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

Nature Protocols volume 11, pages 1081–1100 (2016)Cite this article

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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.

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Figure 1: Schematic diagram of BS-seq, MAB-seq and caMAB-seq.
Figure 2
Figure 3: Schematic diagram of base-resolution mapping methods for oxidized methylcytosines.
Figure 4: Bioanalyzer electropherogram of sequencing libraries of MAB-seq/caMAB-seq.
Figure 5: Base-resolution 5fC/5caC maps and affinity-enrichment-based 5fC/5caC maps at the Tchp–Git2 locus in mouse ESCs.

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Acknowledgements

H.W. was supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research and is currently supported by the National Human Genome Research Institute (R00HG007982). X.W. was supported by the China Scholarship Council. Y.Z. is an investigator of the Howard Hughes Medical Institute.

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Author notes

  1. Hao Wu

    Present address: Present address: Department of Genetics, the Epigenetics Program, University of Pennsylvania, Philadelphia, Pennsylvania, USA.,

  2. Hao Wu and Xiaoji Wu: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute, Boston, Massachusetts, USA

    Hao Wu, Xiaoji Wu & Yi Zhang

  2. Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA

    Hao Wu, Xiaoji Wu & Yi Zhang

  3. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Hao Wu, Xiaoji Wu & Yi Zhang

  4. Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts, USA

    Hao Wu, Xiaoji Wu & Yi Zhang

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H.W. and X.W. performed experiments and carried out data analysis. H.W., X.W. and Y.Z. wrote the manuscript.

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Correspondence to Yi Zhang.

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Wu, H., Wu, X. & Zhang, Y. Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq. Nat Protoc 11, 1081–1100 (2016). https://doi.org/10.1038/nprot.2016.069

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