*EASTBIO* Importance of kinetochore-driven cohesin 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 cell1. 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 Down syndrome. The risk of producing a faulty egg increases with the age of the female but the underlying causes remain unknown. Our knowledge of the basic biological pathways that direct chromosome segregation during meiosis is sparse. This project will uncover fundamental mechanisms of chromosome segregation during meiosis. It will employ fission yeast, a simple unicellular eukaryote that allows basic molecular mechanisms of meiosis, which is highly conserved, 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 pericentromere is the chromosomal region surrounding the centromere which 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 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. Specifically, the student will:

1. Develop tools that abolish kinetochore driven cohesin loading in fission yeast to determine how pericentromeric cohesin influences meiotic recombination, cohesion and chromosome segregation.

Work in budding yeast identified a conserved patch on the Scc4 subunit of the cohesin loader that targets the complex to centromeres2. The student will mutate the equivalent conserved region on the fission yeast cohesin loader and assess its importance in cohesin enrichment at the pericentromere. Similar approaches will generate mutations in the kinetochore subunits onto which the cohesin loader docks, informed by studies on budding yeast. The student will use established genetic assays including CRISPR-Cas9-mediated genome editing to create a library of specific point mutants. These mutants will be analysed alongside heterochromatin mutants using genomic techniques including chromatin immunoprecipitation followed by high throughput sequencing. The student will have the opportunity to gain experience with computational analysis of sequencing data.

Mutants that affect cohesin enrichment at the pericentromere will be subjected to functional assays to determine the exact function of kinetochore-driven and heterochromatin-dependent cohesin association in meiotic chromosome segregation. The student will use advanced live cell imaging methods to examine fission yeast cells carrying markers of interest (e.g. chromosome labels and fluorescent cohesin proteins) and undergoing meiosis3. The findings from this aim will determine the relative contribution of kinetochore-driven and heterochromatin-driven cohesin loading to pericentromeric cohesin enrichment in fission yeast.

2. Establish a synthetic cohesin-rich domain to dissect functions of individual components

To uncover the function of cohesin regulators, independent of the endogenous centromere, an orthogonal tethering system will be developed to generate a synthetic cohesin-rich domain on a chromosome arm. A novel and orthogonal system from bacteria will be developed to target cohesin regulators to chromatin in fission yeast. It will be of particular interest to understand how this synthetic cohesin-rich domain impacts meiotic recombination.

3. Identify novel pericentromere regulators that influence meiosis

A functional genomics screen carried out in the Marston lab (unpublished) identified genes hypothesised to work at the pericentromere and with functions in chromosome segregation. Functional assays will determine if this is the case and identify their interactions with other pericentromere regulators. This will include live cell microscopy, proteomics and bioinformatics. Specific follow up experiments will be designed to functionally characterise interesting factors from the screen.