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

Lead Research Organisation: University of Dundee


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.

Technical Summary

We have previously established techniques for performing quantitative timelapse proteomics of chromatin passing through interphase of the cell cycle. We showed that inhibition of DNA replication had unexpectedly large system-wide effects on the chromatin proteome. The aim of the current project is to use this technology to understand at a systems level how mitotic chromosome structure is established and how it is influenced by, and how it influences, the way that DNA replication occurs. We will perform timelapse proteomics of chromatin passing from G2 into mitosis and back into G1. We will use recently-developed methods to gain functional insight into proteins loaded and unloaded on chromatin at specific stages. We will select a few proteins from this proteomic analysis that have not previously been implicated in mitotic chromosome biology and study their function in detail. A range of assays will be used, including RNAi knockdown in tissue-culture cells and biochemical studies in Xenopus egg extracts. We will block DNA replication in a variety of ways and force unreplicated DNA to pass into mitosis. We will then determine how this effects the chromatin proteome and the way DNA is replicated in the following cell cycle. We will inhibit various activities related to the Anaphase Promoting Complex and determine how this changes chromatin proteins on mitotic exit. We will investigate how this affects the licensing of replication origins and subsequent DNA replication. These experiments will give us a comprehensive systems-level view of the dynamic chromosome proteome during passage through mitosis and the way the interphase and mitotic chromatin organisation is coordinated. This will help explain how epigenetic information about DNA organisation during interphase is interpreted and maintained during mitosis.

Planned Impact

The people who benefit from our work will primarily be researchers interested in the molecular mechanisms of cell cycle progression, chromatin and chromosome organisation. The results will also be of interest to systems modellers interested in the dynamics of subcellular components, especially those that vary with the cell cycle. Outside academic research, the work will be relevant to biotechnology and pharmaceutical companies interested in developing drugs or assay systems in the fields of cell cycle, genetic stability, cancer and aging. Access to our data allows the community to see our results, and prevents needless waste of research resources and duplication of effort. Agencies that fund work in these fields may benefit from accessing our proteomic data and timelapse profiles. In addition, we will share our research with the general public through a range of public engagement activities. The maintenance of genetic and epigenetic stability during the cell division cycle is fundamental to many natural and disease states, including cancer, aging and congenital defects. The work relates to fundamental biology, not to any specific disease state, and therefore it is hard to predict precisely how the results will be exploited. However, two obvious possibilities are that proteins identified in our work could become markers (biochemical or genetic) for specific disease states or could become new drug targets for the treatment of disease. For example, Bod1, which we discovered in our previous screen as a novel chromosome-associated protein, appears to be responsible for a familial genetic defect and we are currently collaborating with other groups on this. We are also involved in the development of anti-cancer drugs that target the replication licensing system, which we characterised in earlier work. However, translating these potential discoveries into the clinic is a very long-term strategy, and we would anticipate would take 10 years or more. As well as including our validated proteomics data in peer-reviewed publications, we will submit the proteomics data to the EBI-PRIDE database. We will also provide the data on our personal lab web pages. Heatmaps, timelapse images, and other image-based data will be exposed to the public on our web pages via the OMERO database. In line with our previous track records, we plan to engage in collaboration with other groups as it becomes appropriate during the course of the project. As we discover specific proteins involved in the processes related to the project, we will contact scientists who have experience and reagents related to those proteins and establish collaborations to undertake further functional analysis. As described above, the major routes of exploitation of the data we generate is publication in scientific journals and submission to public databases. These outputs will potentially drive translational research relevant to cancer and aging in the biotechnology and pharmaceutical sectors. One specific resource that will be generated by the project is novel antibodies raised against human and Xenopus proteins. Once these antibodies have been published we will provide them free on request to the research community. As described in more details in the Impact Plan and in section 1 of the Case for Support, both applicants have a strong track record in publication, collaboration, making presentations to scientific and general audiences, commercial exploitation of research outputs and sharing science with the general public.


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Description We have made new observations about the way that mitotic chromosomes are organised and how they are accurately segregated during mitosis. Major changes occur to chromatin on exit from mitosis that influence where replication origins are licensed. We have shown that licensing represents the loading of double hexamers of Mcm2-7 at sites that can function as replication origins during the subsequent S phase. We have shown that licensing can be regulated by a novel protein Idas that can heterodimeriue with the major licensing regulator geminin. We have also shown the existence of additional regulators of origin licensing that are active in late G1.
We have also made the exciting finding that hypoxia plays an important role, via the PHD1 hydroxylase, in regulating centrosome maturation and hence accurate chromosome segregation.
Exploitation Route Hypoxia is potential an important modulator of cell cycle progression and genome stability
Sectors Healthcare