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Effect of the simultaneous knockdown of TET1, TET2 and TET3 on the transcriptome of HEK293 cells

ABSTRACT: We compared TET triple knockdown cells to control cells treated with non-targeting siRNAs to determine global gene expression changes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE67348 | GEO | 2015/07/16

SECONDARY ACCESSION(S): PRJNA279568

REPOSITORIES: GEO

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Effect of the simultaneous knockdown of TET1, TET2 and TET3 on the transcriptome of HEK293 cells

Project description:We compared TET triple knockdown cells to control cells treated with non-targeting siRNAs to determine global gene expression changes. TET triple knockdown

2015-07-16 | E-GEOD-67348 | biostudies-arrayexpress

Effect of the simultaneous knockdown of TET1, TET2 and TET3 on DNA methylation levels of HEK293 cells

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Endogenous epitope-tagging of Tet1, Tet2 and Tet3 identifies TET2 as a naive pluripotency marker.

Project description:Tet1, Tet2, and Tet3 encode DNA demethylases that play critical roles during stem cell differentiation and reprogramming to pluripotency. Although all three genes are transcribed in pluripotent cells, little is known about the expression of the corresponding proteins. Here, we tagged all the endogenous Tet family alleles using CRISPR/Cas9, and characterised TET protein expression in distinct pluripotent cell culture conditions. Whereas TET1 is abundantly expressed in both naïve and primed pluripotent cells, TET2 expression is restricted to the naïve state. Moreover, TET2 is expressed heterogeneously in embryonic stem cells (ESCs) cultured in serum/leukemia inhibitory factor, with expression correlating with naïve pluripotency markers. FACS-sorting of ESCs carrying a Tet2Flag-IRES-EGFP reporter demonstrated that TET2-negative cells have lost the ability to form undifferentiated ESC colonies. We further show that TET2 binds to the transcription factor NANOG. We hypothesize that TET2 and NANOG co-localise on chromatin to regulate enhancers associated with naïve pluripotency genes.

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Reduced representation bisulfite sequencing (RRBS) of WT, Tet1-/-, Tet2-/-, Tet1-/-:Tet2-/- (DKO), and Tet1-/-:Tet2-/-:Tet3-/- (TKO) murine embryonic stem under normal culture conditions.

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

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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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Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis.

Project description:Dioxygenases of the TET (Ten-Eleven Translocation) family produce oxidized methylcytosines, intermediates in DNA demethylation, as well as new epigenetic marks. Here we show data suggesting that TET proteins maintain the consistency of gene transcription. Embryos lacking Tet1 and Tet3 (Tet1/3 DKO) displayed a strong loss of 5-hydroxymethylcytosine (5hmC) and a concurrent increase in 5-methylcytosine (5mC) at the eight-cell stage. Single cells from eight-cell embryos and individual embryonic day 3.5 blastocysts showed unexpectedly variable gene expression compared with controls, and this variability correlated in blastocysts with variably increased 5mC/5hmC in gene bodies and repetitive elements. Despite the variability, genes encoding regulators of cholesterol biosynthesis were reproducibly down-regulated in Tet1/3 DKO blastocysts, resulting in a characteristic phenotype of holoprosencephaly in the few embryos that survived to later stages. Thus, TET enzymes and DNA cytosine modifications could directly or indirectly modulate transcriptional noise, resulting in the selective susceptibility of certain intracellular pathways to regulation by TET proteins.

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Effect of TET1 and TET3 overexpression on the transcriptome of HEK293 cells

Project description:We compared TET1 and TET3 overexpressing cells to uninduced cells with endogenous levels of the respective transcript to determine global gene expression changes. TET1 overexpression, TET3 overexpression

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Effect of TET1 and TET3 overexpression on the transcriptome of HEK293 cells

Project description:We compared TET1 and TET3 overexpressing cells to uninduced cells with endogenous levels of the respective transcript to determine global gene expression changes.

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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

Project description: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 contribution 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 and may constitute another layer of epigenetic regulation.

2020-07-24 | PXD019514 | Pride

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