ERC FUNDED PROJECTS

Most hereditary diseases in humans are genetically complex, resulting from combinations of mutations in multiple genes. However synthetic interactions between genes are very difficult to identify in population studies because of a lack of statistical power and we fundamentally do not understand how mutations interact to produce phenotypes. C. elegans is a unique animal in which genetic interactions can be rapidly identified in vivo using RNA interference, and we recently used this system to construct the first genetic interaction network for any animal, focused on signal transduction genes. The first objective of this proposal is to extend this work and map a comprehensive genetic interaction network for this model metazoan. This project will provide the first insights into the global properties of animal genetic interaction networks, and a comprehensive view of the functional relationships between genes in an animal. The second objective of the proposal is to use C. elegans to develop and validate experimentally integrated gene networks that connect genes to phenotypes and predict genetic interactions on a genome-wide scale. The methods that we develop and validate in C. elegans will then be applied to predict phenotypes and interactions for human genes. The final objective is to dissect the molecular mechanisms underlying genetic interactions, and to understand how these interactions evolve. The combined aim of these three objectives is to generate a framework for understanding and predicting how mutations interact to produce phenotypes, including in human disease.

1 100 000 €

Start date: 2008-09-01, End date: 2014-04-30

Faithful repair of double stranded DNA breaks (DSBs) is essential, as they are at the origin of genome instability, chromosomal translocations and cancer. Cells repair DSBs through different pathways, which can be faithful or mutagenic, and the balance between them at a given locus must be tightly regulated to preserve genome integrity. Although, much is known about DSB repair factors, how the choice between pathways is controlled within the nuclear environment is not understood. We have shown that nuclear architecture and non-random genome organization determine the frequency of chromosomal translocations and that pathway choice is dictated by the spatial organization of DNA in the nucleus. Nevertheless, what determines which pathway is activated in response to DSBs at specific genomic locations is not understood. Furthermore, the impact of 3D-genome folding on the kinetics and efficiency of DSB repair is completely unknown. Here we aim to understand how nuclear compartmentalization, chromatin structure and genome organization impact on the efficiency of detection, signaling and repair of DSBs. We will unravel what determines the DNA repair specificity within distinct nuclear compartments using protein tethering, promiscuous biotinylation and quantitative proteomics. We will determine how DNA repair is orchestrated at different heterochromatin structures using a CRISPR/Cas9-based system that allows, for the first time robust induction of DSBs at specific heterochromatin compartments. Finally, we will investigate the role of 3D-genome folding in the kinetics of DNA repair and pathway choice using single nucleotide resolution DSB-mapping coupled to 3D-topological maps. This proposal has significant implications for understanding the mechanisms controlling DNA repair within the nuclear environment and will reveal the regions of the genome that are susceptible to genomic instability and help us understand why certain mutations and translocations are recurrent in cancer

1 999 750 €

Start date: 2017-03-01, End date: 2022-02-28

The architecture of DNA in the cell nucleus is an emerging epigenetic key contributor to genome function. We recently developed 4C technology, a high-throughput technique that combines state-of-the-art 3C technology with tailored micro-arrays to uniquely allow for an unbiased genome-wide search for DNA loci that interact in the nuclear space. Based on 4C technology, we were the first to provide a comprehensive overview of long-range DNA contacts of selected loci. The data showed that active and inactive chromatin domains contact many distinct regions within and between chromosomes and genes switch long-range DNA contacts in relation to their expression status. 4C technology not only allows investigating the three-dimensional structure of DNA in the nucleus, it also accurately reconstructs at least 10 megabases of the one-dimensional chromosome sequence map around the target sequence. Changes in this physical map as a result of genomic rearrangements are therefore identified by 4C technology. We recently demonstrated that 4C detects deletions, balanced inversions and translocations in patient samples at a resolution (~7kb) that allowed immediate sequencing of the breakpoints. Excitingly, 4C technology therefore offers the first high-resolution genomic approach that can identify both balanced and unbalanced genomic rearrangements. 4C is expected to become an important tool in clinical diagnosis and prognosis. Key objectives of this proposal are: 1. Explore the functional significance of DNA folding in the nucleus by systematically applying 4C technology to differentially expressed gene loci. 2. Adapt 4C technology such that it allows for massive parallel analysis of DNA interactions between regulatory elements and gene promoters. This method would greatly facilitate the identification of functionally relevant DNA elements in the genome. 3. Develop 4C technology into a clinical diagnostic tool for the accurate detection of balanced and unbalanced rearrangements.

1 225 000 €

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

"Eukaryotic nuclei are organised into functional compartments, – local microenvironments that are enriched in certain molecules or biochemical activities and therefore specify localised functional outputs. Our study seeks to unveil fundamental principles of co-regulation of genes in a chromo¬somal compartment and the preconditions for homeostasis of such a compartment in the dynamic nuclear environment. The dosage-compensated X chromosome of male Drosophila flies satisfies the criteria for a functional com¬partment. It is rendered structurally distinct from all other chromosomes by association of a regulatory ribonucleoprotein ‘Dosage Compensation Complex’ (DCC), enrichment of histone modifications and global decondensation. As a result, most genes on the X chromosome are co-ordinately activated. Autosomal genes inserted into the X acquire X-chromosomal features and are subject to the X-specific regulation. We seek to uncover the molecular principles that initiate, establish and maintain the dosage-compensated chromosome. We will follow the kinetics of DCC assembly and the timing of association with different types of chromosomal targets in nuclei with high spatial resolution afforded by sub-wavelength microscopy and deep sequencing of DNA binding sites. We will characterise DCC sub-complexes with respect to their roles as kinetic assembly intermediates or as representations of local, functional heterogeneity. We will evaluate the roles of a DCC- novel ubiquitin ligase activity for homeostasis. Crucial to the recruitment of the DCC and its distribution to target genes are non-coding roX RNAs that are transcribed from the X. We will determine the secondary structure ‘signatures’ of roX RNAs in vitro and determine the binding sites of the protein subunits in vivo. By biochemical and cellular reconstitution will test the hypothesis that roX-encoded RNA aptamers orchestrate the assembly of the DCC and contribute to the exquisite targeting of the complex."

2 482 770 €

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

"Ageing is the gradual and irreversible breakdown of living systems associated with the advancement of time, which leads to an increase in vulnerability and eventual mortality. Despite recent advances in ageing research, the intrinsic complexity of the ageing process has prevented a full understanding of this process, therefore, ageing remains a grand challenge in contemporary biology. In AGELESS, we will tackle this challenge by uncovering the molecular mechanisms of halted ageing in a unique model system, the bats. Bats are the longest-lived mammals relative to their body size, and defy the ‘rate-of-living’ theories as they use twice as much the energy as other species of considerable size, but live far longer. This suggests that bats have some underlying mechanisms that may explain their exceptional longevity. In AGELESS, we will identify the molecular mechanisms that enable mammals to achieve extraordinary longevity, using state-of-the-art comparative genomic methodologies focused on bats. We will identify, using population transcriptomics and telomere/mtDNA genomics, the molecular changes that occur in an ageing wild population of bats to uncover how bats ‘age’ so slowly compared with other mammals. In silico whole genome analyses, field based ageing transcriptomic data, mtDNA and telomeric studies will be integrated and analysed using a networks approach, to ascertain how these systems interact to halt ageing. For the first time, we will be able to utilize the diversity seen within nature to identify key molecular targets and regions that regulate and control ageing in mammals. AGELESS will provide a deeper understanding of the causal mechanisms of ageing, potentially uncovering the crucial molecular pathways that can be modified to halt, alleviate and perhaps even reverse this process in man."

1 499 768 €

Start date: 2013-01-01, End date: 2017-12-31

As diploid organisms inherit one gene copy from each parent, a gene can be expressed from both alleles (biallelic) or from only one allele (monoallelic). Although transcription from both alleles is detected for most genes in cell population experiments, little is known about allele-specific expression in single cells and its phenotypic consequences. To answer fundamental questions about allelic transcription heterogeneity in single cells, this research program will focus on single-cell transcriptome analyses with allelic-origin resolution. To this end, we will investigate both clonally stable and dynamic random monoallelic expression across a large number of cell types, including cells from embryonic and adult stages. This research program will be accomplished with the novel single-cell RNA-seq method developed within my lab to obtain quantitative, genome-wide gene expression measurement. To distinguish between mitotically stable and dynamic patterns of allelic expression, we will analyze large numbers a clonally related cells per cell type, from both primary cultures (in vitro) and using transgenic models to obtain clonally related cells in vivo. The biological significance of the research program is first an understanding of allelic transcription, including the nature and extent of random monoallelic expression across in vivo tissues and cell types. These novel insights into allelic transcription will be important for an improved understanding of how variable phenotypes (e.g. incomplete penetrance and variable expressivity) can arise in genetically identical individuals. Additionally, the single-cell transcriptome analyses of clonally related cells in vivo will provide unique insights into the clonality of gene expression per se.

1 923 060 €

Start date: 2015-07-01, End date: 2020-06-30

Anopheles gambiae mosquitoes are the major vectors of malaria, a disease with devastating consequences for human health. Novel methods for controlling the natural vector populations are urgently needed, given the evolution of insecticide resistance in mosquitoes and the lack of novel insecticidals. Understanding the processes at the bases of mosquito biology may help to roll back malaria. In this proposal, we will target mosquito reproduction, a major determinant of the An. gambiae vectorial capacity. This will be achieved at two levels: (i) fundamental research, to provide a deeper knowledge of the processes regulating reproduction in this species, and (ii) applied research, to identify novel targets and to develop innovative approaches for the control of natural populations. We will focus our analysis on three major players of mosquito reproduction: male accessory glands (MAGs), sperm, and spermatheca, in both laboratory and field settings. We will then translate this information into the identification of inhibitors of mosquito fertility. The experimental activities will be divided across three objectives. In Objective 1, we will unravel the role of the MAGs in shaping mosquito fertility and behaviour, by performing a combination of transcriptional and functional studies that will reveal the multifaceted activities of these tissues. In Objective 2 we will instead focus on the identification of the male and female factors responsible for sperm viability and function. Results obtained in both objectives will be validated in field mosquitoes. In Objective 3, we will perform screens aimed at the identification of inhibitors of mosquito reproductive success. This study will reveal as yet unknown molecular mechanisms underlying reproductive success in mosquitoes, considerably increasing our knowledge beyond the state-of-the-art and critically contributing with innovative tools and ideas to the fight against malaria.

1 500 000 €

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

In our quest to fully understand the processes that shape the genomic variation of species, describing variation of the past is a fundamental objective. However, the origins and the extent of great ape variation, the genomic description of extinct primate species and the genomic footprints of introgression events all remain unknown. Even today, and in contraposition to human evolutionary biology, the almost null presence of ancient great ape samples has precluded a comprehensive exploration of such diversity. Here, I present two approaches that will expose great ape diversity throughout time and will allow me to compare the genomic impact of introgression events across lineages. First, I would like to take advantage of ancient ape samples that will provide us with a direct view of the genomes of extinct populations. Second, I would like to exploit current and recent diversity to indirectly access the parts of extinct ape genomes that became hybridized with current species in the past. For the latter, we will analyse hundreds of non-invasive samples taken from present-day great apes as well as historical specimens. Altogether, this information will enable me to decipher novel genomes that until now have been lost in time. In this way, I will be able to properly understand the origins and dynamics of genomic variants and to study how admixture has contributed to today´s adaptive landscape. By completing this proposal and performing analogies to the human lineage, fundamental insights will be revealed about (i) the spatial-temporal history of our closest species and (ii) the functional consequences of introgressed events. On top of that, these results will help to annotate functional consequences of novel mutations in the human genome. In so doing, a fundamental insight will be provided into the evolutionary history of these regions and into human mutations with multiple repercussions in the understanding of evolution and human biology.

1 896 875 €

Start date: 2020-06-01, End date: 2025-05-31

Regulation of gene expression plays a key role in the ability of bacteria to rapidly adapt to changing environments and to colonize extremely diverse habitats. The relatively recent discovery of a plethora of small regulatory RNAs and the beginning of their characterization has unravelled new aspects of bacterial gene expression. First, the expression of many bacterial genes responds to a complex network of both transcriptional and post-transcriptional regulators. However, the properties of the resulting regulatory circuits on the dynamics of gene expression and in the bacterial adaptive response have been poorly addressed so far. In a first part of this project, we will tackle this question by characterizing the circuits that are formed between two widespread classes of bacterial regulators, the sRNAs and the two-component systems, which act at the post-transcriptional and the transcriptional level, respectively. The study of sRNAs also led to major breakthroughs regarding the basic mechanisms of gene expression. In particular, we recently showed that repressor sRNAs can target activating stem-loop structures located within the coding region of mRNAs that promote translation initiation, in striking contrast with the previously recognized inhibitory role of mRNA structures in translation. The second objective of this project is thus to draw an unprecedented map of non-canonical translation initiation events and their regulation by sRNAs. Overall, this project will greatly improve our understanding of how bacteria can so rapidly and successfully adapt to many different environments, and in the long term, provide clues towards the development of anti-bacterial strategies.

1 999 754 €

Start date: 2019-09-01, End date: 2024-08-31

The aim of the BEEHIVE project is to generate novel insight into HIV biology, evolution and epidemiology, leveraging next-generation high-throughput sequencing and bioinformatics to produce and analyse whole-genomes of viruses from approximately 3,000 European HIV-1 infected patients. These patients have known dates of infection spread over the last 25 years, good clinical follow up, and a wide range of clinical prognostic indicators and outcomes. The primary objective is to discover the viral genetic determinants of severity of infection and set-point viral load. This primary objective is high-risk & blue-skies: there is ample indirect evidence of polymorphisms that alter virulence, but they have never been identified, and it is not known how easy they are to discover. However, the project is also high-reward: it could lead to a substantial shift in the understanding of HIV disease. Technologically, the BEEHIVE project will deliver new approaches for undertaking whole genome association studies on RNA viruses, including delivering an innovative high-throughput bioinformatics pipeline for handling genetically diverse viral quasi-species data (with viral diversity both within and between infected patients). The project also includes secondary and tertiary objectives that address critical open questions in HIV epidemiology and evolution. The secondary objective is to use viral genetic sequences allied to mathematical epidemic models to better understand the resurgent European epidemic amongst high-risk groups, especially men who have sex with men. The aim will not just be to establish who is at risk of infection, which is known from conventional epidemiological approaches, but also to characterise the risk factors for onwards transmission of the virus. Tertiary objectives involve understanding the relationship between the genetic diversity within viral samples, indicative of on-going evolution or dual infections, to clinical outcomes.

2 499 739 €

Start date: 2014-04-01, End date: 2019-03-31

Identifying risk factors for diseases that can be prevented or delayed by early intervention is of major importance, and numerous genetic, lifestyle, anthropometric and clinical risk factors were found for many different diseases. Another source of potentially pertinent disease risk factors is the human microbiome - the collective genome of trillions of bacteria, viruses, fungi, and parasites that reside in the human gut. However, very few microbiome disease markers were found to date. Here, we aim to develop risk prediction tools based on the human microbiome that predict the likelihood of an individual to develop a particular condition or disease within 5-10 years. We will use a cohort of >2200 individuals that my group previously assembled, for whom we have clinical profiles, gut microbiome data, and banked blood and stool samples. We will invite people 5-10 years after their initial recruitment time, profile disease status and blood markers, and develop algorithms for predicting 5-10 year onset of Type 2 diabetes, cardiovascular disease, and obesity, using microbiome data from recruitment time. To increase the likelihood of finding microbiome markers predictive of disease onset, we will develop novel experimental and computational methods for in-depth characterization of microbial gene function, the metabolites produced by the microbiome, the underexplored fungal microbiome members, and the interactions between the gut microbiota and the host adaptive immune system. We will then apply these methods to >2200 banked samples from cohort recruitment time and use the resulting data in devising our microbiome-based risk prediction tools. In themselves, these novel assays and their application to >2200 samples should greatly advance the microbiome field. If successful, our proposal will identify new disease risk factors and risk prediction tools based on the microbiome, paving the way towards using the microbiome in early disease detection and prevention.

2 500 000 €

Start date: 2019-03-01, End date: 2024-02-29

The centromere is one of the most important chromosomal elements. It is required for proper chromosome segregation in mitosis and meiosis and readily recognizable as the primary constriction of mitotic chromosomes. Proper centromere function is essential to ensure genome stability; therefore understanding centromere identity is directly relevant to cancer biology and gene therapy. How centromeres are established and maintained is however still an open question in the field. In most organisms this appears to be regulated by an epigenetic mechanism. The key candidate for such an epigenetic mark is CENH3 (CENP-A in mammals, CID in Drosophila), a centromere-specific histone H3 variant that is essential for centromere function and exclusively found in the nucleosomes of centromeric chromatin. Using a biosynthetic approach of force-targeting CENH3 in Drosophila to non-centromeric DNA, we were able to induce centromere function and demonstrate that CENH3 is sufficient to determine centromere identity. Here we propose to move this experimental setup across evolutionary boundaries into human cells to develop improved human artificial chromosomes (HACs). We will make further use of this unique setup to dissect the function of targeted CENH3 both in Drosophila and human cells. Contributing centromeric components and histone modifications of centromeric chromatin will be characterized in detail by mass spectroscopy in Drosophila. Finally we are proposing to develop a technique that allows high-resolution mapping of proteins on repetitive DNA to help further characterizing known and novel centromere components. This will be achieved by combining two independently established techniques: DNA methylation and DNA fiber combing. This ambitious proposal will significantly advance our understanding of how centromeres are determined and help the development of improved HACs for therapeutic applications in the future.

1 755 960 €

Start date: 2013-02-01, End date: 2019-01-31

Embryogenesis is the temporal unfolding of cellular processes: proliferation, migration, differentiation, morphogenesis, apoptosis and functional specialization. These processes are well understood in specific tissues, and for specific cell types. Nevertheless, our systematic knowledge of the types of cells present in the developing and adult animal, and about their functional and lineage relationships, is limited. For example, there is no consensus on the number of cell types, and many important stem cells and progenitors remain to be discovered. Similarly, the lineage relationships between specific cell types are often poorly characterized. This is particularly true for the mammalian nervous system. We have developed (1) a reliable high-throghput method for sequencing all transcripts in 96 single cells at a time; and (2) a system for high-throughput phylogenetic lineage reconstruction. We now propose to characterize embryogenesis using a shotgun approach borrowed from genomics. Tissues will be dissected from multiple stages and dissociated to single cells. A total of 10,000 cells will be analyzed by RNA sequencing, revealing their functional cell type, their lineage relationships, and their current state (e.g. cell cycle phase). The novel approach proposed here will bring the powerful strategies pioneered in genomics into the field of developmental biology, including automation, digitization, and the random shotgun method. The data thus obtained will bring clarity to the concept of ‘cell type’; will provide a first catalog of mouse brain cell types with deep functional annotation; will provide markers for every cell type, including stem cells; and will serve as a basis for future comparative work, especially with human embryos.

1 496 032 €

Start date: 2010-11-01, End date: 2015-10-31

The metabolism of cancer cells is altered to meet cellular requirements for growth, providing novel means to selectively target tumorigenesis. While extensively studied, our current view of cancer cellular metabolism is fundamentally limited by lack of information on variability in metabolic activity between distinct subcellular compartments and cells. We propose to develop a spatio-temporal fluxomics approach for quantifying metabolic fluxes in the cytoplasm vs. mitochondria as well as their cell-cycle dynamics, combining mass-spectrometry based isotope tracing with cell synchronization, rapid cellular fractionation, and computational metabolic network modelling. Spatio-temporal fluxomics will be used to revisit and challenge our current understanding of central metabolism and its induced adaptation to oncogenic events – an important endeavour considering that mitochondrial bioenergetics and biosynthesis are required for tumorigenesis and accumulating evidences for metabolic alterations throughout the cell-cycle. Our preliminary results show intriguing oscillations between oxidative and reductive TCA cycle flux throughout the cell-cycle. We will explore the extent to which cells adapt their metabolism to fulfil the changing energetic and anabolic demands throughout the cell-cycle, how metabolic oscillations are regulated, and their benefit to cells in terms of thermodynamic efficiency. Spatial flux analysis will be instrumental for investigating glutaminolysis - a ‘hallmark’ metabolic adaptation in cancer involving shuttling of metabolic intermediates and cofactors between mitochondria and cytoplasm. On a clinical front, our spatio-temporal fluxomics analysis will enable to disentangle oncogene-induced flux alterations, having an important tumorigenic role, from artefacts originating from population averaging. A comprehensive view of how cells adapt their metabolism due to oncogenic mutations will reveal novel targets for anti-cancer drugs.

1 481 250 €

Start date: 2017-02-01, End date: 2022-01-31

Celiac disease affects at least 1% of the world population. Its onset is triggered by gluten, a common dietary protein, however, its etiology is poorly understood. More than 80% of patients are not properly diagnosed and they therefore do not follow a gluten-free diet, thereby increasing their risk for disease-associated complications and early death. A better understanding of the disease biology would improve the diagnosis, prevention, and treatment of celiac disease. This project investigates the disease mechanisms in celiac disease by using predisposing genes and genetic variants as disease initiating factors. Specifically, it will investigate if long, intergenic non-coding RNAs (lincRNAs) are causally involved in celiac disease pathogenesis by regulating protein-coding genes and pathways associated with the disease. This project is based on two important observations by my group: (1) Our genetic studies, which led to identifying 39 celiac disease risk loci, suggest that the mechanism underlying the disease is largely governed by dysregulation of gene expression. (2) We uncovered a previously unrecognized role for lincRNAs that provides clues as to exactly how genetic variation causes disease, as this class of biologically important RNA molecules regulate gene expression. The research will be performed in CD4+ T cells, a severely affected cell type in disease pathology. I will first use celiac disease-associated protein-coding genes to delineate their regulatory pathways and then study the transcriptional programs of lincRNAs present in celiac disease loci. Next I will combine the information and investigate if the expressed lincRNAs modulate the pathways and affect T cell function, thereby discovering if lincRNAs are a missing link between non-coding genetic variation and protein-coding genes. Our findings may well lead to potential therapeutic targets and provide a solid scientific basis for new diagnostic markers, particularly biomarkers, based on genetics.

2 319 914 €

Start date: 2013-02-01, End date: 2018-11-30

The idea of harnessing living organisms for treating human diseases is not new but, so far, the majority of the living vectors used in human therapy are viruses which have the disadvantage of the limited number of genes and networks that can contain. Bacteria allow the cloning of complex networks and the possibility of making a large plethora of compounds, naturally or through careful redesign. One of the main limitations for the use of bacteria to treat human diseases is their complexity, the existence of a cell wall that difficult the communication with the target cells, the lack of control over its growth and the immune response that will elicit on its target. Ideally one would like to have a very small bacterium (of a mitochondria size), with no cell wall, which could be grown in Vitro, be genetically manipulated, for which we will have enough data to allow a complete understanding of its behaviour and which could live as a human cell parasite. Such a microorganism could in principle be used as a living vector in which genes of interests, or networks producing organic molecules of medical relevance, could be introduced under in Vitro conditions and then inoculated on extracted human cells or in the organism, and then become a new organelle in the host. Then, it could produce and secrete into the host proteins which will be needed to correct a genetic disease, or drugs needed by the patient. To do that, we need to understand in excruciating detail the Biology of the target bacterium and how to interface with the host cell cycle (Systems biology aspect). Then we need to have engineering tools (network design, protein design, simulations) to modify the target bacterium to behave like an organelle once inside the cell (Synthetic biology aspect). M.pneumoniae could be such a bacterium. It is one of the smallest free-living bacterium known (680 genes), has no cell wall, can be cultivated in Vitro, can be genetically manipulated and can enter inside human cells.

2 400 000 €

Start date: 2009-03-01, End date: 2015-02-28

Faithful chromosome segregation in all eukaryotes relies on centromeres, the chromosomal sites that recruit kinetochore proteins and mediate spindle attachment during cell division. Fundamental to centromere function is a histone H3 variant, CenH3, that initiates kinetochore assembly on centromeric DNA. CenH3 is conserved throughout most eukaryotes; its deletion is lethal in all organisms tested. These findings established the paradigm that CenH3 is an absolute requirement for centromere function. My recent findings undermined this paradigm of CenH3 essentiality. I showed that CenH3 was lost independently in four lineages of insects. These losses are concomitant with dramatic changes in their centromeric architecture, in which each lineage independently transitioned from monocentromeres (where microtubules attach to a single chromosomal region) to holocentromeres (where microtubules attach along the entire length of the chromosome). Here, I aim to characterize this unique CenH3-deficient chromosome segregation pathway. Using proteomic and genomic approaches in lepidopteran cell lines, I will determine the mechanism of CenH3-independent kinetochore assembly that led to the establishment of their holocentric architecture. Using comparative genomic approaches, I will determine whether this kinetochore assembly pathway has recurrently evolved over the course of 400 million years of evolution and its impact on the chromosome segregation machinery. My discovery of CenH3 loss in holocentric insects establishes a new class of centromeres. My research will reveal how CenH3 that is essential in most other eukaryotes, could have become dispensable in holocentric insects. Since the evolution of this CenH3-independent chromosome segregation pathway is associated with the independent rises of holocentric architectures, my research will also provide the first insights into the transition from a monocentromere to a holocentromere.

1 497 500 €

Start date: 2018-04-01, End date: 2023-03-31

The fitness landscape, the representation of how the genotype manifests at the phenotypic (fitness) levels, may be among the most useful concepts in biology with impact on diverse fields, including quantitative genetics, emergence of pathogen resistance, synthetic biology and protein engineering. While progress in characterizing fitness landscapes has been made, three directions of research in the field remain virtually unexplored: the nature of the genotype to phenotype of standing variation (variation found in a natural population), the shape of the fitness landscape encompassing many genotypes and the modelling of complex genetic interactions in protein sequences. The current proposal is designed to advance the study of fitness landscapes in these three directions using large-scale genomic experiments and experimental data from a model protein and theoretical work. The study of the fitness landscape of standing variation is aimed at the resolution of an outstanding question in quantitative genetics: the extent to which epistasis, non-additive genetic interactions, is shaping the phenotype. The second aim of characterizing the global fitness landscape will give us an understanding of how evolution proceeds along long evolutionary timescales, which can be directly applied to protein engineering and synthetic biology for the design of novel phenotypes. Finally, the third aim of modelling complex interactions will improve our ability to predict phenotypes from genotypes, such as the prediction of human disease mutations. In summary, the proposed study presents an opportunity to provide a unifying understanding of how phenotypes are shaped through genetic interactions. The consolidation of our empirical and theoretical work on different scales of the genotype to phenotype relationship will provide empirical data and novel context for several fields of biology.

1 998 280 €

Start date: 2019-01-01, End date: 2023-12-31

The three-dimensional organization of the genome, which strikingly correlates with gene activity, is critical for many cellular processes. The evolution of molecular techniques has allowed us to unveil chromatin structure at an unprecedented resolution. The most intriguing chromatin structures observed in animals are TADs (Topologically Associating Domains), which represent the functional and structural chromatin domains demarcating the genome. Structural proteins such as insulators proteins, on the other hand, have been shown to play crucial roles in mediating the formation of TADs. However, major structural factors relevant to chromatin structure are still waiting to be discovered in land plants. My preliminary work shows that TADs are widely distributed across the rice genome, and motif sequence analysis suggests the enrichment of plant-specific transcription factors at TAD boundaries, which jointly give rise to an exciting hypothesis that these proteins might be the long-sought-after insulators in land plants. By using various state-of-the-art molecular and computational tools, this timely project aims to fill a huge gap in plant functional genomics and substantially advance our understanding of three-dimensional chromatin structure. This project consists four major aims, which collectively will uncover the identities of plant insulator proteins and generate insights into the dynamics of structural chromatin domains during stress adaptation. Aim 1 will identify and characterize the stability and plasticity of functional chromatin domains in the rice genome during temperature stress adaptation. Aim 2 will identify insulator elements and other structural features of chromatin packing in the Marchantia polymorpha genome from a structural genomics approach. Aim 3 will establish the role of candidate proteins as plant insulators. Lastly, Aim 4 will generate functional insights into the molecular mechanism by which plant insulators shape the three-dimensional genome.

1 498 216 €

Start date: 2018-01-01, End date: 2022-12-31

Proper and controlled expression of genes is essential for normal cell growth. Chromatin modifying enzymes play a fundamental role in the control of gene expression and their deregulation is often linked to cancer. In recent years chromatin modifiers have been considered key targets for cancer therapy and this demands a full understanding of their biological functions. Previous biochemical and structural studies have focused on the identification of chromatin modifying enzymes and characterization of their substrate specificities and catalytic mechanisms. However, a comprehensive view of the biological processes, signaling pathways and regulatory circuits in which these enzymes participate is missing. Protein arginine methyltransferases (PRMTs), which methylate histones and are evolutionarily conserved from yeast to human, constitute an example of chromatin modifying enzymes whose functional and regulatory networks remain unexplored. I propose to use complementary state-of-the-art genomic and proteomic approaches in order to identify the protein networks and cellular pathways that are linked to PRMTs. In parallel, I will identify novel regulatory circuits and define the molecular mechanisms that control methylation of specific histone arginine residues. I will utilize the yeast S. cerevisiae as a model organism because it allows genetic, biochemical and genomic approaches to be combined. Most importantly, many of the pathways and mechanisms in yeast are highly conserved and therefore, the findings from this study will be pertinent to human and other eukaryotic organisms. Establishing a global cellular wiring diagram of PRMTs will serve as a paradigm for other chromatin modifiers and is imperative for assessing the efficacy of these enzymes as therapeutic targets.

1 498 279 €

Start date: 2011-01-01, End date: 2016-06-30

"Our cells receive tens of thousands of different DNA lesions per day. Failure to repair these lesions will lead to cell death, mutations and genome instability, which contribute to human diseases such as neurodegenerative disorders and cancer. Efficient recognition and repair of DNA damage, however, is complicated by the fact that genomic DNA is packaged, through histone and non-histone proteins, into a condensed structure called chromatin. The DNA repair machinery has to circumvent this barrier to gain access to the damaged DNA and repair the lesions. Our recent work suggests that chromatin-modifying enzymes (CME) help to overcome this barrier at sites of DNA damage. However, the identity of these CME, their mode of action and interconnections with DNA repair pathways remain largely enigmatic. The aim of this project is to systematically identify and characterize the CME that operate during DNA repair processes in both yeast and human cells. To reach this goal we will use a cross-disciplinary approach that combines novel and cutting-edge genomics approaches with bioinformatics, genetics, biochemistry and high-resolution microscopy. Epigenetics-IDentifier (Epi-ID) will be used as a tool to unveil novel CME, whereas RNAi-interference and genetic interaction mapping studies will pinpoint the CME that may potentially regulate repair of DNA damage. A series of functional assays will eventually characterize their role in distinct DNA repair pathways, focusing on those that counteract DNA strand breaks and replication stress. Together these studies will provide insight into how CME assist cells to repair DNA damage in chromatin and inform on the relevance of CME to maintain genome stability and counteract human diseases."

1 999 575 €

Start date: 2014-03-01, End date: 2019-02-28

Epigenetic mechanisms play an important role in regulating and maintaining the functionality of cells and have been implicated in a wide range of human diseases. Histone proteins that form the protein core of nucleosomes are subject to a bewildering array of covalent and structural modifications, which can repress, permit, or promote transcription. These modifications can be added and removed by specialized complexes that are recruited by other covalent modifications, by transcription factors, or by the transcriptional machinery. Advances in genomics led to comprehensive mapping of the ``epigenome'' in a range of tissues and organisms. These maps established the tight connection between histone modifications and transcription programs. These static charts, however, are less successful at uncovering the underlying mechanisms, logic, and function of histone modifications in establishing and maintaining transcriptional programs. Our premise is that we can answer these basic questions by observing the effect of genetic perturbations on the dynamics of both chromatin state and transcriptional activity. We aim to dissect the chromatin-transcription system in a systematic manner by building on our extensive experience in modeling and analysis, and a unique high-throughput experimental system we established in my lab. We plan to use the budding yeast model organism, which allows for efficient genetic and experimental manipulations. We will combine two technologies: (1) high-throughput measurements of single-cell transcriptional output using fluorescence reporters; and (2) high-throughput immunoprecipitation sequencing assays to map chromatin state. Measuring with these the dynamics of response to stimuli under different genetic backgrounds and using advanced stochastic network models, we will chart detailed mechanisms that are opaque to current approaches and elucidate the general principles that govern the interplay between chromatin and transcription.

2 396 450 €

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

Cells are continuously exposed to insults that can break or chemically modify their DNA. To protect the DNA, cells have acquired an arsenal of repair mechanisms. Proper repair of DNA damage is essential for organismal viability and disease prevention. What is often overlooked is the fact that the eukaryotic nucleus contains many different chromatin domains that can each influence the dynamic response to DNA damage. Different chromatin environments are defined by specific molecular and biophysical properties, which could necessitate distinct chromatin responses to ensure safe DNA damage repair. The aim of this proposal is to understand how diverse chromatin domains, and in particular the dense heterochromatin environment, shape the dynamic chromatin response to DNA damage. I recently developed locus-specific DNA damage systems that allow for in-depth analysis of chromatin domain-specific repair responses in Drosophila tissue. I will employ these systems and develop new ones to directly observe heterochromatin-specific dynamics and repair responses. I will combine these systems and state-of-the art chromatin analysis with high-resolution live imaging to dissect the DNA damage-associated heterochromatin changes to determine their function in repair -kinetics, -dynamics and -pathway choice. Deciphering the chromatin dynamics that regulate DNA damage repair in heterochromatin will have broad conceptual implications for understanding the role of these dynamics in other essential nuclear processes, such as replication and transcription. More importantly, understanding how chromatin proteins promote repair will be important in determining how cancer-associated mutations in these chromatin proteins impact genetic instability in tumours in the long run.

1 499 404 €

Start date: 2019-12-01, End date: 2024-11-30

Cell-state switching in cancer allows cells to transition from a proliferative to an invasive and drug-resistant phenotype. This plasticity plays an important role in cancer progression and tumour heterogeneity. We have made a striking observation that cancer cells of different origin can switch to a common survival state. During this epigenomic reprogramming, cancer cells re-activate genomic enhancers from specific regulatory programs, such as wound repair and epithelial-to-mesenchymal transition. The goal of my project is to decipher the enhancer logic underlying this canalization effect towards a common survival state. We will then employ this new understanding of enhancer logic to engineer synthetic enhancers that are able to monitor and manipulate cell-state switching in real time. Furthermore, we will use enhancer models to identify cis-regulatory mutations that have an impact on cell-state switching and drug resistance. Such applications are currently hampered because there is a significant gap in our understanding of how enhancers work. To tackle this problem we will use a combination of in vivo massively parallel enhancer-reporter assays, single-cell genomics on microfluidic devices, computational modelling, and synthetic enhancer design. Using these approaches we will pursue the following aims: (1) to identify functional enhancers regulating cell-state switching by performing in vivo genetic screens in mice; (2) to elucidate the dynamic trajectories whereby cells of different cancer types switch to a common survival cell-state, at single-cell resolution; (3) to create synthetic enhancer circuits that specifically kill cancer cells undergoing cell-state switching. Our findings will have an impact on genome research, characterizing how cellular decision making is implemented by the cis-regulatory code; and on cancer research, employing enhancer logic in the context of cancer therapy.

1 999 660 €

Start date: 2017-06-01, End date: 2022-05-31

Cellular clearance is a fundamental process required by all cells in all species. Important physiological processes, such as aging, and pathological mechanisms, such as neurodegeneration, are strictly dependent on cellular clearance. In eukaryotes, most of the cellular clearing processes occur in a specialized organelle, the lysosome. This project is based on a recent discovery, made in our laboratory, of a gene network, which we have named CLEAR, that controls lysosomal biogenesis and function and regulates cellular clearance. The specific goals of the project are: 1) the comprehensive characterization of the mechanisms underlying the CLEAR network, 2) the thorough understanding of CLEAR physiological function at the cellular and organism levels, 3) the development of strategies and tools to modulate cellular clearance, and 4) the implementation of proof-of-principle therapeutic studies based on the activation of the CLEAR network in murine models of human lysosomal storage disorders and of neurodegenerative diseases, such as Alzheimers s and Huntington s diseases. A combination of genomics, bioinformatics, systems biology, chemical genomics, cell biology, and mouse genetics approaches will be used to achieve these goals. Our goal is to develop tools to modulate cellular clearance and to use such tools to develop therapies to cure human disease. The potential medical relevance of this project is very high, particularly in the field of neurodegenerative disease. Therapies that prevent, ameliorate or delay neurodegeneration in these diseases would have a huge impact on human health.

2 100 000 €

Start date: 2010-03-01, End date: 2015-02-28

The 3D organization of chromosomes within the nucleus is of great importance to control gene expression. The cohesin complex plays a key role in such higher-order chromosome organization by looping together regulatory elements in cis. How these often megabase-sized looped structures are formed is one of the main open questions in chromosome biology. Cohesin is a ring-shaped complex that can entrap DNA inside its lumen. However, cohesin’s default behaviour is that it only transiently entraps and then releases DNA. Our recent findings indicate that chromosomes are structured through the processive enlargement of chromatin loops, and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. The goal of this proposal is two-fold. First, we plan to investigate the mechanism by which chromatin loops are formed, and secondly we wish to dissect how looped structures are maintained. We will use a multi-disciplinary approach that includes refined genetic screens in haploid human cells, chromosome conformation capture techniques, the tracing in vivo of cohesin on individual DNA molecules, and visualization of chromosome organization by super-resolution imaging. With unbiased genetic screens, we have identified chromatin regulators involved in the formation of chromosomal loops. We will investigate how they drive loop formation, and also whether cohesin’s own enzymatic activity plays a role in the enlargement of loops. We will study whether and how these factors control the movement of cohesin along individual DNA molecules, and whether chromatin loops pass through cohesin rings during their formation. Ultimately, we plan to couple cohesin’s linear trajectory along chromatin to the 3D consequences for chromosomal architecture. Together our experiments will provide vital insight into how cohesin structures chromosomes.

1 998 375 €

Start date: 2018-04-01, End date: 2023-03-31

The vast majority of genetic diseases are complex traits, conditioned by multiple genetic and environmental factors. Yet our understanding of the genetics underlying such traits in humans remains extremely limited, due largely to the statistical complexity of inferring the effects of allelic variants in a genetically diverse population. Novel tools for the dissection of the genetic architecture of complex traits, therefore, can be most effectively developed in model organisms, where the contribution of individual alleles can be quantitatively determined in controlled genetic backgrounds. We have previously established the yeast Saccharomyces cerevisiae as a model for complex traits by unravelling complex genetic architectures that govern quantitative phenotypes in this organism. We achieved this by pioneering approaches that have revealed crucial information about the complexity of the underlying genetics. Here we propose to advance to the next level of complex trait dissection by developing systematic, genome-wide technologies that aim to identify all of the variants underlying a complex trait in a single step. In particular, we will investigate traits involved in mitochondrial function, which are both clinically relevant and highly conserved in yeast. Our combination of genomic technologies will allow us to: 1) systematically detect, with maximal sensitivity, the majority of genetic variants (coding and non-coding) that condition these traits; 2) quantify the contributions of these variants and their interactions; and 3) evaluate the strengths and limitations of current methods for dissecting complex traits. Taken together, our research will yield fundamental insights into the genetic complexity of multifactorial traits, providing valuable lessons and establishing novel genomic tools that will facilitate the investigation of complex diseases.

2 499 821 €

Start date: 2012-11-01, End date: 2017-10-31

Vertebrates contain hundreds of different cell types which maintain phenotypic identity by a combination of epigenetic programming and genomic regulation. Systems biology approaches are now used in a number of laboratories to determine how transcription factors and chromatin marks pattern the human genome. Despite high conservation of the cellular and molecular function of many mammalian transcription factors, our recent experiments in matched mouse and human tissues indicates that most transcription factor binding events to DNA are very poorly conserved. A hypothesis that could account for this apparent divergence is that the larger regional pattern of transcription factor binding may be conserved. To test this, (1) we are characterizing the global transcriptional profile, chromatin state, and complete genomic occupancy of a set of tissue-specific transcription factors in hepatocytes of strategically chosen mammals; (2) to further identify the precise mechanistic contribution of cis and trans effects, we are comparing transcription factor binding at homologous regions of human and mouse DNA in a mouse line that carries human chromosome 21. Together, these projects will provide insight into the general principles of how transcriptional networks are evolutionarily conserved to regulate cell fate specification and function using a clinically important cell type as a model.

960 000 €

Start date: 2008-10-01, End date: 2013-09-30

Over 100 types of distinct modifications are catalyzed on RNA molecules post-transcriptionally. In an analogous manner to well-studied chemical modifications on proteins or DNA, modifications on RNA - and particularly on mRNA - harbor the exciting potential of regulating the complex and interlinked life cycle of these molecules. The most abundant modification in mammalian and yeast mRNA is N6-methyladenosine (m6A). We have pioneered approaches for mapping m6A in a transcriptome wide manner, and we and others have identified factors involved in encoding and decoding m6A. While experimental disruption of these factors is associated with severe phenotypes, the role of m6A remains enigmatic. No single methylated site has been shown to causally underlie any physiological or molecular function. This proposal aims to establish a framework for systematically deciphering the molecular function of a modification and its underlying mechanisms and to uncover the physiological role of the modification in regulation of a cellular response. We will apply this framework to m6A in the context of meiosis in budding yeast, as m6A dynamically accumulates on meiotic mRNAs and as the methyltransferase catalyzing m6A is essential for meiosis. We will (1) aim to elucidate the physiological targets of methylation governing entry into meiosis (2) seek to elucidate the function of m6A at the molecular level, and understand its impact on the various steps of the mRNA life cycle, (3) seek to understand the mechanisms underlying its effects. These aims will provide a comprehensive framework for understanding how the epitranscriptome, an emerging post-transcriptional layer of regulation, fine-tunes gene regulation and impacts cellular decision making in a dynamic response, and will set the stage towards dissecting the roles of m6A and of an expanding set of mRNA modifications in more complex and disease related systems.

1 402 666 €

Start date: 2016-11-01, End date: 2021-10-31

. Genome stability is one of the most important features in maintaining tissue homeostasis throughout the human lifespan. The research presented here will dissect how the insulator protein CCCTC-binding factor (CTCF), a ubiquitous 11 zinc finger transcription factor, controls the stability of the mammalian genome during ageing. . In Aim 1, we will elucidate how CTCF and tissue-specific master regulators maintain the functional stability of the genome during healthy ageing by developing a novel protocol to map simultaneously transcription and open chromatin in isolated hepatocyte nuclei. Using this protocol, we will explore how CTCF binding stabilizes cellular homeostasis during ageing by knocking down CTCF in vivo, both in isolation and simultaneously with knock down of liver-specific master regulators. . In Aim 2, we will reveal the molecular mechanisms underlying CTCF binding sites as susceptibility loci for somatic mutations. We will profile the mutations in open chromatin of single nuclei immediately following acute exposure to a chemical mutagen; comparing how the pattern of mutations in CTCF bound regions changes across an allelic series of CTCF knockdown mice will reveal how CTCF binding shapes the stability of the genome towards mutations. . These integrated strategies develop and deploy powerful, cutting-edge experimental approaches to reveal novel aspects of how CTCF binding stabilises the mammalian genome during healthy ageing as well as during mutagenesis.

2 488 251 €

Start date: 2019-09-01, End date: 2024-08-31

"Mutation and recombination are fundamental biological processes that determine adaptability of populations. The mutation rate reflects the equilibrium between the need to adapt, the burden of mutation load, the “cost of fidelity”, and random drift that determines a lower limit in achievable fidelity. Recombination fulfills an essential mechanistic role during meiosis, ensuring proper chromosomal segregation. Recombination affects the rate of creation and loss of favorable haplotypes, imposing 2nd-order selection pressure on modifiers of recombination. It is becoming apparent that recombination and mutation rates vary between individuals, and that these differences are in part inherited. Both processes are therefore “evolvable”, and amenable to genomic analysis. Identifying genetic determinants underlying these differences will provide insights in the regulation of mutation and recombination. The mutational load, and in particular the number of lethal equivalents per individual, remains poorly defined as epidemiological and molecular data yield estimates that differ by one order of magnitude. A relationship between recombination and fertility has been reported in women but awaits confirmation. Population structure (small effective population size; large harems), phenotypic data collection (systematic recording of > 50 traits on millions of cows), and large-scale SNP genotyping (for genomic selection), make cattle populations uniquely suited for genetic analysis. DAMONA proposes to exploit these unique resources, combined with recent advances in next generation sequencing and genotyping, to: (i) quantify and characterize inter-individual variation in male and female mutation and recombination rates, (ii) map, fine-map and identify causative genes underlying QTL for these four phenotypes, (iii) test the effect of loss-of-function variants on >50 traits including fertility, and (iv) study the effect of variation in recombination on fertility."

2 258 000 €

Start date: 2013-03-01, End date: 2018-02-28

98% of the human genome is non-protein coding raising the question of the role of the dark matter of the genome. It is now admitted that pervasive transcription generates thousands of noncoding transcripts that regulate gene expression and have broad impacts on development and disease. Among the long non coding (lnc)RNAs, antisense transcripts have been poorly studied despite their putative regulatory importance. Several functional examples include X-chromosome inactivation, maintenance of pluripotency and transcriptional regulation. However, no systematic study has yet addressed the comprehensive functional description of human antisense ncRNA, mainly because of technological issues and their low abundance. Indeed, in budding yeast S. cerevisiae, our group showed the existence of an entire class of antisense regulatory lncRNA extremely sensitive to RNA decay pathways, impinging their study so far. The roles for yeast antisense lncRNAs in shaping the epigenome raises important questions: What are the molecular and biochemical mechanisms by which antisense lncRNAs carry out their functions and are they functionally conserved in human cells? We propose that the dark side of the non-coding genome is another layer of gene regulation complexity that needs to be deciphered. With this proposal, we aim to draw the first exhaustive catalog of human antisense lncRNA in various cell types and tissues using up to date High throughput technologies and bioinformatics pipelines. Second, we propose to determine the functional role of antisense lncRNA on genome expression and stability in the context of cellular stress and cancer. We anticipate that powerful and modern genetic tools such DNA-mediated gene inactivation (ASO) and TALEN approaches will allow precise antisense genes manipulation never achieved so far. Our project is strongly supported by preliminary data indicating an unexpected large number of hidden antisense lncRNA in human cells controlled by RNA decay pathways.

1 998 884 €

Start date: 2014-12-01, End date: 2019-11-30

Regulation of gene expression lies at the heart of fundamental biological processes, such as the formation of different cell types inside an embryo or responses to environmental stimuli. Living cells ensure that the right genes are expressed at the right time and place by carefully controlling every RNA molecule inside a cell from its ‘birth’ by transcription to its final ‘death’ by degradation. While vast efforts strive to understand the first part of this process – transcription, studies of RNA degradation have been more limited. Current knowledge largely relies on small-scale investigation of key – but anecdotal – cases, while technical and experimental difficulties limit its large-scale analysis. Therefore, we still lack a systematic and predictive understanding of RNA degradation: technologies to globally measure it, the molecular mechanisms involved, its functional and physiological implications and models to decode and predict it. Transcriptional silencing makes early embryos an ideal system to study RNA degradation and uncover its basic concepts, as I propose here. Aim 1 will decipher how genomic information within native RNA sequences determines their degradation in embryos. Aim 2 will develop the technology to investigate RNA degradation at single-cell resolution, and uncover its regulation within arising embryonic cell populations. Aim 3 will reveal the molecular implementation of the regulatory code of RNA degradation and determine its physiological roles that underlie the massive degradation of maternal mRNAs – a key regulatory event and a main developmental transition in early embryos of all animals. This work will uncover new principles of RNA degradation in early development and elicit its mechanisms and functions using the zebrafish as an in vivo model system. The assays and models to be developed will be broadly applicable to study RNA degradation in diverse contexts, ranging from disease mechanisms to engineering of RNA- protein interactions.

1 500 000 €

Start date: 2020-03-01, End date: 2025-02-28

Branching morphogenesis, the process by which branched organs such as the lung, prostate, kidney or mammary gland are generated, is a paradigmatic example of complex developmental processes bridging multiple scales. The mechanisms through which given molecular signals and cellular behaviours give rise to a robust organ structure remains a fundamental and open question, for which theoretical methods are needed. Our experience in modelling cytoskeletal mechanics, stem cell dynamics and branching processes puts us in a unique position to tackle this fascinating problem, by combining systems biology and biophysical approaches at multiple scales. In particular, we will focus on: 1. Understanding how stochastic rules lead to robust morphogenetic outputs at the organ scale, and which constraints and optimal design principles they impose on physiological function. 2. Characterizing at the cellular scale the bi-directional feedbacks coordinating fate choices of stem/progenitor cells and niche signals during the extensive remodelling events that branching morphogenesis entails. 3. Developing at the subcellular and cellular scale an integrated mechanochemical theory of pattern formation in branched organs, to understand the coordination of mechanical forces and chemical signals defining their global structure. Towards these goals, we will combine analytical and numerical tools with data analysis methods, to reach a quantitative understanding of the emergent mechanisms driving branching morphogenesis. We will challenge our theoretical predictions with published datasets available for different organs, as well as design specific experimental tests in collaboration with experimental biology groups. This will allow us to compare and contrast different systems, and extract generic classes of design principles of organogenesis across length scales. With this, we expect to generate novel insights of broad relevance for the fields of systems, computational and developmental biology.

1 452 604 €

Start date: 2020-07-01, End date: 2025-06-30

Cells use circuits of interacting proteins to respond to their environment. In the past decades, molecular biology has provided detailed knowledge on the proteins in these circuits and their interactions. To fully understand circuit function requires, in addition to molecular knowledge, new concepts that explain how multiple components work together to perform systems level functions. Our lab has been a leader in defining such concepts, based on combined experimental and theoretical study of well characterized circuits in bacteria and human cells. In this proposal we aim to find novel principles on how circuits resist fluctuations and errors, and how they can be controlled by drugs: (1) Why do key regulatory systems use bifunctional enzymes that catalyze antagonistic reactions (e.g. both kinase and phosphatase)? We will test the role of bifunctional enzymes in making circuits robust to variations in protein levels. (2) Why are some genes regulated by a repressor and others by an activator? We will test this in the context of reduction of errors in transcription control. (3) Are there principles that describe how drugs combine to affect protein dynamics in human cells? We will use a novel dynamic proteomics approach developed in our lab to explore how protein dynamics can be controlled by drug combinations. This research will define principles that unite our understanding of seemingly distinct biological systems, and explain their particular design in terms of systems-level functions. This understanding will help form the basis for a future medicine that rationally controls the state of the cell based on a detailed blueprint of their circuit design, and quantitative principles for the effects of drugs on this circuitry.

2 261 440 €

Start date: 2010-03-01, End date: 2015-02-28

"The spatial organization of the genome inside the cell nucleus is tissue-specific and has been linked to several nuclear processes including gene activation, gene silencing, genomic imprinting, gene co-regulation, genome maintenance, DNA replication, DNA repair, chromosomal translocations and X chromosome inactivation. In fact, just about any nuclear/genome function has a spatial component that has been implicated in its control. We know surprisingly little about chromosome conformation and spatial organization or how they are established. The extent to which they are a cause or consequence of genome functions are current topics of considerable debate, however emerging data from my group and many other groups world-wide indicate that nuclear location and organization are drivers of genome functions, which in cooperation with other features including epigenetic marks, non-coding RNAs and trans-factor binding bring about genome control. Thus, genome spatial organization can be considered on a par with other epigenetic features that together contribute to overall genome control. The classical paradigm of early mammalian development arguably represents the most dramatic and yet least understood process of genome reprogramming, where a single cell undergoes a series of divisions to ultimately give rise to the hundreds of different cell types found in a mature organism. Study of pre-implantation embryo development is hindered by the very nature of the life form, composed of extremely low cell numbers at each stage, which severely limits the options for investigation. My lab has recently developed a novel technique called single cell Hi-C, which has the power to detect tens of thousands of simultaneous chromatin contacts from a single cell. In this application I propose to apply this technology to study chromosome structure and genome organization during mouse pre-implantation development along with single cell transcriptome analyses from the same cells."

2 401 393 €

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

The structure and role of the DNA molecule raise fascinating questions regarding its dynamics, i.e. not only the tri-dimensional reorganisation associated with functional events at short time-scale, but also the structural changes, i.e. rearrangements, that occur in the chromosome over generations. It is increasingly obvious that the physical properties of both the chromosomes and their environment the nucleoplasm, the nuclear periphery, cytoskeleton, etc. are playing important roles in the dynamic changes observed. For instance, we recently showed that chromosome movements during mid-prophase of meiosis in budding yeast result from a trans-acting force generated at the level of the global cytoskeleton network, suggesting that extranuclear mechanical trans-acting signals could also regulate chromosomal metabolism in other ways. Our objectives are to make important contributions to the understanding of the mechanical and functional interplay between the cytoskeleton, the nuclear periphery, and chromosomes through in vitro and in vivo interdisciplinary approaches. We will investigate three questions of fundamental importance: i) the potential transmission and function of mechanical forces from the cytoskeleton to chromatin during interphase, ii) the physical principles that govern chromosome reorganization under mechanical force in vitro, and iii) the global chromatin dynamics during the fundamental S phase and its impact on genome stability. We will use a combination of high-resolution imaging, micromanipulation, and high-throughput molecular techniques (chromosome conformation capture and ChIP-Seq) to reach our goals. Most of these studies will be performed in budding yeast, but will have repercussions in our understanding of higher eukaryotes metabolism.

1 497 000 €

Start date: 2011-06-01, End date: 2017-05-31

Anxiety disorders include different forms of pathological fear and anxiety and rank among the most common health concerns in human medicine. Millions of people become affected every year, and many of them do not respond to treatments. Anxiety disorders are heritable, but genetically complex. As a result, traditional gene mapping methods in the human population with prominent locus and allelic heterogeneity have not succeeded. Similarly, rodents have provided some insights into the circuitry of anxiety, but naturally occurring versions do not exist and gene deletion studies have not provided adequate models. To break through and identify new anxiety genes, I propose a novel and unique approach that resorts to man s best friend, dog. Taking advantage of the exaggerated genetic homogeneity characteristic of purebred dogs, recent genomics tools and the existence of naturally occurring heritable behaviour disorders in dogs can remedy the current lack of a suitable animal model of human psychiatric disorders. I propose to collect and perform a genome-wide association study in four breed-specific anxiety traits in dogs representing the three major forms of human anxiety: compulsive pacing and tail-chasing, noise phobia, and shyness corresponding to human OCD, panic disorder and social phobia, respectively. Canine anxiety disorders respond to human medications and other phenomenological studies suggest a share biological mechanism in both species. The proposed research has the potential to discover new genetic risk factors, which eventually will shed light on the biological basis of common neuropsychiatric disorders in both dog and human, provide insight into etiological mechanisms, enable identification of individuals at high-risk for adverse health outcomes, and facilitate development of tailored treatments.

1 381 807 €

Start date: 2010-10-01, End date: 2015-09-30

Once a new drug target is discovered, screening techniques are applied to detect prospective hits. However, which hit should be taken to the next level of development? This decision is most crucial as it entails huge financial commitments of the subsequent drug optimization. In consequence drugs are only developed against diseases that promise short-term profit. Chemogenomic profiling allows to compile parameters characterizing binding of drug candidates that achieve optimal interference with protein function. Membrane proteins demand different properties than viral ones. Either high isoform selectivity, promiscuous family-wide binding or efficient resistance tolerance is desired. This calls for very different ligand binding characteristics, requiring either enthalpy-/entropy-driven binding, rigid shape complementarity or pronounced residual mobility at the binding site. Interaction kinetics determine on/off-rates and the time a drug spends with its target. Their correct adjustment is essential for drug efficacy. At present chemogenomic binding parameters are rarely available and their correlation with the required target properties is hardly understood. We want to compile a knowledge base from congeneric protein-ligand series to correlate structural, thermodynamic, interaction-kinetic and dynamic behaviour to predict the qualities a lead must meet to optimally address a target. Our studies involve crystal structure analyses, microcalorimetry, molecular dynamics simulations, site-directed mutagenesis and interaction kinetics. This provides a comprehensive picture to productively change our current understanding of drug-protein binding to move from a current trial-and-error to a more efficient rational approach. It provides the opportunity to also consider orphan drugs and address neglected diseases.

1 754 615 €

Start date: 2011-05-01, End date: 2016-04-30

The DNA within our cells is constantly being damaged by both environmental and endogenous agents; of the many forms of DNA damage, the DNA double-strand break (DSB) is considered to be most dangerous. Correct processing of DSBs is not only essential for maintaining genomic integrity but is also required in specific biological processes, such as meiotic recombination and V(D)J recombination, in which DNA breaks are deliberately generated. In animals, defects in the proper response to DSBs can thus have different outcomes: cancer predisposition, embryonic lethality, or compromised immunity. Many genes that play a role in the processing of DSBs have been identified over the past decades, mainly by cloning genes that are responsible for specific human genomic instability or immune deficiency syndromes, and by genetic approaches using unicellular eukaryotes and rodent cell lines. It is, however, evident that many components required in higher eukaryotes are not yet known and the identification of those will be a major challenge for future research. Here, we will for the first time systematically test all genes encoded by an animals genome directly for their involvement in the cellular response to DSB in both somatic and germline tissues: we will use our recently developed transgenic animal models (C. elegans) that visualizes repair of a single localized genomic DNA break in genome wide RNAi screenings to identify (and then characterize) the complement of genes that are required to keep our genome stable, and when mutated can predispose humans to cancer. In parallel, we will study the cellular response to single DNA breaks that are artificially generated during different stages of gametogenesis, as well as address the developmental consequences of DSB induction during the earliest stages of embryonic development – an almost completely unexplored area in the field of genome instability and DNA damage responses.

1 060 000 €

Start date: 2008-05-01, End date: 2014-04-30

Cancers develop in very heterogeneous tissue environments. They depend on the tumor microenvironment (TME) for sustained growth, metastasis, and therapy resistance. Stromal cells are genetically stable and they have less likelihood to develop resistance than cancer cells. Therefore, targeting the TME represents an attractive approach for treating cancer. In order to develop new therapeutic strategies to reprogram the TME and inhibit tumor growth and resistance, it is essential to understand in detail the molecular mechanisms of the interactions between cancer and stromal cell populations. However, current methods to study these interactions require complete dissociation of the tumor, exposing the cells to severe stress and affecting dramatically gene expression patterns. Here, I propose to use Dual Ribosome Profiling (DualRP), a system that I recently developed, to study cell interactions in the TME. DualRP is an approach that allows not only simultaneous analysis of gene expression in two interacting cell populations in vivo, but also is able to uncover metabolic limitations in tumors. I aim to apply DualRP to mouse xenograft models where cancer cells interact with non-transformed fibroblasts and I’ll explore the combined response of both populations to cancer therapy. Moreover, I’ll utilize mouse genetic models tailored for DualRP to study cancer cell and macrophages/endothelial cells interactions. I will employ a combination of mouse genetic models, biochemical tools, deep sequencing, and bioinformatics. These studies will provide insight into how gene expression and metabolic programs define the interaction between cancer and stromal cells to promote tumor growth and metastasis, identify potential targets for therapeutic intervention, and provide maps of cell interactions in vivo. Therefore, this research has the potential to significantly advance our understanding of the molecular and metabolic mechanisms underlying the complex cell interactions in the TME.

1 499 375 €

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

The aim of this project is to understand the dynamics of protein-DNA interactomes underlying circadian oscillators in mammals, and how these shape circadian transcriptional output programs. Specifically our goal is to solve a fundamental issue in circadian biology: the phase specificity problem underlying circadian gene expression. We have taken a challenging and original multi-disciplinary approach in which molecular biology experiments will be tightly interlinked with computational analyses and biophysical modeling. The approach will generate time resolved protein-DNA interactomes in mouse liver for several key circadian repressors at unprecedented resolution. These experiments will be complemented with chromosome conformation capture (3C) experiments to monitor how looping interactions and 3D genome structure rearrange during the circadian cycle, which will inform on how circadian transcription networks use long-range gene regulatory mechanisms. Novel computational algorithms based on biophysical principles will be developed and implemented to optimally analyze interactome and 3C datasets. For the latter, statistical models from polymer physics will be used to reconstruct the chromatin networks and interaction maps from the 3C data. At the detailed level of individual cells, we will investigate transcription bursts, and how those are involved in the control of circadian gene expression. In particular we will exploit high temporal resolution bioluminescence reporters using a biophysical model of transcription coupled with a Hidden Markov Model (HMM). Through our innovative approach, we expect that the data generated and state-of-the-art analyses performed will lead novel insight into the role and mechanics of circadian transcription in controlling circadian outputs in mammals.

1 500 000 €

Start date: 2011-03-01, End date: 2016-02-29

Immediately after fertilization, mammalian genomes undergo a dramatic reshaping of the epigenome as the embryo transitions from the zygote into the pluripotent cells primed for lineage commitment. This is best exemplified by DNA methylation reprogramming, as the gametic patterns are largely erased, and the embryonic genome undergoes a wave of de novo DNA methylation. Moreover, once DNA methylation patterns are established, mechanisms faithfully maintain the mark across cell division. Thus, there is latent potential for DNA methylation deposited in the early embryo to exhibit a lifelong effect. DNA methylation is a modification that is typically associated with gene repression at repetitive elements and at a minority of protein coding genes. I previously described the regulation of the Zdbf2 gene in mice, which is programmed during the de novo DNA methylation program. Challenging the paradigm, in this case DNA methylation is required for activation of a gene via antagonism of the polycomb-group of silencing proteins. If the DNA methylation fails to occur, the gene stays silent throughout life, resulting in a reduced growth phenotype. For my proposed research I will utilize both a cell-based system that recapitulates these early embryonic events as well as an in vivo mouse model to investigate the extent and mechanisms of non-canonical DNA methylation functions. I plan to use a combinatorial approach of genomics, genetics, and proteomics in order to ascertain novel insights into DNA methylation-based regulation. Furthermore, I plan to employ precision epigenome editing tools to address the locus-specific impact of DNA methylation. Ultimately, I strive to gain a clear understanding of the profound epigenetic consequences of DNA methylation on this window of development, which occurs in the first week of mouse embryogenesis, and the second of human, but the repercussions of which can ripple throughout life.

1 495 480 €

Start date: 2020-01-01, End date: 2024-12-31

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

Current anti-angiogenesis based anti-tumor therapy relies on starving tumors by blocking their vascular supply via inhibition of growth factors. However, limitations such as resistance and toxicity, mandate conceptually distinct approaches. We will explore an entirely novel and long-overlooked strategy to discover additional anti-angiogenic candidates, based on the following innovative concept: ¿rather than STARVING TUMORS BY BLOCKING THEIR VASCULAR SUPPLY, we intend TO STARVE BLOOD VESSELS BY BLOCKING THEIR METABOLIC ENERGY SUPPLY¿, so that new vessels cannot form and nourish the growing tumor. This project is a completely new research avenue in our group, but we expect that it will offer refreshing long-term research and translational opportunities for the field. Because so little is known on endothelial cell (EC) metabolism, we will (i) via a multi-disciplinary systems-biology approach of transcriptomics, proteomics, computational network modeling, metabolomics and flux-omics, draw an endothelio-metabolic map in angiogenesis. This will allow us to identify metabolic regulators of angiogenesis, which will be further validated and characterized in (ii) loss and gain-of-function studies in various angiogenesis models in vitro and (iii) in vivo in zebrafish (knockdown; zinc finger nuclease mediated knockout), providing prescreen data to select the most promising candidates. (iv) EC-specific down-regulation (miR RNAi) or knockout studies of selected candidates in mice will confirm their relevance for angiogenic phenotypes in a preclinical model; and ultimately (v) a translational study evaluating EC metabolism-targeted anti-angiogenic strategies (pharmacological inhibitors, antibodies, small molecular compounds) will be performed in tumor models in the mouse.

2 365 224 €

Start date: 2011-05-01, End date: 2016-04-30

"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

Recent genetic studies have identified hundreds of susceptibility genes for common human diseases but genetic effects are small and identifying underlying mechanisms remains challenging. Rodent models offer significant advantages for analysis of disease phenotypes. Advances in genome resources and gene targeting have increased the attractiveness of the rat model for genetic studies but progress has been hampered by absence of relevant rat genome sequences. We recently sequenced the genome of the spontaneously hypertensive rat (SHR) and will shortly have completed the Wistar Kyoto (WKY) rat sequence. The SHR genome contains over 750 genes that are completely or partly deleted, or have a frameshift in their open reading frame. These sequence variants, along with variants controlling dysregulated gene expression that we characterised previously, most likely include the major determinants of SHR cardiovascular and metabolic disease phenotypes. We shall determine the functional consequences of these variants by creating and phenotyping transgenic and knockout rats on the SHR and WKY genetic backgrounds, using transposon-mediated transgenesis and zinc-finger nuclease-mediated gene deletion recently shown to be highly efficient in rats. Genes will be prioritised for study by statistical and informatic analyses using our extensive physiological, gene expression and linkage data in these rat strains, and by comparative analysis with data from human genome-wide association studies. Confirmed rat disease genes will be tested for conserved functions in humans. These proposals provide a systematic route to elucidating the molecular and functional basis of disease phenotypes in SHR and WKY rats, and for translating these findings to advance understanding of common human diseases.

2 476 108 €

Start date: 2011-06-01, End date: 2017-05-31

Animals constantly face complex environments consisted of multiple fluctuating cues. Accurate detection and efficient integration of such perplexing information are essential as animals’ fitness and consequently survival depend on making the right behavioral decisions. However, little is known about how multifaceted stimuli are integrated by neural systems, and how this information flows in the neural network in a single-neuron resolution. Here we aim to address these fundamental questions using C. elegans worms as a model system. With a compact and fully-mapped neural network, C. elegans offers a unique opportunity of generating novel breakthroughs and significantly advance the field. To study functional dynamics on a network-wide scale with an unprecedented single-neuron resolution, we will construct a comprehensive library of transgenic animals expressing state-of-the-art optogenetic tools and Calcium indicators in individual neurons. Moreover, we will study the entire encoding process, beginning with the sensory layer, through integration in the neural network, to behavioral outputs. At the sensory level, we aim to reveal how small sensory systems efficiently encode the complex external world. Next, we will decipher the design principles by which neural circuits integrate and process information. The optogenetic transgenic animals will allow us interrogating computational roles of various circuits by manipulating individual neurons in the network. At the end, we will integrate the gathered knowledge to study how encoding eventually translates to decision making behavioral outputs. Throughout this project, we will use a combination of cutting-edge experimental techniques coupled with extensive computational analyses, modelling and theory. The aims of this interdisciplinary project together with the system-level approaches put it in the front line of research in the Systems Biology field.

1 498 400 €

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

In eukaryotes, the complex regulation of temporal- and tissue-specific gene expression is controlled by the binding of transcription factors to enhancers, which in turn interact with the promoter of their target gene(s) via the formation of a chromatin loop. Despite their importance, the properties governing enhancer function and enhancer-promoter loops in the context of the three-dimensional organisation of the genome are still poorly understood. My recent work suggests that (i) developmental genes are often regulated by multiple enhancers, sometimes located at great linear distances, (ii) the spatio-temporal activity of a large fraction of those enhancers remains unknown, (iii) enhancer-promoter interactions are usually established before the target gene is expressed and are largely stable during embryogenesis, and (iv) stable interactions seem to be associated with the presence of paused RNA Polymerase II at the promoter before gene activation. Building upon these results, we propose to advance to the next level in the dissection of enhancer-promoter interaction functionality in the context of Drosophila embryogenesis. Specifically, we will address three important questions: (i) What determines the specificity of promoter-enhancer interactions in a complex genome? (ii) Are enhancer-promoter interactions tissue-specific, and what are the drivers of this specificity? (iii) Are all enhancer-promoter interactions functional, and how does the activity of an enhancer relate to the expression of the gene it interacts with? To this end, my group will apply an interdisciplinary approach, combining state-of-the-art methods in genetics and genomics, including novel single-cell techniques, using Drosophila embryogenesis as a model system. Our results will provide a unique view of the functionality of enhancer-promoter interactions in a developing embryo, a significant step towards understanding the link between chromatin organisation and transcription regulation.

1 770 375 €

Start date: 2018-05-01, End date: 2023-04-30