TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells

Abstract

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 prominent bivalent promoter hypermethylation without an overall corresponding gene expression decrease in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taken together with further genome-wide methylation and TET1 and DNMT3B ChIP-Seq analysis, we conclude that the TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.

The contribution of active and passive mechanisms of 5mC and 5hmC removal in human T lymphocytes is differentiation‐ and activation‐dependent

“…The fact that DNA methylation has the tendency to accumulate to transcriptional inactive genes [2], and at enhancers that are not actively bound by transcription factors (a process that would be extremely deleterious for genes encoding for effector cytokines) was already proposed with the discovery that DNA-binding factors were necessary and sufficient to induce local demethylation, and that loss of transcription factor binding was conversely sufficient for DNA methylation [43,44]. This is also in line with recent findings showing that the activity of TET proteins is important to safeguard promoters from de novo methylation in embry-onic stem cells, thereby preserving appropriate lineage-specific transcription upon differentiation [45,46]. Similarly, TET1 was shown to prevent the spreading of DNA methylation at CpG islands [28], and also to be required in germ cells to provide efficient protection from aberrant DNA methylation, thereby stabilizing the acquired epigenetic landscape after reprogramming events [47].…”

The Aging Metabolome—Biomarkers to Hub Metabolites

“…It is also quite intriguing that high levels of Tet1 and 5hmC are associated with transcriptionally permissive histone bivalent domain (H3K27me3 marks). [ 73 ] Relationship between αKG levels and distribution of 5hmC and Tet enzymes in the aging genome remain to be elucidated. In conjunction with Tet enzymes, enzymes that mediated histone demethylation, JMJD2 (Jumanji domain‐containing protein 2), and JARID1 (Jumonji, AT rich interactive domain 1) families of the N ε ‐trimethyl‐lysine demethylases are also Fe (II) and αKG dependent.…”

Identification of DNA motifs that regulate DNA methylation

“…We reasoned that, if the identified motifs are important for recruiting the enzymes, these motifs would be enriched around the binding sites of the recruited enzymes. To this end, we have collected all the available ChIP-seq experiments of TET and DNMT enzymes [45][46][47][48] . Indeed, at the center of TET1 ChIP-seq peaks in hESC H1 cells 46 , the UM sites occur 26.7 times of expected counts (see details in Methods), whereas MM motifs occur roughly same (1.4 times) as the expected counts ( Figure 2C, first panel from the left).…”

“…To this end, we have collected all the available ChIP-seq experiments of TET and DNMT enzymes [45][46][47][48] . Indeed, at the center of TET1 ChIP-seq peaks in hESC H1 cells 46 , the UM sites occur 26.7 times of expected counts (see details in Methods), whereas MM motifs occur roughly same (1.4 times) as the expected counts ( Figure 2C, first panel from the left). This observation is consistent with the previous reports that TETs can be recruited to specific locus by DNA binding factors 40,44 .…”