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RNA-seq of TET1 knockout, catalytic mutant and wild type mESCs

ABSTRACT: RNA-seq of TET1 knockout, catalytic mutant and wild type mESCs

PROVIDER: PRJEB47563 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR6747440.fastq.gz	Fastqsanger.gz
	ERR6747441.fastq.gz	Fastqsanger.gz
	ERR6747442.fastq.gz	Fastqsanger.gz
	ERR6747443.fastq.gz	Fastqsanger.gz
	ERR6747444.fastq.gz	Fastqsanger.gz

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RNA-seq of TET1 knockout, catalytic mutant and wild type mESCs

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TET1 interactome in mESCs (via ChIP-MS)

Project description:Precise regulation of DNA methylation in mammals is critical for genome stability and epigenetic regulation. The discovery of the ten-eleven translocation (TET) proteins catalyzing the oxidation from 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized the perspective on the complexity and regulation of DNA modifications. Despite accumulating knowledge about the role of TET1, it remains unclear to what extent these can be attributed to its catalytic activity. Here, we use genome engineering and quantitative multi-omics approaches to dissect the role and mechanism of TET1 in mESCs. Our study identifies TET1 as an essential interaction hub for multiple chromatin modifying complexes and as a global regulator of histone modifications. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. Moreover, we show that the establishment of H3K9me3 and H4K20me3 at ERV1, ERVK, and ERVL is mediated by TET1 independent of DNA demethylation. We provide evidence that repression of endogenous retroviruses depends on the interaction between TET1 and SIN3A. In summary, we demonstrate that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs.

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Dissection of TET1 catalytic and non-catalytic functions reveals DNA demethylation independent gene and ERV regulation

Project description:Surveillance of DNA methylation in mammals is critical for genome stability and epigenetic regulation. The discovery of the ten-eleven translocation (TET) proteins catalyzing the oxidation from 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized the understanding of DNA methylation dynamics. Interestingly, in recent years evidence accumulated that TET1 also harbours non-catalytic functions. However, the role and mechanism of TET1 DNA demethylation independent functions still remain poorly understood. Here, we use genome engineering and quantitative multi-omics approaches to dissect the non-catalytic role of TET1. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. To gain insights into possible mechanisms by which TET1 regulates transcription independent of DNA demethylation, we asked if the loss of TET1 is accompanied by changes in the histone modificaiton landscape. To this end, we compared the relative abundances of core histone modifications between Tet1 KO, Tet1 CM and WT mESCs using quantitative LC-MS/MS analysis. Surprisingly, we observed a profound global reduction of pH4Kac and H4K20me3 as well as H3K27me3 in Tet1 KO mESC. Vice versa, the monomethylation states of the latter two residues, H3K27me1 and H4K20me1 were significantly increased in Tet1 KO. Similar to the results from the transcriptome data, most of these changes were specific to Tet1 KO cells.

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Evaluation of TET1 non-catalytic functions reveals DNA demethylation independent gene and ERV regulation

Project description:Surveillance of DNA methylation is critical for genome stability and epigenetic regulation in mammals. The discovery of the ten-eleven translocation (TET) proteins catalyzing the oxidation from 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized the understanding of DNA methylation maintenance and remodeling. Interestingly in recent years evidence accumulated that TET1 also harbours non-catalytic functions. However, the role and mechanism of TET1 DNA demethylation independent functions still remain poorly understood. Here, we use genome engineering, mass spectrometry and quantitative multi-omics approaches to dissect the non-catalytic role of TET1. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. Our study identifies TET1 as a scaffold protein and a global regulator of histone modifications. Moreover, we show that the establishment of H3K9me3 and H4K20me3 at ERV1, ERVK and ERVL is mediated by TET1 independent of DNA demethylation. Finally, we provide evidence that repression of endogenous retroviruses depends on the interaction between TET1 and SIN3A. In summary, we found that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs.

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Project description:In this study: (1) we characterized the Tet1 non-catalytic functions in the regulation of ESC gene expression programs by performing transcriptomic analysis of Tet1 wild type (WT), Tet1 catalytic mutant (Mut) and Tet1 knockout (KO) mouse ESCs by RNA-seq to identify differentially expressed genes. (2) We mapped the genome-wide occupancy of endogenously FLAG-tagged Tet1-WT and Tet1-Mut in ESCs by CUT&Tag using a specific antibody against FLAG. (3) We determined how the genome-wide occupancy of the epigenetic modifiers: Ezh2, Sin3a, Chd4 and the enrichment of histone marks: H3K27me3, H3K4me3 and H3K27ac, are affected in Tet1-KO ESCs versus Tet1-WT and Tet1-Mut by CUT&Tag or CUT&RUN. (4) We analyzed methylation levels and distribution in Tet1-WT, Tet1-Mut and Tet1-KO ESCs by WGBS, to confirm Tet1 non-catalytic targets with no differential methylation. (5) We explored whether Tet1 non-catalytic functions play a role in chromatin accessibility by performing ATAC-seq in Tet1-WT, Tet1-Mut and Tet1-KO ESCs.

2022-01-25 | GSE176389 | GEO

Gene expression profile of Tet1 knockout mouse embryonic stem cells (mESCs)

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TET1 Displays Catalytic and Non-catalytic Functions in the Adult Mouse Cortex [RNA-seq]

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