Mechanisms of gene regulation by CSL-Notch

Lead Research Organisation: University of Cambridge
Department Name: Physiology Development and Neuroscience

Abstract

The development and health of all animals, including humans, depends on them generating and maintaining the correct balance of tissues. Each tissue consists of cells with distinct characteristics and these must be made and maintained in the right proportions. Two fundamental principles underlie this: the cells must be able to communicate with each other and, in response to this between-cell communication, each must switch the activity of their genes to produce the appropriate repertoire of characteristics for their function. Our research focuses on one important pathway of cell communication, so-called Notch pathway, and we aim to discover how signals through this pathway cause specific switches in gene activities to ensure the correct cell characteristics are made. This is important not only for understanding the normal process of animal development and health, but also for diseases such as cancer that arise through inappropriate Notch signals.

Genes consist of a code, generated from the 4 different letters that make up the DNA of our genetic material, and they are further packaged in the cell to make them more or less accessible to the machinery that reads this code. There must be underlying rules that determine (1) whether or not the Notch signal is able to access a particular gene and (2) whether that access results in a productive outcome from the gene. The rules are likely to rely on information from the DNA code and from the way that the gene is packaged in each type of cell. One of our goals is to find out what these rules are. To achieve this we will take a combination of computational (code analysis) and biological (surveying the genes) approaches. For our model we use the fruitfly, as all these processes are similar across species, so we can use this simple insect to learn about the mechanisms that are relevant to humans as well. In addition, the problem we are investigating, the rules governing gene access and usage, are fundamental to biology. The tools and knowledge that we generate through our investigations are therefore likely to have widespread relevance for deciphering the genetic code.

Technical Summary

Notch signalling, which mediates communication between cells, is essential at multiple steps in the development and maintenance of tissues. As aberrations in its regulation also contribute to many diseases, such as cancers, understanding the mechanisms at the heart of this pathway is of widespread importance for the development and health of all animals, including humans. Different cohorts of genes are regulated in response to Notch activity, depending on the cell-type. Here we propose to take an interdisciplinary approach to determine the molecular mechanisms that distinguish which genes can be accessed by the key Notch pathway DNA-binding protein, CSL, and whether that access results in productive change in gene expression when Notch is activated. In doing so we aim to address two fundamental and interrelated questions, taking an interdisciplinary and systematic approach:
1. Which aspects of the DNA sequence and chromatin landscape determine the sites that CSL occupies?
2. What co-factors are recruited with CSL, and how do they affect the transcriptional outcome of its association with DNA?
Our investigations into the mechanisms of gene regulation by CSL/Notch will not only be relevant for predicting the actions of this important pathway but will also provide a paradigm for the development of novel tools that can contribute to the understanding of transcription factor function in general.

Planned Impact

Beneficiaries:
-Industry involved in pharmaceutical research and drug development.
-Business, industrial and public sector recruiting graduate level staff.
-The general public and schools, through our involvement in public engagement.

Benefits to industry will come from the scientific results and the methodologies we develop:
1) Notch pathway is a major target for cancer and other therapeutics. Increased knowledge about the mechanisms can lead to novel approaches for targeting the pathway and can be important in informing about unforeseen side effects. For example, our results should improve the ability to "read" better the types of targets that could be activated in a particular disease context, important because the outcomes can be very different (e.g. tumour promoting versus tumour suppressing). They could also identify novel protein:protein interactions that would be a good substrate for drug developments. Benefit is likely to be realized in the longer term 5-10 years and it would impact especially on enhancing quality of health.

2) The physicochemical modelling approaches for predicting protein-DNA interactions will provide a new tool for all sectors, including the commercial sector. Its utility could be widespread, as predictability of transcription factor target sites is one of the bottlenecks to deciphering genomic information. In addition, such protein structural approaches lend themselves to subsequent modelling and development of small compounds that interfere with those molecular interactions. For example, the prototype for a leading anti-migraine drug (Zomig) was originally discovered by our collaborator, Prof Glen, using similar computational methodologies to study membrane-bound receptors. Initial benefits (deployment of the technology) could be within 5 years, if good predictive tools for protein:DNA recognition are a successful outcome. Thus, ultimate benefits would be to economic competitiveness of UK and to enhancing quality of health.

Benefits to business, industrial and public sector recruiting graduate level staff will come from the development of relevant research sills and professional skills:
The project's interdisciplinary nature ensures that the staff will acquire a broad range of technical skills (sophisticated computational approaches, high-end genomic and molecular biology techniques, exposure to physical chemistry) which will be applicable in wide range of life sciences, pharmaceutical, computational employment. Further gains come from our international collaboration, enhancing the skills training. We note that our staff already contribute to other sectors e.g. design and implementation of computational backend at Wikipedia, enterprise programming for ING. Alongside technical skills, staff will at the same time develop generic professional skills e.g. presentational skills; writing skills; data handling, including statistics; generic computational skills; project management. Evidence of our track record in this aspect comes from subsequent employment of staff from our groups (e.g. investment banking, parliamentary advisor, publishing, venture capital advisor).

Benefits to the general public and schools, through our involvement in public engagement:
We have a strong track record in communicating modern scientific methodologies and mathematical approaches to the wider community e.g. working with Plus Magazine --a maths magazine for the lay public, part of the Millennium Mathematics Project. We further were contributing to projects at Central Saint Martins School of Art in London, where the aim has been to translate scientific principles into design models. These activities will be continued and extended to encourage scientific understanding and to extend the concepts from our research into other fields. The University of Cambridge encourages and provides excellent support for public dissemination of research through the Office of Community Affairs.

Publications

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Bray S (2016) Notch signalling in context in Nature Reviews Molecular Cell Biology

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Bray SJ (2018) Notch after cleavage. in Current opinion in cell biology

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Housden BE (2014) Visualizing Notch signaling in vivo in Drosophila tissues. in Methods in molecular biology (Clifton, N.J.)

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Li J (2014) Notch signaling assays in Drosophila cultured cell lines. in Methods in molecular biology (Clifton, N.J.)

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Simón R (2014) Drosophila p53 controls Notch expression and balances apoptosis and proliferation. in Apoptosis : an international journal on programmed cell death

 
Description Proteins that regulate transcription bind to specific sequences in the DNA. We have been investigating the binding characteristics of a key transcription factor in an intercellular signaling patwhay, CSL, by using computational approaches combined with in vivo testing of the in silico predictions. These revealed novel characteristics of CSL binding that help explain the range of binding observed in vivo.
2. The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target selection and gene activation in each context. We identified key aspects of chromatin organisation and dynamics that underpin the selective and rapid response to Notch signalling.
3. Investigating whether the regulatory landscape could be shaped by employing different types of repressor, we have decoded the contributions made by 3 such factors.
Exploitation Route new mechanism for transcriptional regulation could lead to development of novel inhibitors
the strategy used for modeling of transcription factor binding more widely adopted.
methods used to obtain chromatin state models are available for others to use
data depostitied in public repositories can be used by others and for meta analysis
Sectors Pharmaceuticals and Medical Biotechnology

 
Description MRC Programme Grant (Programming)
Amount £1,669,548 (GBP)
Funding ID MR/L007177/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 05/2014 
End 04/2019
 
Title Chromatin state model 
Description method to partition chromatin into different activity states 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2015 
Provided To Others? Yes  
Impact Model of chromatin states for two cell lines computational method to implement for other cell lines underpinned Skalska et al, 2015 The EMBO Journal (2015) 34: 1889-1904 
URL https://github.com/rstojnic/notch-chromatin
 
Title Chromatin state model 
Description Model of chromatin states based on profiles of histone modifications 
Type Of Material Computer model/algorithm 
Year Produced 2015 
Provided To Others? Yes  
Impact generated a model of chromatin states in different cell types to map the conditions needed for gene responsive enhnancers. Underpinned publication Skalska et al; The EMBO Journal (2015) 34: 1889-1904 
URL https://github.com/rstojnic/notch-chromatin
 
Title Corepressor binding profiles 
Description Genome-wide binding profiles for Notch pathway co-repressors 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Data will be made available in 2017 
 
Title Genomewide changes in H3K56ac in Notch active cells 
Description Changes in Genome-wide profile of H3K56 acetylation when Notch is activated 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Gene Expression Omnibus (GEO) accession numbers GSE66220, GSE66219 Data for profile of H3K56ac from 2 cell lines before and after Notch activation is accessible to the community Underpinned the publication Skalska et al, The EMBO Journal (2015) 34: 1889-1904 
 
Title Genomewide profile of Su(H)/CSL binding in two cell lines 
Description Genomewide profile of Su(H)/CSL binding in two cell lines 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Gene Expression omnibus (GEO) accession numbers GSE66226 and GSE66225 Data from genome wide binding profiles of Su(H)/CSL binding availale to the community. Underpinned the publication Skalska et al, The EMBO Journal (2015) 34: 1889-1904 
 
Description CSL Dynamics 
Organisation University of Cincinnati
Country United States 
Sector Academic/University 
PI Contribution Formulated the hypothesis and carrying out the in vivo experiments
Collaborator Contribution Providing structural advice and making biophysical measurements
Impact Is multidisciplinary, collaborator is a structural biologist
Start Year 2014
 
Description Computational CSL binding 
Organisation University of Cambridge
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution WE provided the biological context and background for the project and tested the predictions
Collaborator Contribution They performed the computational simulations
Impact Research article: Torella R, Li J, Kinrade E, Cerda-Moya G, Contreras AN, Foy R, Stojnic R, Glen RC, Kovall RA, Adryan B, et al. Nucleic Acids Res 42(16):10550-10563 The work formed the basis of the PhD thesis of Rubben Torella Conference proceedings: Torella RF, Bray SJ, Adryan B, Glen RC ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY. 243. 25 Mar 2012. ISSN 0065-7727
Start Year 2009
 
Description Mathematical modelling of transcrition factor binding 
Organisation University of Cambridge
Department Cambridge Systems Biology Centre (CSBC)
Country United Kingdom 
Sector Academic/University 
PI Contribution A Researcher has spent 6 months in our group learning techniques and acquiring data as a result of his Centenary Grant Award (MRC: G1002110/1).
Start Year 2012
 
Description Talk for electrical engineers Genomes, lab work and microelectronics 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact questions indicated that ideas about the overlap between gene regulation and microelectronics had been communicated.

audience asked about ways these ideas could be developed
Year(s) Of Engagement Activity 2013
 
Description contribution to on-line and paper articles for general public (e.g. Slovak Big Issue) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact articles sparked responses and questions from the general public

correspondence and on-line responses indicate that articles have altered readers perspectives on topics such as regenerative medicine
Year(s) Of Engagement Activity 2013