Transition-metal-catalyzed C-S bond formation: recent developments and pharmaceutical applications.

Despite being considered a stable epigenetic modification [–], increasing evidence suggests that DNA methylation establishment is more dynamic and heterogeneous than previously anticipated [–]. Assessing DNA methylation heterogeneity in bulk cells requires high-coverage amplicon bisulfite sequencing analyses [–]. These analyses reveal the combinatorial patterns of DNA methylation across individual DNA molecules, referred to as epialleles [, , , ]. Previous studies have demonstrated that epiallele analysis can capture the oscillatory behavior of DNA methylation since epiallele patterns reflect the degree and the relative tendency of specific CpG sites to be methylated within a locus [–, , , ]. Notably, we previously reported a high degree of epipolymorphism in brain development, suggesting that the epiallele composition may serve as an epigenetic “identity card” that distinguishes specific brain regions and cell types [, , ]. However, it remains unclear whether each epiallele configuration is clonally transmitted through cell divisions or whether they are dynamically re-established over successive generations. Specifically, it is unknown whether a single cell preserves its epiallele composition in its progeny or whether daughter cells progressively acquire a broader spectrum of epialleles as a consequence of DNA methylation plasticity. To address these questions, we isolated putative single cells and expanded them into individual clones, ensuring that the observed epiallele composition originated from a putative single progenitor cell. By tracking epiallele trajectories at previously characterized loci [, ] and across different genomic regions, we observed that cell expansion does not lead to the fixation of a single (or few) epiallele configuration, which likely reflects epiallele clonal transmission. Instead, it is associated with the reemergence of diverse epiallele profiles with dominant epialleles preserved across cell divisions. Moreover, in some cases, the relative frequencies of epialleles subtly shift, leading each clone to establish a new stable methylation equilibrium. Notably, the tendency of the single CpG sites to be methylated or not methylated remained conserved, supporting a model in which DNA methylation dynamics generate structured epiallele diversity while preserving locus-specific CpG methylation tendencies. In this framework, epigenetic heterogeneity emerges as a controlled system, shaped by the average methylation level of the specific genomic region and independent of the specific genomic context. To unravel the trajectories that epiallele profiles follow from a putative single cell, we designed an experimental system consisting of different conditions (Fig. ). We diluted a confluent U251 “mother” plate (MP, ~4 million cells) to generate putative single, separated cells that were grown until they formed single clones, here defined as diluted mother plate (DMP). Several well-separated clones were then collected from the DMP. Several well-isolated clones were collected the collected from the DMP and expanded independently until reaching ~20,000, ~40,000, ~200,000 cells, and full confluence (~4 million cells), referred to as 20k, 40k, 200k, and confluent clones (CC), respectively. In parallel, the remaining unpicked clones from the DMP (approximately 50–100 clones) were pooled and collected as a reference population (here defined as Pool). DNA methylation was analyzed across these experimental conditions at four levels of progressively increasing resolution: (i) average methylation levels across all analyzed CpG sites; (ii) methylation level at each single-CpG site; (iii) epiallele classes distributions, grouping molecules by the number of methylated CpGs irrespective of position; and (iv) full epiallele profiles, accounting for the specific positional arrangement of methylated and unmethylated CpGs along individual DNA molecules. Assuming that DNA methylation is not bimodally distributed, regions displaying intermediate methylation levels are expected to be the most informative for assessing epiallele diversity and DNA methylation heterogeneity, compared with loci showing either very low or very high methylation. On this basis, we initially focused on the and genes as model loci, as they exhibit intermediate DNA methylation levels [, , , ] together with highly reproducible epiallele distributions [, , , ]. We subsequently extended the same analytical framework to additional genomic regions and contexts, including the and promoters, SRR1 and SRR2 enhancers, intron, LINE1 and Sat2 repetitive elements, and late-replicating regions in the context of solo-WCGW sites. Schematic representation of the experimental setup. In the mother plate (MP), each colored sphere represents a cell. In the diluted mother plate (DMP), each colored sphere represents a group of cells derived from the same mother cell (clone). Clones from the DMP were picked and plated individually into single wells to grow