Dynamic DNA methylation: In the right place at the right time

Abstract

The classical model of cytosine DNA methylation (the presence of 5-methylcytosine, 5mC) regulation depicts this covalent modification as a stable repressive regulator of promoter activity. However, whole-genome analysis of 5mC reveals widespread tissue- and cell type-specific patterns and pervasive dynamics during mammalian development. Here we review recent findings that delineate 5mC functions in developmental stages and diverse genomic compartments as well as discuss the molecular mechanisms that connect transcriptional regulation and 5mC. Beyond the newly appreciated dynamics, regulatory roles for 5mC have been suggested in new biological contexts, such as learning and memory or aging. The use of new single-cell measurement techniques and precise editing tools will enable functional analyses of 5mC in gene expression, clarifying its role in various biological processes.

Figure 1.

DNA methylation signatures of developmental stages and tumors - primordial germ cells and preimplantation: low global 5mC level; postimplantation: global gain of 5mC. late fetal development: local demethylation at embryonically active regulatory sequences; postnatal development: gain of 5mC at embryonically active regulatory sequences, loss of 5mC at regulatory sequences active in differentiated tissues. neuron subtypes A and B: brain specific accumulation of mCH, which abundance is inversely correlated with transcription activity; aged tissues: PMDs in late-replication regions resulted from progressive methylation loss. Dashed line indicates a small percentage of CGI shows hyper-methylation in aged tissues (67). trophectoderm: low global 5mC level similar to preimplantation embryo; placenta and tumors: pervasive PMDs and CGI hypermethylation. The color gradient in the background indicates the increase of 5mC level from bottom to top of each panel.

Figure 2.

The function and regulation of DNA methylation in diverse genomic contexts. (A) Gene promoter CpG islands are protected from 5mC by a CXXC domain containing proteins. De novo DNA methyltransferase DNMT3A activity is inhibited by H3K4me3. At distal regulatory elements, 5mC can either prohibit the binding of 5mC-sensitive TFs, or interact with TFs with preference for methylated binding sequence. DNA methylation at CTCF binding sites regulates chromatin conformation by modulating CTCF binding. In mouse embryonic stem cells (mESCs, top), DNMT3B is recruited to transcribed regions by H3K36me and establishes gene body methylation. In post-mitotic neurons (bottom), 5mCH is bound by MeCP2. (B) Transcription outputs are correlated with 5mC and 5hmC levels of enhancers, promoters and gene bodies.

Figure 3.

Comparison of direct fusion (A) and SunTag protein scaffold (B) based 5mC editing tools. SunTag system allows local amplification of effector (DNMT3A) concentration and independent control of dCas9 and DNMT3A expression levels to minimize off-target de novo methylation caused by un-tethered DNMT3A. Schematics were drawn on the basis of mC editing tools developed by Liu et al. (56), (A) and Pflueger et al. (56, 58) (B). NLS: nuclear localization sequences. GB1: solubility tag (protein G B1 domain). SunTag based mC editing tool (dCas9Sun-D3A) shows comparable on-target methylation at on-target promoters as the direct fusion (dCas9-D3A), but much lower off-target methylation.