Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPR(mt)

Abstract

Organisms respond to mitochondrial stress through the upregulation of an array of protective genes, often perpetuating an early response to metabolic dysfunction across a lifetime. We find that mitochondrial stress causes widespread changes in chromatin structure through histone H3K9 di-methylation marks traditionally associated with gene silencing. Mitochondrial stress response activation requires the di-methylation of histone H3K9 through the activity of the histone methyltransferase met-2 and the nuclear co-factor lin-65. While globally the chromatin becomes silenced by these marks, remaining portions of the chromatin open up, at which point the binding of canonical stress responsive factors such as DVE-1 occurs. Thus, a metabolic stress response is established and propagated into adulthood of animals through specific epigenetic modifications that allow for selective gene expression and lifespan extension.

Figure 1. Neuronal polyglutamine expression induces a lin-65 dependent activation of the UPR^mt

(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 blots analyses.

Figure 2. The C. elegans histone H3K9me1/2 methyltransferase met-2 is required for LIN-65 nuclear accumulation upon 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 Day 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.

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 represents 10 μm. (B) Quantification of H3K9me2 level. The genotypes and treatments are as in (A). H3K9me2 intensity is normalized to DAPI intensity. (*** denotes p < 0.0001; ns denotes p > 0.05 via t-test, error bars indicate SEM, n ≥ 15 nuclei) (C) Representative of 3 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 represents 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 (*** denotes 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. (*** denotes p < 0.0001 via mann-whitney test, error barsindicate SEM, n ≥ 25 nuclei)

Figure 4. DVE-1 nuclear distribution upon 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 Day 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). (*** denotes 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 Figure 4F. (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.

Figure 5. Gene expression changes upon cco-1 RNAi treatment is strongly dependent upon 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 Day1, 2 and 3 adult animals grown on EV or EV+cco-1 RNAi from hatch were collected for qPCR. (* denotes p < 0.05, ** denotes p < 0.01 via t-test, error bar indicates the SEM from three biological replicates)

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.

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 UPR^mt 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 UPR^mt. DVE-1 and LIN-65 accumulate in the nucleus (Figure 2A-C and Figure 4B-D). Animals are long-lived, the UPR^mt 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 (Figure 2A and Figure 4B), reduced UPR^mt 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 UPR^mt induction (Figure 3A and Figure S1B) and partial suppression of increased lifespan (Figure 6C). (E) Loss of atfs-1 during mitochondrial stress results in suppressed UPR^mt 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 (Figure 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 UPR^mt 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 UPR^mt targets. This remodeling works in parallel to the relocation of mitochondrial specific transcription factor ATFS-1 to initiate UPR^mt and regulate longevity.