Notes on the role of dynamic DNA methylation in mammalian development

Abstract

It has been nearly 40 y since it was suggested that genomic methylation patterns could be transmitted via maintenance methylation during S phase and might play a role in the dynamic regulation of gene expression during development [Holliday R, Pugh JE (1975) Science 187(4173):226-232; Riggs AD (1975) Cytogenet Cell Genet 14(1):9-25]. This revolutionary proposal was justified by "... our almost complete ignorance of the mechanism for the unfolding of the genetic program during development" that prevailed at the time. Many correlations between transcriptional activation and demethylation have since been reported, but causation has not been demonstrated and to date there is no reasonable proof of the existence of a complex biochemical system that activates and represses genes via reversible DNA methylation. Such a system would supplement or replace the conserved web of transcription factors that regulate cellular differentiation in organisms that have unmethylated genomes (such as Caenorhaditis elegans and the Dipteran insects) and those that methylate their genomes. DNA methylation does have essential roles in irreversible promoter silencing, as in the monoallelic expression of imprinted genes, in the silencing of transposons, and in X chromosome inactivation in female mammals. Rather than reinforcing or replacing regulatory pathways that are conserved between organisms that have either methylated or unmethylated genomes, DNA methylation endows genomes with the ability to subject specific sequences to irreversible transcriptional silencing even in the presence of all of the factors required for their expression, an ability that is generally unavailable to organisms that have unmethylated genomes.

Keywords: DNA methylation; development; differentiation.

Fig. 1.

CpG and m⁵C densities at mammalian promoters. (A) Strongly bimodal distribution of CpG densities in the region spanning –1,000 to +1,000 relative to TSS with nearly constant m⁵C density across all CpG densities in adult human brain DNA (7) and adult HMECs (39). (B) Nearly identical methylation densities across all promoter regions (defined as in A) in genomes from human brain and from HMECs.

Fig. 2.

Transcription-dependent methylation transitions. (A) The NANOG and OCT4 pluripotency genes have been reported to be regulated by reversible DNA methylation of their promoters and to be completely methylated in nonexpressing tissues (10). Both genes have very CpG-poor promoters, and methylation analysis by paired-end sequencing (8) shows that a substantial number of cells from normal adult mammary tissue show completely unmethylated promoters for both genes. Terminal green CpG dinucleotides are methylated and internal green CpG dinucleotides are unmethylated, whereas terminal red CpGs are unmethylated and internal CpGs are unmethylated. None of the cells express either gene. The presence of unmethylated promoters in the nonexpressing population requires that promoter methylation cannot be the mechanism of repression. Note that the primate-specific AluSx1 retrotransposon 5′ of the NANOG TSS is heavily methylated whereas the proximal promoter is largely unmethylated. (B) Cartoon representation of transcription-dependent methylation and demethylation. (Top) CpG-poor promoters are postulated to acquire partial methylation when not expressed, as in the case of the NANOG and OCT4 genes in A; the binding of transcription factors leads to a loss of methylation when the gene is activated (–17). Under this model, changes in methylation density are a result of transcriptional activation rather than a cause. (Bottom) Constitutive demethylation of CpG-dense/CpG island promoters in both expressing and nonexpressing cell types.

Fig. 3.

Faithful transmission of genomic methylation patterns with advancing age in mouse brain DNA. The paternal differentially methylated region or imprinting control region (DMR/ICR) undergoes de novo methylation in male but not female germ cells; this results of the paternal allele of Igf2 and the maternal allele of H19 in mesodermal tissues of embryonic and fetal mice. Both genes are silenced in adult tissues but the Igf2 promoters remain unmethylated. Methylation patterns across the region are very similar throughout the lifespan of the animals, and this trend holds true for the large majority of the genome. Methylation patterns are from the data described in ref. .