Target capture bisulfite sequencing (tcBS-seq) to evaluate clonal methylation inheritance [BiSulfite-seq]

DNA methylation is considered a stable epigenetic mark due to its presumed long-term inheritance through cell divisions. Here, we perform high-throughput bisulfite sequencing on clonally derived cell lines to quantitatively measure mitotic methylation inheritance at the nucleotide level. We find that although DNA methylation is generally faithfully maintained at hypo- and hypermethylated sites, this is not the case at intermediately methylated CpGs. Low fidelity intermediate methylation is interspersed throughout the genome and within genes with no or low transcriptional activity. Moreover, we determine that the probabilistic changes that occur at intermediately methylated sites are due to DNMT1 rather than DNMT3A/3B activity. The observed lack of clonal inheritance at intermediately methylated sites challenges the concept of DNA methylation as a consistently stable epigenetic mark. To test whether DNMT3A/3B are mechanistically responsible for the probabilistic gain of methylation at intermediately methylated CpG sites, we generated four MEF lines (A, B, C, and D) from sibling E13.5 Dnmt3aflox/flox3bflox/flox mouse embryos and used recombinant TAT-CRE protein to induce the double knockout (DKO) in vitro. We subsequently performed tcBS-seq with four DKO and four matched control cell lines.