Wu, HaoWu, XiaojiShen, LiZhang, Yi2017-08-152014Wu, Hao, Xiaoji Wu, Li Shen, and Yi Zhang. 2014. “Single-Base Resolution Analysis of Active DNA Demethylation Using Methylase-Assisted Bisulfite Sequencing.” Nature Biotechnology 32, no. 12: 1231–1240. doi:10.1038/nbt.3073.1087-0156http://nrs.harvard.edu/urn-3:HUL.InstRepos:33758653Active 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 the active DNA demethylation cascade and reveals a regulatory role of 5fC/5caC excision repair in this step-wise process.en-USJournal Article2015-06-05Yi Zhang, Xiaoji Wu, Li Shen, Hao Wu2017-08-1510.1038/nbt.3073