Word - Babraham_Institute - Discovery Biology for Biomedicine


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Word - Babraham_Institute - Discovery Biology for Biomedicine

  1. 1. PhD Student Opportunities 2011 The Babraham Institute is an international focus for innovative research in post-genomics studying gene function in cells, organs and systems, supported principally by the Research Councils. It is a recognised postgraduate teaching Department of the University of Cambridge. Starting October 2011 a number of Research Council Quota studentships will be available at Babraham leading to a University of Cambridge PhD degree. These studentships can be awarded for up to 4 years. In addition, we will be continuing a new Babraham European Studentship scheme, funded jointly by the Babraham Institute and the Cambridge Home and European Scholarship Scheme (CHESS), which will provide full funding for three further 3 year studentships. Please see our website (www.babraham.ac.uk) and the BBSRC website (http://www.bbsrc.ac.uk/funding/training/eligibility.pdf) for details of eligibility and funding. Non-EU nationals must find funding for academic fees and personal support. In cases where applicants must find their own funding, we will require evidence that the level of funding is at least equal to the standard BBSRC/MRC PhD funding package. Students will join a thriving scientific community situated on an attractive parkland campus near Cambridge. Our 70 students are all members of Cambridge Colleges and participate fully in University social and academic life (www.biomed.cam.ac.uk/gradschool/). Details of our interactive scientific programmes can be found on www.babraham.ac.uk. The Institute is fully equipped for state-of-the-art biological research including: innovative molecular biology, stem cell manipulation and transgenics, epigenetics, structural studies on chromatin, real-time laser scanning confocal microscopy, calcium imaging, fluorescence sorting of cells, gene targeting and knockouts, mouse models of disease, mouse behavioural testing, proteomics and lipidomics. The Institute PhD Recruitment Day will be held on WEDNESDAY 19th JANUARY 2011 to which selected students will be invited to attend interviews, discuss their research interests and view the Institute’s facilities. Full details of potential projects and supervisors are given below; our supervisors welcome informal enquiries. Potential projects (supervisor/title): Michael Coleman (michael.coleman@bbsrc.ac.uk): Mechanisms regulating mitochondrial trafficking in axons Anne Corcoran (anne.corcoran@bbsrc.ac.uk): Does non-coding RNA regulate nuclear structure? Sarah Elderkin (sarah.elderkin@bbsrc.ac.uk): Understanding regulation of Polycomb Repressor Complex 1 in embryonic stem cell self renewal and cellular proliferation Peter Fraser (peter.fraser@bbsrc.ac.uk): Long non-coding RNAs and their role in nuclear compartmentalization, genome organization and epigenetic regulation of gene expression Jon Houseley (jon.houseley@bbsrc.ac.uk): Investigating the roles of non-coding RNA in meiotic recombination Gavin Kelsey (gavin.kelsey@bbsrc.ac.uk): Gender-specificity of imprinting or How sex dictates gene expression
  2. 2. Nicholas Ktistakis (nicholas.ktistakis@bbsrc.ac.uk): Functional proteomics of autophagy Llew Roderick (llew.roderick@bbsrc.ac.uk): Signalling to chromatin to control cardiac hypertrophic gene transcription Len Stephens (len.stephens@bbsrc.ac.uk): Unraveling the signals that coordinate pathogen killing in phagosomes using a genome-wide RNAi screen Martin Turner (martin.turner@bbsrc.ac.uk): Novel pathways for the regulation of lymphocyte development Patrick Varga-Weisz (patrick.varga-weisz@bbsrc.ac.uk): Muscling into chromatin: Role for myosin and actin in chromatin remodeling Marc Veldoen (marc.veldhoen@bbsrc.ac.uk): The maintenance and function of lymphocytes in the skin Sonja Vermeren (sonja.vermeren@bbsrc.ac.uk): Analysing ARAP3 in angiogenesis Michael Wakelam (michael.wakelam@bbsrc.ac.uk): Regulation of mTOR signalling by phospholipase D during ageing Heidi Welch (heidi.welch@bbsrc.ac.uk): Targeting Rac-GEF activity Travel expenses will be paid to those invited to attend our Institute Recruitment Open Day. Applicants should submit a full Curriculum Vitae with a covering letter indicating the two projects in which they are most interested, in order of preference, and arrange for two referees to write to the Institute on their behalf before the deadline; please include your contact details for 3-19 January. Our website also provides a checklist of the information required to be provided in your application before it can be considered. Incomplete applications will not be considered. Please send your applications to: Ms Caroline Coursol, Graduate Studies Programme, The Babraham Institute, Babraham, Cambridge CB22 3AT, Tel: 01223 496324, Fax: 01223 496046 or email babraham.graduate@bbsrc.ac.uk by Friday 17th December 2010 An Equal opportunities employer. An Institute supported by the Biotechnology and Biological Sciences Research Council www.babraham.ac.uk Checklist of Information required for application - Full Curriculum Vitae including o Nationality and Residence in UK information o Details of Schooling including GCSE and A Level results (or other qualifications) o Details of University Education including courses taken and results of any examinations to date o Degree result (if already known)
  3. 3. o Details of any lab based projects or laboratory placements o Details of any industrial placements o Details of any previous employment - Covering Letter giving the details of the two projects in which you are most interested and your reasons for choosing them. These projects should be chosen from different Group Leaders. - The names and contact information of the two referees you have asked to write to the Institute supporting your application for a PhD position - Your contact details between 3rd and 19th January 2011 -------------------------------------------------------------------------------------------------------------------------- Projects for 2011 (full description) Michael Coleman (michael.coleman@bbsrc.ac.uk) Mechanisms regulating mitochondrial trafficking in axons This project will investigate mechanisms that regulate the trafficking of mitochondria along axons. Axons face unique intracellular transport challenges as they must deliver proteins and organelles to their sites of function many centimeters from their origins, and remove them to sites of degradation when they become non-functional. Mitochondrial dysfunction contributes to several neurodegenerative disorders including Parkinson’s disease and peripheral neuropathies. We have developed live imaging methods and new software (Andrews, Gilley and Coleman, submitted) to quantify mitochondrial transport both in primary culture and explanted tissue. By combining these methodologies with Babraham’s extensive resources of PI3 kinase inhibitors and subunit-specific genetically modified mice, we will test the hypothesis that a specific PI3 kinase subunit regulates axonal transport of mitochondria. We will then use appropriate inhibitors or mouse strains to investigate how this is altered in ageing and disease. Our group works at the forefront of studies into axon degeneration mechanisms, having identified a mutant protein that greatly delays axon degeneration (Mack et al., 2001) and a wild-type protein that influences the same pathway (Gilley and Coleman, 2010; Coleman and Freeman, 2010). Recently we studied the roles of axon pathology in Alzheimer’s disease (Adalbert et al., 2009) and Huntington’s disease as well as developing new methods for imaging axonal structure and transport that will be used in this project. References 1. Andrews, S., Gilley, J. & Coleman, M.P. Difference Tracker: ImageJ plugins for fully- automated analysis of multiple axonal transport parameters J Neurosci. Meth. (submitted) 2. Coleman, M.P. and Freeman, M.R. (2010) Wallerian degeneration, WldS and Nmnat. Ann Rev Neurosci. 33: 245-67. 3. Gilley, J. & Coleman, M.P. (2010) Endogenous Nmnat2 is an essential survival factor for maintenance of healthy axons. PLOS Biol 8: (1) e1000300. 4. Adalbert, R., et al (2008) Severely dystrophic axons at amyloid plaques remain continuous and connected to viable cell bodies. Brain 132: 402-16.
  4. 4. 5. Mack, T.G.A.,, et al (2001) Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene Nature Neuroscience 4: 1199-1206 Anne Corcoran (anne.corcoran@bbsrc.ac.uk) Does non-coding RNA regulate nuclear structure? Recombination of multiple genes in the immunoglobulin and T cell receptor DNA loci generates the vast repertoire of antibodies and T cell receptors required for a functional immune system. The focus of our research is to understand the chromatin remodelling mechanisms that regulate these antigen receptor loci, to facilitate V(D)J recombination in lymphocytes. Since these DNA loci are the largest in the genome, containing hundreds of genes that must colocalise, they have evolved several dynamic processes, including nuclear relocalisation (movement within the nucleus), histone modification, non-coding RNA transcription, 3D DNA looping (to bring distal genes together). Thus they also provide an excellent paradigm for chromatin regulation of all multigene loci. In particular, eukaryotic genomes have recently been shown to produce an enormous number of conserved non- coding RNAs, which are thought to play a vital role in many nuclear processes, including developmental regulation of gene expression, gene silencing and formation of functional nuclear substructures. However, the mechanisms are poorly understood. We have shown that large non-coding RNA transcripts are generated in the immunoglobulin heavy chain (Igh) locus prior to V(D)J recombination and are strongly implicated in regulation of recombination. This project will determine the function of these transcripts. In particular we will test the hypothesis that these transcripts contribute to movement of the Igh locus and formation of nuclear substructures that facilitate V(D)J recombination. We will also investigate a putative role for these transcripts in trafficking of key histone modifying enzymes to targets in the Igh. State-of-the-art techniques including high-throughput fluorescence in situ hybridization (FISH), chromatin immunoprecipitation (CHIP), genome- wide next generation sequencing (NGS) and bioinformatics will be used. References 1. Bolland et al 2004, Nature Immunology 5: 630-637 2. Bolland et al 2007, Mol Cell Biol 27:5523-33 3. Featherstone et al 2010, J Biol Chem 285: 9327-38 Sarah Elderkin (sarah.elderkin@bbsrc.ac.uk) Understanding regulation of Polycomb Repressor Complex 1 in embryonic stem cell self renewal and cellular proliferation Polycomb-group (PcG) repressor proteins are key epigenetic regulators involved in both establishing gene expression patterns and maintaining long-term cellular memory. Maintenance of cellular gene expression memory is an important process in regulation of embryonic stem cell self renewal, cell identity, cell proliferation and tumor development (Sparmann and van Lohuzin 2006). PcG proteins form large multi-protein complexes. The Polycomb Repressor Complex 1 (bmiPRC1) containing the Bmi1 subunit has been shown to be an E3 ubiquitin ligase that modifies chromatin by mono-ubiquitylation of histone H2A lysine 119. This epigenetic modification has been associated with gene repression (Wang et al. 2004). In mammalian cells multiple paralogues for the core PRC1 subunits have been identified. Recently we have identified a novel PRC1-like complex melPRC1 which contains the Bmi1 paralogue Mel-18.
  5. 5. Additionally we have found that melPRC1 mediates gene repression through mono- ubiquitylation of H2A lysine 119 is regulated by phosphorylation (Elderkin et al. 2007). We are particularly interested in understanding how epigenetic modifying PRC1-like complexes are potentially regulated by different signalling pathways and how specific genomic loci are silenced by different polycomb complexes to maintain specific transcription profiles in mammalian cells. The PhD project in our laboratory will involve understanding mechanistically how different genomic loci are regulated by specific polycomb complexes in embryonic stem cells. This project will provide strong training in embryonic stem cell and epigenetics research, protein and chromatin biochemistry, molecular biology and bioinformatics. Peter Fraser (peter.fraser@bbsrc.ac.uk) Long non-coding RNAs and their role in nuclear compartmentalization, genome organization and epigenetic regulation of gene expression We have identified a number of novel, long non-coding RNAs (ncRNA) that are enriched in, or restricted to, the nuclei of cells. This project will use various state-of-the-art fluorescence in situ hybridization (FISH) and imaging techniques to investigate the nuclear positioning and potential compartmentalization of these ncRNAs (Nagano et al., 2008). The applicant will use RNA TRAP (Carter et al., 2002; Nagano et al., 2008) adapted for high-throughput sequencing to comprehensively investigate and identify in vivo chromatin/gene targets of the most interesting of these ncRNAs. Potential ncRNA roles in chromatin modification, packaging, locus sequestration and nuclear genome organization (Nagano and Fraser, 2009) will be investigated using ChIP, 3C, 4C and Hi-C techniques. The successful applicant will join have the enthusiasm and intellectual qualities needed to generate and analyze large datasets, with the aim to understand novel gene regulatory mechanisms. References 1. Nagano T, Mitchell JA, Sanz LA, Pauler FM, Ferguson-Smith AC, Feil R and Fraser P (2008) The Air non-coding RNA epigenetically silences transcription by targeting G9a to chromatin. Science 322,1717-1720. 2. Carter, D., Chakalova, L., Osborne, C.S., Dai, Y-F, Fraser, P. (2002) Long-range chromatin regulatory interactions in vivo. Nat. Genet., 32,623-626. 3. Nagano T, Fraser P. (2009) Emerging similarities in epigenetic gene silencing by long noncoding RNAs. Mamm Genome 20:557–562 Jon Houseley (jon.houseley@bbsrc.ac.uk) Investigating the roles of non-coding RNA in meiotic recombination Only a tiny fraction of the human genome encodes proteins, but recent studies show that almost the entire genome is transcribed into RNA. This means that many more genes produce RNA than produce proteins, and the key aim of my research is to find functions for these non-protein coding RNAs. We are particularly interested in the potential roles of non-coding RNAs in coordinating genome rearrangements. Although genomes are often thought of as largely unchanging, they undergo extensive recombination during meiosis, the specialised cell division required
  6. 6. for sexual reproduction. These recombination events are vital for successful meiosis and are responsible for most of the mixing of parental traits in children. We and others have found evidence that non-protein coding RNAs and their associated enzymes can influence DNA recombination. This raises the fascinating possibility that non- coding RNAs can influence genome changes, and therefore that organisms have the ability to control how traits are assorted between progeny during reproduction. Furthermore, studies of meiotic recombination are medically important as cells that undergo too little recombination or recombination in the wrong place end up aneuploid, leading to miscarriages and conditions such as Down Syndrome. The aim of this PhD project is to investigate the roles of non-coding RNAs in coordinating meiotic recombination. It will particularly focus on the changes in chromosome structure that precede recombination and the importance of these changes to the initiation of recombination. The realisation that cells produce so much non-coding RNA provides new areas of fundamental biology to explore, and non-coding RNAs are of considerable pharmaceutical interest. This project will provide much sought-after experience in this fast-moving area of biology, which should be very beneficial to the successful candidate's future scientific career. Further reading 1. Houseley, J., Kotovic, K., El Hage, A. & Tollervey, D. Trf4 targets ncRNAs from telomeric and rDNA spacer regions and functions in rDNA copy number control. Embo J 26, 4996- 5006 (2007). 2. Kobayashi, T. & Ganley, A. R. Recombination regulation by transcription-induced cohesin dissociation in rDNA repeats. Science 309, 1581-1584 (2005). 3. Houseley, J. & Tollervey, D. The many pathways of RNA degradation. Cell 136, 763-776 (2009). 4. Nowacki, M. et al. RNA-mediated epigenetic programming of a genome-rearrangement pathway. Nature 451, 153-158 (2008). 5. Wahls, W. P., Siegel, E. R. & Davidson, M. K. Meiotic recombination hotspots of fission yeast are directed to loci that express non-coding RNA. PLoS ONE 3, e2887 (2008). Gavin Kelsey (gavin.kelsey@bbsrc.ac.uk) Gender-specificity of imprinting or How sex dictates gene expression Genomic imprinting is a form of epigenetic regulation that results in the alleles of certain genes being expressed with a legacy of their parental origin, and is profoundly important for embryonic growth and development in mammals. This parental-allele dependent gene activity is determined by primary imprint marks (at imprinting control regions, ICRs) which are laid down in gametes and stably maintained in somatic cells (Reik & Walter, 2001). A fundamental question is what is the mechanism responsible for germline-specific marking? Most known ICRs are marked by DNA methylation in oocytes, fewer acquire methylation specifically in male germ cells. Our work has identified components necessary for targeting DNA methylation to ICRs in oocytes (Chotalia et al., 2009), and shown that: it requires the ICR promoter to be quiescent; it depends on transcription across the ICR from an upstream promoter; and is likely to require an appropriate set of histone modifications (e.g., lack of H3K4methylation; Ciccone et al., 2009). Transcription through ICRs may down-regulate ICR
  7. 7. promoters to be methylated and/or establish a permissive histone modification pattern. Although we have a model for maternal germline marking, we also need to account for why these sequences are resistant to methylation in male germ cells. This project addresses the crucial question of gender-specificity in imprint establishment. We shall apply the logic underlying female germline ICR marking to male germ cells at the time of methylation establishment to discover why these ICRs are specifically protected. Analysis will be done at several imprinted loci using FACS-purified populations of male germ cells. Genome-wide studies employing next generation sequencing could be undertaken to extend to other sequences methylated specifically in oocytes that we have identified in genome-wide studies. The results of these analyses will lead to mechanistic studies, utilising transgenic, knock-down or targeting approaches, to examine the role of specific factors implicated in protection from methylation. References 1. Chotalia et al., (2009) Transcription is required for establishment of germline methylation marks at imprinted genes. Genes & Dev. 23:105-117. 2. Ciccone et al., (2009) KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints. Nature 461:415-418. 3. Reik & Walter (2001) Genomic imprinting: parental influence on the genome. Nat. Rev. Genet. 2:21-32. Nicholas Ktistakis (nicholas.ktistakis@bbsrc.ac.uk) Functional proteomics of autophagy Autophagy is an important cellular response to nutrient availability and stress. It is frequently altered in many disease models including cancer and neurodegeneration. Autophagy is also required for life span extension. Autophagosomes are novel organelles formed during autophagy that mediate the autophagic response. We have recently proposed that autophagosomes are formed within omegasomes, novel membrane compartments enriched in phosphatidylinositol 3-phosphate and connected to the endoplasmic reticulum. Previous work in my lab has led to the isolation of omegasomes and the characterization of some of the proteins that are found there. This project will explore the function of these proteins during autophagosome formation. References 1. Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, Griffiths G and N.T. Ktistakis 2008. J Cell Biol 182: 685-701 2. Burman C, Ktistakis NT. 2010 FEBS Lett 584: 1302-1312 Special Issue on Autophagy, N. Mizushima ed. 3. Burman C, Ktistakis NT. 2010 Seminars in Immunopathology, in press. Llew Roderick (llew.roderick@bbsrc.ac.uk) Signalling to chromatin to control cardiac hypertrophic gene transcription Cardiovascular diseases including heart failure and cardiac hypertrophy are the greatest causes of morbidity and mortality in the UK. Although, hypertrophy (heart muscle growth without cell division) induced by injury, genetic factors or prolonged hypertension are a precursor of heart failure, cardiac hypertrophy induced by exercise or pregnancy, for
  8. 8. example, improve cardiac function. Moreover, whereas, physiological hypertrophy is reversible, pathological hypertrophy is not. Understanding the mechanisms underlying the induction and progression of cardiac hypertrophy as well as the differences between the beneficial and the pathological forms of hypertrophy are therefore key steps to the development of future therapy. Research in our laboratory has pinpointed nuclear-localised calcium signals acting via specific transcription factors including NFAT as key to the induction of hypertrophy (Mol Cell 2009). Although modulation of histone acetylation is now also known to play a key role in the activation of hypertrophic gene phosphorylation, the role of other epigenetic modifications including methylation and phosphorylation is not determined. We hypothesise that histone methylation is instrumental in the transcriptional reprogramming. Moreover, we predict that different hypertrophic stimuli cause a characteristic signature of histone modifications and that this code determines the hypertrophic phenotype. To test these hypotheses, using animal and cell models of hypertrophy, the student will analyse the effect of pathological and physiological hypertrophic stimuli on methylation of histones of different hypertrophic gene markers (pathological and physiological), investigate the role of epigenetic modifiers in this process (histone methylases/demethylases) and analyse the signal transduction pathways involved. Reference 1. Higazi, D.R., Fearnley, C.J., et al., (2009) Endothelin-1-stimulated InsP3-induced Ca2+ release is a nexus for hypertrophic signaling in cardiac myocytes. Mol Cell, 33, 472-482. Len Stephens (len.stephens@bbsrc.ac.uk) Unraveling the signals that coordinate pathogen killing in phagosomes using a genome- wide RNAi screen Phagocytes such as neutrophils and macrophages are responsible for the detection, phagocytosis and destruction of many bacterial and fungal pathogens. These pathogens are detected by a range of cell-surface receptors that either recognise characteristic foreign molecules or host-derived complexes specifically decorating the pathogen surface. Once engaged the pathogens are drawn in and engulfed by the process of phagocytosis that culminates in the pathogen being sealed inside the phagocyte in a structure called the phagosome. Once created the phagosome changes rapidly, other intracellular membranes fuse and digestive enzymes, ions and reactive oxygen species (ROS) are pumped inside to kill and digest the contents. These processes are crucial to health, a number of life-threatening diseases result either from their dysfunction or from pathogens capable of evading this process. We have shown that a key event coordinating this process is the production of the signaling lipid phosphatidylinositol 3-phosphate (PI3P) in the phagosomal membrane by the class III PI3K, VPS341,2 . This event leads to the movement of a ROS-generating protein complex, via the ability of one of its components (p40PHOX) to specifically bind PI3P through its PX domain, to the cytosolic surface of the phagosome and hence drive transport of ROS into the lumen2,3 . In keeping with this, mutations in the PX domain of p40PHOX, that prevent PI3P binding, cause a human immunodeficiency syndrome4 and in mice reduce their ability to kill pathogenic bacteria such as Staphylococcus aureus5,6 . Furthermore, it is likely that there are further PI3P binding proteins involved in this pathway. Despite our detailed understanding of components of this pathway, the signals between the pathogen-recognition receptors and the engagement of VPS34 with the phagosome are
  9. 9. unknown. VPS34 is also known to be a regulator of other important cell responses, such nutrient-stimulated cell growth and intracellular trafficking of proteins, in organisms from yeast to man. In none of these settings, however, do we have a detailed molecular understanding of how VPS34 activity is controlled. Hence an explanation of how VPS34 is regulated during phagocytosis would have very broad significance. We have model cell lines that can phagocytose defined pathogens and express a fluorescent GFP-PX domain reporter construct that will dynamically localize to phagosomes in response to the accumulation of PI3P. Using fluorescence microscopy it is possible to visualize and quantify the accumulation of the reporter around phagosomes as an indirect measure of VPS34 activity in this compartment. In the context of our existing robotic liquid handling machines, high-throughput confocal microscope and image analysis software it is possible to perform such assays in 96-well tissue culture plates very rapidly. Hence we propose to use a genome-wide RNAi library (Dharmacon, 22,000 targets) to test the effects of individually suppressing the expression of all human genes on the ability of the reporter to decorate phagosomes. We expect a wide variety of hits, including activators and inhibitors and both novel and known molecules, that will need to be validated, classified and prioritized for further study as potential members of the network of molecules that control VPS34 and the maturation of phagosomes. References 1. Anderson, K.E., et al. CD18-dependent activation of the neutrophil NADPH oxidase during phagocytosis of Escherichia coli or Staphylococcus aureus is regulated by class III but not class I or II PI3Ks. Blood 112, 5202-5211 (2008). 2. Ellson, C.D., et al. Phosphatidylinositol 3-phosphate is generated in phagosomal membranes. Curr Biol 11, 1631-1635 (2001). 3. Ellson, C.D., et al. PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40(phox). Nat Cell Biol 3, 679-682 (2001). 4. Matute, J.D., et al. A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity. Blood 114, 3309-3315 (2009). 5. Ellson, C., Davidson, K., Anderson, K., Stephens, L.R. & Hawkins, P.T. PtdIns3P binding to the PX domain of p40phox is a physiological signal in NADPH oxidase activation. Embo J 25, 4468-4478 (2006). 6. Ellson, C.D., et al. Neutrophils from p40phox-/- mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing. J Exp Med 203, 1927-1937 (2006). 7. Herre, J., et al. Dectin-1 uses novel mechanisms for yeast phagocytosis in macrophages. Blood 104, 4038-4045 (2004). Martin Turner (martin.turner@bbsrc.ac.uk) Novel pathways for the regulation of lymphocyte development B lymphocytes develop in the mammalian bone marrow and T lymphocytes develop in the thymus after passing through a well-characterised series of intermediate stages. The progression and phenotype of cells can be assessed by their cell surface phenotype and their transcriptional profile. This type of approach has led to the emergence of a concept of transcriptional networks controlling lymphocyte development, with one or a few
  10. 10. transcription factors acting as “master regulators”. Work from our group has placed emphasis on post-transcriptional processes and their impact on the development and function of lymphocytes. Post-transcriptional processes affect the handling of RNA after transcription and can influence the expression of genes by altering the stability of the mRNA or by changing the rate at which the mRNA is translated into protein. Small RNAs called microRNAs are one aspect of this level of control. RNA binding proteins are also key regulators of mRNA stability and translation. In the project the student will examine the role and mechanism of RNA binding protein function using conditional gene expression mouse models of lymphocyte development. Reference 1. Hodson et al. Nature Immunology 2010 Aug; 11(8):717-24 and comment in Nat Immunol. 2010 Aug; 11(8):697-8. Patrick Varga-Weisz (patrick.varga-weisz@bbsrc.ac.uk) Muscling into chromatin: Role for myosin and actin in chromatin remodeling Actin and myosin are force-generating molecules that have many important functions in cell mobility and cell shape. Over the last few years, it has become clear that these factors also have roles in the nucleus and are important cofactors for transcription. What these proteins do in the nucleus precisely and how they exert their function is largely an enigma. Actin and actin-related proteins are stable components of several chromatin remodeling factors. We will study what is the link between myosin and actin-containing chromatin remodeling factors in transcription regulation, DNA replication and repair. Our hypothesis is that myosin targets remodeling factors in response to environmental signals to control chromatin shape and position. We will combine yeast genetics with genome-wide binding maps to analyze the role of myosin in nuclear processes. We will use chromosome conformation capture technology and live cell imaging to map how the myosin-actin interaction dynamically shapes chromosomes in the living cell. We will study how the interaction between myosin and chromatin is regulated. Initially, these studies will be done in yeast, but if successful, we will test principles in mammalian cells in culture. References 1. Fission yeast Iec1-ino80-mediated nucleosome eviction regulates nucleotide and phosphate metabolism. Hogan CJ, Aligianni S, Durand-Dubief M, Persson J, Will WR, Webster J, Wheeler L, Mathews CK, Elderkin S, Oxley D, Ekwall K, Varga-Weisz PD. Mol Cell Biol. 2010 Feb;30(3):657-74. 2. Regulation of higher-order chromatin structures by nucleosome-remodelling factors. Varga-Weisz PD, Becker PB. Curr Opin Genet Dev. 2006 Apr;16(2):151-6. Review. Marc Veldoen (marc.veldhoen@bbsrc.ac.uk) The maintenance and function of lymphocytes in the skin The mammalian host is engaged in continuous mutual communication with its environment, which provide invaluable signals that maintain a healthy state of being. These interactions often involve small chemical messenger molecules some of which can be derived from the
  11. 11. abundantly present and diverse comensal microbiota that occupy the epithelial sites, the skin and gastro-intestinal tract (Hooper et al., 2002). But interestingly, other factors can be more directly derived from the physical world around us, e.g. the direct exposure to sunlight activates a cholesterol precursor, resulting in the production of cholecalciferol (Vitamin D3). Its converted products, not only regulate concentration of calcium and phosphate in the blood stream but can also function as a cytokine, enhancing the body's defensive mechanisms against invading microorganisms (Adams and Hewison, 2010). Epithelial sites, contain their own specialised subsets of lymphocytes. These cells are thought to have specialised functions in host-defence mechanisms, epithelial barrier formation and in the maintenance of a healthy microbiota (Swamy et al., 2010). Preliminary results in our lab indicate that a close relationship exists between some populations of lymphocytes in the skin and external factors that together impact the maintenance of a healthy and well functioning epithelial barrier. The successful applicant will study the development and maintenance of skin lymphocytes under carefully managed conditions, and will investigate the consequences of altered environmental circumstances on the functioning of the epithelial barrier as well as the microbial composition. References 1. Adams, J.S., and Hewison, M. (2010). Update in vitamin D. J Clin Endocrinol Metab 95, 471-478. 2. Hooper, L.V., Midtvedt, T., and Gordon, J.I. (2002). How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annu Rev Nutr 22, 283- 307. 3. Swamy, M., Jamora, C., Havran, W., and Hayday, A. (2010). Epithelial decision makers: in search of the 'epimmunome'. Nat Immunol 11, 656-665. Sonja Vermeren (sonja.vermeren@bbsrc.ac.uk) Analysing ARAP3 in angiogenesis A vascular system is crucial for the provision of nutrients and oxygen to the developing organism. Initially, precursors fuse to form a primitive vascular plexus as well as the dorsal aorta by a process termed vasculogenesis. Later, this primitive plexus is remodelled by angiogenesis to form a mature network of arteries and veins, which are connected by capillaries [1]. This involves the formation of new vessels by sprouting, by splitting of existing vessels, and by pruning of the pre-existing primitive plexus. Angiogenesis requires tight co-ordination of multiple important processes including cell growth, differentiation and motility. Outside of development, angiogenesis is also important for a number of pathological situations, one example being tumour growth. ARAP3 is a GTPase activating protein for the small GTPases RhoA and Arf6, which is regulated by phosphoinositide 3-OH kinase (PI3K) and the small GTPase Rap [2-4]. The catalytic function of PI3Ka is absolutely required for angiogenesis [5]. We showed recently in vivo and ex vivo that PI3K signalling through ARAP3 is essential for developmental angiogenesis [6]. However, Rap has also been shown to be required for angiogenesis [7]. Aim of this project is to address, whether ARAP3 might act as a co-incidence detector for signals from Rap and PI3K, to regulate Arf and Rho GTPases in angiogenesis. To address this, you will analyse angiogenesis in a variety of assays both in vitro and in vivo, focusing on
  12. 12. sprouting of new vessels, which in turn depends on cell motility. You will use primary endothelial cells in which ARAP3 has been knocked down retrovirally using siRNA to perform motility and sprouting assays. In addition, you will analyse sprouting angiogenesis in murine hindbrain and retina samples. The successful candidate should be a highly motivated and enthusiastic individual. He or she will join a young and dynamic group. You will acquire diverse skills in the fields of cell biology, molecular biology, immunohistochemistry, imaging, as well as in vitro assays. References 1. Adams & Alitalo (2007) Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol 8,464. 2. Krugmann et al. (2002) Identification of ARAP3, a PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoaffiinity matrices. Mol Cell 9, 95. 3. Krugmann et al. (2004) ARAP3 is a PI3K and Rap-regulated GAP for RhoA. Curr Biol 14, 1380. 4. Craig et al. (2010) ARAP3 binding to phosphatidylinositol-(3,4,5)-trisphosphate depends on N-terminal tandem PH domains and adjacent sequences. Cell Signal 22, 257. 5. Graupera et al. (2008) Angiogenesis selectively requires the p110a isoform of PI3K to control endothelial cell migration. Nature 453, 662. 6. Gambardella et al. (2010) PI3K signalling through ARAP3 is essential for developmental angiogenesis. submitted. 7. Chrzanowska-Wodnicka et al. (2008) Defective angiogenesis, endothelial migration, proliferation, and MAPK signaling in Rap1b-deficient mice. Blood 111, 2647. Michael Wakelam (michael.wakelam@bbsrc.ac.uk) Regulation of mTOR signalling by phospholipase D during ageing Rapamycin is a potent antifungal metabolite that inhibits proliferation of mammalian cells and possesses immunosuppressive properties. The mammalian target of Rapamycin, mTOR, is a serine/threonine kinase that is present in all eukaryote organisms, and has been implicated in regulating life span. During development, mTOR primarily regulates growth, but in the adult, where there is relatively little growth, mTOR controls ageing. mTOR integrates signals received from growth factors, nutrients, stress and the energy status of cells, to regulate cellular growth, autophagy, proliferation and shape. Aberrant mTOR activity results in the initiation and progression of several age-related diseases. These include human cancers, the development of autoimmune disorders such as rheumatoid arthritis and Parkinson’s disease, cardiac hypertrophy and type II diabetes. Consequently, mTOR inhibitors such as Rapamycin may be useful therapeutic agents for several age- related human diseases. The Ras-related small G protein, Rheb, activates mTOR and has been shown to induce oncogenic transformation in vitro, and to produce the rapid development of lymphomas and prostate tumours in vivo. The tumourigenic activity of Rheb is dependent on mTOR activity. Rheb activates mTOR in response to growth factors and nutrients by two distinct mechanisms; (1) Rheb binds to an endogenous inhibitor of mTOR, FKBP38, thereby antagonising the inhibition of mTOR by FKBP38, and (2) Rheb binds to and activates
  13. 13. phospholipase D1 (PLD1), thereby elevating the levels of the lipid second messenger phosphatidic acid (PA), a direct activator of mTOR. Mammalian cells express a second isoform of phospholipase D, PLD2, which has also been reported to also regulate mTOR. However, in contrast to PLD1, PLD2 does not appear to be regulated by Rheb, but instead binds to Raptor, an mTOR-interacting protein that is required for mTOR signalling. The aim of this studentship is to: (1) investigate the physiological significance of the Rheb/PLD1/mTOR and PLD2/Raptor/mTOR pathways in regulating mTOR signalling in response to growth factors, nutrients, stress and the energy status of the cell; (2) determine if the tumourigenic activity of Rheb is also dependent on PLD activity; and (3) elucidate the mechanism(s) of PLD/PA regulation of mTOR activity and the importance of these pathways in controlling autophagy. These aims will be achieved through the use of a set of novel isoform-specific PLD1 and PLD2 inhibitors in established model cell lines, and the use of primary cells isolated from mice that no longer express PLD1 and/or PLD2. The possible importance of PLD acting as a scaffolding protein in regulating these pathways will be investigated by transfection of catalytically inactive PLD1 or PLD2 into cells isolated from the knockout mice. Plan B will focus upon determining the molecular regulation of the mTOR complex in vitro – the proteins and lipids are available in the lab. Heidi Welch (heidi.welch@bbsrc.ac.uk) Targeting Rac-GEF activity The small G protein Rac is a key regulator of cytoskeletal structure (and hence cell shape, adhesion, motility, phagocytosis, regulated secretion), gene expression and oxygen radical formation. Dysregulated Rac activity is implicated in a broad range of human disorders ranging from cancer metastasis to inflammatory conditions. Hence, the ability to control Rac activity with drugs would be useful tool in the fight against a range of clinically important human pathologies. However, targeting of Rac activity itself does not seem a good idea, because of the plethora of functional roles which Rac fulfils and the therefore likely devastating side-effects of any such direct drugs. Like all small G proteins, Rac is activated by guanine-exchange factors (GEFs), and just like Rac itself, Rac-GEFs are implicated in a variety of human diseases, mainly immune disorders, cancer and developmental disorders. Rac-GEFs greatly outnumber Rac isoforms, and different types of GEFs couple Rac to different signalling pathways. Therefore, it seems more promising to target the activities of specific Rac-GEFs rather than Rac activity itself. For activation of Rac, the catalytic domain of Rac-GEFs forms a transient protein / protein interaction with Rac. Largely due to structural complexity, this interaction between GEFs and Rac has, until recently, not received much attention as a potential target for novel therapeutics. However, several groups have recently succeeded in identifying small molecule inhibitors that target this interaction specifically, notably NSC23766 and EHT1864 (Gao Y et al, 2004, PNAS 18:7618; Shutes A, et al, 2007, 282:35666), although the potency of these early compounds is low. Our lab focuses on one particular family of Rac-GEFs that we discovered a few years ago, the P-Rex family (Welch HC et al, 2002, Cell 108:809). The P-Rex family controls the function of
  14. 14. neutrophils (cell of the innate immune system that clear bacterial and fungal infections; Welch HC et al, 2005, Curr Biol) and of Purkinje neurons (cells in the cerebellum which regulate fine motor control; Donald S et al, 2008, PNAS 105:4483). There is also exciting recent evidence for P-Rex family involvement in cancer metastasis (Fine et al, 2009, Science 325:1261; Qin et al, 2009, Oncogene 28:1853; and unpublished results). Hence, P-Rex family Rac-GEFs seem valid targets for the development of novel therapeutics. We have very recently embarked on developing inhibitors for the P-Rex family, starting with a basic modeling approach to identify possible candidate compounds that may inhibit the P- Rex-dependent activation of Rac. Quite surprisingly, we have obtained very encouraging preliminary results in vitro, notably regarding the potency of our compounds (unpublished). This PhD project is the detailed characterisation of these early lead P-Rex inhibitors for their potency and specificity in vitro and in vivo. It involves in vitro and in vivo GEF assays with a range of GEFs (P-Rex, isolated P-Rex domains, other types of GEFs) and substrates (Rac and other GTPases) as well as assays for Rac-dependent cell functions in transfected cell lines (e.g. cell shape, motility, invasion assays) and in primary cells, such as isolated neutrophils that either express or lack P-Rex. Depending on the data obtained on specificity and potency of the compounds in the early phases of characterisation, there is also a possibility of further testing of the inhibotors in murine models of inflammatory disorders and metastasis later on. This project has the potential to move P-Rex inhibition towards a translation phase and render the compounds commercially exploitable for the development of anti- inflammatory or anti-metastatic drugs in the future.