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. 2011 Jan;29(1):68-72.
doi: 10.1038/nbt.1732. Epub 2010 Dec 12.

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

Chun-Xiao Song  1 , Keith E Szulwach, Ye Fu, Qing Dai, Chengqi Yi, Xuekun Li, Yujing Li, Chih-Hsin Chen, Wen Zhang, Xing Jian, Jing Wang, Li Zhang, Timothy J Looney, Baichen Zhang, Lucy A Godley, Leslie M Hicks, Bruce T Lahn, Peng Jin, Chuan He
Affiliations

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

Chun-Xiao Song et al. Nat Biotechnol. 2011 Jan.

Abstract

In contrast to 5-methylcytosine (5-mC), which has been studied extensively, little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types. 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 recognized. 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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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturebiotechnology/.

Figures

Figure 1
Figure 1
Selective labeling of 5-hmC in genomic DNA. (a) The hydroxyl group of 5-hmC in duplex DNA can be glucosylated by β-GT to form β-glucosyl-5-hydroxymethylcytosine (5-gmC) using UDP-Glu as a cofactor. (b) An azide group can be installed onto 5-hmC using chemically modified UDP-Glu (UDP-6-N3-Glu), which in turn can be labeled with a biotin moiety using click chemistry for subsequent detection, affinity purification and sequencing.
Figure 2
Figure 2
MS characterization of 5-hmC-, N3-5-gmC- and biotin-N3-5-gmC-containing 11-mer DNA in a model experiment. (a) MALDI-TOF of 5-hmC-, N3-5-gmC- and biotin-N3-5-gmC- containing 11-mer DNA, respectively, with the calculated molecular weight and observed molecular weight indicated. (b) Corresponding reactions of the β-GT–catalyzed formation of N3-5-gmC and the subsequent copper-free click chemistry to yield biotin-N3-5-gmC in duplex DNA. Reactions were performed in duplex DNA with the complementary strand; however, MS monitored the single-stranded DNA containing the modification.
Figure 3
Figure 3
Quantification of 5-hmC in various cell lines and tissues. (a) Dot-blot assay of avidin-HRP detection and quantification of mouse cerebellum genomic DNA containing biotin-N3-5-gmC. Top row: 40 ng of biotin-labeled samples using UDP-6-N3-Glu. Bottom row: 40 ng of control samples using regular UDP-Glu without biotin label. The exact same procedures were followed for experiments in both rows. P7, P14 and P21 represent postnatal day 7, 14 and 21, respectively. (b) Amounts of 5-hmC are shown in percentage of total nucleotides of mouse genome. *, P < 0.05, Student’s t-test; means ± s.e.m. for n = 4 experiments. (c) Dot-blot assay of avidin-HRP detection and quantification of genomic DNA samples from four cell lines (from same blot as in a), except that each dot contains 700 ng DNA. (d) Amounts of 5-hmC are shown in percentage of total nucleotides of the genome; means ± s.e.m. for n = 4 experiments. The dashed line indicates the limit of detection (~0.004%).
Figure 4
Figure 4
Genome-wide distribution of 5-hmC in adult mouse cerebellum and gene-specific acquisition of intragenic 5-hmC during mouse cerebellum development. (a) Genome-scale reproducibility of 5-hmC profiles and enrichment relative to genomic DNA and control-treated DNA in adult mouse cerebellum. Heatmap representations of read densities have been equally scaled and then normalized based on the total number of mapped reads per sample. Data are derived from a single lane of sequence from each condition. Control, UDP-Glu treated without biotin; Input, genomic DNA; 5-hmC, UDP-6-N3-Glu treated with biotin incorporated. (b) Metagene profiles of 5-hmC and input genomic DNA reads mapped relative to RefSeq transcripts expressed at different levels in adult mouse cerebellum. RefSeq transcripts were divided into four equally sized bins based on gene expression level and 5-hmC or input genomic DNA reads falling in 10-bp bins centered on transcription start sites or end sites. The reads were summed and normalized based on the total number of aligned reads (in millions). Input genomic DNA reads were mapped to each of the four gene expression level bins and are plotted here in black. The profiles completely overlap and so are collectively referred to as ‘Input’. (c) Proximal and intragenic enrichment of 5-hmC relative to surrounding regions in adult and P7 mouse cerebellum. Reads from 5-hmC-captured samples and input genomic DNA were summed in 10-bp intervals centered on either TSS or txEnds and normalized to the total number of aligned reads from each sample (in millions). (d) Enrichment of pathways associated with age-related neurodegenerative diseases in genes acquiring intragenic 5-hmC in adult mice relative to P7 mice. Shown are the number of genes that acquired 5-hmC in adult cerebellum and the number of genes expected based on the total number of genes associated with that pathway in mouse. **, P < 10−10; *, P < 10−5.
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Comment in

  • Profiling the sixth base.
    Rusk N. Rusk N. Nat Methods. 2011 Jul;8(7):532-3. doi: 10.1038/nmeth0711-532b. Nat Methods. 2011. PMID: 21850733 No abstract available.

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