title: Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq.
abstract: A complete understanding of the function of the ten-eleven translocation (TET) family of dioxygenase-mediated DNA demethylation requires new methods to quantitatively map oxidized 5-methylcytosine (5mC) bases at high resolution. We have recently developed a methylase-assisted bisulfite sequencing (MAB-seq) method that allows base-resolution mapping of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), two oxidized 5mC bases indicative of active DNA demethylation events. In standard bisulfite sequencing (BS-seq), unmodified C, 5fC and 5caC are read as thymine; thus 5fC and 5caC cannot be distinguished from C. In MAB-seq, unmodified C is enzymatically converted to 5mC, allowing direct mapping of rare modifications such as 5fC and 5caC. By combining MAB-seq with chemical reduction of 5fC to 5hmC, we also developed caMAB-seq, a method for direct 5caC mapping. Compared with subtraction-based mapping methods, MAB-seq and caMAB-seq require less sequencing effort and enable robust statistical calling of 5fC and/or 5caC. MAB-seq and caMAB-seq can be adapted to map 5fC/5caC at the whole-genome scale (WG-MAB-seq), within specific genomic regions enriched for enhancer-marking histone modifications (chromatin immunoprecipitation (ChIP)-MAB-seq), or at CpG-rich sequences (reduced-representation (RR)-MAB-seq) such as gene promoters. The full protocol, including DNA preparation, enzymatic treatment, library preparation and sequencing, can be completed within 6-8 d.
authors.affiliation: Howard Hughes Medical Institute, Boston, Massachusetts, USA.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.|Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Wu
authors.affiliation: Howard Hughes Medical Institute, Boston, Massachusetts, USA.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.|Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Xiaoji
authors.initials: X
authors.lastname: Wu
authors.affiliation: Howard Hughes Medical Institute, Boston, Massachusetts, USA.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.|Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D044503:5-Methylcytosine / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D015254:DNA Modification Methylases / Q000378:metabolism; D051379:Mice; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000494:pharmacology
chemical_list: C560973:5-formylcytosine; D013447:Sulfites; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D015254:DNA Modification Methylases; C042345:hydrogen sulfite