Regulation of Neural Crest development by transcriptional pausing

For a fertilised egg to develop into a multicellular organism such as a tadpole or human many changes have to happen. These changes have to occur at defined times in development and in specific places such that, for example, cells that will form the heart will do so at the right time and in the right place. For these changes to occur the cells in the embryo have to be able to know where and when to begin to specialize into that particular cell type. We are studying how cells can do this. We are looking at this question using an important cell type in the embryo called the Neural Crest. Neural Crest (NC) cells contribute to the formation of many organs in the body such as the nervous system, the cartilage and muscle of the face, the heart and the pigment cells in the skin. They are therefore of importance for normal development as errors in their development are the cause of many birth defects. NC derived cells can also give rise to cancer. As the NC contribute to so many different tissues they could possibly act as a source of stem cells for regeneration of damaged organs. Determining how the NC are formed will therefore contribute to our understanding of development and possibly in the long run lead to advances in stem cell research and regeneration as well as our understanding of cancer. We propose to use embryos of Xenopus laevis (the African Clawed Frog) to do our experiments. Xenopus has many advantages as a model organism for studies in cell and developmental biology. They are relatively cheap and easy to keep. Large numbers of embryos can be obtained when required and these embryos develop quickly outside the mother. In addition Xenopus is an established experimental system with methods for studying gene function in gain-of-function and loss-of-function experiments in whole embryos and parts of the embryos grown in culture. Xenopus has been one of the major model organisms for research into the NC. In previous work our lab tested thousands of small molecules looking for any which affect the pigment pattern of the tadpole. Pigment cells are formed from the NC so the idea was that any compounds, which affected pigment, could be doing so by affecting the NC. We have identified one compound, Leflunomide, which does just this. In addition, as pigment cells also give rise to melanoma, a dangerous skin cancer, we looked to see if Leflunomide could inhibit cancer growth. This also turned out to be the case. Leflunomide is therefore potentially an important compound in helping to understand NC development and combating melanoma. In this application we are further looking at how leflunomide affects NC development and using it to investigate a novel way in which NC and stem cell growth is controlled.

The Neural Crest (NC) are pluripotent cells that arise at the border between the ectoderm and the neural plate. They give rise to tissues such as craniofacial muscle, peripheral and enteric nerves and pigment cells. In adults NC derived tissues can give rise to neuroblastoma and melanoma. We have shown the drug Leflunomide can interfere with NC development and melanoma progression. Leflunomide is an inhibitor of transcriptional elongation, which is a critical part of mRNA synthesis. Recently both transcriptional pausing and elongation have been shown to playing a crucial role in regulating gene expression. We propose that Leflunomide sensitive transcriptional elongation and/or pausing plays a role in NC specification. We will test this using the Xenopus whole embryo and animal cap assays. We will determine the crucial time at which Leflunomide acts to inhibit NC development. We will then determine the role of genes involved in transcriptional pausing and elongation in more detail using morpholinos to knockdown their function in animal caps. In addition we will carry out RNA-seq and ChIP-seq on Leflunomide treated whole embryos and animal caps. These experiments will identify genome-wide those genes sensitive to Leflunomide treatment due to their being paused or sensitive to regulation of transcriptional elongation and which are required for NC development. Finally transcriptional pausing and elongation have been shown to be dependent upon the chromatin organisation around the promoters of many genes. We will use bioinformatic approaches to look at the chromatin configuration around the 5' regions of candidate genes. We will look at the profile of H3K4me3, H3K27me3 and Pol II around genes identified to be sensitive to Leflunomide and involved in NC development.

THE SCHOOL OF BIOLOGICAL SCIENCES AT UEA IS COMMITTED TO SIGNIFICANT GROWTH IN ENGAGEMENT AND ENTERPRISE. Leflunomide is an already FDA-approved drug used for treating patients with Rheumatoid Arthritis. Our exciting and novel research has discovered a potential new use for this drug to treat melanoma. The impact of this discovery is likely to be quite profound as it identifies a novel regulatory mechanism (transcriptional elongation and RNA Polymerase II pausing) as potential targets for drug discovery. Further research to fully understand the mechanism of action of Leflunomide and the role of transcriptional elongation and pausing in cell differentiation and cancer is therefore of high importance. We will utilise the Press Office at UEA and the media to maximise the impact of our work and to disseminate it to the scientific community and general public. Communications and Engagement: 1. I have previously publicised my work in the National and local newspapers and made it accessible to the general public. For example, when our paper on identifying a novel MMP inhibitor was published it garnered media attention from all around the world. Articles appeared in The Daily Telegraph, Daily Mirror, Metro, Liverpool Daily Post, The Northern Echo, EDP, BBC Online, Manchester Evening News, Reuters and numerous others including many websites and blogs. I was also interviewed on RTE Radio (Dublin). I will aim to publicise work from my lab when papers are published and grants are funded. We are certain that the work that has led up to this grant application will attract a lot of media attention (it has just been resubmitted after revision to Nature). The work arising from this new project will also be disseminated using the UEA Press Office. 2. As a lecturer at UEA my lab routinely runs a demonstration on Xenopus development at all University open days, Groups of prospective students and their parents are shown a short powerpoint presentation and a poster outlining what we do and why we are so interested in Development. The groups are also given the opportunity to observe live Xenopus embryos and tadpoles under a microscope. It is always gratifying to see how they react to visualising live tadpoles with beating hearts down the microscope. I have encouraged my PhD students to do lessons in local schools and have developed with them interesting presentations around the topic of Developmental Biology. Collaboration: As can be seen from the grant application this project involves a number of international collaborations at the academic level. The collaboration with Dr Richard White and Prof Len Zon at Harvard Medical School is an ongoing one. The collaboration with Dr Gert Veenstra who has expertise in Xenopus RNA-seq and ChIP-seq currently lacking from my lab is a new one I have fostered by e-mail and personal discussions at a recent meeting. I will continue to manage all these collaborations and coordinate with my colleagues regarding any publicity, which may come from the grant. Exploitation and Application: We will be seeking to exploit the research. Specific partnership agreements are already in place for related projects. We will have regular meetings with our enterprise and engagement officer to discuss results and work up opportunities. To protect the research we will have IP and MTA agreements with new collaborators, drawn up by our research contracts office. Capability: All personnel, particularly the PDRAs will be expected to engage with the impact agenda. Other staff involved will be our web development officer, our media office who will advise and assist with press releases and our School Enterprise and Engagement staff. I have a track record of such activities and have sought training from staff development programmes.