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

该项目的中心目标是更好地了解确保活细胞内的遗传物质得到适当和有效利用的分子机制。遗传物质的正确利用对生命的方方面面都至关重要，从单细胞有机体对周围环境的感知和反应能力，到促进受精卵发育为新生儿的过程。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。该项目将包括大量本科生作为关键的科学贡献者参与。12名学生将在国际和平研究所的实验室进行实验：这些学生将在科学过程的各个方面接受国际和平研究所的指导，包括实验设计和实施、在科学会议上展示研究成果以及对科学文献进行批判性评估。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。大约另外100名学生将在实验室课程的背景下开展这一项目的一部分：这些学生将学习尖端实验方法，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。在真核细胞中，大多数DNA驻留在细胞核内，通过与一些蛋白质结合而高度致密，形成一种称为染色质的结构，其基本单位被称为核小体。虽然核小体对细胞至关重要，但它们也是细胞因子的物理障碍，细胞因子的功能需要获得DNA。在基因转录过程中，一种称为FACT的复合体被招募到基因中，以帮助核小体的分解，以促进RNA聚合酶II(Pol II)的转录，以及在Pol II通过后促进核小体的重新组装。之前PI的实验室使用酿酒酵母模型系统发现了核小体一侧的一个区域，称为ISGI区域，转录后需要从几个基因中适当地分离出事实。在这个项目中，PI的团队将使用全基因组的方法来研究ISGI区域在控制整个酵母基因组中的事实-染色质相互作用以及在促进其他染色体过程中的作用。还将进行实验，以识别和表征影响FACT与基因相互作用能力的其他核小体区域和蛋白质。最后，将使用基于CRISPR的方法同时识别帮助POL II消除其路径上的非核小体障碍的蛋白质，并识别促进Cas9-gRNA定位到目标基因组位置的因素。由于这个项目中研究的因素和过程在进化过程中是高度保守的，这项研究的发现将为所有真核生物，包括人类的相关过程提供新的线索。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.