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.

  • Protocol
  • Published:

Simultaneously measuring the methylation of parent and daughter strands of replicated DNA at the single-molecule level by Hammer-seq

Nature Protocols volume 16, pages 2131–2157 (2021)Cite this article

Subjects

Abstract

The stable maintenance of DNA methylation patterns during mitotic cell division is crucial for cell identity. Precisely determining the maintenance kinetics and dissecting the exact contributions of relevant regulators requires a method to accurately measure parent and daughter strand DNA methylation at the same time, ideally at the single-molecule level. Recently, we developed a method referred to as Hammer-seq (hairpin-assisted mapping of methylation of replicated DNA) that fulfils the above criteria. This method integrates 5-ethynyl-2′-deoxyuridine (EdU) labeling of replicating DNA, biotin conjugation and streptavidin-based affinity purification, and whole-genome hairpin bisulfite sequencing technologies. Hammer-seq offers the unique advantage of simultaneously measuring the methylation status of parent and daughter strands within a single DNA molecule, which makes it possible to determine maintenance kinetics across various genomic regions without averaging effects from bulk measurements and to assess de novo methylation events that accompany methylation maintenance. Importantly, when combined with mutant cell lines in which mechanisms of interest are disrupted, Hammer-seq can be applied to determine the functional contributions of potential regulators to methylation maintenance, with accurate kinetics information that cannot be acquired with other currently available methods. Hammer-seq library preparation requires ~100 ug EdU-labeled genomic DNA as input (~15 million mammalian cells). The whole protocol, from pulse labeling to library construction, can be completed within 2–3 d, depending on the chasing time.

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: Schematic overview of Hammer-seq.
Fig. 2: Quality control of key steps of Hammer-seq with DNA electrophoresis.
Fig. 3: Click reaction system optimization.
Fig. 4: Size distribution of example Hammer-seq libraries determined with a bioanalyzer.
Fig. 5: Illustration of hairpin removal and original sequence recovery.
Fig. 6: Calculation of the maintenance ratio and de novo methylation frequency with Hammer-seq data.

Similar content being viewed by others

Data availability

Figure 6a–c,e and Tables 2 and 3 have associated raw data which have been uploaded to the NCBI Gene Expression Omnibus (GSE131098). More specifically, Fig. 6a,e and Tables 2 and 3 are associated with GSM3763438/39/42/43/46, Fig. 6b is associated with GSM4132515/6/7/8/9, and Fig. 6c is associated with GSM4132541/GSM4633219 (see Table 4 for details about the datasets and their corresponding accession numbers).

Code availability

Adapted HBS-tools and custom script tools used in this protocol are available from https://github.com/realzhang/hammer-seq.

References

  1. Schubeler, D. Function and information content of DNA methylation. Nature 517, 321–326 (2015).

    Article  CAS  Google Scholar 

  2. Li, E. & Zhang, Y. DNA methylation in mammals. Cold Spring Harb. Perspect. Biol. 6, a019133 (2014).

    Article  Google Scholar 

  3. Jones, P. A. & Liang, G. Rethinking how DNA methylation patterns are maintained. Nat. Rev. Genet. 10, 805–811 (2009).

    Article  CAS  Google Scholar 

  4. Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21 (2002).

    Article  CAS  Google Scholar 

  5. Araujo, F. D., Knox, J. D., Szyf, M., Price, G. B. & Zannis-Hadjopoulos, M. Concurrent replication and methylation at mammalian origins of replication. Mol. Cell. Biol. 18, 3475–3482 (1998).

    Article  CAS  Google Scholar 

  6. Gruenbaum, Y., Szyf, M., Cedar, H. & Razin, A. Methylation of replicating and post-replicated mouse L-cell DNA. Proc. Natl Acad. Sci. USA 80, 4919–4921 (1983).

    Article  CAS  Google Scholar 

  7. Kappler, J. W. The kinetics of DNA methylation in cultures of a mouse adrenal cell line. J. Cell Physiol. 75, 21–31 (1970).

    Article  CAS  Google Scholar 

  8. Xu, C. & Corces, V. G. Nascent DNA methylome mapping reveals inheritance of hemimethylation at CTCF/cohesin sites. Science 359, 1166–1170 (2018).

    Article  CAS  Google Scholar 

  9. Adams, R. L. The relationship between synthesis and methylation of DNA in mouse fibroblasts. Biochim. Biophys. Acta 254, 205–212 (1971).

    Article  CAS  Google Scholar 

  10. Liang, G. et al. Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. Mol. Cell. Biol. 22, 480–491 (2002).

    Article  CAS  Google Scholar 

  11. Woodcock, D. M. et al. Delayed DNA methylation is an integral feature of DNA replication in mammalian cells. Exp. Cell. Res. 166, 103–112 (1986).

    Article  CAS  Google Scholar 

  12. Charlton, J. et al. Global delay in nascent strand DNA methylation. Nat. Struct. Mol. Biol. 25, 327–332 (2018).

    Article  CAS  Google Scholar 

  13. Ming, X. et al. Kinetics and mechanisms of mitotic inheritance of DNA methylation and their roles in aging-associated methylome deterioration. Cell Res. 30, 980–996 (2020).

    Article  CAS  Google Scholar 

  14. Sirbu, B. M., Couch, F. B. & Cortez, D. Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA. Nat. Protoc. 7, 594–605 (2012).

    Article  CAS  Google Scholar 

  15. Laird, C. D. et al. Hairpin-bisulfite PCR: assessing epigenetic methylation patterns on complementary strands of individual DNA molecules. Proc. Natl Acad. Sci. USA 101, 204–209 (2004).

    Article  CAS  Google Scholar 

  16. Zhao, L. et al. The dynamics of DNA methylation fidelity during mouse embryonic stem cell self-renewal and differentiation. Genome Res. 24, 1296–1307 (2014).

    Article  CAS  Google Scholar 

  17. Presolski, S. I., Hong, V. P. & Finn, M. G. Copper-catalyzed azide-alkyne click chemistry for bioconjugation. Curr. Protoc. Chem. Biol. 3, 153–162 (2011).

    Article  Google Scholar 

  18. Li, Y. et al. Stella safeguards the oocyte methylome by preventing de novo methylation mediated by DNMT1. Nature 564, 136–140 (2018).

    Article  CAS  Google Scholar 

  19. Tigges, J. et al. The hallmarks of fibroblast ageing. Mech. Ageing Dev. 138, 26–44 (2014).

    Article  CAS  Google Scholar 

  20. Sturm, G. et al. Human aging DNA methylation signatures are conserved but accelerated in cultured fibroblasts. Epigenetics 14, 961–976 (2019).

    Article  Google Scholar 

  21. Urich, M. A., Nery, J. R., Lister, R., Schmitz, R. J. & Ecker, J. R. MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing. Nat. Protoc. 10, 475–483 (2015).

    Article  CAS  Google Scholar 

  22. Gowher, H. & Jeltsch, A. Mammalian DNA methyltransferases: new discoveries and open questions. Biochem. Soc. Trans. 46, 1191–1202 (2018).

    Article  CAS  Google Scholar 

  23. Wang, Q. et al. Imprecise DNMT1 activity coupled with neighbor-guided correction enables robust yet flexible epigenetic inheritance. Nat. Genet. (2020).

  24. Hansen, R. S. et al. Sequencing newly replicated DNA reveals widespread plasticity in human replication timing. Proc. Natl Acad. Sci. USA 107, 139–144 (2010).

    Article  CAS  Google Scholar 

  25. Xu, C. & Corces, V. G. Resolution of the DNA methylation state of single CpG dyads using in silico strand annealing and WGBS data. Nat. Protoc. 14, 202–216 (2019).

    Article  CAS  Google Scholar 

  26. Vaisvila, R. et al. EM-seq: detection of dna methylation at single base resolution from picograms of DNA. Preprint at bioRxiv https://doi.org/10.1101/2019.12.20.884692 (2019).

  27. Mu, Z. M., Le, X. F., Vallian, S., Glassman, A. B. & Chang, K. S. Stable overexpression of PML alters regulation of cell cycle progression in HeLa cells. Carcinogenesis 18, 2063–2069 (1997).

    Article  CAS  Google Scholar 

  28. Sun, M. A., Velmurugan, K. R., Keimig, D. & Xie, H. HBS-Tools for hairpin bisulfite sequencing data processing and analysis. Adv. Bioinformatics 2015, 760423 (2015).

    Article  Google Scholar 

  29. Chuang, L. S. et al. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. Science 277, 1996–2000 (1997).

    Article  CAS  Google Scholar 

  30. Ferry, L. et al. Methylation of DNA Ligase 1 by G9a/GLP recruits UHRF1 to replicating DNA and regulates dna methylation. Mol. Cell 67, 550–565 e5 (2017).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Chinese Ministry of Science and Technology (2018YFE0203300), the China National Science Foundation (31771429) and the Chinese Academy of Sciences (XDB39000000 and QYZDY-SSW-SMC031). Z.Z. is sponsored by the Youth Innovation Promotion Association of Chinese Academy of Sciences (2017133).

Author information

Authors and Affiliations

  1. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Xuan Ming, Bing Zhu & Zhuqiang Zhang

  2. College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China

    Bing Zhu

Authors
  1. Xuan Ming
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bing Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhuqiang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.M. designed and optimized the Hammer-seq experiments and Z.Z. designed the bioinformatics analysis pipeline. Both authors participated in manuscript preparation.

Corresponding author

Correspondence to Zhuqiang Zhang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

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

Related links

Key reference using this protocol

Ming, X. et al. Cell Res. 30, 980–996 (2020): https://doi.org/10.1038/s41422-020-0359-9

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ming, X., Zhu, B. & Zhang, Z. Simultaneously measuring the methylation of parent and daughter strands of replicated DNA at the single-molecule level by Hammer-seq. Nat Protoc 16, 2131–2157 (2021). https://doi.org/10.1038/s41596-020-00488-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-020-00488-9

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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