Targeted DNA demethylation of the Arabidopsis genome using the Human TET1 catalytic domain.
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ABSTRACT:
PROVIDER: PRJNA429659 | ENA |
REPOSITORIES: ENA
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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.
2022-08-11 | PXD028566 | Pride
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:DNA methylation is important for silencing genes and transposable elements. Changes in DNA methylation can be heritable and thus, the loss or gain of methylation can lead to the formation of stable epialleles. A well characterized example of a stable epiallele in plants is fwa-4, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing upregulation of FWA and a heritable late flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the Human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA upregulation, and a heritable late flowering phenotype. Additional ZF-TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCAS9 based targeted demethylation system using the TET1cd and a modified SunTag system and demonstrate its ability to target demethylation at and activate the expression of FWA and CACTA1. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons.
2018-02-18 | GSE109115 | GEO
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.
2013-09-23 | GSE50761 | GEO
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.
2022-07-14 | GSE183465 | GEO
Project description: Not available
| S-EPMC5834696 | biostudies-literature
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.
2017-02-09 | GSE74744 | GEO
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.
2022-08-31 | PXD028850 | Pride
Project description:TET1 oxidizes methylated cytosine into 5-hydroxymethylcytosine, resulting in regulation of DNA methylation and gene expression. Full length TET1 (TET1FL) has a CXXC domain that binds to unmethylated stretches of CpG dinucleotides known as CpG islands (CGIs). This CXXC domain allows TET1 to protect CGIs from aberrant methylation but it also limits its ability to regulate genes outside of CGIs. Here we report a novel isoform of TET1 (TET1ALT) that has a unique transcription start site from an alternate promoter in intron 2, yielding a protein with a unique translation start site. Importantly, TET1ALT lacks the CXXC domain but retains the catalytic domain. TET1ALT is silenced in ESCs but becomes activated in embryonic and adult tissues while TET1FL is expressed in ESCs, but repressed in adult tissues. Overexpression of TET1ALT shows production of 5-hydroxymethylcytosine with distinct (and weaker) effects on DNA methylation or gene expression when compared to TET1FL. TET1ALT (but not TET1FL) is aberrantly activated in multiple cancer types including breast cancer, lung cancer and lymphomas. Our data suggest that the predominantly expressed isoform of TET1 in cancer cells does not protect from CGI methylation and likely mediates dynamic site specific demethylation outside of CGIs.
2017-05-12 | GSE93617 | GEO
Project description:Investigation of TET1 non-catalytic functions in mESCs cultured in serum + LIF (primed)
2022-07-29 | E-MTAB-10937 | biostudies-arrayexpress