Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine

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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.

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Figure 1: Selective labeling of 5-hmC in genomic DNA.
Figure 2: MS characterization of 5-hmC-, N3-5-gmC- and biotin-N3-5-gmC-containing 11-mer DNA in a model experiment.
Figure 3: Quantification of 5-hmC in various cell lines and tissues.
Figure 4: Genome-wide distribution of 5-hmC in adult mouse cerebellum and gene-specific acquisition of intragenic 5-hmC during mouse cerebellum development.

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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.

Author information

C.H., C.-X.S. and P.J. designed the experiments with help from Y.F. and B.T.L. Experiments were performed by C.-X.S., K.E.S., Y.F., C.Y. and Q.D. with the help of W.Z. and X.J.; Q.D. and J.W. carried out the chemical synthesis; K.E.S., X.L., Y.L. and P.J. provided the mouse cerebellum, mouse aNSC and fly samples, and performed deep sequencing; C.-H.C., L.Z., T.J.L. and L.A.G. helped with the mouse ESC, human HeLa, human HEK and related samples; B.Z. and L.M.H. performed the mass spectrometry analysis from HeLa cells. C.H., C.-X.S. and P.J. wrote the paper. All authors discussed the results and commented on the manuscript.

Correspondence to Peng Jin or Chuan He.

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