Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation

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Methylation at the 5′ position of cytosine in DNA has important roles in genome function and is dynamically reprogrammed during early embryonic and germ cell development1. The mammalian genome also contains 5-hydroxymethylcytosine (5hmC), which seems to be generated by oxidation of 5-methylcytosine (5mC) by the TET family of enzymes that are highly expressed in embryonic stem (ES) cells2, 3, 4. Here we use antibodies against 5hmC and 5mC together with high throughput sequencing to determine genome-wide patterns of methylation and hydroxymethylation in mouse wild-type and mutant ES cells and differentiating embryoid bodies. We find that 5hmC is mostly associated with euchromatin and that whereas 5mC is under-represented at gene promoters and CpG islands, 5hmC is enriched and is associated with increased transcriptional levels. Most, if not all, 5hmC in the genome depends on pre-existing 5mC and the balance between these two modifications is different between genomic regions. Knockdown of Tet1 and Tet2 causes downregulation of a group of genes that includes pluripotency-related genes (including Esrrb, Prdm14, Dppa3, Klf2, Tcl1 and Zfp42) and a concomitant increase in methylation of their promoters, together with an increased propensity of ES cells for extraembryonic lineage differentiation. Declining levels of TETs during differentiation are associated with decreased hydroxymethylation levels at the promoters of ES cell-specific genes together with increased methylation and gene silencing. We propose that the balance between hydroxymethylation and methylation in the genome is inextricably linked with the balance between pluripotency and lineage commitment.

At a glance


  1. Distribution of 5-hydroxymethylcytosine in the mouse genome.
    Figure 1: Distribution of 5-hydroxymethylcytosine in the mouse genome.

    a, The specificities of the antibodies used in this study were confirmed by dot blot and (h)MeDIP using PCR fragments containing 5hmC, 5mC or C. b, Immunofluorescence co-staining of J1 ES cells with antibodies against 5hmC (green) and 5mC (red). Grey scale images of the two modifications are shown separately. Staining for 5mC is particularly strong in pericentromeric heterochromatin (arrows), contrary to 5hmC. Scale bar, 10µm. c, Examples of hMeDIP-Seq and MeDIP-Seq profiles at a genomic region on Chr2 in J1 ES cells. d, Relative enrichment (log2 bound/unbound) of 5hmC and 5mC in repetitive sequences in J1 and E14 ES cells and E14 EBs. e, Enrichment of 5hmC and 5mC in single-copy genomic features. Values in d and e represent means of two biological replicates with the ends of the error bars corresponding to the individual data points. f, Validation of the presence of 5hmC in CGIs using glucMS-qPCR (grey bars represent mean±s.d.). Selected CGIs (black bars, upper panel) were tested for the presence of 5hmC at particular MspI sites (grey vertical line). Genomic coordinates of the left-most base pairs of each region: Ctnna3 (chr10, 63044495); Zfp64 (chr2, 168750875); Bend3 (chr10, 43230661); EG240055 (also known as Neurl1b: chr17, 26567975).

  2. Genetic relationship between methylation and hydroxymethylation.
    Figure 2: Genetic relationship between methylation and hydroxymethylation.

    a, Thin layer chromatography separation of radioactively end-labelled bases from MspI-digested genomic DNA, showing reduced levels of 5hmC (arrowheads) in methylation- and TET-deficient ES cells. b, glucMS-qPCR validation of genomic regions specifically enriched for 5mC or 5hmC in wild-type (WT) J1, Np95−/− and TKO ES cells (bars represent mean±s.d.). Genomic regions were selected on the basis of (h)MeDIP-Seq profiles of wild-type ES cells. c, Examples of (h)MeDIP-Seq profiles in wild type, Np95−/− and Tet1/2 KD ES cells. 5hmC profiles are relatively similar, whereas 5mC distribution is significantly altered in Np95−/− cells, but less so in Tet1/2 knockdown(Tet1/2 KD) cells. Shadowed areas highlight regions of altered 5hmC and/or 5mC enrichment. d, Relative enrichment at promoters, exons and 5′ regions of LINE1 elements in J1, Np95−/− and Tet1/2 KD ES cells. Np95 deficiency causes depletion of both 5hmC and 5mC in all three regions, whereas Tet1/2 KD causes preferential reduction of 5hmC at exons and LINE1 promoters, which leads to increased 5mC enrichment in these regions. Values represent means of two biological replicates with the ends of the error bars corresponding to the individual data points.

  3. Strand specificity and sequence context of methylation and hydroxymethylation.
    Figure 3: Strand specificity and sequence context of methylation and hydroxymethylation.

    a, Enrichment of dinucleotide sequences present in the central 200bp of 5hmC and 5mC regions separated into biased and unbiased fragments. CG dinucleotides are enriched in unbiased regions, as expected from its symmetric nature. Biased regions are enriched for CH dinucleotides, indicating extensive non-CpG (hydroxy)methylation. b, Strand bias measurements, which represent the overall level of asymmetric methylation in the genome. Depletion of NP95 increases strand bias in the 5hmC and 5mC profiles due to reduced CpG methylation. Knockdown of Tet1/2 decreases strand bias in the 5mC profile, as expected if reduction of 5hmC at CpGs leads to an accumulation of 5mC at the same sites. Values represent means of two biological replicates with the ends of the error bars corresponding to the individual data points. c, BS-Seq13 validation of the (h)MeDIP data. Percentages of methylated CpGs present in 5hmC-enriched peaks containing low or high 5mC levels are plotted (left) showing the symmetrical nature of CpG methylation (in both biased and unbiased peaks). Conversely, CpH methylation in biased peaks is asymmetric in nature (right). Error bars represent 95% confidence intervals. d, Validation of asymmetric methylation by bisulphite sequencing of biased and unbiased (h)MeDIP-Seq peaks. Filled squares represent methylated/hydroxymethylated cytosines and empty squares represent unmodified cytosines. Bisulphite sequencing confirms the asymmetric nature of methylation in biased regions (middle and bottom) and reveals extensive non-CpG methylation, whereas the unbiased region (top) contains mostly CpG methylation.

  4. Gene expression and promoter methylation in ES cells and during differentiation.
    Figure 4: Gene expression and promoter methylation in ES cells and during differentiation.

    a, Relationship between 5hmC and 5mC levels at gene promoters and expression of downstream genes measured by RNA-Seq in J1 ES cells. Significance levels are relative to all promoters (**P<0.001, ***P<0.0001 throughout the figure). b, Relationship between 5hmC, 5mC and presence/absence of H3K4me3 and H3K27me3 at gene promoters (data from ref. 14). c, qRT-PCR validation of genes downregulated upon Tet1/2 KD across three biological replicates and expression level changes in the same genes from ES to EB differentiation (values are mean±s.d.). d, Induction of a shRNA targeting Tet1 in a stable ES cell line also results in the downregulation of the genes in c. Restoring TET1 expression leads to recovery in expression of these genes (values are mean±s.d.). e, Co-immunostaining of mock and Tet1/2 KD ES cells for 5hmC (red) and CDX2 (green). Scale bar, 20µm. Cells were scored for presence of 5hmC and CDX2 expression (n = 1,120 and 1,209 for mock and Tet1/2 KD cells, respectively; values are percentage of cells±95% confidence interval). f, KD/WT ratios for promoter 5mC and 5hmC shows that downregulated genes, and in particular qRT-PCR-validated ones, suffer methylation changes different from the pool of all genes. Genes downregulated upon ES to EB differentiation have increased 5mC enrichment levels and decreased 5hmC. g, Examples of 5hmC and 5mC profiles of two genes in J1 and E14 ES cells, Tet1/2 KD and E14 EBs, and corresponding quantification of 5mC and 5hmC levels by glucMS-qPCR (the MspI site used is indicated by the grey arrowhead). A considerable reduction in 5hmC levels is detected upon both Tet1/2 KD and differentiation into EBs, with a concomitant increase in 5mC.

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Sequence Read Archive


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Author information

  1. These authors contributed equally to this work.

    • Gabriella Ficz &
    • Miguel R. Branco


  1. Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge CB22 3AT, UK

    • Gabriella Ficz,
    • Miguel R. Branco,
    • Stefanie Seisenberger,
    • Fátima Santos,
    • Timothy A. Hore,
    • C. Joana Marques &
    • Wolf Reik
  2. Bioinformatics Group, The Babraham Institute, Cambridge CB22 3AT, UK

    • Felix Krueger &
    • Simon Andrews
  3. Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK

    • Wolf Reik
  4. Present address: Genetics Department, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal.

    • C. Joana Marques


G.F. and M.R.B. designed and performed experiments and analysed data. S.S. established the (h)MeDIP-Seq protocol and performed bisulphite sequencing. F.S. performed immunostainings. T.A.H. carried out qRT-PCR and glucMS-qPCR analyses. C.J.M. established the inducible Tet1 shRNA ES cell line. F.K. and S.A. performed bioinformatic analyses. W.R. designed and directed the study. G.F., M.R.B. and W.R. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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All sequencing files have been deposited at the EBI Sequence Read Archive under the accession number ERP000570 (http://www.ebi.ac.uk/ena/data/view/ERP000570).

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