The close relationship between the timing of telomere clustering and initiation of chromosome pairing during meiotic prophase has led to the suggestion that the bouquet like arrangement of telomeres on the Nuclear Envelope (NE) is a key step in the homology search. It is our goal to describe the mechanism of bouquet formation and the molecules needed to bring it about. Fission yeast has a prominent bouquet that persists throughout meiotic prophase, a limited number of chromosomes (1n=3), and the telomeres cluster on the NE in association with the Spindle Pole Body (SPB). These characteristics make Schizosaccharomyces pombe an ideal organism to investigate the mechanism of bouquet formation by molecular, genetic and cytological approaches. In a pilot genetic screen, we have isolated meiotic mutants that have defective organization of telomeres (dot). We may have mutants in two potential members of the telomere attachment complex, Spo3, a transmembrane protein, and dot5, a gene required for heterochromatin association with the NE. To test this idea, we will clone dot5, use cytological approaches to localize Spo3 and Dot5 proteins in the nucleus during the bouquet stage, and study their behavior in living cells as telomeres cluster and in fixed cells at an ultrastructural level, and use biochemical and molecular approaches to identify interacting proteins. Finally, we will initiate a new genetics screen based on the cytology of dot phenotypes and insertional mutagenesis, to identify other components of the complex. We will use dot mutants such as dot5 and dot 4-550 that are defective in karyogamy to determine whether SPBs fuse together at karyogamy and whether karyogamy is required to maintain the bouquet. Dot2 is deficient in karyogamy and bouquet maintenance because it has multiple miniSPBs. We will determine whether the dot2 phenotype is due to overexpression of SPB proteins. Finally, we will investigate telomere clustering in living cells using deconvolution light microscopy and mutants to dissect function. This kinetic analysis will allow us to distinguish between various models of telomere clustering. Since meiosis is an evolutionarily conserved process, what we learn about the bouquet stage in fission yeast should be applicable to other eukaryotes, such as human, where problems in meiotic prophase lead to chromosome missegregation, aneuploidy, birth defects or aborted fetuses.
| Li, Fei; Huarte, Maite; Zaratiegui, Mikel et al. (2008) Lid2 is required for coordinating H3K4 and H3K9 methylation of heterochromatin and euchromatin. Cell 135:272-83 |
| Madrid, Alexis S; Mancuso, Joel; Cande, W Zacheus et al. (2006) The role of the integral membrane nucleoporins Ndc1p and Pom152p in nuclear pore complex assembly and function. J Cell Biol 173:361-71 |
| Tang, Xie; Jin, Ye; Cande, W Zacheus (2006) Bqt2p is essential for initiating telomere clustering upon pheromone sensing in fission yeast. J Cell Biol 173:845-51 |
| Li, Fei; Goto, Derek B; Zaratiegui, Mikel et al. (2005) Two novel proteins, dos1 and dos2, interact with rik1 to regulate heterochromatic RNA interference and histone modification. Curr Biol 15:1448-57 |
