Investigating the role of VHL in homology-directed double strand break repair

The DNA in our body, our genetic code, is constantly being damaged; utraviolet light, reactive oxygen, errors during duplication of DNA and many other "accidents"; it is likely that there are up to 1 million DNA lesions in each cell per day. About 10 of these are particularly deleterious double strand breaks, that could cause loss of large pieces of DNA. In addition, there are natural processes that involve generation double strand breaks, for example during development of our immune system. It is therefore unsurprising that we have a elaborate set of proteins at work to detect and repair such damage. This process has been studied extensively in cell culture, and it has been shown that there several independent pathways repairing a variety of different defects, if one fails, backup pathways are often available to step in. Importantly, accumulating mis- or unrepaired DNA damage is thought to be responsible for increasing rates of cancer with age, and even aging itself.

We are studying the role of the human Von Hippel Lindau (VHL) gene in a model organism, the zebrafish. This gene is most famous for its role in an alarm signal that is activated when oxygen levels are too low (hypoxic- or HIF signaling), VHL functions to keep this pathway switched off. However, the gene has a number of less-studied roles that are independent of hypoxic signaling. Interestingly, in zebrafish, we found that the function of human VHL has been split over two genes, named Vhl and Vhl-like (Vll). Importantly, we found that in zebrafish one of the homologs, Vhl, has the "classical" role in hypoxic signaling whereas the other, Vll, does not. Recently, we established a novel and unique reporter system that is able to detect defects in DNA repair in young fish embryos, using a simple fluorescent readout. When using this reporter, we realised that the second gene, Vll, functions in DNA double strand break repair. We already verified that this function can also be exerted by human VHL in zebrafish embryos, thus it is not an evolutionary novelty that is only present in lower vertebrates.

The study of the human VHL/zebrafish Vll gene will improve our understanding of how cells stably maintain their genome sequence. Genome stability is important not only to allow proper transmission of genomic information through the generations, but is also important to prevent the development of cancer. Cancer usually results from cells that have aquired defects in growth-regulating genes, and we suspect that loss of VHL function could promote the formation of such defects.

Defects in DNA repair could render cells vulnerable to particular chemical agents. During the study of Vll function we may identify such compounds and they might in the future be useful in delaying the growth of tumors that lack VHL function.

The human Von Hippel Lindau (VHL) gene was first isolated as a human tumour suppressor gene. An important step in the elucidation of its function was made when it was shown that it was an essential negative regulator of the hypoxic (a.k.a. Hypoxia Inducible Factor (HIF) signalling pathway). VHL is part of a complex that causes proteasomal breakdown of HIF. However, this is not the only function of VHL and over the years numerous other have been identified.
We have used the zebrafish to study the function of the VHL gene. In zebrafish the functions of human VHL have been split over two genes, which we named vhl and vhl-like (vll). Interestingly, we found that the fish vhl gene has an important role in HIF regulation, as mutants we made by reverse genetics show all hallmarks of an inappropriate hypoxic response under normoxic conditions.
The role of vll was intially enigmatic, null mutants that we created in this gene were viable and fertile. However, using a unique and novel in vivo reporter for genome stability that we created, we discovered that the vll gene is important for maintaining genome stability. Subsequent experiments showed that vll is important for homology directed double strand break repair: both homologous recombination and single strand annealing are deficient in the vll mutant.
We now want to define this function better and place it within the hierarchy of the double strand break repair pathway. The fact that the function of human VHL is and advantage as it the multitude of functions of human VHL make it difficult to determine the precise consequences of a lack of the DNA repair function of VHL. As three groups with complementing expertise in zebrafish, cell culture and DNA repair, we will address the molecular mechanisms behind the function of zebrafish vll and human VHL in DNA repair and determine its relative importance with respect to other DNA repair genes, like BRCA2 and ATM.

NC3Rs
Genome sequence and development indicate that fish and mammalian animals models are in fact closely related and thus fish studies are highly relevant. This is important for refinement/replacement, because fish have lower level of neurophysiological development (for instance, an adult zebrafish brain is approximately 1/40th of a mouse brain). We are mainly using embryos which do not count as animal experiments by EU law.
Several thousand chemical compounds may require registration with a risk assessment based on substance-specific data for their toxic properties as a result of REACH legislation. Our LOH model is likely to be very useful such toxicology tests, as it can identify chemicals that -for whatever reason- promote genome instability,. Such a test could easily be integrated with existing tests, eg a Fish Acute Embryological Toxicity, currently in use for such purposes, and might partially replace rat/mouse experiments.
We will promote our work and our model through scientific publications, conferences, and by directly contacting interested parties once more data is available (~2nd yr)

Patients with VHL disease
In the further future, our research will help patients suffering from VHL disease, as our work will highlight unexpected vulnerabilities of VHL mutant cells, that could go on to be translated into novel clinical approaches. In case of strongly encouraging results we will contact clinical collaborators directly, we will promote our work directly by inviting VHL patients to an open day/evening.

Other cancer patients, and our understanding of aging
DNA repair is linked to both cancer and aging, our work/models may also indicate ways in which other cancer might be treated. If for instance BRCA2 mutant cells are vulnerable to loss of Vll/VHL it might indicate that VHL/Vll might be a clinical target is such tumors. We have created a viable fish model with a DNA repair defect. This might become a model to study the role of double strand break repair in aging.

Public engagement/outreach
Through my association with the Bateson centre, Biomedical science and the Sheffield Cancer Research Centre we have been involved in various outreach activities, we have the availability of a "Zebrafish Pod", an exhibition stand that allows members of the public to experience zebrafish research and the use of Green Fluorescent Protein. In future events we will of course use our results to promote our work on the VHL gene Our work is an excellent example of basic science with a clear view on translation with high visual appeal,
I plan to do at least 5 Understanding Animal Research, Science Week, or Teacher-training event each year. In addition, I will participate in university open days, eg Researchers Night.

Industry
Our genome stability reporter has unique strengths as a toxicological test model. When we have additional data to prove its functionality we liaise with our university commercialisation team and contact companies offering toxicology tests (eg. the DHI group; ~yr 2) who already offer a fish embryo acute toxicology test. Such an addition, will allow companies to highlight potential carcinogens in one simple test together with the aforementioned test. This will save costs and improve the quality/depth of their tests.

Training and career development
I am actively involved in promoting career development of my PhD students and postdocs. Several of my former PhD students have taken postdoc positions in prestigious institutes and one of my post-docs recently secured a Graves fellowship in Sheffield. For Dr Elworthy, a very talented scientist, this will this will be a stepping stone towards independence, and to start writing his own grants. The project will offer him the right mix of new skills that he will develop but at the same time exploit his skills in fish/molecular biology. If industrial opportunities arise, he will be a point of call, this will provide important commercial contacts.