ERC FUNDED PROJECTS

In eukaryotes, silencing small (s)RNAs, including micro (mi)RNAs and small interfering (si)RNAs, regulate many aspects of biology, including cell differentiation, development, hormonal responses, or defense. In particular, many plant and metazoan miRNAs play crucial roles in embryonic/post-embryonic development; the precise timing and localization of their expression is thus crucial to their action. Hence, specific miRNA repertoires underlie specific cell identities, and deviations from such repertoires often have deleterious consequences such as cancer. Many miRNAs also help organisms to adapt to stress, thus, given their importance in virtually all aspects of biology, understanding how, when and where miRNAs exert their actions is of paramount importance. To date, however, the few approaches to miRNA-mediated silencing in whole organisms have not taken into account the exquisite definition, in space and time, of their biogenesis and action, leading to an inaccurate view of the biology of these molecules at the systems level. Using the root system of the model plant Arabidopsis thaliana, we propose to explore, at single-cell and subcellular resolution levels, the biology of the main miRNA effector protein, ARGONAUTE 1 (AGO1) in intact tissues. Using a combination of state-of the-art technologies for single-cell forward genetics, protein purification and RNA/polysome profiling, we will establish a functional spatiotemporal map of the root AGO1-sRNAome and identify cell-specific modifiers of sRNA biogenesis and action. As a complement to the above approaches, chemical genetics will isolate small molecules allowing direct and specific manipulation of AGO1-dependent sRNA pathways in planta. RNA silencing modifier compounds will also accelerate forward and reverse approaches of RNA silencing in plants with sensitized genetic backgrounds, and uncover novel connections between miRNA/siRNA and physiological or metabolic pathways.

2 251 600 €

Start date: 2013-07-01, End date: 2018-06-30

Comparative genomics revealed that ~5% of the human genome is conserved among mammals. This fraction is likely functional, and could harbor pathogenic mutations. We have shown (Nature 2002, Science 2003) that more than half of the constrained fraction of the genome consists of Conserved Non-Coding sequences (CNCs). Model organisms provided evidence for enhancer activity for a fraction of CNCs; in addition another fraction is part of large non-coding RNAs (lincRNA). However, the function of the majority of CNCs is unknown. Importantly, a few pathogenic mutations in CNCs have been associated with genetic disorders. We propose to i) perform functional analysis of CNCs, and ii) identify the spectrum of pathogenic CNC mutations in recognizable human phenotypes. The aims are: 1. Functional genomic connectivity of CNCs 1a. Use 4C in CNCs in various cell types and determine their physical genomic interactions. 1b. Perform targeted disruption of CNCs in cells and assess the functional outcomes. 2. Pathogenic variation of CNCs 2a. Assess the common variation in CNCs: i) common deletion/insertions in 350 samples by aCGH of all human CNCs; ii) common SNP/small indels using DNA selection and High Throughput Sequencing (HTS) of CNCs in 100 samples. 2b. Identify likely pathogenic mutations in developmental syndromes. Search for i) large deletions and duplications of CNCs using aCGH in 1500 samples with malformation syndromes, 1000 from spontaneous abortions, and 500 with X-linked mental retardation; and ii) point mutations in these samples by targeted HTS. The distinction between pathogenic and non-pathogenic variants is difficult, and we propose approaches to meet the challenge. 3. Genetic control (cis and trans eQTLs) of expression variation of CNC lincRNAs, using 200 samples.

2 353 920 €

Start date: 2010-07-01, End date: 2015-06-30

Although pioneered by human geneticists as a potential solution to the challenging problem of finding the genetic basis of common human diseases, genome-wide association studies (GWAS) have, owing to advances in genotyping and sequencing technology, become an obvious general approach for studying the genetics of natural variation. They are particularly useful when inbred lines are available because once these lines have been genotyped they can be phenotyped multiple times, making it possible (as well as extremely cost-effective) to study many different traits in many different environments, while replicating the phenotypic measurements to reduce environmental noise. Here we propose to continue our groundbreaking GWAS work in the model plant Arabidopsis thaliana. We will explore the limits of the approach, moving beyond marker-trait linkage disequilibrium to full sequence information. We will carry out GWAS of important life-history traits using over 1000 inbred A. thaliana lines for which nearly complete sequence information is available. The GWAS will be complemented by linkage mapping in F2 crosses to eliminate confounding linkage disequilibrium, associations will be verified experimentally, and confirmed causal polymorphisms will be added to the model in an iterative manner in order to create an increasingly refined genotype-phenotype map.

2 183 956 €

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

In eukaryotic cells, the higher-order organization of genomes is functionally important to ensure correct execution of gene expression programs. For instance, as cells differentiate into specialized cell types, chromosomes undergo diverse structural and organizational changes that affect gene expression and other cellular functions. However, how this process is achieved is still poorly understood. The elucidation of the mechanisms that control the spatial architecture of the genome and its contribution to gene regulation is a key open issue in molecular biology, relevant for physiological and pathological processes. Increasing evidence indicated that large-scale folding of chromatin may affect gene expression by locating genes to specific nuclear subcompartments that are either stimulatory or inhibitory to transcription. Nuclear periphery (NP) and nucleolus are two important nuclear landmarks where repressive chromatin domains are often located. The interaction of chromosomes with NP and nucleolus is thought to contribute to a basal chromosome architecture and genome function. However, while the role of NP in genome organization has been well documented, the function of the nucleolus remains yet elusive. To fully understand how genome organization regulates chromatin and gene expression states, it is necessary to obtain a comprehensive functional map of genome compartmentalization. However, so far, only domains associating with NP (LADs) have been identified and characterized while nucleolar-associated domains (NADs) remained under-investigated. The aim of this project is to fill this gap by developing methods to identify and characterize NADs and analyse the role of the nucleolus in genome organization, moving toward the obtainment of a comprehensive functional map of genome compartmentalization for each cell state and providing novel insights into basic principles of genome organization and its role in gene expression and cell function that yet remain elusive.

2 500 000 €

Start date: 2018-09-01, End date: 2023-08-31

Elements operating in the context of a system generate results that are different from the simple addition of the results of each element. This notion is one of the basic tenants of systems science. In systems biology/medicine complex (disease) phenotypes arise from multiple interacting factors, specifically proteins. Yet, the biochemical and mechanistic base of complex phenotypes remain elusive. An array of powerful genomic technologies including GWAS, WGS, transcriptomics, epigenetic analyses and proteomics have identified numerous factors that contribute to complex phenotypes. It can be expected that over the next few years, genetic factors contributing to specific complex phenotypes will be comprehensively identified, while their interactions will remain elusive. The project “Proteomics 4D: The proteome in context “explores the concept, that complex phenotypes arise from the perturbation of modules of interacting proteins and that these modules integrate seemingly independent genomic variants into a single biochemical response. We will develop and apply a generic technology to directly measure the composition, topology and structure of wild type and genetically perturbed protein modules and relate structural changes to their functional output. This will be achieved by a the integration of quantitative proteomic and phosphoproteomic technologies determining molecular phenotypes, and hybrid structural methods consisting of chemical cross-linking and mass spectrometry, cryoEM and computational data integration to probe structural perturbations. The project will focus initially on the structural and functional effects of cancer associated mutations in protein kinase modules and then generalize to study perturbed modules in any tissue and disease state. The resources supporting this technology will be disseminated to catalyze a broad transformation of biology and molecular medicine towards the analysis of the proteome as a modular entity, the proteome in context.

2 208 150 €

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

Transposable elements (TEs) account for more than two thirds of the human genome. They can inactivate genes, provide novel coding functions, sprinkle chromosomes with recombination-prone repetitive sequences, and modulate cellular gene expression through a wide variety of transcriptional and posttranscriptional influences. As a consequence, TEs are considered as essential motors of evolution yet they are occasionally associated with disease, causing about one hundred Mendelian disorders and possibly contributing to several human cancers. As expected for such genomic threats, TEs are subjected to tight epigenetic control imposed from the very first days of embryogenesis, in part owing to their recognition by sequence-specific RNA- and protein-based repressors. It is generally considered that the evolutionary selection of these TE controllers reflects a simple host-pathogen arms race, and that their action results in the early and permanent silencing of their targets. We have recently uncovered new evolutionary evidence and obtained genomic and functional data that invalidate this dual assumption, and suggest instead that transposable elements and their epigenetic controllers establish species-specific transcriptional networks that play critical roles in human development and physiology. The general objective of the present proposal is to explore the breadth of this phenomenon, to decipher its mechanisms, to unveil its functional implications, and to probe how this knowledge could be exploited for basic research, biotechnology and clinical medicine.

2 500 000 €

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