Genetic analysis of DNA methylation in Neurospora crassa

粗糙脉孢菌 DNA 甲基化的遗传分析

• 批准号: 8254188
• 负责人: Andrew David Klocko
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8254188
• 关键词: Animal Model; Animals; Automobile Driving; Bacterial DNA; Binding; Cancer Patient; Carrier Proteins; Cell Nucleus; Chemicals; Chimeric Proteins; Chromosome Segregation; Chromosomes; Co-Immunoprecipitations; Cytoplasm; DNA; DNA Methylation; DNA Methylation Regulation; DNA Modification Process; Data; Defect; Development; Developmental Process; Eukaryota; Fungal DNA; Fungi Model; Gene Silencing; Genes; Genome; Genomics; Goals; Green Fluorescent Proteins; Growth and Development function; Heterochromatin; Histones; Human; Human Development; Individual; Lysine; Maintenance; Methylation; Methyltransferase; Modeling; Modification; Molds; Mutate; Neurospora; Neurospora crassa; Nuclear; Organism; Pathway interactions; Point Mutation; Prevention strategy; Protein Methyltransferases; Proteins; Regulation; Role; Site-Specific DNA-Methyltransferase (Adenine-Specific); System; Testing; Western Blotting; Work; X Inactivation; alpha Karyopherins; base; cell growth; cellular development; genetic analysis; mutant; novel; nucleocytoplasmic transport; release factor; research study; tumor progression

The establishment and maintenance of heterochromatin and DNA methylation is essential for the proper growth and development of all organisms. Specifically, heterochromatin and DNA methylation are essential for X- chromosome inactivation, chromosome segregation, and parasitic gene silencing in humans. However, the mechanisms controlling formation of heterochromatin, and subsequent DNA methylation, are presently unclear. In order to fully understand these critical human developmental processes, the mechanisms underlying the formation of heterochromatin and DNA methylation must be discerned. Heterochromatic regions of the genome are distinguished from their actively transcribed euchromatic counterparts by several covalent modifications. Namely, heterochromatic DNA and associated histone proteins are methylated. While several proteins have been identified that catalyze the methylation of DNA and histones, the regulation of these methyltransferase proteins is only now being appreciated. Studies in the model organism Neurospora crassa, a filamentous fungus, have been critical to understanding the formation of heterochromatin. Neurospora shares conserved DNA methylation machinery with humans, but unlike many higher eukaryotes, this methylation machinery is simple yet dispensable. Recent work has identified a protein that is known to be critical for nuclear transport as being important to establish both histone and DNA methylation, indicating that this protein has a critical role in regulating the methylation machinery. This proposal will focus on characterizing the role of the nuclear transport protein in DNA methylation. DamID experiments using translational fusions to the bacterial DNA Adenine Methylase (dam) gene will analyze the genomic localization of this protein. The influence of this nuclear transport protein on the global activity of the methylation machinery will be analyzed by western blotting experiments with tagged components of the DNA methylation machinery. Translational fusions of this protein to Green Fluorescent Protein (GFP) will analyze the cellular localization of this protein. Co-immunoprecipitation experiments will analyze the ability of this nuclear transport protein to interact with the H3K9me3 machinery. Moreover, the ability of the methylation machinery to influence the genomic localization of the nuclear transport protein will be investigated. Lastly, known interactors of the nuclear transport protein will be examined for their role in DNA methylation. By characterizing the genes required for the regulation of the DNA methylation machinery, we will be able to understand how heterochromatin is properly established in humans. In addition, identifying putative genes that could become mutated for the progression of cancer is essential to develop treatments or prevention strategies for cancer patients.

异染色质和 DNA 甲基化的建立和维持对于所有生物体的正常生长和发育至关重要。具体来说，异染色质和DNA甲基化对于人类X染色体失活、染色体分离和寄生基因沉默至关重要。然而，控制异染色质形成和随后的 DNA 甲基化的机制目前尚不清楚。为了充分了解这些关键的人类发育过程，必须弄清楚异染色质和 DNA 甲基化形成的机制。基因组的异染色质区域通过几种共价修饰与活跃转录的常染色质区域区分开来。即，异染色质 DNA 和相关组蛋白被甲基化。虽然已经鉴定出几种催化 DNA 和组蛋白甲基化的蛋白质，但这些甲基转移酶蛋白质的调节作用现在才被人们认识到。对模式生物粗糙脉孢菌（一种丝状真菌）的研究对于理解异染色质的形成至关重要。脉孢菌与人类共享保守的 DNA 甲基化机制，但与许多高等真核生物不同，这种甲基化机制简单但可有可无。最近的工作发现了一种已知对核运输至关重要的蛋白质，它对于建立组蛋白和 DNA 甲基化也很重要，表明该蛋白质在调节甲基化机制中具有关键作用。该提案将重点关注核转运蛋白在 DNA 甲基化中的作用。 DamID 实验使用细菌 DNA 腺嘌呤甲基酶 (dam) 基因的翻译融合来分析该蛋白质的基因组定位。该核转运蛋白对甲基化机制整体活性的影响将通过使用 DNA 甲基化机制的标记组件进行蛋白质印迹实验来分析。该蛋白与绿色荧光蛋白 (GFP) 的翻译融合将分析该蛋白的细胞定位。免疫共沉淀实验将分析该核转运蛋白与 H3K9me3 机制相互作用的能力。此外，还将研究甲基化机制影响核转运蛋白基因组定位的能力。最后，将检查已知的核转运蛋白相互作用因子在 DNA 甲基化中的作用。通过表征 DNA 甲基化机制调节所需的基因，我们将能够了解异染色质如何在人类中正确建立。此外，识别可能因癌症进展而发生突变的基因对于制定癌症患者的治疗或预防策略至关重要。