Distribution and evolution of methylation profiles in mouse brain cells.

The methylation profiles generated during the early stages of mouse brain development play a critical role in the gene expression regulation program; given the large number of different cellular population in murine brain, is the great importance the study of the cell to cell heterogeneity in terms of DNA methylation. In this work, I performed an in-depth single molecule methylation analysis in order to assess the cell to cell methylation heterogeneity analyzing the epialleles composition at the D-aspartate oxidase (ddo) putative regulatory region, that is a gene implicated in a correct neurodevelopment and that undergoes a strong methylation changes during the early stages of mouse development. I found that brain methylation heterogeneity is generated and develops in an extremely conserved fashion, giving rise to a deterministically regulated distribution of different epialleles, distinct for each stage and cell type, evoking the possible existence of a novel, cell population based, combinatorial code of CpG methylation. Importantly, rapid epialleles remodeling toward mature neuronal and glial patterns was observed in ES cells population upon neural differentiation. The high degree of epipolimorphism, detected also in pure cell populations, supports the existence of mechanisms oriented to maintain the epiallele patterns in a dynamic equilibrium involving continuously occurring methylation and demethylation events in each single cell. The interplay between contiguous CpGs differing in methylation susceptibility likely underlies specific epiallele frequency and dynamics in a spatial-specific manner. The present data on Ddo gene provide a proof of principle that employment of high coverage single molecule methylation analysis, may potentially reveal unprecedented mechanisms underlying methylation establishment, changes and alterations within cell populations in development and diseases, unpredictable by classical methylation analyses.