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

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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.