Genetic analysis of DNA methylation in Neurospora crassa

异染色质和DNA甲基化的建立和维持对于所有生物的正常生长和发育是必不可少的。具体地说，异染色质和DNA甲基化是人类X染色体失活、染色体分离和寄生虫基因沉默的关键。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，必须弄清异染色质形成和DNA甲基化的潜在机制。基因组的异染色区通过几个共价修饰与其活跃转录的常染色区相区别。也就是说，异染色质DNA和相关的组蛋白是甲基化的。虽然已经确定了几种蛋白质可以催化DNA和组蛋白的甲基化，但对这些甲基转移酶蛋白质的调节直到现在才被认识到。模式生物粗糙脉孢霉是一种丝状真菌，对理解异染色质的形成至关重要。脉孢子虫与人类共享保守的DNA甲基化机制，但与许多高等真核生物不同的是，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，它对建立组蛋白和DNA甲基化都很重要，表明该蛋白质在调节甲基化机制中具有关键作用。这项提案将重点描述核运输蛋白在DNA甲基化中的作用。利用细菌DNA腺嘌呤甲基酶(DAM)基因的翻译融合的DAMID实验将分析该蛋白质的基因组定位。这种核转运蛋白对甲基化机制整体活性的影响将通过对DNA甲基化机制的标记成分进行Western blotting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这种核运输蛋白与H3K9me3机械相互作用的能力。此外，还将研究甲基化机制影响核运输蛋白基因组定位的能力。最后，将检查已知的核运输蛋白的相互作用因子在DNA甲基化中的作用。通过描述调控DNA甲基化机制所需的基因，我们将能够理解异染色质是如何在人类体内正确建立的。此外，识别癌症进展过程中可能发生突变的假定基因对于开发癌症患者的治疗或预防策略至关重要。

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.