ERC FUNDED PROJECTS

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 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

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

Over the past decade small RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) have been shown to carry pivotal roles in post-transcriptional regulation and genome protection and to play an important part in various physiological processes in animals. miRNAs can be found in a very wide range of animals yet their functions were studied almost exclusively in members of the Bilateria such as insects, nematodes and vertebrates. Hence studying their function in representatives of non-bilaterian phyla such as Cnidaria (sea anemones, corals, hydras and jellyfish) is crucial for understanding the evolution of miRNAs in animals and can provide important insights into their roles in the ancient ancestor of Cnidaria and Bilateria. The sea anemone Nematostella vectensis is an excellent model for such a study since it can be grown in large numbers throughout its life cycle in the lab and because well-established genetic manipulation techniques are available for this species. Our preliminary results indicate that miRNAs in Nematostella frequently have a nearly perfect match to their messenger RNA (mRNA) targets, resulting in cleavage of the target. This mode of action is common for plant miRNAs, but is very rare in Bilateria. This finding together with my recent discovery of a Nematostella homolog of HYL1, a protein involved in miRNA biogenesis in plants, raises the exciting possibility that the miRNA pathway existed in the common ancestor of plants and animals. Here I suggest to bring together an array of advanced biochemical and genetic methods such as gene knockdown, transgenesis, high throughput sequencing and immunoprecipitation in order to obtain - for the first time - a deep understanding of the biogenesis and mechanism of action of small RNAs in Cnidaria. This will provide a novel way to understand the evolution of this important molecular pathway and to evaluate its age and ancestral form.

1 499 587 €

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

The CRISPR-Cas system has been extensively studied for its ability to cleave DNA. In contrast, studies of the ability of the system to acquire and integrate new DNA from invaders as a form of prokaryotic adaptive immunity, have lagged behind. This delay reflects the extreme enthusiasm surrounding the potential of using the system’s cleavage capabilities as a genome editing tool. However, the enormous potential of the adaptation process can and should arouse a similar degree of enthusiasm. My lab has pioneered studies on the CRISPR adaptation process by establishing new methodologies, and applying them to demonstrate the essential role of the proteins and DNA elements, as well as the molecular mechanisms, operating in this process. In this project, I will establish novel platforms for studying adaptation and develop them into biotechnological applications and research tools. These tools will allow me to identify the first natural and synthetic inhibitors of the adaptation process. This, in turn, will provide genetic tools to control adaptation, as well as advance the understanding of the arms race between bacteria and their invaders. I will also harness the adaptation process as a platform for diversifying genetic elements for phage display, and for extending phage recognition of a wide range of hosts. Lastly, I will provide the first evidence for an association between the CRISPR adaptation system and gene repression. This linkage will form the basis of a molecular scanner and recorder platform that I will develop and that can be used to identify crucial genetic elements in phage genomes as well as novel regulatory circuits in the bacterial genome. Together, my findings will represent a considerable leap in the understanding of CRISPR adaptation with respect to the process, potential applications, and the intriguing evolutionary significance.

2 000 000 €

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

Transcription factors (TF) regulate genome function by controlling gene expression. Comprehensive characterization of the in vivo binding of TF to the DNA in relevant primary models is a critical step towards a global understanding of the human genome. Recent advances in high-throughput genomic technologies provide an extraordinary opportunity to develop and apply systematic approaches to learn the underline principles and mechanisms of mammalian transcriptional networks. The premise of this proposal is that a tractable set of rules govern how cells commit to a specific cell type or respond to the environment, and that these rules are coded in regulatory elements in the genome. Currently our understanding of the mammalian regulatory code is hampered by the difficulty of directly measuring in vivo binding of large numbers of TFs to DNA across multiple primary cell types and their natural response to physiological stimuli. Here, we overcome this bottleneck by systematically exploring the genomic binding network of 1. All relevant TFs of key hematopoietic cells in both steady state and under relevant stimuli. 2. Follow the changes in TF networks as cells differentiate 3. Use these models to engineer cell states and responses. To achieve these goals, we developed a new method for automated high throughput ChIP coupled to sequencing (HT-ChIP-Seq). We used this method to measure binding of 40 TFs in 4 time points following stimulation of dendritic cells with pathogen components. We find that TFs vary substantially in their binding dynamics, genomic localization, number of binding events, and degree of interaction with other TFs. The analysis of this data suggests that the TF network is hierarchically organized, and composed of different types of TFs, cell differentiation factors, factors that prime for gene induction, and factors that bind more specifically and dynamically. This proposal revisits and challenges the current understanding of the mammalian regulatory code.

1 500 000 €

Start date: 2012-10-01, End date: 2017-09-30

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

A novel type of defense system was recently identified in bacteria: the CRISPR array and its associated gene products (Cas). The system inserts short DNA sequences, called spacers, derived from foreign nucleic acid molecules in between direct repeats, thus forming the CRISPR array. The transcribed spacers eventually serve as molecular guides for Cas proteins that monitor and destroy nucleic acids having sequences similar to those spacers. Thorough mapping of the functional components and regulators of the system in a single model organism will be extremely valuable for understanding its mechanism of action. Studying the interactions between bacteria and phages should highlight the evolutionary role of the system and its consequences for shaping ecological systems. These insights will lead to novel ways of exploiting the system to improve molecular biology tools, to protect fermenting bacteria from phage spoilage, to equip phages with anti-CRISPR warfare to fight bacteria, and to prevent horizontal gene transfer between pathogens. Here, I intend to systematically seek out new roles of the system and to identify fundamental mechanisms and components that allow the system to function efficiently. I will address fundamental questions such as how the system avoids sampling self DNA into the CRISPR array. In addition, I will pursue two revolutionary possibilities. One, that the CRISPR/Cas system is not merely an adaptive defense system against phages, but that one of its roles is to serve as molecular machinery for silencing specific harmful genes by generating small silencing RNAs without the need for Cas proteins. The other is to test the system’s ability to prevent horizontal gene transfer of antibiotic resistance genes in an effort to study the system’s ecological value, potentially for applicative uses. My proposed studies will allow deeper understanding of the system, and enable breakthroughs from both basic and applicative aspects of the CRISPR field studies.

1 499 000 €

Start date: 2013-12-01, End date: 2018-11-30

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

1 461 576 €

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