Meiotic prophase regulation and achiasmate chromosome segregation in Caenorhabditis elegans By Christina Marie Glazier A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology in the Graduate Division of the University of California, Berkeley Committee in Charge: Professor Abby Dernburg, Chair Professor Georjana Barnes Professor Rebecca Heald Professor Marvalee Wake Spring 2015 Abstract Meiotic prophase regulation and achiasmate chromosome segregation in Caenorhabditis elegans by Christina Marie Glazier Doctor of Philosophy in Molecular and Cell Biology University of California, Berkeley Professor Abby Dernburg, Chair Meiosis is the specialized cell division by which sexually reproducing organisms produce haploid gametes. In order to reduce the chromosome complement by half, chromosomes must undergo pairing, synapsis, and crossover formation, followed by two rounds of chromosome division. All of these mechanisms depend on the proper establishment, maintenance, and remodeling of sister chromatid cohesion. Sister chromatid cohesion is mediated by cohesin complexes, which associate with newly replicated sister chromatids. Cohesion is required for all major aspects of meiosis: formation of the synaptonemal complex, induction of DNA double- strand breaks, and the repair of breaks to form crossovers, in addition to the regulated release of cohesion to enable segregation. During meiosis, cohesin complexes incorporate specialized subunits and are subject to different regulation, compared to mitotically dividing cells. The functions and regulation of cohesion during meiosis remain poorly characterized, and a major goal of my thesis work has been to address these important questions. Wapl is a widely conserved regulator of cohesin. It has been implicated in antagonizing the association of cohesin complexes with DNA to facilitate cohesin removal during mitosis. However, its role in meiotic chromosome dynamics has not been investigated in any detail. To better understand the roles and regulation of sister chromatid cohesion in meiosis, I have focused on the Caenorhabditis elegans Wapl homolog, WAPL-1. I found that C. elegans WAPL-1 promotes faithful mitotic chromosome segregation, as in other organisms. I also found that WAPL-1 affects cohesin dynamics during meiosis, contributes to DNA double-strand break repair, and is regulated by the meiosis-specific kinase, CHK-2. A second component of my thesis work examines the behavior of achiasmate chromosomes during meiosis. When early meiotic events fail to establish the requisite crossovers, the resulting achiasmate chromosomes often missegregate, or nondisjoin. Chromosome nondisjunction can result in aneuploid gametes, which has disastrous consequences for the developing embryo. To accurately detect autosomal nondisjunction in single embryos, I developed a fragment length polymorphism (FLP) assay. I used this approach to analyze chromosome segregation in oocyte meiosis in wildtype animals at different ages, and in mutants with elevated frequencies of achiasmate chromosomes. I found that nondisjunction occurred asymmetrically, yielding a higher frequency of monosomic than trisomic embryos. I also found evidence that germline apoptosis protects C. elegans hermaphrodites from increased nondisjunction as they age. Together, the results of these studies further illuminate meiotic prophase regulation and achiasmate chromosome segregation. 1 . Dedicated to those who imparted on me four pieces of advice required for the successful completion of this dissertation. To my father, who, through his own actions and stories of my Nana and Papa, reminds me to work hard. To my mother, who teaches me to stand up for others, as well as myself. To my sweet brother, who reminds me to be kind. And to my Lola and Lolo, who taught me that happiness is sadness. i Table of Contents Chapter 1: Introduction ............................................................................................................... 1 1.1 Meiosis, a specialized cell division ....................................................................................... 1 1.2 Early meiotic processes ensure proper chromosome segregation ......................................... 2 1.3 Chromosome nondisjunction and its affects on human health ............................................. 8 1.4 The model organism Caenorhabditis elegans ....................................................................... 9 Chapter 2: WAPL-1 is regulated by the meiotic kinase, CHK-2, during meiotic prophase 13 2.1 Introduction and summary of results .................................................................................. 13 2.2 Results ................................................................................................................................. 14 2.3 Discussion ........................................................................................................................... 23 2.4 Materials and Methods ........................................................................................................ 25 Chapter 3: Achiasmate chromosome segregation .................................................................... 39 3.1 Introduction and summary of results .................................................................................. 39 3.2 Results ................................................................................................................................. 41 3.3 Discussion ........................................................................................................................... 49 3.4 Materials and Methods ........................................................................................................ 51 Chapter 4: Concluding remarks and future perspectives ....................................................... 57 4.1 Review of findings .............................................................................................................. 57 4.2 Implication of findings ........................................................................................................ 59 4.3 Remaining questions ........................................................................................................... 60 References .................................................................................................................................... 63 Appendix A C. elegans strains and plasmids ............................................................................ 68 Appendix B Mass spectrometry data tables ............................................................................. 70 ii List of Tables Table 1 List of C. elegans strains. ................................................................................................ 68 Table 2 List of plasmids. .............................................................................................................. 69 Table 3 C. elegans proteins identified by mass spectrometry following immunoprecipitation of endogenous WAPL-1. ........................................................................................................... 70 Table 4 List of phosphorylated amino acids identified by mass spectrometry following in vitro phosphorylation of recombinant WAPL-1. ........................................................................... 82 Table 5 List of phosphorylated amino acids identified by mass spectrometry following purification of WAPL-1 from wildtype C. elegans lysate .................................................. 104 iii List of Figures Figure 1.1 Schematic of restoration of diploidy through sexual reproduction and early meiotic events .................................................................................................................................... 11 Figure 1.2 Schematic of chromosome nondisjunction at Meiosis I ............................................. 12 Figure 2.1 The WAPL-1 isoform A is the primary transcribed and translated isoform .............. 29 Figure 2.2 WAPL-1 localizes to mitotically dividing nuclei by immunofluorescence ............... 30 Figure 2.3 WAPL-1 is present during meiotic prophase, but sensitive to immunofluorescence protocol ................................................................................................................................. 31 Figure 2.4 wapl-1 mutants display phenotypes associated with mitotic defects ......................... 32 Figure 2.5 Immunoprecipitation of WAPL-1 identifies mitotic cohesin complex proteins and DNA repair proteins as WAPL-1 interactors ........................................................................ 33 Figure 2.6 wapl-1 mutants display defects in DNA DSB repair .................................................. 34 Figure 2.7 wapl-1 mutants display defects in inhibition of SCC-1 .............................................. 35 Figure 2.8 A candidate screen identified CHK-2 as a WAPL-1 regulator .................................. 36 Figure 2.9 Misregulation of WAPL-1 in chk-2 causes defects in COH-3/4 loading ................... 37 Figure 2.10 Mutation of WAPL-1 CHK-2 consensus motifs reduces WAPL-1 phosphorylation in vitro, but does not affect WAPL-1 regulation in the germline ......................................... 38 Figure 3.1 Schematic of fragment length polymorphism protocol and mating scheme .............. 53 Figure 3.2 The fragment length polymorphism assay assesses autosomal nondisjunction