All eukaryotic cells depend on faithful cell division to give rise to daughter cells that contain the correct number of chromosomes. Chromosome segregation errors during meiosis in germ cells can lead to aneuploid gametes, which can cause infertility, genetic disorders such as Down Syndrome and miscarriages. Additionally, chromosome segregation errors during mitosis can lead to aneuploid somatic cells, which are a hallmark of cancer. Despite the importance of tightly regulating these two types of cell division, the mechanisms that ensure fidelity during these processes remain active areas of investigation. Proteins that have been shown to be important regulators of fidelity during meiosis in Caenorhabditis elegans (C. elegans) include ZHP-3 and PCH-2. ZHP-3 is a putative SUMO or ubiquitin ligase required for crossover recombination, but whether or not ZHP-3 is regulated to promote recombination remains poorly understood. PCH-2 is a conserved AAA+ ATPase required for timely coordination of the meiotic prophase events pairing, synapsis and recombination, but the mechanism for how it accomplishes this coordination is currently unknown. Additionally, PCH-2 has been shown to be required for both silencing and activation of the spindle assembly checkpoint during mitosis, but how it regulates the checkpoint is still an active area of investigation. Here, I use C. elegans as a model system to investigate the molecular mechanisms of these two proteins during meiotic and mitotic chromosome segregation. In Chapter 1, I investigate whether ZHP-3 is phosphorylated by a conserved DNA damage kinase, CHK-1, to determine if this phosphorylation event is required for ZHP-3’s localization or function during meiosis. I find that while ZHP-3 is phosphorylated by CHK-1 kinase in vitro, mutation of these phosphorylation sites in vivo does not affect ZHP-3’s localization to meiotic chromosomes, or its function in promoting crossover recombination. In Chapter 2, I investigate a different regulator of meiosis, PCH-2, to determine how it coordinates meiotic prophase events and whether it acts on meiotic HORMADS to accomplish this. I find that mutations in PCH-2 genetically interact with mutations in the meiotic HORMADS HTP-3 and HIM-3, suggesting that these proteins work together to coordinate meiotic prophase events. Additionally, I find that PCH-2 acts on HTP-3 to coordinate pairing and synapsis, but acts on HIM-3 during recombination, indicating that the meiotic HORMADS are cleanly delegated to regulate specific meiotic prophase events. Finally, in Chapter 3, I explore PCH-2’s role during somatic cell division and investigate how PCH-2 regulates the strength of the spindle assembly checkpoint. I find that PCH-2 is more enriched in P1 cells compared to AB cells in the early C. elegans embryo, potentially explaining why cells that give rise to the germline have stronger checkpoints than somatic cells. Additionally, we find that this enrichment depends on cell fate factors and PCH-2’s adaptor protein CMT-1. Taken together, these results contribute mechanistic insight for how these proteins promote fidelity during meiosis and mitosis by determining post-translational modifications of meiotic recombination factors (Chapter 1), identifying potential substrates of meiotic prophase regulators (Chapter 2), and demonstrating how spindle checkpoint regulators are differentially inherited in specific cell types to give rise to differences in spindle checkpoint strength (Chapter 3).

Diversity in the animal kingdom is established through mutations, gene flow, and sexual reproduction. During gamete production, genetic material is exchanged between maternal and paternal chromosomes via a process called crossover (CO) recombination. In C. elegans, recombination between homologs is essential for proper segregation during anaphase I. During this process, double strand breaks are enzymatically induced via SPO-11 and repaired through the CO pathway. Thus, loss of CO promoting factors, such as MSH-4, MSH-5 and ZHP-3, should lead to persistent DNA damage and an increase in activation of DNA damage induced apoptosis, a process dependent on the conserved apoptosis activator, EGL-1. However, Silva et al showed that loss of these CO-promoting factors fails to activate DNA damage induced apoptosis (32). Further, these factors are hypothesized to be required for initiation of the DNA-damage checkpoint since apoptosis fails to activate in msh-4, msh-5, and zhp-3 mutants after IR radiation. We present an alternate explanation of these results: When a conserved upstream regulatory region of egl-1 is mutated, msh-5 and zhp-3 mutants exhibit an increase in apoptosis dependent on the DNA damage checkpoint proteins, HUS-1 and CEP-1. Also, a feedback mechanism involving PCH-2, a AAA+ ATPase that is activated by defects in recombination is not required for DDR dependent apoptotic regulation. This is in contrast to the requirement of PCH-2 for synaptic mutants to activate germline apoptosis. Based on these results, we hypothesized that when CO recombination is specifically disrupted, DNA damage induced apoptosis is inhibited, potentially to favor repair over removal. Our goal was to understand the regulation of apoptosis during the process of CO recombination and to identify a novel regulatory factor(s) that interacts with an egl-1 regulatory region (bc373) to limit apoptosis when CO recombination is defective. We tested a variety of factors including NHR-6, NHR-72, NHR-91, and screened 98 oogenic transcription factors.

All sexually reproducing organisms rely on the events of meiosis, the cell division that produces gametes such as sperm and eggs. In order to ensure a proper chromosome complement in successive generations and to promote genetic diversity, meiotic chromosomes will align with their counterparts, synapse, and undergo recombination, the means of genetic exchange. Failure to accurately complete these steps during meiosis results in devastating consequences for progeny.

The mechanisms that execute and monitor the genetic interactions in meiosis are an exciting area of study in the field. In particular, understanding the importance of chromatin localization and dissecting the interactions of chromatin and various associated meiotic proteins are critical to our understanding of meiosis. Additionally, checkpoints exist that monitor proper completion of meiotic events, but the control of these checkpoints is not fully understood.

Using a variety of cytological and genetic techniques in C. elegans, the work here addresses the importance of perinuclear localization of chromatin during meiosis. A class of nuclear membrane-associated proteins called the LEM proteins are involved in perinuclear tethering of chromatin. The three LEM proteins in C. elegans have differential effects on meiotic fidelity. Loss of one of these proteins, LEM-2, causes impaired repair and processing of double-stranded DNA breaks, which are necessary for recombination. It also induces meiotic errors that result in elevated levels of cell death in the germline. Loss of another one of these proteins, EMR-1, also affects meiotic DNA repair but less severely than LEM-2. Finally, LEM-3 appears to be required for a meiotic checkpoint that monitors synapsis.

This thesis also provides insight into the regulatory mechanisms that trigger germ cell death when errors in meiosis activate checkpoints. A conserved pro-apoptotic protein, EGL-1, is necessary for the two checkpoints to induce apoptosis, but its requirement for each checkpoint depends on the manner in which they are activated. Experiments also reveal that CED-13, a largely uncharacterized protein with homology to EGL-1, also has a role in meiotic checkpoints. Moreover, my studies indicate that recombination negatively regulates egl-1 mediated germline apoptosis, potentially as a mechanism to promote repair over removal of defective meiotic nuclei.

Accurate chromosome segregation in meiosis and mitosis is essential for avoiding aneuploidy, a hallmark of cancer cells. In meiosis, proper chromosome segregation relies upon the events of meiotic prophase: pairing, synapsis, and recombination between homologous chromosomes. After meiotic recombination, chromosome architecture must be remodeled to form bivalents, the structures that promote proper homolog portioning at meiosis I. How recombination is coupled to chromosome remodeling remains unclear. Here, we show that the conserved ZHP-3 protein, required for crossover formation, has ubiquitin ligase activity in vitro, indicating it may serve to coordinate meiotic recombination with changes in chromosome architecture. Furthermore, we identify ZHP-3 as a substrate of both the MPK-1 (MAP) and CHK-1 kinases in vitro. MAP kinase integrates developmental processes with meiotic chromosome dynamics—it may phosphorylate ZHP-3 to coordinate and regulate these events. Finally, in mitosis, the spindle checkpoint regulates the fidelity of mitotic segregation by delaying the onset of anaphase until all chromosomes are properly attached to the mitotic spindle. Here, we identify PCH-2Trip13 as a novel component of the spindle checkpoint in C. elegans. We show that through the CMT-1p31comet protein, PCH-2Trip13 regulates the amount of Mad2 that localizes to mitotic chromosomes, suggesting that PCH-2Trip13 may regulate the strength or robustness of the spindle checkpoint response. Furthermore, we show that the requirement for PCH-2Trip13 in MAD-2 recruitment to kinetochores is conserved in human epithelial RPE1 cells. Together, these data help to elucidate the mechanisms by which multiple cell types ensure proper chromosome segregation in both