SUMO proteomics in C. elegans; germ line, meiosis and the DNA damage response

Proteins need to be regulated to properly fulfil their function in cells and living organisms. Specialized sets of proteins are required to convey a variety of signals within living cells. Such signalling proteins confer signals by attaching distinct sets of molecules to their target proteins. The attachment of such molecules, referred to as modification serves as a 'cellular language'. Modern molecular biology research focuses on such signalling proteins and tries to address their importance in modifying the activity of their targets. Modifications that confer signals such as phosphorylation or ubiquitylation have been known for a long time. However, only in the past decade, it became clear that sumoylation provides an important way of intracellular communication. The Hay laboratory elaborated important molecular details pertaining to basic biochemical reactions that are needed for SUMO modification. SUMO by itself is a sizable protein, that is attached to certain residues of its target proteins and several proteins are needed to confer sumoylation.
So called 'systems approaches' aim to provide a comprehensive analysis and understanding of the 'SUMO language'. Such analysis requires the identification, of all SUMO modified proteins, and a detailed analysis of how and to which extend SUMO modification changes in response to various physiological and pathophysiological conditions. First experiments to assess the global role of SUMO modification were done in with cell cultures. While those systems have obvious experimental advantages, these cells do not necessarily reflect the behaviour of cells in intact organisms. This is why we plan to use the small roundworm C. elegans as a model system to globally asses the extend, and importance of sumoylation in a living organism.
C. elegans was chosen as the simplest possible animal system. Studies are facilitated by the simplicity of the organism at the developmental and anatomical level, by the ease of its maintenance, as well as by the availability of methods that allow for manipulating its genetic information procedures necessary to study the importance of SUMO modification. Despite of its simplicity, C. elegans is a multicellular organism that shares many fundamental genetic programs with humans. Thus results obtained in the C. elegans system are likely to be applicable to humans and working with worms does not raise ethical concerns.
As part of our studies we want to express modified versions of SUMO in the worm, such that we can best apply the advanced methods we have developed to isolate all SUMO modified proteins. To identify those proteins, but also to assess the extent of SUMO modification we will use a method we recently developed allowing for the identification and quantification of thousands of worm proteins. We not only want to find all SUMO modified proteins, but also want to assess the importance of SUMO modification. To start with this, we chose an important biological processes extensively studied in our labs. 1) Meiosis is a specialised cell division, needed for the generation of germ cells. During this division the duplicated set of chromosomes occurring in normal cells is reduced to just one copy. The subsequent fusion of germ cell restores a duplicated set of chromosomes one coming from the father, another one coming from the mother. Not only this, during meiosis a process referred to as recombination also mixes individual chromosomes such that chromosomes contain elements from both grandparents. Recombination ensures genetic diversity. 2) Recombination pertains to the breaking and linking DNA molecules. Recombination is used when DNA damaging agents such as ionizing irradiation breaks chromosomes. We already have know that SUMO modification is important for meiosis and recombination and we now want perform a systematic analysis of this. Our studies are important to understand how the genetic information is passed on from one generation to the next.

SUMO is a small protein modifier that can be covalently attached to proteins in an analogous way to ubiquitin. To address the prevalence and relevance of SUMO conjugation in a living organism we propose to use the roundworm, Caenorhabditis elegans as a model system. We plan to characterise the SUMO (SMO-1 in C. elegans) proteome using high accuracy mass spectrometry. We aim to employ several strategies to purify the SMO-1 modified C. elegans proteome. Furthermore, we will exploit recently developed mass spectrometry based methods to determine the actual target residues of SUMO modification. The dynamic changes in the SMO-1 proteome in response to ionising radiation and tissue-specific SMO-1 sub-proteomes will be assessed taking advantage of our recent adaptation of the SILAC (stable isotope labelling with amino acids) technique for use in nematodes. We aim to provide a comprehensive description of the SMO-1 proteome in a multicellular organism. These studies will be complemented by functional studies that will focus on both the global role of the SMO-1 conjugation pathway, as well as the specific role of SMO-1 conjugates. As to our analysis we will particularly focus on DNA double strand break repair, on meiotic recombination and on general meiosis progression. New SMO-1 modified targets will be functionally followed up using a battery of C. elegans genetics and cytology based approaches. Generating knockin- constructs that lack critical SMO-1 modification sites will allow for assessing the importance of sumolylation. Overall, our project combines high accuracy mass spectrometry with the C. elegans model system to provide a global understanding of the dynamic SMO-1 proteome and to efficiently test the functional consequences of SMO-1 modification in vivo.

Our research project is predominately interest driven and we thus try to do excellent basic research. We vigorously promote the use of model organisms to study processes relevant to human disease. The nematode model allows for easy experimentation and cost effectiveness, which cannot be achieved in transgenic mouse models. Importantly, we make the case of systematically and globally scanning for SUMO modification at the level of a multicellular organism, and to systematically scan for phenotypes associated with SUMO modification. Phenotypes will be assessed at the level of an entire organism. Such high throughput analysis cannot be done in any mammalian model. In the past many results stemming from C. elegans research could be directly verified in mammalian systems and we are confident in suggesting that this will continue to be the case. C. elegans developed into a premier model to study meiosis and is also widely regarded in the apoptosis and DNA response fields. Also we point out that results originating from C. elegans research, such as RNAi, have developed from basic discovery to clinical application in less than ten years. Equally, generally used techniques such as the exploitation of GFP were first demonstrated in the worm. Besides, our approach is technology driven and we therefore expect to impact on developing improved mass spectrometry based procedures as part of our research.
In the long term we aim to not only increase our knowledge of how sumoylation globally affects signalling, but also to examine how to directly translate our results to benefit patients in the future. While it would be unrealistic within a three year project grant that funds a single postdoctoral researcher to immediately aim at translating our expected results into vertebrate transgenic models, we are nevertheless very well placed in Dundee to do this in the long term to more directly contribute to human well being and healthy aging. The Hay lab is set up to undertake biochemical studies to employ tissue culture based systems to translate our results into mammalian systems. Roland is part of the recently founded Dundee SCILLS Institute, which is focusing on signalling conferred by ubiquitin like modifiers such as SUMO. Since the foundation of this institute a spin-off, Ubiquigent, focused on marketing key reagents in the field was already founded. Dundee College of Life Sciences is unique in having an entire department dedicated to drug screening. Thus, if we hit on a conserved and important regulatory networks impinging on SUMO regulation, devising and conducting appropriate high throughput assays and screens is an achievable and realistic goal. Indeed Ronald advises several large UK pharmaceutical companies in conducting such screens further strengthening our position to ensure that our results achieve the maximal impact. Furthermore, the Gartner and Hay labs are part of the Cancer Research UK funded Dundee Cancer Centre. This centre brings academic scientists, medical scientists, and medical doctors together in one forum, providing us with access to local tissue banks and patient databases.
Besides the direct benefits of our research, we contribute to UK higher education. Indeed, we act in the strong belief that competitive academic research cannot and should not be separate from teaching. Thus both Ronald and myself take part in undergraduate and graduate teaching and we are also involved in multiple outreach activities to explain our science to lay people. Finally, employing talented postdoc from Argentina will foster cultural and economic links ultimately strengthening the UK. It is evident that Argentina is a rapidly expanding market, not only in the economic sense, but also as to expanding its research and educational base. To sustain the leading position of the UK as an knowledge based society it is important to foster interactions with rapidly developing countries at all levels.