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TET1 catalytic activity is required for reprogrammign of imprinting control regions and the patterning of sperm-specific hypomethylated regions [RNA-seq: Female PGCs]

ABSTRACT: DNA methylation erasure is required for mammalian primordial germ cell reprogramming. TET enzymes iteratively oxidize 5-methylcytosine to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxycytosine to facilitate active genome demethylation. Whether these bases are required to promote relication coupled dilution or active base excision repair during germline reprogramming remains unresolved due to the lack of genetic models that decouple TET1 activities. Here, we generated two mouse lines expressing catalytically inactive TET1 (Tet1-HxD) and TET1 that stalls at 5hmC (Tet1-V). This experiment was done to conduct transcriptiomic analysis of embryonic day (E)14.5 female primordial germ cells (PGCs) of WT, Tet1-HxD, Tet1-V, and Tet1-KO mice.

ORGANISM(S): Mus musculus

PROVIDER: GSE253636 | GEO | 2024/02/27

REPOSITORIES: GEO

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Project description:Enzymatic erasure of DNA methylation in mammals involves iterative 5-methylcytosine (5mC) oxidation by the ten-eleven translocation (TET) family of DNA dioxygenase proteins. As the most abundant form of oxidized 5mC, the prevailing model considers 5-hydroxymethylcytosine (5hmC) as a key nexus in active DNA demethylation that can either indirectly facilitate replication-dependent depletion of 5mC by inhibiting maintenance DNA methylation machinery (UHRF1/DNMT1), or directly be iteratively oxidized to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) and restored to cytosine (C) through thymine DNA glycosylase (TDG)-mediated 5fC/5caC excision repair. In proliferative somatic cells, to what extent TET-dependent removal of 5mC entails indirect DNA demethylation via 5hmC-induced replication-dependent dilution or direct iterative conversion of 5hmC to 5fC/5caC is unclear. Here we leverage a catalytic processivity stalling variant of human TET1 (TET1.var: T1662E) to decouple the stepwise generation of 5hmC from subsequent 5fC/5caC generation, excision and repair. By using a CRISPR/dCas9-based epigenome-editing platform, we demonstrate that 5fC/5caC excision repair (by wild-type TET1, TET1.wt), but not 5hmC generation alone (by TET1.var), is requisite for robust restoration of unmodified cytosines and reversal of somatic silencing of the methylation-sensitive, germline-specific RHOXF2B gene promoter. Furthermore, integrated whole-genome multi-modal epigenetic sequencing reveals that hemi-hydroxymethylated CpG dyads predominantly resist replication-dependent depletion of 5mC on the opposing strand in TET1.var-expressing cells. Notably, TET1.var-mediated 5hmC generation is sufficient to induce similar levels of differential gene expression (compared to TET1.wt) without inducing major changes in unmodified cytosine profiles across the genome. Our study suggests 5hmC alone plays a limited role in driving replication-dependent DNA demethylation in the presence of functional DNMT1/UHRF1 mechanisms, but can regulate gene expression as a bona fide epigenetic mark in proliferative somatic cells.

Dataset Information

TET1 catalytic activity is required for reprogrammign of imprinting control regions and the patterning of sperm-specific hypomethylated regions [RNA-seq: Female PGCs]

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