Plant microRNAs in WT and SIDT1 KO mice liver
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE132152 | GEO | 2020/10/19
REPOSITORIES: GEO
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Project description: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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Project description: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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