GENETIC ASPECTS OF DNA METHYLATION

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， 在X染色体失活和基因调控中， 那些受基因印记影响的人小鼠DNA的过度表达 甲基转移酶（MTase）基因可诱导肿瘤转化， 培养的细胞，MTase活性的工程降低可以减缓肿瘤 进步，大量的间接证据表明， 肿瘤抑制基因的异常甲基化可导致癌症。 在 此外，有证据表明甲基化在“基因组防御”中起作用。 真菌、植物和动物中的系统。 的长期目标 本研究旨在阐明DNA的功能和控制 甲基化在真核生物利用一个杰出的模型 真菌Neurospora crassa。 甲基化是甲基化 链孢霉，这有助于遗传分析的这一进程和 分析甲基化所需的工具和材料就在手边。 具体目标是：1。来识别控制 DNA甲基化的建立和维持。 序列要求 将进一步定义，并将测试新模型。2.以隔离 结合DNA序列的蛋白质，所述DNA序列发出DNA甲基化信号和/或 甲基化，并通过分离和破坏来确定其功能 对应的基因。 3.识别其他交易行为 甲基化机器的组成部分。 甲基化基因， 已经鉴定的和通过插入鉴定的其他基因 诱变将被分离和表征。 4.阐明本 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，曲古抑菌素A（TSA），一种组蛋白， 去乙酰化酶抑制剂，导致区域DNA甲基化丢失。 替代模型将通过以下方式进行测试： 与未甲基化，甲基化但TSA不敏感， 和TSA敏感序列;测试TSA诱导的 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 H4对甲基化的影响;测试DNA甲基化是否影响组蛋白 乙酰化 5.为了阐明复制与 时间和DNA甲基化。甲基化突变体和我们现有的方法 研究脉孢菌复制的方法将用于研究 无论是DNA甲基化还是组蛋白低乙酰化， DNA的复制

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.