ERC FUNDED PROJECTS

Epigenetic inheritance entails transmission of phenotypic traits not encoded in the DNA sequence and, in the most extreme case, Transgenerational Epigenetic Inheritance (TEI) involves transmission of memory through multiple generations. Very little is known on the mechanisms governing TEI and this is the subject of the present proposal. By transiently enhancing long-range chromatin interactions, we recently established isogenic Drosophila epilines that carry stable alternative epialleles, defined by differential levels of the Polycomb-dependent H3K27me3 mark. Furthermore, we extended our paradigm to natural phenotypes. These are ideal systems to study the role of Polycomb group (PcG) proteins and other components in regulating nuclear organization and epigenetic inheritance of chromatin states. The present project conjugates genetics, epigenomics, imaging and molecular biology to reach three critical aims. Aim 1: Analysis of the molecular mechanisms regulating Polycomb-mediated TEI. We will identify the DNA, protein and RNA components that trigger and maintain transgenerational chromatin inheritance as well as their mechanisms of action. Aim 2: Role of 3D genome organization in the regulation of TEI. We will analyze the developmental dynamics of TEI-inducing long-range chromatin interactions, identify chromatin components mediating 3D chromatin contacts and characterize their function in the TEI process. Aim 3: Identification of a broader role of TEI during development. TEI might reflect a normal role of PcG components in the transmission of parental chromatin onto the next embryonic generation. We will explore this possibility by establishing other TEI paradigms and by relating TEI to the normal PcG function in these systems and in normal development. This research program will unravel the biological significance and the molecular underpinnings of TEI and lead the way towards establishing this area of research into a consolidated scientific discipline.

2 500 000 €

Start date: 2018-11-01, End date: 2023-10-31

Malaria parasite infection in humans has been called “the strongest known force for evolutionary selection in the recent history of the human genome”, and I hypothesize that a similar statement may apply to the mosquito vector, which is the definitive host of the malaria parasite. We previously discovered efficient malaria-resistance mechanisms in natural populations of the African malaria vector, Anopheles gambiae. Aim 1 of the proposed project will implement a novel genetic mapping design to systematically survey the mosquito population for common and rare genetic variants of strong effect against the human malaria parasite, Plasmodium falciparum. A product of the mapping design will be living mosquito families carrying the resistance loci. Aim 2 will use the segregating families to functionally dissect the underlying molecular mechanisms controlled by the loci, including determination of the pathogen specificity spectra of the host-defense traits. Aim 3 targets arbovirus transmission, where Anopheles mosquitoes transmit human malaria but not arboviruses such as Dengue and Chikungunya, even though the two mosquitoes bite the same people and are exposed to the same pathogens, often in malaria-arbovirus co-infections. We will use deep-sequencing to detect processing of the arbovirus dsRNA intermediates of replication produced by the RNAi pathway of the mosquitoes. The results will reveal important new information about differences in the efficiency and quality of the RNAi response between mosquitoes, which is likely to underlie at least part of the host specificity of arbovirus transmission. The 3 Aims will make significant contributions to understanding malaria and arbovirus transmission, major global public health problems, will aid the development of a next generation of vector surveillance and control tools, and will produce a definitive description of the major genetic factors influencing host-pathogen interactions in mosquito immunity.

2 307 800 €

Start date: 2013-03-01, End date: 2018-02-28

The eukaryotic genome is packaged into large-scale chromatin structures that occupy distinct domains in the nucleus and this organization is now seen as a key contributor to genome functions. Two key functions of the genome can take advantage of nuclear organization: regulated gene expression and the propagation of a stable genome. To understand these fundamental processes, we have chosen to use yeast as a model system that allows genetics, molecular biology and advanced live microscopy approaches to be combined. Budding yeast have been very powerful to demonstrate that gene position can play an active role in regulating gene expression. Distinct subcompartments dedicated to either gene silencing or activation of specific genes are positioned at the nuclear periphery. To gain insight into the mechanisms underlying this sub-compartmentalization, we will address three complementary issues: - What are the mechanisms involved in the establishment and maintenance of silent nuclear compartments? - How and why are some activated genes recruited to the nuclear periphery? - What are the relationships between repressive and activating nuclear compartments? Concerning the maintenance of genome integrity, recent advances in yeast highlight the importance of nuclear architecture. However, how nuclear organization influences the formation and processing of DNA lesions remain poorly understood. We will focus on two main questions: - How and where in the nucleus are double strand breaks recognized, processed, and repaired? - Where do breaks or gaps resulting from replicative stress at 'fragile sites' arise in the nucleus and how does nuclear organization influence their stability? We hope to gain a better understanding of the mechanisms presiding nuclear organization and its importance for genome functions. These mechanisms are likely to be conserved and will be subsequently tested in higher eukaryotic cells.

1 000 000 €

Start date: 2008-09-01, End date: 2014-05-31

Transcriptional regulation of genes in eukaryotic cells requires a complex and highly regulated interplay of chromatin environment, epigenetic status of target sequences and several different transcription factors. Eukaryotic genomes are tightly packaged within nuclei, yet must be accessible for transcription, replication and repair. A striking correlation exists between chromatin topology and underlying gene activity. According to the textbook view, chromatin loops bring genes into direct contact with distal regulatory elements, such as enhancers. Moreover, we and others have shown that genomes are organized into discretely folded megabase-sized regions, denoted as topologically associated domains (TADs), which seem to correlate well with transcription activity and histone modifications. However, it is unknown whether chromosome folding is a cause or consequence of underlying gene function. To better understand the role of genome organization in transcription regulation, I will address the following questions: (i) How are chromatin configurations altered during transcriptional changes accompanying development? (ii) What are the real-time kinetics and cell-to-cell variabilities of chromatin interactions and TAD architectures? (iii) Can chromatin loops be engineered de novo, and do they influence gene expression? (iv) What genetic elements and trans-acting factors are required to organize TADs? To address these fundamental questions, I will use a combination of novel technologies and approaches, such as Hi-C, CRISPR knock-ins, ANCHOR tagging of DNA loci, high- and super-resolution single-cell imaging, genome-wide screens and optogenetics, in order to both study and engineer chromatin architectures. These studies will give groundbreaking insight into if and how chromatin topology regulates transcription. Thus, I anticipate that the results of this project will have a major impact on the field and will lead to a new paradigm for metazoan transcription control.

1 500 000 €

Start date: 2016-06-01, End date: 2021-05-31

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

"Diatoms are the most successful group of eukaryotic phytoplankton in the modern ocean. Recently completed whole genome sequences have revealed a wealth of information about the evolutionary origins and metabolic adaptations that may have led to their ecological success. A major finding is that they have acquired genes both from their endosymbiotic ancestors and by horizontal gene transfer from marine bacteria. This unique melting pot of genes encodes novel and largely unexplored capacities for metabolic management. The project will address the current gap in knowledge about the physiological functions of diatom gene products and about the evolutionary mechanisms that have led to diatom success in contemporary oceans. We will exploit genome-enabled approaches to pioneer new research topics addressing: 1. How has diatom evolution enabled interactions between chloroplasts and mitochondria that have provided diatoms with physiological and metabolic innovations? 2. What are the relative contributions of DNA sequence variation and epigenetic processes in diatom adaptive dynamics? By combining these questions, we will uniquely be able to identify sentinel genes that have driven major physiological and metabolic innovations in diatoms, and will explore the mechanisms that have selected and molded them during diatom evolution. We will focus our studies largely on diatom responses to nutrients, in particular nitrate and iron, and will exploit the advantages of Phaeodactylum tricornutum as a model diatom species for reverse genetics. The proposed studies will revisit textbook understanding of photosynthesis and nitrogen metabolism, and will refine hypotheses about why diatoms dominate in contemporary ocean settings. By placing our studies in evolutionary and ecological contexts, in particular by examining the contribution of epigenetic processes in diatoms, our work will furthermore provide insights into how the environment selects for fitness in phytoplankton."

2 423 320 €

Start date: 2012-06-01, End date: 2017-05-31

Understanding the mechanisms underlying the initiation and regulation of transcription remains one of the most fundamental questions in biology. Much of what we know about the transcription process was inferred from experiments on a handful of genes. As these experiments are not realistically scalable, corresponding computational methods building on these findings have emerged; however, these are not accurate enough for annotation of genomes. The limitations reflect that we have no accurate universal model describing transcription initiation; to a large extent, our understanding is based on case stories. Recently, high-throughput methods have been developed to chart the TSS landscape with nucleotide resolution. Using these data, I have dissected promoters at nucleotide level and found patterns that explain the transcription initiation rate for individual nucleotides. The objective for this work is to extend this to the first universal model for how cells select core promoters and associated TSSs. This will have two counterparts: i)prediction of TSSs from DNA sequence given a region of accessible DNA, and ii)prediction of DNA accessibility based on DNA sequences and dynamic epigenetic factors. Such a model will be a corner stone of future experimental and computational transcriptome and gene regulation studies.

812 399 €

Start date: 2008-09-01, End date: 2013-08-31

In eukaryotes, the complex regulation of temporal- and tissue-specific gene expression is controlled by the binding of transcription factors to enhancers, which in turn interact with the promoter of their target gene(s) via the formation of a chromatin loop. Despite their importance, the properties governing enhancer function and enhancer-promoter loops in the context of the three-dimensional organisation of the genome are still poorly understood. My recent work suggests that (i) developmental genes are often regulated by multiple enhancers, sometimes located at great linear distances, (ii) the spatio-temporal activity of a large fraction of those enhancers remains unknown, (iii) enhancer-promoter interactions are usually established before the target gene is expressed and are largely stable during embryogenesis, and (iv) stable interactions seem to be associated with the presence of paused RNA Polymerase II at the promoter before gene activation. Building upon these results, we propose to advance to the next level in the dissection of enhancer-promoter interaction functionality in the context of Drosophila embryogenesis. Specifically, we will address three important questions: (i) What determines the specificity of promoter-enhancer interactions in a complex genome? (ii) Are enhancer-promoter interactions tissue-specific, and what are the drivers of this specificity? (iii) Are all enhancer-promoter interactions functional, and how does the activity of an enhancer relate to the expression of the gene it interacts with? To this end, my group will apply an interdisciplinary approach, combining state-of-the-art methods in genetics and genomics, including novel single-cell techniques, using Drosophila embryogenesis as a model system. Our results will provide a unique view of the functionality of enhancer-promoter interactions in a developing embryo, a significant step towards understanding the link between chromatin organisation and transcription regulation.

1 770 375 €

Start date: 2018-05-01, End date: 2023-04-30

Our project is to understand the role of non coding (nc)RNA in the regulation of epigenetic landscape and gene expression. RNA interference pathway is absent in the budding yeast but recent works from our laboratory showed the existence of an original ncRNA-dependent pathway that controls gene expression in S. cerevisiae. We characterized a cryptic unstable ncRNA mediating the transcriptional silencing of the transposon Ty1 through histone methylation. Furthermore, unpublished data suggest the existence of subtelomeric ncRNAs that might control telomere metabolism and promoter-associated ncRNAs that mediate repressive epigenetic marks. We propose that a class of unstable ncRNA mediates genome expression and fluidity through histone modifications. Following 2 directions, our aim is to systematically identify these ncRNAs (A) and further characterize their regulatory mechanisms (B). (A) First, we aim to identify the regulatory ncRNAs by performing genome-wide approaches in strains accumulating these regulatory ncRNAs. We envisage developing protocols to analyze the cryptic transcriptome using deep sequencing technologies. (B) Second, we will further characterize the previously identified regulatory ncRNAs controlling repetitive regions (Ty1 transposon and telomeric repeats) but also gene expression. Through a range of experimental procedures from living cell biology (Fluorescence Immuno Hybridization), biochemical approaches (RNA-TRAP) and genetic, we will determine the dynamics of the regulatory ncRNA within the cell, the associated proteins that regulate their activities and the chromatin defects resulting from their expression. Our aim is to extensively describe the RNAi-like regulation in S. cerevisiae, that we anticipate to be broadly conserved in other eukaryotes.

1 735 524 €

Start date: 2009-12-01, End date: 2014-11-30

In Eukaryotes, cellular identity and tissue-specific functions are linked to the epigenetic landscape and the multi-scale architecture of the genome. The packing of DNA into nucleosomes at the ~100 bp scale and the organization of whole chromosomes into functional territories within the nucleus are well documented. At an intermediate scale, chromosomes are organised in megabase to sub-megabase structures called Topologically Associating Domains (TADs). Critically, TADs are highly correlated to patterns of epigenetic marks determining the transcriptional state of the genes they encompass. Until now, the lack of efficient technologies to map chromosome architecture and epigenetic marks at the single-cell level have limited our understanding of the molecular actors and mechanisms implicated in the establishment and maintenance of the multi-scale architecture of chromosomes and epigenetic states, and the interplay between this architecture and other nuclear functions such as transcription. The overall aim of EpiScope is to unveil the functional, multi-scale, 3D architecture of chromatin at the single-cell level while preserving cellular context, with a toolbox of groundbreaking high-performance microscopies (Hi-M). Hi-M will use unique combinations of multi-focus and single-molecule localization microscopies with novel DNA labeling methods and microfluidics. Hi-M will enable the study of structure-function relationships within TADs of different chromatin types and correlate single-cell variations in epigenomic patterns to 3D conformations with genomic specificity and at the nanoscale. Finally, Hi-M will be used to develop a novel high-throughput, high-content method to unveil the full pairwise distance distribution between thousands of genomic loci at the single cell level and at multiple length-scales. Our findings and technologies will shed new light into the mechanisms responsible for cellular memory, identity and differentiation.

1 999 780 €

Start date: 2017-09-01, End date: 2022-08-31

Palaeogenomics is the nascent discipline concerned with sequencing and analysis of genome-scale information from historic, ancient, and even extinct samples. While once inconceivable due to the challenges of DNA damage, contamination, and the technical limitations of PCR-based Sanger sequencing, following the dawn of the second-generation sequencing revolution, it has rapidly become a reality. Indeed, so much so, that popular perception has moved away from if extinct species’ genomes can be sequenced, to when it will happen - and even, when will the first extinct animals be regenerated. Unfortunately this view is naïve, and does not account for the financial and technical challenges that face such attempts. I propose an exploration of exactly what the limits on genome reconstruction from extinct or otherwise historic/ancient material are. This will be achieved through new laboratory and bioinformatic developments aimed at decreasing the cost, while concomitantly increasing the quality of genome reconstruction from poor quality materials. In doing so I aim to build a scientifically-grounded framework against which the possibilities and limitations of extinct genome reconstruction can be assessed. Subsequently genomic information will be generated from a range of extinct and near-extinct avian and mammalian species, in order to showcase the potential of reconstructed genomes across research questions spanning at least three different streams of research: De-extinction, Evolutionary Genomics, and Conservation Genomics. Ultimately, achievement of these goals requires formation of a dedicated, closely knit team, focusing on both the methodological challenges as well as their bigger picture application to high-risk high-gain ventures. With ERC funding this can become a reality, and enable palaeogenomics to be pushed to the limits possible under modern technology.

2 000 000 €

Start date: 2016-04-01, End date: 2021-03-31

Polycomb group (PcG) and trithorax group (trxG) genes were discovered in Drosophila melanogaster as repressors and activators of Hox genes, a set of transcription factors that specify the antero-posterior axis of the body plan. PcG and trxG proteins form multimeric complexes that are required to maintain their expression state after the initial transcriptional regulators disappear from the embryo. Subsequent work led to a better understanding of their mechanisms of action. Moreover, PcG and trxG genes have also been identified in vertebrates, where they regulate Hox genes, they are involved in cell proliferation, stem cell identity and cancer, genomic imprinting in plants and mammals and X inactivation. PcG and trxG components form multimeric complexes. Some trxG and PcG components possess methyltransferase activities directed toward specific lysines of histone H3, whereas other trxG and PcG proteins interpret these histone marks. Recent studies have described the genomewide distribution of PcG proteins and of their related histone modification in Drosophila and other species. However, the PcG recruitment code to their target chromatin is still not understood, and the mechanism of PcG-mediated gene silencing is unclear. The formation of subnuclear silencing compartments might contribute to the stable repression of transcription. Drosophila PcG proteins have a speckled nuclear distribution and the number of these so-called PcG bodies is progressively reduced during development. We showed that multiple PREs can associate in the nucleus to enhance the strength of PcG-mediated silencing. However, we do not know how frequent is this clustering process and how important it is functionally at a genomewide level. Our project will tackle these questions by using a combination of genetics, developmental biology, cell biology, genomics and bioinformatic approaches, with the aim to gain an integrated understanding of the role of Polycomb and trithorax in biology

2 200 000 €

Start date: 2009-09-01, End date: 2015-08-31

There is no living organism that does not contain transposons, and they make up a significant fraction, and in some instances, the vast majority of the genome in a given species. Owing to their proliferation propensity, these mobile genetic elements can create genetic variability providing selective benefits, but they also have a mutagenic potential. Therefore, host genomes have evolved epigenetic surveillance mechanisms to recognize and silence transposons. If maintenance of silencing is rather well understood, little is know about how the host recognize transposons as non-self elements and initiate silencing as they invade the genome. Also, although several examples indicate that a variety of environmental factors can reverse transposon silencing, how such factors interfere with the silencing machinery is largely unknown. The proposed research project will make use of our recent discovery of active endogenous retrotransposons in Arabidopsis to decipher genetically the mechanisms involved in initiating silencing of a transposon. In parallel, we will characterize how DNA-methylation associated silencing can be efficiently re-established once it has been lost, and use a genome-wide approach to determine the extent of this phenomenon. Finally, we intend to determine the genome-wide impact of a stress on transposon silencing and to genetically identify and characterize the molecular bases of stress-induced release of silencing at transposons. Our studies have the potential to bring general insights into how transposons have been so successful in colonizing host genomes, how they are kept under tight control and can be unleashed thereby contributing to genome plasticity and environmental adaptation.

1 490 876 €

Start date: 2010-10-01, End date: 2015-09-30

Drug resistance is limiting our ability to treat most infectious diseases and forms of cancer. Indeed this relentless evolution is the major driver of treatment failure for diseases that are responsible for over half of the global disease related mortality. Yet, the underlying principles that guide this evolutionary response are poorly understood, in particular with regards to understanding the impact of multidrug treatment. LimitMDR will characterize evolutionary trajectories leading to multidrug resistance in response to individual and combination drug treatment through the execution of large-scale adaptive evolution experiment with two bacterial pathogens followed by genome sequencing and phenotyping. This effort will enable testing of contrasting hypotheses regarding the evolution of multidrug resistance in response to combination treatment. We will characterize the cause-and-effect of resistance and sensitivity mutations identified in our global data set and map comprehensive fitness landscapes of mutations accumulated during drug resistance evolution to understand the evolutionary dynamics underlying resistance evolution. To accomplish these bold goals we shall develop novel multiplexed methodologies enabling unprecedented scale of construction and phenotypic testing of identified mutations. While genetic epistasis is considered of key importance to resistance evolution most studies focus on mutations within an individual gene. Through the development of a novel experimental approach we shall elucidate complex epistatic interaction networks between mutations accumulated during resistance evolution. Finally, we will conduct mechanistic studies to uncover the mechanisms of collateral sensitivity. These studies will shed light on this underappreciated phenomenon, which is of critical relevance to drug discovery and the evolution of drug resistance. In conclusion LimitMDR will develop groundbreaking novel methodologies and scientific insights that will c

1 492 453 €

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

Meiosis is an essential stage in the life cycle of sexually-reproducing organisms. Indeed, meiosis is the specialized cell division that reduces the number of chromosomes from two sets in the parent to one set in gametes, while fertilization restores the original chromosome number. Meiosis is also the stage of development when genetic recombination occurs, thus being the heart of Mendelian heredity. Increasing our knowledge on meiotic mechanisms, in addition to its intrinsic interest, may have also important implications for agriculture and medicine. In the last decade Arabidopsis emerged as one of the prominent models in the field of meiosis. Indeed, the meiotic field benefits greatly from a multi-model approach with several kingdoms represented, highlighting both conserved mechanisms and variation around the theme. Arabidopsis did not emerge only as a representative of its phylum, but is also a very good model to study meiosis in general, notably because of the possibility of large scale genetic studies and the availability of large mutant collections and wide range of molecular and cytological tools. In this project we aim to use original approaches to decipher much further meiotic mechanisms, by isolating a large number of novel genes and characterizing their functions in an integrated manner. To identify new meiotic functions, we will use innovative genetic approaches. The first work package is based on a new suppressor screen strategy, taking advantage of a unique and favourable situation in Arabidopsis. The second is an unprecedented screen that exploits the fact that we can now synthesize haploids in a higher eukaryote. The third work package aims to fully exploit the available transcriptome data. In the fourth work package we will use these new genes to deeply decipher the meiotic mechanisms in an integrated manner.

1 492 663 €

Start date: 2012-01-01, End date: 2016-12-31

MicroRNAs (miRNAs) are 20-22 nt non-coding RNAs that regulate gene expression post transcriptionally via base pairing to complementary target mRNAs. They have fundamental importance for development and stress adaptation in plants and animals. Although a molecular frame work for miRNA biogenesis, degradation and action has been established, many aspects of this important gene regulatory pathway remain unknown. This project explores four main points. First, we propose to use genetic approaches to identify factors required for translational repression by miRNAs in plants. This mode of action was until recently thought to occur only exceptionally in plants. My post doctoral work showed that it occurs in many miRNA-target interactions. The mechanism remains unknown, however, leaving open a fertile area of investigation. Second, we wish to test specific hypotheses regarding the in vivo role of miRNA mediated endonucleolysis of mRNA targets. Long believed to serve exclusively as a degradation mechanism, we propose to test whether this process could have important functions in biogenesis of long non-coding RNA derived from mRNAs. Third, my postdoctoral work has provided unique material to use molecular genetics to explore pathways responsible for miRNA degradation, an aspect of miRNA biology that only now is emerging as being of major importance. Finally, our unpublished results show that plant miRNAs and their associated effector protein Argonaute (AGO) are associated with membranes and that membrane association is crucial for function. This is in line with similar data recently obtained from different animal systems. We propose to use genetic, biochemical and cell biological approaches to clarify to which membrane compartment AGO and miRNAs are associated, how they are recruited to this compartment, and what the precise function of membrane association is. These innovative approaches promise to give fundamental new insights into the inner workings of the pathway.

1 459 011 €

Start date: 2012-01-01, End date: 2016-12-31

Some 150 years after the emergence of genetics, epigenetic mechanisms are increasingly understood to be fundamental players in phenotype transmission and development. In addition, epigenetic alterations are now linked to several human diseases including cancers. A common feature of many epigenetic phenomena, for which X-chromosome inactivation (XCI) is the paradigm, is the implication of non-coding RNAs (ncRNAs). Regulatory ncRNAs belong to 2 major classes: (i) long ncRNAs, which can be transcribed from a single strand as well as in the opposite orientation when they may overlap with either protein-coding or non-coding genes. Both sense (Xist) and antisense (Tsix) ncRNAs control the initiation of XCI; and (ii) short ncRNAs, such as si- or miRNAs, which interfere, through different pathways, with gene function. The aim of this project is to gain insights into the regulation and function of ncRNAs in the control of gene expression program, using XCI as a model system. We propose to combine molecular genetics, embryology and cell biology to (1) decipher the transcriptional control of Xist and the coordinate regulation of the Xist/Tsix sense/antisense tandem in relation to developmental programs; (2) functionally characterise a novel ncRNA on the X chromosome which nests several miRNAs and for which preliminary data suggest a role in XCI and (3) develop a system to extend our knowledge of the regulatory stages of XCI in human through the use of human embryonic stem cells. Our comprehensive analysis of the function and regulation of ncRNAs in XCI has important implications for our understanding of the numerous diseases associated with abnormal patterns of inactivation and is a critical prerequisite to any subsequent therapeutic approaches. This project is in absolute adequacy with the future “Epigenetic and Cell Fate “ host centre co-headed by Prs. Lalande and Viegas-Pequignot, a large-scale initiative expected to strengthen French and European research in Epigenetics.

1 220 000 €

Start date: 2009-04-01, End date: 2014-03-31

The control of DNA based processes in biology is crucial for an organism s life, development, reproduction and evolution. In fact, deregulation of gene expression, of the replication program, of DNA repair or recombination has often disastrous consequences. The eukaryotic genome is structurally and functionally organized into chromatin by a highly complex mixture of proteins that control access to the DNA. In particular, heterochromatin proteins play a prominent role in this control. Genetics, biochemistry and molecular biology identified many players involved in eukaryotic DNA biology and provided everything we currently know about it. However the full composition of a given locus remains largely obscure and just like any object under scientific study, knowing the composition is an absolute pre-requisite for a full understanding of its features. Consequently, the nature of the interactions between chromatin structural components and DNA machineries is poorly understood and the elaboration of models from rather incompletely characterized systems can be misleading. During my post-doctorate, I have developed an unbiased approach for the in vivo purification of chromatin proteins in a locus specific manner, and I intend to apply this new technology in combination with a genetic approach. Monitoring the consequence of heterochromatin protein loss on the full composition of regulated loci will allow a deeper understanding of the role these proteins in the regulation of eukaryotic genome biology.

1 707 500 €

Start date: 2010-01-01, End date: 2014-12-31

Genomic DNA represents the blueprint of life: it instructs solutions to challenges during life cycles of organisms. Curiously DNA in higher organisms is mostly non-protein coding (e.g. 97% in human). The popular “junk-DNA” hypothesis postulates that this non-coding DNA is non-functional. However, high-throughput transcriptomics indicates that this may be an over-simplification as most non-coding DNA is transcribed. This pervasive transcription yields two molecular events that may be functional: 1.) resulting long non-coding RNA (lncRNA) molecules, and 2.) the act of pervasive transcription itself. Whereas lncRNA sequences and functions differ on a case-by-case basis, RNA polymerase II (Pol II) transcribes most lncRNA. Pol II activity leaves molecular marks that specify transcription stages. The profiles of stage-specific activities instruct separation and fidelity of transcription units (genomic punctuation). Pervasive transcription affects genomic punctuation: upstream lncRNA transcription over gene promoters can repress downstream gene expression, also referred to as tandem Transcriptional Interference (tTI). Even though tTI was first reported decades ago a systematic characterization of tTI is lacking. Guided by my expertise in lncRNA transcription I recently identified the genetic material to dissect tTI in plants as an independent group leader. My planned research promises to reveal the genetic architecture and the molecular hallmarks defining tTI in higher organisms. Environmental lncRNA transcription variability may trigger tTI to promote organismal responses to changing conditions. We will address the roles of tTI in plant cold response to test this hypothesis. I anticipate our findings to inform on the fraction of pervasive transcription engaging in tTI. My proposal promises to advance our understanding of genomes by reconciling how the transcription of variable non-coding DNA sequences can elicit equivalent functions.

1 499 952 €

Start date: 2018-02-01, End date: 2023-01-31

Polycomb Group (PcG) proteins are pivotal for the specification and maintenance of cell identity by preventing inappropriate gene activation. They function mostly through the regulation of chromatin structure and, in particular, the post-translational modification of histones. Although the enzymatic activities of the main PcG complexes has been described, lots remain to be discovered in terms of how these chromatin modifying activities are regulated. Genome wide analysis uncovered genes that are targeted by PcG proteins in various model cell lines, however it still very unclear how PcG proteins are targeted to a specific set of genes depending on the cell type. Finally, PcG proteins are frequently fund deregulated in diseases among which cancer but whether the alteration of their expression is a causative event to pathologies requires further investigation. This proposal is focused on the Polycomb Repressive Complex 2 (PRC2) whose function is pivotal to the polycomb machinery. In the first two aims of this proposal, we will investigate mechanistically how transcription factors, non-coding RNAs and chromatin structure might independently or in conjunction establish the conditions conducive to gene targeting by PRC2 and regulate its activity. In the third and fourth aims of this proposal, we will investigate what is the function of PRC2 during tumorigenesis and cell reprogramming and how its function is regulated during these processes.

1 499 815 €

Start date: 2012-02-01, End date: 2017-01-31

RNA interference (RNAi) mediates chromatin modifications in fungi, plants, drosophila and vertebrates. In the fission yeast Schizosaccharomyces pombe, where the molecular mechanisms are the most detailed, it is believed that small interfering RNAs (siRNAs) guide the RNA-Induced Transcriptional Silencing (RITS) complex to specific chromatin regions to trigger formation of heterochromatin. Although it is clear that siRNAs are required for RITS localization to chromatin, important events of RITS siRNA-dependent recruitment remain to be elucidated. Additionally, critical aspects of the following RITS-driven heterochromatin formation mechanism need to be clarified. Genetic data indicate that among proteins required for assembly of heterochromatin some act very early in this process to the point that they should interact directly or indirectly with RITS. However, no physical connection between RITS and these enzymes has yet been found. This project proposes to address these fundamental aspects of RNAi-mediated heterochromatin assembly in S.pombe, by coupling interdisciplinary and innovative approaches to classical protein affinity purifications, yeast molecular genetics and chromatin techniques. The first part of the project will concentrate on the characterization of an unprecedented physical connection between RITS and a chromatin-modifying activity. In parallel, we propose to develop two complementary approaches to identify new proteins that physically and/or genetically interact with RITS. Finally, thanks to a UV light-induced crosslinking technology, we intend to study RITS recruitment to chromatin by analyzing its direct interaction with nucleic acids both in vitro and in vivo. As the control of chromatin structure by RNAi is evolutionary conserved, our studies have the potential to bring general insights into how RNAi-based chromatin modifications take place and are regulated in the kingdom of eukaryotes.

904 940 €

Start date: 2009-01-01, End date: 2013-12-31

Enhancers control the correct spatio-temporal activation of gene expression. A comprehensive characterization of the properties and regulatory activities of enhancers as well as their target genes is therefore crucial to understand the regulation and dysregulation of differentiation, homeostasis and cell type specificity. Genome-wide chromatin assays have provided insight into the properties and complex architectures by which enhancers regulate genes, but the understanding of their mechanisms is fragmented and their regulatory targets are mostly unknown. Several factors may confound the inference and interpretation of regulatory enhancer activity. There are likely many kinds of regulatory architectures with distinct levels of output and flexibility. Despite this, most state-of-the-art genome-wide studies only consider a single model. In addition, chromatin-based analysis alone does not provide clear insight into function or activity. This project aims to systematically characterize enhancer architectures and delineate what determines their: (1) restricted spatio-temporal activity; (2) robustness to regulatory genetic variation; and (3) dynamic activities over time. My work has shown enhancer transcription to be the most accurate classifier of enhancer activity to date. This data permits unprecedented modeling of regulatory architectures via enhancer-promoter co-expression linking. Careful computational analysis of such data from appropriate experimental systems has a great potential for distinguishing the different modes of regulation and their functional impact. The outcomes have great potential for providing us with new insights into mechanisms of transcriptional regulation. The results will be particularly relevant to interpretation of regulatory genetic variations. Ultimately, knowing the characteristics and conformations of enhancer architectures will increase our ability to link variation in non-coding DNA to phenotypic outcomes like disease susceptibility.

1 436 293 €

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

Eukaryotic chromosomes are condensed into several hierarchical levels of complexity: DNA is wrapped around core histones to form nucleosomes, nucleosomes form a higher-order structure called chromatin, and chromatin is subsequently organized by long-range contacts. The conformation of chromatin at these three levels greatly influences DNA transcription. One class of chromatin regulatory proteins called insulator factors set up boundaries between heterochromatin and euchromatin and generate long-range loops. In Drosophila, three types of insulators (Su(Hw), dCTCF and BEAF) have been shown to regulate transcription and organize chromatin at the higher level by the formation of long-range interactions that were proposed to be mediated by the coalescence of several insulator proteins into clusters (insulator bodies). Our research aims to unravel the mechanism by which insulator bodies dynamically regulate chromatin structure and transcription by using single-molecule biophysics and quantitative modeling. On one hand, we will apply novel super-resolution fluorescence microscopy methods to investigate the structure and assembly dynamics of insulator bodies in single cells throughout the cell cycle and the role of their regulatory partners. On the other hand, we will reconstitute the looping activity of insulators in vitro and apply single-molecule manipulation methods to gain detailed insights into the molecular mechanisms involved in defining and regulating chromatin organization by insulators. This project has the potential to impact our understanding of several fundamental cellular processes: transcription regulation, cell-cycle dynamics, higher-order chromatin organization, and cell differentiation. The methods developed here will be directly applicable to the investigation of other nuclear super-structures, such as transcription and replication factories and Polycomb bodies, and thus will impact other research areas, such as DNA replication, transcription and cell division.

1 500 000 €

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