Cytosine methylation dynamics during normal cell differentiation

Abstract

A major mediator of epigenetic regulation of gene expression in the mammalian genome is the methylation of cytosines in the context of cytosine-guanine (CG) dinucleotides. Our current appreciation of how cytosine methylation exerts its effects is based on limited information, as prior techniques only study a few loci simultaneously. In order to look at methylation changes on a whole-genome level, we have developed a technique called the HELP assay.;The HELP assay digesting genomic DNA with the methylation-sensitive restriction enzyme HpaII and its methylation-insensitive isoschizomer MspI, amplifies fractions <2 kb in size by ligation-mediated PCR, and enriches the unmethylated fraction of the genome. Products generated by this technique were hybridized to an oligonucleotide microarray that covers 6.2 Mb of the mouse genome in a proof-of-principle experiment. This experiment shows that the HELP assay allows both intragenomic and intergenomic comparisons of cytosine methylation.;Having demonstrated feasibility of the HELP assay, we applied it to characterize DNA methylation dynamics of cellular differentiation in vivo (different stages of spermatogenic cells) and in vitro (human ES cells and their differentiated derivatives: mesenchymal stem cells and erythroid progenitor cells). Our results have shown methylation differences between different cell types in genomic regions that are not demonstrated in previous studies. Specifically, we showed that CG dinucleotide-depleted promoters and intergenic regions are more prone to methylation changes, thus more informative in distinguishing different cell types via their methylation patterns.;These results emphasize the importance of using comprehensive, and non-biased assays in order to get clearer picture of the dynamics of DNA methylation patterns in physiology.