Two conserved epigenetic regulators prevent healthy ageing

Abstract

It has long been assumed that lifespan and healthspan correlate strongly, yet the two can be clearly dissociated1,2,3,4,5,6. Although there has been a global increase in human life expectancy, increasing longevity is rarely accompanied by an extended healthspan4,7. Thus, understanding the origin of healthy behaviours in old people remains an important and challenging task. Here we report a conserved epigenetic mechanism underlying healthy ageing. Through genome-wide RNA-interference-based screening of genes that regulate behavioural deterioration in ageing Caenorhabditis elegans, we identify 59 genes as potential modulators of the rate of age-related behavioural deterioration. Among these modulators, we found that a neuronal epigenetic reader, BAZ-2, and a neuronal histone 3 lysine 9 methyltransferase, SET-6, accelerate behavioural deterioration in C. elegans by reducing mitochondrial function, repressing the expression of nuclear-encoded mitochondrial proteins. This mechanism is conserved in cultured mouse neurons and human cells. Examination of human databases8,9 shows that expression of the human orthologues of these C. elegans regulators, BAZ2B and EHMT1, in the frontal cortex increases with age and correlates positively with the progression of Alzheimer’s disease. Furthermore, ablation of Baz2b, the mouse orthologue of BAZ-2, attenuates age-dependent body-weight gain and prevents cognitive decline in ageing mice. Thus our genome-wide RNA-interference screen in C. elegans has unravelled conserved epigenetic negative regulators of ageing, suggesting possible ways to achieve healthy ageing.

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Fig. 1: A genome-wide screen of C. elegans identifies genes that regulate age-related loss of 5-HT and dopamine.
Fig. 2: Deletion of baz-2 or set-6 delays age-related behavioural decline and extends lifespan in C. elegans.
Fig. 3: BAZ-2 and SET-6 repress mitochondrial function.
Fig. 4: Knockout of Baz2b improves spatial learning and memory abilities in old mice.

Data availability

The raw sequence data generated here have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA554977. Source Data for Figs. 14 and Extended Data Figs. 110 are provided with the paper.

Code availability

All custom code used to generate figures is available at https://github.com/SHYKON-YIN/nature.

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Acknowledgements

We thank M-m. Poo and X. Shi for critical reading of the manuscript; Q. Sun, Y.-J. Cai and Y.-Z. Li for generating Baz2b-knockout mice; the C. elegans Genetics Center (funded by the National Institutes of Health (NIH) Office of Research Infrastructure Programs P40 OD010440) for providing strains; members of C.-y. Li’s laboratory for helping with mouse behavioural assays; M. Sun for helping to prepare the H3K9 ChIP–seq DNA libraries; and the Institute of Neuroscience Molecular and Cellular Biology Core Facility, the Optical Imaging Facility, and the Animal Facility (Chinese Academy of Sciences) for technical support. This work was supported by grants to S.-Q.C. (from the National Natural Science Foundation of China, grants 31925022, 91949206 and 81527901; and from the National Key R&D Program of China, grant 2018YFC2000400); the Strategic Priority Research Program of the Chinese Academy of Science (grant XDB32020100); the Shanghai Municipal Science and Technology Major Project (grant 2018SHZDZX05); and grants to L.J. (from the National Key R&D Program of China, grant 2018YFA0507300; and the National Natural Science Foundation of China, grant 31771455).

Author information

J.Y. conducted HPLC assays, imaging assays and most of the behavioural assays; performed co-immunoprecipitation assays and western blots to detect the H3K9 methylation level in C. elegans; prepared the mRNA samples for RNA-seq; conducted experiments in mammalian cells; and analysed the gene-expression profile from human brain gene-expression datasets. J.Y. and X.-J.L. performed the genome-wide RNAi screen as well as lifespan and stress-resistance assays. S.-Y.C. conducted the RT–qPCR assays in C. elegans and the qPCR-based determination of mitochondrial DNA/nuclear DNA ratios; detected the ATP level and OCR in C. elegans; conducted UPRmt activation assays; and performed behavioural and lifespan assays related to UPRmt. S.-Y.C. and X.-J.L. performed the lifespan and H2O2 stress resistance assays in mutants of different longevity pathways. X.-J.L. carried out assays of male mating behaviour. S.-G.Y. analysed the ChIP–seq data and the RNA-seq data of C. elegans, and helped to analyse gene-expression levels from human brain datasets. X.C. conducted the in vitro histone methyltransferase assay and ChIP–seq assays in C. elegans. Z.-Y.L. performed the co-immunoprecipitation and ChIP–qPCR assays in mammalian cells. Z.-Y.L. and S.-W.Y. measured the OCRs of isolated mitochondria from mice. G.G. carried out co-immunoprecipitation of SET-6 and BAZ-2. X.-L.K. and Z.C. helped to prepare samples for ChIP–seq assays in C. elegans. J.-A.Y. performed part of the assays for ATP detection in mouse neurons. Z.-Y.L. and D.-Y.L. performed mouse behavioural assays. Q.J. helped with the BSR and ESR assays. P.H. supervised the ChIP–seq data analysis. L.J. supervised experiments regarding epigenetics and analysis of the data. S.-Q.C. conceived the project. S.-Q.C., L. J., J.Y., S.-Y.C. and S.-G.Y. designed the study, analysed the data, and wrote the manuscript with input from all authors.

Correspondence to Lubin Jiang or Shi-Qing Cai.

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Peer review information Nature thanks Cole Haynes and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Categories and coexpression network of genome-wide RNAi screening hits.

a, Effects of representative positive clones on BAS-1 protein levels in Pbas-1::bas-1::gfp transgenic worms. White arrows indicate NSM neurons. Scale bar, 20 µm. Images are representative of three independent experiments. b, Effect of each screening hit on the expression of BAS-1::GFP. The GFP fluorescence in NSM neurons of worms individually treated with the 59 screening hits, also shown in Supplementary Table 1, was normalized to that in worms treated with the control RNAi. P values were determined by unpaired two-tailed t-test (see Supplementary Table 1 for numbers of tested worms and exact P values). c, Categories of genes corresponding to the 59 screening hits identified by RNAi. d, Coexpression network of screening hits and their partners. The network was constructed with GeneMANIA. Black and grey dots indicate screening hits and their partners, respectively. Blue and orange dots show screening hits whose human homologues are involved in age-related neurodegeneration and cell senescence, respectively. e, Pharyngeal pumping in TU3401 worms (which express a dsRNA channel, SID-1, in neurons) after RNAi treatment in the presence of 20 μM 2′-deoxy-5-fluorouridine (FUDR). The genes from the top 20 screening hits were examined. Numbers of tested worms are shown beneath the bars. Data shown are means ± s.e.m.; *P < 0.05; **P < 0.05; ***P < 0.001; ns, not significant (one-way ANOVA with Dunnett's test; see Supplementary Information for exact P values)Source Data.

Extended Data Fig. 2 BAZ-2 and SET-6 regulate age-related decline in BAS-1 expression.

a, Expression patterns of BAZ-2 and SET-6. Scale bar, 30 μm. Images are representative of three independent experiments. b, Representative fluorescence images (left) and quantitative analysis (right) of BAZ-2 and SET-6 expression. We quantified only those cells whose nuclear morphology was clearly visualized with DAPI. Scale bar, 10 μm. n = 14 and 18 worms for BAZ-2 and SET-6, respectively. c, d, Representative western blots (c) and quantitative analysis (d) of age-related changes in endogenous SET-6 and BAZ-2 protein levels in genome-edited baz-2GFP::FLAG;set-6GFP::HA worms. n = 5 independent experiments. Tubulin expression is used as a reference. For gel source data, see Supplementary Fig. 2. In d, each data point represents the result of one independent experiment. e, Fluorescence images (left) and quantitative analysis (right) of BAS-1 expression in baz-2 and set-6 mutant worms. Quantitative analysis of BAS-1 levels was performed by measuring GFP fluorescence intensity in the soma of NSM neurons. White arrows indicate NSM neurons. Scale bar, 15  μm. The numbers of tested worms are shown beneath the bars. All data shown are means ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (d, Kruskal–Wallis test; e, one-way ANOVA with Dunnett’s test; see Supplementary Information for exact P values). Source Data

Extended Data Fig. 3 Deletion of baz-2 or set-6 extends lifespan and enhances stress resistance via mechanisms related to dietary restriction and mitochondrial function.

ac, Percentage survival of N2, baz-2, set-6, and baz-2;set-6 worms under oxidative (a), ultraviolet (b) and heat (c) stress. d, Lifespan curves of daf-16, daf-16;baz-2, and daf-16;set-6 mutant worms (left) and their abilities to resist to oxidative stress (right). e, Lifespan curves of eat-2, eat-2;baz-2, and eat-2;set-6 mutant worms (left) and their abilities to resist to oxidative stress (right). f, Lifespan curves of clk-1, clk-1;baz-2, and clk-1;set-6 mutant worms (left) and their abilities to resist to oxidative stress (right). In af, for oxidative-stress assays, data shown are means ± s.e.m.; one-way ANOVA with Dunnett’s test; numbers of independent experiments are shown beneath the bars. For heat-shock, ultraviolet-stress and lifespan assays, data represent the sum of animals in multiple experiments; two-sided log-rank test. The numbers of independent experiments and of tested hermaphrodites are indicated in parentheses. In all assays, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (see Supplementary Information for exact P values). Source Data

Extended Data Fig. 4 Epigenetic regulators BAZ-2 and SET-6 localize at the promoter region of target genes.

a, Co-immunoprecipitation of BAZ-2 and SET-6 using genome-edited baz-2GFP::FLAG;set-6GFP::HA worms (left) or transgenic worms expressing BAZ-2::FLAG and SET-6::HA (right). Images are representative of four independent experiments. For gel source data, see Supplementary Fig. 3. b, Representative western blots (left) and quantitative analysis (right) of H3K9 methylation levels in N2, baz-2, set-6 and baz-2;set-6 worms. Normalized H3K9 methylation levels were calculated by normalizing the ratio of H3K9 methylation and histone 3 levels to that of N2 worms. For gel source data, see Supplementary Fig. 4. c, Peaks of BAZ-2- and SET-6-binding sites in the region −1,000 bp to +1000 bp around the transcription start site (TSS). Only those peaks with a fold change of more than 2 are plotted. The y-axis indicates the average read coverage normalized to the number of uniquely mapped reads per million per genomic bin (bins = 1,000). d, Pie chart showing the distribution of overlapping BAZ-2- and SET-6-binding sites in genomic features. TTS, transcription termination site. e, f, ChIP-qPCR analysis of endogenous BAZ-2 (e) or SET-6 (f) enrichment at nuclear genes encoding mitochondrial proteins in genome-edited baz-2GFP::FLAG and set-6GFP::HA worms. ChIP-qPCR data from N2 worms were used as a control. In b, e, f, the numbers of independent experiments are shown beneath the bars; data are means ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001 (b, one-way ANOVA with Dunnett’s test; e, f, two-tailed t-test; see Supplementary Information for exact P values). Source Data

Extended Data Fig. 5 BAZ-2 and SET-6 regulate the expression of nuclear genes encoding mitochondrial proteins.

a, Scatter plots of mRNA-seq data for N2 versus baz-2 mutant worms (left) and N2 versus set-6 mutant worms (right). The x- and y-axes represent the log10-transformed transcripts per million clean tags (TPM) expression values of N2 (x-axis), baz-2 (left, y-axis) and set-6 (right, y-axis) animals. Differentially expressed genes (DEGs) were defined through the parameters of false discovery rate (FDR) ≤ 0.001 and |log2Ratio| ≥ 1. b, RT–PCR analysis of changes in the expression of nuclear genes encoding mitochondrial proteins in baz-2 and set-6 mutant worms at day 1 (left) or day 7 (right) of adulthood. The numbers of independent experiments are shown beneath the bars. c, Western blots (left) and quantitative analysis (right) of nDNA-encoded ATP5A and mtDNA-encoded MTCO1 proteins. n = 3 independent experiments. For gel source data, see Supplementary Fig. 5. In b, c, data shown are means ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (one-way ANOVA with Dunnett’s test; see Supplementary Information for exact P values). Source Data

Extended Data Fig. 6 BAZ-2 and SET-6 repress gene expression by altering the H3K9me3 level of target genes.

a, Heatmaps showing gene expression (left) and occupation by BAZ-2 and SET-6 at DNA regions (middle and right) of 450 differentially expressed genes. The enrichment of genes was analysed at DAVID Bioinformatics Resources; a hypergeometric test was performed using mitochondrial and ribosome genes from cluster 2 for statistical analysis. bd, Average-read-coverage profiles of the cluster-2 differentially expressed genes for ChIP analysis of H3K9me3 (b), H3K9me2 (c) and H3K9me1 (d) from N2, baz-2 and set-6 mutant worms at day 2 (A2) and day 7 (A7) of adulthood. RPM, reads per million mapped reads. e, ChIP–seq profiles of H3K9me1, H3K9me2 and H3K9me3 at the indicated DNA regions of representative cluster-2 genes, cyc-2.1 on chromosome (Chr.) IV and gst-36 on chromosome X. The ChIP–seq analysis of H3K9me3 was carried out twice, with averages shown. Source Data

Extended Data Fig. 7 Human homologues of BAZ-2 and SET-6.

a, Diagram showing the similarity between C. elegans BAZ-2 and SET-6 and their human homologues. aa, amino acids; ANK, ankyrin repeats; BROMO, bromo domain; DDT, ‘DNA-binding homeobox and different transcription factors’ domain; MBD, methyl-CpG binding domain; PHD, plant homeodomain; SET, Su(var.)3-9, Enhancer-of-zeste, Trithorax domain; b, Alignment of conserved domains in BAZ-2 or SET-6 with those in their mammalian homologues. Identical and conservative residues are highlighted in black and in grey, respectively.

Extended Data Fig. 8 Mammalian Baz2b and Ehmt1 have a conserved role in repressing mitochondrial function.

a, b, Transcription levels of BAZ2A and EHMT2 in the prefrontal cortex of human brains at different ages. Expression values in a (n = 30 samples) are from the dataset GSE1572 (see ref. 8), and in b (n = 145 samples) are from brain samples without neurodegenerative disease in the dataset GSE44772 (see ref. 9). Pearson’s r correlation coefficient was used for statistical testing. c, Co-immunoprecipitation of BAZ2B and EHMT1 in HEK293T cells. Images are representative of three independent experiments. For gel source data, see Supplementary Fig. 6. d, ChIP-qPCR analysis of BAZ2B or EHMT1 enrichment at nuclear genes encoding mitochondrial proteins in HEK293T cells. Immunoglobulin G (IgG) antibody was used as a control. eg, Effects of downregulation of mouse Baz2b or Ehmt1 by short hairpin RNAs (shRNAs) on the transcription of mitochondria-related genes (e), ATP level (f) and oxygen-consumption rate (g) in primary mouse cerebellar neurons. F, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP); NC, negative control; O, oligomycin; R, rotenone. hi, Oxygen-consumption rate (h) and ATP content (i) in primary mouse cortical neurons overexpressing (OE) mouse Baz2b or Ehmt1. Graphical OCR data are representative of three independent experiments. In di, sample numbers are shown beneath the bars. Data shown are means ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001 (d, two-tailed t-test, eh, one-way ANOVA with Dunnett’s test; i, Kruskal–Wallis test; see Supplementary Information for exact P values). Source Data

Extended Data Fig. 9 Effects of deleting Baz2b on mitochondrial function and mouse behaviour.

a, Diagram showing the generation of Baz2b−/− (KO) mice. UTR, untranslated repeat; WT, wild-type. Protospacer-adjacent motif (PAM) sequences are highlighted in green; the substitution site is highlighted in red. b, Western blot analysis of Baz2b protein level in samples from WT, Baz2b+/ and Baz2b−/− mice. Images are representative of three independent experiments. For gel source data, see Supplementary Fig. 7. c, Oxygen-consumption rates (OCRs) of mitochondria isolated from 12-month-old WT, Baz2b+/ and Baz2b−/− male mice. A, antimycin; F, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP); O, oligomycin; R, rotenone. n = 4 mice per group. P values were determined by one-way ANOVA with Dunnett’s test. d, e, Spontaneous locomotion of old (d) and young (e) mice in an open field test. The numbers of tested mice are shown beneath the bars. f, Escape latency in Barnes maze trials during training days for young WT, Baz2b+/ and Baz2b−/− mice. Numbers of tested mice are indicated in parentheses. In all assays, data shown are means ± s.e.m. Source Data

Extended Data Fig. 10 Expression level of BAZ2B and EHMT1 correlates positively with the progression of Alzheimer’s disease in humans.

a, Expression level of BAZ2B (left) and EHMT1 (right) at different Braak stages in the prefrontal cortex of brains with Alzheimer’s disease. b, Expression level of BAZ2B (left) and EHMT1 (right) at different stages of frontal atrophy in the prefrontal cortex of brains with Alzheimer’s disease. ce, Correlations between the expression levels of BAZ2B (x-axes) and selected nuclear genes (encoding mitochondrial proteins; y-axes) in the prefrontal cortex of brains with Alzheimer’s disease. fh, Correlations between the expression levels of EHMT1 (x-axes) and selected nuclear genes (encoding mitochondrial proteins; y-axes) in the prefrontal cortex of brains with Alzheimer’s disease. In ah, expression values (n = 390 samples) of examined genes are from the dataset GSE44772, using Pearson’s r correlation coefficient for statistical testing. i, Proposed working model for the epigenetic regulation of mitochondrial function and healthy ageing. Source Data

Supplementary information

Supplementary Information

This file contains statistical test results, lists of RT-PCR primers and uncropped gel images.

Reporting Summary

Supplementary Table 1

Positive genes obtained by the RNAi screen.

Supplementary Table 2

Overlapping genes that bind to BAZ-2 and SET-6.

Supplementary Table 3

Common differentially expressed genes in baz-2 and set-6 mutant worms.

Supplementary Table 4

The efficiency of Baz2b and Ehmt1 shRNAs in primary mouse neurons.

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Yuan, J., Chang, S., Yin, S. et al. Two conserved epigenetic regulators prevent healthy ageing. Nature 579, 118–122 (2020). https://doi.org/10.1038/s41586-020-2037-y

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