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

研究精子特异性组蛋白变体在转录调控中对线虫精子生育能力至关重要的机制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。将多能生殖细胞分化为活动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，获得紧密致密的精子DNA。在初步研究中，发现了第一个已知的线虫精子核碱性蛋白，一种称为hTAS-1的组蛋白过氧化氢变异体，并发现它是实现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突变体中既有激活基因，也有抑制基因。值得注意的是，HTAS-1突变体中被激活的基因通过配子形成进程、卵母细胞成熟、排卵、精子信号传递和精子迁移行为来发挥形成生殖细胞的功能。因此，该项目的中心假设是，hTAS-1的掺入调节了协同工作的基因的表达，从而实现了精子的最佳受精率。为了测试这一点，将对生殖细胞进展、卵母细胞发育和精子行为进行评估，以确定每种因素如何导致HTAS-1突变体的生育力下降。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的掺入位置。为了阐明HTAS-1是如何激活和抑制转录的，将利用质谱学的方法对HTAS-1上的PTMS进行表征，以了解HTAS-1的功能是如何在基因靶点上调节的。这个职业项目的研究具有重要意义，因为它通过掺入精子特异性组蛋白变体来阐明转录调节机制，组蛋白变体在保守的发育过程-精子产生中发挥作用。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。它具有重要而广泛的影响，因为它有助于旧金山州立大学未被充分代表的少数族裔和女性学生的职业发展，旧金山州立大学是一所为少数族裔服务的本科生机构。为了提高多元文化学生的基本和科学写作技能，朱博士将开发一门科学计划书写作研究生课程，通过与英语系和旧金山州立大学学习辅助中心的合作，协调基本写作技能的辅导。因此，该项目将学生的参与纳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。

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.