Epigenetic regulation of gene expression by the exoribonuclease pacman

Development of an organism from egg to adult requires sets of genes to be switched on and off at particular times and in the correct order. If genes are not switched off when necessary, cells can continue to multiply in an uncontrolled way leading to cancer. As well as being important in cancer, gene regulation is crucial in controlling the balance between stem cell self-renewal and pathways to cell specialisation which are required to form the particular cells and tissues in the body. Since stem cells have a vast potential in regenerative medicine for the replacement of defective tissue, the understanding of gene control is crucial for harnessing the potential of these cells. Therefore studying the mechanisms whereby genes are switched off (as well as on) is vitally important for providing basic knowledge that has potential to lead to novel therapeutics. Using the fruit fly Drosophila as a model organism, we have recently discovered that an enzyme named Pacman is involved in the growth and differentiation of imaginal discs, which form adult structures such as wings and legs. Imaginal discs are similar to stem cells in that they carry the information to build the adult tissue. We have shown that Pacman normally affects the production of a protein called Simjang (Korean for 'strong heart') which in turn controls a gene silencing complex (the NuRD complex) which shuts down parts of the chromosome, preventing genes from being turned on. This gene silencing complex is important because it is known to be involved in many critical cellular events including tissue regeneration, formation of blood cells, ageing and spread of cancer cells. This is the first time that an enzyme involved in degradation of messenger RNA has been shown to be directly affecting a gene silencing complex. The aim of this project is to understand how the Pacman uses Simjang to control particular sets of genes involved in growth and differentiation. Our hypothesis is that, in normal cells, simjang messenger RNA is somehow 'tagged' for destruction so that not much of Simjang protein is made. This means that there is not enough Simjang to turn on the gene silencing complexes. These gene silencing complexes, when activated, normally act as 'brakes' to prevent growth and differentiation of the wing disc. Therefore when the brake is not pressed by Simjang, these genes are not turned on, allowing normal growth and development of the tissue. When the pacman gene is mutated, there is no (or little) degradation of simjang RNA, resulting in more Simjang protein, which turns on the gene silencing complexes (i.e. presses the brake) therefore switching on genes that prevent tissue growth and development. In this project we aim to test this hypothesis, find out the details of this gene control pathway and identify the genes which act as brakes to prevent growth and development. The mechanism of gene regulation which forms the basis of this proposal is entirely novel; as yet no research group has found this link between RNA degradation and gene silencing. Since all the proteins involved are similar in Drosophila and humans the new 'control module' we have discovered is likely to be relevant to gene regulation in humans. Since Pacman is known to be important in other important cellular events such as wound healing, migration of cell sheets, and male fertility, this work may also shed light on the molecular mechanisms of these processes in other tissues. Therefore the insights we gain during this project may help us to improve treatment for cancer and other diseases and also help us to understand the ways that tissues grow and develop. This project will therefore provide valuable insights into a new method of gene regulation which can be used in the development of new therapeutics.

Pacman/Xrn1 is a highly conserved cytoplasmic exoribonuclease which is known to play a crucial role in gene regulatory events such as control of mRNA stability, RNA interference and regulation via microRNAs. Using Drosophila as a model system, we have shown that mutations in pacman result in larvae with small imaginal wing discs and complete lethality at the early pupal stage. These results demonstrate that pacman is involved in regulation of growth and differentiation of wing discs. Furthermore, our data suggest that pacman targets a particular set of transcripts in this tissue and that the effect is tissue specific. To determine the particular set of transcripts targeted by Pacman, we carried out microarray analysis, supported by quantitative RT-PCR, on mRNA extracted from 3rd instar imaginal wing discs. We have shown that rather few mRNAs are expressed at substantially higher levels in the mutant discs compared to wild-type. Comparison of pre-mRNA and mRNA levels of these top-ranked transcripts identified one transcript, simjang/p66, which is post-transcriptionally regulated by Pacman. Simjang is highly conserved, and is known to be a component of the NuRD histone deacetylation complex which is involved in epigenetic gene silencing. This is the first time that a direct link has been shown between RNA stability and chromatin modification. The overall aim of the project is to understand how Pacman acts via Simjang to control growth and differentiation in the imaginal disc. Our hypothesis is that Pacman normally degrades simjang RNA resulting low levels of Simjang and lack of activation of the NuRD complex at a number of critical sites in the Drosophila genome. The resulting relief of silencing of particular sets of genes allows activation of other genes controlling growth and differentiation of the wing disc. The complementary techniques to be used to test this hypothesis will make use of the range of tools and reagents available in the Drosophila model system.

Who will benefit from this research? This project is primarily 'basic, blue-sky research' which nevertheless is likely to lead to new insights important for new therapeutics in the future. Beneficiaries in the public sector already include clinicians, who are benefiting from the knowledge and techniques developed from our current BBSRC-funded research. Since the focus of the proposed project is gene regulation, it is relevant for many fields of clinical research. Other potential beneficiaries include Industrial collaborators and the general public, particularly school children. How will they benefit from this research? Clinicians will benefit from the proposed research because the project addresses the regulation of gene expression during growth and development. Since control of epigenetic gene silencing is important in many new therapeutics and in cancer, this project will provide fundamental insights which will be relevant to their research. A greater understanding of the role of Pacman/Xrn1 in controlling gene silencing is likely to benefit clinicians who are seeking new cancer therapies. There is already significant interest in epigenetic transcriptional regulation by chromatin remodelling complexes since HDAC chemical inhibitors have shown potential as anti-cancer agents. Since we have previously shown that pacman/xrn-1 is involved in RNA interference, these studies are also of strategic relevance since they may lead to new technologies for post-genomic research and gene therapy. The skills and expertise gained by the post-doc during the project will also benefit clinicians. The cross-fertilization between the BBSRC-funded research of my group and local clinicians/scientists has already resulted in collaborative projects. For example, local clinicians have already benefited from our expertise in microarray analysis in a joint project on the use of microRNAs as biomarkers in certain leukaemias. This research, which is funded by a small grant, has already shown excellent promise and we hope to develop a suitable biomarker within the next two years. In addition, in an existing collaboration with Dr. Liz James, from the University of Brighton, we are using our research skills to help her and her industrial partners to develop new therapeutics for wound healing, which involve seeding of cells on novel biomaterials. Therefore, the continuation of these collaborations, aided by the proposed BBSRC grant, is likely to enhance the quality of life in the UK. The research skills gained by both the post-doc and the research technician will be valuable to their future careers and applicable to Biomedical Industries. In addition, the post-doc will be in a position to teach high-level techniques to PhD students including clinical PhD and MD students. The proposed project will therefore benefit the biomedical knowledge of future NHS clinicians. Summary of Planned Impact activities 1. We will continue to give seminars/presentations on our work to general audiences, where applicable. This will include, for example, the annual South Coast RNA network meeting in November of each year and which is attended by potential Industrial sponsors. 2. We will present our work to clinical colleagues at Networking events organised by the University of Sussex Business and Enterprize office. This includes the local Sussex Cancer Network. 3. We will continue to talk about our research to schoolchildren during Open Days and by appointment. 4. We will seek Industrial sponsorship of our work through the London Technology Network. 5. We plan to build on our existing collaborative work with colleagues at the Universities of Sussex and Brighton, and also with clinical colleagues at local hospitals. 6. We aim to use our existing Industrial Enterprize funding from the University of Sussex to develop our novel ideas on cancer biomarkers with a view to licencing our product to a Pharmaceutical company.