Evolved Readers of 5-Hydroxymethylcytosine-containing CpG Duplex Combinations in Mammalian DNA

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Cytosine modifications are the central regulatory elements of mammalan genomes, and exist in palindromic CpG dyads. However, it is poorly understood, how specific combinations of such modifications in the two strands of CpGs (“CpG duplex modifications”) act as unique chemical signals in chromatin regulation. As a basis for answering this question, it is essential to understand, where specific CpG duplex modifications are located in mammalian genomes. We have recently developed the first reader protein of the CpG duplex modification hmC/mC (hmC = 5-hydroxymethylcytosine). In the present project, we will evolve novel reader proteins of the TET-generated CpG duplex modifications hmC/hmC and hmC/C by bacterial surface display of methyl-CpG-binding domain (MBD) proteins, and employ them for the enrichment, sequencing and mapping of these modifications in the context of mESC cells and the mouse brain. In the life cycle of cytosine modifications, hmC/hmC is the next TET-generated oxidation product of the initial hmC/mC product, whereas hmC/C is the direct passive demethylation product of hmC/hmC. These modifications thus represent the logic next steps after the design of our first reader, and complete the series of frequent hmC-containing CpG duplex modifications that involve only the frequent cytosine nucleobases hmC, mC, or C. Global and local comparison of the newly obtained maps with existing maps of other regulatory elements will provide first clues to their functions, such as their involvement in gene expression regulation, chromatin opening, and potential crosstalk to histone modifications. These studies will set an important basis for a deeper understanding of how hmC/hmC and hmC/C modulate mC-based pathways of chromatin regulation, and how they may act as unique signals in previously unknown pathways.