TET1 (HUES8 WT hESCs) and DNMT3B (HUES8 WT and HUES8 TET TKO hESCs) ChIP-Seq

The TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish due to challenges of distinguishing global versus locus-specific effects. Here we show that TET1/2/3 triple knockout (TKO) human embryonic stem cells (hESCs) exhibit preferential hypermethylation at bivalent promoters without corresponding gene expression changes in undifferentiated hESCs. In the absence of the TET proteins, abnormal accumulation of DNMT3B at bivalent promoters results in hypermethylation and impaired gene activation upon differentiation. Broadly, the competitive balance between the TET proteins and de novo methyltransferases at bivalent promoters could facilitate rapid changes of their methylation state to either activate or silence transcription in a cell-lineage and gene dependent manner. ChIP-Seq was performed for TET1 (WT hESCs) and DNMT3B (WT and TKO hESCs). WT and TKO hESCs were cultured in standard hESC media, as described above. ~5 × 107 cells were fixed, washed and snap-frozen according to the Cell Fixation protocol from Active Motif (http://www.activemotif.com/documents/1848.pdf). Immunoprecipitation and DNA sequencing was done by Active Motif.