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. 2011 May 19;473(7347):343-8.
doi: 10.1038/nature10066. Epub 2011 Apr 13.

TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity

Kristine Williams  1 , Jesper Christensen, Marianne Terndrup Pedersen, Jens V Johansen, Paul A C Cloos, Juri Rappsilber, Kristian Helin
Affiliations

TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity

Kristine Williams et al. Nature. .

Abstract

Enzymes catalysing the methylation of the 5-position of cytosine (mC) have essential roles in regulating gene expression and maintaining cellular identity. Recently, TET1 was found to hydroxylate the methyl group of mC, converting it to 5-hydroxymethyl cytosine (hmC). Here we show that TET1 binds throughout the genome of embryonic stem cells, with the majority of binding sites located at transcription start sites (TSSs) of CpG-rich promoters and within genes. The hmC modification is found in gene bodies and in contrast to mC is also enriched at CpG-rich TSSs. We provide evidence further that TET1 has a role in transcriptional repression. TET1 binds a significant proportion of Polycomb group target genes. Furthermore, TET1 associates and colocalizes with the SIN3A co-repressor complex. We propose that TET1 fine-tunes transcription, opposes aberrant DNA methylation at CpG-rich sequences and thereby contributes to the regulation of DNA methylation fidelity.

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Figures

Figure 1
Figure 1
Identification of TET1 target genes. a, Western blot showing TET1, OCT4 and NANOG levels for control-transfected (shScr) and TET1-depleted (shTet1#3 and shTet1#5) mouse ES cells. b, Examples of TET1 ChIP-seq results in control or Tet1 knockdown ES cells. ChIP-seq was performed using both an anti-N- and anti-C-terminal TET1 antibody (Tet1-N and Tet1-C). y-axis of binding profiles denotes number of sequence tag reads. c, Left panel, mean distribution of tags across gene bodies for TET1 ChIP-seq in control and TET1 knockdown cells. Right panel, diagram illustrating the overall distribution of TET1 binding sites into TSS (±1 kb), promoter (−1 to −5 kb), exon, intron and intergenic regions. d, Venn diagram illustra5ng the overlap of TET1 target genes using anti-TET1-N and -C antibodies. e, f, Histograms showing promoter CpG density, divided into high-, intermediate- or low-density CpG promoters (HCP, ICP or LCP) as defined in ref. 25 (e) or distribution of H3K4me3 (K4) and H3K27me3 (K27)25 (f) for all genes or for TET1 target genes. g, Overlay of TET1 target genes with active genes (RNA polymerase II binding and H3K79me2), non-productive (RNA polymerase II binding, no H3K79me2) and inactive (no RNA polymerase II binding or H3K79me2), .
Figure 2
Figure 2
Hydroxymethylcytosine localizes to TSS and gene body. a, Examples of hmC DIP-seq results in mouse ES cells. ChIP-seq profiles of TET1 are included for comparison. b, Diagram illustrating the overall distribution of hmC into TSS (±1 kb), promoter (−1 to −5 kb), exon, intron and intergenic regions. c, The mean distribu5on of tags across gene bodies for hmC, mC and IgG. d, Almost a third (28%) of hmC positive TSSs showed a more than twofold reduction in hmC signal in mouse ES cells depleted of TET1. e, DIP-qPCR was performed in control mouse ES cells, Tet1 knockdown cells (shTet1#3 and shTet1#5), and Dnmt TKO cells as indicated. f, Overlay of genes positive for hmC at the TSS with TET1 target genes using FDR cut-off values of 0.01 or 0.1 in the ChIP-seq analysis. g, Overlay of hmC positive genes with active genes (RNA polymerase II binding and H3K79me2), non-productive (RNA polymerase II binding, no H3K79me2) and inactive (no RNA polymerase II binding or H3K79me2), . h, Distribution of high-, intermediate- or low density CpG promoters (HCP, ICP or LCP) for all genes or hmC-positive genes. i, Plot illustrating the genome-wide correlation of TET1, hmC and mC signal intensity (rpm, reads per million) with CpG density. All error bars denote s.d., n = 3.
Figure 3
Figure 3
Knockdown of Tet1 in ES cells affects transcription. a, Microarray analyses were performed in control (shScr) and Tet1 knockdown cells (shTet1#4 and shTet1#5) in triplicates. Venn diagram showing overlap between TET1-bound genes, and genes up- or downregulated by both shRNAs using a cut-off of FDR < 0.05. b, qRT–PCR validation of selected genes. c, Genes that were found upregulated or downregulated by Tet1 knockdown show similar regulation in Dnmt TKO ES cells. All error bars denote s.d., n = 3.
Figure 4
Figure 4
TET1 interacts with SIN3A. a, Peptides identified by mass spectrometry from anti-Flag and tandem anti-Flag–HA purification of Flag–HA–TET1 and Flag–HA–TET2 stably expressed in 293 cells. The presented proteins are all part of the SIN3A complex. b, Antibodies specific for TET1, SIN3A and c-Myc (negative control) were used for immunoprecipitation (IP) and western blot (WB) using nuclear extracts from mouse ES cells. Input represents 8%. c, Examples of SIN3A and TET1 ChIP-seq results in mouse ES cells. d, Diagram illustrating the overall distribution of SIN3A binding sites into TSS (±1 kb), promoter (−1 to −5 kb), exon, intron and intergenic regions. e, Mean distribution of tags across gene bodies for SIN3A and TET1. f, Venn diagram illustrating a significant (P < 10–8) overlap between TET1 and SIN3A target genes (FDR < 0.01). g, ChIP-qPCR in control or Tet1 knockdown cells (shTet1#4 and shTet1#5). h, Left panel, western blot illustrating knockdown efficiencies of TET1 and SIN3A. Right panel, genes that are upregulated by Tet1 knockdown are also de-repressed by Sin3a knockdown. All error bars denote s.d., n = 3.
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References

    1. Fouse SD, et al. Promoter CpG methylation contributes to ES cell gene regulation in parallel with Oct4/Nanog, PcG complex, and histone H3 K4/K27 trimethylation. Cell Stem Cell. 2008;2:160–169. - PMC - PubMed
    1. Meissner A, et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature. 2008;454:766–770. - PMC - PubMed
    1. Mohn F, et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell. 2008;30:755–766. - PubMed
    1. Saxonov S, Berg P, Brutlag DL. A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proc. Natl Acad. Sci. USA. 2006;103:1412–1417. - PMC - PubMed
    1. Takai D, Jones PA. Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc. Natl Acad. Sci. USA. 2002;99:3740–3745. - PMC - PubMed

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