Natural Duplex Readers of Cytosine Modifications in Mammalian DNA

在这个项目中，我们将开发方法来深入了解哺乳动物DNA的两条CpG链中TET产生的胞嘧啶修饰（“CpG双链修饰”）的特定组合如何作为独特的化学信号在染色质调控中发挥作用。鉴于对称CpG甲基化在胚胎发育、细胞分化和癌症发展中的关键功能，如果不深入了解TET产生的、在基因组中共存的替代性CpG双链体修饰的个体作用，就无法理解这些过程。我们将利用大肠杆菌介导的cDNA文库定向进化进行蛋白质组分析实验。coli表面展示和FACS筛选，用于发现TET产生的CpG双链修饰的阅读器和反阅读器。我们将通过使用先进的合成DNA探针设计的钓鱼/蛋白质组学来补充这些研究。对于具有高度相关性的蛋白质，我们将进行SELEX和相互作用分析，以揭示其选择性并找到其功能的初步线索。对于选定的候选者，我们将进一步进行更深入的跟踪研究，以揭示已确定的相互作用的生物学意义。我们的实验将为我们和其他人的后续项目提供许多起点，并为更深入地了解TET产生的胞嘧啶修饰的特定组合如何作为哺乳动物染色质调控中的独特信号奠定基础。

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.