Regulation of Genomic and Epigenomic Stability at CpG Sites

CpG 位点基因组和表观基因组稳定性的调控

• 批准号: 8098984
• 负责人: ALFONSO BELLACOSA
• 金额: $ 37.19万
• 依托单位: FOX CHASE CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8098984
• 关键词: Acetylation; Active Sites; Adenine; Affect; Arabidopsis; Base Excision Repairs; Biochemical; Cardiovascular system; Cell Differentiation process; Cell Line; Chromatin; Colorectal Cancer; Complex; CpG Island Methylator Phenotype; CpG Islands; Cytosine; DNA; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA Repair; DNA Repair Enzymes; DNA Sequence; Deaminase; Deamination; Defect; Development; Diagnosis; Disease; Embryo; Employment; Enhancers; Epigenetic Process; Excision; Fertilization; Fibroblasts; Frequencies; Genome; Genome Stability; Genomics; Germ Lines; Goals; Grant; Hereditary Disease; Histone H3; Human; Human Genetics; Hypermethylation; In Vitro; Intestinal Neoplasms; Intestines; Knock-in Mouse; Lead; Light; Link; Liver; Malignant Neoplasms; Mammalian Cell; Mammals; Measures; Methylation; Mismatch Repair; Modeling; Modification; Mus; Mutagenesis; Mutant Strains Mice; Mutation; Organism; Pattern; Phenotype; Physiological; Process; Regulation; Retinoic Acid Receptor; Risk; Role; Site; Surveys; Testing; Thymine; Tissues; Transcription Coactivator; Tumor Suppressor Genes; Uracil; Work; Zebrafish; cancer diagnosis; cancer genetics; cancer therapy; chromatin modification; demethylation; developmental disease; embryonic stem cell; epigenomics; in vitro activity; in vivo; mammalian genome; mouse model; novel strategies; outcome forecast; prevent; promoter; public health relevance; repair enzyme; stem cell biology; tumor; tumorigenesis; ward

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.

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的重要表观遗传修饰，包括 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，因为甲基化和非甲基化胞嘧啶都有自发脱氨基的倾向（前者较高），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癌症和人类遗传疾病中近三分之一的突变。为了维持 CpG 位点的基因组稳定性，MED1（也称为 MBD4）和 TDG 会去除有问题的胸腺嘧啶或尿嘧啶。生物体还在 CpG 位点建立并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。我们意外地发现胚胎致死性与 TDG 无效性相关，并发现 TDG 是某些富含 CpG 的启动子处 DNA 去甲基化和其他表观遗传状态（例如组蛋白 H3 乙酰化）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，以防止发育缺陷、诱变和肿瘤发生。具体目标是： 1) 描述发育过程中 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正常发育是否需要 TDG 催化活性，并评估 ES 细胞体外分化和小鼠胚胎成纤维细胞表型所需的 TDG 功能。我们还将通过 ChIP-seq 研究 TDG 启动子占据与基因组规模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：遗传定义的细胞系（TDG、MED1 或两者缺陷）中的 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 之间在避免突变和甲基化改变方面的体内合作。 3)评估MED1和TDG在肿瘤发生中的作用。使用 Min 小鼠模型，我们将评估 MED1 和 TDG 在肠道肿瘤发生中的作用。我们还将分析人类癌症中 TDG 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之间的新联系，以及使用 DNA 修复机制来影响 DNA 去甲基化。 公共健康相关性：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 CpA 或 TpG 的突变，还是其称为甲基化的生理修饰的变化。该项目重点研究两种 DNA 修复酶 MED1/MBD4 和 TDG，它们可以防止 CpG 突变和甲基化改变，从而预防癌症和其他疾病。这些研究可能会带来癌症诊断和治疗的新策略。