Dataset Information

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

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 steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation.

SUBMITTER: Mulholland CB

PROVIDER: S-EPMC7374584 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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

Mulholland Christopher B CB Traube Franziska R FR Ugur Enes E Parsa Edris E Eckl Eva-Maria EM Schönung Maximilian M Modic Miha M Bartoschek Michael D MD Stolz Paul P Ryan Joel J Carell Thomas T Leonhardt Heinrich H Bultmann Sebastian S

Scientific reports 20200721 1

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 tra ...[more]

PMID: 32694513

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Project description:Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeodomain-containing protein NANOG, which has an essential role in establishing ground state pluripotency during somatic cell reprogramming. Whereas the genomic occupancy of NANOG has been extensively investigated, comparatively little is known about NANOG-associated proteins and their contribution to the NANOG-mediated reprogramming process. Using enhanced purification techniques and a stringent computational algorithm, we identify 27 high-confidence protein interaction partners of NANOG in mouse embryonic stem cells. These consist of 19 previously unknown partners of NANOG that have not been reported before, including the ten-eleven translocation (TET) family methylcytosine hydroxylase TET1. We confirm physical association of NANOG with TET1, and demonstrate that TET1, in synergy with NANOG, enhances the efficiency of reprogramming. We also find physical association and reprogramming synergy of TET2 with NANOG, and demonstrate that knockdown of TET2 abolishes the reprogramming synergy of NANOG with a catalytically deficient mutant of TET1. These results indicate that the physical interaction between NANOG and TET1/TET2 proteins facilitates reprogramming in a manner that is dependent on the catalytic activity of TET1/TET2. TET1 and NANOG co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in embryonic stem cells, and TET1 binding is reduced upon NANOG depletion. Co-expression of NANOG and TET1 increases 5-hydroxymethylcytosine levels at the top-ranked common target loci Esrrb and Oct4 (also called Pou5f1), resulting in priming of their expression before reprogramming to naive pluripotency. We propose that TET1 is recruited by NANOG to enhance the expression of a subset of key reprogramming target genes. These results provide an insight into the reprogramming mechanism of NANOG and uncover a new role for 5-methylcytosine hydroxylases in the establishment of naive pluripotency.

Project description:Background:Pluripotency is proposed to exist in two different stages: Naive and Primed. Conventional human pluripotent cells are essentially in the primed stage. In recent years, several protocols have claimed to generate naive human embryonic stem cells (hESCs). To the best of our knowledge, none of these protocols is currently recognized as the gold standard method. Furthermore, the consistency of the resulting cells from these diverse protocols at the molecular level is yet to be shown. Additionally, little is known about the principles that govern the metabolic differences between naive and primed pluripotency. In this work, using a computational approach, we tried to shed light on these basic issues. Results:We showed that, after batch effect removal, the transcriptome data of eight different protocols which supposedly produce naive hESCs are clustered consistently when compared to the primed ones. Next, by integrating transcriptomes of all hESCs obtained by these protocols, we reconstructed p-hESCNet and n-hESCNet, the first metabolic network models representing hESCs. By exploiting reporter metabolite analysis we showed that the status of NAD + and the metabolites involved in the TCA cycle are significantly altered between naive and primed hESCs. Furthermore, using flux variability analysis (FVA), the models showed that the kynurenine-mediated metabolism of tryptophan is remarkably downregulated in naive human pluripotent cells. Conclusion:The aim of the present paper is twofold. Firstly, our findings confirm the applicability of all these protocols for generating naive hESCs, due to their consistency at the transcriptome level. Secondly, we showed that in silico metabolic models of hESCs can be used to simulate the metabolic states of naive and primed pluripotency. Our models confirmed the OXPHOS activation in naive cells and showed that oxidation-reduction potential vary between naive and primed cells. Tryptophan metabolism is also outlined as a key pathway in primed pluripotency and the models suggest that decrements in the activity of this pathway might be an appropriate marker for naive pluripotency.

Dataset Information

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

Publications

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

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