TET-deficient knockout cells transit between pluripotent states and exhibit precocious germ cell differentiation
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ABSTRACT:
PROVIDER: PRJNA1142943 | ENA |
REPOSITORIES: ENA
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Project description:TET1, TET2 and TET3 are DNA demethylases with critical roles during early embryonic development and cell differentiation. To assess the contributions of TET proteins to cell function during early development, single and compound knockouts of Tet genes in mouse pluripotent embryonic stem cells (ESCs) were generated. ESCs lacking all alleles of Tet1 and Tet2, or all alleles of Tet1, Tet2 and Tet3 form epiblast-like cells and epiblast stem cells in culture, indicating that TET proteins are not required to transit between naïve, formative and primed pluripotency. Moreover, in differentiation protocols, ESCs with double-knockouts of Tet1 and Tet2 or triple-knockouts of Tet1, Tet2 and Tet3 do not differentiate normally , fail to activate somatic gene expression and retain expression of pluripotency transcription factors. Therefore, TET1 and TET2, but not TET3 act redundantly to facilitate somatic differentiation. Importantly, ESCs with double knockouts of Tet1 and Tet2, or triple knockouts of Tet1, Tet2 and Tet3 cells do differentiate into primordial germ cell-like cells (PGCLCs). Furthermore, PGCLC differentiation of Tet1, Tet2 double knockouts, or Tet1, Tet2, Tet3 triple knockouts occurs with high efficiency in the presence or absence of PGC-promoting cytokines. Moreover, acquisition of a PGCLC transcriptional programme occurs more rapidly in the absence of TET proteins. These results establish TET proteins as key regulators enabling epiblast cells to choose between somatic and germline fates. The functions of Tet1 and Tet2, but not Tet3 are required to enable epiblast cells to undergo somatic differentiation and in the absence of TET protein function, epiblast cell differentiation defaults to the germline.
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Project description:DNA methylation at 5-position of cytosine (5mC) is one of the best studied epigenetic modifications that plays important roles in diverse biological processes. Iterative oxidation of 5mC by the Ten-eleven translocation (Tet) family of proteins generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), which can be further processed by DNA repair proteins leading to DNA demethylation. Functional characterization of the Tet proteins has been complicated by the redundancy between the three Tet proteins. Using the CRISPR/Cas9 technology, we have generated mouse embryonic stem cells (ESCs) deficient for all three Tet proteins (TKO). Whole genome bisulphite sequencing (WGBS) analysis revealed that Tet-mediated DNA demethylation mainly occurs distal enhancers as well as promoters that significantly overlap with 5hmC, 5fC and 5caC. Characterization of the Tet TKO ESCs revealed a function for Tet proteins in cell fate restriction as Tet TKO ESCs tend to adopt both primed pluripotent stem cell-like state and 2-cell embryo-like state. In addition, Tet TKO ESCs exhibit elongated telomeres. Thus, our study reveals a role of Tet proteins not only in DNA demethylation, but also in cell fate restriction and telomere maintenance. 2 samples for WGBS and 2 samples for RNA-seq
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