Natural Duplex Readers of Cytosine Modifications in Mammalian DNA

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In this project, we will develop methodology to obtain insights into how specific combinations of TET-generated cytosine modifications in the two CpG strands of mammalian DNA (“CpG duplex modifications”) act as unique chemical signals with roles in chromatin regulation. Given the key functions of symmetric CpG methylation in embryonic development, cell differentiation and cancer development, these processes cannot be understood without insights into the individual roles of TET-generated, alternative CpG duplex modifications that co-exist in genomes. We will conduct proteome profiling experiments by employing directed evolution of cDNA libraries by E. coli surface display and FACS screening, for discovering readers and antireaders of TET-generated CpG duplex modifications. We will complement these studies by fishing/proteomics with advanced synthetic DNA probe designs. For identified proteins with high relevance, we will conduct SELEX and interaction analyses to reveal their selectivities and find first clues to their functions. For selected candidates, we will further conduct more in-depth follow-up studies to reveal the biological significance of the identified interactions. Our experiments will afford numerous starting points for follow-up projects by us and others, and set the basis for a deeper understanding of how specific combinations of TET-generated cytosine modifications act as unique signals in mammalian chromatin regulation.