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

研究一种精子特异性组蛋白变异在秀丽隐杆线虫生殖能力重要的转录调控中的机制，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。多能生殖细胞向可移动精子的分化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，使精子DNA紧密压缩。在初步研究中，第一个已知的秀丽隐杆线虫精子核基本蛋白，一种名为HTAS-1的组蛋白H2A变体被鉴定出来，并被发现是最佳生育能力所必需的。DNA微阵列分析的初步数据表明，与野生型动物相比，htas-1突变体中存在激活和抑制基因。引人注目的是，在htas-1突变体中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来形成生殖细胞。因此，这个项目的中心假设是HTAS-1的结合调节了基因的表达，这些基因在精子中协同工作以达到最佳生育能力。为了验证这一点，将评估生殖细胞的进展、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突变体的生育能力下降的。为了确定HTAS-1转录调控的直接靶点，Chu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结合的位点。为了阐明HTAS-1如何激活和抑制转录，将使用质谱法开始表征HTAS-1上的PTMs，以了解HTAS-1的功能如何在基因靶点上被调节。这个CAREER项目的研究意义重大，因为它阐明了通过结合精子特异性组蛋白变体的转录调节机制，该变体在保守的发育过程中起作用，即精子产生。该项目将定义进化保守机制，以调节DNA包装的变化如何改变对精子形成重要的基因的表达。它具有重要而广泛的影响，因为它有助于旧金山州立大学（San Francisco State University）这个为少数族裔服务的本科院校中未被充分代表的少数族裔和女性学生的职业发展。为了提高多元文化学生的基础写作和科学写作技能，朱博士将与旧金山州立大学英语系和学习援助中心合作，开发一门科学提案写作研究生课程，并与基础写作技能辅导相协调。因此，该项目整合了学生参与染色质生物学和生殖的基础研究，并为促进他们在科学方面的长期专业发展提供了资源。

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.