Dynamique and biological function of deoxycytosine methylation – 5metdC

Deoxycytosine methylation (5mdC) is an epigenetic mark that has emerged as a powerful means by which cells regulate both chromatin structure and gene expression. It also plays a key role in genetic plasticity, as the process of 5mdC erasure can give rise to mutations. In eukaryotes, cytosine methylation is mediated by three types of conserved DNA methyltransferases (MTs). While symmetrically methylated CpG sites targeted by the Dnmt1 (maintenance) and Dnmt3 (de novo) methylases have been the focus of intense study, the Dnmt2 family of enzymes remains enigmatic. Dnmt2 is the most conserved MT in that it is present in a single gene copy even in species that do not have the two other MTs. However, its ability to methylate DNA is still controversial, and its major identified activity to date is tRNA methylation. In fact, organisms that possess this enzyme as their only MT show very low or undetectable levels of 5mdC. Nevertheless, a dual function for Dnmt2 on both RNA and DNA may provide an attractive explanation for its presence as the only MT in diverse species as well as its widespread evolutionary conservation.

Recently, we have made a critical breakthrough in our knowledge of the Dnmt2 enzyme in the fission yeast S. pombe. While fission yeast cells habor an ortholog of Dnmt2 (Pmt1), previous studies reported conflicting evidence regarding the existence of 5mdC in the genome. We have now conclusively demonstrated for the first time the presence of Dnmt2-dependent 5-deoxycytosine methylation in this organism. By combining liquid chromatography-tandem mass spectrometry (LC/MS) with nuclear magnetic resonance (NMR), we unambiguously identified a methyl on carbon 5 of the deoxyribose in genomic DNA. Importantly, we showed that this mark requires Dnmt2/Pmt1 and that it is actively removed by deamination. This modification is dynamic, as it is linked to cell cycle progression and regulated by nutritional cues. Finally, we found that the deregulation of 5mdC formation gives rise to genome instability. Building on this work, we propose a multidisciplinary and innovative project to investigate the regulation and function of 5-deoxycytosine methylation mediated by the highly conserved Dnmt2 enzyme. As part of these studies, we will also address the regulation of the dual activities of Pmt1 on tRNA and DNA. First, we will determine the mechanisms that control cytosine methylation and evaluate the sites of 5mdC formation in the genome. Next, we will identify the pathways by which cells erase 5mdC as well as the mutational landscape that arises as a result of the pattern of DNA methylation. Finally, we will investigate the functions of 5mdC, assessing both immediate effects on gene regulation and genome stability as well as long-term impacts on cellular fitness and genome evolution.

Our results will bring original insight to our understanding of the epigenetic modifications that are brought about by the Dnmt2 methyltransferase and their genetic consequences. Interestingly, the presence of Dnmt2 among distinct taxa suggests an adaptive value that may be crucial in adverse and changing environments, and our work will pave the way to understanding these functions. Moreover, our analysis of the “costs” of DNA methylation with regard to mutation frequency and spectrum will provide a unique perspective into the forces that shape genome evolution during normal and pathological contexts. As DNA methylation is integral to a variety of processes ranging from differentiation to ageing, our investigation of new mechanisms and roles for this modification will lead to novel breakthroughs into the function of this vital and versatile epigenetic mark.