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Functional genomic analysis of WT, Tet1-/-, Tet2-/-, Tet1-/-:Tet2-/- (DKO) murine embryonic stem cells

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Mus musculus

PROVIDER: GSE122814 | GEO | 2019/06/26

REPOSITORIES: GEO

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Functional genomic analysis of WT, Tet1-/-, Tet2-/-, Tet1-/-:Tet2-/- (DKO) murine embryonic stem cells

Project description:Functional genomic analysis of WT, Tet1-/-, Tet2-/-, Tet1-/-:Tet2-/- (DKO) murine embryonic stem cells

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RNA-seq of WT, Tet1-/-, Tet2-/-, Tet1-/-:Tet2-/- (DKO), and Tet1-/-:Tet2-/-:Tet3-/- (TKO) murine embryonic stem cells following six days of LIF withdrawal.

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Project description:RRBS was used to investigate the methylation status of CpG dinucleotides in different Tet knockouts.

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DAZL regulates Tet1 translation in murine embryonic stem cells.

Project description:Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3β and MEK (so-called 2i conditions) pushes ESC cultures toward a more homogeneous naïve pluripotent state, but the molecular underpinnings of this naïve transition are not completely understood. Here, we demonstrate that DAZL, an RNA-binding protein known to play a key role in germ-cell development, marks a subpopulation of ESCs that is actively transitioning toward naïve pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5-hydroxylation of methyl-cytosine, and enhances Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely stunts 2i-mediated TET1 induction and hydroxymethylation. Our results provide insight into the regulation of the acquisition of naïve pluripotency and demonstrate that DAZL enhances TET1-mediated cytosine hydroxymethylation in ESCs that are actively reprogramming to a pluripotent ground state.

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Distinct roles of the methylcytosine oxidases Tet1 and Tet2 in mouse embryonic stem cells.

Project description:Dioxygenases of the Ten-Eleven Translocation (TET) family are 5-methylcytosine oxidases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products in DNA. We show that Tet1 and Tet2 have distinct roles in regulating 5hmC in mouse embryonic stem cells (mESC). Tet1 depletion diminishes 5hmC levels at transcription start sites (TSS), whereas Tet2 depletion is predominantly associated with decreased 5hmC in gene bodies. Enrichment of 5hmC is observed at the boundaries of exons that are highly expressed, and Tet2 depletion results in substantial loss of 5hmC at these boundaries. In contrast, at promoter/TSS regions, Tet2 depletion results in increased 5hmC, potentially because of the redundant activity of Tet1. Together, the data point to a complex interplay between Tet1 and Tet2 in mESC, and to distinct roles for these two proteins in regulating promoter, exon, and polyadenylation site usage in cells.

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Gene expression profiles of Tet1 knockout (T1KO), Tet2 knockout (T2KO) and Tet1/Tet2 double knockout (DKO) mouse embryonic stem cells.

Project description:Global gene expression profile of single and double mutant mouse ES cells were compared to wt ES cells. Two male Tet1 KO, one male Tet2 KO, two male double KO, two female double KO, two male WT and two female WT mouse ES cells were compared. Global gene expression profile of single and double knockout mouse embryonic stem cells were compared to that of wild type mouse ES cells. All used ES lines were derived from C57/BL/6 mixed background mice. RNA from feeder free mutant mouse ES cells was competetively hybridized against RNA from WT ES cells. Same sex lines were compared. Two independent ES line of each genotype were used, with the exception of Tet2 KO ES cells where only one male line was used.

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Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells.

Project description:TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.

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