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 centromere

Centromeres are defined epigenetically in the majority of eukaryotes by the presence of chromatin containing the centromeric histone H3 variant CENP-A. Most species have a single gene encoding a centromeric histone variant whereas C. elegans has two: HCP-3 (also known as CeCENP-A) and CPAR-1. Prior RNAi replacement experiments showed that HCP-3 is the functionally dominant isoform, consistent with CPAR-1 not being detectable in embryos. GFP::CPAR-1 is loaded onto meiotic chromosomes in diakinesis and is enriched on bivalents until meiosis I. Here we show that GFP::CPAR-1 signal loss from chromosomes precisely coincides with homolog segregation during anaphase I. This loss of GFP::CPAR-1 signal reflects proteolytic cleavage between GFP and the histone fold of CPAR-1, as CPAR-1::GFP, in which GFP is fused to the C-terminus of CPAR-1, does not exhibit any loss of GFP signal. A focused candidate screen implicated separase, the protease that initiates anaphase by cleaving the kleisin subunit of cohesin, in this cleavage reaction. Examination of the N-terminal tail sequence of CPAR-1 revealed a putative separase cleavage site and mutation of the signature residues in this site eliminated the cleavage reaction, as visualized by retention of GFP::CPAR-1 signal on separating homologous chromosomes at the metaphase-anaphase transition of meiosis I. Neither cleaved nor uncleavable CPAR-1 were centromere-localized in mitosis and instead localized throughout chromatin, indicating that centromere activity has not been retained in CPAR-1. Although the functions of CPAR-1 and of its separase-dependent cleavage remain to be elucidated, this effort reveals a new substrate of separase and provides an in vivo biosensor to monitor separase activity at the onset of meiosis I anaphase.

Centromeres are epigenetically defined by the centromere-specific histone H3 variant CENP-A. Specialized loading machinery, including the histone chaperone HJURP/Scm3, participates in CENP-A nucleosome assembly. However, Scm3/HJURP is missing from multiple lineages, including nematodes, with CENP-A-dependent centromeres. Here, we show that the extended N-terminal tail of Caenorhabditis elegans CENP-A contains a predicted structured region that is essential for centromeric chromatin assembly; removal of this region prevents CENP-A loading, resulting in failure of kinetochore assembly and defective chromosome condensation. By contrast, the N-tail mutant CENP-A localizes normally in the presence of endogenous CENP-A. The portion of the N-tail containing the predicted structured region binds to KNL-2, a conserved SANTA domain and Myb domain-containing protein (referred to as M18BP1 in vertebrates) specifically involved in CENP-A chromatin assembly. This direct interaction is conserved in the related nematode Caenorhabditis briggsae, despite divergence of the N-tail and KNL-2 primary sequences. Thus, the extended N-tail of CENP-A is essential for CENP-A chromatin assembly in C. elegans and partially substitutes for the function of Scm3/HJURP, in that it mediates a direct interaction between CENP-A and KNL-2. These results highlight an evolutionary variation on centromeric chromatin assembly in the absence of a dedicated CENP-A-specific chaperone/targeting factor of the Scm3/HJURP family.