Regulation of Genomic and Epigenomic Stability at CpG Sites

描述（由申请人提供）：该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MED1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中一种重要的表观遗传修饰，由CpG位点的胞嘧啶形成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的倾向——前者的倾向更高——分别产生胸腺嘧啶和尿嘧啶。事实上，据估计，近三分之一的癌症和人类遗传疾病突变是由CpG位点的脱氨引起的。为了维持CpG位点，MED1（也称为MBD4）和TDG的基因组稳定性，去除有害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哺乳动物和癌症中，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零合子相关的胚胎致命性，并发现TDG是一些富含cpg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，MED1和TDG促进CpG位点的基因组和表观基因组稳定性，以抵御发育缺陷、突变和肿瘤发生。具体目的是：1)描述发育过程中对TDG的需求及其在DNA去甲基化和染色质修饰中的作用。我们将使用敲入菌株确定TDG的催化活性是否需要正常发育，并评估体外胚胎干细胞分化和小鼠胚胎成纤维细胞表型需要TDG的哪些功能。我们还将通过ChIP-seq研究启动子占用TDG与基因组尺度上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)通过测量基因定义细胞系（TDG、MED1或两者都缺乏）的：G:T和G:U错配修复、单突变和MED1-TDG双突变小鼠的突变频率和甲基化改变，评估MED1和TDG在体内避免突变和甲基化改变方面的合作。3)评价MED1和TDG在肿瘤发生中的作用。利用Min小鼠模型，我们将评估MED1和TDG在肠道肿瘤发生中的作用。我们还将分析TDG在人类癌症中的变化。这些研究将揭示CpG位点的基因组和表观基因组稳定性与DNA修复机制影响DNA去甲基化之间的新联系。

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.