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Series GSE228704 Query DataSets for GSE228704
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 [human ChIP-seq]
Organism Homo sapiens
Experiment type Genome binding/occupancy 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.
Overall design ChIP-sequencing of a flag-tagged catalytic domain of human TDG with a catalytic N140A mutation (3XFLAG-TDG-N140A-CD) and endogenous MBD3 in HEK293, HepG2, MCF-7, and IMR90 cell lines.
Contributor(s) Sapozhnikov DM, Szyf M
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Submission date Apr 01, 2023
Last update date Jun 30, 2023
Contact name Moshe Szyf
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
Platforms (1)
GPL24676 Illumina NovaSeq 6000 (Homo sapiens)
Samples (19)
GSM7135340 HEK293 FLAG-TDG-N140A-CD replicate 1
GSM7135341 HEK293 FLAG-TDG-N140A-CD replicate 2
GSM7135342 HEK293 FLAG-TDG-N140A-CD replicate 3
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
BioProject PRJNA951252

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Supplementary file Size Download File type/resource
GSE228704_RAW.tar 4.0 Mb (http)(custom) TAR (of NARROWPEAK)
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Raw data are available in SRA
Processed data provided as supplementary file

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