Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

N6-methyldeoxyadenine is a transgenerational epigenetic signal for mitochondrial stress adaptation

Nature Cell Biology volume 21, pages 319–327 (2019)Cite this article

Subjects

Abstract

N6-methyldeoxyadenine (6mA), a major type of DNA methylation in bacteria, represents a part of restriction-modification systems to discriminate host genome from invader DNA1. With the recent advent of more sensitive detection techniques, 6mA has also been detected in some eukaryotes2,3,4,5,6,7,8. However, the physiological function of this epigenetic mark in eukaryotes remains elusive. Heritable changes in DNA 5mC methylation have been associated with transgenerational inheritance of responses to a high-fat diet9, thus raising the exciting possibility that 6mA may also be transmitted across generations and serve as a carrier of inheritable information. Using Caenorhabditis elegans as a model, here we report that histone H3K4me3 and DNA 6mA modifications are required for the transmission of mitochondrial stress adaptations to progeny. Intriguingly, the global DNA 6mA level is significantly elevated following mitochondrial perturbation. N6-methyldeoxyadenine marks mitochondrial stress response genes and promotes their transcription to alleviate mitochondrial stress in progeny. These findings suggest that 6mA is a precisely regulated epigenetic mark that modulates stress response and signals transgenerational inheritance in C. elegans.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Mitochondrial perturbation transmits stress adaptation to progeny.
Fig. 2: H3K4me3 is required for the inheritance of mitochondrial stress adaptation.
Fig. 3: DNA 6mA mediates the transmission of mitochondrial stress adaptation.
Fig. 4: 6mA marks ATFS-1-mediated mitochondrial stress response genes to promote stress adaptation in progeny.
Fig. 5: 6mA responds to mitochondrial stress and improves animal fitness.

Similar content being viewed by others

Data availability

Deep-sequencing (ChIP-seq, MeDIP-seq and RNA-seq) data that support the findings of this study have been deposited in the GEO under accession codes GSE85835 (H3K4me3 ChIP-seq), GSE118268 (6mA MeDIP-seq) and GSE118175 (RNA-seq). Previously published ATFS-1 ChIP-seq and atfs-1 mutant microarray data that were re-analysed here are under the accession codes GSE63803 (ATFS-1 ChIP-seq)13 and GSE38196 (atfs-1 mutant microarray)17.

References

  1. Heyn, H. & Esteller, M. An adenine code for DNA: a second life for N6-methyladenine. Cell 161, 710–713 (2015).

    Article  CAS  Google Scholar 

  2. Greer, E. L. et al. DNA methylation on N6-adenine in C. elegans. Cell 161, 868–878 (2015).

    Article  CAS  Google Scholar 

  3. Zhang, G. et al. N6-methyladenine DNA modification in Drosophila. Cell 161, 893–906 (2015).

    Article  CAS  Google Scholar 

  4. Koziol, M. J. et al. Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications. Nat. Struct. Mol. Biol. 23, 24–30 (2016).

    Article  CAS  Google Scholar 

  5. Wu, T. P. et al. DNA methylation on N(6)-adenine in mammalian embryonic stem cells. Nature 532, 329–333 (2016).

    Article  CAS  Google Scholar 

  6. Liu, J. et al. Abundant DNA 6mA methylation during early embryogenesis of zebrafish and pig. Nat. Commun. 7, 13052 (2016).

    Article  CAS  Google Scholar 

  7. Yao, B. et al. DNA N6-methyladenine is dynamically regulated in the mouse brain following environmental stress. Nat. Commun. 8, 1122 (2017).

    Article  Google Scholar 

  8. Xiao, C. L. et al. N(6)-methyladenine DNA modification in the human genome. Mol. Cell 71, 306–318.e307 (2018).

    Article  CAS  Google Scholar 

  9. Ng, S. F. et al. Chronic high-fat diet in fathers programs beta-cell dysfunction in female rat offspring. Nature 467, 963–966 (2010).

    Article  CAS  Google Scholar 

  10. Wang, S. & Kaufman, R. J. The impact of the unfolded protein response on human disease. J. Cell Biol. 197, 857–867 (2012).

    Article  CAS  Google Scholar 

  11. Yoneda, T. et al. Compartment-specific perturbation of protein handling activates genes encoding mitochondrial chaperones. J. Cell Sci. 117, 4055–4066 (2004).

    Article  CAS  Google Scholar 

  12. Houtkooper, R. H. et al. Mitonuclear protein imbalance as a conserved longevity mechanism. Nature 497, 451–457 (2013).

    Article  CAS  Google Scholar 

  13. Nargund, A. M., Fiorese, C. J., Pellegrino, M. W., Deng, P. & Haynes, C. M. Mitochondrial and nuclear accumulation of the transcription factor ATFS-1 promotes OXPHOS recovery during the UPR(mt). Mol. Cell 58, 123–133 (2015).

    Article  CAS  Google Scholar 

  14. Liu, Y., Samuel, B. S., Breen, P. C. & Ruvkun, G. Caenorhabditis elegans pathways that surveil and defend mitochondria. Nature 508, 406–410 (2014).

    Article  CAS  Google Scholar 

  15. Greer, E. L. et al. Transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans. Nature 479, 365–371 (2011).

    Article  CAS  Google Scholar 

  16. Greer, E. L. et al. Members of the H3K4 trimethylation complex regulate lifespan in a germline-dependent manner in C. elegans. Nature 466, 383–387 (2010).

    Article  CAS  Google Scholar 

  17. Nargund, A. M., Pellegrino, M. W., Fiorese, C. J., Baker, B. M. & Haynes, C. M. Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. Science 337, 587–590 (2012).

    Article  CAS  Google Scholar 

  18. R Core Team R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2013).

  19. Stouder, C. & Paoloni-Giacobino, A. Transgenerational effects of the endocrine disruptor vinclozolin on the methylation pattern of imprinted genes in the mouse sperm. Reproduction 139, 373–379 (2010).

    Article  CAS  Google Scholar 

  20. Burdge, G. C. et al. Dietary protein restriction of pregnant rats in the F0 generation induces altered methylation of hepatic gene promoters in the adult male offspring in the F1 and F2 generations. Br. J. Nutr. 97, 435–439 (2007).

    Article  CAS  Google Scholar 

  21. Marinus, M. G. & Morris, N. R. Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12. J. Bacteriol. 114, 1143–1150 (1973).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Christensen, J. et al. RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3. Cell 128, 1063–1076 (2007).

    Article  CAS  Google Scholar 

  23. Dillin, A. et al. Rates of behavior and aging specified by mitochondrial function during development. Science 298, 2398–2401 (2002).

    Article  CAS  Google Scholar 

  24. Lee, S. S. et al. A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nat. Genet. 33, 40–48 (2003).

    Article  CAS  Google Scholar 

  25. Copeland, J. M. et al. Extension of Drosophila life span by RNAi of the mitochondrial respiratory chain. Curr. Biol. 19, 1591–1598 (2009).

    Article  CAS  Google Scholar 

  26. Liu, X. et al. Evolutionary conservation of the clk-1-dependent mechanism of longevity: loss of mclk1 increases cellular fitness and lifespan in mice. Genes Dev. 19, 2424–2434 (2005).

    Article  CAS  Google Scholar 

  27. Melo, J. A. & Ruvkun, G. Inactivation of conserved C. elegans genes engages pathogen- and xenobiotic-associated defenses. Cell 149, 452–466 (2012).

    Article  CAS  Google Scholar 

  28. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Article  CAS  Google Scholar 

  29. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  Google Scholar 

  30. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  Google Scholar 

  31. Ramirez, F., Dundar, F., Diehl, S., Gruning, B. A. & Manke, T. deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res. 42, W187–W191 (2014).

    Article  CAS  Google Scholar 

  32. Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    Article  CAS  Google Scholar 

  33. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    Article  CAS  Google Scholar 

  34. Eden, E., Navon, R., Steinfeld, I., Lipson, D. & Yakhini, Z. GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinform. 10, 48 (2009).

    Article  Google Scholar 

  35. Wickham, H. ggplot2: Elegant Graphics For Data Analysis (Springer, New York, 2009).

Download references

Acknowledgements

We thank G. Ruvkun and Q. Liu for critical suggestions and proofreading. We thank J. Zhu for providing cell line and technical support, C. Haynes, the National Bio-Resource Project and the Caenorhabditis Genetics Center (funded by NIH Office of Research Infrastructure Programs grant no. P40 OD010440) for providing strains. This work was supported by the National Natural Science Foundation of China (grant nos.91854205, 31422033, and 31471381) the Ministry of Science and Technology of China (National Key Research and Development Program of China grant no. 2017YFA0504000973 and 973 grant no. 2013CB910104), Peking-Tsinghua Center for Life Sciences and an HHMI International Research Scholar Award (grant no. 55008739) to Y.Liu. C. Ma is supported in part by the Postdoctoral Fellowship of Peking-Tsinghua Center for Life Sciences.

Author information

Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Beijing Key Laboratory of Cardiometabolic Molecular Medicine and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China

    Chengchuan Ma, Rong Niu, Tianxiao Huang, Li-Wa Shao, Yong Peng, Wanqiu Ding, Chuan-Yun Li, Aibin He & Ying Liu

  2. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Chengchuan Ma, Li-Wa Shao & Yong Peng

  3. Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    Ye Wang, Guifang Jia & Chuan He

  4. Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA

    Chuan He

  5. Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA

    Chuan He

Authors
  1. Chengchuan Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rong Niu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tianxiao Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Li-Wa Shao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yong Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wanqiu Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ye Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Guifang Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chuan He
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Chuan-Yun Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Aibin He
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Ying Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.M. and Y.L. designed the experiments. C.M., R.N. and L.-W.S. carried out the worm experiments. Y.W., G.J. and C.H. performed the 6mA mass spectrometry analysis. Y.P. and A.H. performed H3K4me3 ChIP-seq analysis. T.H., W.D. and C.-Y.L. carried out MeDIP-seq analysis. T.H. performed RNA-seq analysis. C.M. and Y.L. wrote the manuscript.

Corresponding author

Correspondence to Ying Liu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Antimycin induces developmental delay in C. elegans.

a-d, Wild-type worms were treated with 0 μM (a), 0.96 μM (b), 1.92 μM (c), or 2.88 μM (d) antimycin. Images were taken 57 h after freshly laid eggs were treated with antimycin. e, Adult percentage of wild-type worms treated with different doses of antimycin. Adult worms were counted 57 h after freshly laid eggs were treated with antimycin. f, Time course experiment showing percentage of adult in untreated or 1.92 μM antimycin-treated animals. g, Representative images showing developmental stage of untreated or antimycin-treated animals. All experiments have been repeated independently for three times with similar results . n = 3 biologically independent experiments (e,f), >100 animals per experiment (e,f). Data in f are presented as mean ± s.e.m. Statistical source data are provided in Supplementary Table 7.

Supplementary Figure 2 Inter/transgenerational inheritance of stress adaptation is a general feature of mitochondrial dysfunction.

a,b, ATP content of worms in each generation in the intergenerational (a) and transgenerational (b) experiment. c-g, Intergenerational inheritance experiment tested in wild-type animals treated with TTFA (c), sodium azide (d), or 0.32 μM (e) 0.64 μM (f) and 1.28 μM (g) tunicamycin. n = 3 biologically independent experiments (a-g), ~ 10,000 animals per experiment (a,b), >100 animals per experiment (c-g). Data are presented as mean ± s.e.m. Statistical analysis was performed by paired two-tailed t-test; ns, not significant, P > 0.05. P-values are calculated based on comparison between single and sequential exposure groups. Statistical source data are provided in Supplementary Table 7.

Supplementary Figure 3 Inheritance of mitochondrial stress adaptation is not mediated by H3K9, H3K27 and H3K36 methylation.

Intergenerational and transgenerational inheritance tested in met-2 (a,b), jmjd-3.1 (c,d) or met-1 (e,f) mutants. n = 3 biologically independent experiments, >100 animals per experiment. Data are presented as mean ± s.e.m. Statistical analysis was performed by paired two-tailed t-test; ns, not significant, P > 0.05. Statistical source data are provided in Supplementary Table 7. P-values are calculated based on comparison between single and sequential exposure groups in line graphs.

Supplementary Figure 4 DAMT-1 promotes global induction of 6mA upon mitochondrial stress.

a, Genotyping of damt-1 mutant. b, Representative fluorescent images of hsp-6p::gfp or damt-1; hsp-6p::gfp worms fed on control or spg-7 RNAi. Scale bar: 100 μm. c, Genotyping of damt-1 germline rescued worms. d, Intergenerational inheritance tested in damt-1; pie-1p::damt-1 animals. e, Quantification of global 6 mA level in (Fig. 3c) by UHPLC–QqQ-MS/MS. f, Dot blot of untreated wild-type worms, or worms treated with 1.92 μM antimycin, 90 μM TTFA, 4.62 μM NaN3, or 0.64 μM tunicamycin. g, Dot blot of sequential tunicamycin-treated wild-type animals. h, Total RNA and genomic DNA were extracted and tested with DNA 6 mA antibody on dot blot. The result shows that the signals on dot blot probed with DNA 6mA antibody is not due to any contamination of RNA m6A. i, Quantification of total RNA m6A level upon antimycin treatment by UHPLC–QqQ-MS/MS. j, DNA dot blot of wild-type animals fed on dam- dcm- E.coli and treated with or without antimycin. k, Schematic representation of CRISPR–Cas9-mediated knockout of damt-1 in worms. l, Dot blot (top) and intergenerational inheritance (bottom) of damt-1 Cas9 knockout animals. m, in vitro methyltransferase assay tested in nuclear lysates from untreated or antimycin-treated wild-type animals or damt-1 mutants. Worms for dot blot were collected when they reached L4 stage. Data in a-c, f-h, j and l represent results from three independent assays. n = 3 biologically independent experiments (d,i,l,m), >100 animals per experiment (d, l bottom), ~ 100,000 animals per experiment (i,m). Graph data are presented as mean ± s.e.m. Statistical analysis was performed by paired (d,l) or unpaired (i,m) two-tailed t-test; ns, not significant, P > 0.05. Statistical source data are provided in Supplementary Table 7. Uncropped dot blot figures are presented in Supplementary Figure 6.

Supplementary Figure 5 6mA modulates mitochondrial stress response genes and damt-1.

a, GO term analysis of specific peaks in 6 mA MeDIP-seq comparing antimycin-treated vs. untreated wild-type animals. MeDIP-seq was performed twice and the data shown represent pooled results from two assays. b, GO term analysis of RNA-seq, which compares genes enriched from P0 to F2 specifically in wild-type animals, but not in damt-1 mutants, after a single antimycin exposure at P0. Two independent RNA-seq analyses were performed. c,e,g, MeDIP-qPCR analysis of 6mA occupancies on innate immune genes clec-9 and clec-80 (c), damt-1 (e), and heat shock stress or ER stress response gene hsf-1 or xbp-1 (g). d,f, qPCR analysis of mRNA levels of innate immune genes clec-9 and clec-80 (d) and damt-1 (f) with or without antimycin treatment. h, MeDIP-qPCR analysis of 6 mA occupancies on mitochondrial stress response gene or control gene mrpl-2, in untreated or antimycin-treated damt-1 mutants and their recovered progeny. i, qPCR analysis of mRNA levels of mitochondrial stress response gene atfs-1 and hsp-6 in untreated or antimycin-treated damt-1 mutants and their recovered progeny. j, Representative fluorescent images of sdhb-1p::mtGFP transgenic animals raised on control E. coli OP50 or Pseudomonas GRm0260. Scale bar, 50 μm. k, Intergenerational inheritance of mitochondrial stress adaptation tested in wild-type worms raised on control E. coli OP50 or Pseudomonas GRm0260. l, Mean lifespan of untreated or antimycin-treated wild-type animals, damt-1 or atfs-1 mutants, and their recovered progeny. n = 3 biologically independent experiments (c-i, k-l), ~ 100,000 animals (c,e,g,h), ~ 1000 animals (d,f,i), >100 animals (k), ~ 100 animals (l) per experiment. Graph data are presented as mean ± s.e.m. Statistical analysis was performed by paired (h,i,k) or unpaired (c-g, l) two-tailed t-test; one-sided Fisher test for MeDIP-seq and RNA-seq GO term analysis (a,b); ns, not significant, P > 0.05. Statistical source data are provided in Supplementary Table 7. Uncropped genotyping gel figures are presented in Supplementary Figure 6.

Supplementary Figure 6 Uncropped dot and gel figures.

a, Dot blot of wild-type worms with single antimycin exposure at P0, relative to Fig. 3c. b, Dot blot of damt-1 mutant worms treated with antimycin, relative to Fig. 3d. c, Dot blot of spr-5 mutant worms with single antimycin exposure at P0, relative to Fig. 3f. d, Dot blot of spr-5;damt-1 mutant worms with single antimycin exposure at P0, relative to Fig. 3i. e, Dot blot of wild-type worms with sequential or single antimycin exposure, relative to Fig. 4e. f, Dot blot of antimycin-treated wild-type worms or atfs-1 mutants, relative to Fig. 4l. g, Dot blot of antimycin-treated fly S2 cells or dissected ovaries, relative to Fig. 5a. h, Dot blot of wild-type worms, or clk-1, isp-1, phb-2 or spg-7 mutants, relative to Fig. 5d. i, Genotyping of damt-1 mutant, relative to SI-4a. j, Genotyping of damt-1 germline rescued worms, relative to SI-4c. k, Dot blot of antimycin, TTFA, NaN3 or tunicamycin treated wild-type worms, relative to SI-4f. l, Dot blot of wild-type worms with sequential tunicamycin treatment, relative to SI-4g. m, Total RNA and genomic DNA were extracted and tested with DNA 6 mA antibody on dot blot, relative to SI-4h. o, Dot blot of antimycin-treated wild-type worms or CRISPR-cas9 damt-1 knockout worms, relative to SI-4j. Boxes indicate the cropped sections used in the corresponding figures.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ma, C., Niu, R., Huang, T. et al. N6-methyldeoxyadenine is a transgenerational epigenetic signal for mitochondrial stress adaptation. Nat Cell Biol 21, 319–327 (2019). https://doi.org/10.1038/s41556-018-0238-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-018-0238-5

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing