Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing

Nat Biotechnol. 2014 Dec;32(12):1231-40. doi: 10.1038/nbt.3073. Epub 2014 Nov 2.

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.

Publication types

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

MeSH terms

  • 5-Methylcytosine / chemistry
  • Animals
  • Base Sequence
  • Chromatin / genetics*
  • Cytosine / analogs & derivatives
  • Cytosine / chemistry
  • DNA Methylation / genetics*
  • DNA Repair / genetics
  • Embryonic Stem Cells / chemistry
  • Gene Expression Regulation / genetics
  • High-Throughput Nucleotide Sequencing / methods*
  • Methyltransferases / chemistry
  • Methyltransferases / genetics*
  • Mice
  • Thymine DNA Glycosylase / genetics

Substances

  • 5-carboxylcytosine
  • 5-formylcytosine
  • Chromatin
  • 5-hydroxymethylcytosine
  • 5-Methylcytosine
  • Cytosine
  • Methyltransferases
  • Thymine DNA Glycosylase

Associated data

  • GEO/GSE62631