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，存在于回文CpG二聚体中。然而，对于CPGS的两条链中这种修饰的特定组合(“CpG双链修饰”)如何在染色质调节中充当独特的化学信号，人们还知之甚少。作为回答这个问题的基础，了解特定的CpG双链修饰在哺乳动物基因组中的位置是至关重要的。我们最近开发了第一个CpG双链修饰的阅读器蛋白HMC/MC(HMC=5-羟甲基胞嘧啶)。在本项目中，我们将通过甲基CpG结合结构域(MBD)蛋白的细菌表面展示来进化Tet产生的CpG双链修饰HMC/HMC和HMC/C的新的阅读器蛋白，并将它们用于mESC细胞和小鼠脑中这些修饰的浓缩、测序和定位。在胞嘧啶修饰的生命周期中，HMC/HMC是最初的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，这些修改代表了我们第一个阅读器设计后的逻辑下一步，并完成了一系列频繁的含有HMC的CpG双链修改，这些修改只涉及频繁的胞嘧啶核苷酸碱基HMC、MC或C。将新获得的图谱与其他调控元件的现有图谱进行全局和局部比较，将为了解它们的功能提供第一条线索，例如它们参与基因表达调控、染色质打开，以及可能与组蛋白修饰的串扰。这些研究将为更深入地理解HMC/HMC和HMC/C如何调节基于MC的染色质调节通路，以及它们如何在先前未知的通路中作为独特信号发挥作用奠定重要基础。

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.