Oxidation of 5-methylcytosine: DNA damage and epigenetic reprogramming

描述（由申请人提供）：所有生物体的DNA都会发生持续性损伤，除非损伤得到正确修复，否则可能导致基因突变或表观遗传变化。修饰的碱基5-甲基胞嘧啶（5 mC）可以发生脱氨基和氧化，导致遗传突变和表观遗传扰动。我们已经证明，氧化损伤产物，5-羟甲基胞嘧啶（5 hmC），抑制蛋白质的结合，选择性地结合到甲基化的DNA和维护甲基转移酶DNMT 1不识别5 hmC时，甲基化DNA复制后。因此，5 hmC的形成可以遗传地改变复制细胞中的表观遗传模式。然而，新出现的证据表明，5 mC向5 hmC的转化也可能是干细胞分化所必需的酶表观遗传重编程途径的一部分，并且5 hmC在大多数（如果不是全部）人类癌细胞中不存在。在本申请中，我们提出了一系列的研究，以进一步了解这种假定的DNA去甲基化途径在人类神经干细胞，小鼠胚胎干细胞和一系列的人类癌症干细胞和建立的细胞系。我们希望了解DNA去甲基化如何依赖于DNA损伤修复途径，以及DNA损伤修复和DNA去甲基化如何纠缠在一起。在第一个目标中，我们提出了一种新的稳定同位素标记方法，这将使我们能够跟踪DNA复制，甲基化和羟基化以及通过碱基切除修复途径切除可能的中间体的动态过程。在第二个目标中，我们建议使用一系列创新的新方法来识别和量化可能作用于途径中间体的酶活性，进一步定义该途径的未知部分。在第三个目标中，我们提出了一些新的质谱方法，以确定蛋白质结合的途径的中间体，并测量特定的DNA中间体，包括5 hmC，如何影响的特异性和幅度的DNA-蛋白质相互作用。拟议的研究将允许在前所未有的水平上检查这一重要的去甲基化途径。所获得的信息对于理解再生医学背景下正常干细胞的生物学至关重要，并且了解该途径如何在人类癌细胞中变得有缺陷可以为新型靶向化疗提供新的见解。

DESCRIPTION (provided by applicant): The DNA of all organisms undergoes persistent damage that can result in genetic mutations or epigenetic changes unless the damage is correctly repaired. The modified base, 5-methylcytosine (5mC), can undergo both deamination and oxidation resulting in both genetic mutations and epigenetic perturbation. We have demonstrated that the oxidation damage product, 5-hydroxymethylcytosine (5hmC), inhibits the binding of proteins that selectively bind to methylated DNA and that the maintenance methyltransferase DNMT1 does not recognize 5hmC when methylating DNA following DNA replication. Therefore, the formation of 5hmC could heritably alter epigenetic patterns in replicating cells. Emerging evidence indicates, however, that the conversion of 5mC to 5hmC could also be part of an enzymatic epigenetic reprogramming pathway essential for stem cell differentiation and that 5hmC is absent from most if not all human cancer cells. In this application, we propose a series of studies to further understand this putative DNA demethylation pathway in human neural stem cells, mouse embryonic stem cells and in a series of human cancer stem cells and established cell lines. We wish to understand how DNA demethylation relies upon DNA damage repair pathways, and how DNA damage repair and DNA demethylation might become entangled. In the first aim, we propose a novel stable isotope labeling method that will allow us to follow the dynamic processes of DNA replication, methylation and hydroxylation and excision of possible intermediates by the base excision repair pathway. In the second aim, we propose to use a series of innovative new methods to identify and quantify enzymatic activities that might act on pathway intermediates, further defining as yet unknown parts of this pathway. In the third aim, we propose some novel mass spectrometry approaches to identify proteins that bind to intermediates of the pathway and to measure how specific DNA intermediates, including 5hmC, affect the specificity and magnitude of the DNA-protein interactions. The proposed studies will allow examination of this important demethylation pathway at an unprecedented level. The information obtained is essential for understanding the biology of normal stem cells within the context of regenerative medicine, and understanding how the pathway becomes defective in human cancer cells could provide new insights into novel targeted chemotherapy.