Genetic Aspects of DNA Methylation

描述(申请人提供)：DNA甲基化是高等真核生物正常发育所必需的，部分原因可能是DNA甲基化在基因组防御、基因组印迹和其他沉默过程中的作用，如X染色体失活。DNA甲基化异常与肿瘤发生、印记障碍、脆性X综合征、糖尿病和神经疾病(如双相综合症、阿尔茨海默氏症、自闭症和精神分裂症)有关。DNA甲基化在粗糙脉孢菌中是必不可少的，这有助于遗传学研究。这项拟议研究的总体目标是通过利用这一杰出的模型系统来阐明真核生物中DNA甲基化的机制。这项工作将得到宝贵的资源和工具的推动，这些资源和工具包括：1.高效的基因打靶和表位标记程序；2.覆盖约80%基因组的基因敲除菌株的集合；3.用于反向遗传学的突变过程，RIP(重复诱导点突变)；4.结合亚硫酸氢盐甲基化分析、染色质免疫沉淀和DAMID方法的高效高通量测序；5.鉴定蛋白质及其修饰的敏感蛋白质组学(质谱学)和免疫学方法。拟议研究的一个重要目标是确定DNA甲基化机制的所有组成部分，并阐明它们之间的相互关系。重要的是要确定要甲基化的序列是如何定位的，并扩大我们对异染色质形成和DNA甲基化所需下游步骤的机械理解(目标1和2)。这项研究的另一个总体目标是通过识别和描述调节甲基化模式的机制来促进我们对DNA甲基化调控的理解(目标3)。最后一个目标是提高我们对与DNA甲基化一起作用以沉默序列的机制的理解(目标4)。该项目的具体目标是：1.开发新的和现有的DIM(甲基化缺陷)突变体，以阐明DNA甲基化的机制。2.测试异染色质元件在沉默的建立和/或维持中的作用。3.鉴定DNA甲基化和沉默的修饰物。4.鉴定无DNA甲基化缺陷的沉默突变体。

DESCRIPTION (provided by applicant): DNA methylation is required for normal development of higher eukaryotes, presumably in part because of its role in genome defense, genomic imprinting and in other silencing processes such as X-chromosome inactivation. Abnormal DNA methylation has been implicated in tumorigenesis, imprinting disorders, Fragile-X syndrome, diabetes and neurologic disorders (e.g. bipolar syndrome, Alzheimer's, autism and schizophrenia). DNA methylation is dispensable in the fungus Neurospora crassa, facilitating genetic studies. The overall goal of the proposed research is to elucidate the mechanism of DNA methylation in eukaryotes by taking advantage of this outstanding model system. The work will be facilitated by valuable resources and tools including: 1. Efficient procedures for gene targeting and epitope-tagging; 2. A collection of gene knockout strains covering ~80% of the genome; 3. The mutagenic process, RIP (repeat-induced point mutation), for reverse-genetics; 4. Efficient high throughput sequencing coupled with bisulfite methylation analysis, chromatin immunoprecipitation and DamID methodology; 5. Sensitive proteomic (mass spectrometry) and immunological methods for identification of proteins and their modifications. An important goal of the proposed research is to identify all the components of the DNA methylation machinery and to elucidate their interrelationships. It is important to determine how sequences to be methylated are targeted and to expand our mechanistic understanding of the downstream steps required for heterochromatin formation and DNA methylation (Aims 1 & 2). Another general goal of the research is to advance our understanding of the regulation of DNA methylation by identifying and characterizing the mechanisms that modulate methylation patterns (Aim 3). A final goal is to improve our understanding of mechanisms that work alongside DNA methylation to silence sequences (Aim 4). Specific aims of the project are: 1. To exploit new and existing dim (defective in methylation) mutants to elucidate the mechanism of DNA methylation. 2. To test elements of heterochromatin for roles in the establishment and/or maintenance of silencing. 3. To characterize modifiers of DNA methylation and silencing. 4. To characterize silencing mutants that do not show DNA methylation defects.