In contrast to 5-methylcytosine (5-mC), which has been studied extensively1,2,3, little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types4,5. Here we present a method for determining the genome-wide distribution of 5-hmC. We use the T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment and sequencing of 5-hmC–containing DNA fragments in mammalian genomes. Using this method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized4. We also find a gene expression level–dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $20.83 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Gene Expression Omnibus
Klose, R.J. & Bird, A.P. Genomic DNA methylation: the mark and its mediators. Trends Biochem. Sci. 31, 89–97 (2006).
Reik, W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447, 425–432 (2007).
Gal-Yam, E.N., Saito, Y., Egger, G. & Jones, P.A. Cancer epigenetics: modifications, screening, and therapy. Annu. Rev. Med. 59, 267–280 (2008).
Kriaucionis, S. & Heintz, N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324, 929–930 (2009).
Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009).
Yi, C., Yang, C.G. & He, C. A non-heme iron-mediated chemical demethylation in DNA and RNA. Acc. Chem. Res. 42, 519–529 (2009).
Hausinger, R.P. FeII/alpha-ketoglutarate-dependent hydroxylases and related enzymes. Crit. Rev. Biochem. Mol. Biol. 39, 21–68 (2004).
Ito, S. et al. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466, 1129–1133 (2010).
Ko, M. et al. Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature 468, 839–843 (7 Nov 2010).
Loenarz, C. & Schofield, C.J. Oxygenase catalyzed 5-methylcytosine hydroxylation. Chem. Biol. 16, 580–583 (2009).
Huang, Y. et al. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS ONE 5, e8888 (2010).
Jin, S.G., Kadam, S. & Pfeifer, G.P. Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Res. 38, e125 (2010).
Flusberg, B.A. et al. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat. Methods 7, 461–465 (2010).
Josse, J. & Kornberg, A. Glucosylation of deoxyribonucleic acid. III. alpha and beta-glucosyl transferases from T4-infected Escherichia coli. J. Biol. Chem. 237, 1968–1976 (1962).
Lariviere, L. & Morera, S. Structural evidence of a passive base-flipping mechanism for beta-glucosyltransferase. J. Biol. Chem. 279, 34715–34720 (2004).
Kolb, H.C., Finn, M.G. & Sharpless, K.B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40, 2004–2021 (2001).
Speers, A.E. & Cravatt, B.F. Profiling enzyme activities in vivo using click chemistry methods. Chem. Biol. 11, 535–546 (2004).
Sletten, E.M. & Bertozzi, C.R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem. Int. Ed. 48, 6974–6998 (2009).
Baskin, J.M. et al. Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl. Acad. Sci. USA 104, 16793–16797 (2007).
Ning, X., Guo, J., Wolfert, M.A. & Boons, G.J. Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast huisgen cycloadditions. Angew. Chem. Int. Ed. 47, 2253–2255 (2008).
Munzel, M. et al. Quantification of the sixth DNA base hydroxymethylcytosine in the brain. Angew. Chem. Int. Ed. 49, 5375–5377 (2010).
Lyko, F., Ramsahoye, B.H. & Jaenisch, R. DNA methylation in Drosophila melanogaster. Nature 408, 538–540 (2000).
Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–770 (2008).
Lister, R. et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322 (2009).
Edwards, J.R. et al. Chromatin and sequence features that define the fine and gross structure of genomic methylation patterns. Genome Res. 20, 972–980 (2010).
Skene, P.J. et al. Neuronal MeCP2 is expressed at near histone-octamer levels and globally alters the chromatin state. Mol. Cell 37, 457–468 (2010).
Lim, J. et al. A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration. Cell 125, 801–814 (2006).
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).
Maunakea, A.K. et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466, 253–257 (2010).
Wu, S.C. & Zhang, Y. Active DNA demethylation: many roads lead to Rome. Nat. Rev. Mol. Cell Biol. 11, 607–620 (2010).
Silva, J. et al. Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol. 6, e253 (2008).
Szulwach, K.E. et al. Cross talk between microRNA and epigenetic regulation in adult neurogenesis. J. Cell Biol. 189, 127–141 (2010).
Donnelly, M.I. et al. An expression vector tailored for large-scale, high-throughput purification of recombinant proteins. Protein Expr. Purif. 47, 446–454 (2006).
Crain, P.F. Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry. Methods Enzymol. 193, 782–790 (1990).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Thomas, P.D. et al. PANTHER: a library of protein families and subfamilies indexed by function. Genome Res. 13, 2129–2141 (2003).
Thomas, P.D. et al. Applications for protein sequence-function evolution data: mRNA/protein expression analysis and coding SNP scoring tools. Nucleic Acids Res. 34, W645–650 (2006).
Jung, M.E. & Miller, S.J. Total synthesis of isopavine and intermediates for the preparation of substituted amitriptyline analogs—facile routes to substituted dibenzocyclooctatrienes and dibenzocycloheptatrienes. J. Am. Chem. Soc. 103, 1984–1992 (1981).
We would like to thank S. Warren for the helpful discussion and critical reading of the manuscript. This study was supported partly by the US National Institutes of Health (GM071440 to C.H. and NS051630/MH076090/MH078972 to P.J.) and the University of Chicago.
A patent application has been filed for the technology disclosed in this publication.
Supplementary Tables 1–5, Supplementary Methods and Supplementary Figs. 1–12 (PDF 5662 kb)
About this article
Thymine DNA glycosylase recognizes the geometry alteration of minor grooves induced by 5-formylcytosine and 5-carboxylcytosine
Chemical Science (2019)
Genome-wide DNA methylomic differences between dorsolateral prefrontal and temporal pole cortices of bipolar disorder
Journal of Psychiatric Research (2019)
Simple and Efficient Room-Temperature Release of Biotinylated Nucleic Acids from Streptavidin and Its Application to Selective Molecular Detection
Analytical Chemistry (2019)
ACS Chemical Biology (2019)
Science Advances (2019)