Analysis of nuclear architecture during meiosis in yeast

All the cells of your body contain a central region called the nucleus, which is separated from the rest of the cell by a membrane. The nucleus contains the main genetic material required for coding life. This genetic material, DNA, is organised into various groups known as chromosomes. Humans have 23 different types of chromosome, each of which carries thousands of genes in a very tightly compact form. Like most animals and plants, there are two copies of each chromosome per nucleus in humans. One copy came from each parent during sexual reproduction. When cells divide to create growth, chromosomes are duplicated and then split in two, so that each new cell receives two copies of every chromosome type. During sexual reproduction a specialised type of cell division, that involves two nuclear divisions, is used so each new cell (or gamete) contains only one copy of each chromosome. This type of cell division is called meiosis. During meiosis the chromosomes are specially arranged to ensure that, by the end, one of each type is passed into the new cells. Rearranging the chromosomes involves the two copies (originating from different parents) lining up next each other so that they are paired. During chromosome pairing, special joints are made between the two chromosomes called crossovers. Crossovers are important to generate diversity and to hold the paired chromosomes together until just the right time for nuclear division. It is extremely important that crossovers only happen between two chromosomes of the same type rather than between chromosomes of different types. Crossovers between different types of chromosome cause a reorganisation of genetic material that can lead to inviable products of meiosis. We recently published data suggesting that one way chromosomes avoid making the wrong connections is through nuclear compartmentalisation. We think of the nucleus as a spherical sack, the space of which is filled with chromosomes. Nuclear compartmentalisation refers to the idea that not all parts of all chromosomes can access any part of the nuclear volume at the same time. It is well known, for example, that the ends of chromosomes, telomeres, bind the nuclear membrane. This means that chromosomes ends cannot move into the central space of the nucleus, so telomeres are unlikely to accidentally crossover with the middle section of another chromosome. In our recent publication we showed data and a model suggesting that this attachment of telomeres to the nuclear membrane is essential to maintain proper nuclear compartmentalisation. The aim of this project is to find out which genes produce the proteins that hold the telomeres on the outer edge of the nucleus. We will look at the position of telomeres using special fluorescent dyes and a microscope that can create three/dimensional images. We will use one colour dye, linked to a protein trans/membrane domain, to highlight the surface of the nuclear membrane. A second colour will be use to highlight a specific region of a chromosome (e.g. the telomere) by binding to a DNA sequence we can integrate anywhere we want it. We plan to develop a third colour reporter so that we can view two chromosome regions simultaneously. Using these markers we will be able to look at the position of telomeres relative to the nuclear membrane. We will test various mutant forms of yeast (a very convenient and efficient model organism) to see if the gene we have mutated was needed to ensure normal localisation of the telomeres. Based on other published work we already have a good idea which genes to mutate. Once we know which genes are required to maintain normal nuclear architecture, we will undertake tests to find out how mutating them disrupts other aspects of meiosis. In particular, we will use a well-developed genetic test to find out if chromosome regions that do not normally come into contact now make crossovers that will damage chromosome structure.

The reorganisation of chromosomes within the nucleus of meiotic cells is vital to the accuracy of chromosome segregation. For accurate nuclear division in the first meiotic division, homologous chromosomes must be paired and joined by crossovers. During chromosome pairing there is a radical shift in nuclear architecture from centromeres being clustered near the spindle pole body, to a random distribution. Telomeres are attached to the nuclear envelope and move from a random distribution to being briefly clustered before separating again. One role of chromosome pairing is to reduce the chances of unwanted interactions between non/allelic sites during crossover production. Such ectopic events cause chromosome rearrangements that are deleterious to daughter cells. Recent genetic data supports the view that localisation of telomeres to the nuclear envelope is a contributing factor in partitioning different chromosome domains. Such telomere led compartmentalisation of the nucleus reduces the risk of ectopic interactions. The goals of this project are to determine which proteins are required to position telomeres in the nuclear membrane and to test the idea that localisation provides protection against ectopic interactions. A candidate approach will be used to determine the role of proteins on organising chromosomes in meiotic cells. Based on information from mitotic studies, various genes will be deleted, and their influence on nuclear architecture will be assessed cytologically. Mitotic studies suggest a significant amount of redundancy in the system of localising telomeres and there is also evidence of cell cycle specificity. So, regulation may be similar, but not identical in meiotic and mitotic cells. The main assay will focus on the use of fluorescently tagged proteins that illuminate the nuclear envelope in one colour and the specific chromosome domains in different colours. Three-dimensional microscopy will be used to determine the position of chromosome domains (eg telomere, centromere, interstitial regions) with respect to the nuclear envelope. Preliminary data using a membrane-spanning domain fused to RFP and lacR/GFP indicates that we can identify position in three/dimensions, and detect differences between cells at different stages of meiosis. Introducing a third marker using CFP will permit simultaneous identification of different chromosome domains providing the advantage of internal controls, eg to compare telomere and centromere locality in the same cells. The information from cytological analysis will be used to test the hypothesis that telomere lead compartmentalisation of the nucleus inhibits ectopic interactions. We will test whether or not losing gene function influences the frequency of ectopic interactions. This will be done by a well-established assay using experimental reporter cassettes integrated into various sites of the yeast genome. The assessment of ectopic interactions will be extended to include natural repeated sequences in the yeast genome that could crossover during meiosis, with deleterious effects. These experiments will provide a clear view on how nuclear architecture protects the cell from unwanted chromosomal interactions. Finally, the project will involve a proteomic based screen to look for new meiosis specific proteins involved in organising the nucleus. This will be done in the main by TAP/tagging proteins identified to be important in earlier experiments. Little is known about the network of proteins required to establish and maintain nuclear architecture, thus this work has the potential to identify as yet uncharacterised complex members. One meiosis specific candidate protein to study is Ndj1, which binds telomeres and influences their position in the nuclear envelope. This protein is available in an HA/tagged version, which will be used in coimmuno/precipitation experiments to look for interacting partners.