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.
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Data availability
All relevant data are available and/or included with the manuscript as Source Data or Supplementary Information. RNA-sequencing data have been uploaded to the Gene Expression Omnibus (GEO) database with accession number GSE138502.
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Acknowledgements
We thank M. Malinouski and T. Mullins for assistance with the reconfiguration of the Biosorter; G. Csankovszki for antibodies, C. elegans RNAi feeding clones and comments; B. Braeckman for the N2jrIs2[Prpl-17::Grx-1-roGFP2] strain; J. Nandakumar for HeLa (EM-2-11ht) cells; the Caenorhabditis Genetics Center (funded by National Institutes of Health Infrastructure Program P40 OD010440) for C. elegans strains; the DNA Sequencing Core (BRCF), R. Tagett, W. Wu and the Bioinformatics Core of University of Michigan for RNA sequencing and data analysis; K. Wan for protein purification; R. Sawarkar and J. Labbadia for important suggestions; Jakob laboratory members for comments on the manuscript and J. Bardwell for critically reading the manuscript. Mass spectrometry was performed by MS Bioworks. This work was supported by NIH grants GM122506 and AG046799 as well as the Priority Program SPP 1710 of the Deutsche Forschungsgemeinschaft (Schw823/3-2) to U.J., a NIH T32 Career Training in the Biology of Aging grant to D.B., a NIH T32 Career Training in the Biology of Aging grant and a Bright Focus ADR Fellowship (A2019250F) to B.J.O., and the National Natural Science Foundation of China (31470737) to Y.C.
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Contributions
D.B. conceived and conducted most experiments, performed data analysis and wrote the manuscript; D.K. conceived experiments and initiated work with the BioSorter; Y.Z. performed the in vitro methyltransferase assays; K.U. performed the reverse thiol trapping and prepared samples for mass spectrometry analysis; B.J.O. assisted with RNAi experiments and western blots for methylation marks in C. elegans; L.X. performed siRNA in HeLa cells; M.K. built and operated the lifespan instrument; A.K. assisted with worm sorting and produced brood size data; Y.-T.L. purified mammalian proteins; Y.D. conceived experiments and provided material; S.Q. and Y.C. conceived experiments; U.J. conceived experiments, conducted data analysis and wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 In vivo readout of endogenous redox states at different stages during C. elegans lifespan and sorting parameters of oxidized and reduced subpopulations.
a, Microscopy 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. Data points that are not significantly different from each other (P > 0.05) are labelled with the same letter. Data are mean ± s.e.m; n, number of worms; one-way ANOVA with Tukey correction. b, The Grx–roGFP2 ratio (A405/A488) was calculated using the partial profiling feature (pp) configured to analyse extinction and emission data from 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 opacity (extinction) and length (time of flight) was gated as R1.
Extended Data Fig. 2 Sorting efficiency and lifespan of L2ox and L2red subpopulations.
a–d, Microscopy analysis of the Grx1–roGFP2 ratio of individual worms (dots) previously sorted into L2ox, L2mean and L2red subpopulations. n, number of worms; one-way ANOVA with Tukey correction. e–i, Survival curves of sorted L2ox, L2mean and L2red worms. For n numbers, P values (log-rank test) see Extended Data Table 2. Insets, Grx1–roGFP2 ratio of individual worms (dots), assessed by fluorescence microscopy after sorting. n, number of worms. For P values (two-sided unpaired t-test) see Extended Data Table 2.
Extended Data Fig. 3 Physiological properties of L2ox and L2red sorted worms.
a, Length measurements of L2ox and L2red worms (symbol) from nose to tail tip immediately after sorting. No significant difference; P = 0.4735 (unpaired two-sided t-test). b, Brood size of L2ox and L2red worms, measured at the indicated time points. n, number of worms. No significant difference within a single age; P = 0.6532 (two-way ANOVA). c–f, Basal respiration (c), maximal (d) and spare (e) respiratory capacity and basal rates of flux through glycolysis (f) of L2ox and L2red worms. n = 3 independent sorting experiments. ECAR, extracellular acidification rate; OCR, oxygen consumption rate. P = 0.9469 (c), P = 0.7784 (d), P = 0.7904 (e) and P = 0.7925 (f); two-sided unpaired t-test. g, Survival of L2ox, L2mean and L2red worms 20 h after heat shock. n = 5 independent sorting experiments; two-sided unpaired t-test. The connected data points represent data from the same sorting experiment. The survival of L2ox is set to 1. All data are mean ± s.e.m.
Extended Data Fig. 4 Gene expression profiles of L2ox and L2red.
a, Steady-state transcript levels of selected oxidative stress-related genes in L2ox and L2red worms. n, number of independent sorting experiments; unpaired two-sided t-test. Data are mean ± s.e.m. b, Volcano plot showing fold changes versus P values for the transcriptomes of L2ox and L2red subpopulations. DEGs (P ≤ 0.05) are represented by red dots (see Methods for statistical definition of DEGs). Data were collected from four independent sorting experiments. c, GSEA 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 or phenotype associated with genes known to have 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 L2ox that intersect with H3K4me3 peak signals within their 5′ region (500 bp upstream and downstream from the transcription start site). The H3K4me3 chromatin immunoprecipitation (ChIP) datasets were generated from L3-staged N2 worms: ChIP chip, GEO entry GSE30789 in d and chromatin immunoprecipitation followed by sequencing (ChIP–seq), GEO entry GSE28770 in e, indicating that these marks are set during larval development. Hypergeometric probability: d, P = 0.064; e, P = 2.786 × 10−6. In f, g, Venn diagrams show the overlap among upregulated (f) or downregulated (g) gene sets in L2ox and downregulated or upregulated set-9(rw5) and set-26(tm2467) gene sets (GEO entry: GSE100623). See Supplementary Table 1 for datasets in d–g.
Extended Data Fig. 5 Redox sensitivity of in vivo H3K4m3e3 levels and in vitro histone methyltransferase complex activity.
a, Global H3K4me3 levels in the sorted L2ox and L2red worms. A representative western blot using antibodies against H3K4me3 is shown. b, c, Quantification of global H3K27ac (b) and H3K27me3 (c) levels by western blot. n = 3 independent sorting experiments. P = 0.3793 (b) and P = 0.0905 (c); unpaired two-sided t-test. Data represent mean ± s.e.m. d–f, 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 COMPASS subunits (SET domain of MLL1–WDR5–ASH2L–RBBP5). Reaction rates were derived from the first 20 min of the linear range. h–j, In vitro histone methyltransferase assays of core COMPASS subunits, consisting of purified GST–WDR5 (WDR5), GST–ASH2L (ASH2L), GST–RBBP5 (RBBP5) and either GST–MLL1 SET domain or untagged MLL1 SET domain (h), GST–SET1A SET domain (i) or GST–SET1B SET domain (j). Superscript ox indicates that the protein was pre-treated with either 1 mM (+) or 2 mM (++) H2O2 for 30 min before the activity assay. DTT was added after the H2O2 treatment. n = 3 independent experiments; one-way ANOVA with Sidak correction. Data are mean ± s.e.m. k, The MLL1 SET domain was treated with either 2 mM DTT, 2 mM H2O2 or 2 mM H2O2, followed by 4 mM DTT. Catalase was used to quench the H2O2. The proteins were denatured and thiols were modified with NEM before loading onto non-reducing SDS–PAGE to prevent non-specific thiol oxidation. The proteins were visualized by silver staining. M, marker. l, MLL1 SET domain treated with either 2 mM DTT, 2 mM H2O2 or 2 mM H2O2, followed by 4 mM DTT. All reduced protein thiols were then labelled with the 500-Da thiol-reactive compound AMS, causing a 500-Da mass decrease per oxidized thiol—detectable on reducing SDS–PAGE. M, protein marker. m, Cysteine oxidation state in MLL1 SET domain after treatment with either 2 mM DTT or 2 mM H2O2 followed by NEM labelling 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 domain. For blot and gel source images, see Supplementary Figs. 1 and 3. o, Sequence alignment of the SET domain. All cysteines 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 Fig. 6 Effects of H3K4me3 downregulation 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 two-sided t-test. 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 two generations using western blot analysis. c, H3K4me3 levels in N2jrIs2[Prpl-17::Grx1-roGFP2] worms treated with control RNAi, ash-2 RNAi or set-2 RNAi for two 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 two-sided t-test. h, Representative survival curves of N2jrIs2[Prpl-17::Grx1-roGFP2] worms treated with ash-2 or set-2 RNAi for two generations and treated with 1 mM paraquat (PQ) for 10 h at the L2 larval stage. For n numbers, repetitions and statistics (log-rank), see Extended Data Table 4. Data in a, d–g, represent mean ± s.e.m. For blot source images, see Supplementary Figs. 1, 3.
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Supplementary Figures
Supplementary Figure 1-3: Full gel and blot images.
Supplementary Table
Supplementary Table 1: RNA-sequencing data including DEGs and GSEA analysis related to Extended Data Fig. 3b and c. Data used for Venn diagrams and pie charts in Fig. 3b, Extended Data Fig. 3d-g are also included.
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Bazopoulou, D., Knoefler, D., Zheng, Y. et al. Developmental ROS individualizes organismal stress resistance and lifespan. Nature 576, 301–305 (2019). https://doi.org/10.1038/s41586-019-1814-y
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DOI: https://doi.org/10.1038/s41586-019-1814-y
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