NIHRDataInsights/HRCSData huggingface dataset:
Title: RNA helicases as regulators of the response of B-cells to programmed DNA rearrangements
Funder: Wellcome Trust

Abstract: In mature B-cells, the generation of antibodies of different isotypes relies on class switch recombination (CSR) mechanisms. Although crucial for adaptive immunity, CSR imposes challenges to genome integrity as it involves programmed induction of DNA double-strand breaks (DSBs) in rapidly proliferating B-cells. A key player in the cellular response to CSR DSBs is the protein kinase ataxia telangiectasia mutated (ATM). Evidence suggests RNA-dependent mechanisms control the DNA damage response by ATM, although there is very limited understanding of how these function in B-cells and which RNA-binding proteins (RBPs) are required for CSR. This proposal aims to define the critical roles of RNA helicases as integrators of ATM signals to control class switch recombination mechanisms in B-cells. I propose an integrative approach combining genomic and proteomic methodologies together with ex vivo B-cell differentiation systems and conditional gene-targeting in mice. I will determine RNA helicase-dependent mechanisms controlling the cell-cycle and CSR DSB-repair and investigate their roles in B-cell immune responses. This work will provide new insight into RNA helicase-mediated pathological mechanisms resulting in CSR deregulation and the development of immune disease or B-cell lymphomagenesis.

Lay abstract: B-lymphocytes are cells of the immune system responsible for the production of antibodies (specialized proteins called immunoglobulins), which recognize specific pathogens. B-cell development occurs concomitantly with mechanisms of DNA cut-and-paste occurring at the genomic loci that encode for immunoglobulins (referred to as gene rearrangements). Gene rearrangements diversify the antibody response to ensure that a wide range of pathogens can be efficiently targeted. However, breaks on the DNA molecule are dangerous to the stability of the B-cell genome and must be tightly controlled to limit the risk for immunodeficiency and lymphoid malignancy. Recent studies implicate a role for RNA and RNA-binding proteins (RBPs) in these processes. This proposal aims to explore the role of RNA remodelling enzymes, known as RNA helicases, in B-cell development and immunoglobulin gene rearrangements. This work will provide new insight into the role of RNA helicases in B-cells, with implications for immune disease and B-cell lymphomagenesis.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Role of AMPK in Nutrient Sensing and in Cancer
Funder: Wellcome Trust

Abstract: Firstly, we will investigate the role of AMP-activated protein kinase (AMPK) in glucose sensing. Our recent work in collaboration with Shengcai Lin (Xiamen University) shows that mammalian AMPK is activated by glucose deprivation via a pathway independent of the canonical AMP/ADP-dependent pathway. AMPK appears instead to sense the levels of the glycolytic intermediate fructose-1,6-bisphosphate (FBP), with the effect being transmitted from the likely receptor, aldolase, to LKB1 and AMPK via LAMTOR1 and AXIN. We will investigate the possibility that this mechanism adjusts the levels of glycolytic enzymes according to glucose availability, via reciprocal regulation of AMPK and TORC1 at the lysosomal surface. Secondly, we will screen metabolites from animal cells to identify novel regulators of AMPK that bind the Allosteric Drug and Metabolite (ADaM) site. Thirdly, we will pursue our hypothesis that AMPK is a glycogen sensor that determines the final size of glycogen particles and also ensures that glycogen stores are rapidly replenished whenever they become depleted. Finally, we will investigate why some subunit isoforms of AMPK are amplified in human cancers, while others are either mutated or deleted, or unaffected. We will also investigate whether AMPK inhibitors might be useful adjuncts to chemotherapy with DNA-damaging drugs like etoposide.

Lay abstract: The applicant has shown that the protein AMPK senses the energy state of living cells (like the system in a mobile phone that monitors battery charge), and adjusts cellular metabolism accordingly. For example, AMPK senses energy shortage in muscle during exercise, and is responsible for many of the short- and long-term adaptations that occur following exercise, many of which have health benefits. Several chronic human diseases, such as obesity or Type 2 diabetes, can be regarded as disorders of energy balance; AMPK is also crucial here, which is why some existing anti-diabetic drugs work partly by switching it on. The applicant has recently uncovered new unsolved questions regarding AMPK: (1) How does it sense availability of carbohydrate independently of cellular energy? (2) Does it sense other cellular metabolites that bind in the so-called “ADaM” site on the protein? (3) Can it sense availability of glycogen, the main intracellular reserve of carbohydrate? (4) Why are some genes encoding AMPK subunits amplified in human cancers? Answers to these questions will yield insights relevant to major human dieases. For example, they might suggest that AMPK inhibitors would enhance the effect of drugs currently used for chemotherapy in cancer, reducing their unwanted side effects.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Replication through structure-prone DNA – mechanism and impact on genome stability
Funder: Wellcome Trust

Abstract: This proposal is aimed at understanding how repetitive and structure-prone sequences are replicated accurately. Repetitive DNA comprises over half of the human genome. Repeat instability due to unusual DNA secondary structures causes many neurodegenerative diseases and is a diagnostic and prognostic cancer marker. While genetic studies revealed that repeats impair DNA replication and that replication can induce repeat instability, mechanistic insight is lacking. It is therefore crucial to understand how repeats are accurately replicated in normal tissues and how they become unstable in disease. First, I will determine the nature of replication-dependent DNA structures. Building on my recent advances in sequence-specific in vitro replication, I will delineate the effects of DNA secondary structures on replication dynamics, including DNA unwinding, DNA synthesis, Okazaki fragment maturation and replisome stability. Finally, I will discover factors that enable faithful replication of structure-prone DNA using a combination of biochemistry, candidate genetics and proteomics. As a long-term goal, I aim to elucidate the interplay between replication and repair pathways, such as mismatch repair. Altogether, these aims will define the relationship between DNA sequence, structure and replication and may lead to identification of novel factors that modulate the formation and resolution of toxic DNA structures.

Lay abstract: Each dividing cell in our body must produce two accurate copies of its genome. Certain regions of the genome are more difficult to copy than others, with repetitive sequences being especially challenging. Genetic alterations within repeats often occur in cancer and give rise to heritable disorders such as Fragile X syndrome. It is therefore important to understand how repeats are copied accurately in normal cells and how this goes wrong in disease. This proposal is aimed at understanding how replication deals with repetitive sequences. I will use a recent state-of-the-art technique which recreates the entire process of DNA replication in a test tube. I will identify the factors that are required to prevent replication errors and reveal how they work. The expected results will provide insight into the process of DNA replication in health and may improve our understanding, diagnosis and treatment of diseases such as Fragile X and cancer.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Dissecting robustness mechanisms via signalling adaptation and trafficking plasticity during developmental and pathological cell migration
Funder: Wellcome Trust

Abstract: Robustness mechanisms are ubiquitous in biological systems where they act to ensure a stable outcome despite intrinsic (genetic) and extrinsic (environmental) variabilities. However, the underlying mechanisms are often unclear. In my proposed research, I will investigate the mechanistic basis of how systems deal with change in the context of cell migration during development, immune responses, and cancer invasion. Cell migration models I will use for my study include: the posterior lateral line primordium (pLLP) migration in development and macrophage recruitment during wound immune response in zebrafish, and invasion of cultured cancer cells.The main goals of the proposed original project are: (1) Characterizing the molecular mechanisms and network architectural designs that enable robust adaptation during cell migration in a multicellular organism, (2) Analysing the requirement of regulated membrane trafficking switching during chemotactic adaptation, and (3) Quantitatively defining the role of dynamic buffering in breast cancer spreading.To achieve these goals, an interdisciplinary approach will be taken, combining live imaging, genetics, biochemistry and phosphoproteomics. I will generate datasets bridging molecular, cellular, and tissue scales to obtain a systems level understanding of adaptive responses. This work will have broad implications for understanding chemotactic adaptation in development, physiology and disease.

Lay abstract: Biological systems are reliable because they can produce stable outcomes even when changes are experienced. However, how this is brought about is still unclear. Here, I will investigate the problem using moving cells. Cells move in response to attractant signals to form organs at the correct places, mediate healing of wounds, and during pathological conditions including cancer spreading. Thus, reliable cell movement can be both beneficial and detrimental to health and fitness. To model the various cell movement processes, I will use the zebrafish, which is transparent and facilitates observation of activities inside cells in real time. In combination, cancer cell lines will be used to address situations where reliability may be undesirable. I will uncover the molecular underpinnings of how cells interpret changes in the environment and respond rapidly and appropriately. This will allow us to develop strategies to increase or decrease cell movement reliability depending on the context.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Mechanistic understanding of the reading and writing of DNA methylation
Funder: Wellcome Trust

Abstract: DNA methylation guides transcription and the formation of heterochromatin, and is commonly dysregulated in a number of diseases. DNA methylation is inextricably linked to the chromatin environment; DNA methyltransferases bind directly to nucleosomes and histone post-translational modifications alter methyltransferase binding and catalysis. Despite the critical role of DNA methylation, few molecular details are available to describe how it is controlled within intact nucleosomes. Biochemical and structural studies to date have used short DNA fragments or linear peptide sequences to infer control mechanisms of DNA methyltransferases. I will build on my expertise and use chemical and catalytic approaches to create site-specifically modified nucleosomes. I will use single-particle cryo-electron microscopy to study individual nucleosome-DNA methylation protein complexes and combine these findings with biochemical and biophysical approaches to help explain how these proteins spatiotemporally deposit and maintain DNA methylation. I aim to investigate the structure of the complete DNA methylation pathway; from the initial recruitment of factors to modified chromatin, to the correct loading and catalysis of DNA methylation sites, to subsequent maintenance of nucleosome bound hemi-methylated DNA. This work will transform our understanding of how DNA methylation is deposited in chromatin and how this can become dysregulated in disease.

Lay abstract: If DNA is the cell’s instruction manual, then methylation of this DNA creates the tabs that help to direct how the information is read and interpreted. DNA methylation is associated with turning off genes and compacting DNA, and is faulty in many different diseases, including cancer. DNA methyl tabs are placed on DNA by molecular machines, but we do not understand how DNA methylation is deposited and maintained throughout our lifetime. So far we have been limited to looking at small fragments of the bigger picture, by studying artificial snapshots of small pieces of DNA and the molecular machines involved in placing DNA methyl tabs. To help understand this process further we will look at more complete structures of the molecular machines that deposit DNA methylation. By striving to recreate the true environment where these reactions occur in the cell, we will better explain how these fascinating molecular machines work.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Molecular basis of the oxidative stress response and its regulation
Funder: Wellcome Trust

Abstract: Reactive oxygen species (ROS) are obligatory by-products of aerobic living conditions and important signalling molecules. Excessive amounts of ROS can be deleterious for cells: they can damage proteins, membranes and the DNA. They are the cause and consequence of multiple human diseases. Complex pathways have evolved to maintain the redox balance and, if necessary, trigger apoptosis to protect cells from damage accumulation. Despite their importance in human health and disease, the molecular mechanisms of ROS-regulated gene expression and cell survival decisions under oxidative conditions are poorly understood, with several key questions remaining. What are the molecular mechanisms underlying redox stress response in higher eukaryotes? What are the safety mechanisms that protect cells from thriving under oxidative stress and how are these mechanisms hijacked in pathological conditions? To answer these questions, I will elucidate the key steps in the redox stress response using a combination of biophysical and structural techniques. This work will focus on: i) the regulation of the major transcription factor implicated in the redox stress response; ii) the activation of the detoxification machinery; iii) the safety mechanisms that govern cell fate under oxidative stress.

Lay abstract: Reactive oxygen species (ROS) are highly reactive molecules produced by cells during fundamental processes (e.g. metabolism; when cells convert food into energy) or after exposure to external factors (e.g. pollution). Although ROS play an important role in many cell functions (e.g. cell-to-cell communication), high levels of ROS can damage cells and alter genetic information. Therefore, cells need to control ROS levels and ensure that they remain at a healthy level. This is called maintaining the ‘redox balance’. Problems in maintaining this balance are a cause and a consequence of several human diseases, including cancer and neurodegenerative diseases. Drugs that maintain the redox balance may help to treat ROS-related diseases. Despite their importance in human health and disease, the mechanisms underlying the redox balance remain poorly understood. I want to discover how cells detect and respond to changes to the redox balance, and how these responses are disrupted, especially in cancer.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Replisome unloading: mechanism and importance for cell biology
Funder: Wellcome Trust

Abstract: Cell division requires the accurate duplication of all genetic information. DNA replication is precisely regulated as mistakes can lead to severe consequences, such as genetic disease, cancer and premature ageing. Surprisingly, despite over six decades of research, little is known about how eukaryotic replication is completed and the replisome disassembled. It is crucial that we understand this essential stage of the replication reaction so we can fully comprehend the consequences of its deregulation and to be able to exploit this for benefit of human health. Importantly, the execution and regulation of this process has never been studied in human cells. This proposal will elucidate how replisome disassembly is accomplished and coordinated in human cells, identify and characterise novel regulators of replisome disassembly and to determine the consequences of its disruption for cell biology. To deliver these answers we will combine biochemical approaches using Xenopus laevis egg extract model system and cell biology in immortalized human cell lines. The outcomes of this work will be a first description of replisome disassembly in human cells, providing a much better understanding of the mechanism of this process and demonstration the importance of its precise execution for genome stability.

Lay abstract: Despite six decades of research into the understanding of the fundamental process of DNA replication, little is known about how eukaryotic replication is completed and the replication machinery disassembled. It is crucial that we understand this essential stage of replication so we can fully comprehend the consequences of its deregulation and to be able to exploit it in the future for the benefit of human health. My group discovered the first elements of this disassembly mechanism using model systems. We will use our unique expertise to answer many of the questions that emerged from our pioneering work. This proposal will elucidate how replisome disassembly is accomplished and coordinated in human cells, identify and characterise novel regulators of replisome disassembly and determine the consequences of its disruption for cell biology. The outcomes of this proposal will lay a base for future investigations into targeting this process for therapy of human diseases.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Regulation of SUMO in DNA double-strand break repair.
Funder: Wellcome Trust

Abstract: Proper repair of DNA double-strand breaks (DSBs) is vital to immunological health, cancer protection and healthy ageing. The cellular response to this form of damage is highly co-ordinated by a series of diverse post-translational modifications. Recently we, and others, demonstrated a vital role for the small-ubiquitin like modifiers (SUMOs) in the DSB response. Despite an emerging picture of the mammalian SUMO-proteome our understanding of the regulation of SUMOylation following DNA damage is superficial and our current knowledge points to a largely passive process. Contrary to the current prevailing view of SUMOylation we hypothesise that the regulation of this modification in the DSB response is highly coordinated. We suggest this regulation is fundamental to correct DNA repair. Our aims are to identify mechanisms of SUMO enzyme cascade initiation, to establish how SUMO proteases perform the vital role of maintaining the balance of SUMO conjugates during DNA damage signalling, and to describe the features of a novel subclass of SUMO target modification site critical to the cellular response to DSBs. In achieving these aims we anticipate this programme will underpin future developments relevant to cancer treatment and healthy ageing by identifying new areas of regulation and cross-talk in the DNA repair process.

Lay abstract: Our cells protect their DNA from long-term damage by actively repairing it. This process is vital to healthy ageing and without it cancer can arise. Cells have several rapid response mechanisms to call their internal repair services to damaged DNA. Recently we have found out that one of these mechanisms uses tiny proteins called SUMOs. We think SUMO might act like sticky tape and call the repair services by sticking them to each other and to the damaged region. However there are fundamental aspects of the way that SUMO is involved that we don’t currently understand. How does the cell initiate the ‘sticking’ processes? Too much sticky would be dangerous to the repair process, so how do cells stop this? What determines where the sticky goes? In this programme we aim to address how SUMO is directed to damaged DNA, how the amount of SUMO at damage sites is controlled, and whether special mechanisms exist to put it in the right place. The regulation of SUMO is an understudied aspect of DNA repair so the knowledge we will gain is likely to provide the basis for new developments in medicine relevant to cancer and healthy ageing.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: The interplay between the oxygen sensors PHDs and the cell cycle
Funder: Wellcome Trust

Abstract: Oxygen is essential for aerobic respiration in metazoa and most organisms have evolved ways of adapting to decreased oxygen concentration (hypoxia), which can occur both in physiological and pathological situations. In mammals, the response to hypoxia involves three Prolyl Hydroxylase enzymes (PHDs 1-3), which can regulate stability of the transcription factor, HIF1-alpha, by site-specific proline hydroxylation. We recently discovered that PHD1 similarly controls the stability of Cep192, a critical component of the centrosome, thereby affecting formation of the mitotic spindle, which in turn inhibits cell division and prevents formation of cilia. This unexpected discovery links oxygen sensing with regulation of the cell cycle. We hypothesise that sensing oxygen levels is critical for the control of cell cycle progression in vivo, particularly within tissues, where oxygen levels can vary significantly. Our collaborative project will undertake a detailed investigation of the molecular mechanisms responsible for the physiological regulation of cell division in response to oxygen levels in human cells and tissues. We will study the interplay between PHD enzymes and their targets and determine how this modulates the activities of components of the cell cycle machinery. Further, we will investigate how such interactions may contribute to human kidney disease.

Lay abstract: Oxygen is essential for life in multicellular organisms. Animals have evolved mechanisms to cope with decreased oxygen concentration (hypoxia), which can occur both in physiological and pathological situations. In humans, sensing and responding to changes in oxygen involves a class of proteins (PHDs), that use oxygen to modify a specific protein, called hypoxia inducible factor (HIF), which is a master regulator of genes that helps cells handle low oxygen levels. We recently discovered that these oxygen sensing PHD proteins also target proteins required for the key process of cell division. This unexpected finding suggests that sensing oxygen is crucial for regulating cell division, which we propose may be particularly important in tissues, where oxygen concentration can vary. This collaborative project will build on our discovery by studying in detail the molecular mechanisms that connect oxygen sensing enzymes and their targets to regulate cell cycle progression. We will test the hypothesis that the sensing of oxygen levels regulates cell division in human tissues and investigate how dysregulation of these mechanisms may contribute to human disease, with a major focus on diseases of the kidney, where PHD enzymes are known to play important roles.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS