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. 2015 Nov;12(11):1047-50.
doi: 10.1038/nmeth.3569. Epub 2015 Sep 7.

Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale

Bo Xia  1   2 , Dali Han  3   4 , Xingyu Lu  3   4 , Zhaozhu Sun  1   2 , Ankun Zhou  1   2 , Qiangzong Yin  5 , Hu Zeng  1   2 , Menghao Liu  1   2 , Xiang Jiang  1   2 , Wei Xie  5 , Chuan He  3   4   6   7 , Chengqi Yi  1   2   6   7
Affiliations

Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale

Bo Xia et al. Nat Methods. 2015 Nov.

Abstract

Active DNA demethylation in mammals involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). However, genome-wide detection of 5fC at single-base resolution remains challenging. Here we present fC-CET, a bisulfite-free method for whole-genome analysis of 5fC based on selective chemical labeling of 5fC and subsequent C-to-T transition during PCR. Base-resolution 5fC maps showed limited overlap with 5hmC, with 5fC-marked regions more active than 5hmC-marked ones.

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Conflict of interest statement

Competing financial interests

B.X., A.Z. and C.Y. are co-inventors on a filed patent (WO2015043493) for the labeling strategies and sequencing methods reported herein.

Figures

Figure 1
Figure 1. Cyclization labeling of 5fC and fC-CET
(a) Scheme for Friedländer Reaction. (b) AI-mediated cyclization labeling of 5fC and subsequent conjugation of biotin via click chemistry. (c) Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) Mass Spectrometry characterization of the AI-mediated labeling of 5fC in a 9-mer DNA oligo. Calculated and observed MS are shown. (d) Sanger sequencing results of the 5fC-containing 76-mer DNA. AI labeling caused C-to-T transition during PCR (indicated with the red inverted triangle). (e) Schematic diagram of fC-CET. Genomic DNA was sequentially labeled with AI, conjugated to biotin for pull-down enrichment, washed with NaOH to remove 5fC-null strands, cleaved from beads with DTT and ligated to adaptors. Such DNA was then subjected to next generation sequencing, and C-to-T transitions were specifically searched for to define 5fC sites in the whole-genome. (f) Fold enrichment of spike-in controls using qPCR. Values represent fold enrichment over the input (n = 3), normalized to the C-Ref sequence (reference with only regular Cs). 5xC mix: PCR-amplified DNA with 70% dCTP, 15% dmCTP, 10% dhmCTP and 5% dcaCTP; Single 5fC: synthetic DNA with single 5fC site; 10% 5fC: PCR-amplified DNA with 10% dfCTP (within dCTP).
Figure 2
Figure 2. fC-CET reveals base-resolution 5fC maps in the whole-genome
(a) Genome browser view of representative 5fC-enriched regions in the Nanog gene. The data shown here represents results from two biological replicates. Results from hmC-Seal in the same region were also plotted for comparison. (b) Venn diagram showing that fC-CET detected 5fC-marked regions largely overlap with hmC-Seal detected 5hmC regions. (c,d) Overall distribution of 5fC sites in genomic elements of wild-type mESCs (c) and their relative enrichment (d).
Figure 3
Figure 3. 5fC represents a more active marker than 5hmC
(a) Venn diagram of 5fC sites detected by fC-CET and 5hmC sites detected by TAB-seq in wild-type mESCs, showing limited overlap of 5fC and 5hmC on the single-base level. (b–d) Heatmaps of the abundance of 5hmC (horizontal) and 5mC (longitudinal) for the TAB-seq-detected 5hmC sites (b) and fC-CET-detected 5fC sites in wild-type (c) or TDG null (d) mESCs. (e) A representative view showing that 5fC-marked sites exhibit lower 5mC abundance compared to 5hmC. 5mC and 5hmC data were shown as mean value of two biological replicates; 5fC data represents results from two biological replicates. (f) Normalized read densities of 5fC (blue, fC-CET) and 5hmC (grey, hmC-Seal) at H3K4me1-, H3K27ac-, p300- and Tet1-enriched regions in wild-type mESCs.
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