Regulation of Genomic and Epigenomic Stability at CpG Sites

描述（由申请人提供）：本项目的总体目标是了解两种相关的哺乳动物碱基切除修复酶MED 1和TDG如何通过防止突变和甲基化模式改变来维持CpG位点的基因组和表观基因组稳定性。DNA甲基化是哺乳动物基因组的重要表观遗传修饰，包括在CpG位点处由胞嘧啶形成5-甲基胞嘧啶。DNA甲基化增加了突变的风险，因为甲基化和未甲基化的胞嘧啶都有自发脱氨基的倾向，前者更高，分别产生胸腺嘧啶和尿嘧啶。事实上，据估计，CpG位点的脱氨基作用导致了癌症和人类遗传疾病中近三分之一的突变。为了保持CpG位点的基因组稳定性，MED 1（也称为MBD 4）和TDG去除了引起问题的胸腺嘧啶或尿嘧啶。生物体还在CpG位点建立和调节适当的染色质状态/DNA甲基化模式（表观基因组稳定性）。表观基因组稳定性的改变发生在克隆的哺乳动物以及癌症中，因此对干细胞生物学和癌症诊断/预后/治疗都有影响。我们意外地发现了与TDG无效接合相关的胚胎致死性，并发现TDG是某些富含CpG的启动子的DNA去甲基化和其他表观遗传状态（如组蛋白H3乙酰化）的调节所必需的。这些观察结果表明，MED 1和TDG促进CpG位点的基因组和表观基因组稳定性的模型，以抵御发育缺陷，诱变和肿瘤发生。具体目的是：1）研究TDG在发育过程中的需求及其在DNA去甲基化和染色质修饰中的作用。我们将确定是否TDG催化活性是需要正常的发展，使用一个敲入株和评估TDG功能所需的ES细胞体外分化和小鼠胚胎成纤维细胞的表型。我们还将通过ChIP-seq研究TDG的启动子占用率与基因组规模上的甲基化模式之间的关系;并确定TDG是否参与受精后父本基因组的去甲基化。2)通过测量以下指标，评价MED 1和TDG在避免突变和甲基化改变方面的体内协作：遗传定义细胞系（TDG、MED 1或两者缺陷）中的G：T和G：U错配修复、单突变和MED 1-TDG双突变小鼠的突变频率和甲基化改变。3)评价MED 1和TDG在肿瘤发生中的作用。使用Min小鼠模型，我们将评估MED 1和TDG在肠道肿瘤发生中的作用。我们还将分析人类癌症中的TDG改变。这些研究将阐明CpG位点的基因组和表观基因组稳定性之间的联系，以及DNA修复机制对DNA去甲基化的影响。 公共卫生相关性：基因组中的一些DNA序列，称为CpG，在癌症中经常发生改变，无论是在突变为CpA或TpG方面，还是在称为甲基化的生理修饰方面。该项目的重点是两种DNA修复酶，MED 1/MBD 4和TDG，它们可以防止CpG突变和甲基化改变，从而抵御癌症和其他疾病。这些研究可能会为癌症的诊断和治疗带来新的策略。

DESCRIPTION (provided by applicant): The overall goal of this project is to understand how two related mammalian base excision repair enzymes, MED1 and TDG, maintain genomic and epigenomic stability at CpG sites, by preventing both mutations and altered methylation patterns. DNA methylation is an important epigenetic modification of the mammalian genome consisting in the formation of 5-methylcytosine from cytosine at CpG sites. DNA methylation increases the risk of mutations, because both methylated and unmethylated cytosines have a tendency - higher for the former - to spontaneously deaminate, generating thymine and uracil, respectively. Indeed, deamination at CpG sites is estimated to cause nearly one-third of all mutations in cancer and human genetic diseases. In order to maintain genomic stability at CpG sites, MED1 (also known as MBD4) and TDG, remove the offending thymine or uracil. Organisms also establish and regulate the proper chromatin states/DNA methylation patterns at CpG sites (epigenomic stability). Alterations in epigenomic stability occur in cloned mammals as well as in cancer and thus have implications for both stem cell biology and cancer diagnosis/prognosis/treatment. We made the unexpected discovery of embryonic lethality associated with TDG nullizygosity and found that TDG is required for DNA demethylation at some CpG-rich promoters and modulation of other epigenetic states, such as histone H3 acetylation. These observations suggest a model in which MED1 and TDG promote both the genomic and epigenomic stability of CpG sites, in order to ward off against developmental defects, mutagenesis and tumorigenesis. The Specific Aims are: 1) Characterize the requirement of TDG during development and its role in DNA demethylation and chromatin modification. We will determine whether TDG catalytic activity is required for normal development using a knock-in strain and assess which TDG function is required for ES cell differentiation in vitro and for phenotypes in mouse embryo fibroblasts. We will also study by ChIP-seq the relationship between promoter occupancy by TDG and methylation patterns on a genomic scale; and determine whether TDG is involved in demethylation of the paternal genome after fertilization. 2) Evaluate the in vivo cooperation between MED1 and TDG in avoidance of mutations and altered methylation, by measuring: G:T and G:U mismatch repair in genetically defined cell lines (deficient in TDG, MED1 or both), mutation frequency and altered methylation of single- and MED1-TDG double-mutant mice. 3) Evaluate the role of MED1 and TDG in tumorigenesis. Using the Min mouse model, we will evaluate the role of MED1 and TDG in intestinal tumorigenesis. We will also analyze TDG alterations in human cancer. These studies will shed light on the emerging link between genomic and epigenomic stability at CpG sites and the employment of the DNA repair machinery to effect DNA demethylation. PUBLIC HEALTH RELEVANCE: Some DNA sequences in the genome, known as CpG, are frequently altered in cancer, both in terms of mutation to CpA or TpG, and in terms of change of their physiological modification called methylation. This project focuses on two DNA repair enzymes, MED1/MBD4 and TDG that prevent both CpG mutations and alterations in methylation, thus warding off against cancer and other diseases. These studies may lead to novel strategies for the diagnosis and treatment of cancer.