Infidelity of Cytosine Methylation and Human Cancer

描述（由申请人提供）：新出现的数据表明，人类癌症的发展是一个多步骤的过程，不仅由遗传损伤驱动，而且由表观遗传变化驱动。表观遗传信号的扰动可导致肿瘤抑制基因表达的丧失或癌基因的不适当表达。表观遗传控制的分子基础涉及CpG二核苷酸和蛋白质中胞嘧啶残基的酶促甲基化，其选择性地结合甲基化DNA序列，启动改变局部染色质结构和转录活性的相互作用蛋白质的级联反应。虽然人类肿瘤中的表观遗传扰动列表每天都在增长，但表观遗传信号在肿瘤发展中变得混乱的机制在很大程度上尚未探索。在最初的资助期间，我们发现炎症损伤引起的氧化损伤和卤化可能导致DNA甲基化的丧失。这种竞争性的更新应用程序的重点是探索卤嘧啶可以改变表观遗传信号的机制。 在本申请的五个目的中，我们提出1）在凝胶电泳迁移率变动测定中进一步绘制甲基结合蛋白及其DNA结合位点与核苷类似物和纯化的甲基结合蛋白之间的关键接触点，2）检查结构，5-氯胞嘧啶（5CIC）和5-氯尿嘧啶（5CIU）的动力学和编码潜力-使用高场同位素编辑的NMR和聚合酶延伸测定法检测含有寡核苷酸的CpG二核苷酸，3）使用最近开发的质量标记测定法检测CpG二核苷酸中胞嘧啶残基对氯化反应的反应性，4）检查5-氯嘧啶指导甲基化酶介导的甲基化或抑制甲基化的能力，和5）检查5CIC残基抑制转录和诱导哺乳动物细胞中可遗传的表观遗传变化的能力。这项提议的结果可能会对DNA损伤可能导致可遗传的表观遗传改变的机制提供重要的新见解，这些表观遗传改变在许多人类肿瘤的发展中至关重要。

DESCRIPTION (provided by applicant): Emerging data suggests that the development of cancer in man is a multi-step process driven by not only genetic damage but also epigenetic changes. Perturbations in epigenetic signals can result in the loss of tumor suppressor expression or inappropriate oncogene expression. The molecular basis of epigenetic control involves the enzymatic methylation of cytosine residues in CpG dinucleotides and proteins that selectively bind to methylated DNA sequences, initiating a cascade of interacting proteins that alter local chromatin structure and transcriptional activity. While the list of epigenetic perturbations in human tumors grows daily, the mechanisms by which epigenetic signals become scrambled in tumor development are largely unexplored. In the initial funding period, we discovered that oxidative damage and halogenation arising from inflammatory damage could result in loss of DNA methylation. This competing renewal application focuses on exploration of the mechanisms whereby halopyrimidines could alter epigenetic signals. In the five aims of the current application, we propose to 1) further map critical contact points between methyl-binding proteins and their DNA-binding sites with nucleoside analogs and purified methyl-binding proteins in gel electrophoretic mobility shift assays, 2) examine the structure, dynamics and coding potential of 5-chlorocytosine (5CIC) and 5-chlorouracil (5CIU)-containing oligonucleotides using high field isotope edited NMR and polymerase extension assays, 3) examine the reactivity of cytosine residues in a CpG dinucleotide toward chlorination reactions using a recently developed mass-tagging assay, 4) examine the capacity of 5-chlorpyrimidines to direct methylase-mediated methylation or to inhibit methylation, and 5) to examine the capacity of 5CIC residues to inhibit transcription and induce heritable epigenetic changes in mammalian cells. This proposal's results will likely shed important new light on mechanisms by which DNA damage could result in heritable epigenetic alterations critical in the development of many human tumors.