Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq

Nat Protoc. 2016 Jun;11(6):1081-100. doi: 10.1038/nprot.2016.069. Epub 2016 May 12.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • 5-Methylcytosine / metabolism
  • Animals
  • Cell Line
  • Cytosine / analogs & derivatives
  • Cytosine / metabolism
  • DNA / chemistry*
  • DNA / genetics*
  • DNA / metabolism
  • DNA Methylation / drug effects*
  • DNA Modification Methylases / metabolism
  • Mice
  • Sequence Analysis, DNA / methods*
  • Sulfites / pharmacology

Substances

  • 5-formylcytosine
  • Sulfites
  • 5-Methylcytosine
  • Cytosine
  • DNA
  • DNA Modification Methylases
  • hydrogen sulfite