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. 2011 May 19;473(7347):394-7.
doi: 10.1038/nature10102. Epub 2011 May 8.

Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells

William A Pastor  1 , Utz J Pape, Yun Huang, Hope R Henderson, Ryan Lister, Myunggon Ko, Erin M McLoughlin, Yevgeny Brudno, Sahasransu Mahapatra, Philipp Kapranov, Mamta Tahiliani, George Q Daley, X Shirley Liu, Joseph R Ecker, Patrice M Milos, Suneet Agarwal, Anjana Rao
Affiliations

Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells

William A Pastor et al. Nature. .

Abstract

5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC). 5hmC and TET proteins have been implicated in stem cell biology and cancer, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the 'poised' chromatin signature found at developmentally-regulated genes in ES cells.

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Figures

Figure 1
Figure 1. Comparison of 5hmC enrichment methodsa
a, The GLIB method. Glucose is added to 5hmC by BGT, oxidized with sodium periodate to yield aldehydes, and reacted with the aldehyde reactive probe (ARP), yielding two biotins at the site of every 5hmC. b, 5hmC is converted to CMS by sodium bisulphite. c–f, Precipitation of PCR amplicons containing (1) varying amounts of 5hmC by GLIB methodology (c), anti-CMS methodology (d), or anti-5hmC antibody (e); or (2) varying amounts of 5mC by anti-5mC antibody (f). pAb, polyclonal antibody; mAb, monoclonal antibody. Between 1 and 6 independent experiments per method, mean percentage input precipitated ± s.d. is indicated. g, Overlap between HERGs identified by the GLIB and anti-CMS methodologies. Left panel, number of HERGs; right panel, number of base pairs contained within HERGs.
Figure 2
Figure 2. Genomic distribution of 5hmC or 5mC enriched regions of the genome
a, Correlation of HERG or MERG density on each chromosome (y-axis) with gene density in the same chromosome (x-axis). Density is defined as frequency divided by chromosome length. b, c, Both HERGs and MERGs are enriched in transcribed regions (b), whereas HERGs are preferentially enriched at enhancers and the start sites of genes (c). The percentage of HERGs or MERGs mapping to the indicated genomic feature (darker bar) is compared with the percentage of randomly chosen sequences mapping to that feature (lighter bar). 5′ UTR, 5′ untranslated region. TSS, transcription start site (−800 bp to +200 bp relative to start of transcription). See Supplementary Methods for detailed definition of how HERGs or MERGs were classified as mapping to genomic features. d, Distribution of HERGs and MERGs relative to the TSS. The centre of each HERG was plotted relative to the nearest TSS in 1,000 bp increments from −10 kb to +10 kb surrounding the TSS.
Figure 3
Figure 3. Properties of HERGs at transcription start sites
a, The percentage of genes with 5hmC at the TSS (blue and red bars) reported to contain histone H3 trimethylation (left) or PRC components (right) at their promoters is compared to the fraction of all genes (grey bars) with these promoter marks. Number of genes in each category is indicated. b, HERGs are enriched at the TSSs of genes with low expression in ES cells. All genes were ranked by level of expression in ES cells and sorted into deciles from lowest to highest. The per cent of genes within the decile category with 5-hmC enriched at the TSS (left) or within gene bodies (right) are shown for each methodology. The first five deciles, which are comprised of genes lacking statistically significant expression, are pooled and averaged in this analysis. c, HERGs are enriched at the TSS of genes upregulated upon differentiation to embryoid bodies (EB). The percentage of genes with 5-hmC at their TSS (blue bars) that are substantially upregulated or downregulated upon differentiation to EB is compared with the percentage of total genes similarly regulated (grey bars). Number of genes in each category is indicated. d, Overlap between genes with 5hmC at the TSS and genes positively or negatively regulated by Tet1 (ref. 8).
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References

    1. Tahiliani M, et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009;324:930–935. - PMC - PubMed
    1. Kriaucionis S, Heintz N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science. 2009;324:929–930. - PMC - PubMed
    1. Szwagierczak A, Bultmann S, Schmidt CS, Spada F, Leonhardt H. Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res. 2010;38:e181. - PMC - PubMed
    1. Ito S, et al. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature. 2010;466:1129–1133. - PMC - PubMed
    1. Ko M, et al. Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature. 2010;468:839–843. - PMC - PubMed

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