Simultaneous mapping of active DNA demethylation and sister chromatid exchange in single cells

Abstract

To understand mammalian active DNA demethylation, various methods have been developed to map the genomic distribution of the demethylation intermediates 5-formylcysotine (5fC) and 5-carboxylcytosine (5caC). However, the majority of these methods requires a large number of cells to begin with. In this study, we describe low-input methylase-assisted bisulfite sequencing (liMAB-seq ) and single-cell MAB-seq (scMAB-seq), capable of profiling 5fC and 5caC at genome scale using ∼100 cells and single cells, respectively. liMAB-seq analysis of preimplantation embryos reveals the oxidation of 5mC to 5fC/5caC and the positive correlation between chromatin accessibility and processivity of ten-eleven translocation (TET) enzymes. scMAB-seq captures the cell-to-cell heterogeneity of 5fC and 5caC and reveals the strand-biased distribution of 5fC and 5caC. scMAB-seq also allows the simultaneous high-resolution mapping of sister chromatid exchange (SCE), facilitating the study of this type of genomic rearrangement. Therefore, our study not only establishes new methods for the genomic mapping of active DNA demethylation using limited numbers of cells or single cells but also demonstrates the utilities of the methods in different biological contexts.

Non‐canonical Bases in the Genome: The Regulatory Information Layer in DNA

“…[98,99] Of interest in this context are several analyses of genomic fdC profiles showing alink between the presence of fdC and epigenetic tuning at regulatory elements, such as enhancers and promoters. [100][101][102][103][104][105][106] However, due to the correlative and snapshot-like nature of this kind of analysis, the data do not provide information about whether the presence of fdC at these sites is permanent or transient. A single cell analysis revealed high cell-to-cell heterogeneity of the fdC profiles, [105,106] which may reflect fast turnover of fdC.…”

“…[100][101][102][103][104][105][106] However, due to the correlative and snapshot-like nature of this kind of analysis, the data do not provide information about whether the presence of fdC at these sites is permanent or transient. A single cell analysis revealed high cell-to-cell heterogeneity of the fdC profiles, [105,106] which may reflect fast turnover of fdC. This establishes fdC as ar ather short-lived demethylation intermediate (frequent methylation/demethylation cycles).…”

Advances in the Profiling of Single‐Cell DNA Modifications

“…In another report, single‐cell methylase‐assisted bisulfite sequencing (scMAB‐seq) method was reported to profile 5fCand 5caC at the single‐cell level . The single‐cell method is adopted from MAB‐seq in combination with scRRBS and PBAT‐based strategy .…”

“…TET dioxygenase-mediated active demethylation [18] [14] 5mC PBAT(preamplification with tagging) 1.0-2.2 million CpG sites Limited below 100 mESC,HL60,K562 scBS-seq [13] 5mC PBAT(preamplification) 3.7 million CpG sites Limited below 100 mESC, mouse oocyte snmC-seq [15] 5mC PBAT(preamplification with tagging) 5% of the genome 100-1000 Mouse neuronal cell, human neuronal cells sci-MET [17] 5mC Combinational cellular indexing 0.8% of the genome Above 1000 Human neuronal cell scAba-seq [27] 5hmC Aba-seq combined with Cel-seq 44 000 5hmCpG sites 100-500 mESC, mouse oocyte CLEVER-seq [31] 5fC C-T chemistry combined with MALBAC 3500-7000 5fCpG sites Limited below 100 Mouse early embryo scMAB-seq [34] 5fC/5caC MAB-seq combined with scRRBS/scBS-seq Refer to scRRBS/scBS-seq Limited below 100 mESC, mouse zygote www.advancedsciencenews.com www.small-methods.com 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) and restored to unmodified cytosines through the base excision repair (BER) pathway. [19][20][21] As mentioned above, the three oxidized methylcytosines may play unique regulatory roles in gene expression in addition to serving as intermediates of active demethylation .…”

“…Small Methods 2019, 3,1900137 In another report, single-cell methylase-assisted bisulfite sequencing (scMAB-seq) method was reported to profile 5fCand 5caC at the single-cell level. [34] The single-cell method is adopted from MAB-seq [35] in combination with scRRBS [11] and PBATbased strategy. [13] The M.SssI CpG methyltransferase methylated all the unmodified cytosines before bisulfite treatment, and only 5fCand 5caC can be deaminized and read as "T" in subsequent sequencing.…”