DNA methylation plays important roles in genomic imprinting, X-chromosome inactivation, regulation of gene expression and maintenance of epigenetic memory. Two catalytically active de novo
DNA methyltransferases, Dnmt3a and Dnmt3b, establish DNA methylation patterns by adding methyl groups onto unmethylated DNA. Dnmt3l, a close homolog of Dnmt3a and Dnm3b, lacks the catalytic domain but interacts with unmethylated H3K4. This interaction recruits and activates methylation activity of Dnmt3a/b [6
]. When cells divide, another DNA methyltransferase, Dnmt1, maintains the pattern of DNA methylation by converting the hemimethylated DNA synthesized during replication to fully methylated state [7
]. Deletion of Dnmt1 or Dnmt3b results in embryonic lethality, whereas Dnmt3a knockout animal die around postnatal day 30 (P30), collectively indicating the essential role of DNA methyltransferases during development [11•
]. In the classical view of DNA methylation, gene silencing is achieved through inhibiting transcription factor binding to DNA by methylation at the promoter CpG sites and recruitment of methyl-CpG binding proteins (MBDs), which further recruit HDAC repressor complexes, collectively resulting in a repressive state of the chromatin. Even though, most CpG islands overlap with proximal promoters and mediate gene silencing, DNA methylation in intergenic regions and gene bodies is widespread, indicating the importance of distal-promoter methylation and its role in tissue-specific gene expression [13
]. During early embryogenesis, DNA methylation pattern is established gradually upon fertilization and development of the zygote. At first, methylation patterns of both paternal and maternal genomes are largely removed, owing to Dnmt1 being excluded from the nucleus [17
]. Subsequently, de novo
DNA methyltransferases begin to re-establish DNA methylation patterns during implantation and subsequent germ layer and cell type differentiation [11•
]. The genome-wide eradication of DNA methylation in pre-implantation embryos, followed by re-methylation to establish DNA methylation patterns, is an important process for setting up the pluripotency in early embryonic stem cells [18
]. In continuously self-renewing stem cells, genes that regulate differentiation need to be repressed in a stable manner, and this repression needs to be heritable once cells undergo division. Therefore, DNA methylation as well as histone modifications, work in combination to regulate stem cell self-renewal, differentiation, as well as reprogramming, including de-differentiation and trans-differentiation.
DNA methylation and histone modifications play important roles in stem cell differentiation along the neural lineage. During neural development, neurogenesis always precedes gliogenesis, and this correlates with tight spatiotemporal changes in the epigenetic landscape within the genome. Specifically, numerous neuronal genes, in addition to being inhibited by PRC2, are also inhibited by the action of REST (RE1 silencing transcription factor) in undifferentiated stem cells and in nonneuronal cells in the developing embryo. REST exerts its effects through RE1 binding site on promoters of target genes, and by recruiting histone modifiers and chromatin-binding proteins [20
]. Highly expressed REST in ESCs and NPCs preferably binds to neuronal genes to prevent premature neuronal differentiation by maintaining neuronal lineage genes in a poised state. The dissociation of REST from its target genes is also concomitant with REST repression as NPCs differentiate into neurons [21
]. However, the action of REST on NPCs and non-neuroectodermal lineages differ fundamentally. In ESCs and NPCs, REST recruits corepressor complex that consists of CoREST, HDAC, mSin3A and MeCP2 to the RE1 site. The HDAC within the complex plays a crucial role of maintaining the reversible repression of neuronal genes in ESCs and NPCs. Although the REST complex found in non-neuroectodermal cells is composed of similar core regulators, the chromatin state of neuronal genes is strikingly different compared to that of ESCs and NPCs. The REST complex in non-neuroectodermal cells has a higher association with H3K9 methyltransferase (G9A), SUV39H1 and the H3K4 demethylase, JARID1C. This REST-associated complex found in non-neuroectodermal cells induces permanent silencing of neuronal genes via H3K9 methylation, DNA methylation and H3K4 demethylation [20
]. On the contrary, the inactive – but, poised – neuronal promoters in stem cells are not associated with DNA methylation or histone H3K9 methylation. The transcription of neuronal genes takes place once NPCs commit to a neuronal lineage and the expression of REST is turned off, followed by the removal of the REST repressor complex from neuronal gene promoters.