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5-Hydroxymethylcytosine Profiling in Human DNA

  • John P. Thomson
  • Colm E. Nestor
  • Richard R. MeehanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1589)

Abstract

Since its “re-discovery” in 2009, there has been significant interest in defining the genome-wide distribution of DNA marked by 5-hydroxymethylation at cytosine bases (5hmC). In recent years, technological advances have resulted in a multitude of unique strategies to map 5hmC across the human genome. Here we discuss the wide range of approaches available to map this modification and describe in detail the affinity based methods which result in the enrichment of 5hmC marked DNA for downstream analysis.

Keywords:

5-hydroxymethylcytosine 5hmC 5mC DNA immunoprecipitation and enrichment HmeDIP Epigenetics 

Notes

Acknowledgements

We thank Dr. Jamie Hackett for his role in initially developing the hmeDIP protocol presented here. Research in RRMs lab is supported by the Medical Research Council, the BBSRC and IMI-MARCAR: the Innovative Medicine Initiative Joint Undertaking (IMI JU) under grant agreement number 115001 (MARCAR project. URL: http://www.imi-marcar.eu/).

References

  1. 1.
    Kriaucionis S, Heintz N (2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324:929–930CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Shen L, Zhang Y (2013) 5-hydroxymethylcytosine: generation, fate, and genomic distribution. Curr Opin Cell Biol 25(3):289–296CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Song CX, Yi C, He C (2012) Mapping recently identified nucleotide variants in the genome and transcriptome. Nat Biotechnol 30:1107–1116CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L et al (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324:930–935CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Thomson JP, Hunter JM, Lempiainen H, Muller A, Terranova R, Moggs JG, Meehan RR (2013) Dynamic changes in 5-hydroxymethylation signatures underpin early and late events in drug exposed liver. Nucleic Acids Res 41:5639–5654CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Thomson JP, Lempiainen H, Hackett JA, Nestor CE, Muller A, Bolognani F, Oakeley EJ, Schubeler D, Terranova R, Reinhardt D et al (2012) Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome. Genome Biol 13:R93CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Robinson MD, Stirzaker C, Statham AL, Coolen MW, Song JZ, Nair SS, Strbenac D, Speed TP, Clark SJ (2010) Evaluation of affinity-based genome-wide DNA methylation data: effects of CpG density, amplification bias, and copy number variation. Genome Res 20:1719–1729CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Globisch D, Munzel M, Muller M, Michalakis S, Wagner M, Koch S, Bruckl T, Biel M, Carell T (2010) Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS One 5:e15367CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Nestor CE, Ottaviano R, Reddington J, Sproul D, Reinhardt D, Dunican D, Katz E, Dixon JM, Harrison DJ, Meehan RR (2012) Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes. Genome Res 22:467–477CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Laird A, Thomson JP, Harrison DJ, Meehan RR (2013) 5-hydroxymethylcytosine profiling as an indicator of cellular state. Epigenomics 5:655–669CrossRefPubMedGoogle Scholar
  11. 11.
    Thomson JP, Moggs JG, Wolf CR, Meehan RR (2013) Epigenetic profiles as defined signatures of xenobiotic exposure. Mutat Res Genet Toxicol Environ Mutagen 764–765:3–9PubMedGoogle Scholar
  12. 12.
    Ficz G, Gribben JG (2014) Loss of 5-hydroxymethylcytosine in cancer: cause or consequence? Genomics 104(5):352–357CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Nestor C, Ruzov A, Meehan R, Dunican D (2010) Enzymatic approaches and bisulfite sequencing cannot distinguish between 5-methylcytosine and 5-hydroxymethylcytosine in DNA. Biotechniques 48:317–319CrossRefPubMedGoogle Scholar
  14. 14.
    Huang Y, Pastor WA, Zepeda-Martinez JA, Rao A (2012) The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine. Nat Protoc 7:1897–1908CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Cui L, Chung TH, Tan D, Sun X, Jia XY (2014) JBP1-seq: a fast and efficient method for genome-wide profiling of 5hmC. Genomics 104:368–375CrossRefPubMedGoogle Scholar
  16. 16.
    Robertson AB, Dahl JA, Ougland R, Klungland A (2012) Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads. Nat Protoc 7:340–350CrossRefPubMedGoogle Scholar
  17. 17.
    Song CX, Szulwach KE, Fu Y, Dai Q, Yi C, Li X, Li Y, Chen CH, Zhang W, Jian X et al (2011) Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat Biotechnol 29:68–72CrossRefPubMedGoogle Scholar
  18. 18.
    Serandour AA, Avner S, Oger F, Bizot M, Percevault F, Lucchetti-Miganeh C, Palierne G, Gheeraert C, Barloy-Hubler F, Peron CL et al (2012) Dynamic hydroxymethylation of deoxyribonucleic acid marks differentiation-associated enhancers. Nucleic Acids Res 40:8255–8265CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Szulwach KE, Li X, Li Y, Song CX, Wu H, Dai Q, Irier H, Upadhyay AK, Gearing M, Levey AI et al (2011) 5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging. Nat Neurosci 14:1607–1616CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Wang T, Wu H, Li Y, Szulwach KE, Lin L, Li X, Chen IP, Goldlust IS, Chamberlain SJ, Dodd A et al (2013) Subtelomeric hotspots of aberrant 5-hydroxymethylcytosine-mediated epigenetic modifications during reprogramming to pluripotency. Nat Cell Biol 15:700–711CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Booth MJ, Branco MR, Ficz G, Oxley D, Krueger F, Reik W, Balasubramanian S (2012) Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science 336:934–937CrossRefPubMedGoogle Scholar
  22. 22.
    Yu M, Hon GC, Szulwach KE, Song CX, Zhang L, Kim A, Li X, Dai Q, Shen Y, Park B et al (2012) Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149:1368–1380CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Booth MJ, Ost TW, Beraldi D, Bell NM, Branco MR, Reik W, Balasubramanian S (2013) Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat Protoc 8:1841–1851CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Thomson JP, Hunter JM, Nestor CE, Dunican DS, Terranova R, Moggs JG, Meehan RR (2013) Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques. Nucleic Acids Res 41:e206CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Ficz G, Branco MR, Seisenberger S, Santos F, Krueger F, Hore TA, Marques CJ, Andrews S, Reik W (2011) Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473:398–402CrossRefPubMedGoogle Scholar
  26. 26.
    Wu H, D’Alessio AC, Ito S, Wang Z, Cui K, Zhao K, Sun YE, Zhang Y (2011) Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. Genes Dev 25:679–684CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Szwagierczak A, Bultmann S, Schmidt CS, Spada F, Leonhardt H (2010) Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res 38:e181CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Matarese F, Carrillo-de Santa Pau E, Stunnenberg HG (2011) 5-hydroxymethylcytosine: a new kid on the epigenetic block? Mol Syst Biol 7:562CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • John P. Thomson
    • 1
  • Colm E. Nestor
    • 2
  • Richard R. Meehan
    • 1
    Email author
  1. 1.MRC Human Genetics Unit, Institute of Genetics and Molecular MedicineThe University of EdinburghEdinburghUK
  2. 2.The Centre for Individualized Medication, Linköping University Hospital, Linköping UniversityLinköpingSweden

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