Oxidation of 5-methylcytosine: DNA damage and epigenetic reprogramming

描述(申请人提供)：所有生物体的DNA都会遭受持续性损伤，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学变化。修饰后的碱基5-甲基胞嘧啶(5mC)可发生脱氨基和氧化反应，导致基因突变和表观遗传扰动。我们已经证明了氧化损伤产物5-羟甲基胞嘧啶(5hmC)抑制了选择性与甲基化DNA结合的蛋白质的结合，并且维持甲基转移酶DNMT1在DNA复制后甲基化DNA时不识别5hmC。因此，5hmC的形成可以遗传地改变复制细胞的表观遗传模式。然而，新的证据表明，5mC到5hmC的转换也可能是对干细胞分化至关重要的酶促表观遗传重编程途径的一部分，5hmC在大多数(如果不是全部)人类癌细胞中缺失。在这一应用中，我们提出了一系列研究，以进一步了解这一假定的DNA去甲基化途径在人类神经干细胞、小鼠胚胎干细胞以及一系列人类癌症干细胞和已建立的细胞系中。我们希望了解DNA去甲基化是如何依赖于DNA损伤修复途径的，以及DNA损伤修复和DNA去甲基化是如何纠缠在一起的。在第一个目标中，我们提出了一种新的稳定的同位素标记方法，使我们能够跟踪DNA复制、甲基化和羟化的动态过程，以及通过碱基切除修复途径切除可能的中间产物。在第二个目标中，我们建议使用一系列创新的新方法来鉴定和量化可能作用于途径中间体的酶活性，进一步定义这一途径的未知部分。在第三个目标中，我们提出了一些新的质谱学方法来鉴定与该途径的中间体结合的蛋白质，并测量特定的DNA中间体，包括5hmC，如何影响DNA-蛋白质相互作用的特异性和大小。拟议的研究将使人们能够在前所未有的水平上检查这一重要的去甲基化途径。所获得的信息对于在再生医学的背景下理解正常干细胞的生物学是必不可少的，了解该途径如何在人类癌细胞中变得有缺陷可以为新的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.