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

胞嘧啶修饰是哺乳动物基因组的中心调控元件，并且存在于回文CpG二联体中。然而，人们对CpG的两条链中的这种修饰的特定组合（“CpG双链体修饰”）如何在染色质调节中充当独特的化学信号知之甚少。作为回答这个问题的基础，了解特定的CpG双链修饰位于哺乳动物基因组中的何处是至关重要的。 我们最近开发了第一个CpG双链修饰hmC/mC（hmC = 5-羟甲基胞嘧啶）的阅读器蛋白。在本项目中，我们将通过细菌表面展示甲基-CpG结合域（MBD）蛋白来进化TET产生的CpG双链修饰hmC/hmC和hmC/C的新型阅读器蛋白，并将其用于mESC细胞和小鼠脑中这些修饰的富集、测序和作图。在胞嘧啶修饰的生命周期中，hmC/hmC是初始hmC/mC产物的下一个TET生成的氧化产物，而hmC/C是hmC/hmC的直接被动去甲基化产物。因此，这些修饰代表了我们的第一个阅读器设计之后的逻辑下一步，并完成了一系列频繁的含hmC的CpG双链体修饰，这些修饰仅涉及频繁的胞嘧啶核碱基hmC、mC或C。新获得的地图与其他调控元件的现有地图的全局和局部比较将提供它们的功能的第一线索，例如它们参与基因表达调控、染色质开放和对组蛋白修饰的潜在串扰。这些研究将为更深入地了解hmC/hmC和hmC/C如何调节基于mC的染色质调控途径，以及它们如何在以前未知的途径中作为独特的信号奠定重要基础。

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.