Stability of cell fate is intimately linked with the stability of the epigenetic information. Contrary to numerous posttranslational histone modifications that can either have a relatively short half-life (histone acetylation, phosphorylation, ribosylation) or their modification /de-modification cycle is mechanistically well understood (histone methyltransferases vs histone demethylases); DNA methylation (5-methylcytosine, 5mC) has been historically considered to be very stable thus providing a heritable form of epigenetic information.
Recent advances in genome-wide profiling of DNA methylation have led to accumulation of large amount of new data detailing changes of DNA methylation patterns in various cell types during development and in the course of differentiation. This revealed that DNA methylation patterns are potentially more dynamic than previously anticipated. Apart from differentiation and development, which typically represent proliferative cell systems; limited information is available as to whether DNA methylation patterns observed in non-proliferative (post-mitotic) cells are truly stable or rather represent a snapshot of a dynamic equilibrium state with ongoing methylation/remethylation cycles. This idea has been further supported by the discovery of the Tet family dioxygenases and the existence of the 5mC/5hmC/5fC/5caC oxidative DNA demethylation pathway (Tahiliani et al, Science 2009; Kriaucionis et al, Science 2009).
To address this question, we would like to employ a novel chemical biology approach to label genomic regions undergoing DNA methylation–demethylation cycles.
The aim of the proposed project is to take advantage of a chemical derivative of S-adenosyl methionine (SAM) - a cofactor and a methyl group donor in the methylation reaction carried out by DNA methyltransferases (Dnmts) (for a review see Tomkuviene et al, Curr Opin Biotech 2019 and Deen et al, Angew Chem 2017). Upon delivery into cells (cultured cells, oocytes, early mouse embryos), the methylation reaction carried out by the endogenous Dnmts will transfer the modified methyl group to the target DNA. Using Click chemistry, these DNA regions can be subsequently either visualised for microscopy or purified and subjected to sequencing).
Using this chemical biology approach, we will uncover regions of de novo DNA methylation activity (and DNA de/methylation turnover) in cultured proliferating or post-mitotic cells and also in the context of oocytes and early mouse embryos.