Meiosis is the specialized cell division that generates eggs and sperm. Errors in meiosis are extremely common: up to 30% of all human eggs have the wrong number of chromosomes and this results in miscarriages, infertility and birth defects such as Down’s syndrome. However, the underlying reasons remain unclear. The risk of producing a faulty egg increases with the age of the female but the underlying causes remain unknown. This project will uncover molecular pathways that are important for accurate chromosome segregation during meiosis. It will use budding 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.
During meiosis, chromosomes undergo two consecutive rounds of chromosome segregation: maternal and paternal chromosomes are separated in meiosis I, while sister chromatids are separated during meiosis II. This requires that cohesin, the protein complex that holds duplicated sister chromatids together, is lost in a step-wise manner during meiosis. Cohesin is lost from chromosome arms during meiosis I but protected in the region surrounding the centromere, known as the pericentromere. The protection of pericentromeric cohesin relies on a conserved protein, known as shugoshin. Shugoshin associates with and protects pericentromeric cohesin until meiosis II. At this point, shugoshin must be inactivated but how this is achieved is unknown. Our recent work has identified SUMOylation as an important mechanism regulating shugoshin inactivation. SUMO (small ubiquitin-like modifier) is a protein that is attached covalently to target proteins post-translationally by SUMO ligase enzymes. SUMOylation is known to have an important role in regulating chromosome segregation during meiosis also in C. elegans. This project will address the mechanism by which SUMOylation regulates the two-step segregation of chromosomes during meiosis.
Aim 1: When and where is shugoshin SUMOylated occur during meiosis?
Shugoshin is a chromatin and cohesin-associated protein, localizing at pericentromeres from the time of DNA replication until the time at which sister chromatids segregate during meiosis II. The position and timing of shugoshin SUMOylation will be determined using established biochemical methods in highly synchronised meiotic yeast cells or carrying mutations known to abolish shugoshin association with chromosomes.
Aim 2: What is the function of shugoshin SUMOylation during meiosis?
SUMOylation can alter protein-protein interactions, localization or stability. To understand how shugoshin SUMOylation affects its function, a version of shugoshin lacking its SUMOylation sites will be analysed. Live cell microscopy assays will visualize cohesin and chromosome segregation. Chromatin Immunoprecipitation followed by next generation sequencing (ChIP-Seq) will determine how shugoshin SUMOylation affects its association with chromosomes.
Aim 3: Which proteins are SUMOylated during meiosis?
SUMOylation is an important regulatory mechanism during meiosis but the targets remain unknown. To identify proteins that are SUMOylated/deSUMOylated during meiotic chromosome segregation, all SUMOylated proteins in yeast will be isolated biochemically and analysed by mass spectrometry. This global analysis will identify groups of proteins that undergo this important modification and stimulate further cell biological analysis.
1. Pelisch F, Tammsalu T, Wang B, Jaffray EG and Gartner T and Hay, R.T. (2017) SUMO-dependent protein network regulates chromosome congression during oocyte meiosis. Molecular Cell, 65:66-77.
2. Marston AL (2015). Shugoshins: tension-sensitive pericentromeric adaptor proteins safeguarding chromosome segregation. Molecular and Cellular Biology. 35, 634-48.
3. Verzijlbergen KF, Nerusheva OO, Kelly D, Kerr A, Clift D, de Lima Alves F, Rappsilber J and Marston AL (2014) Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere. eLife 3, e01374.
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