Mechanisms of establishment and functional significance of DNA demethylation during erythroid differentiation.

Erythroid differentiation is associated with global DNA demethylation, but a complete methylome was lacking in the erythroid lineage. We have generated allele-specific base resolution methylomes of primary basophilic erythroblasts (BasoEs) and compared these with 8 other cell types. We found that DNA demethylation during differentiation from hematopoietic stem/progenitor cells (HSPCs) to BasoEs occurred predominantly in intergenic sequences and in inactive gene bodies causing the formation of partially methylated domains (PMDs) in 74% of the BasoE methylome. Moreover, differentially methylated regions (DMRs) between HSPCs and BasoEs occurred mostly in putative enhancer regions and were most often associated with GATA, EKLF, and AP1 binding motifs. Surprisingly, promoters silent in both HSPCs and BasoEs exhibited much more dramatic chromatin changes during differentiation than activated promoters. Unmethylated silent promoters were often associated with active chromatin states in highly methylated domains (HMDs) but with polycomb-repression in PMDs, indicating that silent promoters are generally regulated differently in HMDs and PMDs. We show that long PMDs replicate late, but that short PMDs replicate early and therefore that the partial methylation of DNA after replication during erythroid expansion occurs throughout S phase of the cell cycle. We propose that baseline maintenance methylation following replication decreases during erythroid differentiation resulting in PMD formation and that the presence of HMDs in the BasoE methylome results from transcription-associated DNA methylation of gene bodies. We detected ∼700 large allele-specific DMRs that were enriched in single-nucleotide polymorphisms, suggesting that primary DNA sequence might be a determinant of DNA methylation levels within PMDs.