Volume 165, Issue 5, Pages 1197-1208 (May 2016) Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPRmt  Ye Tian, Gilberto Garcia, Qian Bian, Kristan K. Steffen, Larry Joe, Suzanne Wolff, Barbara J. Meyer, Andrew Dillin  Cell  Volume 165, Issue 5, Pages 1197-1208 (May 2016) DOI: 10.1016/j.cell.2016.04.011 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Neuronal Polyglutamine Expression Induces a lin-65-Dependent Activation of the UPRmt (A) Representative photomicrographs demonstrating: (a) bright field image of aligned, wild-type worms; (b) YFP expression in the neurons; (c) hsp-6p::gfp expression in wild-type (WT) animals; (d) hsp-6p::gfp is induced in the intestine in rgef-1p::Q40::yfp animals; (e) hsp-6p::GFP suppression in atfs-1 animals; (f) hsp-6p::GFP suppression in lin-65 animals. Arrows indicate the posterior region of the intestine where hsp-6p::gfp is induced or suppressed. See also Figure S1. (B) Immunoblots of GFP expression in hsp-6p::gfp; rgef-1p::Q40::yfp animals in the presence or absence of atfs-1 or lin-65 mutation. Anti-tubulin serves as a loading control. ∗Indicates a cleaved band from Q40::YFP that is recognized by GFP antibody. (C) Representative photomicrographs of WT, atfs-1, and lin-65 animals grown on EV or cco-1 RNAi from hatch. Day 2 of adulthood animals were used for imaging and western blot analyses. Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 The C. elegans Histone H3K9me1/2 Methyltransferase met-2 Is Required for LIN-65 Nuclear Accumulation on cco-1 RNAi (A) Representative photomicrographs of lin-65p::lin-65::mCherry animals in a WT or met-2 mutant background grown on EV, cco-1 RNAi, or cco-1+lin-65 double RNAi bacteria from hatch (as indicated). Images were taken at day 1 of adulthood. Arrows indicate intestinal nuclei. (B) Immunoblot of LIN-65::HA expression in animals grown on EV and EV+cco-1 RNAi from hatch as indicated. Animals were collected at days 1, 2, and 3 of adulthood. Anti-tubulin serves as a loading control. (C) Immunoblot of LIN-65::HA expression in WT or met-2 mutants grown on EV or cco-1 RNAi (as indicated) from hatch. Animals were collected at day 2 of adulthood. Anti-tubulin serves as a loading control. See also Figure S2. Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Mitochondrial Stress Induced Chromatin Reorganization (A) Representative maximal intensity projection images of H3K9me2 immunostaining of intestinal nuclei in day 1 adult WT, met-2, or lin-65 mutant animals grown on EV or cco-1 RNAi from hatch (as indicated). H3K9me2 (red); DAPI (grey). Scale bar, 10 μm. (B) Quantification of H3K9me2 level. The genotypes and treatments are as in (A). H3K9me2 intensity is normalized to DAPI intensity. ∗∗∗p < 0.0001; ns denotes p > 0.05 via t test. Error bars indicate SEM, n ≥ 15 nuclei. (C) Representative of three center images of DAPI staining with lmn-1p::emr-1::gfp of intestinal nuclei in day 1 adult animals grown on EV or cco-1 RNAi from hatch as indicated. lmn-1p::emr-1::gfp (green); DAPI (grey). Scale bar, 10 μm. (D) Quantification of the intestinal nuclear size at day 1 adulthood using lmn-1p::emr-1::gfp as a marker. Animals grown on EV or cco-1 RNAi from hatch. ∗∗∗p < 0.0001 via t test. Error bars indicate SEM, n ≥ 20 nuclei. (E) Quantification of the distribution of DAPI staining signal in intestinal nuclei at day 1 of adulthood in animals grown on EV or cco-1 RNAi from hatch. The distribution of fractions from the top four bins are shown as boxplots. ∗∗∗p < 0.0001 via Mann-Whitney test. Error bars indicate SEM, n ≥ 25 nuclei. See also Figure S3. Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 DVE-1 Nuclear Distribution on Mitochondrial Stress Is Dependent on lin-65 and met-2 (A) Representative photomicrographs of day 1 adult animals expressing both lin-65p::lin-65::mCherry and dve-1p::dve-1::gfp grown on cco-1 RNAi from hatch. (B) Representative photomicrographs of day 1 dve-1p::dve-1::gfp animals in wild-type, lin-65 and met-2 background grown on EV or cco-1 RNAi from hatch. (C) Immunoblot of GFP expression in WT animals grown on EV or cco-1 RNAi from hatch. Animals were collected at days 1, 2, and 3 of adulthood. Anti-tubulin serves as a loading control. (D) Immunoblot of GFP expression in day 1 adult animals in WT, lin-65 or met-2 mutant background grown on EV or cco-1 RNAi from hatch. Anti-tubulin serves as a loading control. (E) Quantification of the number of intestinal nuclei with DVE-1 puncta structure per worm. The genotypes and treatments are as in (B). ∗∗∗p < 0.0001 via t test. Error bars indicate SEM, n ≥ 30 worms. See also Figure S4D. (F) Representative photomicrographs of DVE-1::GFP punctate structure in one intestinal nucleus. DVE-1::GFP (green); DAPI (blue). (G) The intensity profile of both DAPI and DVE-1::GFP across the white line as shown in (F). (H) Quantification of the distribution of DVE-1::GFP signal in nuclei with DAPI staining. DVE-1::GFP animals grown on cco-1 RNAi from hatch. The distribution of fractions of all three bins are shown as boxplots (n ≥ 30 nuclei). (I) Representative photomicrographs of DVE-1::GFP expression in animals grown on: (a) cco-1 RNAi for 5 days from hatch; (b) cco-1 RNAi for 3 days from hatch and then transferred to cco-1+lin-65 double RNAi for 2 more days; (c) cco-1+lin-65 double RNAi from L4, F1 generations were examined. See also Figure S5. Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Gene Expression Changes on cco-1 RNAi Treatment Is Strongly Dependent on lin-65 and met-2 (A) Venn diagram of numbers of differentially expressed genes in wild-type, lin-65, and met-2 mutant animals grown on cco-1 RNAi for 2 days. Genes with an adjusted p value < 0.05 were selected as differentially expressed genes. See also Table S2. (B) Quantitative PCR of hsp-6 and timm-23 mRNA level. Synchronized days 1, 2, and 3 adult animals grown on EV or EV+cco-1 RNAi from hatch were collected for qPCR. ∗p < 0.05, ∗∗p < 0.01 via t test. Error bar indicates the SEM from three biological replicates. See also Figure S6 and Table S2. Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Mitochondrial Stress-Induced LIN-65 Nuclear Translocation Is Partially Dependent on clpp-1 and dve-1 but Independent of atfs-1 (A) Representative photomicrographs of day 1 adult animals expressing lin-65p::lin-65::mCherry grown on RNAi from hatch as indicated. (B) Immunoblot of against HA-tag in LIN-65::HA animals grown on RNAi bacteria from hatch (as described in A). Animals were collected at day 1 of adulthood. Anti-tubulin serves as a loading control. (C) Survival analyses of lin-65(n3441) animals on EV or EV+cco-1 RNAi. See Table S1 for lifespan statistics. (D) Survival analyses of met-2(ok2307) animals on EV or atfs-1 RNAi. See Table S1 for lifespan statistics. (E) Survival analyses of met-2(ok2307) and atfs-1 RNAi-treated animals under mitochondrial stress condition with cco-1 RNAi. See Table S1 for lifespan statistics. See also Figure S7. Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Model for Mitochondrial Stress Signaling Pathway (A) Under non-stressed conditions, MET-2 produces H3K9me1/2 histone subunits in the cytoplasm. ATFS-1 translocates to the mitochondria and is degraded. DVE-1 and LIN-65 do not accumulate in the nucleus and the UPRmt is not induced, animals are normal lived and the nucleus is not compacted (Figure 3A). (B) During mitochondrial stress, MET-2 continues to produce H3K9me2 histone subunits, ATFS-1 now translocates to the nucleus to induce UPRmt. DVE-1 and LIN-65 accumulate in the nucleus (Figures 2A–2C and 4B–4D). Animals are long-lived, the UPRmt is induced, nuclei become compacted (Figure 3A), and H3K9me2 levels remain unchanged (Figure 3B). (C) Loss of met-2 during mitochondrial stress results in reduced nuclear H3K9me2 levels, nuclei that are less compacted (Figure 3A), reduced DVE-1 and LIN-65 nuclear accumulation (Figures 2A and 4B), reduced UPRmt induction (Figure S1B), and partial suppression of increased lifespan (Figure 6E). (D) Loss of lin-65 during mitochondrial stress results in reduced nuclear H3K9me2 levels (Figure 3B), nuclei that are less compacted (Figure 3A), reduced DVE-1 nuclear accumulation (Figure 4B), reduced UPRmt induction (Figures 3A and S1B), and partial suppression of increased lifespan (Figure 6C). (E) Loss of atfs-1 during mitochondrial stress results in suppressed UPRmt induction (Figure 1A) and partial suppression of lifespan extension (Figure 6E). This is independent of compacted nuclei (Figure S7B) and nuclear accumulation of DVE-1 and LIN-65 (Figures 6A and 6B). (F) Loss of both met-2 and atfs-1 during mitochondrial stress results in nuclei that are less compacted, reduced nuclear accumulation of DVE-1 and LIN-65, no UPRmt induction, and complete suppression of lifespan extension. (Figure 6E). (G) During mitochondrial stress, LIN-65 and DVE-1 accumulate in the nucleus, chromatin becomes remodeled, and DVE-1 is able to form puncta at the loose regions of the chromatin, activating transcription of UPRmt targets. This remodeling works in parallel to the relocation of mitochondrial-specific transcription factor ATFS-1 to initiate UPRmt and regulate longevity. Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure S1 lin-65 Is Specifically Required for UPRmt Induction, Related to Figure 1 (A) Representative photomicrograph of lin-65(uth2) suppressed hsp-6p::gfp induction in animals expressing rgef-1p::Q40::yfp. (B) lin-65(n3441) and met-2(ok2307) strongly attenuated hsp-6p::gfp induction in animals grown on either cco-1 RNAi or phb-2 RNAi from hatch. Quantification of the hsp-6p::GFP mean fluorescence intensity using COPAS worm sort system. (∗∗∗ denotes p < 0.0001 via t test; error bars indicate SEM, n ≥ 100 worms). (C) lin-65(n3441) is not required for tunicamycin induced hsp-4p::gfp induction. Synchronized L4 animals were treated with 25ng/μl tunicamycin (Tm) or DMSO for 4 hr at 20°, prior to imaging. (D) lin-65(n3441) is not required for heat shock induced hsp-16.2p::gfp induction. Synchronized day 1 of adulthood animals were placed at 34°C for 20 min and allowed to recover for 6 hr prior to imaging. Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure S2 lin-65 Is Mainly Expressed in the Intestine and Neurons, Related to Figure 2 (A) Representative photomicrograph of lin-65p::mCherry transgenic animals. lin-65 is expressed in several head neurons and throughout intestine. (B) Quantitative PCR of lin-65 mRNA level. Synchronized day1, day2 and day3 adult animals grown on empty vector (EV) or EV+cco-1 RNAi from hatch were collected for qPCR. (∗ denotes p < 0.05, ns denotes p > 0.05 via t test, error bar indicates the SEM from three biological replicates). (C) Representative photomicrograph of lin-65(n3441); hsp-6p::gfp; rgef-1p::Q40::yfp animals with or without lin-65p::lin-65::mCherry rescue. (D) Representative photomicrograph of lin-65p::lin-65::mCherry transgenic animals. (E) Immunoblots of HA-epitope tagged expression of lin-65p::lin-65::HA transgenic animals grown on empty vector, EV+cco-1 RNAi and cco-1+lin-65 RNAi as indicated. Animals were collected at day 1 of adulthood. Anti-tubulin serves as a loading control. Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure S3 cco-1 RNAi Does Not Change the Number of Intestinal Nuclei or Their DNA Content, Related to Figure 3 (A) Representative photomicrographs of H3K9me2 immunostaining of the germline of wild-type, met-2(ok2307), lin-65(n3441), and set-25(n5021) mutant animals. H3K9me2 (Red); DAPI (Grey). (B) Quantification of the number of intestinal nuclei at day 1 of adulthood in animals expressing elt-2::GFP grown on either empty vector or cco-1 RNAi from hatch. No significant difference in nuclei numbers was observed in cco-1 RNAi treated animals. (ns denotes p > 0.05 via t test, error bar indicates SEM, n ≥ 10 worms). (C) Quantification of the DNA content of intestinal nuclei at day 1 of adulthood in animals grown on empty vector or cco-1 RNAi from hatch. Maximal DAPI fluorescence intensity was measured for each condition. No significant difference in DNA content was observed in cco-1 RNAi treated animals. (ns denotes p > 0.05 via t test, error bar indicates SEM, n ≥ 15 nuclei). Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure S4 lin-65 Functions Cell Autonomously in the Intestine to Regulate DVE-1 Nuclear Distribution on Mitochondrial Stress, Related to Figure 4 (A) Representative photomicrographs of dve-1p::dve-1::gfp transgenic animals in lin-65(n3441) background grown on cco-1 RNAi with or without lin-65p::lin-65::mCherry rescue. (B) Representative photomicrographs of dve-1p::dve-1::gfp; rgef-1p::Q40::yfp transgenic animals in lin-65(n3441) background grown on cco-1 RNAi with or without gly-19p::lin-65::mCherry rescue. (C) Quantitative PCR of dve-1 mRNA level. Synchronized day1, day2 and day3 adult animals grown on empty vector (EV) or EV+cco-1 RNAi from hatch were collected for qPCR. (ns denotes p > 0.05 via t test, error bar indicates the SEM from three biological replicates). (D) Representative photomicrographs of dve-1p::dve-1::gfp in the most proximal two intestinal nuclei in wild-type, lin-65(n3441), and met-2(ok2307) animals grown on empty vector or cco-1 RNAi. DVE-1 punctate structures formed in the nuclei of wild-type animals on cco-1 RNAi treatment. Nuclei are highlighted with white circles. (E) Representative photomicrographs of H3K9me2 immunostaining of an intestinal nucleus in dve-1p::dve-1::gfp animals grown on cco-1 RNAi. H3K9me2 signals are not colocalized with DVE-1::GFP puncta. Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure S5 Loss of set-25 Results in DVE-1::GFP Nuclear Accumulation but Is Not Required for cco-1 RNAi-Induced Longevity, Related to Figure 4 (A) Representative photomicrographs of day 1 dve-1p::dve-1::gfp transgenic animals in wild-type and set-25(n5021) mutant background. (B) Survival analysis of set-25(n5021) animals on EV or cco-1 RNAi. set-25 mutants have no effect on lifespan grown on empty vector (normal condition) or cco-1 RNAi (mitochondrial stress condition). See Table S1 for lifespan statistics. Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure S6 Heatmaps for Genes Differentially Expressed under cco-1 RNAi Condition in Wild-Type Animals, Related to Figure 5 (A) Log2 fold-change in mean expression values are represented for 1312 genes identified as differentially expressed in wild-type animals treated with cco-1+empty vector RNAi relative to empty vector alone; corresponding values for lin-65(n3441) and met-2(ok2307) mutants are displayed. See also Table S2. (B) Dispersion values, used for statistical testing, for the 1312 differentially expressed genes in wild-type animals (plotted in the same order as in (A)), as assigned by DESeq, reflect the relationship between each gene’s mean expression and variance among replicates. See also Table S2. Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure S7 dve-1 RNAi Affects LIN-65 Protein Stability and atfs-1 RNAi Induces LIN-65 Accumulation in the Absence of Mitochondrial Stress, Related to Figure 6 (A) Immunoblot of HA fusion protein expressed by lin-65p::lin-65::HA in wild-type animals grown on empty vector, atfs-1 RNAi, clpp-1 RNAi, or dve-1 RNAi from hatch. Animals were collected at Day 1 of adulthood. Anti-tubulin serves as a loading control. (B) atfs-1 is not required for chromatin compaction induced by cco-1 RNAi. Representative maximal intensity projection images of DAPI immunostaining of intestinal nuclei in Day 2 adult wild-type animals grown on empty vector, cco-1 and atfs-1 RNAi from hatch (as indicated). DAPI (Blue), Scale bar represents 10 μm. Cell 2016 165, 1197-1208DOI: (10.1016/j.cell.2016.04.011) Copyright © 2016 Elsevier Inc. Terms and Conditions