The dynamics of DNA methylation in normal and cancer cells

正常细胞和癌细胞中 DNA 甲基化的动态

• 批准号: 8019997
• 负责人: Gregory Adam Horwitz
• 金额: $ 5.3万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8019997
• 关键词: Aging; Binding; Biological Assay; Carcinogenesis Mechanism; Cell Differentiation process; Cell physiology; Characteristics; Chromatin; Chromatin Structure; Colon Adenocarcinoma; Coupled; CpG dinucleotide; DNA; DNA Binding Domain; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA Restriction Enzymes; Data; Epigenetic Process; Essential Genes; Fingers; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genome; Genomic Imprinting; Genomics; Goals; Human; Hypermethylation; In Vitro; Knock-out; Lead; Maintenance; Malignant Neoplasms; Mammalian Cell; Mammals; Measurement; Methylation; Methyltransferase; Missense Mutation; Mus; Mutation; Nephroblastoma; Normal Cell; Pattern; Peptides; Phenotype; Play; Proliferating Cell Nuclear Antigen; Promoter Regions; Property; Proteins; Recruitment Activity; Regulation; Research Proposals; Role; Shotguns; Site; Southern Blotting; Testing; Tissue-Specific Gene Expression; Ubiquitin; Vertebrates; X Inactivation; base; bisulfite; cancer cell; cancer type; carcinogenesis; cell type; cellular pathology; demethylation; embryonic stem cell; gain of function; in vivo; insight; knock-down; loss of function; malignant breast neoplasm; mammalian genome; mutant; preference; research study; small hairpin RNA

DESCRIPTION (provided by applicant): Genomic patterns of DNA methylation in mammals play a critical role in gene regulation and chromatin organization. DNA methylation is important in a diverse range of cellular functions and pathologies, including tissue-specific gene expression, cell differentiation, genomic imprinting, X chromosome inactivation, regulation of chromatin structure, aging, and carcinogenesis. CG methylation is the most common type found in mammalian genomes and it is maintained by the maintenance DNA methyltransferase called DNMT1, which acts on newly synthesized DNA at replication forks and shows a preference for hemimethylated CG sites. UHRF1 contains a methyl DNA binding domain that shows strong preferential binding to hemimethylated CG sites. DNMT1 is recruited to replication foci in part through its interaction with proliferating cell nuclear antigen (PCNA), and in part by directly interacting with UHRF1. How methylated DNA is maintained at sites of gene silencing in normal and transformed cancer cells is still relatively unclear. UHRF1 binds hemimethylated DNA through its SRA domain; however, it is unclear what roles the various other domains of UHRF1 play in maintaining DNA methylation. Our preliminary data has revealed a previously unidentified site of methylation on DNMT1. We believe this might be another mechanism for regulating the maintenance DNA methylation. The goal of this project is to understand in greater detail how methylated DNA is maintained at sites of gene silencing in normal and transformed cancer cells. UHRF1 with mutations of the ubiquitin like domain, PHD, SRA, and the RING finger motif, respectively, will be stably expressed in murine embryonic stem (ES) cells with a targeted disruption of UHRF1. Various assays will be employed to help elucidate the roles these domains play in maintaining DNA methylation. Synthesized methylated peptides of DNMT1 will also be tested for direct binding to the UHRF1 PHD domain in vitro compared to unmethylated peptides. The function of methylated DNMT1 will also be examined by performing knockout and knockdown of the methyltransferase required for methylating DNMT1. Finally, an shRNA screen in murine ES cells will be developed to identify other genes required for DNA methylation. There is growing evidence that the DNA methylome undergoes characteristic changes in the majority of cancers examined thus far. This is highly relevant to mechanisms of carcinogenesis, since mis-regulated DNA methylation patterns can promote abnormal gene expression and metastatic properties of numerous cancer cell types. The long-term goal of these studies is to determine what role DNA methylation is playing in the transformation of a normal cell to a cancer phenotype.

描述（由申请人提供）：哺乳动物 DNA 甲基化的基因组模式在基因调控和染色质组织中发挥着关键作用。 DNA 甲基化在多种细胞功能和病理学中发挥着重要作用，包括组织特异性基因表达、细胞分化、基因组印记、X 染色体失活、染色质结构调节、衰老和癌变。 CG 甲基化是哺乳动物基因组中最常见的类型，由称为 DNMT1 的维持 DNA 甲基转移酶维持，该酶作用于复制叉处新合成的 DNA，并显示出对半甲基化 CG 位点的偏好。 UHRF1 包含一个甲基 DNA 结合域，显示出与半甲基化 CG 位点的强烈优先结合。 DNMT1 部分通过与增殖细胞核抗原 (PCNA) 相互作用，部分通过直接与 UHRF1 相互作用而被招募到复制灶。 在正常和转化的癌细胞中，甲基化 DNA 如何维持在基因沉默位点仍相对不清楚。 UHRF1 通过其 SRA 结构域结合半甲基化 DNA；然而，目前尚不清楚 UHRF1 的其他各个结构域在维持 DNA 甲基化方面发挥什么作用。 我们的初步数据揭示了 DNMT1 上先前未识别的甲基化位点。 我们相信这可能是调节维持 DNA 甲基化的另一种机制。 该项目的目标是更详细地了解正常和转化癌细胞中基因沉默位点上甲基化 DNA 是如何维持的。 分别具有泛素样结构域、PHD、SRA 和环指基序突变的 UHRF1 将在鼠胚胎干 (ES) 细胞中稳定表达，并靶向破坏 UHRF1。 将采用各种测定来帮助阐明这些结构域在维持 DNA 甲基化中所发挥的作用。 与未甲基化肽相比，还将在体外测试 DNMT1 合成甲基化肽与 UHRF1 PHD 结构域的直接结合。 还将通过对 DNMT1 甲基化所需的甲基转移酶进行敲除和敲低来检查甲基化 DNMT1 的功能。 最后，将开发小鼠 ES 细胞中的 shRNA 筛选，以鉴定 DNA 甲基化所需的其他基因。 越来越多的证据表明，迄今为止检查的大多数癌症中，DNA 甲基化组都发生了特征性变化。 这与致癌机制高度相关，因为错误调控的 DNA 甲基化模式可以促进多种癌细胞类型的异常基因表达和转移特性。 这些研究的长期目标是确定 DNA 甲基化在正常细胞向癌症表型的转变中发挥什么作用。