Proteomic functional and systems analysis of mitotic chromosome organisation and its interdependence with DNA replication

Most cells in the human body contain a single copy of the DNA containing all the genetic information required to direct the development and maintenance of the individual. Although they all contain copies of the same genetic information, different cell types express different genes. For example, a skin cell expresses a different set of genes to a liver cell, which makes the cells behave differently. The reason that different cells express different genes is usually because different cell types have different proteins bound to their genes or because the genes have different chemical modifications. These modifications are called epigenetic modifications. When a cell divides, it typically creates two daughter cells that express the same genes as did the mother cell. This is because when cells copy their DNA prior to cell division, they also copy the epigenetic modifications that control which genes are expressed. Just prior to cell division, in a process call mitosis, cells compact their DNA into small bundles called chromosomes so that the two copies can be separated and each sent to one of the two daughter cells. The purpose of the proposed work is to understand how the DNA is correctly compacted during mitosis, and to understand how the epigenetic information is maintained during the compaction, so that cells behave consistently from one cell division to the next. As well as representing an unexplored area of basic biology, the proposed work has significant implications for human health, as defects in the maintenance of genetic and epigenetic information is an important cause of disease, including cancer, aging and congenital defects. We will address the problem using technology that we have been developing over the last 6 years. This involves the use of frog egg extracts that support the major events of the cell division cycle, including the copying of DNA and its segregation during mitosis, in a test tube. Because these reactions take place outside a cell, the system is easy to manipulate biochemically and we can isolate DNA and proteins bound to it in a very gentle way without having to disrupt other cellular structures. Using this system we will isolate DNA and the proteins bound to it at different times as the extract passes through mitosis. We will identify and quantify all the proteins associated with the DNA using a technology called mass spectrometry. By looking at proteins that are loaded and unloaded on chromatin at specific stages of mitosis, we will obtain information about their likely function. We will use this information to investigate in detail the role that a few selected proteins play in this function. We will also disrupt different stages of the process - for example by preventing the copying of DNA that normally occurs before mitosis - to see how the system responds to stresses that living cells might encounter. Again we will use mass spectrometry to identify proteins that respond to these stresses and then perform more detail biochemical experiments to understand their function. These experiments will give us a comprehensive view of the way that DNA-associated proteins change as cells pass through mitosis and will give us new insights into how the epigenetic information is maintained during mitosis to ensure that cells behave in a consistent manner from one cell division to the next. The results will help explain how epigenetic information may be disrupted during cellular aging or in diseases such as cancer.