title: Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing.
abstract: 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 the active DNA demethylation cascade and reveals a regulatory role of 5fC/5caC excision repair in this step-wise process.
authors.affiliation: 1] Howard Hughes Medical Institute, Boston, Massachusetts, USA. [2] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA. [3] Harvard Stem Cell Institute, Boston, Massachusetts, USA. [4] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Wu
authors.affiliation: 1] Howard Hughes Medical Institute, Boston, Massachusetts, USA. [2] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA. [3] Harvard Stem Cell Institute, Boston, Massachusetts, USA. [4] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Xiaoji
authors.initials: X
authors.lastname: Wu
authors.affiliation: 1] Howard Hughes Medical Institute, Boston, Massachusetts, USA. [2] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA. [3] Harvard Stem Cell Institute, Boston, Massachusetts, USA. [4] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Shen
authors.affiliation: 1] Howard Hughes Medical Institute, Boston, Massachusetts, USA. [2] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA. [3] Harvard Stem Cell Institute, Boston, Massachusetts, USA. [4] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry / Q000031:analogs & derivatives; D000818:Animals; D001483:Base Sequence; D002843:Chromatin / Q000235:genetics*; D003596:Cytosine / Q000031:analogs & derivatives / Q000737:chemistry; D019175:DNA Methylation / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D053595:Embryonic Stem Cells / Q000737:chemistry; D005786:Gene Expression Regulation / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D044502:Thymine DNA Glycosylase / Q000235:genetics
chemical_list: C560974:5-carboxylcytosine; C560973:5-formylcytosine; D002843:Chromatin; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D008780:Methyltransferases; D044502:Thymine DNA Glycosylase