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