Developmental ROS individualizes organismal stress resistance and lifespan

Abstract

A central aspect of aging research concerns the question of when individuality in lifespan arises¹. Here we show that a transient increase in reactive oxygen species (ROS), which occurs naturally during early development in a subpopulation of synchronized Caenorhabditis elegans, sets processes in motion that increase stress resistance, improve redox homeostasis and ultimately prolong lifespan in those animals. We find that these effects are linked to the global ROS-mediated decrease in developmental histone H3K4me3 levels. Studies in HeLa cells confirmed that global H3K4me3 levels are ROS-sensitive and that depletion of H3K4me3 levels increases stress resistance in mammalian cell cultures. In vitro studies identified SET1/MLL histone methyltransferases as redox sensitive units of the H3K4-trimethylating complex of proteins (COMPASS). Our findings implicate a link between early-life events, ROS-sensitive epigenetic marks, stress resistance and lifespan.

Extended Data Figure 1.. In vivo read-out of endogenous redox states at different stages during C. elegans lifespan and sorting parameters of oxidized and reduced subpopulations.

(a) Microscopic analysis of the Grx1-roGFP2 ratio of individual N2jrIs2[Prpl-17::Grx1-roGFP2] worms (symbol) cultivated at 15 °C and imaged at the indicated time points. Means (bars) of every two sequential time points that are not significantly different from each other (P > 0.05) share the same letter. Data represent mean ± SEM; n, number of animals; one-way ANOVA with Tukey correction. (b) The roGFP2 ratio (405/488) was calculated using the partial profiling feature (pp) configured to analyze extinction and emission data from each 488 nm and 405 nm lasers that sequentially excited each worm. (c) A population of N2jrIs2[Prpl-17::Grx1-roGFP2] at the L2 stage separated based on their opacity (extinction) and length (time of flight) was gated as R1.

Extended Data Figure 2.. Sorting efficiency and lifespan of L2^ox and L2^red subpopulations.

(a-d) Microscopic analysis of the Grx1-roGFP2 ratio of individual worms (symbol) previously sorted into L2^ox, L2^mean and L2^red subpopulations. n = animals; one-way ANOVA with Tukey correction. (e-i) Survival curves of sorted L2^ox, L2^mean and L2^red worms. For n numbers, P values (log rank test) see Extended Table 2. (Inserts) The Grx1-roGFP2 ratio of individual worms (symbol), as assessed by fluorescence microscopy after sorting is shown. n = animals. For P values (unpaired t-test, two sided) see Extended Table 2.

Extended Data Figure 3.. Physiological properties of L2^ox and L2^red sorted worms.

(a) Length measurements of L2^ox and L2^red worms (symbol) from nose to tail tip immediately after sorting. No significant difference; P = 0.4735 (unpaired t-test, two-sided). (b) Brood size of L2^ox and L2^red worms, measured at the indicated time points. n = animals. No significant difference within a single age; P = 0.6532 (two – way ANOVA). (c) Basal respiration, (d) maximal and (e) spare respiratory capacity and (f) basal rates of flux through glycolysis (ECAR) of L2^ox and L2^red worms. n = 3 independent sorting experiments. P = 0.9469 (c), P = 0.7784 (d), P = 0.7904 (e), P = 0.7925 (f); unpaired t-test, two-sided. (g) Survival of L2^ox and L2^red worms 20 hours after heat shock. n = 5 independent sorting experiments; unpaired t-test, two-sided. The data points connected represent data from the same sorting experiment. The survival of L2^ox is set to 1. All data represent mean ± SEM.

Extended Data Figure 4.. Gene expression profiles of L2^ox and L2^red.

(a) Steady-state transcript levels of selected oxidative stress-related genes in L2^ox and L2^red worms. n, independent sorting experiments; unpaired t-test, two sided. Data represent mean ± SEM. (b) Volcano plot showing fold changes versus P values for the transcriptomes of L2^ox and L2^red subpopulations. Differentially expressed genes (DEGs, P ≤ 0.05, changes ≥ log₂ ± 0.6) are represented by red dots (see methods for statistical definition of DEGs). Data were collected from 4 independent sorting experiments. (c) Gene Set Enrichment Analysis of the 327 DEGs. Normalized enrichment scores (see methods for calculation) are represented by the bar graph. Terms (for summary, see Supplementary Table 1) indicating origin/process/phenotype associated with genes known to play a role in the process are shown on the left. Some terms (*) have been merged and are represented as a single category bar for simplicity (for detailed values, see Supplementary Table 1). (d, e) Percentage of DEGs identified in L2^ox that intersect with H3K4me3 peak signals within their 5’ region (500 bp upstream and downstream from the transcription start site). The H3K4me3 ChIP data sets were generated from L3-staged N2 worms (ChIP–chip, GEO entry: GSE30789) in (a) and ChIP-seq, GEO entry: GSE28770) in (b), indicating that these marks are set during larval development. Hypergeometric probability for (a): P = 0.064 and (b): P = 2.786 × 10^-6. (c) and (d) Venn diagrams show the overlap among up-regulated (c) or down-regulated (d) gene sets in L2^ox ]and down – or up – regulated set-9(rw5) and set-26(tm2467) gene sets (GEO entry: GSE100623). See Supplementary Table 1 for data sets in (a) – (d).

Extended Data Figure 5.. Redox sensitivity of in vivo H3K4m3e3 levels and in vitro histone methyltransferase complex activity.

(a) Global H3K4me3 levels in the L2^ox and L2^red sorted worms. A representative westernblot using antibodies against H3K4me3 is shown. Quantification of global H3K27ac (b) and H3K27me3 (c) levels by westernblot. n = 3 independent sorting experiments. P = 0.3793 (b) and P = 0.0905 (c); unpaired t-test, two-sided. Data represent mean ± SEM. Global H3K4me3 (d), ASH2L (e) and MLL1 (f) levels in HeLa cells before and after H₂O₂ treatment, as assessed by western blot. (g) Time course of the in vitro methyltransferase reaction for core HMC (MLL1-WDR5-ASH2L-RBBP5). Reaction rates were derived from the first 20 min of the linear range. In vitro histone methyltransferase assays of core HMC members, consisting of purified GST-WDR5 (WDR5), GST-ASH2L (ASH2L)_, GST-RBBP5 (RBBP5) and either GST-MLL1_SET or untagged MLL1_SET (h), GST-SET1A_SET (i) or GST-SET1B_SET (j). Superscript OX indicates that the protein was pre-treated with either 1 mM (+) or 2 mM (++) H₂O₂ for 30 min prior to the activity assay. DTT was added after the H₂O₂ treatment. n = 3 independent experiments; one - way ANOVA with Sidak correction. Data represent mean ± SEM. (k) MLL1_SET was treated with either 2 mM DTT, 2 mM H₂O₂ or 2 mM H₂O₂ followed by 4 mM DTT-treatment. Catalase was used to quench the H₂O₂. The proteins were denatured and thiols were modified with NEM prior to loading onto non-reducing SDS-PAGE to prevent non-specific thiol oxidation. The proteins were visualized by silver staining. M, marker. (l) MLL1_SET treated with either 2 mM DTT, 2 mM H₂O₂ or 2 mM H₂O₂ followed by 4 mM DTT-treatment was analyzed. All reduced protein thiols were then labeled with the 500 Da thiol-reactive AMS, causing a 500-Da mass decrease per oxidized thiol detectable on reducing SDS-PAGE. (m) Cysteine oxidation state in MLL1_SET after treatment with either 2 mM DTT or 2 mM H₂O₂ followed by NEM labeling as assessed by LC-MS/MS. The peptide containing Cys3967 could not be detected. (n) Schematic representation of the redox sensitivity of the MLL1_SET. For blot and gel source images, see Supplementary Fig. 1 and 3. o) Sequence alignment of the SET-domain. All cysteines present in MLL1 are shown in bold, and the five absolutely conserved cysteines are highlighted in yellow. Cysteines shown to be involved in zinc coordination are marked with an asterisk. NCBI protein blast and Clustal Omega Multiple Sequence Alignment, Clustal O (1.2.4) were used.

Extended Data Figure 6.. Effects of H3K4me3 down-regulation on heat shock response and endogenous redox state.

(a) ASH-2 and SET-2 transcript levels of N2jrIs2[Prpl-17::Grx1-roGFP2] worms treated with ash-2 or set-2 RNAi for 2 generations. n = 6 (ash-2) and n = 2 (set-2) independent experiments; unpaired t-test, two-sided. E.V., empty vector. (b) ASH-2 protein levels in N2jrIs2[Prpl-17::Grx1-roGFP2] worms treated with control RNAi or ash-2 RNAi for 2 generations using westernblot analysis. (c) H3K4me3 levels in N2jrIs2[Prpl-17::Grx1-roGFP2] worms treated with control RNAi, ash-2 RNAi or set-2 RNAi for 2 generations. (d) Transcript levels of selected heat shock genes after heat shock treatment of N2jrIs2[Prpl-17::Grx1-roGFP2] worms treated with the indicated RNAi. n = 3 independent experiments; one-way ANOVA with Bonferroni correction. (e) Transcript levels of selected heat shock genes in set-2 or wdr-5.1 mutants before and after heat shock treatment. n = 3 independent experiments; one-way ANOVA with Bonferroni correction. (f) ASH2L levels following ash2L siRNA treatment of HeLa cells. n = 2 independent experiments. (g) Grx1-roGFP2 ratios of L2 larval worms treated with ash-2 RNAi, set-2 RNAi, wdr-5.1 RNAi or the empty vector were measured using the BioSorter. n = 4 (ash-2, set-2) and n = 3 (wdr-5.1) independent sorting experiments; unpaired t-test, two-sided. (h) Representative survival curves of N2jrIs2[Prpl-17::Grx1-roGFP2] worms treated with ash-2 or set-2 RNAi for 2 generations, and treated with 1 mM PQ for 10 hours at the L2 larval stage. For n numbers, repetitions and statistics (log-rank), see Extended Data Table 4. Data in (a), (d), (e-g) represent mean ± SEM. For blot source images, see Supplementary Fig. 1 and 3.

Figure 1.. Endogenous redox state in an age-synchronized population of C. elegans larvae.

(a) Distribution of Grx1-roGFP2 ratios of a L2-staged N2jrIs2[Prpl-17::Grx1-roGFP2] population. L2 worms with Grx1-roGFP2 ratios between 2 and 3 standard deviations above (red line, insert: R2, red, L2^ox) or below (blue line, insert: R4, blue, L2^red) the mean were sorted and compared to animals with mean Grx1-roGFP2 ratios (green line, insert: R3, green, L2^mean). Insert: axes are redrawn to scale. n = 15,599 animals. (b) Representative microscopic analysis of the Grx1-roGFP2 ratio of individual worms (symbols) of the L2^ox, L2^mean and L2^red subpopulations. n = animals; One-way ANOVA with Tukey correction. The experiment was repeated 4 more times with similar results (see Extended Data Figure 2a–d). (c) Longitudinal analysis of the redox state. L2^ox and L2 ^red-sorted were cultivated at 20°C and the Grx1-roGFP2 ratio of animals (symbol) in each subpopulation was determined microscopically at the indicated time points. n = animals; Mann-Whitney U test, two - sided. Data in (b) and (c) represent mean ± SEM.

Figure 2.. Oxidized L2 subpopulations show increased stress resistance and longer lifespan.

Experiments were performed with N2jrIs2[Prpl-17::Grx1-roGFP2] animals sorted into L2^ox, L2^mean and L2^red subpopulations. (a) Representative survival curves of sorted worms that survived heat shock treatment. (b) Representative survival curves of sorted worms cultivated on NGM plates supplemented with 2 mM PQ. (c) Representative survival curves of sorted worms. See Extended Data Fig. 2e–i for repetitions. (Insert) The Grx1-roGFP2 ratio of individual worms (symbol) after sorting is shown. n = animals; unpaired t-test, two sided. (d) Representative survival curves of a non-sorted (mixed) worm population treated at the L2 stage with either nothing, 1 mM PQ or 10 mM NAC for 10 hours. (Insert) The Grx1-roGFP2 ratio of individual worms (symbol) after treatment is shown. n animals; one-way ANOVA with Tukey correction. Data in inserts represent mean ± SEM. (e-f) Representative survival curves of a L2^red (e) or L2^ox subpopulation (f) after a 10 h-treatment with either nothing, 1 mM PQ or 10 mM NAC. The specific sorting events, number of individuals, repetitions and statistical analysis (log-rank) for each of the data sets shown in this figure can be found in Ext. Data Tables 1–3.

Figure 3.. H3K4me3 - A redox sensitive histone modification involved in stress gene expression and resistance.

(a) Heat shock gene transcripts in sorted subpopulations before and after heat shock. n = 3 independent sorting experiments; two-way ANOVA with Tukey correction. (b) Venn diagram of upregulated genes in L2^ox and ash-2 RNAi worms (complete list in Supplementary Table 1). (c) Quantification of global H3K4me3 levels in L2^ox and L2^red worms. n = 7 independent sorting experiments; unpaired t-test, two-sided. (d) Quantification of H3K4me3 levels in HeLa cells before and after H₂O₂ treatment. n = 3 independent experiments; one-way ANOVA with Dunnett correction. (e) In vitro histone methyltransferase assays of core HMC members; ^ox: pre-treated with 1 mM (+) or 2 mM (++) H₂O₂ for 30 min prior to activity assay. Thiol-reducing agent dithiothreitol (DTT) was added after the H₂O₂ treatment. n = 3 independent experiments; one-way ANOVA with Sidak correction. (f) Reverse thiol trapping of oxidized and reduced MLL1_SET. A 500-Da mass increase per oxidized thiol can be detected on non-reducing SDS-PAGE. (g) Heat shock survival of C. elegans N2 wild-type, wdr-5, set-2, rbr-2 and set2/rbr-2 mutants after 48 hours (n = 3 independent experiments; one-way ANOVA with Dunnett correction) or N2jrIs2[Prpl-17::Grx1-roGFP2] worms treated with ash-2 or control RNAi after 24 hours (n = 5 independent experiments; unpaired t-test, two-sided. E.V. empty vector). (h) Heat shock survival of ASH2L-siRNA treated HeLa cells. n = 5 independent experiments; two-way ANOVA with Tukey correction. (i) Transcript levels of heat shock genes after 30 min heat stress treatment of ASH2L siRNA treated HeLa cells. N.T., non-targeting. n = 3 independent experiments; unpaired t-test, two-sided. Data in (a), (c), (d), (e) and (g-i) represent mean ± SEM. For blot and gel source images, see Supplementary Fig. 1–3.

Figure 4.. An intrinsically oxidizing environment confers increased stress resistance via down-regulation of global H3K4me3 levels.

Survival of N2jrIs2[Prpl-17::Grx1-roGFP2] worms treated with ash-2 (a) or set-2 (b) RNAi, sorted into L2^ox and L2^red and measured 24 hours after heat shock. n = 3 (a) and n = 4 (b) independent experiments; two-way ANOVA with Tukey correction. Data represent mean ± SEM. Representative survival curves of N2jrIs2[Prpl-17::Grx1-roGFP2] worms treated with ash-2 (c) or set-2 (d) RNAi and sorted into L2^ox and L2^red. For n numbers, repetitions and statistics (log-rank) in (c) and (d), see Extended Data Tables 2 and 4.