Importance of kinetochore-driven cohesion loading at a heterochromatic pericentromere for accurate chromosome segregation during meiosis

Meiosis is the specialized cell division that generates gametes, which have half the number of chromosomes of the parental cell. Human meiosis is extremely error-prone: up to 30% of all human eggs have the wrong number of chromosomes, causing miscarriages and birth defects such as Downs syndrome. Moreover, the risk of producing a faulty egg increases with the age of the female. However, the underlying causes remain unknown. Furthermore, our knowledge of the basic biological pathways that direct chromosome segregation during meiosis is extremely limited. This project aims to uncover fundamental mechanisms of chromosome segregation during meiosis. Since meiosis is highly conserved, this project will employ fission yeast, a simple unicellular eukaryote that allows basic molecular mechanisms to be dissected in detail. The knowledge gained will inform future work aimed at identifying the causes of defective egg formation in humans and the influence of ageing.
The project will focus on the role of the pericentromere, the chromosomal region surrounding the centromere. This region plays several important and specialized functions in directing chromosome segregation during meiosis. Pericentromeres influence meiotic recombination, regulate the linkages between chromosomes and direct the orientation of chromosome attachment to microtubules. A key and conserved feature of the pericentromere, that underlies all of these functions, is that it is highly enriched in cohesin, the protein complex that links the newly duplicated chromosomes together after DNA replication. In budding yeast, a dedicated pathway directs cohesin loading to the centromere to enrich the pericentromere. However, budding yeast centromeres are unusual since they lack the "silent" heterochromatin, typically associated with centromeres of many other eukaryotes, including humans. In contrast, fission yeast pericentromeres are heterochromatic, moreover, this pericentromeric heterochromatin is known to be required for cohesin enrichment. This leads to the hypothesis that more complex pericentromeres recruit cohesin through two independent pathways: kinetochore-driven and heterochromatin-driven association. The goal of this project is use fission yeast to understand how the interplay between kinetochore-driven and heterochromatin-driven cohesin association contributes to the specialized function of the pericentromere in chromosome segregation during meiosis.
Work in budding yeast identified a conserved patch on the Scc4 subunit of the cohesin loader that targets the complex to centromeres. The equivalent conserved region on the fission yeast cohesin loader will be mutated and its importance in cohesin enrichment at the pericentromere will be assessed. Similar approaches will generate mutations in the kinetochore subunits onto which the cohesin loader docks, informed by studies on budding yeast. These mutants will be subjected to functional assays to determine the exact function of kinetochore-driven and heterochromatin-dependent cohesin association in meiotic chromosome segregation. Advanced live cell imaging methods will be used to examine fission yeast cells carrying markers of interest and undergoing meiosis will be used, alongside molecular biological assays, such as chromatin immunoprecipitation.
A recent functional genomics screen carried out in the Marston lab (unpublished) surveyed essentially all non-essential genes of fission yeast for roles in meiotic chromosome segregation. Several of the genes newly found to have roles in meiosis are hypothesized to work at the pericentromere. Functional assays will be carried out to determine if this is the case and to identify their interactions with other pericentromere regulators. This will include live cell microscopy, proteomic approaches and bioinformatics analysis. Specific follow up experiments will be designed to functionally characterise the most interesting factors from the screen.