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

该项目的中心目标是提供对分子机制的更好理解，以确保活细胞内的遗传物质得到正确有效的利用。遗传物质的适当利用对生命的各个方面都至关重要，从单细胞生物感知和响应周围环境的能力到促进新生儿从受精卵发育的过程。因此，这项研究将有助于我们理解具有广泛生物学重要性的基本问题。该项目将包括大量本科生作为主要科学贡献者的参与。12名学生将在PI的实验室进行实验：这些学生将在PI的科学过程的各个方面得到指导，包括实验设计和执行，在科学会议上的研究报告，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。另外大约100名学生将在实验课程的背景下进行部分项目：这些学生将学习尖端的实验方法，并将参与与当前基因编辑技术的使用和伦理考虑相关的讨论。在真核细胞中，大部分DNA位于细胞核内，并通过与许多蛋白质结合形成称为染色质的结构，其基本单位称为核小体。尽管核小体对细胞至关重要，但它们也代表了需要进入DNA才能发挥功能的细胞因子的物理障碍。在基因转录过程中，一种称为FACT的复合物被招募到基因上，以协助核小体的拆卸，以促进RNA聚合酶II （RNA Polymerase II, Pol II）的转录，并在Pol II通过后促进核小体的重新组装。PI实验室先前使用酿酒酵母模型系统的工作确定了核小体一侧的一个区域，称为ISGI区域，需要在转录后将FACT与几个基因适当分离。在这个项目中，PI的团队将使用全基因组方法来研究ISGI区域在整个酵母基因组中控制fact -染色质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协助Pol II去除其路径中的非核小体障碍的蛋白质和鉴定促进Cas9-gRNA定位到目标基因组位点的因子。由于本项目研究的因素和过程在整个进化过程中都是高度保守的，因此这项研究的结果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。该奖项反映了美国国家科学基金会的法定使命，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。

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.