GENETIC ASPECTS OF DNA METHYLATION

DNA 甲基化的遗传方面

• 批准号: 6329666
• 负责人: Eric U. SELKER
• 金额: $ 33.42万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6329666
• 关键词: DNA binding protein; DNA methylation; DNA replication; Neurospora; acetylation; affinity chromatography; fungal genetics; fungal proteins; genetic transcription; high performance liquid chromatography; immunoprecipitation; laboratory rabbit; methyltransferase; molecular cloning; nucleic acid sequence; point mutation; pseudogenes; regulatory gene; ribosomal DNA; transcription factor

In many organism, including humans, DNA is modified by methylation of certain cytosines. DNA methylation is essential in mammals, and is used in X-chromosome inactivation and in the regulation of genes, such as those subject to genomic imprinting. Over-expression of the mouse DNA methyltransferase (MTase) gene can induce tumorigenic transformation of cultured cells, engineered reduction of MTase activity can slow tumor progression, and considerable circumstantial evidence suggests that aberrant methylation of tumor suppressor genes can cause cancer. In addition, there is evidence that methylation serves in "genome defense" systems in fungi, plants and animals. The long-term goal of the research proposed here is to elucidate the function and control of DNA methylations in eukaryotics by taking advantage of an outstanding model system the fungus Neurospora crassa. Methylation is dispensable in Neurospora which facilitates genetic analyses of this process and the tools and materials required to dissect methylation are at hand. Specific aims are: 1. To identify the cis-acting signals that control establishment and maintenance of DNA methylation. Sequence requirements will be further defined and new models will be tested. 2. To isolate proteins that bind DNA sequences that signal DNA methylation and/or are methylated, and to determine their function by isolation and disruption of the corresponding genes. 3. To identify other trans-acting components of the methylation machinery. Methylation genes that we have already identified and additional genes identified by insertional mutagenesis will be isolated and characterized. 4. To elucidate the role of histone acetylation in the control of DNA methylation. These studies are based on our finding that Trichostatin A (TSA), a histone deacetylase inhibitor, causes regional loss of DNA methylation. Alternate models will be tested by: assessing the acetylation of histones associated with unmethylated, methylated but TSA-insensitive, and TSA-sensitive sequences; testing whether TSA-induced loss of methylation depends on transcription; testing the function of candidate cis-acting elements; testing the effect of mutations in histones H3 and H4 on methylation; testing whether DNA methylation affects histone acetylation. 5. To elucidate the relationship between replication timing and DNA methylation. Methylation mutants and methods that we have developed to study replication in Neurospora will be used to investigate whether either DNA methylation or histone hypoacetylation cause late replication of DNA.

在包括人类在内的许多生物体中，DNA是通过甲基化修饰的 某些胞嘧啶。DNA甲基化在哺乳动物中是必不可少的，并被用于 在X染色体失活和基因调控方面，例如 那些受到基因组印记影响的人。小鼠DNA的高效表达 甲基转移酶(MTase)基因可诱导肝癌细胞发生致瘤转化 培养细胞，工程降低MTase活性可以减缓肿瘤 进展，而且相当多的间接证据表明 肿瘤抑制基因的异常甲基化可能导致癌症。在……里面 此外，有证据表明甲基化在“基因组防御”中起作用。 真菌、植物和动物的系统。中国的长期目标是 这里提出的研究是为了阐明DNA的功能和控制 真核生物中的甲基化：利用一个杰出的模型 系统的真菌粗糙脉孢菌。甲基化是不必要的。 脉孢子虫，促进了这一过程的遗传分析和 分析甲基化所需的工具和材料近在咫尺。 具体目标是：1.识别控制顺式作用的信号 DNA甲基化的建立和维持。顺序要求 将进一步定义，并将测试新型号。2.隔离 结合发出DNA甲基化信号的DNA序列的蛋白质和/或 甲基化，并通过分离和破坏来确定其功能 相应的基因。3.找出其他反式行为 甲基化机制的组成部分。我们拥有的甲基化基因 已确定的基因和通过插入的 诱变作用将被分离和鉴定。4.澄清 组蛋白乙酰化在DNA甲基化控制中的作用。这些 研究是基于我们的发现，曲古抑素A(TSA)，一种组蛋白 脱乙酰酶抑制剂，导致DNA甲基化的区域性丢失。 替代模型将通过以下方式进行测试：评估乙酰化 组蛋白与未甲基化、甲基化但对TSA不敏感， 和TSA敏感序列；测试TSA是否导致 甲基化依赖于转录；测试候选基因的功能 顺式作用元件；测试组蛋白H3和H3突变的影响 H4甲基化；测试DNA甲基化是否影响组蛋白 乙酰化。5.阐明复制之间的关系 时间和DNA甲基化。甲基化突变体和我们拥有的方法 为研究脉孢子菌的复制而开发的将用于研究 DNA甲基化或组蛋白低乙酰化是否会导致迟发 复制DNA。