Meiotic chromosome segregation in C. elegans : discovering a new look for CENP-A

In this dissertation, I use the nematode, Caenorhabditis elegans, to understand mechanisms involved in chromosome segregation during meiosis, by exploring the role that known mitotic kinetochore proteins play during meiotic segregation. During mitosis, the histone-H3 variant CENP-A is known to be the nucleosomal subunit at the centromere that is responsible for directing kinetochore assembly. In cells lacking CENP-A, chromosomes are unable to segregate leading to aneuploidy and cell-death. Interestingly, I demonstrate that during meiosis CeCENP-A no longer directs outer kinetochore assembly. Furthermore, in embryos depleted of CeCENP-A, meiotic chromosome segregation appears completely normal, whereas subsequent mitotic divisions completely fail. Outer kinetochore components localize to a cup-like structure, which likely is involved in aligning chromosomes at the metaphase plate. I speculate that this new mechanism for chromosome segregation in meiosis may be a requirement to facilitate proper segregation of recombined chromosome pairs. During these meiotic studies, I discovered a striking CeCENP-A cleavage event, which I spend the remainder of this dissertation describing, and postulate that this cleavage event may be involved in maintenance of the centromere during mitotic divisions. Centromere specification is thought to be propagated by an epigenetic mark produced by CENP-A. The mechanism for how CENP-A achieves this mark is unknown. In a variety of studies presented here, I show that CeCENP-A is a substrate for Separase-mediated cleavage and discuss its possible implications on maintaining the epigenetic mark. This cleavage event is best demonstrated during meiosis in embryos expressing N- terminally tagged GFP::CeCENP-A. During mitosis, I show that Separase is unable to cleave centromeric CeCENP-A under wild-type conditions. However, in the absence of the kinetochore, centromeric CeCENP-A is cleaved, indicating that the kinetochore protects CeCENP-A while non- centromeric CeCENP-A is susceptible to cleavage. Worms expressing an uncleavable mutant form of CeCENP-A show an increase in embryonic lethality, and worms solely expressing a pre-cleaved form of CeCENP-A completely lose CeCENP-A localization and function, resulting in complete embryonic lethality. These data suggest that CeCENP-A cleavage may be a way to inactivate CeCENP-A loading. I propose that cleavage of improperly loaded CeCENP-A onto chromosome arms may be a mechanism used by the cell to ensure that the epigenetic mark for CeCENP-A loading remains strictly at the centromer