DNA methylation patterns propagate in a non-clonal manner leading to cell-to-cell epialleles heterogeneity

It remains debated whether the combination of methylated and unmethylated CpGs, defined as epialleles, propagates in a clonal or, instead, in an oscillatory manner during cell division or in interphase. Using DAO and DDO as model genes, we here analyzed epiallele profiles in single-cell-derived clones determining whether epialleles are faithfully copied from mother to daughter cells or a heterogeneous epiallele set arises, leading to cell-to-cell methylation variability. Our findings revealed that: i) Epialleles are not clonally inherited and a wide set is restored. ii) Despite the non-clonal inheritance, each clone acquired a distinctive epiallele distribution that differed in frequencies from the bulk cells epialleles. iii)The clones epiallele distribution remains consistent even increasing cell numbers, suggesting the establishment of a new dynamic equilibrium over time. Overall, we found that DNA methylation at the single-molecule level undergoes continuous oscillatory changes, generating a wide range of epialleles and leading to the cell-to-cell heterogeneity.