TET2 binds regions of open chromatin with enhancer features in ES cells. (A) Venn diagram showing overlap of called peaks in TET2-N or FLAG M2 ChIP-seq experiments (left) as well as average ChIP signal from replicate samples (right). High- and low-confidence TET2 binding sites are defined, respectively, as regions showing evidence of TET2 binding in both peak sets (high) or only supported by a called positive region in one peak set (low). (B) Heat maps of ChIP-seq signal from wild-type TET2 or TET2 with two copies of a FLAG tag (2xFL). Tracks of H3K27ac and EP300 enrichment as well as regions of DHS in ES cells are also shown. The vertical axis contains all high-confidence TET2 binding sites defined in A, sorted by decreasing EP300 read counts. The horizontal axis is centered on TET2 peaks. (C) Histogram showing overlap of high-confidence TET2 binding sites with regions of DHS in ES cells. Random matched control regions (Control) were generated with same size, orientation, and distance relative to gene bodies. (D) Histogram showing overlap as C but with random matched control regions relative to DHS. The percentage of CpG islands, proximal (±250 bp) or distal (±1500 bp) transcription start sites (TSS), EP300 binding sites, H3K27ac, and H3K4me1 enriched domains, Gene body, and CTCF binding sites, overlapping TET2 high-confidence binding sites are shown. Data indicate the number of TET2 BS overlapping a given region set divided by the total number of loci in the region set (e.g., 175,237 DHS in ES cells) and fold enrichment over matched control are stated (red). (E) Representative ChIP-seq tracks showing a TET2-bound region upstream of the Zfp281 gene in ES cells. (F) Pie chart showing the distribution of high-confidence TET2 binding sites with respect to the indicated genomic regions. Each TET2 binding site is counted only once and excluded when moving clockwise from proximal promoters (e.g., 8% of TET2 high-confidence BS overlap with DHS but does not overlap with any of the preceding genomic elements). Blue hues indicate the fraction of TET2-bound regions with regulatory potential at promoter-distal sites (∼90% of all TET2-bound regions). (G) Pie chart as in F showing average change in DNA methylation (5mC and 5hmC) upon TET2 loss in ES cells (whole-genome bisulfite sequencing data [WGBS] from Hon et al. 2014) in high-confidence TET2 binding sites and flanking regions (±250 bp). Only CpG sites covered by more than 10 reads were included in the analysis. Each TET2 binding site is counted only once and excluded when moving clockwise. Red hues indicate the fraction (∼93%) of TET2-bound regions showing DNA methylation changes consistent with loss of TET2 catalytic activity (gain or 5mC or loss of 5hmC, or both). Regions in B–F enriched for DHS, EP300, H3K27ac, H3K4me1, and CTCF sites in ES cells were experimentally determined in the ENCODE Project (The ENCODE Project Consortium 2011).
