A systems biology study of E2F and NF-kappaB cross-talk

We have previously used microscopy to study the important stress signalling system called NF-kappaB, which is present in virtually all mammalian cell types. This system has been shown to be important in inflammatory diseases such as arthritis and asthma, as well as cancer. We showed that NF-kappaB, uses the frequency of movements into and out of the nucleus to determine which genes are switched on. We have found that a transcription factor (a protein which binds to and controls the switching on of other genes) called E2F interacts with the NF-kappaB signalling system. E2F is well known as the principal switch that allows cells to begin to make another copy of their DNA prior to cell division (in a repeating process called the cell cycle). It seems that E2F stops an NF-kappaB signal when there is a high level of this protein inside cells. We found that E2F holds a key NF-kappaB protein called p65 in the nucleus, when it would normally be in the cytoplasm. When NF-kappaB is stimulated, the presence of a high level of E2F causes a delay before oscillations in NF-kappaB movement begin. We wish to investigate 1) the levels of E2F and NF-kappaB proteins at different stages of cell division and which of these proteins bind to each other in the cell. For this we will use mass spectrometry-based measurement of exact protein concentrations and will measure protein interactions using either microscopy or antibodies that allow the precipitation of proteins that bind to each other. Throughout this process we will use and develop a mathematical model of this complicated system. We have already built mathematical models of the NF-kappaB system and we will link these with existing mathematical models of the cell cycle. The mathematical models will be used to predict and interpret the experimental results. The work will therefore allow a new understanding of the relationship between the basic processes of inflammatory signalling (NF-kappaB) and cell division (E2F) which will be important for understanding normal and disease-related inflammation and cell division.

We will develop a systems biology programme to study the dynamics of the interactions between the cell cycle/E2F and NF-kappaB systems. We have used cell imaging to characterise a novel physical and functional interaction between the the cell cycle G1/S regulatory transcription factor E2F-1 and the NF-kappaB p65-p50 complex. This interaction mediates differential transcriptional control regulating cellular inflammatory signalling, cell cycle and apoptosis. Increased levels of E2F-1 sequester p65 in the nucleus, reducing NF-kappaB responses to TNFalpha. We will couple our existing deterministic mathematical model for the NF-kappaB system with the most appropriate elements from previously published models of the G1-S transition to develop a predictive mathematical model of the dynamic links between the E2F and NF-kappaB systems. We will use centrifugal elutriation for larger scale cell cycle synchronisation to enable quantitative proteomic and ChIP analyses. We will study the interactions of E2Fs 1,2 and 3 with different NF-kappaB family members to establish whether the different G1-S E2Fs have different interactions with the NF-kappaB system. A preliminary mathematical model has predicted that higher E2F-1 levels modulate IKK activation following NF-kappaB signalling. We will test the hypothesis that this occurs through the DNA damage-regulated Chk1 and Chk2 kinases. We will make BAC constructs to study the expression and protein localisation of fluorescent reporter-tagged E2F and E2F target genes. We will focus on the regulation of the G1/S cell cycle control genes, Cyclin D1, and Skp2. This will investigate the hypothesis that the E2F and NF-kappaB transcription factors piggy-back onto each others DNA binding sites acting as hybrid transcription factors. We will use transcription analysis from the BAC constructs in single cells to quantify transcriptional noise and variation to investigate the role that this may play in variability of cell cycle timing.

The application of systems biology offers great potential for a better understanding of cell signalling and decision-making pathways. This in turn creates the opportunity for the identification of better drug targets and more efficacious modes of treatment of disease. This application is relevant to two important processes in human and animal disease. The first is inflammation and innate immunity, where NF-kappaB is a critically important regulator. The second is cell division, where E2F regulates the G1 to S phase transition in the cell cycle. We show for the first time the critical nature and importance of the interaction between these processes. This is important for applied research and is of relevance to healthcare and to the pharmaceutical industry. In this respect we have ongoing collaborations with AstraZeneca. Dr John Unitt has been a collaborator for several years and is a co-author on several of our papers including two Science papers. He is currently a member of the advisory board for our BBSRC SABR project. He has expressed significant interest in this new project and it is likely that the results will be of interest and relevance to AstraZeneca and other pharmaceutical companies. At the same time all the pharmaceutical companies are looking for developments in systems biology that prove its utility for new drug target identification and validation. We will at all stages in this project consider whether the results are of commercial value and will seek to establish specific collaborations with pharmaceutical companies when appropriate. The major technologies being used in this project are bio-imaging based. This is an important and currently growing area. We have close relationships with instrumentation companies and in particular with AstraZeneca and Hamamatsu Photonics with whom MW has collaborated for 13 and 17 years respectively. This involves the loan and testing of equipment and the exchange of ideas, for new developments in microscopy and detection. Both companies (together with 4 other instrumentation companies) currently sponsor the BBSRC SABR project through support for an annual training course that is held in June or July. This years course is 6-10 July 2009. The companies provide speakers for the course from Germany and Japan, the loan of demonstration equipment and financial support. While the course is focused on providing training for the staff on the SABR project, places are made available to staff on other systems biology projects from around the country. Representatives of each of the instrumentation companies are on the scientific advisory board for the BBSRC SABR project. The present project will serve to support these collaborations by extending our interest in bio-imaging to include cell cycle analysis. One of the major spin-off advantages of live cell imaging research is that the pictures and time-lapse movies of cells are very visual and therefore are a good resource for developing Public Understanding of Science materials and presentations. MW has regularly given public lectures at specific meetings (e.g. ASE). In 2006 a team from the centre for Cell Imaging (led by Prof. M. White and including the postdoc on this grant Dr J. Ankers when he was a student) presented an exhibit entitled 'The Language of Cells' at the Royal Society exhibitions in London, Glasgow and at 'Science Day' at Buckingham Palace. We will put in an application to present a new exhibit at the Royal Society Summer Exhibition in 2010, the 350th anniversary of the Royal Society. The plans for the new exhibit are being led by Dr J. Ankers.