Natural Duplex Readers of Cytosine Modifications in Mammalian DNA

在这个项目中，我们将开发方法学来深入了解哺乳动物DNA的两条CpG链中Tet生成的胞嘧啶修饰的特定组合(“CpG双重修饰”)如何作为独特的化学信号在染色质调节中发挥作用。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作用，如果不深入了解基因组中共存的Tet产生的替代CpG双链修饰的个体作用，就无法理解这些过程。我们将利用基因文库的定向进化，通过大肠杆菌表面展示和流式细胞仪筛选进行蛋白质组谱实验，以发现Tet产生的CpG双链修饰的阅读器和反阅读器。我们将通过先进的合成DNA探针设计，通过渔业/蛋白质组学补充这些研究。对于识别出的高度相关的蛋白质，我们将进行SELEX和相互作用分析，以揭示它们的选择性，并找到它们功能的第一线索。对于选定的候选人，我们将进一步进行更深入的后续研究，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大量的起点，并为更深入地理解Tet产生的胞嘧啶修饰的特定组合如何在哺乳动物染色质调节中作为独特信号作用奠定基础。

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.