TALE-based Decoding of 5-Hydroxymethylcytosine by Selective Modification Response

DNA中的5-甲基胞嘧啶（mC）是基因表达的中心调控元件。最近，发现了10 - 11易位（TET）蛋白将mC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），其中后两个核碱基可以通过碱基切除修复被C取代。这为DNA的主动去甲基化和动态表观遗传修饰提供了一个模型，对基因表达调控和疾病具有至关重要的意义。此外，hmC本身越来越被认为是一种表观遗传标记，因为它与关键表观遗传蛋白的相互作用不同，并且在基因组和细胞类型中表现出独特的分布。揭示hmC的生物学功能需要简单而有效的方法，可以用原子分辨率描述其区域模式（分型）和更广泛的概况（分析）。然而，这种检测发展的瓶颈是一个长期存在的难题，即只有典型的核碱基，而不是表观遗传的核碱基，可以用可编程的方式（通过沃森-克里克杂交）识别。这将当前的hmC类型/分析限制为相对复杂或分辨率较差的方法。为了克服这一困境，我们最近引入了DNA识别扩展可编程性的概念。这是基于可编程转录激活因子样效应物（TALEs），它由具有四个典型核碱基的个体选择性的串联重复组成。我们已经确定了TALE重复序列，为C、mC和hmC提供了额外的选择性，这使我们能够基于工程TALE的dna结合，开发出第一个基因组体外检测，用于直接、可编程地检测单个表观遗传核碱基。在这个项目中，我们将通过选择性修改响应的概念扩展这种方法。我们将采用选择性酶促糖基化hmC（到β -葡萄糖基-hmC， ghmC）来显著增加hmC与所有其他核碱基之间的大小差异。我们将进一步设计非特异性结合C、mC、hmC、fC和caC的TALE重复序列，但不与ghmC结合，从而实现TALE的选择性结合。我们将定义将这些TALE重复序列整合到全长TALE中的最佳参数，并建立基于亲和富集和qPCR的hmC分型/分析方法。我们将用当前的一种方法对该试验的关键分析参数进行基准测试。这将为hmC的分型/谱分析提供一种简单且完全有效的方法，具有原子分辨率、高序列分辨率和定量水平分析，这将使人们对hmC在正常和疾病过程中基因表达调控中的作用有新的认识。

5-methylcytosine (mC) in DNA is a central regulatory element of gene expression. Recently, ten-eleven translocation (TET) proteins have been discovered to oxidize mC to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC), of which the latter two nucleobases can be excised and replaced with C via base excision repair. This provides a model for the active demethylation and thus the dynamic epigenetic modification of DNA, with paramount importance for gene expression regulation and disease. Moreover, hmC is increasingly recognized as an epigenetic mark per se, since it interacts differently with key epigenetic proteins and exhibits unique distribution across genomes and cell types. Unravelling the biological functions of hmC requires simple and effective methods that can delineate both its regional patterns (typing) and broader profiles (profiling) in genomes with atomic resolution. However, a bottleneck for the development of such assays is the long-standing dilemma that only canonical, but not epigenetic nucleobases can be recognized in a programmable manner (by Watson-Crick hybridization). This limits current hmC typing / profiling to comparably complicated or poorly resolved approaches. To overcome this dilemma, we have recently introduced the concept of expanded programmability of DNA recognition. This is based on programmable transcription-activator-like effectors (TALEs) that consist of concatenated repeats with individual selectivities for the four canonical nucleobases. We have identified TALE repeats that provide additional selectivities for C, mC, and hmC, which allowed us to develop the first genomic in vitro assay for the direct, programmable detection of single epigenetic nucleobases, based on DNA-binding by engineered TALEs. In this project, we will extend this approach by the concept of selective Modification Response. We will employ selective enzymatic glucosylation of hmC (to beta-glucosyl-hmC, ghmC) to significantly increase the size-difference between hmC and all other nucleobases. We will further design TALE repeats that nonspecifically bind to C, mC, hmC, fC, and caC, but not to ghmC, enabling selective binding of TALEs. We will define optimal parameters for the integration of these TALE repeats into full-length TALEs and establish an hmC typing / profiling assay based on affinity enrichment and qPCR. We will benchmark key analytical parameters of this assay with the one of current approaches. This will provide a simple and fully validated assay for the typing / profiling of hmC with atomic resolution, high sequence resolution and quantitative level analysis that will enable new insights into the role of hmC in gene expression regulation in both normal and disease processes.