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Base Resolution Analysis of 5-Hydroxymethylcytosine in the Mammalian Genome

ABSTRACT: The study of 5-hydroxylmethylcytosines (5hmC), the sixth base of the mammalian genome, as an epigenetic mark has been hampered by a lack of method to map it at single-base resolution. Previous affinity purification-based methods could not precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach for mapping 5hmC at base resolution. Application of this new method to the embryonic stem cells not only confirms widespread distribution of 5hmC in mammalian genome, but also reveals a strong sequence bias and strand asymmetry at sites of 5hmC. Additionally, the relative abundance of 5hmC varies significantly depending on the types of functional sequences, suggesting different mechanisms for 5hmC deposition and maintenance. Furthermore, we observe high levels of 5hmC and reciprocally low levels of 5mC at transcription factor binding sites, revealing a dynamic DNA methylation process at cis-regulatory elements.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE36173 | GEO | 2012/05/17

SECONDARY ACCESSION(S): PRJNA156839

REPOSITORIES: GEO

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Base Resolution Analysis of 5-Hydroxymethylcytosine in the Mammalian Genome

Project description:The study of 5-hydroxylmethylcytosines (5hmC), the sixth base of the mammalian genome, as an epigenetic mark has been hampered by a lack of method to map it at single-base resolution. Previous affinity purification-based methods could not precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach for mapping 5hmC at base resolution. Application of this new method to the embryonic stem cells not only confirms widespread distribution of 5hmC in mammalian genome, but also reveals a strong sequence bias and strand asymmetry at sites of 5hmC. Additionally, the relative abundance of 5hmC varies significantly depending on the types of functional sequences, suggesting different mechanisms for 5hmC deposition and maintenance. Furthermore, we observe high levels of 5hmC and reciprocally low levels of 5mC at transcription factor binding sites, revealing a dynamic DNA methylation process at cis-regulatory elements. Base resolution sequencing of 5 hydroxymethylcytosine in human and mouse embryonic stem cells

2012-05-16 | E-GEOD-36173 | biostudies-arrayexpress

A Sensitive Approach to Map Genome-wide 5-Hydroxymethylcytosine and 5-Formylcytosine at Single-base Resolution

Project description:Mapping genome-wide 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) at single-base resolution is important to understand their biological functions. We present a cost-efficient mapping method that combines 5hmC-specific restriction enzyme PvuRts1I with a 5hmC enrichment method. The sensitive method enables detection of low abundant 5hmC sites, providing a more complete 5hmC landscape than available bisulfite-based methods. This method generated the first genome-wide 5fC map at single-base resolution. Parallel analyses revealed that 5hmC and 5fC existed with lower abundance and more dynamically in non-CpG context than in CpG context. In the genic region, distribution of 5hmCpG and 5fCpG differed from 5hmCH and 5fCH (H=A, T, C). 5hmC and 5fC were distributed distinctly at regulatory protein-DNA binding sites, depleted in permissive transcription factor binding sites, and enriched at active and poised enhancers. This sensitive bisulfite-conversion free method can be applied to biological samples with limited starting material or low abundance of cytosine modifications. Sensitive mapping of genome-wide 5-hydroxymethylcytosine and 5-formylcytosine in mouse embryonic stem cell at single-base resolution by combining 5-hydroxymethylcytosine specific restriction enzyme PvuRts1I and 5-hydroxymethylcytosine enrichment method (selective chemical labeling or SEAL)

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Project description:Active DNA demethylation in mammals involves TET-mediated iterative oxidation of 5-methylcytosine (5mC)/5-hydroxymethylcytosine (5hmC) and subsequent excision repair of highly oxidized cytosine bases 5-formylcytosine (5fC)/5-carboxylcytosine (5caC) by Thymine DNA glycosylase (TDG). However, quantitative and high-resolution analysis of active DNA demethylation activity remains challenging. Here we describe M.SssI methylase-assisted bisulfite sequencing (MAB-seq), a method that directly maps 5fC/5caC at single-base resolution. Genome-wide MAB-seq allows systematic identification of 5fC/5caC in Tdg-depleted embryonic stem cells, thereby generating a base-resolution map of active DNA demethylome. A comparison of 5fC/5caC and 5hmC distribution maps indicates that catalytic processivity of TET enzymes correlates with local chromatin accessibility. MAB-seq also reveals strong strand asymmetry of active demethylation within palindromic CpGs. Integrating MAB-seq with other base-resolution mapping methods enables quantitative measurement of cytosine modification states at key transitioning steps of active demethylation pathway, and reveals a regulatory role of 5fC/5caC excision repair in active DNA demethylation cascade. Analysis of 5fC/5caC excision repair-dependent active DNA demethylome by MAB-seq in mouse embryonic stem cells.

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Project description:5-Hydroxymethylcytosine (5hmC) is an important epigenetic mark that can regulate gene expression. While some methods were developed to detect 5hmC, direct genome-wide mapping of 5hmC at base resolution are still highly desirable. Herein, we proposed a single-step deamination sequencing (SSD-seq) method for the genome-wide mapping of 5hmC at single-base resolution. This method capitalizes on a screened engineered human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A) protein to produce differential deamination activity toward cytosine (C), 5-methylcytosine (5mC), and 5hmC. In SSD-seq, an engineered A3A protein (eA3A-v10) can adequately deaminate C and 5mC, but not 5hmC. The original C and 5mC in DNA are deaminated by eA3A-v10 to form uracil (U) and thymine (T), both of which are read as T during sequencing. However, 5hmC is resistant to the deamination by eA3A-v10 and is still read as C during sequencing. Therefore, the remaining C in the sequence reads manifests the original 5hmC. Applying SSD-seq to generate a base-resolution map of 5hmC in human lung tissue, we found that 5hmC was almost entirely confined to CpG dinucleotides. The base-resolution map of 5hmC from human lung tissue generated by SSD-seq correlated strongly with that generated by prior ACE-seq. Taken together, the SSD-seq method is single-step, bisulfite-free and does not require DNA glycosylation or chemical treatment, which offers a valuable tool for the direct and quantitative detection of 5hmC in genomes at single-base resolution.

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Project description:Cytosine base modifications 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) are present in mammalian DNA. Here, reduced bisulfite sequencing is developed for quantitatively sequencing 5fC at single-base resolution. This method is then applied with oxidative bisulfite sequencing to gain a map of 5mC, 5hmC and 5fC in mouse embryonic stem cells. 12 samples, reduced representation bisulphite treatment: 4 replicates each for bisulphite (BS), oxidative BS (oxBS) and reduced BS (redBS) for the detection of 5mC, 5hmC and 5fC. Mouse (strain B6C) embryonic stem cells.

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