5-methylcytosine turnover: Mechanisms and therapeutic implications in cancer

Abstract

DNA methylation at the fifth position of cytosine (5mC) is one of the most studied epigenetic mechanisms essential for the control of gene expression and for many other biological processes including genomic imprinting, X chromosome inactivation and genome stability. Over the last years, accumulating evidence suggest that DNA methylation is a highly dynamic mechanism driven by a balance between methylation by DNMTs and TET-mediated demethylation processes. However, one of the main challenges is to understand the dynamics underlying steady state DNA methylation levels. In this review article, we give an overview of the latest advances highlighting DNA methylation as a dynamic cycling process with a continuous turnover of cytosine modifications. We describe the cooperative actions of DNMT and TET enzymes which combine with many additional parameters including chromatin environment and protein partners to govern 5mC turnover. We also discuss how mathematical models can be used to address variable methylation levels during development and explain cell-type epigenetic heterogeneity locally but also at the genome scale. Finally, we review the therapeutic implications of these discoveries with the use of both epigenetic clocks as predictors and the development of epidrugs that target the DNA methylation/demethylation machinery. Together, these discoveries unveil with unprecedented detail how dynamic is DNA methylation during development, underlying the establishment of heterogeneous DNA methylation landscapes which could be altered in aging, diseases and cancer.

Keywords: 5-Methylcytosine; DNA methylation; DNMT; TET; cancer; dynamics.

FIGURE 1

Cytosine methylation and oxidation cycle. In mammals, DNA methyltransferases (DNMTs) are responsible for the addition of a methyl group to the C5 position of cytosines to form 5-methylcytosines (5 mC). DNMT3A and DNMT3B drive de novo methylation during development, while DNMT1 is involved in maintaining DNA methylation by copying methylation patterns onto the newly replicated DNA strand. DNA methylation can be reversed in a passive way through replication-dependent dilution in absence of DNMT1. DNA demethylation can also occur actively, through Ten eleven translocation (TET) proteins that oxidize successively 5 mC into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5 fC) and 5-carboxylcytosine (5caC) and the replacement of the oxidized bases 5 fC and 5caC by an unmodified cytosine through thymine DNA glycosylase (TDG)-mediated abasic site (AP) formation and base excision repair (BER).

FIGURE 2

Cytosines undergo dynamic methylation turnover. (A). In a cell population, steady-state DNA methylation is reached when overall DNA methylation rates (orange) are equivalent to demethylation ones (blue). Given an identical steady-state, CpGs are influenced by different methylation (de novo) and demethylation (active oxidation and passive dilution) rate combinations called DNA methylation turnover. (B). Example of DNA methylation turnover depending on CpGs location in the genome, based on Ginno et al. (2020). In euchromatic context, CpGs at regulatory regions including enhancers and promoters present low steady-state DNA methylation levels associated with increased demethylation rates (blue) compared to methylation ones (orange). High rates of passive demethylation are observed at promoters while active demethylation is more important at enhancers. CpGs in heterochromatic regions have high steady-state methylation which tends to be associated with low DNA methylation turnover. However, CpGs located at gene bodies are also highly methylated but tend to have a higher turnover.

FIGURE 3

DNA methylation turnover in heterochromatin versus euchromatin. Transcriptionally silent genes are found in highly-condensed chromatin regions called heterochromatin and marked by repressive histone marks H3K9me3, H3K27me3 and H4K20me3. DNA methylation levels in heterochromatin are high and DNA methylation turnover rates are usually low. CpGs in heterochromatic regions are highly methylated by DNMTs and inaccessible by TET proteins. On the opposite, transcriptionally active genes are associated with a less-condensed, nucleosome-depleted and accessible chromatin known as euchromatin. In transcriptionally active regions, gene promoters are enriched in H3K4me3 and H3K27ac, enhancers in H3K4me1 and H3K27ac and gene bodies in H3K9Ac and H3K36me. Highly methylated CpGs within highly transcribed genes, gene bodies and nearby regulatory regions (enhancers and promoters) are subjected to high methylation turnover rates while hypermethylated CpGs within intergenic regions have lower TET and DNMT engagement. Transcription factors binding can regulate both methylation by DNMTs and demethylation by TETs.