Abstract
5-hydroxymethylcytosine (5hmC) was recently identified as an abundant epigenetic mark in mammals. Subsequent research has implicated 5hmC in normal mammalian development and disease pathogenesis in humans. Many of the techniques commonly used to assay for canonical 5-methylcytosine (5mC) cannot distinguish between 5hmC and 5mC. The development of antibodies specific to 5hmC has allowed for specific enrichment of DNA fragments containing 5hmC. Hydroxymethylated DNA immunoprecipitation (hmeDIP) has become an invaluable tool for determining both locus-specific and genome-wide profiles of 5hmC in mammalian DNA. Here, we describe the use of hmeDIP to characterize the relative abundance of 5hmC at loci in mammalian DNA.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kriaucionis S, Heintz N (2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324:929–930
Tahiliani M, Koh KP, Shen Y et al (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324:930–935
Globisch D, Munzel M, Muller M et al (2010) Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS One 5:e15367
Jin SG, Wu X, Li AX et al (2011) Genomic mapping of 5-hydroxymethylcytosine in the human brain. Nucleic Acids Res 39:5015–5024
Nestor CE, Ottaviano R, Reddington J et al (2012) Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes. Genome Res 22:467–477
Szwagierczak A, Bultmann S, Schmidt CS et al (2010) Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res 38:e181
Ito S, D’Alessio AC, Taranova OV et al (2010) Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466:1129–1133
Koh KP, Yabuuchi A, Rao S et al (2011) Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Cell Stem Cell 8:200–213
Huang Y, Pastor WA, Shen Y et al (2010) The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS One 5:e8888
Jin SG, Kadam S, Pfeifer GP (2010) Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Res 38:e125
Nestor C, Ruzov A, Meehan R et al (2010) Enzymatic approaches and bisulfite sequencing cannot distinguish between 5-methylcytosine and 5-hydroxymethylcytosine in DNA. Biotechniques 48:317–319
Ficz G, Branco MR, Seisenberger S et al (2011) Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473:398–402
Wu H, D’Alessio AC, Ito S et al (2011) Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. Genes Dev 25:679–684
Stroud H, Feng S, Morey Kinney S et al (2011) 5-Hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells. Genome Biol 12:R54
Matarese F, Carrillo-de Santa Pau E, Stunnenberg HG (2011) 5-Hydroxymethylcytosine: a new kid on the epigenetic block? Mol Syst Biol 7:562
Mohn F, Weber M, Schubeler D et al (2009) Methylated DNA immunoprecipitation (MeDIP). Method Mol Biol 507:55–64
Weber M, Davies JJ, Wittig D et al (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37:853–862
Kinney SM, Chin HG, Vaisvila R et al (2011) Tissue-specific distribution and dynamic changes of 5-hydroxymethylcytosine in mammalian genomes. J Biol Chem 286:24685–24693
Acknowledgements
We thank Dr. James P. Reddington for critical reading of this manuscript and Raffaele Ottaviano for helpful advice. We thank members of the Meehan lab for advice on the development and application of 5hmC protocols. Work in RM’s lab is supported by the Medical Research Council, by the BBSRC, and by the Innovative Medicines Initiative Joint Undertaking (IMI JU) under grant agreement number 115001 (MARCAR project). URL: http://www.imi-marcar.eu/.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Nestor, C.E., Meehan, R.R. (2014). Hydroxymethylated DNA Immunoprecipitation (hmeDIP). In: Stockert, J., Espada, J., Blázquez-Castro, A. (eds) Functional Analysis of DNA and Chromatin. Methods in Molecular Biology, vol 1094. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-706-8_20
Download citation
DOI: https://doi.org/10.1007/978-1-62703-706-8_20
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-705-1
Online ISBN: 978-1-62703-706-8
eBook Packages: Springer Protocols