ERC FUNDED PROJECTS

"The attine fungus-growing ants are prime models for understanding phenotypic adaptations in social evolution and symbiosis. The mutualism has many hallmarks of advanced cooperation in its mating system commitments and functional complementarity between multiple symbiont partners, but potential conflicts between sexes and castes over reproductive priorities, and between hosts and symbionts over symbiont mixing have also been documented. With collaborators at BGI-Shenzhen and the Smithsonian Institution my group has obtained six reference genomes representing all genus-level branches of the higher attine ants and a lower attine outgroup. With collaborators in Denmark and Australia we have pioneered proteomic approaches to understand the preservation of sperm viability in spite of sperm competition and the enzymatic decomposition of plant substrates that the ants use to make their fungus gardens grow. Here, I propose an integrated study focusing on four major areas of attine ant biology that are particularly inviting for in depth molecular approaches: 1. The protein-level networks that secure life-time (up to 20 years) sperm storage in specialized ant-queen organs and the genetic mechanisms that shape and adjust these “sexual symbiome” networks. 2. The ant-fungal symbiome, i.e. the dynamics of fungal enzyme production for plant substrate degradation and the redistribution of these enzymes in fungus gardens through fecal deposition after they are ingested but not digested by the ants. 3. The microbial symbiome of ant guts and other tissues with obligate bacterial mutualists, of which we have identified some and will characterize a wider collection across the different branches of the attine ant phylogeny. 4. The genome-wide frequency of genomic imprinting and the significance of these imprints for the expression of caste phenotypes and the regulation of potential reproductive conflicts."

2 290 102 €

Start date: 2013-05-01, End date: 2018-04-30

I propose to study how eukaryotic cells generate autophagosomes, organelles bounded by a double membrane. These are formed during autophagy and mediate the degradation of cytoplasmic substances within the lysosomal compartment. Autophagy thereby protects the organism from pathological conditions such as neurodegeneration, cancer and infections. Many core factors required for autophagosome formation have been identified but the order in which they act and their mode of action is still unclear. We will use a combination of biochemical and cell biological approaches to elucidate the choreography and mechanism of these core factors. In particular, we will focus on selective autophagy and determine how the autophagic machinery generates an autophagosome that selectively contains the cargo. To this end we will focus on the cytoplasm-to-vacuole-targeting pathway in S. cerevisiae that mediates the constitutive delivery of the prApe1 enzyme into the vacuole. We will use cargo mimetics or prApe1 complexes in combination with purified autophagy proteins and vesicles to reconstitute the process and so determine which factors are both necessary and sufficient for autophagosome formation, as well as elucidating their mechanism of action. In parallel we will study selective autophagosome formation in human cells. This will reveal common principles and special adaptations. In particular, we will use cell lysates from genome-edited cells in combination with purified autophagy proteins to reconstitute selective autophagosome formation around ubiquitin-positive cargo material. The insights and hypotheses obtained from these reconstituted systems will be validated using cell biological approaches. Taken together, our experiments will allow us to delineate the major steps of autophagosome formation during selective autophagy. Our results will yield detailed insights into how cells form and shape organelles in a de novo manner, which is major question in cell- and developmental biology.

1 999 640 €

Start date: 2016-03-01, End date: 2021-02-28

The biomembrane is a prerequisite of life. It enables the cell to maintain a controlled environment and to establish electrochemical gradients as rapidly accessible energy stores. Biomembranes also provide scaffold for organisation and spatial definition of signal transmission in the cell. Crystal structures of membrane proteins are determined with an increasing pace. Along with functional studies integral studies of individual membrane proteins are now widely implemented. The BIOMEMOS proposal goes a step further and approaches the function of the biomembrane at the higher level of membrane protein complexes. Through a combination of X-ray crystallography, electrophysiology, general biochemistry, biophysics and bioinformatics and including also the application of single-particle cryo-EM and small-angle X-ray scattering, the structure and function of membrane protein complexes of key importance in life will be investigated. The specific targets for investigation in this proposal include: 1) higher-order complexes of P-type ATPase pumps such as signalling complexes of Na+,K+-ATPase, and 2) development of methods for structural studies of membrane protein complexes Based on my unique track record in structural studies of large, difficult structures (ribosomes and membrane proteins) in the setting of a thriving research community in structural biology and biomembrane research in Aarhus provides a critical momentum for a long-term activity. The activity will take advantage of the new possibilities offered by synchrotron sources in Europe. Furthermore, a single-particle cryo-EM research group formed on my initiative in Aarhus, and a well-established small-angle X-ray scattering community provides for an optimal setting through multiple cues in structural biology and functional studies

2 444 180 €

Start date: 2010-04-01, End date: 2015-03-31

Brain structure determines function. Disentangling regional microstructural properties and understanding how these properties constitute brain function is a central goal of neuroimaging of the human brain and a key prerequisite for a mechanistic understanding of brain diseases and their treatment. Using magnetic resonance (MR) imaging, previous research has established links between regional brain microstructure and inter-individual variation in brain function, but this line of research has been limited by the non-specificity of MR-derived markers. This hampers the application of MR imaging as a tool to identify specific fingerprints of the underlying disease process. Exploiting state-of-the-art ultra-high field MR imaging techniques, I have recently developed two independent spectroscopic MR methods that have the potential to tackle this challenge: Powder averaged diffusion weighted spectroscopy (PADWS) can provide an unbiased marker for cell specific structural degeneration, and Spectrally tuned gradient trajectories (STGT) can isolate cell shape and size. In this project, I will harness these innovations for MR-based precision medicine. I will advance PADWS and STGT methodology on state-of-the-art MR hardware and harvest the synergy of these methods to realize Cell-specific in-vivo MORPHOMETRY (C-MORPH) of the intact human brain. I will establish novel MR read-outs and analyses to derive cell-type specific tissue properties in the healthy and diseased brain and validate them with the help of a strong translational experimental framework, including histological validation. Once validated, the experimental methods and analyses will be simplified and adapted to provide clinically applicable tools. This will push the frontiers of MR-based personalized medicine, guiding therapeutic decisions by providing sensitive probes of cell-specific microstructural changes caused by inflammation, neurodegeneration or treatment response.

1 498 811 €

Start date: 2018-12-01, End date: 2023-11-30

"Translational research aims at applying mechanistic understanding in the development of "precision medicine", which depends on precise diagnostic tools and therapeutic approaches. Cancer therapy is experiencing a switch from non-specific, cytotoxic agents towards molecularly targeted and rationally designed compounds with the promise of greater efficacy and fewer side effects. The two cell-cycle kinases CDK4 and CDK6 normally facilitate cell-cycle progression but are abnormally activated in certain cancers. CDK6 is up-regulated in hematopoietic malignancies, where it is the predominant cell-cycle kinase. The importance of CDK4/6 for tumor development is underscored by the fact that the US FDA selected inhibitors of the kinase activity of CDK4/6 as "breakthrough of the year 2013". Our recent findings suggest that the effects of the inhibitors may be limited as CDK6 is not only involved in cell-cycle progression: ground-breaking research in my group and others has shown that CDK6 is involved in regulation of transcription in a kinase-independent manner thereby driving the proliferation of leukemic stem cells and tumor formation. We have now identified mutations in CDK6 that convert it from a tumor promoter into a tumor suppressor. This unexpected outcome is accompanied by a distinct transcriptional profile. Separating the tumor-promoting from the tumor suppressive functions may open a novel therapeutic avenue for drug development. We aim at understanding which domains and residues of CDK6 are involved in rewiring the transcriptional landscape to pave the way for sophisticated inhibitors. The idea of turning a cancer cell's own most potent weapon against itself is novel and would represent a new paradigm for drug design. Finally, the understanding of CDK6 functions in tumor promotion and maintenance will also result in better patient stratification and improved treatment decisions for a broad spectrum of cancer types."

2 497 520 €

Start date: 2016-09-01, End date: 2021-08-31

Cancer and other diseases are driven by genomic alterations initiated by DNA breaks. Within our genomes, some regions are particularly prone to breakage, and these are known as common fragile sites (CFSs). CFSs are present in every person and are frequently sites of oncogenic chromosomal rearrangements. Intriguingly, despite their fragility, many CFSs are well conserved through evolution, suggesting that these regions have important physiological functions that remain elusive. My previous background in genome editing, proteomics and replication-born DNA damage has given me the tools to propose an ambitious and comprehensive plan that tackles fundamental questions on the biology of CFSs. First, we will perform a systematic analysis of the function of CFSs. Most of the CFSs contain very large genes, which has made technically difficult to dissect whether the CFS role is due to the locus itself or to the encoded gene product. However, the emergence of the CRISPR/Cas9 technology now enables the study of CFSs on a more systematic basis. We will pioneer the engineering of mammalian models harbouring large deletions at CFS loci to investigate their physiological functions at the cellular and organism levels. For those CFSs that contain genes, the cDNAs will be re-introduced at a distal locus. Using this strategy, we will be able to achieve the first comprehensive characterization of CFS roles. Second, we will develop novel targeted approaches to interrogate the chromatin-bound proteome of CFSs and its dynamics during DNA replication. Finally, and given that CFS fragility is influenced both by cell cycle checkpoints and dNTP availability, we will use mouse models to study the impact of ATR/CHK1 pathway and dNTP levels on CFS instability and cancer. Taken together, I propose an ambitious, yet feasible, project to functionally annotate and characterise these poorly understood regions of the human genome, with important potential implications for improving human health.

1 499 711 €

Start date: 2016-05-01, End date: 2021-04-30

The goal of the proposed research is to examine how the independent and combined effects of childhood adiposity (assessed by body mass index [BMI]; kg/m2) height, change in BMI and height, and pubertal timing from the ages of 7 to 13 years are associated with the risk of cancer incidence in adulthood. Greater body size (adipose tissue and different types of lean tissue) reflecting past or ongoing growth may increase the risk of cancer in individuals as greater numbers of proliferating cells increase the risk that mutations leading to the subsequent development of cancer occur. As childhood is a period of growth, it is plausible that it is of particular relevance for the early establishment of the risk of cancer. Data from the Copenhagen School Health Records Register, which is based on a population of schoolchildren born between 1930-1983 and contains computerised weight and height measurements on >350.000 boys and girls in the capital city of Denmark, as well as data from other cohorts will be used. Survival analysis techniques and the newly developed Dynamic Path Analysis model will be used to examine how body size (BMI and height) at each age from 7 to 13 years as well as change in body size during this period is associated with the risk of multiple forms of cancer in adulthood with a simultaneous exploration of the effects of birth weight and pubertal timing. Additionally, potential effects of childhood and adult health and social circumstances will be investigated in sub-cohorts with this information available. Results from this research will demonstrate if childhood is a critical period for the establishment of the risk for cancer in adulthood and will lead into mechanistic explorations of the associations at the biological level, investigations into associations between childhood body size and mortality and contribute to developing improved definitions of childhood overweight and obesity that are based upon long-term health outcomes.

1 199 998 €

Start date: 2012-02-01, End date: 2017-01-31

Background: Acrylamide is a chemical formed in many commonly consumed foods and beverages. It is neurotoxic, crosses the placenta and has been associated with restriction of fetal growth in humans. In animals, acrylamide causes heritable mutations, tumors, developmental toxicity, reduced fertility and impaired growth. Therefore, the discovery of acrylamide in food in 2002 raised concern about human health effects worldwide. Still, epidemiological studies are limited and effects on health of prenatal exposure have never been evaluated. Research gaps: Epidemiological studies have mostly addressed exposure during adulthood, focused on cancer risk in adults, and relied on questionnaires entailing a high degree of exposure misclassification. Biomarker studies on prenatal exposure to acrylamide from diet are critically needed to improve exposure assessment and to determine whether acrylamide leads to major diseases later in life. Own results: I have first authored a prospective European study showing that prenatal exposure to acrylamide, estimated by measuring hemoglobin adducts in cord blood, was associated with fetal growth restriction, for the first time. Objectives: To determine the effects of prenatal exposure to acrylamide alone and in combination with other potentially toxic adduct-forming exposures on the health of children and young adults. Methods: Both well-established and innovative biomarker methods will be used for characterization of prenatal exposure to acrylamide and related toxicants in blood from pregnant women and their offspring in prospective cohort studies with long-term follow-up. Risk of neurological disorders, impaired cognition, disturbed reproductive function and metabolic outcomes such as obesity and diabetes will be evaluated. Perspectives: CHIPS project will provide a better understanding of the impact of prenatal exposure to acrylamide from diet on human health urgently needed for targeted strategies for the protection of the health.

1 499 531 €

Start date: 2018-07-01, End date: 2023-06-30

Inheritance of DNA sequence and its proper organization into chromatin is fundamental for eukaryotic life. The challenge of propagating genetic and epigenetic information is met in S phase and entails genome-wide disruption and restoration of chromatin coupled to faithful copying of DNA. How specific chromatin structures are restored on new DNA and transmitted through mitotic cell division remains a fundamental question in biology central to understand cell fate and identity. Chromatin restoration on new DNA involves a complex set of events including nucleosome assembly and remodelling, restoration of marks on DNA and histones, deposition of histone variants and establishment of higher order chromosomal structures including sister-chromatid cohesion. To dissect these fundamental processes and their coordination in time and space with DNA replication, we have developed a novel technology termed nascent chromatin capture (NCC) that provides unique possibility for biochemical and proteomic analysis of chromatin replication in human cells. I propose to apply this innovative cutting-edge technique for a comprehensive characterization of chromatin restoration during DNA replication and to reveal how replication timing and genotoxic stress impact on final chromatin state. This highly topical project brings together the fields of chromatin biology, DNA replication, epigenetics and genome stability and we expect to make groundbreaking discoveries that will improve our understanding of human development, somatic cell reprogramming and complex diseases like cancer. The proposed research will 1) identify and characterize novel mechanisms in chromatin restoration and 2) address molecularly how replication timing and genotoxic insults influence chromatin maturation and final chromatin state.

1 692 737 €

Start date: 2011-11-01, End date: 2017-04-30

One of the most dramatic transitions in biology is the oocyte-to-zygote transition. This refers to the maturation of the female germ cell or oocyte, which undergoes two rounds of meiotic chromosome segregation and, following fertilization, is converted to a mitotically dividing embryo. We aim to establish an innovative research program that addresses fundamental questions about the molecular processes controlling the mammalian oocyte-to-zygote transition to ensure faithful inheritance of genomes from one generation to the next. We are taking an interdisciplinary approach combining germ cell and chromosome biology with cell cycle and epigenetic studies to understand how maternal factors regulate chromosome segregation in oocytes and chromatin organization in the zygote. A molecular understanding of key players regulating these processes is a requisite step for investigating how their deterioration contributes to maternal age-related aneuploidy and infertility. Aneuploidy is the leading cause of mental retardation and spontaneous miscarriage. The current trend towards advanced maternal age has increased the frequency of trisomic fetuses by 71% in the past ten years. A better understanding of mammalian meiosis is therefore relevant to human reproductive health. A special feature of the female germ line is that meiotic DNA replication occurs in the embryo but oocytes remain arrested until the first meiotic division is triggered months (mouse) or decades (human) later. The longevity of oocytes poses a challenge for the cohesin complex that must hold together sister chromatids from DNA synthesis until chromosome segregation. We specifically aim to: 1) elucidate how sister chromatid cohesion is maintained in mammalian oocytes, 2) identify mechanisms regulating cohesion in young and aged oocytes, and 3) investigate how the inheritance of genetic and resetting of epigenetic information is coordinated with cell cycle progression at the oocyte-to-zygote transition.

1 499 738 €

Start date: 2014-02-01, End date: 2019-01-31

Inflammation is a response to noxious stimuli and initiates tissue repair. If resolution fails, however, chronic inflammation develops, which drives tissue damage in many diseases including autoimmunity, cancer and infections. Inflammatory processes are increasingly being appreciated as tightly integrated with metabolic pathways. The molecular crosstalk occurs on different levels including secreted metabolites and cytokines. I hypothesise that this interface of metabolism and inflammation represents a functional rheostat that shapes tissue damage and disease. Here, I propose to analyse the metabolic and inflammatory processes in a mouse model of chronic viral hepatitis. I chose this model to explore the inflammatory rheostat because the liver is the central organ for metabolism and a hotspot for receiving, processing and distributing local and systemic signals. Cutting-edge technologies including deep sequencing, quantitative proteomics and metabolomics will let us create longitudinal multi-dimensional maps of virus-induced alterations. Paired with immunological, virological and pathological analyses, I expect to identify novel regulatory nodes between metabolism and inflammation. Within our systems-wide experiments and supported by preliminary results, we will specifically focus on the immunomodulatory roles of the metabolite bile acids and oxidative metabolism. These as well as other candidates will be investigated by genetic and pharmacological perturbations in cell culture and in mouse models. Bioinformatics integration of the orthogonal profiling kinetics is expected to reveal novel properties of the molecular networks mediating between metabolism and inflammation. This proposed cross-disciplinary approach aims to improve our understanding of the crosstalk of metabolism and inflammation. The results of this project may be relevant to viral hepatitis in man and bear broader implications for other inflammatory diseases.

1 701 011 €

Start date: 2016-04-01, End date: 2021-03-31

Targeted therapy (TT) is frequently used to treat metastatic cancer. Although TT can achieve effective tumor control for several months, durable treatment responses are rare, due to emergence of aggressive, drug-resistant clones (RCs) with high metastatic competence. Tumor heterogeneity and plasticity result in multifaceted resistance mechanisms and targeting RCs poses a daunting challenge. To better understand the clinical emergence of RCs, my work focuses on the poorly understood events during TT-induced tumor regression. We recently reported that during this phase drug-responsive cancer cells release a therapy-induced secretome, which remodels the tumor microenvironment (TME) and propagates disease relapse by promoting the survival of drug-sensitive cells and stimulating the outgrowth of RCs. Consequently, intervening with combination therapies during the tumor regression period has the potential to prevent the clinical emergence of RCs in the first place. Here, we outline strategies to (1) understand how RCs emerge and (2) to leverage our findings on the TME remodeling for combination therapies. First, we will develop a novel and innovative parental clone-lookup method, that will allow us to identify and isolate treatment-naïve, parental clones (PCs) that gave rise to RCs. In functional experiments, we will assess (i) whether PCs were already resistant before or developed resistance during TT, (ii) whether PCs have a higher susceptibility to develop resistance than random clones, and (iii) the mechanistic basis for metastatic competence in different clones. Second, we will study the TT-induced TME remodeling, focusing on the effects on tumor vasculature and immune cells. We will utilize our results to target PCs and RCs by combining TT in the phase of tumor regression with other therapies, such as immunotherapies. Our study will provide new mechanistic insights into the biological processes during tumor regression and aims for novel therapeutic strategies.

1 500 000 €

Start date: 2018-03-01, End date: 2023-02-28

Chronic Systemic Inflammation (CSI) resulting from systemic release of inflammatory cytokines and activation of the immune system is responsible for the progression of several debilitating diseases, such as Psoriasis, Arthritis and Cancer. Initially localised diseases can result in CSI with subsequent systemic spread to distant organs, a key patho-physiological phase responsible for major morbidity and even mortality. Despite the importance of CSI, a complete understanding of the molecular mechanisms, signalling pathways and cell types involved, as well as the chronological evolution of the systemic inflammatory response is still elusive. The classical approach to study inflammation has focused on investigating individual cell types or organs in the pathogenesis of a single disease, thereby neglecting important organ cross-talk and systemic interactions. Furthermore, understanding the temporal and spatial kinetics modulating the inflammatory response requires a detailed study of interactions between different immune and non-immune organs at various time points during disease progression in the context of the whole organism. The aim of this research proposal is to substantially advance our understanding of whole organ physiology in relation to systemic inflammation as a cause or/and consequence of disease with the focus on Psoriasis/Joint Diseases and Cancer Cachexia. The goal is to elucidate the molecular mechanisms at the cellular and systemic level, and to decipher endocrine interactions and cross-talks between distant organs. Various model systems ranging from cell cultures to genetically engineered mouse models to human clinical samples will be employed. Genomic, proteomic and metabolomic data will be combined with functional in vivo assessment using mouse models to understand the multi-faceted role of systemic inflammation in chronic human diseases, such as Inflammatory Skin/Joint disease and Cachexia, a deadly systemic manifestation of Cancer.

2 499 875 €

Start date: 2018-06-01, End date: 2023-05-31

The evolutionary arms race has optimized and shaped the way animals attend to relevant sensory stimuli in an ever-changing environment. This is a complex task, because the vast majority of sensory experiences are not relevant. In humans, attentional disorders are a serious public health concern because of its high prevalence, but its causes are mostly unknown. In this proposal, I will explore the neuronal mechanisms used by the nervous system to attend visual cues to enable appropriate behaviors. We will combine cutting edge imaging techniques, optogenetic interventions, behavioral read outs and targeted connectomics to study the neuronal transformations of the mouse Superior Colliculus (SC), an evolutionary conserved midbrain area known to process sensorimotor transformations and to be involved in the allocation of attention. First, this work will reveal a detailed description of visual representation in the SC, focusing on understanding how defined retinal information-streams, like motion and color, contribute to these properties. Second, we will characterize sensorimotor transformations instructed by the SC. The combination of the previous two objectives will determine mechanisms of visual saliency and sensory driven attention (“bottom-up” attention). Finally, we will explore the neuronal mechanisms of attention by studying the modulatory effect of higher brain areas (“top-down” attention) on sensory transformation and multisensory integration in the SC. Taken together, this proposal aims to understand principles underlying sensorimotor transformation and build a framework to study attention in health and disease.

1 446 542 €

Start date: 2017-12-01, End date: 2022-11-30

G protein-coupled receptors make up both the largest membrane protein and drug target families. DE-ORPHAN aims to determine the close functional context; specifically physiological agonists and signaling pathways; and provide the first research tool compounds, of orphan peptide receptors. Determination of physiological agonists (aka de-orphanization), by high-throughput screening has largely failed. We will introduce a new research strategy: 1) developing highly innovative bioinformatics methods for handpicking of all orphan receptor targets and candidate ligand screening libraries; and 2) employing a screening technique that can measure all signaling pathways simultaneously. The first potent and selective pharmacological tool compounds will be identified by chemoinformatic design of focused screening libraries. We will establish the ligands’ structure-activity relationships important for biological activity and further optimization towards drugs. The first potent and selective Gs- and G12/13 protein inhibitors will be designed by structure-based re-optimization from a recent crystal structure of a Gq-inhibitor complex, and applied to determine orphan receptor signaling pathways and ligand pathway-bias. They will open up for efficient dissection of important signaling networks and development of drugs with fewer side effects. DE-ORPHANs design hypotheses are based on unique computational methods to analyze protein and ligand similarities and are founded on genomic and protein sequences, structural data and ligands. The interdisciplinary research strategy applies multiple ligands acting independently but in concert to provide complementary receptor characterization. The results will allow the research field to advance into studies of receptor functions and exploitation of druggable targets, ligands and mechanisms. Which physiological insights and therapeutic breakthroughs will we witness when these receptors find their place in human pharmacology and medicine?

1 499 926 €

Start date: 2015-05-01, End date: 2020-04-30

The application of antimicrobial compounds produced by hosts or defensive symbionts to counter the effects of diseases has been identified in a number of organisms, but despite extensive studies on their presence, we know essentially nothing about why antimicrobials do not trigger rampant resistance evolution in target parasites. In stark contrast to virtually any other organism, fungus-farming termites have evolved a sophisticated agricultural symbiosis that pre-dates human farming by 30 million years without suffering from specialised diseases. I will capitalise on recent pioneering work in my group on proximate evidence for antimicrobial defences in the termites, their fungal crops, and their complex gut bacterial communities, by proposing to develop the farming symbiosis as a major model to test three novel concepts that may account for the evasion of resistance evolution. First, the antimicrobial compounds may have properties and evolve in ways that preclude resistance evolution in pathogens. Second, resistance is only possible towards individual compounds and not natural antimicrobial cocktails. Third, pathogens can only successfully invade and proliferate if they bypass several consecutive lines of defence, analogous to the six hallmarks of metazoan defence against cancer development. Addressing these concepts will allow fundamental insights into the remarkable success of complementary symbiont contributions to defence, and they will clarify the forces of multilevel natural selection that have allowed long-lived insect societies to evolve sustainability. Documenting and understanding these disease management principles is fundamentally important for several branches of evolutionary biology, and strategically important for adjusting human practices for future antimicrobial stewardship.

1 998 809 €

Start date: 2018-06-01, End date: 2023-05-31

"DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. In cancer increased aberrant DNA methylation is believed to work as a silencing mechanism for tumor suppressor genes such as INK4A, RB1 and MLH1. The high frequency of abnormal DNA methylation found in cancer might be due to the inactivation of a proofreading and/or fidelity system regulating the correct patterns of DNA methylation. Currently we have very limited knowledge about such mechanisms. In this research proposal, we will focus on elucidating the biological function of a novel protein family, which catalyzes the conversion of 5-methyl-cytosine (5-mC) to 5-hydroxymethyl cytosine (5-hmC). By catalyzing this reaction the TET proteins most likely work as DNA demethylases, and they might therefore have a role in regulating DNA methylation fidelity. Interestingly, accumulated data has in the last 2 years shown that TET2 is one of the most frequently mutated genes in various hematological cancers. We propose to investigate the molecular mechanisms by which TET2 regulates normal hematopoiesis, how its inactivation leads to hematopoietic malignancies and how the protein contributes to the regulation of DNA methylation patterns and transcription. Furthermore, we propose several experimental approaches for identifying proteins required for the recruitment of TET proteins to target genes and to analyze their role in the regulation of DNA methylation patterns and in cancer. Finally, we will investigate the potential functional role of 5-hmC and explore the potential mechanisms by which this modification could be erased. We expect to provide new insights into the biology of DNA methylation, hydroxymethylation and contribute to unravel the roles of TET proteins in normal physiology and cancer."

2 298 000 €

Start date: 2012-07-01, End date: 2017-06-30

DNA-protein cross-links (DPCs) are common DNA lesions caused by endogenous, environmental, and chemotherapeutic agents. Cells are susceptible to these lesions during S phase, as DPCs impede replication fork progression and are likely to induce genomic instability, a cause of cancer and aging. Despite its relevance to human health, the repair of DPCs is poorly understood. Research on DPC repair has mainly involved testing cellular responses to compounds such as formaldehyde, but these agents induce a wide variety of DNA lesions, and conflicting results have been reported. To overcome these obstacles, I have developed the first in vitro system that recapitulates replication-coupled DPC repair. In this system, a plasmid containing a site-specific DPC is replicated in Xenopus egg extracts. Using this approach, I demonstrated that DPC repair requires DNA replication. When a replication fork encounters a DPC, the DPC is degraded into a peptide-adduct, which allows replication bypass by translesion DNA synthesis. Importantly, these experiments identified a novel proteolytic pathway whose activity is regulated by replication. This in vitro system now provides a powerful means to identify and characterize the different factors that participate in S phase DPC repair. I speculate that for DPC processing to occur, the protein-adduct must first be detected, then marked for degradation and ultimately degraded. Using a series of complementary strategies, which will take advantage of the in vitro system combined with proteome and genome wide approaches, I seek to uncover the different players that participate in each of these events. This project will enable a detailed mechanistic outlook of a complex multi-step reaction that has not been feasible to achieve using existing methodologies. It will also improve our understanding of how DPCs impact genomic stability and the consequences of not repairing these lesions for human health.

1 498 856 €

Start date: 2017-01-01, End date: 2021-12-31

Understanding the mechanisms underlying the initiation and regulation of transcription remains one of the most fundamental questions in biology. Much of what we know about the transcription process was inferred from experiments on a handful of genes. As these experiments are not realistically scalable, corresponding computational methods building on these findings have emerged; however, these are not accurate enough for annotation of genomes. The limitations reflect that we have no accurate universal model describing transcription initiation; to a large extent, our understanding is based on case stories. Recently, high-throughput methods have been developed to chart the TSS landscape with nucleotide resolution. Using these data, I have dissected promoters at nucleotide level and found patterns that explain the transcription initiation rate for individual nucleotides. The objective for this work is to extend this to the first universal model for how cells select core promoters and associated TSSs. This will have two counterparts: i)prediction of TSSs from DNA sequence given a region of accessible DNA, and ii)prediction of DNA accessibility based on DNA sequences and dynamic epigenetic factors. Such a model will be a corner stone of future experimental and computational transcriptome and gene regulation studies.

812 399 €

Start date: 2008-09-01, End date: 2013-08-31

Computational, structure-based, drug design offers insight at an atomic resolution, which is commonly not attainable by experimental means. Detailed calculations on protein-ligand interactions help to rationalize and predict experimental findings. Accurate and efficient calculations of binding free energies is essential in this respect. In addition, knowledge concerning the enthalpic and entropic contributions are highly relevant to determine novel drug design strategies and to understand the underlying principles of ligand binding. Currently available methods to address ligand affinity either do not include all relevant contributions to the binding free energy, or are too computationally demanding to be applied straightforwardly. In addition, calculations on enthalpy and entropy for drug design purposes are very rare, due to the difficulty in calculating these accurately. This proposal describes the research that leads the way to new, standard applications to be used in drug design processes in academia and industry. Furthermore, we propose to investigate the enthalpic and entropic contributions to ligand binding. We define a ligand-surroundings enthalpy and entropy, which conveys more information than the experimentally accessible enthalpy and entropy of ligand binding. In support of this research, we will develop new enhanced sampling techniques which not only render the above calculations practically feasible, but which will also find their application in related research questions such as the protein folding problem or the elucidation of protein-protein interactions. The methods described are highly relevant for the pharmaceutical industry, where currently available computational approaches are insufficient to answer the questions of todays drug discovery programmes.

1 485 615 €

Start date: 2011-01-01, End date: 2015-12-31

"Our existence as human beings is based on plants and their products. Worldwide, crops are threatened by pests including biotrophic fungi. Therefore, it is of vital interest to develop new strategies to reduce crop losses and to improve crop plants for the growing world population. Biotrophic plant pathogens employ small secreted molecules, so-called effectors, to overcome plant defence systems and to establish biotrophy. The rapid increase in available genome sequences of biotrophic pathogens and in transcriptomic datasets of their biotrophic stages allow us to identify putative secreted proteinaceous effectors by bioinformatic means. However, our insight into the functions of these effectors is still very limited. In this proposal, the PI´s extensive experience on both the plant host side and the fungal pathogen side of the biotrophic interaction is exploited to develop a workflow for functional, partially robotic-based screens to fill this gap. The combination of screen-deduced functional information with the analysis of effector localisation and specific host interactors will provide the basis for formulating starting hypotheses of effector function. These will then be tested in individual case studies, employing the well established Ustilago maydis-Zea mays as well as the new Ustilago bromivora-Brachypodium distachyon model systems. The project will be conducted at the Max Planck Institute (MPI) for Terrestrial Microbiology in a highly stimulating scientific environment. Linking the dramatic morphological changes and underlying molecular events during biotrophy on the host side to the action of subsets or even single effector proteins will allow the creation of a synthetic effectome. The deep functional understanding of the manipulative toolbox of biotrophs has the potential to facilitate transgenic crop development and will open a new era in the development of sustainable antifungal plant protection strategies."

1 446 316 €

Start date: 2014-02-01, End date: 2019-01-31

Why does a mutation have a deleterious effect when it occurs in one species but shows no apparent consequences on the phenotype when it occurs in another species? What are some of possible explanations on the molecular basis of this phenomenon? Are the computational predictions of the extent of this phenomenon in nature accurate? The present project aims to take a swing at answering, at least partially, these basic questions of epistasis in molecular evolution. Within our work we plan to address these issues using computational approaches, systematic fitness assays of engineered orthologous genotypes and experimental functional assays of specific cases of epistasis identified by evolutionary analysis. By tackling these goals and utilising this array of approaches the projects aims to create a synthesis between theory and experimentation under the confines of a single laboratory that will allow us to study this phenomenon in a systematic fashion on the interface of different fields and methodologies.

1 461 576 €

Start date: 2014-01-01, End date: 2018-12-31

Domesticated crops hardly resemble their wild ancestors, and often trade higher yield in artificially optimized conditions for lower performance in fluctuating environments. Leafcutter ants (genus Atta) provide fascinating parallels with human farmers, harvesting fresh vegetation used as compost to produce domesticated fungal crops that feed massive societies with millions of workers. However, while human agricultural systems are imperiled by rapid global changes, leafcutter ants have managed to grow one type of cultivar from Texas to Argentina, thriving across extreme rainfall and temperature gradients and across diverse climates over millions of years. However, the eco-physiological mechanisms governing this farming resiliency are poorly understood. I propose a new in vitro mapping paradigm to visualize the niche requirements of fungal cultivars. Creating multidimensional landscapes of nutrient availability (e.g. protein, carbohydrates, Na, P) and environmental stress (e.g. temperature, moisture, plant toxins, crop pathogens) I will answer three main questions: 1) What genes and biochemical pathways shape cultivar performance across interacting gradients of nutrition and stress? 2) Do colonies harvest substrates to navigate nutritional contours of cultivar performance maps and avoid production tradeoffs? 3) Do locally adaptive cultivar traits shape the performance of farming societies across regional ecological gradients, and over 60 million years of co-evolutionary crop domestication by farming ants? My cutting-edge approach will deliver transformative advances to the field of eco-physiology, enabling seamless integration between field and laboratory experiments, and providing new ways to visualize evolutionary mechanisms across levels of biological organization from genes to symbiotic partnerships, and from within diverse farming assemblages to across populations spanning entire continents.

1 427 741 €

Start date: 2018-02-01, End date: 2023-01-31

A major goal in biology is to understand how gene regulatory information is encoded by the human genome and how it defines different gene expression programs and cell types. Enhancers are genomic elements that control transcription, yet despite their importance, only a minority of enhancers are known and functionally characterized. In particular, their activity changes during cellular signalling or cell type transitions are largely elusive. Furthermore, fundamental questions about transcriptional co-factors have remained unanswered even though they regulate enhancer activities and have become attractive therapeutic targets, e.g. for cancer treatment. Here, I propose a functional genomics approach in mammalian cells with three specific objectives: First, we will identify and functionally characterize transcriptional enhancers in selected human and mouse cells using the recently developed quantitative enhancer activity assay STARR-seq. Second, we will determine enhancer activity changes quantitatively during steroid hormone signalling, cell differentiation, and malignant transformation to reveal enhancers that are important for these processes. Third, we will systematically dissect the functional relationship of enhancers and transcriptional co-factors. This proposal uses emerging in-house technology to address fundamental questions in enhancer biology and complement the genome-wide profiling of gene expression and chromatin states (e.g. by ENCODE). We will gain insights into the genomic organization of active enhancers and reveal chromatin or sequence features associated with dynamic activity changes. I also expect that we will be able to define co-factor requirements for enhancer function and reveal if different types of enhancers exist. Given our expertise in experimental and computational approaches and STARR-seq, I anticipate that we reach our aims and make major contributions to the understanding of gene regulation in mammals.

1 999 906 €

Start date: 2015-09-01, End date: 2020-08-31