CAREER: Investigating Mechanisms of Histone Variant Function and Regulation that Affect Transcriptional Control and fertility in C. Elegans

职业：研究影响线虫转录控制和生育能力的组蛋白变异功能和调节机制

• 批准号: 0747515
• 负责人: Diana Chu
• 金额: $ 65.5万
• 依托单位: San Francisco State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2013-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0747515&HistoricalAwards=false
• 关键词: CAREER; Investigating; Mechanisms; Histone; Variant

Investigating mechanisms of a sperm-specific histone variant in transcriptional regulation important for fertility in C. elegans sperm are formed during a period where extreme changes in chromatin architecture are coupled with dynamic shifts in gene expression. Differentiation of pluripotent germ cells into mobile sperm requires transcriptional regulation of specific genes. Simultaneously, sperm nuclear basic proteins, such as histone variants, replace somatic histones to achieve tightly compacted sperm DNA. In preliminary studies, the first known C. elegans sperm nuclear basic protein, a histone H2A variant called HTAS-1, was identified and found to be required for optimal fertility. Preliminary data from DNA microarray analyses identified both activated and repressed genes in htas-1 mutants compared with wild-type animals. Strikingly, genes that are activated in htas-1 mutants function in forming germ cells via gamete formation progression, oocyte maturation, ovulation, sperm signaling, and sperm migratory behavior. Therefore, the central hypothesis of this project is that HTAS-1 incorporation modulates the expression of genes that work in concert for optimal fertility in sperm. To test this, germ cell progression, oocyte development, and sperm behavior will be assessed to determine how each contributes to reduced fertility in htas-1 mutants. To identify direct targets of HTAS-1 transcriptional regulation, Dr. Chu and her colleagues will define sites of HTAS-1 incorporation during sperm development using chromatin immunoprecipitation and DNA microarray analysis. To elucidate how HTAS-1 both activates and represses transcription, characterization of PTMs on HTAS-1 will be initiated using mass spectrometry to understand how HTAS-1 function is regulated at gene targets. The research in this CAREER project is significant because it elucidates mechanisms of transcriptional regulation through incorporation of a sperm-specific histone variant that functions in a conserved developmental process, sperm production.The project will define evolutionarily conserved mechanisms that regulate how changes in DNA packaging alter the expression of genes important for sperm formation. It has important and broad impact because it contributes to the career development of underrepresented minority and female students at San Francisco State University, an undergraduate minority-serving institution. To boost both basic and scientific writing skills in multicultural students, Dr. Chu will develop a graduate course in scientific proposal writing that is coordinated with tutoring in basic writing skills through collaboration with the English Department and Learning Assistance Center at SFSU. Thus the project integrates the participation of students into basic research on chromatin biology and reproduction and also provides resources to help promote their long-term professional development in science.

研究精子特异性组蛋白变异体在C.秀丽线虫精子是在染色质结构的极端变化与基因表达的动态变化相结合的时期形成的。多能生殖细胞分化为移动的精子需要特定基因的转录调控。同时，精子核碱性蛋白，如组蛋白变体，取代体细胞组蛋白，以获得紧密压缩的精子DNA。在初步研究中，第一个已知的C。鉴定了秀丽线虫精子核碱性蛋白，一种称为HTAS-1的组蛋白H2 A变体，并发现其是最佳生育力所需的。从DNA微阵列分析的初步数据确定了激活和抑制基因的htas-1突变体相比，野生型动物。引人注目的是，htas-1突变体中被激活的基因通过配子形成过程、卵母细胞成熟、排卵、精子信号传导和精子迁移行为在生殖细胞的形成中发挥作用。因此，该项目的中心假设是HTAS-1掺入调节基因的表达，这些基因共同作用以获得精子中的最佳生育力。为了验证这一点，我们将评估生殖细胞的发育、卵母细胞的发育和精子的行为，以确定它们是如何导致htas-1突变体生育力下降的。为了确定HTAS-1转录调控的直接靶点，朱博士和她的同事将使用染色质免疫沉淀和DNA微阵列分析来确定精子发育过程中HTAS-1掺入的位点。为了阐明HTAS-1如何激活和抑制转录，将使用质谱法开始对HTAS-1上的PTM进行表征，以了解HTAS-1功能如何在基因靶点进行调节。本CAREER项目的研究意义重大，因为它阐明了转录调控机制，通过掺入精子特异性组蛋白变体，在一个保守的发育过程中发挥作用，精子产生。该项目将定义进化上保守的机制，调节DNA包装的变化如何改变对精子形成重要的基因的表达。它具有重要和广泛的影响，因为它有助于代表性不足的少数民族和女性学生在旧金山弗朗西斯科州立大学，本科少数民族服务机构的职业发展。为了提高多元文化学生的基本和科学写作技能，朱博士将开发一个科学提案写作的研究生课程，通过与SFSU的英语系和学习辅助中心合作，与基本写作技能的辅导相协调。因此，该项目将学生的参与融入染色质生物学和生殖的基础研究，并提供资源，帮助促进他们在科学方面的长期专业发展。