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.

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Figure 1: Distribution of 5-hydroxymethylcytosine in the mouse genome.
Figure 2: Genetic relationship between methylation and hydroxymethylation.
Figure 3: Strand specificity and sequence context of methylation and hydroxymethylation.
Figure 4: Gene expression and promoter methylation in ES cells and during differentiation.

Accession codes

Primary accessions

Sequence Read Archive

Data deposits

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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We would like to thank A. Bird for discussions and input, K. Tabbada for help with epigenomics and transcriptomics sequencing, and M. Okano for providing the TKO ES cells. We would like to thank H. Stuart for her dedicated contribution to gene expression analyses, and all Reik lab members for advice and discussion. This work was funded by the BBSRC, MRC and the EU NoE The Epigenome.

Author information

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.

Correspondence to Wolf Reik.

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