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| Status |
Public on Jun 30, 2023 |
| Title |
Bisulfite-free base-resolution sequencing of oxidized cytosines (APOBEC-seq) reveals a ubiquitous role of thymine DNA glycosylase in active gene promoters and an interaction with MBD3/NuRD [mouse APOBEC-seq] |
| Organism |
Mus musculus |
| Experiment type |
Methylation profiling by high throughput sequencing
|
| Summary |
Active DNA demethylation, the enzymatic removal of methyl groups from DNA initially triggered by their successive oxidation, is a fundamental process critical to regulating cellular identity. The major enzymes involved in this process have been recently elucidated, but other important components in this pathway and the independent contributions of oxidized 5-methylcytosine derivatives to the regulation of gene expression remain unclear. A major obstacle to the elucidation of this pathway is the low abundance of these derivatives and limited power of current detection technologies. Here, we first develop a technique for their detection with APOBEC3A conversion and sequencing (APOBEC-seq) to directly discriminate deamination-insensitive oxidized cytosines from methylated or unmethylated counterparts, which now facilitates robust DNA demethylation analysis suitable to interrogate the active DNA demethylation pathway by a comprehensive set of experiments. Our results demonstrate that APOBEC-seq is a powerful tool for the detection of oxidized cytosines, revealing insights about the relationship between oxidized gene re-activation and demethylation as a function of TDG, APEX1, and the MBD family of proteins. We also report a ubiquitous binding of TDG to all active unmethylated and unoxidized promoters – regardless of RNA polymerase subtype – suggesting that, together with TET family of enzymes, the presence of TDG at these regions may safeguard active genes from DNA hypermethylation-induced silencing and may explain the failures of CRISPR/dCas9-based DNA methylation editing tools to introduce persistent DNA hypermethylation at targeted promoters. We also report an interaction between MBD3/NuRD and TDG which recruits TDG to its targets and may shed light on the critical role of MBD3 in development. Finally, we apply APOBEC-seq to profile oxidation in vivo in the mouse cortex and report a dramatic tissue-specific pattern of oxidation in genes and enhancers.
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| |
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| Overall design |
APOBEC-sequencing of ChIP DNA for histone marks, RNA pol2 phosphoSer5, and input.
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| |
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| Contributor(s) |
Sapozhnikov DM, Szyf M |
| Citation missing |
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| |
| Submission date |
Apr 01, 2023 |
| Last update date |
Jun 30, 2023 |
| Contact name |
Moshe Szyf |
| E-mail(s) |
moshe.szyf@mcgill.ca
|
| Phone |
5143987107
|
| Organization name |
McGill University
|
| Department |
Pharmacology & Therapeutics
|
| Street address |
3655 Promenade Sir William Osler, Room 1309
|
| City |
Montreal |
| State/province |
QC Quebec |
| ZIP/Postal code |
H3G1Y6 |
| Country |
Canada |
| |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (12)
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| GSM7135359 |
APOBEC-seq of H3K27ac ChIP DNA from adult mouse cortex animal no. 5 |
| GSM7135360 |
APOBEC-seq of H3K27ac ChIP DNA from adult mouse cortex animal no. 7 |
| GSM7135361 |
APOBEC-seq of H3K27ac ChIP DNA from adult mouse cortex animal no. 9 |
| GSM7135362 |
APOBEC-seq of H3K4me1 ChIP DNA from adult mouse cortex animal no. 5 |
| GSM7135363 |
APOBEC-seq of H3K4me1 ChIP DNA from adult mouse cortex animal no. 7 |
| GSM7135364 |
APOBEC-seq of H3K4me1 ChIP DNA from adult mouse cortex animal no. 9 |
| GSM7135365 |
APOBEC-seq of RNA polymerase II phospho Serine 5 ChIP DNA from adult mouse cortex animal no. 5 |
| GSM7135366 |
APOBEC-seq of RNA polymerase II phospho Serine 5 ChIP DNA from adult mouse cortex animal no. 7 |
| GSM7135367 |
APOBEC-seq of RNA polymerase II phospho Serine 5 ChIP DNA from adult mouse cortex animal no. 9 |
| GSM7135368 |
APOBEC-seq of input DNA from adult mouse cortex animal no. 5 |
| GSM7135369 |
APOBEC-seq of input DNA from adult mouse cortex animal no. 7 |
| GSM7135370 |
APOBEC-seq of input DNA from adult mouse cortex animal no. 9 |
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| This SubSeries is part of SuperSeries: |
| GSE228707 |
Bisulfite-free base-resolution sequencing of oxidized cytosines (APOBEC-seq) reveals a ubiquitous role of thymine DNA glycosylase in active gene promoters and an interaction with MBD3/NuRD |
|
| Relations |
| BioProject |
PRJNA951255 |
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