Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency.

Scientific Reports
Christopher B MulhollandSebastian Bultmann

Abstract

Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent s...Continue Reading

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Citations

Feb 5, 2021·Frontiers in Cell and Developmental Biology·Vera Garcia-OuteiralDiana Guallar
Apr 20, 2021·The Journal of Pathology·Kohei FujikuraLaura D Wood
Jun 11, 2021·Angewandte Chemie·Eva KorytiakováThomas Carell

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Methods Mentioned

BETA
RNA-seq
deamination
PCR
gene knockout
restriction digest
Assay

Software Mentioned

R
DESeq2
HTSFilter
Spectronaut Pulsar X
zUMIs
Ensembl
Biognosys

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