Tracking Ddo epialleles during neural differentiation and brain development

We performed high resolution methylation analysis at single molecule level of the promoter region of D-Aspartate-Oxidase (Ddo) gene during brain development and embryonic stem cell neural differentiation. Single molecule methylation analysis enabled us to establish the effective epiallele composition whithin mixed or pure brain cell populations. In this framework, an epiallele is defined as a specific combination of methylated CpG within Ddo locus and can represent the epigenetic haplotype revealing a cell to cell methylation heterogeneity. By this approach, we found an high degree of polymorphism of methylated alleles (epi-polymorphism) evolving in a remarkably conserved fashion during brain development. The different sets of epialleles mark stage, brain areas and cell type and unravel the possible role of specific CpG in favouring or inhibiting local methylation. Undifferentiated embryonic stem cell showed non-organized distribution of epialleles which apparently originated by stochastic methylation events on individual CpGs. Upon neural differentiation, despite no change in average methylation were detected, epiallele distribution profoundly changed gradually shifting toward organized patterns specific to the glial or neuronal cell types. Our findings provide a deep view of gene methylation heterogeneity in brain cell populations promising to furnish innovative ways to unravel mechanisms underlying methylation patterns generation and alteration in brain diseases.