RUI: Defining Structural Features of C. elegans histone H2A Variants Important for Transcriptional Regulation in Different Cell Types

智力优势：不同类型的细胞的发育和功能依赖于不同组基因的表达。改变不同基因集合的可及性是基因调控的一种方式。基因的可及性可以依赖于将组蛋白H2A变体整合到压缩DNA的核小体中。例如，线虫表达的H2A变体包括进化上保守的H2A.Z(HTZ-1)和精子特异性的HTAS-1。该项目将测试这一假设，即HTZ-1和HTAS-1的结构差异会改变DNA的可及性，从而以细胞类型特有的方式调节基因表达。首先，生物物理方法将定义线虫H2A蛋白的结构特性。其次，荧光共振能量转移(FRET)和生化方法将识别含有H2A、HTZ-1和HTAS-1的核小体在稳定性、DNA解缠和包装方面的差异。在基因突变体中进行的细胞学分析将关联H_2A变异体的掺入如何影响基因表达。在体外和体内用H2A交换HTZ-1和HTAS-1的结构域将确定这些H2 A变体的结构域对于它们的掺入和在不同细胞类型的转录中的作用是重要的。总体而言，这些研究将阐明线虫组蛋白H2A变体的结构特性，这些特性有助于指定不同的细胞类型。更广泛的影响：这项研究将在旧金山州立大学进行，这是一所历史上一直为少数族裔服务的公立机构。生物化学和生物化学专业的本科生和硕士学生将在生物化学、生物物理学和分子遗传学方法学的理论和实践方面接受PI及其在旧金山州立大学和加州大学旧金山分校的合作者的培训，以了解DNA组织和转录调控的基本方面。学生们将分组工作，分享资源、想法和专业知识。在PI的监督下，学生将学会批判性地评估科学研究，计划实验，并解释实验数据，成为独立的批判性思考者。拟议工作提供的跨学科培训和指导将为推进旧金山州立大学不同学生在科学方面的教育和职业生涯提供坚实的基础。

Intellectual Merit: Different types of cells rely on the expression of distinct sets of genes for their development and function. Altering the accessibility of different sets of genes is one way genes can be regulated. The accessibility of genes can rely on incorporating histone H2A variants into the nucleosomes that compact DNA. For example, C. elegans express H2A variants that include the evolutionarily conserved H2A.Z (HTZ-1) and the sperm-specific HTAS-1. This project will test the hypothesis that structural differences in HTZ-1 and HTAS-1 alter DNA accessibility to regulate gene expression in a cell-type specific manner. First, biophysical approaches will define structural properties of C. elegans H2A proteins. Second, fluorescence resonance energy transfer (FRET) and biochemical approaches will identify differences in the stability, DNA unwrapping, and packing of nucleosomes containing H2A, HTZ-1, and HTAS-1. Cytological assays conducted in genetic mutants will correlate how H2A variant incorporation affects gene expression. Swapping domains of HTZ-1 and HTAS-1 with H2A in vitro and in vivo will identify structural domains of these H2A variants that are important for their incorporation and roles in transcription in distinct cell types. Overall, these studies will elucidate the structural properties of C. elegans histone H2A variants that contribute to the specification of different cell types. Broader Impacts: The research will be conducted at San Francisco State University, a historically minority-serving public institution. Undergraduate and master's students from the Biology and Chemistry and Biochemistry programs will be trained by the PI and her collaborators at SFSU and UCSF in the theory and practice of biochemical, biophysical, and molecular genetic methodology to understand fundamental aspects of DNA organization and transcriptional regulation. Students will work in cohorts to share resources, ideas, and expertise. Under the supervision of the PI, students will learn to critically evaluate scientific research, plan experiments, and interpret experimental data to become independent critical thinkers. The interdisciplinary training and mentorship provided by the proposed work will provide a strong foundation to advance the education and careers of SFSU's diverse students in the sciences.