Volume 3, Issue 1, Pages 79-91 (January 2013) A Fasting-Responsive Signaling Pathway that Extends Life Span in C. elegans  Masaharu Uno, Sakiko Honjoh, Mitsuhiro Matsuda, Haruka Hoshikawa, Saya Kishimoto, Tomohito Yamamoto, Miki Ebisuya, Takuya Yamamoto, Kunihiro Matsumoto, Eisuke Nishida  Cell Reports  Volume 3, Issue 1, Pages 79-91 (January 2013) DOI: 10.1016/j.celrep.2012.12.018 Copyright © 2013 The Authors Terms and Conditions

Cell Reports 2013 3, 79-91DOI: (10.1016/j.celrep.2012.12.018) Copyright © 2013 The Authors Terms and Conditions

Figure 1 Aging-Related Genes Are Induced Early in Response to Fasting (A) The sampling scheme is shown. At day 2 of adulthood, wild-type N2 worms were transferred to the plate in the presence or absence of food. At the indicated time, worms were collected and total RNAs were extracted. (B) Expression profiles of genes whose expression was upregulated during 48 hr of fasting. Average expression profiles of two independent experiments are shown (upper). Numbers of upregulated genes, whose expression at the indicated time point was upregulated more than 2-fold as compared to 0 hr fed (both in fed and fasting conditions), are shown (lower). (C) Hierarchical clustering analysis for the total fasting-induced upregulated genes. Venn diagram for fasting-upregulated genes is shown. Blue circle or red circle indicates genes upregulated at 9/12 hr or 18/24/36/48 hr of fasting. (D) Top five terms in the GO analysis for 704 upregulated genes at 9 or 12 hr fasting (upper) and 989 upregulated genes after 18 hr fasting, which were not upregulated until 12 hr (lower), respectively, are shown. (E) Promoter analysis for the upregulated genes at 9 or 12 hr fasting. Top five enriched binding sites are shown. V, vertebrate. (F) Survival curves of control RNAi- (upper left, n = 50 [AL] and 66 [IF]), fos-1 RNAi- (upper right, n = 55 [AL] and 54 [IF]), jun-1 RNAi- (lower, left, n = 59 [AL] and 61 [IF]), or fos-1;jun-1 RNAi- (lower right, n = 67 [AL] and 44 [IF]) treated worms in AL and IF. ∗p < 0.05 log rank test (p = 1.2E-20, 1.6E-9, 6.5E-12, and 7.1E-4 in control RNAi-, jun-1 RNAi- or fos-1 RNAi- treated worms, respectively). Mean life-span extension by IF (±SEM) (lower far left) of three independent experiments is shown. ∗p < 0.05 t test. See also Tables S1, S2, and Figure S1. Cell Reports 2013 3, 79-91DOI: (10.1016/j.celrep.2012.12.018) Copyright © 2013 The Authors Terms and Conditions

Figure 2 KGB-1 Is Required for IF-Induced Longevity and Lies Upstream of AP-1(JUN-1/FOS-1) in the Fasting-Stimulus Response (A) Mean life span (±SEM) (left) and mean life-span extension by IF (±SEM) (right) of four independent experiments are shown. ∗p < 0.05 t test. (B) Survival curves of control RNAi- (left) and kgb-1 RNAi- (middle) treated worms in AL and IF are shown. ∗p < 0.05 log rank test (p = 2.0E-13, and 2.1E-5 in control- (n = 34 [AL] and 29 [IF]) and kgb-1-RNAi- (n = 34 [AL] and 45 [IF]) treated worms, respectively). Mean life-span extension by IF (±SEM) of three independent experiments is shown (right). ∗p < 0.05 t test. (C) Activation of KGB-1 in response to fasting. KGB-1 activation was analyzed by immunoblot analysis at 30 and 60 min (upper left), and at 4.5 and 9 hr (lower). Mean KGB-1 activation (phosphoKGB-1/total KGB-1) ±SEM of two independent experiments is shown (upper right). In these experiments, worms at 2 days adult in the presence of food were collected at time 0 and moved into plate with (food, +) and without food (food, −), respectively. The value at time 0 is set to 1. (D) Phosphorylation of JUN-1 and FOS-1 by KGB-1. 293T cells were transfected with Myc-KGB-1 (WT), KGB-1(K67R) (KR), Myc-KGB-1(S198A and Y200F) (ADF), Flag-JUN-1, and HA-FOS-1 as indicated. See also Figure S2 and Table S2. Cell Reports 2013 3, 79-91DOI: (10.1016/j.celrep.2012.12.018) Copyright © 2013 The Authors Terms and Conditions

Figure 3 The KGB-1 JNK Cascade Is Required for IF-Induced Longevity IF-induced increase in life span was suppressed in mek-1(ks54), mlk-1(km19), and nsy-1(ky400) mutants. Survival curves of N2 (upper left, n = 34 [AI] and 29 [IF]), mek-1(ks54) (upper middle, n = 43 [AL] and 43 [IF]), mlk-1(km19) (upper right, n = 44 [AL] and 38 [IF]), nsy-1(ky400) (lower left, n = 43 [AL] and 33 [IF]), mtk-1(km14) (lower middle, n = 49 [AL] and 48 [IF]), and mlk-1;nsy-1 (lower right, n = 29 [AL] and 50 [IF]) in AL and IF are shown. ∗p < 0.05 log rank test (p = 1.9E-19, 5.4E-5, 3.1E-7, 1.1E-6, 7.0E-10, and 3.6E-4 in N2, mek-1(ks54), mlk-1(km19), nsy-1(ky400), mtk-1(km14), and mlk-1;nsy-1, respectively). Mean life span ±SEM (lower left) and mean life-span extension by IF (±SEM) (lower right) of three independent experiments is shown (bottom). ∗p < 0.05 t test. See also Figure S3 and Table S2. Cell Reports 2013 3, 79-91DOI: (10.1016/j.celrep.2012.12.018) Copyright © 2013 The Authors Terms and Conditions

Figure 4 KGB-1-JUN-1/FOS-1 Signaling Regulates Fasting-Induced Upregulation of DAF-16 Target Genes without Affecting Fasting-Induced DAF-16 Nuclear Translocation (A and D) The expression levels of six DAF-16 target genes (aqp-1, dod-6, hil-1, hsp-12.6, mtl-1, and sod-3) in N2 or kgb-1(km21) (A) and in control RNAi-, fos-1 RNAi-, or jun-1 RNAi-treated worms (D) are shown. At day 2 of adulthood, worms were transferred to the plate in the presence (Fed) or absence (fasting) of food. After incubation for 2 days, worms were collected and total RNA was extracted. The expression levels of six DAF-16 target genes were determined by qRT-PCR. Each value was normalized to act-1, and the value in wild-type (N2) (Fed) was set to 1. Mean mRNA levels ±SEM of three independent experiments are shown. ∗p < 0.05 t test. (B) Representative images of DAF-16 localization (little or no nuclear accumulation [−], weak nuclear accumulation [+], and strong nuclear accumulation [++]) in control RNAi; fasting are shown (left). Percentages of worms with nuclear accumulation of DAF-16::GFP in intestine after 15 hr of fasting were scored in three independent experiments (right). ∗p < 0.05 Fisher’s exact test. (C) Survival curves of N2 (upper left, n = 41 [AL] and 52 [IF]), daf-16(mu86) (upper right, n = 53 [AL] and 50 [IF]), kgb-1(km21) (lower left, n = 59 [AL] and 40 [IF]), or daf-16;kgb-1 (lower right, n = 56 [AL] and 60 [IF]) worms in AL and IF are shown. ∗p < 0.05 log rank test [p = 6.5E-13, 1.4E-6, 5.3E-3, and 4.0E-3 in N2, daf-16(mu86), kgb-1(km21), and daf-16;kgb-1 worms, respectively]. Mean life-span extension by IF (±SEM) of three independent experiments is shown (far right). ∗p < 0.05 t test. See also Figure S4 and Table S2. Cell Reports 2013 3, 79-91DOI: (10.1016/j.celrep.2012.12.018) Copyright © 2013 The Authors Terms and Conditions

Figure 5 The KGB-1/AP-1 Module Plays a Central Role in Mediating Fasting-Induced Gene Expression Changes (A) Scatterplots of fold changes of the fasting-induced upregulated genes for kgb-1(km21) (left), jun-1(gk557) (middle), or daf-16(mu86) (right). A black line indicates (Y – 1) = 0.5 × (X − 1). When the (Y − 1) value becomes less than half of the (X – 1) value in each fasting-induced gene (i.e., each gene below a linear black line), we consider it as “dependent on an indicated mutant.” These “dependent genes” are shown by red crosses in each plot. (B and D) Venn diagram for fasting-induced 539 genes, whose expression is upregulated in response to fasting with statistical significance in wild-type worms, is shown. Blue, red, or green circle indicates KGB-1-, DAF-16-, or JUN-1-dependent genes, respectively (B). Blue, green, or red circle indicates KGB-1-, MEK-1-, or MLK-1-dependent genes, respectively (D). (C) Hierarchical clustering analysis for 539 fasting-induced genes. See also Tables S1, S4, and Figure S5. Cell Reports 2013 3, 79-91DOI: (10.1016/j.celrep.2012.12.018) Copyright © 2013 The Authors Terms and Conditions

Figure 6 The Components of the SCF E3 Ligase Complexes Are Induced by Fasting through the Fasting-Responsive Signaling Pathways and Are Involved in IF-Induced Longevity (A) Immunoblotting analysis (antiubiquitin antibody) to detect protein ubiquitination in the indicated worms. Worms were treated with cul-1 RNAi or control (Ctrl) RNAi in the presence or absence of proteasome inhibitor bortezomib (20 μM). Food + (plus) and – (minus) show fed and fasting (2 days), respectively. Relative ubiquitination levels are shown (lower). The value of lane 5 is set to 1. (B) Immunoblotting analysis (DNP antibody) to detect protein carbonylation in the indicated worms. Total protein extracted from same number of worms is blotted. As an aged animal control, we use 4 days adult grown at 25°C (lane 5). Worms were treated with cul-1 RNAi or control (Ctrl) RNAi. Food + (plus) and – (minus) show fed and fasting (2 days), respectively. Relative protein carbonylation levels are shown (lower). The value of lane 1 is set to 1. (C) Survival curves of control RNAi- (n = 41 [AL] and 52 [IF]), cul-1 RNAi- (n = 55 [AL] and 49 [IF]), cul-6 RNAi- (n = 47 [AL] and 53 [IF]), and cul-1;cul-6 RNAi- (n = 33 [AL] and 31 [IF]) treated worms in AL (upper left) or IF (lower left) are shown. ∗p < 0.05 log rank test. Mean life-span extension by IF (±SEM) of three independent experiments is shown (right). ∗p < 0.05 t test. See also Tables S1, S2, S4, and Figure S6. Cell Reports 2013 3, 79-91DOI: (10.1016/j.celrep.2012.12.018) Copyright © 2013 The Authors Terms and Conditions

Figure S1 Gene Expression Changes in Response to Fasting, Related to Figure 1 (A) Expression profiles of genes, whose expression was downregulated less than half during 48 hr of fasting. Average expression profiles of two independent experiments are shown (left). Numbers of downregulated genes, whose expression at the indicated time point was downregulated less than half as compared to Fed 0 hr (both in Fed and Fasting conditions), are shown (right). (B) Promoter analyses for upregulated genes at 12, 18, 24, 36 or 48 hr fasting, and top ten overrepresented binding sites are shown. V, vertebrate. (C) Survival curves of N2 (left) and jun-1(gk557) (middle) in AL and IF are shown. ∗p < 0.05 log rank test (p = 1.0E-18, and 4.4E-5 in N2 and jun-1(gk557), respectively). Mean life span (±SEM) (right) and mean life-span extension by IF (±SEM) (far right) of three independent experiments is shown. ∗p < 0.05 t test. (D) Survival curves of control RNAi-, fos-1 RNAi-, jun-1 RNAi-, or fos-1;jun-1 RNAi-treated daf-2 mutants (left). Mean life span (±SEM) of three independent experiments is shown (right). Cell Reports 2013 3, 79-91DOI: (10.1016/j.celrep.2012.12.018) Copyright © 2013 The Authors Terms and Conditions

Figure S2 KGB-1 Expression, Related to Figure 2 kgb-1 promoter::gfp transgenic worms are observed. Representative images are shown. Cell Reports 2013 3, 79-91DOI: (10.1016/j.celrep.2012.12.018) Copyright © 2013 The Authors Terms and Conditions

Figure S3 C. elegans p38 MAPKs Are Dispensable for IF-Induced Longevity, Related to Figure 3 IF-induced longevity is not suppressed in the loss-of-function mutants of pmk-1, pmk-2, and pmk-3. Survival curves of pmk-1(km25) (upper left), pmk-2(km37) (upper middle), and pmk-3(ok169) (upper right) in AL and IF are shown. ∗p < 0.05 log rank test (p = 2.3E-14, 1.4E-11, and 1.3E-10 in pmk-1(km25), pmk-2(km37), and pmk-3(ok169), respectively). Mean life-span extension by IF (±SEM) (lower) of three independent experiments is shown. ∗p < 0.05 t test. Cell Reports 2013 3, 79-91DOI: (10.1016/j.celrep.2012.12.018) Copyright © 2013 The Authors Terms and Conditions

Figure S4 JUN-1 Plays a Role in Fasting-Induced Expression of DAF-16 Target Genes, Related to Figure 4 (A) At day 2 of adulthood, worms (wild-type N2 or jun-1 null mutant (gk557) worms) were transferred to the plate in the presence (Fed) or absence (Fasting) of food. After incubation for 2 days, worms were collected and total RNA was extracted. The expression levels of six DAF-16 target genes (aqp-1, dod-6, hil-1, hsp-12.6, mtl-1, and sod-3) were determined by qRT-PCR. Each value was normalized to act-1, and the value in wild-type-fed is set to 1. Mean levels ±SEM of three independent experiments are shown. (B) The promoter regions of six DAF-16 target genes (aqp-1, dod-6, hil-1, hsp-12.6, mtl-1, and sod-3) are shown. Black bars indicate TPA response element (TGACTCA). Closed and open circles indicate DAF-16 binding elements (DBE), TATTTC and CTTATCA, respectively. ∗, one mismatch in TRE or DBE. Positions of TRE in each promoter are shown in Table S3. Cell Reports 2013 3, 79-91DOI: (10.1016/j.celrep.2012.12.018) Copyright © 2013 The Authors Terms and Conditions

Figure S5 KGB-1, JUN-1 and DAF-16 Are Involved in Fasting-Induced Transcriptional Changes, Related to Figure 5 Numbers of genes, whose expression is upregulated more than 2 fold or downregulated less than half during 48 hr of fasting with statistical significance, are shown. Cell Reports 2013 3, 79-91DOI: (10.1016/j.celrep.2012.12.018) Copyright © 2013 The Authors Terms and Conditions

Figure S6 SCF E3 Ligase Complex Components Are Involved in IF-Induced Longevity, Related to Figure 6 (A) Scatter plots of relative gene expression of the fasting-induced upregulated genes for kgb-1(km21) (left), jun-1(gk557) (middle), or daf-16(mu86) (right). Red line indicates the (y – 1) = 0.5 (x – 1). (B) Venn diagram for fasting-induced 539 genes is shown. Blue circle, red one, or green one indicate genes downregulated in kgb-1(km21), jun-1(gk557), or daf-16(mu86) mutants. (C) The gene ontology analysis for 198 genes, whose expression levels after 2 days of fasting in wild-type worms were more than two-fold of those in every mutant of kgb-1, jun-1, and daf-16, is shown. (D) Expression profiles of whole F-box proteins (322 genes, upper) and cullin proteins (6 genes, lower) during fasting in wild-type worms and mutants (daf-16(mu86), jun-1(gk557), and kgb-1(km21)) after 2 days of feeding (Fed) or fasting (Fasting) are shown. (E) RNAi of each of genes encoding cullin proteins (cul-1, 2, 3, 4, 5, and 6) was performed from L4 stage by feeding RNAi. Mean life span ±SEM (upper) and mean life-span extension by IF (±SEM) (bottom) of three or four independent experiments are shown. ∗p < 0.05, t test. (F) At day 2 of adulthood, worms (wild-type N2, kgb-1(km21), jun-1(gk557), and daf-16(mu86)) were transferred to the plate in the presence (Fed) or absence (Fasting) of food. After incubation for 2 days, worms were collected and total RNA was extracted. The expression levels of cul-1 were determined by qRT-PCR. Each value was normalized to act-1, and the value in wild-type (N2) (Fed) is set to 1. Mean levels ±SEM of three independent experiments are shown. Cell Reports 2013 3, 79-91DOI: (10.1016/j.celrep.2012.12.018) Copyright © 2013 The Authors Terms and Conditions