Investigation of the DNA interaction specificity of DNA methyltransferases and demethylases by deep enzymology

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DNA methylation has important roles in the regulation of gene expression, genomic stability, cell differentiation and mammalian development. It is introduced by DNA methyltransferases (DNMTs) and its active removal is triggered by the oxidation of 5-methylcytosine catalyzed by Ten-eleven translocation (TET) methylcytosine dioxygenases. Like many other DNA interacting enzymes, DNMTs and TETs need to identify their target sequences (CpG in most cases) embedded into different DNA sequence contexts, which is a difficult and not well understood task. To investigate the influence of flanking sequences on target sequence recognition and determine the sequence preferences of DNA methyltransferases in great depth, we have developed a novel “Deep Enzymology” approach. In this procedure, a pool of DNA substrates is generated which contain one (modified) target site flanked by 10 random nucleotides on each side. The substrate pool is methylated and the methylation of all sequences is analyzed by bisulfite conversion followed by NGS, thereby providing the methylation state of individual product molecules and their specific flanking sequence. Our previous studies have documented that this approach is very powerful allowing to discover novel and important mechanistic details and that the flanking sequence preferences of DNMTs are correlated with cellular DNA methylation profiles. Here, we plan to use this technology to investigate the detailed DNA flanking sequence preferences of selected enzymes involved in DNA methylation. We plan to investigate human and mouse DNMT3B, DNMT3C (a DNMT3B paralogue identified in mice and rodents) and DNMT1 and determine to what extent the flanking sequence preferences of these enzymes correspond to the sequences of their characteristic biological substrates, which in all cases are specific repetitive elements. Two plant methyltransferases (DRM2 with CHH specificity and CMT3 with CNG specificity) will be studied and the experimental flanking sequence preferences will be compared with DNA methylation profiles in plant cells. We will adapt our method for the analysis of methylcytosine oxidation and apply it to study the flanking sequence preferences of human TET enzymes, which will then be compared with hydroxymethylation in genomic DNA. Finally, we plan to develop a Deep Enzymology workflow to investigate RNA methylation and apply it on DNMT2, a tRNA-Asp methyltransferase with unclear spectrum of additional substrates. We have formed collaborations with leading experts worldwide that will help us to delineate the biological consequences of our findings. We expect that our project will provide seminal insights into the mechanisms of these important enzymes and deepen our understanding of their fundamental role in the process of DNA methylation and demethylation.