Gains of DNA methylation in myeloid terminal differentiation are dispensable for gene silencing but influence the differentiated phenotype

DNA methylation-mediated regulation drives and stabilizes transcription states throughout development. In myeloid differentiation, DNA methylation changes occur predominantly in the direction towards hypomethylation. Also, in vitro differentiation of monocytes to dendritic cells and macrophages is characterized by DNA demethylation. In this study, we identified the existence of methylation changes in the direction of hypermethylation among genes that become repressed during monocyte-to-dendritic cell differentiation. We identified the acquisition of DNA methylation in genes such as CSF3R, FYN, and CX3CR1, but not in others, such as CD14. Analysis of the dynamics of methylation and expression changes of these genes revealed that loss of expression was rapid and was associated with the loss of H3K4me3 and H3K36me3, whereas gains of DNA methylation were progressive and partially concomitant with increases in H3K9me3 and H3K27me3. Inhibition of DNA methyltransferases, with the DNA replication-independent drug nanaomycin A, revealed that there were no effects on expression and H3K4me3 changes, despite the partial impairment of DNA methylation and H3K27me3 acquisition. However, cells treated with the DNA methyltransferase inhibitor showed lower levels of dendritic cell surface markers, suggesting a potential effect on the stability of the differentiated phenotype. Our data give rise to a novel perspective on the functional relevance and mechanisms of the acquisition of DNA methylation in myeloid cell differentiation.