.0 (33 downregulated) proteins. (F) Twenty-five most significant biological processes that are downregulated upon overexpression of *chinmo* in adult neurons. See Figure 7—figure supplement 2 for upregulated biological processes. (**G, H**) Overexpression of *chinmo* in the adult nervous system downregulates genes involved in fat metabolism. RT-PCR quantitation of fold- change in mRNA levels of genes (F) involved in fat metabolism in the head tissue (G) and decapitated body tissue (H) of *3X ElavGS >UAS chinmo* flies that were fed the solvent/ethanol (light blue bars) or RU-486 (pink bars) diet for 10 days. (I) Knockdown of *fasn1* in the adult fat body reduces lifespan more significantly in flies that are fed an AL (compare red solid and dotted lines) compared to flies that are fed a DR diet (compare blue solid and blue dotted lines) conditions (See Figure 7—source data 1 for p values and median and maximum lifespan for additional experimental replicate). (J) Knockdown of *fatp* in the adult fat body reduces lifespan under DR (compare blue solid and blue dotted lines) conditions (See Figure 7—source data 2 for p values and median and maximum lifespan for additional experimental replicate). (K) Overexpression of *Flag FATP* increases lifespan in flies that are fed a DR diet (Compare blue dotted line with blue solid line) more significantly than flies that are fed an AL diet (See Figure 7—source data 3 for p values and median and maximum lifespan for additional experimental replicate). For statistical comparison of survival curves, p values and χ² were calculated with log-rank test. Genotypes of strains used in this figure: (**A-H**) *3X ElavGS >UAS chinmo: P{elav-Switch.O}GS −1A / +; P{elav-Switch.O}GS-3A, P{elav-Switch.O} GSG301/P{w+, UAS-chin::SV40}/+*; (I) *FB GS/+; UAS fasn1^RNAi/+: w¹¹¹⁸; P{w[+mW.hs]=Switch1}106/+; P{y[+t7.7] v[+t1.8]=TRiP.HMS01524}attP2/+;* (J) *FB GS/+; UAS fatp^RNAi/+:w[1118]; P{w[+mW.hs]=Switch1}106/+; P{y[+t7.7] v[+t1.8]=TRiP.HMC04206}attP2/+;* (K) *FB GS/+; UAS Flag fatp/+: w[1118]; P{w[+mW.hs]=Switch1}106/+;P{w+, UAS-Flag fatp} attP2/+*.

Figure 7—source data 1 Lifespan analysis of FBGS >UAS fasn1^RNAi and +/+; UAS fasn1^RNAi strains.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig7-data1-v2.docx
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Figure 7—source data 2 Lifespan analysis of FBGS >UAS fatp^RNAi and +/+; UAS fatp^RNAi strains.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig7-data2-v2.docx
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Figure 7—source data 3 Lifespan analysis of FBGS >UAS Flag fatp and +/+; UAS Flag fatp strains.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig7-data3-v2.docx
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Figure 7—source data 4 Survival proportions of lifespan curves of strains used in Figure 7I–K.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig7-data4-v2.xlsx
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Figure 7—source data 5 Upregulated biological processes from proteomic analysis in Figure 7D–F.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig7-data5-v2.xlsx
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Figure 7—source data 6 Downregulated biological processes from proteomic analysis in Figure 7D–F.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig7-data6-v2.xlsx
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Figure 7—figure supplement 1

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Overexpression of *chinmo* in adult neurons represses transcription of genes involved in fat metabolic processes in the brain within 2 days of induction.

(**A, B**) Quantitation of *chinmo* mRNA levels in RNA extracted from head and decapitated body tissue of *3X ElavGS >UAS chinmo* flies that were fed a solvent (light blue bars) or RU-486 (pink bars) diet for 2 days. (C) Female flies that were fed an RU-486 supplemented diet for 2 days displayed increased levels of Chinmo in neuronal cells, as detected by Chinmo, Woc (nuclear marker), and Dapi staining of dissected adult fly brains. (**D, E**) Overexpression of *chinmo* in the adult nervous system downregulates genes involved in fat metabolism. RT-PCR Quantitation of fold change in mRNA levels of genes involved in fat metabolism in the head tissue (D) and decapitated body tissue (E) of *3X ElavGS >UAS chinmo* flies that were fed a solvent (light blue bars) or RU-486 (pink bars) diet for 2 days. (D) Induction of Chinmo for 2 days was sufficient to lead a statistically significant reduction of seven of the eight genes analyzed in the head tissue. (E) 3 out of 8 genes analyzed also displayed a statistically significant albeit lower reduction in the decapitated body tissue. Expression levels were normalized to *Actin5c*. Values are mean ± SD, n ≥ 3. p Values were calculated by using unpaired t test with Welch’s correction. Genotypes of strains used in this figure: (**A-E**) *3XElavGS > UAS chinmo: P{elav-Switch.O}GS −1A / +; P{elav-Switch.O}GS-3A, P{elav-Switch.O} GSG301/P {w+, UAS-chin::SV40}/+*.

Figure 7—figure supplement 2

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Over expression of *chinmo* in adult neurons upregulates cytoplasmic processes.

Twenty-five most significant biological processes that are upregulated upon overexpression of *chinmo* in adult neurons.

Figure 7—figure supplement 3

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Molecular and survival analysis of strains utilized in Figure 7.

(A) Overexpression of flag Chinmo in the adult brain neurons leads to a reduction in Fatp protein. Western blot analysis of adult fly lysates from 3X ElavGS >*UAS Flag chinmo* flies that were fed an AL diet in presence (+AL) or absence (AL-) of RU-486. The whole fly cell lysates were analyzed by western blotting with Flag (Flag Chinmo), Fatp and Tubulin (normalization control) antibodies. Fatp levels reduced upon overexpression of Flag chinmo. (B) Quantitative RT-PCR of *fasn1* from abdominal tissue of *FB GS >UAS fasn1^RNAi* flies in presence of RU-486 (light red and light blue) or in absence of RU-486 (bars with solid red and blue color) flies that were fed *Ad libitum* (AL) (red) or DR diet (blue) for 10 days. Expression levels were normalized to *Actin*. Values are mean ± SD, n = 6. (C) Quantitative RT-PCR of *fatp* from abdominal tissue of *FB GS >UAS fatp^RNAi* flies in presence of RU-486 (light red and light blue) or in absence of RU-486 (bars with solid red and blue color) flies that were fed Ad libitum (AL) (red) or DR diet (blue) for 10 days. Expression levels were normalized to *Actin*. Values are mean ± SD, n = 6. *Fatp* is more significantly knocked down under DR conditions. (D) Western blot of whole fly lysates from FB GS/+; UAS Flag fatp/+flies that were fed an AL and DR diet in presence (AL+, DR+) and absence (-AL, -DR) of RU-486. The whole fly cell lysates were analyzed by western blotting with Flag (Flag Fatp), Fatp, and Tubulin (normalization control) antibodies. Flag Fatp was detected in AL + and DR +lysates. (**E-G**) Effect of RU-486 on the lifespan of *UAS fasn^RNAi*, *UAS fatp^RNAi*, and *UAS Flag fatp* flies. (E) Crosses were established between *UAS fasn^RNAi* and *w¹¹¹⁸* and the progeny was sorted into four groups (AL-, DR-, AL +, and DR+). Median lifespan in: AL- is 28 days; AL + is 26 days: DR- is 50 days and DR +is 46 days. (F) Crosses were established between *UAS fatp^RNAi* and *w¹¹¹⁸* and the progeny was sorted into four groups (AL-, DR-, AL +, and DR+). Median lifespan in: AL- is 36 days; AL + is 32 days: DR- is 38 days and DR +is 42 days. (G) Crosses were established between *UAS Flag fatp* and *w¹¹¹⁸* and the progeny was sorted into four groups (AL-, DR-, AL + and DR+). Median lifespan in: AL- is 26 days; AL + is 32 days: DR- is 46 days and DR +is 48 days. For statistical comparison of survival curves, p values and Χ² were calculated with log-rank test. Genotypes of strains used in this figure: (A) *3X ElavGS >UAS Flag chinmo: P{elav-Switch.O}GS −1A / +; P{elav-Switch.O}GS-3A, P{elav-Switch.O} GSG301/P{w+, UAS-chin::SV40}/+;* (B) *FBGS /+;UASfasn^RNAi/+: w¹¹¹⁸; P{w [+mW.hs]=Switch1}106/+;P{y[+t7.7]v[+t1.8]=TRiP.HMS01524}attP2/+;*(C) *FBGS/+;UASfatp^RNAi/+:w[1118];P{w[+mW.hs]=Switch1}106/+;P{y[+t7.7]v[+t1.8]=TRiP.HMC04206}attP2/+;* (D) *FBGS/+; UAS Flag fatp/+; w[1118]; P{w [+mW.hs]=Switch1}106/+;P{w+,UAS-Flag fatp}attP2/+;* (E) *UASfasn1^RNAi/+: w¹¹¹⁸; +/+; P {y[+t7.7]v [+t1.8]=TRiP. HMS01524} attP2/+;* (F) *UAS fatp^RNAi/+:w¹¹¹⁸;+/+;P{y[+t7.7]v[+t1.8]=TRiP. HMC04206} attP2/+;* (G) *UAS Flag fatp/+: w¹¹¹⁸;+/+; P{w+, UAS-Flag fatp} attP2 / +*.

Figure 7—figure supplement 3—source data 1 Uncropped western blots with relevant bands labeled for Figure 7—figure supplement 3A and D.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig7-figsupp3-data1-v2.docx
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Figure 7—figure supplement 3—source data 2 Original image files of the unedited for western blots in Figure 7—figure supplement 3A.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig7-figsupp3-data2-v2.zip
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Figure 7—figure supplement 3—source data 3 Original image files of the unedited for western blots in Figure 7—figure supplement 3D.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig7-figsupp3-data3-v2.zip
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Figure 7—figure supplement 3—source data 4 Survival proportions of lifespan curves of strains used in Figure 7—figure supplement 3E–G.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig7-figsupp3-data4-v2.xlsx
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Figure 8 with 1 supplement

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Overexpression of human primary *miR-125b-1* in the adult fat tissue promotes longevity and increases TAG levels under both AL and DR conditions.

(A) Schematic of *Drosophila melanogaster* primary *miR-125* (*pri miR-125*) transcript and the *Homo sapiens* primary *miR-125b-1* (*pri hsa miR-125b-1*) indicating that the processed miRNA sequence in fruit flies and humans is identical. (B) Quantitative RT-PCR of *miR-125* from abdominal tissue of *FB GS >UAS chinmo* flies in presence of RU-486 (bars with red and blue pattern) or in absence of RU-486 (bars with solid red and blue color) in flies that were fed Ad libitum (AL) (red) or DR diet (blue) for 20 days. Expression levels were normalized to SnoR442. Values are mean ± SD, n = 6. (C) Overexpression of *hsa pri miR-125* increases lifespan in flies that are fed an AL or DR diet (Compare red and blue dotted lines with red and blue solid line) (See Figure 8—source data 1A–B for number of flies and median and maximum lifespan). The p values and Χ² values indicate that the increase in lifespan is greater under DR conditions. (D) *FB GS >UAS hsa pri miR-125b-1* flies store increased levels of triglycerides (TAG) when fed an AL or DR diet. Quantitation of TAG stored levels in -RU-486 and +RU-486 fed 20 d old *FBGS >UAS hsa pri miR-125b-1 flies*. (E) Fat bodies/abdomens of female flies were dissected and stained for the content and diameter of lipid droplet (LD) (red are lipid droplets stained with Nile red and blue is Dapi). Scale bar, 25 μm. (F) Quantitation of lipid droplet (LD) diameter in (E). Quantitation of 15 largest LDs in five samples per condition. Error bars represent ± SD. (**G, H**) Overexpression of *hsa pri miR-125* upregulates *fasn1* but not *fatp* under AL conditions. RT-PCR quantitation of mRNA levels of *fasn1* (G) and *fatp* (H) in the fat tissue of *FB GS >UAS hsa pri mir-125* flies that were fed an AL (red and pink bars) or DR diet (dark blue and light blue) under uninduced (red and dark blue bars) and induced (pink and light blue bars) conditions for 20 days. Expression levels were normalized to *Actin5c*. Values are mean ± SD, n ≥ 6. Genotypes of strains used in this figure: (**B-H**) *FBGS/+; UAS has pri miR-125b-1: w¹¹¹⁸; P{w[+mW.hs]=Switch1}106/+; P{v+, UAS-hsa miR-125b-1} VK00033/+*.

Figure 8—source data 1 Lifespan analysis of FBGS >UAS hsa pri miR-125b-1 strain.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig8-data1-v2.docx
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Figure 8—source data 2 Survival proportions of lifespan curves of strains used in Figure 8C.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig8-data2-v2.xlsx
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Figure 8—figure supplement 1

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Effect of RU on lifespan of *UAS pri hsa miR-125b-1>*w¹¹¹⁸ flies.

To test if RU486 affects lifespan of flies expressing the *UAS pri hsa miR-125b-1* transgene. Median lifespan in: AL- is 30 days; AL + is 28 days: DR- is 52 days and DR +is 46 days. Please refer to Figure 8—figure supplement 1—source data 1 for the second replicate of this experiment. Genotype of strain used in this Figure: *+/+; UAS pri has miR-125b-1: w¹¹¹⁸; +/+; P{v+, UAS-hsa miR-125b-1} VK00033/+*.

Figure 8—figure supplement 1—source data 1 Lifespan analysis of UAS has pri miR-125b-1/+ strain.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig8-figsupp1-data1-v2.docx
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Figure 8—figure supplement 1—source data 2 Survival proportions of lifespan curve of strain used in Figure 8—figure supplement 1.: https://cdn.elifesciences.org/articles/62621/elife-62621-fig8-figsupp1-data2-v2.xlsx
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Figure 9

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*miR-125* regulates DR-dependent lifespan extension by post-transcriptionally silencing *chinmo*.

Proposed model summarizing the mechanism by which *miR-125* and *chinmo* regulate lifespan extension by DR. *miR-125* targets *chinmo* mRNA in the brain under AL and DR conditions. In the adult fat tissue, Chinmo transcriptionally represses genes involved in fat metabolism. DR-mediated cytoplasmic relocalization of Chinmo in the fat tissue relieves transcriptional repression of genes involved in fat metabolism, thus increasing lifespan.

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Chinmo is silenced by let-7 and miR-125 in a biphasic manner.

(A) Schematic of a 1.4kb Chinmo 3’UTR fragment indicating number and arrangement of let-7 and miR-125 binding sites. (B) *Chinmo* derepression is significantly higher in let-7 mutant brains dissected from staged pupae (24h and 96h after puparium formation)(red dashed lines indicates the stage at which let-7 represses *chinmo*) (C) *Chinmo* is predominantly regulated by *miR-125* in adult flies as the degree of depression of *chinmo* in *ΔmiR-125* flies is more widespread and higher in *miR-125* mutants than in *Δlet-7* adult fly brains (Day-3 after eclosion)(red dashed lines indicates late pupal stage).

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miR-125 is the predominant miRNA that silences chinmo in adult flies.

(A-D) Confocal images of 3d adult brains immunostained for Chinmo (green). No Chinmo expression was detected in brains of flies harboring either the wild type of the ΔmiR-100 transgene (panels A and B). The level of Chinmo expression in ΔmiR-125 mutants is much higher than in Δlet-7 mutant adult flies (compare panels C and D).

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Antibody	Anti-GFP(chicken)	Thermo Fisher Scientific	A10262, RRID:AB_2534023	1:2500/1:1000
Antibody	Anti-Flag(mouse)	Sigma-Aldrich	F-1804, RRID:AB_262044	1: 2500
Antibody	Anti-Fatp(rabbit)	Gift from B. Mollereau Dourlen et al., 2012		1:200
Antibody	Anti-acetyl lysine	Cell Signaling	#9441; RRID:AB_331805	1:750
Antibody	Anti-chinmo (rat)	Gift from Nick Sokol (Wu et al., 2012)		1:1000
Antibody	Anti-Woc(rabbit)	Gift from Maurizio Gatti (Raffa et al., 2005)		1:1000
Antibody	Anti-La(rabbit)	Gift from S.L. Wolin (Yoo and Wolin, 1994)		1:1000
Antibody	Anti-tubulin(mouse)	Sigma-Aldrich	T9026, RRID:AB_477593	1:4000
Antibody	Anti-dCsp1(mouse)	DSHB	DSHB Cat# ab49, RRID:AB_2307340	1:1000
Antibody	Anti-Syntaxin 1A (mouse)	DSHB	DSHB Cat# 8C3, RRID:AB_528484	1:500
Antibody	Anti-HrsFL(Guinea Pig)	Gift from Hugo Bellen		1:2000
Antibody	Anti-mouse HRP	Jackson Immuno Research, Laboratories, Inc	115-035-003, RRID:AB_10015289	1: 2500
Antibody	Anti-chicken HRP	Sigma-Aldrich	A9046, RRID:AB_258432	1: 2500
Antibody	Anti-Rabbit HRP	Jackson Immuno Research, Laboratories, Inc	111-035-003, RRID:AB_2313567	1: 2500
Antibody	Alexa Fluor 488 Affinipure Donkey anti-chicken IgY	Jackson Immuno Research, Laboratories, Inc	703-545-155, RRID:AB_2340375	1:1000
Antibody	Alexa Fluor 488 Affinipure Donkey anti-Rat IgG	Jackson Immuno Research, Laboratories, Inc	712-545-153, RRID:AB_2340684	1:1000
Antibody	Alexa Fluor 488 Affinipure Donkey anti-Mouse IgG	Jackson Immuno Research, Laboratories, Inc	712-545-151; RRID:AB_2340846	1:1000
Antibody	Goat-anti-Rabbit IgG, Alexa Fluor 568	Thermo Fisher Scientific	A-11036, RRID:AB_10563566	1:1000
Antibody	Alexa Fluor 568 Goat-anti-Mouse IgG	Thermo Fisher Scientific	A-11031, RRID:AB_144696	1:1000
Chemical compound, drug	Nile Red	Sigma-Aldrich	72485	1:250
Other	Normal goat serum	Jackson Immuno Research, Laboratories, Inc	005-000-121, RRID:AB_2336990
Chemical compound, drug	Deacetylase inhibitor cocktail	ApexBio	K1017	1:100
Chemical compound, drug	Phospho Safe Extraction reagent	Merck	71296
Other	Anti-Flag M2-Affinity gel	Sigma-Aldrich	A2220, RRID:AB_10063035	Resin
Peptide, recombinant protein	Flag peptide	Sigma-Aldrich	F4799
Chemical compound, drug	SYBR Premix Ex Taq	Takara Bio	RR420A
Commercial assay or kit	Taqman Fast advanced mix	Thermo Fisher Scientific	4444557
Sequence-based reagent	Taqman microRNA assay for miR-100	Thermo Fisher Scientific	Assay ID 000287
Sequence-based reagent	Taqman probes for let-7–5 p	Thermo Fisher Scientific	Assay ID 000332
Sequence-based reagent	Taqman probes for miR-125–5 p	Thermo Fisher Scientific	Assay ID 000449
Sequence-based reagent	Taqman probes for Sno446	Thermo Fisher Scientific	Assay ID 001742
Sequence-based reagent	Taqman probes for 2S rRNA	ThermFisher Scientific	Assay ID 001766
Other	HyClone CCM3 cell culture media	Cytiva	SH30061.01
Chemical compound,drug	RNAiso Plus	Takara Bio	9109
Chemical compound, drug	Phenol: Chloroform	Sigma-Aldrich	P1944
Transfection construct	Lipofectamine 3000 Transfection Reagent	Thermo Fisher Scientific	L3000015
Other	DNAse I (RNAse-free)	New England BioLabs, Inc	NEB Catalog #: M0303	Enzyme
Commercial assay or kit	Triglyceride reagent	Sigma-Aldrich	T2449
Commercial assay or kit	Free Glycerol reagent	Sigma-Aldrich	F6428
Recombinant DNA reagent	TopoXL cloning kit	Thermo Fisher Scientific	K804010
Commercial assay or kit	High-Capacity Reverse Transcription assay kit	Thermo Fisher Scientific	4368814
Other	SUPERase In RNAse Inhibitor	Thermo Fisher Scientific	AM2696	RNAase inhibitor
Other	dNTP set 100 mM solutions	Thermo Fisher Scientific	R0181	dNTPs
Other	Superscript IV Reverse Transcriptase	Thermo Fisher Scientific	18090050	Enzyme
Other	Phusion High-Fidelity DNA Polymerase	New England BioLabs, Inc	NEB Catalog# M0530S	Enzyme
Recombinant DNA reagent	Quick Ligation Kit	New England BioLabs, Inc	NEB Catalog# M2200L
Genetic reagent (D. melanogaster)	w¹¹¹⁸	Bloomington Drosophila Stock Centre
Genetic reagent (D. melanogaster)	let-7-C^GKI	Sokol et al., 2008
Genetic reagent (D. melanogaster)	let-7-C^KO2	Wu et al., 2012
Genetic reagent (D. melanogaster)	chinmo¹	Zhu et al., 2006
Genetic reagent (D. melanogaster)	P{w+, UAS-Chin::SV40}	Chawla et al., 2016
Genetic reagent (D. melanogaster)	P{w+, UAS-chinmo^{RNAi 148}} VK00033	Chawla et al., 2016
Genetic reagent (D. melanogaster)	P{w+, UAS-hsa miR-125b-1} VK00033
Genetic reagent (D. melanogaster)	P{v+, let-7-C ^ΔmiR-100}attP2	Chawla et al., 2016
Genetic reagent (D. melanogaster)	P{v+, let-7-C ^Δlet-7}attP2	Chawla et al., 2016
Genetic reagent (D. melanogaster)	P{v+, let-7-C^ΔmiR-125}attP2	Chawla et al., 2016
Genetic reagent (D. melanogaster)	P{w+, let-7-Cp^3.3kb::cDNA}VK00033	Chawla and Sokol, 2012
Genetic reagent (D. melanogaster)	3XelavGS	Kind gift from Scott Pletcher
Genetic reagent (D. melanogaster)	P{w+, UAS-FASN1^RNAi-1}attP2	Bloomington Stock 35775
Genetic reagent (D. melanogaster)	P{w+, UAS-FATP^RNAi}attP2	Bloomington Stock 55919
Genetic reagent (D. melanogaster)	P{w+, UAS-Flag FATP::SV40}	This study (Chawla Lab)		G. Chawla (Regional Centre for Biotechnology)
Genetic reagent (D. melanogaster)	P{w+, UAS-Flag Chinmo::SV40}	This study (Chawla Lab)		G. Chawla (Regional Centre for Biotechnology)
Genetic reagent (D. melanogaster)	FB-GS	Bloomington Stock 8151
Genetic reagent (D. melanogaster)	5966 (Gut specific GS)	Kind gift from David Walker
Genetic reagent (D. melanogaster)	Da GS	Kind gift from David Walker
Genetic reagent (D. melanogaster)	UAS GFP	Bloomington Stock 32198
Sequence-based reagent	Primers for RT-PCRs and cloning, see Supplementary file 2	This study (Chawla Lab)		G. Chawla (Regional Centre for Biotechnology)
Recombinant DNA reagent	UAS Flag Chinmo plasmid	This study (Chawla Lab)		G. Chawla (Regional Centre for Biotechnology)
Recombinant DNA reagent	UAS GFP Chinmo plasmid	This study (Chawla Lab)		G. Chawla (Regional Centre for Biotechnology)
Software, algorithm	OASIS	Han et al., 2016	https://sbi.postech.ac.kr/oasis/
Software, algorithm	GraphPad Prism	GraphPad	RRID:SCR_000306
Software, algorithm	GPS-PAIL 2	Deng et al., 2016, Li et al., 2006	http://pail.biocuckoo.org

Additional files

Supplementary file 1 Genotypes used in the study.: https://cdn.elifesciences.org/articles/62621/elife-62621-supp1-v2.docx
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Supplementary file 2 Primers used in the study.: https://cdn.elifesciences.org/articles/62621/elife-62621-supp2-v2.docx
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Supplementary file 3 Downregulated biological process.: https://cdn.elifesciences.org/articles/62621/elife-62621-supp3-v2.xlsx
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Supplementary file 4 Upregulated biological process.: https://cdn.elifesciences.org/articles/62621/elife-62621-supp4-v2.xlsx
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Transparent reporting form: https://cdn.elifesciences.org/articles/62621/elife-62621-transrepform-v2.docx
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Manish Pandey
Sakshi Bansal
Sudipta Bar
Amit Kumar Yadav
Nicholas S Sokol
Jason M Tennessen
Pankaj Kapahi
Geetanjali Chawla

(2021)

miR-125-chinmo pathway regulates dietary restriction-dependent enhancement of lifespan in Drosophila

eLife 10:e62621.

https://doi.org/10.7554/eLife.62621

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let-7 and miR-125 are required for DR-dependent enhancement of lifespan.

Figure 1—source data 1

Figure 1—source data 2

Scheme for generation of experimental strains used in Figures 1 and 2.

Lifespan analysis controls and expression analysis of strains used in Figure 1.

Figure 1—figure supplement 2—source data 1

Figure 1—figure supplement 2—source data 2

Reducing chinmo (ΔmiR-125, chinmoRNAi and ΔmiR-125, chinmo1) levels in miR-125 mutants suppresses the loss of DR-mediated increase in lifespan extension and decrease in triglyceride levels.

Figure 2—source data 1

Figure 2—source data 2

Figure 2—source data 3

Effect of genetic background and/or RU-486 in UAS chinmoRNAi/+ and chinmo1/+ strains used for analysis in Figure 2.

Figure 2—figure supplement 1—source data 1

Figure 2—figure supplement 1—source data 2

Chinmo is nutrient-dependent downstream effector of the DR-pathway.

Figure 3—source data 1

Figure 3—source data 2

Nutrient-dependent regulation of Chinmo in the adult fat tissue.

Export of Chinmo from the nucleus is dependent on deacetylation of Chinmo by dSir2.

Figure 4—source data 1

Figure 4—source data 2

Figure 4—source data 3

Figure 4—source data 4

Figure 4—source data 5

Evaluation of predicted acetylated lysine residues in Chinmo.

Figure 4—figure supplement 1—source data 1

Figure 4—figure supplement 1—source data 2

Chinmo protein and mRNA are upregulated in adult and larval fat tissue upon starvation.

Chinmo co-fractionates with exosomal proteins upon nutrient deprivation.

Figure 5—source data 1

Figure 5—source data 2

Elution of vesicles using gel filtration columns.

Figure 5—figure supplement 1—source data 1

Figure 5—figure supplement 1—source data 2

Ectopic expression of chinmo in adult neurons and fat body mimics miR-125 mutant DR phenotype.

Figure 6—source data 1

Figure 6—source data 2

Lifespan and molecular analysis of strains used in Figure 6.

Figure 6—figure supplement 1—source data 1

Non-autonomous expression of Chinmo in abdominal fat tissue of 3XElavGS > UAS Chinmo flies.

Non-autonomous expression of Chinmo in abdominal fat tissue of 3XElavGS > UAS Flag chinmo flies.

Ectopic expression of chinmo in adult neurons represses transcription of genes involved in fat metabolic processes.

Figure 7—source data 1

Figure 7—source data 2

Figure 7—source data 3

Figure 7—source data 4

Figure 7—source data 5

Figure 7—source data 6

Overexpression of chinmo in adult neurons represses transcription of genes involved in fat metabolic processes in the brain within 2 days of induction.

Over expression of chinmo in adult neurons upregulates cytoplasmic processes.

Molecular and survival analysis of strains utilized in Figure 7.

Figure 7—figure supplement 3—source data 1

Figure 7—figure supplement 3—source data 2

Figure 7—figure supplement 3—source data 3

Figure 7—figure supplement 3—source data 4

Overexpression of human primary miR-125b-1 in the adult fat tissue promotes longevity and increases TAG levels under both AL and DR conditions.

Figure 8—source data 1

Figure 8—source data 2

Effect of RU on lifespan of UAS pri hsa miR-125b-1>w1118 flies.

Figure 8—figure supplement 1—source data 1

Figure 8—figure supplement 1—source data 2

miR-125 regulates DR-dependent lifespan extension by post-transcriptionally silencing chinmo.

Chinmo is silenced by let-7 and miR-125 in a biphasic manner.

miR-125 is the predominant miRNA that silences chinmo in adult flies.

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

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Reducing chinmo (ΔmiR-125, chinmo^RNAi and ΔmiR-125, chinmo¹) levels in miR-125 mutants suppresses the loss of DR-mediated increase in lifespan extension and decrease in triglyceride levels.

Effect of genetic background and/or RU-486 in UAS chinmo^RNAi/+ and chinmo¹/+ strains used for analysis in Figure 2.

Effect of RU on lifespan of UAS pri hsa miR-125b-1>w¹¹¹⁸ flies.