ERC FUNDED PROJECTS

The evolutionary pressures exerted by viruses on their host cells constitute a major force that drives the evolution of cellular antiviral mechanisms. The proposed research is motivated by our interest in the roles of protein-RNA interactions in both prokaryotic and eukaryotic antiviral pathways and will proceed in two directions. The first project stems from our current work on the CRISPR pathway, a recently discovered RNA-guided adaptive defense mechanism in bacteria and archaea that silences mobile genetic elements such as viruses (bacteriophages) and plasmids. CRISPR systems rely on short RNAs (crRNAs) that associate with CRISPR-associated (Cas) proteins and function as sequence-specific guides in the detection and destruction of invading nucleic acids. To obtain molecular insights into the mechanisms of crRNA-guided interference, we will pursue structural and functional studies of DNA-targeting ribonuceoprotein complexes from type II and III CRISPR systems. Our work will shed light on the function of these systems in microbial pathogenesis and provide a framework for the informed engineering of RNA-guided gene targeting technologies. The second proposed research direction centres on RNA-targeting antiviral strategies employed by the human innate immune system. Here, our work will focus on structural studies of major interferon-induced effector proteins, initially examining the allosteric activation mechanism of RNase L and subsequently focusing on other antiviral nucleases and RNA helicases, as well as mechanisms by which RNA viruses evade the innate immune response of the host. In our investigations, we plan to approach these questions using an integrated strategy combining structural biology, biochemistry and biophysics with cell-based functional studies. Together, our studies will provide fundamental molecular insights into RNA-centred antiviral mechanisms and their impact on human health and disease.

1 467 180 €

Start date: 2013-11-01, End date: 2018-10-31

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

1 692 737 €

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

Cardiovascular disease, diabetes and cancer have a dramatic impact on modern society, and in great part are related to uptake of cholesterol and sugar. We still know surprisingly little about the molecular details of the processes that goes on in this essential part of human basic metabolism. This application addresses cholesterol and sugar transport and aim to elucidate the molecular mechanism of cholesterol and sugar uptake in humans. It moves the frontiers of the field by shifting the focus to in vitro work allowing hitherto untried structural and biochemical experiments to be performed. Cholesterol uptake from the intestine is mediated by the membrane protein NPC1L1. Despite extensive research, the molecular mechanism of NPC1L1-dependent cholesterol uptake still remains largely unknown. Facilitated sugar transport in humans is made possible by sugar transporters called GLUTs and SWEETs, and every cell possesses these sugar transport systems. For all these uptake systems structural information is sorely lacking to address important mechanistic questions to help elucidate their molecular mechanism. I will address this using a complementary set of methods founded in macromolecular crystallography and electron microscopy to determine the 3-dimensional structures of key players in these uptake systems. My unpublished preliminary results have established the feasibility of this approach. This will be followed up by biochemical characterization of the molecular mechanism in vitro and in silico. This high risk/high reward membrane protein proposal could lead to a breakthrough in how we approach human biochemical pathways that are linked to trans-membrane transport. An improved understanding of cholesterol and sugar homeostasis has tremendous potential for improving general public health, and furthermore this proposal will help to uncover general principles of endocytotic uptake and facilitated diffusion systems at the molecular level.

1 499 848 €

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

Cells must ensure the integrity of their genome, and failure to do so can lead to mutations and cause disease. A sophisticated molecular network senses genomic lesions and coordinates their faithful repair with other DNA transactions, including transcription and DNA replication. Research over the last years has significantly advanced our understanding of the DNA damage response and continues to provide crucial insights that explain how cells deal with genotoxic stress to avoid malignant transformation. More recently, the intriguing phenomenon of cellular heterogeneity reached into the limelight as it became increasingly clear that significant variability exists between individual cells, even of the same genetic background and cell type. Single cells matter, for instance during cellular transformation or tumor relapse, and cellular variability thus impacts disease development and therapeutic outcome. Its determinants are surprisingly unexplored, however, and have not been studied in context of genome integrity maintenance. The main objective of this project is to systematically assess cellular heterogeneity in genome integrity maintenance and characterize its causes and consequences. Quantitative automated high-content imaging of large cell cohorts will be used to identify hitherto unknown determinants of variability in the cellular responses to genotoxic stress and dissect at the single cell level the variability in (1) the chromatin response to DNA double-strand breaks, (2) the cellular response to replication stress, and (3) the cellular capacity to trigger phase transitions, a newly emerging mechanism of dynamic compartmentalization, at sites of genomic lesions. This project will bridge two thus far independently developed research fields (genome stability and cellular heterogeneity), reveal how cell-to-cell variation impacts cell fate and survival in response to genotoxic stress, and may uncover ways to homogenize this response for improved cancer therapies.

1 500 000 €

Start date: 2017-04-01, End date: 2022-03-31

The scientific goal of this proposal is to contribute to our understanding of RNA-mediated epigenetic mechanisms of genome regulation in eukaryotes. Choosing ciliated protozoa as model organisms gives a wonderful opportunity to study the incredibly complex epigenetic mechanism of programming large-scale developmental rearrangements of the genome. This involves extensive rearrangements of the germline DNA, including elimination of up to 95% of the genome. The massive DNA rearrangement makes ciliates the perfect model organism to study this aspect of germline-soma differentiation. This process is proposed to be regulated by an RNA-mediated homology-dependent comparison of the germline and somatic genomes. Ciliate’s genomic subtraction is one of the most fascinating examples of the use of RNA-mediated epigenetic regulation, and of a specialized RNA interference pathway, to convey non-Mendelian inheritance in eukaryotes. The ‘genome scanning’ model raises many interesting questions, which are also relevant to other RNA-mediated regulation systems. One of the most intriguing is a ‘thermodynamic’ problem: the model assumes that a very complex population of small RNAs representing the entire germline genome can be compared to longer transcripts representing the entire rearranged maternal genome, resulting in the efficient selection of germline-specific scnRNAs, which are able to target DNA deletions in the developing nucleus. How is it possible that the truly enormous number of pairing interactions implied can occur in such a short time, just a few hours? RNA-RNA pairing interactions would probably have to be assisted by a dedicated molecular machinery. This proposal focuses on characterizing proteins and RNAs that can orchestrate the massive genome rearrangements in ciliates.

1 500 000 €

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

RNA silencing is a pan-eukaryotic gene regulation process that involves RNA molecules 19-30nt in length. These molecules are produced by RNAse-III proteins in the Dicer family and engage into sequence-specific regulation of complementary DNA or RNA upon their incorporation into effector complexes. RNA silencing serves essential roles in biology but the molecular bases of its mechanisms are still poorly understood. One major aspect of the proposed project is to decipher genetically the composition of RNA silencing effector complexes and to understand how those complexes orchestrate the regulation of fundamental processes involved in cell differentiation, notably the process of dosage compensation during chromosome X inactivation in mammals. The second aspect is part of our ongoing efforts to understand the implication of small RNAs in plant and animal innate immunity, their impact on pathogen’s fitness and evolution, and how pathogens counteract small RNA-directed immune pathways.

900 000 €

Start date: 2008-08-01, End date: 2012-07-31

Cockayne syndrome is a congenital disease with impaired DNA repair in actively transcribed genes. Affected children show developmental abnormalities and signs of premature aging. Cockayne syndrome is caused by mutations in the Cockayne syndrome complementation group A (CSA) and B (CSB) genes. While CSB encodes a SWI/SNF ATPase that likely assists the stalled RNA polymerase in overcoming lesions, CSA’s detailed role in repair has so far remained elusive. CSA is part of the DDB1-CSA-Cul4-Rbx1 E3 ubiquitin ligase complex. The available data suggest that CSA may function as a substrate adaptor for ubiquitination by the DDB1-Cul4-Rbx1 ligase. I will pursue a novel structure-driven proteomic approach to identify the substrate epitope recognized by the DDB1-CSA-Cul4-Rbx1 E3 ubiquitin ligase. These experiments aim to provide the important missing signal required for recruitment of the ligase complex to sites of stalled RNA polII complexes. Importantly, the CSA ligase, as it arrives at sites of DNA damage, is inactive. Our comprehensive structural and biochemical efforts will thus include the mechanisms of regulation of the CSA ubiquitin ligase activity by activators and inhibitors following recruitment to the repair site. MoBa-CS will not only improve our understanding of CSA’s molecular role in Cockayne syndrome, but also reveal CSA’s mode of action within the essential transcription coupled repair pathway. Understanding the cellular signals overseeing transcription coupled repair will provide important insights into how the pathway is integrated within the overall DNA damage response circuitry of the cell. ERC funding would enable us to pursue an interdisciplinary proteomic and structure based approach in examining DDB1-CSA function.

1 499 235 €

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

Analogous to quality control checks along the assembly line in industrial manufacturing processes, cells possess multiple quality control systems that ensure accurate expression of the genetic information throughout the intricate chain of biochemical reactions. “Nonsense-mediated mRNA decay” (NMD) represents a quality control mechanism that recognizes and degrades mRNAs of which the protein coding sequence is truncated by the presence of a premature termination codon (PTC). By eliminating these defective mRNAs with crippled protein-coding capacity, NMD substantially reduces the synthesis of potentially deleterious truncated proteins. Given that 30 % of all known disease-causing mutations in humans lead to the production of a nonsense mRNA, NMD serves as an important modulator of the clinical manifestations of genetic diseases, and manipulating NMD therefore represents a promising strategy for future therapies of many genetic disorders. However, the underlying molecular mechanisms of NMD are currently not well understood. One goal of our research is to understand at the molecular level how PTCs are recognized and distinguished from correct termination codons and how this recognition of nonsense mRNAs subsequently triggers their rapid degradation. In addition to triggering NMD, we have recently discovered that PTCs in certain immunoglobulin genes can also lead to the transcriptional silencing of the corresponding gene. We now search for the biological relevance of this novel quality control mechanism termed “nonsense-mediated transcriptional gene silencing” (NMTGS) and want to identify the involved molecules and their interactions. Using mainly mammalian cell cultures, we study the effect on the expression of engineered NMD and NMTGS reporter genes upon various treatments of the cells. State-of-the-art biochemical and molecular biology techniques are employed with the goal to further our understanding of these processes and their regulation at the molecular level.

1 300 000 €

Start date: 2008-06-01, End date: 2013-05-31

Cellular life depends on the timely and accurate duplication of chromosomal DNA through semi-conservative replication to sustain genomic integrity and organismal viability. In all domains of life, DNA replication relies on dedicated initiator proteins that recognize and bind specific genomic sites, termed replication origins, to facilitate the loading of ring-shaped replicative helicases onto DNA. While origin recognition by initiators is determined by specific DNA sequences in prokaryotes and in the eukaryote S. cerevisiae, origin specification in higher eukaryotes instead appears to rely on chromatin context and DNA structure. Yet, how initiators help specify replication origins at the molecular level and how their binding sites are established in higher eukaryotes remain foremost and long-standing questions in the field. This research proposal focuses on uncovering the molecular and structural principles for chromosomal binding site selection by the eukaryotic initiator, the origin recognition complex (ORC), in metazoan systems. Employing integrated biochemical, structural, and cell-based approaches, we aim to 1) elucidate how ORC binds DNA and how DNA structural elements contribute to this interaction, 2) determine how nucleosomes are recognized by ORC, and 3) identify auxiliary binding partners of ORC and establish how they contribute to origin specification. The outcomes of our proposed efforts will have far-reaching implications for multiple scientific fields by defining mechanistic links between chromatin architecture and DNA replication initiation, and they will set the foundation to understand at the molecular level how the replication initiation program is altered during cell differentiation and development. Our studies also have significant biomedical relevance, as failure to precisely replicate chromosomal DNA leads to genetic instability, which in turn underpins many human diseases, including cancer and certain developmental disorders.

1 500 000 €

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