NIHRDataInsights/HRCSData huggingface dataset:
Title: Mechanism and regulation of RNA-directed chromatin modification.
Funder: Medical Research Council

Abstract: An understanding of genome regulation is essential for understanding disease. Recent studies have revealed that non-coding (nc)RNAs have widespread roles in genome regulation, including transcriptional silencing via chromatin modification. Despite this, the molecular mechanisms underlying such RNA-directed chromatin modification (RdCM) remain poorly understood. My aim is to understand the mechanism, regulation and roles of RdCM, using the fission yeast Schizosaccharomyces pombe as a model system. Heterochromatin formation at fission yeast centromeres is an excellent paradigm for RdCM in general. Chromatin modification is known to require non-coding centromeric RNAs, processed by the RNAi intereference (RNAi) pathway. However, the mechanism by which RNAi recruits chromatin modifiers has remained obscure. My first objective is to elucidate the mechanism of RdCM by determining the functions of four novel proteins I have identified as being involved in RdCM. One of these proteins, Stc1, plays a key role in linking RNAi to chromatin modification; I will characterise the functions of Stc1 to determine the molecular mechanism by which Stc1 mediates RNAi-dependent chromatin modification. I will also determine the roles of the other three proteins in facilitating RdCM. My second objective is to identify and characterise negative regulators of RdCM. Ectopic chromatin modification can down-regulate essential genes, a cause of cancer. Proper regulation is therefore critical, but despite this, little is known about regulators of RdCM. I will perform genetic screens to identify proteins that negatively regulate RdCM, and determine their mode of action. My third objective is to characterise alternative roles of RdCM. Thus far studies in fission yeast have focussed on the role of RdCM in maintaining constitutive heterochromatin. However, preliminary data suggests that, as in higher eukaryotes, RNAi/RdCM also regulates retroelements and certain genes. I will determine the mechanisms and roles of ncRNA-mediated regulation at these loci, and examine how these pathways may contribute to stress responses. These studies will address specific questions about the mechanism and regulation of RdCM, and broaden the scope of fission yeast as a model for ncRNA-mediated genome regulation. The characterisation of these pathways in yeast and identification of homologous components in other species will form a basis for future studies aimed at understanding analogous pathways in humans. Changes in ncRNAs and defects in RdCM have been linked to numerous diseases, so understanding the mechanisms and regulation of RdCM will help us to understand many diseases associated with mis-regulation of gene expression in humans.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Structural studies on macromolecular complexes in translational control and ribosome biogenesis
Funder: Medical Research Council

Abstract: Translation of messenger RNA (mRNA) into protein on the ribosome is a fundamental process at the core of cellular function. Ribosomes constitute a large protein-RNA assembly that, in eukaryotes, requires ~170 soluble factors during ribosome biogenesis. In eukaryotes, the export of pre-ribosomal particles across the nuclear envelope requires adaptor proteins to recruit components of the nuclear export machinery. Pre-ribosomal particles are only exported once they have reached a particular maturation state and translation does not occur until ribosome maturation is completed in the cytoplasm. The rate of translation of a given mRNA is dependent not only on its availability but also on protein factors bound to the mRNA that can either promote or inhibit translation, sequester mRNAs into stress granules or processing bodies, or otherwise alter the location of mRNAs within the cell. Many transiently expressed mRNAs contain cis-acting sequences in their 3‘ untranslated regions that mark them as unstable and recruit protein factors that activate mRNA turnover. Signalling events can induce binding of other protein factors that protect these mRNAs from degradation. The work proposed here will use a combination of biochemistry and X-ray crystallography to gain insights into the functions of macromolecular complexes involved in translational control of mRNAs and in ribosome biogenesis. Understanding the structures of proteins in different states and/or complexes often provides key insights into their function, and allows hypotheses about the molecular mechanisms of cellular processes to be developed. These hypotheses can then be tested by creating site-directed mutants that are examined in biochemical and cell-based assays. The first project examines how structured cis-acting sequences in certain mRNAs recruit a protein complex known as NF90/NF45. This complex promotes the stability of bound mRNAs and inhibits translation. This complex is also a cellular host factor recruited by hepatitis C virus to its RNA genome and co-localises at viral replication foci in infected hepatocytes. This project aims to understand the molecular basis for RNA recognition by NF90/NF45 in these different systems The second project will address the molecular basis for the final maturation stages in eukaryotic ribosome biogenesis, based initially on studies in budding yeast. Removal and recycling of ribosomal export factors in the cytoplasm is often coupled to the insertion of ribosomal proteins. I will examine complexes of ribosomal processing factors with ribosomal proteins to understand the mechanism behind this functional coupling.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Genetic association analysis in family samples with missing data
Funder: Medical Research Council

Abstract: Family-based association analysis uses samples of cases and their relatives to draw inferences on disease association: most commonly, cases and their parents, or sib pairs (including twins) are used. Their advantages include matching of subjects for genetic and environmental background, and the ability to distinguish the effects of maternally inherited genes from those paternally inherited. Major collections of family data are in progress and require rigorous statistical methods for their epidemiology. This project will extend our previous work on likelihood based analysis of samples with missing data. We will develop and implement methods for distinguishing the effects of genes transmitted by the mother from those carried by her. We will develop proper analysis of quantitative traits when subjects have been sampled on the basis of their trait values. We will devise appropriate methods for analysis of copy number variants in family data. In each of these applications we will allow for missing genotype data, in particular missing parents in nuclear families, in a statistically optimal fashion. These methods will be applied in a number of collaborative studies. We will also translate our methods into a general epidemiological framework to allow more widespead application outside of genetics.


Research activity codes: 2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: GENERIC HEALTH RELEVANCE
Title: Methods for modelling repeated measures in a lifecourse framework
Funder: Medical Research Council

Abstract: There is increasing emphasis in medical research on fetal and childhood antecedents of disease, and how these interact with other exposures throughout the lifecourse to influence later-life conditions. Answering questions about the relative importance of aspects and timing of growth, behaviour and health status for longer term outcomes requires longitudinal data. Analysis of such data must account for dependencies between repeated observations on the same person, including serial autocorrelation (greater correlation among measurements closer together in time). Where there are repeated measures of exposures related to a later-life outcome, standard regression models may be affected by multicollinearity. Measurement error may vary over time, and there will usually be dropout over time (due to death, illness, etc). The proposed research will focus on developing methods for more robust analysis of lifecourse data, by tackling these problems First, we will improve the modelling of trajectories of change over time. We will develop methods for detecting and modelling autocorrelation (e.g., autocorrelation around a growth spurt or a period of illness). The impact of the assumption of Normality and constant variance in modelling growth trajectories will be examined, and methods for re-parameterising the variance function developed. We will then develop (and include in standard statistical software) methods for selecting the best-fitting parameterisation of growth curves. Second, we will tackle problems inherent in modelling the relationship between correlated exposures and later outcomes. The role of partial least squares in reducing the dimension of multiple correlated exposures will be examined. There are often several competing lifecourse hypotheses related to any given problem, and we will develop methods for distinguishing between these. We will develop (and include in standard statistical software) methods for including the uncertainty in the estimation of growth trajectories or latent classes of growth when using them as exposures in a model for later outcome. Finally, we will explore the joint modelling of exposures and outcomes using cross-lagged models, and the impact of modelling the initial conditions of such models. We will compare our methods to others (including simple lifecourse approaches, path analysis) to answer scientifically important questions pertaining to the ALSPAC study. Data from other longitudinal studies available to the co-applicants, and simulated data, will be used to test the generalisibility of our methods and conclusions. We will develop guidelines for deciding when simpler methods are likely to be useful and unbiased, and when more statistically sophisticated methods would be more appropriate.


Research activity codes: 2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: GENERIC HEALTH RELEVANCE
Title: Understanding how cells trigger mitosis
Funder: Medical Research Council

Abstract: We aim to understand what controls the entry to mitosis in human cells. Although many important regulators have been identified it is not known whether we have identified all the necessary factors, nor how they act on each other to make the final decision to enter mitosis. This question is important to the understanding and treatment of cancer because when cells enter mitosis with damaged DNA this can lead to chromosome breakage or loss and consequent aneuploidy, which can clearly contribute to the development of cancer. To provide a definitive answer to this problem we will combine single cell imaging with cell engineering and biochemistry to determine: how Cyclin A promotes mitosis; and how the decision to activate Cyclin B1-Cdk1 is finally made; and how the cell cycle machinery is coordinated in throughout the cell. By mass-spectrometry we have already identified the proteins specifically associated with Cyclin A and Cyclin B1 at each stage of the cell cycle, which has revealed a number of candidates for proteins that could be involved in the control of mitosis and will be pursued. Single cell imaging will provide the necessary temporal resolution to determine when and how cells enter mitosis and we will use FRET probes for CyclinA-Cdk and for Cyclin B1-Cdk1 activity to assay both the exact time when each kinase is turned on, and the kinetics with which it is activated in living cells. In addition to transgenic mouse embryos, recent advances in gene targeting in human cells now allow us to combine the resolution of single cell imaging with biochemical analysis in defined genetic backgrounds. Using gene targeting we will introduce epitope tags and point mutations into specific genes to generate cell lines containing analogue-sensitive kinases, conditionally unstable proteins, and epitope-tagged proteins that will enable us both to assay their level and localisation in living cells and to identify their interaction partners and post-translational modifications by mass spectroscopy. The analogue-sensitive kinases and conditionally unstable proteins will enable us specifically to inactivate a chosen protein at a defined point in the cell cycle, using an ATP-analogues for kinases, and either Cre-mediated excision or an inducible-degron for other proteins, such as phosphatases. With this multi-pronged approach we will be able to identify the molecular mechanisms that control entry to mitosis and how this is coordinated with the unreplicated DNA and damaged DNA checkpoint pathways.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Mass spectrometry at the frontiers of molecular medicine
Funder: Medical Research Council

Abstract: Much progress has been made in obtaining atomic images of the important machineries that govern protein folding, degradation and transport across the cell membrane. Complementary information is required however to probe the dynamic changes in organization and conformation that these protein assemblies undergo. We propose to develop and apply mass spectrometry based approaches to a range of biomolecules that are refractory to conventional structural approaches. We plan to develop our methodology to overcome two of the most challenging characteristics displayed by protein complexes: intrinsic heterogeneity; and association with membranes. The difficulties these pose is exemplified in two systems: the polydisperse small heat shock proteins chaperones that play a crucial role in maintaining protein homeostasis; and the membrane channels and transporters that play an active role in small molecule/drug transport. The small heat shock proteins are implicated in a range of protein deposition diseases, with specific variants directly linked to cataract, desmin-related cardiomyopathy and Charcot-Marie-Tooth disease. The membrane channels and transporters we will study have important implications in type II diabetes, and the oral bio-availability of drugs. Through the basic biomedical research programme outlined here we will probe the molecular properties of these assemblies with a particular focus on monitoring their oligomeric state, heterogeneity, dynamics and conformational changes; and the ways in which these are altered in response to small molecule binding. We plan to implement the results of these fundamental studies to gain insight into the mechanism of ligand binding and its effects on these dynamic properties. We anticipate that the outcomes of this research will be a new means of studying these intractable biomolecules that will be broadly applicable to studying the effects of small molecules in polydisperse and membrane associated assemblies with high impact for biomedical research.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Mechanisms underlying limb regeneration in an adult vertebrate
Funder: Medical Research Council

Abstract: Limb regeneration in salamanders proceeds by formation of the limb blastema, a mound of mesenchymal stem cells at the end of the stump. In earlier work we identified the GPI-anchored cell surface protein Prod 1, a member of the three-finger protein (TFP) superfamily, as a potential determinant of blastemal cell identity. Prod 1 can signal to the interior of cells in part by activating the Epidermal Growth Factor receptor. The secreted newt Anterior Gradient Protein (nAG) interacts with Prod 1 as a ligand-receptor combination. nAG expression is regulated by the nerve supply, not only during limb regeneration but also during limb development. nAG is able to rescue the regeneration of a blastema in the absence of the nerve. The mechanism of action of nAG will be studied at the molecular level in relation to its thioredoxin fold, essential cysteine residue, and possible formation of a disulphide bonded heterodimer with Prod 1. We will also study its activity in relation to confrontations of proximal and distal blastemal cells, since we propose there is a connection between the implementation of positional identity by Prod 1 and growth control by the nerve (nAG). The studies on Prod 1 signalling have led to identification of a point mutant that acts as a dominant negative (residue 59), and we propose to utilise this in transgenic axolotls in order to obtain information about Prod 1 function in regeneration. Prod 1 is the first example of a taxon-specific protein implicated in regeneration. This has important implications for our understanding of both the evolution of regeneration and how to extend regenerative ability in mammals. An expansion group of newt and axolotl TFPs derived from a single salamander-specific sequence has been identified and named the Stef genes. These GPI-anchored proteins provide a focus for efforts to define the taxon-specific aspects of the mechanism of regeneration, and the identification of their putative binding partners will be an important goal in understanding their activity. Our analysis should provide new information about the mechanism of limb regeneration, and about certain critical steps that are required to establish a regenerative outcome to limb amputation.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Using GO to enhance the utility of drosophila data to medical research
Funder: Medical Research Council

Abstract: The aim of this proposal is to improve the accessibility and utility of drosophila research data to scientists involved in medical research. We will achieve this by improving the breadth and depth of drosophila Gene Ontology (GO) annotation with emphasis on annotating orthologs of human genes. We will prioritise drosophila genes relevant to human disease and genes where data about the gene in human is limited. Each gene target will be comprehensively annotated with GO terms based on a manual review of the published literature. In a complementary approach, we will improve consistency of GO annotation by annotating sets of genes with shared functions. We will also improve annotation coverage of all drosophila genes by incorporation of more high-throughput data sets and introducing new annotation strategies such as proven text mining approaches and community annotation. The more comprehensive GO Annotation will improve the accessibility of data on drosophila gene function to the wider research community. In collaboration with FlyBase, we will seek to improve ontology based links between drosophila mutant phenotypes and the defects caused by hereditary diseases, enabling researchers to search for genes related to a particular disease.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: A Structural Investigation of Dephosphorylation in an Hsp90 Regulatory Complex
Funder: Medical Research Council

Abstract: Regulation of the Hsp90 molecular chaperone by post-translational modification is an important but neglected aspect of Hsp90 biology. Since many of the proteins dependent on Hsp90 for activation are oncogenes, an understanding of these regulatory pathways will provide new avenues for the design of chemotherapeutics. For example, phosphorylation and dephosphorylation of the cochaperone Cdc37 are essential steps in the process of activation of Hsp90-dependent kinases. The dephosphorylation step proceeds in cis by the simultaneous binding of Cdc37 and the phosphatase PP5 to Hsp90. The aim of this project is to gain structural data about the interactions within the complex formed by these three proteins. We will use X-ray crystallography in a reductionist approach to obtain atomic resolution detail of domain interactions, and single particle electron microscopy in an holistic approach to determine the structure of the complex as a whole. These results will provide a structural understanding of two important aspects of the biology in this system: (1) regulation of Hsp90 function by the action of the PP5 phosphatase on the phosphorylated cochaperone Cdc37; and (2) the regulation of the phosphatase PP5 by Hsp90 and the role of Hsp90 in PP5 substrate recognition. These results will provide a platform from which drugs that aim to abrogate the interactions involved can be designed.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: UK Stem Cell Bank - Third Phase
Funder: Medical Research Council

Abstract: Following competitive tender to establish the UK Stem Cell Bank (the Bank) in 2002, and successful delivery of all objectives for Phase 1 funding (2003-2005), a Phase 2 grant for this project (2006-2011) was secured by the National Institute for Biological Standards and Control (now a centre of the Health Protection Agency). All objectives are again being met on time and within budgets agreed with sponsors. A description of progress made in Phase 2 is given in the attached Progress Report and in the Annual Reports on its website at www.ukstemcellbank.org.uk. During Phase III, the Bank will focus its activities on supporting the key public health aspects of stem cell therapy and development of human cell-based assays in safety testing of products. Phase 3 will see the Bank coordinating more closely with translational scientific programmes, thus making the Bank?s resources available to provide broad support for the UK?s developing the regenerative medicine clinical community, both academic and commercial,and those developing models for in vitro toxicology and drug discovery. The Bank will work in closer coordination with commercial partners whilst retaining its neutral position and capability to support all stakeholders in this field. At the same time, the Bank aims to sustain and develop its position as the foremost repository of ethically-sourced and well characterised stocks of human stem cell lines banked to international quality standards. The Bank will continue to develop its international role in supporting the development of stem cell therapy through: ? the provision of quality-assured and safety tested cell lines and ancillary products; ? its continued lead on best practice and the setting of international consensus standards; ? its developing training programme for stem cell scientists, technical staff and quality assurance personnel; ? its scientific collaborations and internal research programme to establish robust methodologies for cell expansion, differentiation, preservation and characterisation. Furthermore, the Bank proposes to implement its cost recovery plan to progressively reduce its dependence on Research Council funding during the period of Phase 3. These aims are intended to sustain the leading International profile of the Bank whilst increasing its support for commercial delivery of products and at the same time progressing towards substantially removing the burden of funding which has fallen on its sponsors.


Research activity codes: 5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT);1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE