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

DNA甲基化在基因表达调控、基因组稳定性、细胞分化和哺乳动物发育中具有重要作用。它由DNA甲基转移酶（DNMT）引入，并通过十-十一易位（泰特）甲基胞嘧啶双加氧酶催化的5-甲基胞嘧啶氧化引发其主动清除。与许多其他DNA相互作用酶一样，DNMT和THBG需要识别嵌入不同DNA序列背景中的靶序列（大多数情况下为CpG），这是一项困难且尚未充分理解的任务。为了深入研究侧翼序列对靶序列识别的影响并确定DNA甲基转移酶的序列偏好，我们开发了一种新的“深度酶学”方法。在该程序中，产生DNA底物池，其含有一个（经修饰的）靶位点，每侧接10个随机核苷酸。将底物池甲基化，并通过亚硫酸氢盐转化和NGS分析所有序列的甲基化，从而提供单个产物分子及其特定侧翼序列的甲基化状态。我们以前的研究已经证明，这种方法非常强大，可以发现新的和重要的机制细节，并且DNMT的侧翼序列偏好与细胞DNA甲基化谱相关。在这里，我们计划使用这种技术来研究参与DNA甲基化的选定酶的详细DNA侧翼序列偏好。我们计划研究人类和小鼠DNMT 3B，DNMT 3C（在小鼠和啮齿动物中鉴定的DNMT 3B parasites）和DNMT 1，并确定这些酶的侧翼序列偏好在多大程度上对应于其特征性生物底物的序列，在所有情况下都是特定的重复元件。将研究两种植物甲基转移酶（具有CHH特异性的DRM 2和具有CNG特异性的CMT 3），并将实验侧翼序列偏好与植物细胞中的DNA甲基化谱进行比较。我们将调整我们的甲基胞嘧啶氧化的分析方法，并将其应用于研究人类泰特酶的侧翼序列偏好，然后将其与基因组DNA中的羟甲基化进行比较。最后，我们计划开发一个深度酶学工作流程来研究RNA甲基化，并将其应用于DNMT 2，这是一种tRNA-Asp甲基转移酶，其额外底物的谱不清楚。我们已经与世界各地的顶尖专家建立了合作关系，这将有助于我们描述我们发现的生物学后果。我们希望我们的项目将为这些重要酶的机制提供开创性的见解，并加深我们对它们在DNA甲基化和去甲基化过程中的基本作用的理解。

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.