The role of TET proteins and hydroxymethylcytosine in embryonic stem cells
Kristine Williams (1,2), Jesper Christensen (1,2), Marianne Terndrup Pedersen (1,2), Jens V. Johansen (1,3), Paul A. C. Cloos (1,2), Juri Rappsilber (4), and Kristian Helin (1,2) (1) Biotech Research and Innovation Centre (BRIC) and (2) Centre for Epigenetics, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark. (3) The Bioinformatics Centre, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark. (4) Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, EH9 3JR, UK.
In eukaryotic cells, DNA is tightly packed with histone proteins in a complex structure known as chromatin. The chromatin conformation is dynamic and can be regulated by the addition of covalent modifications to DNA or the histone N-terminal tails. These so called -Y´epigenetic¡ modifications can affect the accessibility of genes and thereby determine transcriptional outcome. In addition, chromatin modifications are implicated in the regulation of genomic stability and cell fate decisions, as well as pathological processes such as the development of cancer. DNA methylation refers to the addition of methyl-groups to cytosines in DNA generating methyl-cytosine (mC). DNA methylation at promoter regions is correlated with transcriptional repression and both the establishment and maintenance of DNA methylation is essential for embryonic development. Recently, it was demonstrated that members of the TET (ten-eleven translocation) family can catalyze hydroxylation of mC, generating hydroxymethylcytosine (hmC). However, the exact biological functions of TET proteins and hmC is still unknown. We performed genome-wide mapping of Tet1 and hmC in mouse embryonic stem cells (ESCs), which have high Tet1 expression. We find that Tet1 localizes to transcription start sites (TSSs) of a large number of genes and is especially enriched in regions with high CpG density. Global mapping of the hmC mark demonstrates that hmC is enriched in gene bodies and TSSs of genes. Furthermore, hmC localizes to regions with higher CpG density that mC. We find a significant overlap between hmC localization and Tet1 binding although not all Tet1 binding sites display enrichment for hmC. Efficient knockdown of Tet1 does not affect ESC proliferation or self-renewal, but leads to both transcriptional activation and repression of genes. In agreement with a role for Tet1 in gene repression, we find that Tet1 associates with the Sin3a co-repressor complex. Genome-wide mapping of Sin3a shows a significant overlap with Tet1 target genes, and ChIP experiments suggest that recruitment of Sin3a is partly dependent on Tet1. Our data suggest that Tet1 has dual roles in in transcriptional regulation, where it antagonizes DNA methylation through hydroxylation at CpG rich regions, and represses the transcription through recruitment of the Sin3a co-repressor complex.