Cell Reports
Volume 32, Issue 11, 15 September 2020, 108155
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A Bisulfite-free Approach for Base-Resolution Analysis of Genomic 5-Carboxylcytosine

https://doi.org/10.1016/j.celrep.2020.108155Get rights and content
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Highlights

  • A bisulfite-free caCLEAR approach for profiling 5caCG sites genome-wide

  • caCLEAR specifically targets only 5caCG sites and avoids whole-genome sequencing

  • Two pluripotency states of mouse ESCs differ in the genomic distribution of 5caCGs

  • 5caCGs tend to distribute in the antisense strand of highly expressed protein genes

Summary

Due to an extreme rarity of 5-carboxylcytosine (5caC) in the mammalian genome, investigation of its role brings a considerable challenge. Methods based on bisulfite sequencing have been proposed for genome-wide 5caC analysis. However, bisulfite-based sequencing of scarcely abundant 5caC demands significant experimental and computational resources, increasing sequencing cost. Here, we present a bisulfite-free approach, caCLEAR, for high-resolution mapping of 5caCGs. The method uses an atypical activity of the methyltransferase eM.SssI to remove a carboxyl group from 5caC, generating unmodified CGs, which are localized by uTOP-seq sequencing. Validation of caCLEAR on model DNA systems and mouse ESCs supports the suitability of caCLEAR for analysis of 5caCGs. The 5caCG profiles of naive and primed pluripotent ESCs reflect their distinct demethylation dynamics and demonstrate an association of 5caC with gene expression. Generally, we demonstrate that caCLEAR is a robust economical approach that could help provide deeper insights into biological roles of 5caC.

Keywords

5-carboxylcytosine
5caC
5-methylcytosine
5mC
demethylation
covalent labeling
C-C bond cleavage
pluripotency states
mouse ESCs

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These authors contributed equally

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