Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing
收藏干细胞与再生医学数据中心2022-02-20 更新2024-03-06 收录
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资源简介：
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.
提供机构：
Harvard Medical School/HHMI
创建时间：
2022-02-20
