Genetic Aspects of DNA Methylation

我们利用真菌粗糙脉孢菌来阐明DNA甲基化的控制和功能，以及 X染色体失活、基因组印迹和正常发育所需的色素遗传过程 哺乳动物。在人类中，DNA甲基转移酶(MTase)的缺失会导致ICF综合征，甲基转移酶(MTase)的缺失会导致ICF综合征。 DNA结合蛋白MeCP2导致Rett综合征，甲基化异常与癌症有关。。 分离出DNA甲基化缺陷(DIM)的脉孢菌突变体使人们对对照和 甲基化在真核生物中的作用。例如，我们将DIM-5鉴定为组蛋白H3MTase 首次证明了组蛋白修饰可以控制DNA甲基化。我们的工作得到了帮助 有价值的资源包括：1.脉孢子虫基因组序列，2.有效的敲打方法 用于定位和生化研究的表位标签系统，4.抗体 对修饰的组蛋白和其他相关表位具有特异性，以及5.强大的分析方法(例如，质量 光谱分析、染色质免疫沉淀和微阵列分析)。我们的大部分努力将集中在 识别和表征与DNA甲基化有关的基因产物。我们会找出有缺陷的突变体 在使用正向和反向遗传学使甲基化DNA沉默方面。后者将利用 基因组敲除计划，这将在未来几年内扰乱大多数脉孢子菌基因。我们将测试 所有的敲除都是为了抑制我们沉默的bar基因。我们还将使用生化方法来 基于我们对引发脱氧核糖核酸序列的鉴定，确定引发从头甲基化的蛋白质(S) 体内的甲基化。在其他工作中，我们将阐明组蛋白的功能、机制和相互关系 与甲基化DNA相关的修饰。我们将确定哪种基因/蛋白质对每一种 观察到这种真菌的组蛋白修饰，并测试不能执行该基因的突变体的表型 修改。我们还将确定组蛋白修饰相互依赖的程度。在……里面 此外，我们将使用‘芯片上的ChlP’来鉴定野生型和突变型脉孢霉的‘表观基因组’。 并将使用蛋白质组学来研究关键的组蛋白 MTase存在于蛋白质复合体中。最后，我们将跟进我们的观察结果，认为组蛋白 去甲基酶影响DNA甲基化。

We exploit the fungus Neurospora crassa to elucidate the control and function of DNA methylation, an pigenetic process required for X chromosome-inactivation, genomic imprinting and normal development in mammals. In humans, loss of a DNA methyltransferase (MTase) causes ICF syndrome, loss of the methyl- DNA binding protein MeCP2 causes Rett syndrome and abnormal methylation is associatedwith cancer. . Isolation of Neurospora mutants defective in DNA methylation (dim) has led to insights into the control and function of methylation in eukaryotes. For example, our identification of DIM-5 as a histone H3 MTase demonstrated for the first time that histone modifications can control DNA methylation. Our work is helped by valuable resources including: 1. the sequence of the Neurospora genome, 2. efficient methods to knock out genes in Neurospora, S.epitope tagging systems for localization and biochemical studies, 4. antibodies specific for modified histones and other relevant epitopes and 5. powerful analytical methods (e.g., mass spectrometry, chromatin immunoprecipitation and microarray analysis). Most of our effort will go towards identifying and characterizing gene products involved in DNA methylation. We will identify mutants defective in silencing methylated DNA using both forward and reverse genetics. The latter will take advantage of the genome knock-out project, which will disrupt most Neurospora genes within the next few years. We will test all knock-outs for de-repression of our silenced bar gene. We will also employ a biochemical approach to identify protein(s) that trigger de novo methylation based on our identification of sequences that trigger DNA methylation in vivo. In other work, we will elucidate the function, mechanism and interrelationships of histone modifications associated with methylated DNA. We will determine which gene/protein is responsible for each observed histone modification in this fungus and test the phenotypes of mutants unable to perform the modifications. We also will determine the extent to which the histone modifications depend on each other. In addition, we will characterize the Neurospora 'epigenome' in wildtype and mutants using a 'ChlP-on-chip' approach with stock and custom microarrays and will use proteomics to investigate whether critical histone MTases are in protein complexes. Finally, we will follow up our observations suggesting that histone demethylases influence DNA methylation.