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

DNA中的5-甲基胞嘧啶(MC)是基因表达的中枢调控元件。最近发现了10-11个易位(Tet)蛋白将MC氧化为5-羟甲基胞嘧啶(HMC)、5-甲酰胞嘧啶(FC)和5-羧基胞嘧啶(CAC)，其中后两个碱基可以被切除，并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至关重要。此外，HMC本身越来越被认为是一个表观遗传标记，因为它与关键的表观遗传蛋白相互作用不同，并在基因组和细胞类型中显示出独特的分布。解开HMC的生物学功能需要简单而有效的方法，既可以描绘其区域模式(分型)，也可以在基因组中以原子分辨率描绘更广泛的图谱(图谱)。然而，这类分析发展的一个瓶颈是长期存在的两难境地，即只能以可编程的方式(通过Watson-Crick杂交)识别标准的、而不是表观遗传的核苷酸碱基。这将当前的HMC分型/分析限制为相对复杂或解析较差的方法。为了克服这一困境，我们最近引入了DNA识别的扩展可编程性的概念。这是基于可编程的转录激活子样效应器(TALE)，它由具有四个典型核苷酸碱基的单独选择性的串联重复组成。我们已经确定了为C、MC和HMC提供额外选择性的TALE重复序列，这使得我们能够开发出第一个基于工程TALE与DNA结合的直接、可编程的单个表观遗传核苷酸碱基的体外基因组检测方法。在这个项目中，我们将通过选择性修改响应的概念来扩展这种方法。我们将使用选择性的酶促糖基化HMC(到β-葡萄糖基-HMC，ghmC)来显著增加HMC与所有其他碱基的大小差异。我们将进一步设计非特定绑定到C、MC、HMC、FC和CAC的TALE重复序列，但不能绑定到ghmC，从而实现TALE的选择性绑定。我们将定义将这些TALL重复序列整合到全长TALL中的最佳参数，并建立基于亲和力浓缩和qPCR的HMC分型/图谱分析。我们将用目前的方法之一对该分析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分析方法，具有原子分辨率、高序列分辨率和定量水平分析，将使人们能够对HMC在正常和疾病过程中的基因表达调控作用有新的见解。

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.