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)26, 27.

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.

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)26, 27. h, Distribution of high-, intermediate- or low density CpG promoters (HCP, ICP or LCP)25 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.

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 complex17. 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.