DNA methylation patterns re-establish during clonal expansion restoring cell-to-cell epialleles heterogeneity.

It is still debated whether the combination of methylated and unmethylated CpGs, defined as epialleles, is stably propagated during cell division or whether epiallele diversity can dynamically emerge over time. By analyzing epiallele profiles in putative single-cell-derived clones, we sought to track the behaviour of DNA methylation profiles through cell divisions to determine whether epialleles are faithfully copied from mother to daughter cells, or instead a heterogeneous epiallele population is generated, giving rise to cell-to-cell methylation variability. Our findings revealed that: i) clonal expansion did not result in the maintenance of a single dominant epiallele configuration; ii) expanding clones progressively re-established a broad repertoire of epialleles similar to that observed in the mixed population; iii) although the major epiallele structures were partially preserved, each clone displayed a distinct epiallele distribution characterized by differences in epiallele frequencies relative to the bulk population. Notably, these clone-specific epiallele distributions remained relatively stable as cell numbers increased during proliferation. Overall, our findings suggest that epiallele diversity reemerges during clonal expansion, consistent with the dynamic behavior of DNA methylation at individual CpG sites and cell-to-cell variability in DNA methylation patterns.