ERC FUNDED PROJECTS

"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

Recent cancer genome analyses have led to the discovery of a process involving massive genome structural rearrangement (SR) formation in a one-step, cataclysmic event, coined chromothripsis. The term chromothripsis (chromo from chromosome; thripsis for shattering into pieces) stands for a hypothetical process in which individual chromosomes are pulverised, resulting in a multitude of fragments, some of which are lost to the cell whereas others are erroneously rejoined. Compelling evidence was presented that chromothripsis plays a crucial role in the development, or progression of a notable subset of human cancers – thus, tumorigensis models involving gradual acquisitions of alterations may need to be revised in these cancers. Presently, chromothripsis lacks a mechanistic basis. We recently showed that in childhood medulloblastoma brain tumours driven by Sonic Hedgehog (Shh) signalling, chromothripsis is linked with predisposing TP53 mutations. Thus, rather than occurring in isolation, chromothripsis appears to be prone to happen in conjunction with (or instigated by) gradually acquired alterations, or in the context of active signalling pathways, the inference of which may lead to further mechanistic insights. Using such rationale, I propose to dissect the mechanism behind chromothripsis using interdisciplinary approaches. First, we will develop a computational approach to accurately detect chromothripsis. Second, we will use this approach to link chromothripsis with novel factors and contexts. Third, we will develop highly controllable cell line-based systems to test concrete mechanistic hypotheses, thereby taking into account our data on linked factors and contexts. Fourth, we will generate transcriptome data to monitor pathways involved in inducing chromothripsis, and such involved in coping with the massive SRs occurring. We will also combine findings from all these approaches to build a comprehensive model of chromothripsis and its associated pathways.

1 471 964 €

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

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

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