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:

Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads

Nature Protocols volume 7, pages 340–350 (2012)Cite this article

Subjects

Abstract

We describe a method for the efficient and selective identification of DNA containing the 5-hydroxymethylcytosine (5-hmC) modification. This protocol takes advantage of two proteins: T4 β-glucosyltransferase (β-gt), which converts 5-hmC to β-glucosyl-5-hmC (β-glu-5-hmC), and J-binding protein 1 (JBP1), which specifically recognizes and binds to β-glu-5-hmC. We describe the steps necessary to purify JBP1 and modify this protein such that it can be fixed to magnetic beads. Thereafter, we detail how to use the JBP1 magnetic beads to obtain DNA that is enriched with 5-hmC. This method is likely to produce results similar to those of other 5-hmC pull-down assays; however, all necessary components for the completion of this protocol are readily available or can be easily and rapidly synthesized using basic molecular biology techniques. This protocol can be completed in less than 2 weeks and allows the user to isolate 5-hmC-containing genomic DNA that is suitable for analysis by quantitative PCR (qPCR), sequencing, microarray and other molecular biology assays.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Overview of 5-hmC enrichment mediated by β-gt and JBP1.
Figure 2: Workflow for JBP1 purification and cross-linking to magnetic beads.
Figure 3: Workflow for JBP1-mediated pull-down of 5-hmC–containing DNA.
Figure 4: Results from high-throughput sequencing of JBP1-enriched DNA.

Similar content being viewed by others

References

  1. Penn, N.W. Modification of brain deoxyribonucleic acid base content with maturation in normal and malnourished rats. Biochem. J. 155, 709–712 (1976).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Cannon-Carlson, S.V., Gokhale, H. & Teebor, G.W. Purification and characterization of 5-hydroxymethyluracil-DNA glycosylase from calf thymus. Its possible role in the maintenance of methylated cytosine residues. J. Biol. Chem. 264, 13306–13312 (1989).

    CAS  PubMed  Google Scholar 

  3. Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kriaucionis, S. & Heintz, N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324, 929–930 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ito, S. et al. Role of Tet proteins in 5mC to 5-hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466, 1129–1133 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Szwagierczak, A., Bultmann, S., Schmidt, C.S., Spada, F. & Leonhardt, H. Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res. 38, e181 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Ko, M. et al. Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature 468, 839–843 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Guo, J.U., Su, Y., Zhong, C., Ming, G.L. & Song, H. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145, 423–434 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wu, H. et al. Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. Genes Dev. 25, 679–684 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wu, H. et al. Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells. Nature 473, 389–393 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Robertson, J., Robertson, A.B. & Klungland, A. The presence of 5-hydroxymethylcytosine at the gene promoter and not in the gene body negatively regulates gene expression. Biochem. Biophys. Res. Commun. 411, 40–43 (2011).

    Article  CAS  PubMed  Google Scholar 

  12. Georgopoulos, C.P. & Revel, H.R. Studies with glucosyl transferase mutants of the T-even bacteriophages. Virology 44, 271–285 (1971).

    Article  CAS  PubMed  Google Scholar 

  13. Kornberg, S.R., Zimmerman, S.B. & Kornberg, A. Glucosylation of deoxyribonucleic acid by enzymes from bacteriophage-infected Escherichia coli. J. Biol. Chem. 236, 1487–1493 (1961).

    CAS  PubMed  Google Scholar 

  14. Gommers-Ampt, J.H. et al. β-D-glucosyl-hydroxymethyluracil: a novel modified base present in the DNA of the parasitic protozoan T. brucei. Cell 75, 1129–1136 (1993).

    Article  CAS  PubMed  Google Scholar 

  15. Borst, P. & Sabatini, R. Base J: discovery, biosynthesis, and possible functions. Annu. Rev. Microbiol. 62, 235–251 (2008).

    Article  CAS  PubMed  Google Scholar 

  16. van Leeuwen, F. et al. β-D-glucosyl-hydroxymethyluracil is a conserved DNA modification in kinetoplastid protozoans and is abundant in their telomeres. Proc. Natl. Acad. Sci. USA 95, 2366–2371 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sabatini, R., Meeuwenoord, N., van Boom, J.H. & Borst, P. Recognition of base J in duplex DNA by J-binding protein. J. Biol. Chem. 277, 958–966 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Cross, M. et al. The modified base J is the target for a novel DNA-binding protein in kinetoplastid protozoans. EMBO J. 18, 6573–6581 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Grover, R.K. et al. O-glycoside orientation is an essential aspect of base J recognition by the kinetoplastid DNA-binding protein JBP1. Angew Chem. Int. Ed. Eng. 46, 2839–2843 (2007).

    Article  CAS  Google Scholar 

  20. Robertson, A.B. et al. A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res. 39, e55 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ficz, G. et al. Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473, 398–402 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. Stroud, H., Feng, S., Morey Kinney, S., Pradhan, S. & Jacobsen, S.E. 5-Hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells. Genome Biol. 12, R54 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Pastor, W.A. et al. Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 473, 394–397 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Flusberg, B.A. et al. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat. Methods 7, 461–465 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Song, C.X., Yu, M., Dai, Q. & He, C. Detection of 5-hydroxymethylcytosine in a combined glycosylation restriction analysis (CGRA) using restriction enzyme Taq(α)I. Bioorg. Med. Chem. Lett. 21, 5075–5077 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Xu, S.Y., Corvaglia, A.R., Chan, S.H., Zheng, Y. & Linder, P. A type IV modification-dependent restriction enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300. Nucleic Acids Res. 39, 5597–5610 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Szwagierczak, A. et al. Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5-hydroxymethylcytosine. Nucleic Acids Res. 39, 5149–5156 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Song, C.X. et al. Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat. Biotechnol. 29, 68–72 (2011).

    Article  CAS  PubMed  Google Scholar 

  29. Nestor, C., Ruzov, A., Meehan, R. & Dunican, D. Enzymatic approaches and bisulfite sequencing cannot distinguish between 5-methylcytosine and 5-hydroxymethylcytosine in DNA. Biotechniques 48, 317–319 (2010).

    Article  CAS  PubMed  Google Scholar 

  30. Studier, F.W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was funded by the Norwegian Research Council and the Norwegian Cancer Society.

Author information

Author notes

  1. Adam B Robertson and John Arne Dahl: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre for Molecular Biology and Neuroscience, Institute of Medical Microbiology, Oslo University Hospital Rikshospitalet, Oslo, Norway

    Adam B Robertson, John Arne Dahl, Rune Ougland & Arne Klungland

  2. Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway

    Arne Klungland

Authors
  1. Adam B Robertson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John Arne Dahl
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rune Ougland
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Arne Klungland
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.B.R., J.A.D. and A.K. initiated the project. A.B.R., J.A.D. and A.K. designed the study. A.B.R., J.A.D. and R.O. designed and conducted the experiments. A.B.R. drafted the manuscript and all the authors participated in writing and editing the manuscript.

Corresponding author

Correspondence to Arne Klungland.

Ethics declarations

Competing interests

The authors have been given a provisional patent for the method described in this protocol.

Supplementary information

Supplementary Note 1

Test oligonucleotide sequences and qPCR primers (DOC 31 kb)

Supplementary Note 2

Sequence of pET28a-JBP1 (DOC 42 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Robertson, A., Dahl, J., Ougland, R. et al. Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads. Nat Protoc 7, 340–350 (2012). https://doi.org/10.1038/nprot.2011.443

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.443

This article is cited by

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