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.
Sperm and oocytes contribute equal but unique complements of DNA to each new life. At the start of this project, there was a limited understanding in C. elegans regarding how sperm or oocyte specification either coordinates with or modifies the basic underlying process of meiosis to give rise to these disparate cell types. Oogenesis produces large, nutrient-rich, and immobile oocytes that do not complete meiosis until after fertilization. In contrast, spermatogenesis results in small, motile `haploid sperm with highly protected chromatin. Studies funded by this award provided a detailed molecular analysis of key landmark events of spermatogenesis and identified spermatogenesis-specific features of meiosis in the model organism C. elegans. We found that, as in many meiotic programs, C. elegans spermatogenesis includes a chromosome aggregation or "karyosome" phase. This extended stage provides a period for chromosome and microtubule remodeling prior to the meiotic divisions. Our analysis identified several gamete-specific features of the meiotic program that may contribute to the differential timing, pace, and mechanics of meiotic progression. Our findings provide a foundation for understanding how differentiation influences meiosis, which is an essential step in identifying universal features required for reproductive success in all organisms (PLoS Genetics, 2009 Aug;5(8):e1000611). In addition to DNA, embryos also receive parent-specific information from sperm and oocytes that include ‘epigentic information’, like histone variants and post-translationally modified histones. Such information is then reprogrammed in the new embryo to ensure proper development. However, it is unclear how many of these marks are established during gamete formation and reprogrammed after fertilization. We define a signature histone variant and post-translational modification profile of sperm chromatin from C. elegans. The fate of these histone marks was then tracked before and after fertilization using immunocytochemistry. We find the sperm-specific histone H2A variant HTAS-1 specifically marks sperm chromatin during late stages of spermatogenesis. Sperm chromatin repackaging involves departure of both H2A ubiquitinated on lysine 119 (H2AK119ub) and H4 acetylated on lysine 16 (H4K16Ac), features also required for mouse sperm chromatin repackaging. Proteomic analysis identified 25 sites differentially modified between C. elegans sperm and embryos, revealing a wide-spread erasure of histone acetylation as well as a retention of histone methylation at sites that mark the transcriptional history of distinct chromatin domains during spermatogenesis. After fertilization, we show HTAS-1 and 9 histone PTM marks distinguish sperm and oocyte chromatin in the newly fertilized oocyte and characterize distinct paternal and maternal histone remodeling events as oocytes transition to embryos. These include retention of HTAS-1, removal of the H2A variant HTZ-1, and differential reprogramming of histone PTMs. Overall, we identified novel and conserved features of paternal chromatin that are specified during spermatogenesis and processed in the embryo. To understand how histone variants influence chromatin structure, we also embarked on a collaboration with Geeta Narlikar at UCSF to understand how histone variants influence nucleosome stability and DNA interaction in vitro. We cloned C. elegans genes encoding histone H3, H2B, and H4 as well as H2A and 3 H2A variants, including HIS-35, HTAS-1, and HTZ-1 and successfully reconstituted nucleosomes. So far, we find C. elegans nucleosomes composed of canonical histone subtypes are similar to those previously characterized in other model systems. Also, in comparison to canonical H2A, the incorporation of histone H2A variants HTAS-1 and HTZ-1 stabilize nucleosomes. Interestingly, HTZ-1 causes more unwrapping of DNA, while HTAS-1 reduced unwrapping. Thus our studies have the potential to reveal how the incorporation of histone H2A variants may influence local DNA interactions and nucleosome stability. This work provided research training for 14 Master’s (7 under-represented minority-URM) and 10 (6 URM) undergraduate students. They conducted cutting-edge molecular techniques, including cytology, confocal microscopy and proteomic analysis. They also attended national scientific meetings to present and receive input on their work. The training they received has helped them pursue careers in biology, including attending graduate school. Further, we developed the Skills for Scientific Proposal Writing course to teach students to develop a scientific proposal based on their own research. We partnered with the Learning Assistance Center on campus to provide writing tutorials for students to help with basic grammar skills and boost performance of students with remedial writing skills. All students developed important critical thinking skills that enabled them to improve their research as well as obtain funding for their work, earning departmental research awards and fellowships as well as the NSF Graduate Research Fellowship. From Fall 2009-Fall 2012 there have been 125 students (79 female, 46 URM) who have completed this course. The course, in terms of format and collaboration with the LAC, is being considered as a model by the University for how to introduce Writing Across the Disciplines (WAC) at the graduate level. Overall, the diverse students at SFSU are benefiting substantially from both the research and the writing programs initiated by this project.
