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| Status |
Public on Feb 10, 2015 |
| Title |
Single base resolution analysis of 5-formyl and 5-carboxyl cytosine reveals the actual promoter DNA methylation dynamics in embryonic stem cells [RRBS] |
| Organism |
Mus musculus |
| Experiment type |
Methylation profiling by high throughput sequencing
|
| Summary |
DNA methylation is catalysed by DNA methyltransferases (DNMTs) and is necessary for a correct embryonic development. On the other hand, the DNA demethylation is mediated by the Ten Eleven Translocation (Tet) proteins through oxidation of 5-methyl cytosine (5mC) to 5-hydroxyl (5hmC), 5-formyl (5fC) and 5-carboxyl (5caC) cytosine, and by the Thymine-DNA glycosylase (TDG) that excises the 5fC and 5caC. In embryonic stem cells (ESCs), gene promoters are maintained in an hypomethylated state, but the dynamics of this phenomenon still remains unknown. Here we present a genome-wide approach, named methylation-assisted bisulfite sequencing (MAB-Seq) that enables single-base resolution mapping of 5fC and 5caC and measuring of their relative abundance. Application of this method to mouse ESCs exposed the presence of 5fcaC residues on the hypomethylated promoters of the expressed genes, revealing an active DNA demethylation mechanism since the loss of TDG leads to an increase of 5fC/5caC. We also show that TDG is actually bound on these regions and that co-localizes and interacts with Tet1. We moreover demonstrate, by reduced representation of bisulfite sequencing (RRBS), that active promoters are actually demethylated by a Tet-dependent mechanism and that Dnmt1 and Dnmt3a are responsible of this DNA methylation. Our work shows the whole-genome map of 5fC and 5caC at single base resolution in ESCs, it demonstrates in detail the DNA methylation dynamics occurring on expressed gene promoters and identifies the key players of this mechanism. Furthermore, we provide a new tool (MAB-Seq) that can be broadly used in all biological contexts for epigenetics study involving identification and quantification of 5fC and 5caC at single base resolution.
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| Overall design |
Methylation-assisted bisulfite sequencing (MAB-Seq) of E14 embryonic stem cells (ESCs), Biotag ChIP-Seq of Tdg and Reduced representation Bisulfite Sequencing (RRBS) in E14 ESCs.
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| Contributor(s) |
Neri F, Incarnato D, Oliviero S |
| Citation(s) |
25660018 |
| Submission date |
Mar 06, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Francesco Neri |
| E-mail(s) |
francesco.neri@unito.it
|
| Organization name |
University of Torino
|
| Street address |
Via Nizza 52
|
| City |
Torino |
| State/province |
Italy |
| ZIP/Postal code |
10126 |
| Country |
Italy |
| |
|
| Platforms (1) |
| GPL16173 |
Illumina HiScanSQ (Mus musculus) |
|
| Samples (6)
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| This SubSeries is part of SuperSeries: |
| GSE55660 |
Single base resolution analysis of 5-formyl and 5-carboxyl cytosine reveals the actual promoter DNA methylation dynamics in embryonic stem cells |
|
| Relations |
| BioProject |
PRJNA240311 |
| SRA |
SRP039505 |
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