Target capture bisulfite sequencing (tcBS-seq) to evaluate clonal methylation inheritance [RNA-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. We devised an experiment to directly assess the fidelity of DNA methylation through cell divisions at the genome-scale, by subcloning populations of cells and performing target DNA capture followed by high-throughput bisulfite sequencing (tcBS-seq) on both the subclones (14 cell lines) and the parent population of cells (2 cell lines). To do so, we established mouse embryonic fibroblasts (MEFs) from two sibling E13.5 mouse embryos and immortalised the cell lines. From these parental lines, we randomly sampled fourteen single cells to establish clonal populations of around 1-2 million cells on which we profiled DNA methylation using tcBS-seq.