Deciphering sumoylation-dependent signalling through systematic loss-of-function analysis in C. elegans

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

Abstract

Proteins are often active in multiprotein complexes and have to be localised into the appropriate cellular compartments. In many cases, proteins are modified and these modifications can regulate their stability, promote physical interactions and specify their localisation, for example. One such modification is sumoylation; where a short polypeptide (SUMO) is transferred onto a protein. The added SUMO on a protein leads to novel interactions or relocalisation.
Sumoylation has been found involved in Huntington‘s disease and viral infection. Some viruses inhibit sumoylation to create a better environment for their multiplication. I discovered that in absence of SUMO, oncogenic pathways, pathways involved in cancer, are more active.
I will investigate sumoylation in vivo by systematic identification of the co-factors involved in this process using C. elegans as a model. The advantages of this model are that the sumoylation process is conserved in C. elegans, and there is a technology called RNA interference (RNAi) that inactivates gene expression. We have built an RNAi library that individually inactivates all the predicted genes of the C. elegans genome. Using this technology I will identify the genes cooperating and regulating the sumoylation process. I will also use classical biochemistry and fluorescence imaging to visualise sub-cellular localisation.

Technical Summary

The canonical Ras and Notch signalling pathways are critical during C. elegans vulva development. Genetic mutations that activate or inhibit these pathways cause characteristic vulval phenotypes. The vulva is a good model to study the Ras and Notch signalling pathways, their interaction (crosstalk), and their transcriptional effects. In Julie Ahringer‘s laboratory, we developed an RNAi feeding library that targets 87% of the predicted genes of the C. elegans genome; currently C. elegans is the only organism where genome-wide RNAi screens can be carried out in the whole organism. Taking advantage of this powerful technology, I performed two genome-wide RNAi screens for genes involved in inhibition of Ras-induced vulval fate. I identified nine new genes and amongst those, three belong to the sumoylation conjugation pathway. Sumoylation inhibits Ras signalling by targeting chromatin remodelling factors, and inhibits Notch signalling by an unknown mechanism.

Sumoylation controls important cellular events such as sub-cellular localisation, cell division, and gene expression. The discovery that SUMO inhibits Ras and Notch pathways raised important questions, such as how SUMO achieves Ras and Notch regulation, and how specific sumoylation can occur? I will study the function and regulation of sumoylation during Ras and Notch signalling using the C. elegans vulva as a model. Four aspects of this process will be studied: targets, effects, specificity and regulation. I will identify SUMO targets, study the effects that sumoylation has on these targets, find E3 ligases/co-factors involved in specific sumoylation, and investigate how sumoylation is regulated.

To understand the process of sumoylation and its regulation, I will carry out systematic RNAi loss-of-function analysis in sensitised backgrounds. I expect to identify SUMO targets and E3 ligases responsible for regulating Notch or RAS signalling. In addition, I will study the effects of sumoylation on candidates ing GFP imaging and biochemistry. GFP fusions will be used to study sub-cellular relocalisation upon sumoylation, and biochemistry to identify novel targets or new E3 ligase activity. The mechanisms of specificity and regulation of sumoylation are still poorly defined and this work will investigate these processes.

Sumoylation is a basic biological process conserved from yeast to human. It has been linked to neurodegenerative diseases and viral infections. It is also likely to be involved in cancer through genomic stability and regulation of oncogenic signalling pathways. This project will help us to get insights into important aspects of sumoylation and maybe develop therapeutic strategies to regulate SUMO functions.
 
Description BBSRC Standard
Amount £490,821 (GBP)
Funding ID BB/J014834/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2012 
End 09/2015
 
Description FP7
Amount £421,000 (GBP)
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 07/2009 
End 07/2013
 
Title anti-set-16, rbbp-5, and bet-1 
Description these are antibodies 
Type Of Material Antibody 
Provided To Others? No  
Impact not yet but will be used in future publications 
 
Description A novel ageing-related pathway regulating ROS homeostasis. 
Organisation University of Manchester
Department Faculty of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We will perform the C. elegans of this project to test the in vivo relevance of the proposed molecular mechanism
Collaborator Contribution The partners have uncovered a novel molecular mechanism that regulates ROS homeostasis in a cell system.
Impact We secured a grant with the BBSRC. This is a multidisciplinary (molecular mechanism, ageing and animal system- C. elegans)
Start Year 2011
 
Description Natural genetic variation and cancer signaling pathways 
Organisation University of Wageningen
Country Netherlands 
Sector Academic/University 
PI Contribution We are performing RNAi on recombinant inbred lines, and analyse the data using a newly developed assay.
Collaborator Contribution Development of new methods and new expertise
Impact multi-discplinary: transcriptomics, proteomics, and computer modeling. Publications are in preparations only since we just started
Start Year 2009
 
Description Manchester Science Festival 'Meet the Scientists' 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We prepared a workshop to show how C. elegans can contribute to improving human health. We explained that genes are conserved and using genetics we can better understand how cancer develop. We used a gane in which they have to identify phenotypes.

We were informed that our workshop was amongst the very popular and we have been asked to participate in another event.
Year(s) Of Engagement Activity 2009