The defining feature of meiosis, the cell division sequence that generates haploid gametes from diploid precursors, is segregation of homologous chromosomes. Homolog segregation requires two fundamental modifications of chromosome architecture to enable bivalents to bi-orient: homologs must be stably connected and sister centromeres must orient to the same pole (mono-orient) so that homologous centromeres can bi- orient. For most eukaryotes, the connections between dyads are provided by chiasmata, which develop from crossovers between homologous chromatids.1-3 However, in Drosophila males, crossingover is absent and chiasmata are replaced by a conjunction complex that consists of Stromalin in Meiosis (SNM) and Mod(mdg4) in Meiosis (MNM).4 How the conjunction complex connects homologs and how it is removed at anaphase I are important unanswered questions that will be addressed in this proposal. SNM is a paralog of the SA/Stromalin cohesin subunit, suggesting that the conjunction complex could be a modified cohesin, a possibility buttressed by indirect evidence that Separase is activated at anaphase I. Proposed experiments will determine whether the core cohesin subunits SMC1 and SMC3 are required for conjunction and whether Separase activity is required for homolog segregation at anaphase I. A second major aim of the proposal is to define the meiotic cohesion apparatus in Drosophila. In most eukaryotes, the meiosis-specific Rec8 subunit is required for all meiosis-specific cohesin functions which include promoting recombination and synapsis and stabilizing chiasmata.2,5 No Rec8 homolog has been identified in Drosophila. Instead, meiotic cohesion depends on three unique meiosis-specific genes, orientation disruptor (ord), sisters on the loose (solo) and sisters unbound (sun) that exhibit nearly identical rec8-like mutant phenotypes. Although they lack apparent cohesin homology, all three genes are required for stable cohesin localization to centromeres and the proteins they encode colocalize with cohesins both on centromeres and synaptonemal complexes.4 Several recent findings suggest that SOLO may be a functional Rec8 ortholog despite its lack of significant primary sequence similarity and that SUN may also be a cryptic cohesin. Tests of these hypotheses are outlined in the proposal along with more open-ended approaches to elucidate how SOLO and SUN function.
The third aim addresses the mechanism of sister centromere mono-orientation, in which sister centromeres align side-by-side and collaborate in forming a single kinetochore.3 In budding and fission yeast, meiosis-specific monopolin proteins are required for mono-orientation. However, the yeast monopolins are not conserved and no similar proteins have been identified in higher eukaryotes. The proposed experiments will follow up preliminary evidence for a role of protein sumoylation in regulating mono-orientation in Drosophila meiosis and will include both biochemical and genetic experiments to identify Drosophila monopolins.
Accurate meiotic chromosome segregation depends upon the establishment, persistence, and timely removal of cohesion between sister chromatids. Chromosome missegregation during meiosis is responsible for a large fraction of infertility, spontaneous miscarriage, and genetic disease in humans, and premature loss of sister chromatid cohesion has been implicated as a major cause of missegregation. Better understanding of how cohesion is established and maintained during meiosis is thus crucial to designing clinical interventions to reduce this disease burden.
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