Epigenetic Mechanisms for the Inheritance of Acquired Mutations

该奖项是根据2009年《美国复苏和再投资法案》(公法111-5)提供资金的。RNA通常被认为是基因表达的管道，但在纤毛原生动物中有一种新的作用模式。PI的实验室已经表明，母体RNA模板既为Oxytricha中的DNA重排提供了组织指南，也提供了一个模板，可以将体细胞生长过程中可能出现的自发突变传递给下一代。这种由RNA引导的DNA修复机会在纤毛虫Oxytricha中是深刻的，它通过全球DNA重排删除了95%的生殖系基因组，严重破坏了它的染色体，然后对剩下的数十万个片段进行排序和重新排序。用于重新排序的程序化信息来自瞬时表达的母体RNA。这意味着，母体基因组中出现的变化，无论是点突变还是新的DNA重排模式，都有机会传递给下一代，绕过通常通过生殖系DNA进行遗传的模式。此外，这些RNA模板中的点突变偶尔转移到F1代，为后天获得的自发体细胞替换提供了一种稳定遗传的机制，而不会改变生殖系基因组。这表明，体细胞大核基因组实际上是一个“表观基因组”，通过前一代产生的模板和信号形成。这项研究将针对3个特定目标，这将在Oxytricha中产生对以下方面的详细见解：1)通过RNA模板对核苷酸替换的表观遗传，通过探索参与RNA引导的DNA修复的可能聚合酶；2)通过程序性DNA缺失对内含子丢失的表观遗传，这将有助于体细胞基因组的发育简化；3)拷贝数变异的表观遗传：第三个特定目标将探索母体模板RNA在调节DNA拷贝数方面的另一个新发现的作用。这为适应环境提供了一种可遗传的非孟德尔机制。广泛的影响：虽然RNA模板的研究及其作为驱动基因组重排的新型合成工具的预期发展可能对分子生物学领域产生重大影响，但这项研究的第一个主要影响将是在一个广泛的新的交叉学科领域对三名独立研究人员(每个人都将是负责三个特定目标之一的负责人)进行科学和专业培训，该领域结合了RNA生物学、DNA重组、表观遗传学、进化、纤毛分子生物学、微生物多样性和基因组学。所有三个具体目标都解决了表观遗传的新机制，进一步发展了Oxytricha作为模式系统。除了培训博士后和博士后，调查员还将指导本科生，这是该项目的核心活动。所有普林斯顿大学的本科生都要完成高强度的初级和高级独立工作，这通常会导致发表论文，而PI在招募女性和少数族裔进入她的实验室和普林斯顿的生态学和进化生物学研究生项目方面有着很好的记录。高中志愿者也将被邀请参加暑期的研究，指导年轻学生的机会将为高级实验室成员提供关键经验，培训其他人进行实验室研究。研究人员还非常积极地开发一种新的出版模式，作为Biology Direct的联合主编，它提供开放的、签署的和发表的同行审查和作者回应，以及开放获取的读者，所有这些都鼓励公开的科学辩论。BIONICAL-DIRECT.com的透明性也提供了一种新的工具，让学生了解发表作品所涉及的步骤。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5). RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa. The PI's lab has shown that maternal RNA templates provide both an organizing guide for DNA rearrangements in Oxytricha and a template that can transmit spontaneous mutations that may arise during somatic growth to the next generation. This opportunity for RNA-guided DNA repair is profound in the ciliate Oxytricha, which deletes 95% of its germline genome through global DNA rearrangements that severely fragment its chromosomes, and then sorts and reorders the hundreds of thousands of pieces remaining. The programmed information for reordering comes from transiently-expressed maternal RNAs. This means that changes that arise in the maternal somatic genome, whether at the level of point mutations or new DNA rearrangement patterns, have an opportunity to be passed on to the next generation, bypassing the usual mode of inheritance via germline DNA. Furthermore, the occasional transfer of point mutations in these RNA templates to the F1 generation provides a mechanism for stable inheritance of acquired, spontaneous somatic substitutions, without altering the germline genome. This suggests the somatic macronuclear genome is really an "epigenome", formed through templates and signals arising from the previous generation. The research will address 3 specific aims, which will yield detailed insight in Oxytricha into the following:1) Epigenetic inheritance of nucleotide substitutions via RNA templates, by exploring possible polymerases involved in RNA-guided DNA repair;2) Epigenetic inheritance of intron loss via programmed DNA deletion, which would contribute to developmental streamlining of a somatic genome;3) Epigenetic inheritance of copy-number variation: The third specific aim will explore an additional newly-discovered role for maternal template RNAs in regulating DNA copy number. This provides a heritable non-Mendelian mechanism for adaptation to the environment.Broader impacts: While the study of RNA templates and their expected development as novel synthetic tools to drive genome rearrangement are likely to have a great impact on the field of molecular biology, the first major impact of this research will be the scientific and professional training of three independent researchers (each will be the lead individual responsible for one of the three specific aims) in a broad new interdisciplinary area that combines RNA biology, DNA recombination, epigenetics, evolution, ciliate molecular biology, microbial diversity and genomics. All three specific aims address novel mechanisms for epigenetic inheritance, further developing Oxytricha as a model system. In addition to the training of doctoral and postdoctoral students, the investigator will also mentor undergraduates, an activity central to this project. All Princeton undergraduates perform intensive junior and senior independent work, often leading to publications, and the PI has a strong record of recruiting women and minorities to her lab and to Princeton's graduate program in Ecology and Evolutionary Biology. High school volunteers will also be invited to participate in research during the summer, and the opportunity to mentor younger students will provide the senior lab members with critical experience in training others to perform laboratory research. The investigator is also very active in developing a new model of publishing, as co-Editor-in-Chief of Biology Direct, which offers open, signed and published peer review and author responses, plus open-access readership, all of which encourage open scientific debate. The transparency of biology-direct.com also provides a novel vehicle for teaching students about the steps involved in publishing their work.