RUI: Investigations on yFACT-genome interactions and other chromatin processes

RUI：yFACT-基因组相互作用和其他染色质过程的研究

• 批准号: 2015806
• 负责人: Andrea Duina
• 金额: $ 48万
• 依托单位: Hendrix College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2015806&HistoricalAwards=false
• 关键词: RUI; Investigations; yFACT; genome; interactions

The central goal of this project is to provide a better understanding of the molecular mechanisms that ensure that the genetic material within living cells is used properly and efficiently. Proper utilization of genetic material is crucial for all aspects of life, ranging from the ability of single-celled organisms to sense and respond to their surroundings to processes that promote the development of a newborn human being from a fertilized egg. Thus, this research will contribute to our understanding of fundamental questions of widespread biological importance. The project will include participation of a large number of undergraduate students as key scientific contributors. Twelve students will be carrying out experiments in the PI’s laboratory: these students will be mentored by the PI in various aspects of the scientific process, including experimental design and execution, presentation of research at scientific meetings, and critical evaluation of scientific literature. These students will also have the opportunity to develop skills required for effective communication of scientific concepts to the general public. Approximately 100 additional students will carry out part of this project in the context of laboratory courses: these students will learn cutting edge experimental approaches and will be engaged in discussions related to the uses and ethical considerations of current gene editing technologies. Within eukaryotic cells, most of the DNA resides within the cell nucleus and is highly compacted through association with a number of proteins to form a structure known as chromatin, the basic unit of which is referred to as the nucleosome. Whereas nucleosomes are of critical importance to cells, they also represent physical obstacles to cellular factors whose functions require access to DNA. During gene transcription, a complex known as FACT is recruited to genes to assist with the disassembly of nucleosomes to facilitate RNA Polymerase II (Pol II) transcription, as well as to promote nucleosome re-assembly in the wake of Pol II passage. Previous work from the PI’s laboratory using the Saccharomyces cerevisiae model system identified a region on the side of the nucleosome, referred to as the ISGI region, required for proper dissociation of FACT from several genes following transcription. In this project, the PI’s team will use genome-wide approaches to study the role of the ISGI region in controlling FACT-chromatin interactions across the entire yeast genome and in promoting other chromosomal processes. Experiments will also be carried out to identify and characterize additional nucleosomal regions and proteins that influence FACT’s ability to interact with genes. Finally, a CRISPR-based approach will be used to simultaneously identify proteins that assist Pol II in removing non-nucleosomal obstacles in its path and to identify factors that promote Cas9-gRNA localization to target genomic sites. Since the factors and processes studied in this project are highly conserved across evolution, findings stemming from this research will shed new light on processes relevant to all eukaryotes, including humans.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

该项目的中心目标是更好地了解分子机制，以确保活细胞内的遗传物质得到正确和有效的利用。 适当利用遗传物质对生命的所有方面都至关重要，从单细胞生物体感知和应对环境的能力到促进受精卵发育成新生儿的过程。 因此，这项研究将有助于我们理解具有广泛生物学意义的基本问题。 该项目将包括大量的本科生作为关键的科学贡献者的参与。 12名学生将在PI的实验室进行实验：这些学生将在科学过程的各个方面得到PI的指导，包括实验设计和执行，在科学会议上介绍研究，以及对科学文献的批判性评价。 这些学生还将有机会发展向公众有效传播科学概念所需的技能。 另外约100名学生将在实验室课程中开展该项目的一部分：这些学生将学习最先进的实验方法，并将参与与当前基因编辑技术的使用和伦理考虑有关的讨论。 在真核细胞内，大部分DNA驻留在细胞核内，并通过与许多蛋白质结合而高度压缩，形成称为染色质的结构，其基本单位称为核小体。 虽然核小体对细胞至关重要，但它们也代表了细胞因子的物理障碍，细胞因子的功能需要接近DNA。 在基因转录过程中，一种称为FACT的复合物被募集到基因中，以帮助核小体的拆卸，从而促进RNA聚合酶II（Pol II）转录，以及在Pol II通过后促进核小体重新组装。 PI实验室之前使用酿酒酵母模型系统进行的工作确定了核小体一侧的一个区域，称为ISGI区域，该区域是转录后FACT与多个基因正确解离所需的。 在这个项目中，PI的团队将使用全基因组方法来研究ISGI区域在控制整个酵母基因组中的FACT-chromatin相互作用和促进其他染色体过程中的作用。 还将进行实验，以确定和表征影响FACT与基因相互作用能力的其他核小体区域和蛋白质。 最后，基于CRISPR的方法将用于同时鉴定有助于Pol II消除其路径中的非核小体障碍的蛋白质，并鉴定促进Cas9-gRNA定位到靶基因组位点的因子。 由于该项目中研究的因素和过程在进化过程中高度保守，因此该研究的发现将为包括人类在内的所有真核生物的相关过程提供新的线索。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。