Quantitative single cell 5hmC sequencing reveals non-canonical gene regulation by non-CG hydroxymethylation

Eb, Fabyanic; Hu, Peng; Q, Qiu; Wang, T; Kn, Berríos; Flournoy, Jennifer; Dr, Connolly; Zhou, Zhaolan; Rm, Kohil; Wu, Hao

doi:10.1101/2021.03.23.434325

Cited by 3 publications

(2 citation statements)

References 45 publications

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“…The whole genome BS-seq (WGBS) and bACE-seq (WG-bACE-seq) experiments were performed as previously described with minor modifications (Fabyanic et al, 2023; Fabyanic et al, 2021). Briefly, ∼15 ng of genomic DNA from each sample was first spiked in with in vitro methylated lambda phage genomic DNA (0.2%) as controls and was then subjected to bisulfite conversion (EZ DNA Methylation-Direct Kit, Zymo Research Cat# D5020).…”

Section: Methods Detailsmentioning

confidence: 99%

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5-hydroxymethylcytosines regulate gene expression as a passive DNA demethylation resisting epigenetic mark in proliferative somatic cells

Wei,

Zhang,

Qiu

et al. 2023

Preprint

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SUMMARYEnzymatic 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-specificRHOXF2Bgene 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 abona fideepigenetic mark in proliferative somatic cells.

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Section: Methods Detailsmentioning

confidence: 99%

“…The pre-processing (read alignment, quality filtering and read deduplication) was performed for BS-seq and bACE-seq datasets as previously described with minor modifications (Fabyanic et al, 2023; Fabyanic et al, 2021). Briefly, demultiplexing of inline barcodes was first performed allowing up to 1-nt mismatch.…”

Section: Quantification and Statistical Analysismentioning

confidence: 99%

5-hydroxymethylcytosines regulate gene expression as a passive DNA demethylation resisting epigenetic mark in proliferative somatic cells

Wei,

Zhang,

Qiu

et al. 2023

Preprint

Self Cite

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An Overview of Global, Local, and Base-Resolution Methods for the Detection of 5-Hydroxymethylcytosine in Genomic DNA

Erlitzki,

Kohli

2024

Methods in Molecular Biology

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Mammalian DNA methylome dynamics: mechanisms, functions and new frontiers

Wei

2022

Development

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DNA methylation is a highly conserved epigenetic modification that plays essential roles in mammalian gene regulation, genome stability and development. Despite being primarily considered a stable and heritable epigenetic silencing mechanism at heterochromatic and repetitive regions, whole genome methylome analysis reveals that DNA methylation can be highly cell-type specific and dynamic within proximal and distal gene regulatory elements during early embryonic development, stem cell differentiation and reprogramming, and tissue maturation. In this Review, we focus on the mechanisms and functions of regulated DNA methylation and demethylation, highlighting how these dynamics, together with crosstalk between DNA methylation and histone modifications at distinct regulatory regions, contribute to mammalian development and tissue maturation. We also discuss how recent technological advances in single-cell and long-read methylome sequencing, along with targeted epigenome-editing, are enabling unprecedented high-resolution and mechanistic dissection of DNA methylome dynamics.

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Quantitative single cell 5hmC sequencing reveals non-canonical gene regulation by non-CG hydroxymethylation

Cited by 3 publications

References 45 publications

5-hydroxymethylcytosines regulate gene expression as a passive DNA demethylation resisting epigenetic mark in proliferative somatic cells

5-hydroxymethylcytosines regulate gene expression as a passive DNA demethylation resisting epigenetic mark in proliferative somatic cells

An Overview of Global, Local, and Base-Resolution Methods for the Detection of 5-Hydroxymethylcytosine in Genomic DNA

Mammalian DNA methylome dynamics: mechanisms, functions and new frontiers

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