DFG funding for the i nvestigation of the DNA interaction specificity of DNA methyltransferases and demethylases by "Deep Enzymology"
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.