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

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

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.

Technical Summary

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.

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/H013725/1 01/02/2010 30/09/2010 £630,089
BB/H013725/2 Transfer BB/H013725/1 01/10/2010 31/07/2013 £467,499
 
Description identification of key roles for the cell cycle regulating transcription factors E2F1 and E2F4 in interacting with the NF-kB signalling system. These interactions result in a repression of NF-kB responses in S-phase (when DNA replication occurs) and an enhanced response at the checkpoint just before cells enter S-phase. The data suggest an important functional interaction and predictive mathematical models of the cell cycle and NF-kB have been coupled together to predict the behaviour of these systems
Exploitation Route The mathematical models will be further developed and part of this has already been done through a PhD studentship

There is great potential to use the coupling between the cell cycle as a paradigm for other coupled systems.

The results are relevant to cancer and chemotherapy responses
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Useful public understanding of science resources that have been used in museums and schools Mathematical models of cell cycle and NF-kB
First Year Of Impact 2010
Sector Education,Healthcare
Impact Types Societal

 
Description Appointed to Pool of Experts for BBSRC Panels, 2012-2013.
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description BBSRC Bioscience for Industry Committee (single meeting for presentation)
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description BBSRC Panel: Enabling Theme 2, the Exploiting New Ways of Working Strategy Panel (ENWW)
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description BBSRC Strategic Tools and Resources Development Fund (sTRDF) Panel
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description External Reviewer for FRISYS-ZBSA (Zentrum fur biosystemanalyse), Freiberg, Germany, 26th May 2011.
Geographic Reach Asia 
Policy Influence Type Membership of a guidance committee
 
Description German Virtual Liver Scientific Advisory Board (42m euros programme )
Geographic Reach Asia 
Policy Influence Type Participation in a advisory committee
URL http://www.virtual-liver.de/wordpress/en/media/handling-biological-complexity-using-systems-approach...
 
Description Invited to attend Research Sponsors' Consultation Meeting (BBSRC &MRC) on Imaging for the biological and biomedical sciences, 14 May 2012.
Geographic Reach National 
Policy Influence Type Membership of a guidance committee
 
Description MRC Microbiome
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description MRC Systems Immunology
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description MRC/BBSRC New Microscopy Initiative
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Member of BBSRC Research Equipment Initiative (REI) Review Panel
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of BBSRC Site visit to Imperial Centre for Systems Biology
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of BBSRC Site visit to Oxford Centre for Systems Biology
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of BBSRC Tools and Resources Development Fund Panel, Feb 2012.
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of BBSRC/EPSRC TDRI Panel
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of MRC Doctoral Training Panel
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of MRC Population and System Medicine Board, 1 April 2012 - 1 April 2016
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Membership of BBSRC Interdisciplinary Systems Biology Strategy Committee
Geographic Reach Asia 
Policy Influence Type Participation in advisory committee
 
Description Wellcome Trust/Wolfson Capital Awards in Biomedical Sciences Committee
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Strategic Lola
Amount £4,160,524 (GBP)
Funding ID BB/K003097/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2013 
End 04/2018
 
Description Temporal manipulation of genetic circuits in single cells
Amount £122,019 (GBP)
Funding ID BB/P027040/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2017 
End 09/2018
 
Title Development of fluorescence correlation spectroscopy for protein quantification and interaction measurement 
Description Tool based on confocal microscopy for measurement of absolute fluorescent protein concentration and measurement of protein dissociation constants in single living cells. They method involves a combination of experimental measurement and mathematical analysis of the data. Further development to consider use with light sheet microscope in collaboration with Zeiss. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact Papers published. Collaboration with Carl Zeiss. 
 
Title Data from: Dynamic NF-?B and E2F interactions control the priority and timing of inflammatory signalling and cell proliferation 
Description  
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
 
Title Mathematical models of NF-kB and inked systems 
Description ODE and stochastic models of the NF-kB system and other modules that couple with it 
Type Of Material Computer model/algorithm 
Provided To Others? Yes  
Impact Multiple citations with other papers being based on our models 
 
Description Carl Zeiss 
Organisation Carl Zeiss AG
Country Germany 
Sector Private 
PI Contribution We have advised Zeiss on trends in bioimaging since 1996. We have provided new data and tested prototype equipment. We have spoken at Zeiss organised meetings. We have given them an opportunity to display Zeiss equipment at our training courses. We have organised symposia that have been supported by Zeiss. We have held expert discussion meetings to review microscopy trends that have involved senior Zeiss staff
Collaborator Contribution Zeiss have made a cash contribution to training courses (received) of £16,250. Zeiss estimate of total value of in-kind staff time for collaboration, training courses and other meetings (including visits of teams from Germany) ?25,000. In addition, Zeiss have also committed over £30,000 in cash and ~£80000 in in kind staff for future training meetings and collaborative visits. Zeiss helped to design the new Systems Microscopy Centre in Manchester and made a 45% discount (value ?350k) for the purchase of equipment in 2011. Zeiss are a formal MICA partner on both Liverpool and Manchester awards from the MRC/BBSRC New Microscopy Initiative. In the award to Manchester they have made a contribution of £614,314 in staff time, development costs and equipment contribution. This involves FCS (developed during this project), luminescence fluorescence imaging, light sheet microscopy and SOFI super-resolution imaging. More recently Zeiss have made a further contribution to our new clinical single cell centre. This includes over £400k in equipment discounts and £25k in cash contribution to training and symposia. Over the years the Zeiss contributions have included them helping us with public understanding of science exhibitions where they loaned equipment, provided support for professional poster preparation and used their delivery services to transport our exhibit materials and equipment to the exhibition venues. This included an exhibition in Buckingham Palace in 2006. Zeiss have sponsored 2-3 meetings per year in Manchester. In 2016 this included a session on light sheet imaging and a session on new confocal imaging technologies. In 2017 they have sponsored an image analysis daya and will sponsor a single cell biology workshop.
Impact Annual training courses MICA collaborative MRC grant MICA collaboration on new single cell centre The relationship with Zeiss has been two way. We have been given the opportunity to be early adopters f new technology and to feedback idease for improvement. We receive very favourable deals on microscope purchases and maintenance contracts. Specific areas of successful collaboration lie in improvements to higher throughput live cell imaging using the confocal microscopes; optimisation of truly dark microscopes for quantitative luminescence imaging and the development of FCS. Multiple workshops organised (2-3 per yeat)