Programmable 5-Methylcytosine Oxidation and Covalent Capture of Genomic Loci for Targeted Proteomics

哺乳动物基因表达受基因组5-甲基胞嘧啶（mC）的动态调控。mC由DNA甲基转移酶引入，而mC通过10 - 11易位（泰特）双加氧酶反复氧化为5-羟甲基胞嘧啶（hmC）、5-甲酰基胞嘧啶（fC）和5-羧基胞嘧啶（caC）控制活性去甲基化。这些氧化的mC衍生物（oxi-mC）本身就是潜在的表观遗传标记，具有与染色质蛋白质相互作用的独特能力。最初的捕捞/蛋白质组学研究与核提取物中的合成DNA提供了第一个相互作用的hmC，fC和caC的配置文件。然而，虽然提供了宝贵的初步线索的功能的基地，这样的实验并不能反映复杂的相互作用网络的染色质，并不能建立从头mC氧化和变化之间的因果关系，在自然染色质的景观在用户定义的基因座在体内，如蛋白质组成和蛋白质修饰。在这个项目中，我们将开发用于在用户定义的基因组位点处的细胞内mC氧化的工具，并结合这些位点的共价捕获用于蛋白质组学研究。这将使1.）直接或间接招募或排斥的蛋白质的发现，以及2.）使发现什么组蛋白和其他蛋白质翻译后修饰可能被写入或删除的mC氧化。我们将设计泰特酶和转录激活因子样效应子（TALE）的融合构建体，并在哺乳动物细胞系中表达它们用于氧化。我们将配备这些结构与环辛炔处理应变促进反电子狄尔斯-阿尔德环加成（SPIEDAC）使用遗传密码扩展。在基因座特异性结合和体内mC氧化、构建体和其他染色质蛋白的甲醛交联以及随后的DNA分离和片段化之后，具有四嗪功能化珠的SPIEDAC将能够完全共价捕获结合的染色质基因座用于蛋白质组学分析。SPIEDAC的共价捕获应克服非共价捕获方法在灵敏度、选择性和严格去除假阳性的能力方面的局限性。我们将首先将此方法应用于热激后应激体形成中涉及的重复SATIII位点，研究这一过程的表观遗传控制机制。之后，我们将靶向三个单基因位点CDKN 2A，BRCA 1和ZAP 70，这些基因位点在不同的细胞类型中参与了mC控制的癌症发展，这些细胞类型涵盖了CpG密度方面的不同启动子类型。从这些研究中，我们预计oxi-mC如何参与调节，直接和间接的蛋白质募集和释放，以及它们如何整合在DNA和蛋白质（组蛋白）修饰之间的串扰中的新见解。我们的方法提供了一个新的途径，发现读者的oxi-mC识别这样的相互作用，在天然染色质和不同的基因座和细胞环境。

Mammalian gene expression is dynamically regulated by genomic 5-methylcytosine (mC). mC is introduced by DNA methyltransferases, whereas iterative oxidation of mC to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC) by ten-eleven translocation (TET) dioxygenases controls active demethylation. These oxidized mC derivatives (oxi-mC) are potential epigenetic markers in their own right, with unique interaction abilities to chromatin proteins. Initial fishing/proteomics studies with synthetic DNAs in nuclear extracts have provided first interaction profiles of hmC, fC and caC. However, though giving invaluable initial clues to the functions of the bases, such experiments do not reflect the complex interaction networks of chromatin, and cannot establish causalities between de novo mC oxidation and changes in the landscape of natural chromatin at user-defined loci in vivo, such as protein composition and protein modifications. In this project, we will develop tools for the intracellular mC oxidation at user-defined genomic loci combined with the covalent capture of these loci for proteomics studies. This will enable 1.) the discovery of proteins that are directly or indirectly recruited or repelled, and 2.) enable the discovery of what histone and other protein posttranslational modifiations may be written or erased by mC oxidiation. We will design fusion constructs of TET enzymes and transcription-activator-like effectors (TALEs) and express them in mammalian cell lines for oxidiation. We will equip these constructs with cyclooctyne handles for strain-promoted inverse electron Diels-Alder cycloaddions (SPIEDAC) using genetic code expansion. After locus-specific binding and mC oxidiation in vivo, formaldehyde crosslinking of the construct and other chromatin proteins followed by DNA isolation and fragmentation, SPIEDAC with tetrazine-functionalized beads will enable the fully covalent capture of the bound chromatin loci for proteomic analyses. Covalent capture by SPIEDAC should overcome limitations of noncovalent capture approaches in view of sensitivity, selectivity and ability for stringent removal of false-positives. We will first apply this approach to the repetitive SATIII locus involved in stress body formation after heat shock, to study epigenetic control mechanism of this process. Afterwards, we will target the three single gene loci CDKN2A, BRCA1 and ZAP70 involved in mC-controlled cancer development in different cell types that cover different promoter types in respect to CpG densities. From these studies, we anticipate new insights into how oxi-mC are involved in regulatory, direct and indirect protein recruitment and release and how they are integrated in the crosstalk between DNA and protein (histone) modifications. Our approach provides a new avenue to the discovery of readers of oxi-mC by identifying such interactions in natural chromatin and in different locus- and cellular contexts.