Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA218854 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR975622_1.fastq.gz | Fastqsanger.gz | |||
| SRR975622_2.fastq.gz | Fastqsanger.gz | |||
| SRR975623_1.fastq.gz | Fastqsanger.gz | |||
| SRR975623_2.fastq.gz | Fastqsanger.gz | |||
| SRR975624_1.fastq.gz | Fastqsanger.gz |
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Project description:Recent large-scale studies have defined genomewide, cell type-specific patterns of DNA methylation, a modification known to be important for regulating gene expression in both normal development and disease states. However, determining the functional significance of specific methylation events remains a challenging problem due to the current lack of targeted methodologies for removing these modifications. Here we describe an approach for efficient targeted demethylation of specific CpGs in human cells using fusions of engineered transcription activator-like effector (TALE) repeat arrays and the TET1 hydroxylase catalytic domain. Using these TALE-TET1 fusions, we demonstrate that modification of certain critical methylated promoter CpG positions can be associated with substantial increases in endogenous human gene expression. Our results delineate a general strategy for defining the functional significance of specific CpG methylation marks in the context of endogenous gene loci and validate new programmable DNA demethylation reagents with broad utility for research and potential therapeutic applications. Bisulfite sequencing of three different loci in three different cell lines (Klf4 in K562s, HBB in K562s and RHOXF2 in 293s and HeLas. Biological triplicates of all samples and controls (off-target and GFP controls).
2013-09-23 | E-GEOD-50761 | biostudies-arrayexpress
Project description:Recent large-scale studies have defined genomewide, cell type-specific patterns of DNA methylation, a modification known to be important for regulating gene expression in both normal development and disease states. However, determining the functional significance of specific methylation events remains a challenging problem due to the current lack of targeted methodologies for removing these modifications. Here we describe an approach for efficient targeted demethylation of specific CpGs in human cells using fusions of engineered transcription activator-like effector (TALE) repeat arrays and the TET1 hydroxylase catalytic domain. Using these TALE-TET1 fusions, we demonstrate that modification of certain critical methylated promoter CpG positions can be associated with substantial increases in endogenous human gene expression. Our results delineate a general strategy for defining the functional significance of specific CpG methylation marks in the context of endogenous gene loci and validate new programmable DNA demethylation reagents with broad utility for research and potential therapeutic applications.
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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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Project description:DNA demethylating agents are epigenetic drugs for the therapy of myeloid leukemias. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects, however, the relationships between these characteristics have not been established yet due to the lack of method to induce only DNA demethylation. Here we show that a fusion protein comprising the methyl-CpG binding domain (MBD) and the catalytic domain of Ten-eleven translocation protein 1 (TET1-CD) globally demethylates and upregulates a number of methylated genes. Gene expression microarray analyses using human embryonic kidney cell line 293T indicate that cells expressing wild-type (wt) TET1 catalytic domain with MBD (MBD-TET1-CDwt) upregulated more genes than ones expressing TET1-CDwt without MBD or catalytically inactive TET1-CD mutant (mut) with MBD (MBD-TET1-CDmut) and their upregulated genes frequently contained CpG islands (CGIs) within ± 1,000 bp of the transcription start site (TSS). Interestingly, 88% of genes upregulated 5-fold or more by MBD-TET1-CDwt were also reactivated after treatment with DNA demethylating agent, 5-azacytidine, suggesting that gene reactivation by both methods is primarily based on DNA demethylation.
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Project description:Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytsosine (5fC), and 5-carboxylcytosine (5caC). 5fC/5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), while some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naïve ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR-domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of PRDM14 expression rapidly removed the 5mC associated with transient elevation of 5hmC at pluripotency-associated genes, germline-specific genes, and imprinted loci but not across the entire genome, which resemble second wave of DNA demethylation in gonadal PGCs. PRDM14 physically interacts with TET1/TET2 and enhances the recruitment of TET1/TET2 at target loci. Knockdown of Tet1/Tet2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors against APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 normally takes place in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs. To investigate the function of TET1/TET2 in transcriptional regulation by PRDM14 in ESCs, we exploited microarray analysis using total mRNA derived from Scramble, Scramble + PRDM14, Tet1/Tet2 KD, Tet1/Tet2 KD + PRDM14 mouse ESC.
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| PRJNA429659 | ENA