Similar Datasets
Project description:Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.
| S-EPMC7897302 | biostudies-literature
Project description:Active DNA demethylation via Ten-eleven Translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of TET’s role in reprogramming. Here, we employ biochemically engineered TET mutants to examine their effects on the reprogramming efficiency of mouse embryonic fibroblasts (MEFs). We show that only TET mutants proficient for oxidation to 5fC/5caC are able to rescue the reprogramming potential of Tet2 -deficient MEFs. This effect correlated with an increased capacity to drive DNA demethylation at reprogramming enhancers, ultimately accelerating chromatin opening in these regions. Together, these experiments demonstrate that DNA demethylation through 5fC/5caC intermediates is critical for iPSC reprogramming.
2021-12-21 | GSE150964 | GEO
Project description:Active DNA demethylation via Ten-eleven Translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of TET’s role in reprogramming. Here, we employ biochemically engineered TET mutants to examine their effects on the reprogramming efficiency of mouse embryonic fibroblasts (MEFs). We show that only TET mutants proficient for oxidation to 5fC/5caC are able to rescue the reprogramming potential of Tet2 -deficient MEFs. This effect correlated with an increased capacity to drive DNA demethylation at reprogramming enhancers, ultimately accelerating chromatin opening in these regions. Together, these experiments demonstrate that DNA demethylation through 5fC/5caC intermediates is critical for iPSC reprogramming.
2021-12-21 | GSE158333 | GEO
Project description:Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency
| PRJNA664926 | ENA
Project description:Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency [ATAC-seq]
| PRJNA634247 | ENA
Project description:Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency [RNA-Seq]
| PRJNA665115 | ENA
Project description:5-Methylcytosine (5mC), the major modified DNA base in mammalian cells, can be oxidized enzymatically to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the Ten-Eleven-Translocation (TET) family of proteins. Whereas 5fC and 5caC are recognized and removed by base excision repair proteins, the 5hmC base accumulates to substantial levels in certain cell types such as brain-derived neurons and is viewed as a relatively stable DNA base. As such, the existence of "reader" proteins that recognize 5hmC would be a logical assumption, and various searches have been undertaken to identify proteins that specifically bind to 5hmC and the other oxidized 5mC bases. However, the existence of definitive 5hmC "readers" has remained unclear and proteins interacting specifically with 5fC or 5caC are also very few. On the other hand, 5hmC is incapable of interacting with a number of proteins that recognize 5mC at CpG sequences, suggesting that 5hmC is an anti-reader modification that may serve to displace 5mC readers from DNA. In this review article, we discuss candidate proteins that may interact with oxidized 5mC bases.
| S-EPMC7007358 | biostudies-literature
Project description:5-methylcytosine (5mC) was long thought to be the only enzymatically created modified DNA base in mammalian cells. The discovery of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine as reaction products of the TET family 5mC oxidases has prompted extensive searches for proteins that specifically bind to these oxidized bases. However, only a few of such "reader" proteins have been identified and verified so far. In this review, we discuss potential biological functions of oxidized 5mC as well as the role the presumed reader proteins may play in interpreting the genomic signals of 5mC oxidation products.
| S-EPMC5294966 | biostudies-literature
Project description:AID/APOBEC family enzymes are best known for deaminating cytosine bases to uracil in single-stranded DNA, with characteristic sequence preferences that can produce mutational signatures in targets such as retroviral and cancer cell genomes. These deaminases have also been proposed to function in DNA demethylation via deamination of either 5-methylcytosine (mC) or TET-oxidized mC bases (ox-mCs), which include 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. One specific family member, APOBEC3A (A3A), has been shown to readily deaminate mC, raising the prospect of broader activity on ox-mCs. To investigate this claim, we developed a novel assay that allows for parallel profiling of activity on all modified cytosines. Our steady-state kinetic analysis reveals that A3A discriminates against all ox-mCs by >3700-fold, arguing that ox-mC deamination does not contribute substantially to demethylation. A3A is, by contrast, highly proficient at C/mC deamination. Under conditions of excess enzyme, C/mC bases can be deaminated to completion in long DNA segments, regardless of sequence context. Interestingly, under limiting A3A, the sequence preferences observed with targeting unmodified cytosine are further exaggerated when deaminating mC. Our study informs how methylation, oxidation, and deamination can interplay in the genome and suggests A3A's potential utility as a biotechnological tool to discriminate between cytosine modification states.
| S-EPMC5570014 | biostudies-literature
Project description:Ten-eleven translocation (TET) family enzymes (TET1, TET2, and TET3) oxidize 5-methylcytosine (5mC) and generate 5-hydroxymethylcytosine (5hmC) marks on the genome. Each TET protein also interacts with specific binding partners and partly plays their role independent of catalytic activity. Although the basic role of TET enzymes is well established now, the molecular mechanism and specific contribution of their catalytic and noncatalytic domains remain elusive. Here, by combining in silico and biochemical screening strategy, we have identified a small molecule compound, C35, as a first-in-class TET inhibitor that specifically blocks their catalytic activities. Using this inhibitor, we explored the enzymatic function of TET proteins during somatic cell reprogramming. Interestingly, we found that C35-mediated TET inactivation increased the efficiency of somatic cell programming without affecting TET complexes. Using high-throughput mRNA sequencing, we found that by targeting 5hmC repressive marks in the promoter regions, C35-mediated TET inhibition activates the transcription of the BMP-SMAD-ID signaling pathway, which may be responsible for promoting somatic cell reprogramming. These results suggest that C35 is an important tool for inducing somatic cell reprogramming, as well as for dissecting the other biological functions of TET enzymatic activities without affecting their other nonenzymatic roles.
| S-EPMC7035619 | biostudies-literature