While the word “turnover” may send the culinary-minded to the kitchen and the business-savvy straight to the accounts books, epigeneticists studying the turnover of DNA methylation have been left between a rock and a hard place! Researchers are in this unappetizing predicament as while we can accurately paint a genome-wide picture of DNA methylation levels, any value for a given CpG is an average and tells us little regarding the relative rates of DNA methylation and demethylation for individual cytosines. However, researchers from the laboratory of Dirk Schübeler (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland) have recently cooked up something very special in response, although we will have to take you through a few of the unique ingredients and essential preparatory steps first!
Average methylation levels generally mask the differences between methylation rates (C -> 5mC) and demethylation rates (5mC -> C), with DNA methylation deriving from the activity of the DNMT3a and DNMT3b de novo DNA methyltransferases and the DNA demethylation deriving from both active demethylation by the ten-eleven translocation methylcytosine dioxygenase (TET)1/2/3 proteins and passive demethylation via imperfect maintenance by DNMT1. To analyze methylation turnover, the team studied DNA methylation over time in mESCs (using both amplicon bisulfite sequencing and SureSelect sequencing) following the loss of active demethylating and de novo methylating activity via genetic ablation and then employed dynamic theoretical modeling to infer methylation/demethylation rates.
So, by taking all these succulent ingredients and treating them with the care and consideration expected from any good chef, what tasty epigenetic morsels did Ginno and colleagues plate up?
- Overall, the study highlights the existence of methylated cytosines with highly variable methylation kinetics throughout the genome
- CpGs with identical steady-state DNA methylation levels possess enzymatic rates that vary as much as two orders of magnitude
- A surprisingly considerable number of sites display high rates of passive demethylation that de novo methylation activity typically masks
- TET proteins significantly influence the rate of DNA demethylation, with a more significant contribution in mESCs when compared to the loss of methylation via DNMT1-mediated imperfect maintenance
- The data suggest that the genomic context dictates the presence of CpGs with different methylation/demethylation rates
- The highest TET-mediated demethylation rates occur at active distal regulatory elements
- Gene transcription coincides with a high turnover of DNA methylation within transcribed gene bodies
- Heterochromatin and euchromatin show opposing turnover rates, with heterochromatin displaying high DNA methylation but low turnover
- Hypermethylated CpGs undergo elevated levels of methylation turnover when positioned close to regulatory regions or within highly active genes
- Regulatory regions generally display a reduction in de novo and maintenance methylation and an increase in active demethylation as a function of transcription factor binding
- However, the data also highlights transcription factor specific influence on methylation kinetics
- Transcription factor binding and positioned nucleosomes inhibit the activity of DNMT1 and DNMT3, while accessibility represents a robust determinant for active demethylation
- These activities likely account for the intricate and cell type-specific patterns of reduced methylation levels observed at regulatory regions
Overall, this succulent study provides evidence against the supposition of DNA methylation as a stable epigenetic mark by providing evidence for the presence of highly dynamic DNA methylation profiles in some areas of the genome.
First author Paul Ginno explains, “Studying methylation as a steady-state process does not reveal actual methylation turnover. By comparing the rates of methylation/demethylation of CpGs in different genomic locations, we found that rates could differ by two orders of magnitude for CpGs with similar steady-state measurements” and group leader Dirk Schübeler continues “It’s the first time that a genome-scale map of DNA methylation kinetics has been done at this level.”
For every single last tasty morsel of data from this Instagramable epigenetic delight, see Nature Communications, May 2020.