Meiosis is a specialized type of cell division used by sexually reproducing organisms to produce haploid gametes from diploid cells. Errors in meiosis in humans result in chromosomal imbalances that result in infertility and birth defects such as Down and Turner syndromes. Meiosis is highly conserved through evolution. The budding yeast, Saccharomyces cerevisiae, has been an excellent model organism in which to study meiosis due to its sophisticated genetics and ease of biochemical, molecular and cytological analyses. Many genes known to cause sterility in mammals were first identified in yeast.
My lab uses a variety of approaches to understand chromosome behavior during yeast meiosis. We are particularly interested in the role that phosphorylation plays in meiotic chromosome segregation. A major difference between meiosis and mitosis is the reduction in chromosome number accomplished by having two rounds of chromosome segregation follow a single round of chromosome duplication. Proper segregation of homologs at Meiosis I requires that the chromosomes be physically connected by a combination of reciprocal crossovers and sister chromatid cohesion. To generate crossovers, programmed double strand breaks (DSBs) are made and repaired preferentially using homologs, as opposed to sister chromatids (interhomolog bias). Recombination is regulated at several different levels by phosphorylation. My lab has focused on two kinases, Mek1, a meiosis-specific kinase required for interhomolog bias and the meiotic recombination checkpoint, and Cdc7-Dbf4, a highly conserved cell cycle kinase that is necessary for DSB formation, cohesin removal, mono-orientation of sister kinetochores at Meiosis I and meiotic progression. To generate unbiased lists of substrates for these two kinases at different stages of meiosis, we have developed a method for doing Stable Isotope Labeling in Cell Culture (SILAC) in sporulating cells. After pre-growing cells in synthetic medium containing either normal or heavy versions of arginine and lysine, the cells are induced to arrest in meiotic prophase, and an analog-sensitive version of either Mek1 or Cdc7 is inhibited in the heavy culture. This allows phosphatases to remove Mek1 or Cdc7-dependent phosphates, which then cannot be replaced due to inactivation of the kinase. The heavy and light cultures are then combined, crude chromatin is isolated and digested with trypsin, and phosphopeptides enriched using immobilized metal affinity chromatography (IMAC). The identity and location of the phosphosites are determined by mass spectrometry. Potential Mek1 or Cdc7-dependent phosphosites are indicated by phosphopeptides that are more abundant in the light culture compared to the heavy.
Now that we have a list of phosphopeptides at different points in meiotic prophase, we are in the process of mutating phosphosites to determine the functional significance of the phosphorylation. In this way, we have found the phosphorylation of the C terminus of the synaptonemal complex protein, Zip1, is essential for regulating the formation of crossovers, as opposed to non-crossovers. Further studies of other phosphorylated proteins should lead to a greater understanding of how meiotic chromosome behavior is regulated.