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

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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.

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Figure 1: MAB-seq strategy and quantitative mapping of active DNA demethylation.
Figure 2: Genome-scale MAB-seq analysis of the mouse genome.
Figure 3: Identification of CpGs associated with distinct processivity of TET-mediated oxidation.
Figure 4: Strand asymmetry of TET/TDG-dependent active DNA demethylation.
Figure 5: Base-resolution mapping analysis reveals distinct distribution patterns of 5fC and 5caC.
Figure 6: Base-resolution mapping of unmodified C and 5mC/5hmC reveals that TDG-mediated 5fC/5caC excision affects active demethylation dynamics.

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Acknowledgements

We thank S. Yamaguchi and F. Lu for generating and characterizing Tet mutant mouse ESCs. We also thank L.M. Tuesta and S. Yamaguchi for critical reading of the manuscript. This project is supported by National Institutes of Health grant U01DK089565. 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 (K99HG007982). X.W. was supported by the China Scholarship Council. Y.Z. is an investigator of the Howard Hughes Medical Institute.

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H.W. and Y.Z. conceived the project. H.W. and X.W. performed experiments and carried out data analysis. L.S. performed sequencing. H.W., X.W. and Y.Z. wrote the manuscript.

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Correspondence to Hao Wu or Yi Zhang.

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Wu, H., Wu, X., Shen, L. et al. Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing. Nat Biotechnol 32, 1231–1240 (2014). https://doi.org/10.1038/nbt.3073

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