Cell competition is the mechanism by which suboptimal cells are removed from a growing tissue through apoptosis. It is driven by a local comparison of cell fitness among neighboring cells. In Drosophila, multiple pathways can modify cell fitness and trigger cell competition, yet they all converge into few downstream signals leading to “loser” fate commitment and cell elimination. Loser fate is controlled by the upregulation of the membrane protein isoforms Flower-lose and to a lesser extent by the downregulation of the ubiquitously expressed isoform Flower-ubi. Moreover, loser cells also express protective signals (including the secreted protein Sparc) that delay their elimination. Current knowledge suggests that loser cell elimination relies on the comparison of the relative concentration among neighboring cells of loser signals (Flower-lose) and the absolute concentration of protective inputs (Sparc). Yet the description of the process is still very qualitative and the dynamics of these components is unknown. I propose to quantitatively dissect the rules leading to loser cell fate commitment. I will first perform live imaging of cell competition in order to correlate the endogenous levels and the dynamics of Flower-lose and Sparc with cell elimination. I will then quantitatively manipulate these components by developing light and chemical inducible gene expression in cell culture and in vivo. This study should shed new light on the process of cell competition and pave the way for a quantitative approach of developmental decision.

Animals can adapt to environmental changes by modifying their innate preferences, generating learned behavior that matches their experiences. Many genes required for learning and memory have been found through candidate gene studies and classical genetic methods. In this study I propose an alternative approach to seek the genetic basis of learning and memory, using natural variation among wild-type C. elegans strains. I will seek two wild-type strains with variations in learning and memory capabilities and identify the underlying genetic differences using quantitative behavior assays and linkage-based mapping techniques. A microfluidic system will be designed to monitor animals’ behavior upon classical conditioning, after pairing the attractive odor propanol with a noxious acidic pH stress of HCl. Upon successful learning and memory acquisition the worms’ attraction to propanol, the conditioned stimulus, will be reduced. Learning and short- and long-term memory phenotypes will be quantified by measuring the reduction in attraction to propanol. I will seek two wild-type strains with natural variability in learning and memory capabilities, and employ Quantitative Trait Locus (QTL) and Chromosome Substitution Strain (CSS) mapping techniques to identify the genetic basis of their behavior variability. I will characterize the identified genes’ function in the context of learning and memory, and seek the neural circuitry underlying the learned preference behavior using established genetic and imaging tools. Combining my approach with new microfluidic technologies and quantitative measurements, I aim to identify relevant genes that were not identified in classical approaches.

Selective attention allows flexible tuning of behavior according to the current behavioral goal. Adaptive changes in the response properties of neurons in the auditory cortex accompany attentional shifts. Yet, the causal mechanisms that enable such attention-related cortical adaptation remain largely unknown. Commonly suggested mechanism speculates that ‘top-down’ inputs, allegedly from the prefrontal cortex, actively modulates the local excitatory-inhibitory balance at the targeted sensory neurons. Here we suggest testing this proposed mechanism using a novel behavioral paradigm and optogenetic stimulation of specific types of neurons. We will train rats to selectively attend to a cued frequency (CueF) while ignoring a distracting frequency (DstrF), an octave or more apart. The tonotopic organization of the auditory cortex will be exploited to allow separate perturbation of the local neural activity at the CueF and DstrF receptive felids. To this end, the light-sensitive cation channel Channelrhodopsin-2 (ChR2) will be introduced into layer 2/3 excitatory neurons by means of in-utero electroporation. This will allow stimulating excitatory microcircuit at either of the receptive fields (CueF/DstrF) and probing the effect of such stimulations on selective attention and on local neural activity. In addition, ‘top-down’ projection neurons to the auditory cortex will be retrogradely transduced with ChR2 to allow stimulating them from outside of the auditory cortex. This will allow probing the causal role different projections to the auditory cortex play at controlling neural mechanisms of selective attention.

Circadian rhythms are thought to have evolved to enable organisms to organise their activities according to the Earth’s predictable daily cycles but data revealing the benefits of circadian rhythms are scarce. More difficult still is explaining why parasites that exclusively live within the bodies of other organisms have a circadian rhythm. For example, the developmental rhythm of many malaria parasite species is coordinated; parasites invade host red blood cells, replicate, and then release their progeny in a timed, synchronized burst. Similarly, there is increasing evidence of circadian rhythms in the effectors and regulators of host immune responses, but it is not known whether this influences protection from disease. What is lacking is an explicit recognition that rhythms in immune defence and parasite development could provide an evolutionary advantage to hosts, parasites or both, as well as recognition that both parties may control each other’s rhythms. We will integrate different perspectives and methods from evolutionary ecology, chronobiology, parasitology, and immunology to investigate the causes and consequences of rhythms for hosts and parasites. Our project uses an established rodent-malaria model system. We will capitalise on the reciprocal feedback from integrating empirical and theoretical approaches to answer the following questions: (A) How are rhythms in parasite development initiated and maintained? Do parasites use their own time-keeping mechanisms to organise development or do they use cues from the host's circadian rhythms? (B) How do rhythms affect the ability of hosts to cope with infection? Do rhythms maximise immune defence or are they a constraint resulting from competing demands on the immune system? (C) How do rhythms affect the survival and transmission of parasites? Do developmental rhythms enable parasites to avoid host immune responses or facilitate the exploitation of circadian driven resource availability? (D) Are rhythms in parasite development and host immune responses adaptive for parasites, hosts, or neither? Does the evolution of parasite rhythms drive the evolution of host rhythms, or vice-versa? Thus our project breaks new ground by elucidating: the role of rhythms in infections, how rhythms may be exploited for clinical benefit, and the adaptive significance of rhythms for hosts and parasites.

MicroRNAs (miRNAs) are essential components of the genetic regulatory networks in plants and animals. Through inactivation of specific mRNAs, especially those encoding transcription factors, they modulate the homeostasis, development and responses to environmental stimuli of multicellular organisms. In order to accomplish its biological functions a spatial/temporal balance between a given miRNA and its targets has to be reached. Thus, a fine-regulation of the miRNA action is critical. In contrast to animals, where multiple regulatory factors modulating miRNA action have been identified, only a few have been found in plants. In this project, I propose two main strategies to elucidate the mechanism regulating miRNAs. First, I will focus on the regulation of miRNAs generation; I developed a novel screening system that allowed me to identify numerous mutants defective in miRNA activity. As part of my research project, I will characterize the isolated mutants, establish their mechanisms of action and explore their relationship with other transcriptional processes. The second strategy will focus on later events on the life of a miRNA. I will study, by genetic and biochemical approaches, the processes that determine the fate of a mature miRNA duplex, a critical regulatory step. The project will be conducted in an integrative way, using multidisciplinary approaches to study miRNA regulation and its relationships with other pathways. My goal is to elucidate the intricate mechanisms regulating the miRNA activity. This knowledge is fundamental to understand how miRNAs affects downstream biological processes in plants and potentially in other organisms.

The liver is strategically located at a crossroads of the systemic circulation, allowing it to carry out its metabolic functions in lipid, carbohydrate and protein generation and in the degradation of toxic or waste products. In homeostasis, the liver also serves as a central immunological shield between the gut and systemic circulation. Pathogens, toxins, and harmless food-derived antigens can enter the liver directly from the gut lumen, and be scanned by liver antigen-presenting cells (APCs) and lymphocytes. Despite such continuous exposure to microbial and non-self molecules, the liver appears to be an immune-privileged organ, which favors the induction of peripheral tolerance rather than immunity. Liver tolerance is achieved by the cooperative action of various tolerogenic liver-resident APCs populations and immune regulatory mediators, which are released locally from liver resident cells. Following liver injury, inflammation is a pathological hallmark feature of chronic hepatitis. Sustained inflammation then promotes liver fibrosis and, as an end stage, liver cirrhosis, autoimmune hepatitis or hepatocellular carcinoma. Although much progress has been made in understanding the role of the liver in immune tolerance, the precise mechanisms, and whether tolerance is maintained during chronic inflammation remain poorly understood. During my studies I will explore the hypothesis that the liver serves as a cardinal immunological organ, responsible for the induction of systemic immune tolerance to food and bacteria antigens. During chronic hepatitis, liver immune tolerance is compromised and systemic food intolerance, inflammation, and autoimmune diseases develop. To experimentally validate this hypothesis, I will use specific mouse models of liver inflammation and antigen presentation ablation and characterize the cellular and molecular mechanisms underlying liver induced tolerance and disease.

Pausing auf RNA polymerase (RNAP) II in the promoter proximal region of a large set of genes is the major rate-limiting step in human gene expression. The aim of this proposed research is to understand the mechanism by which promoter proximal pausing of RNAPII is controlled in human cells. I will achieve this goal by a combination of genome-wide and biochemical methods.
My research will focus on the role of phosphorylation of the RNAPII C-terminal domain (CTD) in the regulation of promoter proximal pausing. I will adapt nascent elongating transcript sequencing (NET-seq), a novel approach that allows RNAPII transcription to be studied genome-wide at single-nucleotide resolution in yeast, to human cells. I will use NET-seq to determine promoter proximal pausing sites of RNAPII across the entire human genome with single-nucleotide precision. I will make use of the high spatial resolution of human NET-seq data to extract the DNA sequences that underlie promoter proximal pausing sites and to reveal pausing motifs. By performing NET-seq with monoclonal antibodies directed against site-specific phospho-modifications of the CTD, I will determine the CTD phosphorylation state of transcriptionally engaged RNAPII at promoter proximal pausing sites. To establish the role of CTD phosphorylation in the regulation of promoter proximal pausing in vivo and to reveal transcription factors that are involved, I will disrupt CTD phosphorylation by CTD kinase depletion and through site-specific mutations of the CTD.
This new approach is critical to understand the way RNAPII CTD phosphorylation functions in the regulation of promoter proximal pausing and how its dysregulation causes cancer.

Intracellular recording of the neuronal transmembrane potential has been crucial to our understanding of the biophysical mechanisms underpinning neuronal computation. Existing intracellular recording techniques, however, do not permit long-term measurements of this potential, being limited by electrode-induced changes in neuronal function and the inherent instability of these recording devices. In practice, intracellular electrical recordings are generally limited to less than one hour, but many important processes occur on timescales of hours to days such as learning and plasticity. While in isolated cases repeated recordings from an identified neuron can be a work-around, this remains particularly challenging in vivo.
Here we propose to develop minimally-invasive intracellular recording electrodes for in vivo recordings by combining two complimentary technologies that have been separately developed by the laboratories of Andreas Schaefer (MPI f. medical research, Heidelberg, Germany) and Nick Melosh (Materials Science & Engineering, Stanford, USA). Schaefer group has described solid-conductor intracellular nananelectrodes (SCINEs) that can be inserted into neuronal membranes to record supra- and sub-threshold neuronal potentials without causing membrane damage or diluting cytoplasmic molecules. Unfortunately, these nanoelectrodes only insert into membranes with low probability and limited duration - both, presumably, due to an unstable membrane-electrode interaction. Melosh group has developed technology that allows inorganic nanostructures to insert and fuse spontaneously and stably into lipid bilayers. This is accomplished by creating a 2-10 nm wide hydrophobic band around the hydrophilic metallic electrode, mimicking the internal hydrophobic region of the lipid bilayer. Upon contact with a lipid membrane, this band fuses into the core of the bilayer, thus allowing the nanostructure to traverse the membrane without disrupting it and providing stable adhesion with the cell. Melosh group has verified integration of such “Stealth Probes” into membranes through atomic force microscopy and intracellular cyclic voltammetry.
Together, we plan to develop biomimetically patterned solid-conductor nanoelectrodes, “stealth SCINEs” (sSCINEs), for non-invasive intracellular recording, with Melosh group focusing on design, and testing of membrane-electrode interfaces, and Schaefer group focusing on assessing sSCINEs for physiology and developing in vivo sSCINE recording preparations. First applications will be mapping stability and plasticity of synaptic properties and substhreshold receptive fields in the mouse olfactory bulb — experiments currently impossible due to limited recording duration of current intracellular recording techniques and the high-dimensional input space.

The eukaryotic genome is spatially organized and highly dynamic. In yeast, several inducible genes are repositioned from the nuclear interior to the nuclear periphery upon activation, where they interact with nuclear pore complexes (NPCs). This interaction is thought to optimize transcription and DNA repair and promote mRNA quality control and export. Interestingly, gene-NPC targeting is accompanied by the formation of gene loops, which correlate with a “memory” of previous transcription. Many factors involved in looping and NPC-targeting also play multiple roles in gene expression, which complicates the interpretation of reverse genetic approaches. An important challenge in the field is to establish direct cause-effect relationships between the three-dimensional folding and positioning of a gene and its activity. To approach this problem, I propose to engineer synthetic, inducible gene loops by using novel optogenetic tools in S. cerevisiae. This will allow to determine whether artificially induced gene looping and NPC-tethering can recapitulate NPC function in terms of transcriptional activation and memory, and facilitation of mRNA export. Additionally, I aim to test whether the synthetic system can bypass the requirement for essential components of the natural NPC-associated gene expression machinery. These experiments will create a powerful basis to address a number of key questions in the field.

Metastasis of any organ-specific cancer type has a poor prognosis and is the cause of 90% of cancer-related deaths. MicroRNAs (miRs), small non-coding RNAs regulating gene expression, are increasingly implicated in cancer progression, including metastasis.
A recent study by Dr. Hanahan’s group (Olson et al, 2009) examined the dynamic expression of miRs during different stages of tumour progression in a mouse model of pancreatic neuroendocrine tumour (PNET), and unveiled unique miR signatures in each stage; suggesting a putative role in cancer progression. One of the miR signatures was detected in a subset of primary PNET and shared common miRs with the liver bona fide metastasis miR signature. The former one, referred to as met-like primary (MLP) signature, suggests that a subset of primary tumours can diverge and acquire enhanced metastatic abilities. Preliminary data suggest that miRs -214, -137, -449, and -199 of the MLP signature might act in a synergistic way to promote metastasis.
The focus of the proposed fellowship is to characterize the synergistic effect of the aforementioned miRs. The contribution of each miR to the synergistic effect will first be determined, and subsequently the critical time-frame/stage for their role during the metastatic process will be elucidated. Furthermore, subsequent experiments will aim to identify target genes and cellular processes altered by miRs. Finally, this study will also examine the prognostic value of the miRs in human PNET cancer.
Understanding the role of miRs in PNET metastasis can provide guidance for future experiments as well as the design of novel therapeutic approaches targeting tumour metastasis.

Do cells think? We imagine that thinking is the product of complex nervous systems. Studies by our team and others, has demonstrated that seemingly simple microbes, such as brewers yeast, display signs of having memory and consequently the rudiments of intelligent behavior. Yeasts grown on a particular sugar typically activate genes that code for enzymes to digest that sugar. We found if yeasts are grown for a long time on glucose, they appear to maintain activation of the genes required to digest glucose, and consequently take much longer to switch on the genes required for growth on an alternative sugar once the glucose is used up. This history-dependent behavior can be interpreted as a rational decision because, for yeast, the constant availability of glucose in the past appears to imply that glucose is likely to become available again in the future. In other words, yeast cells somehow "remember" how long they have been consuming a certain sugar, and they adapt their behavior accordingly. These preliminary results show that cells and thus the biochemical machinery – the genes and proteins – that generate their behavior are influenced by the past. We will set up experiments to first quantify history-dependent behavior at the single-cell level and second to identify genes and specific molecular processes involved in memory. We will specifically focus on epigenetic mechanisms, those that do not involve changes to the genes themselves but to changes in the structure of DNA and the inheritance of particular proteins from cell to cell. Further, to identify why history-dependent behavior may have evolved, we will determine environments (sequential appearances of different sugars) that favor and environments that disfavor history-dependent behavior. Our research will not only reveal new insight into microbial physiology, but may also establish general principles into how previous experience shapes future choices for cells. Our efforts may open up new avenues of research, such as whether complex nervous systems may use similar strategies to store information or whether cancer cells “learn” differently from normal cells, perhaps in a way that could be harnessed to selectively kill these cells.

The identification and characterization of RNAs lacking protein-coding potential has shifted our view from RNA molecules as simple genome information carriers to potential regulators of gene expression. Among them, long non-coding RNAs (lncRNAs) are believed to recruit chromatin-modifying complexes to specific genomic loci and thereby modulate chromatin states and transcriptional permissiveness at such loci. A more detailed understanding of how lncRNAs function and an estimate on the prevalence of lncRNA-mediated regulation of gene expression requires the interacting and regulated genomic loci to be identified. Here, I propose investigations to identify and characterize RNA-chromatin interactions in mouse embryonic stem cells. Differentiation of ES cells involves changes in expression of lncRNAs and presumably of lncRNA-regulated expression programs making it a suitable process to study lncRNA function. To identify lncRNA-chromatin interactions, I will develop a novel technique aimed to unveil transcriptome- and genome-wide RNA-chromatin interactions as well as make use of methods that capture candidate lncRNA-chromatin complexes by tiling antisense-oligos. Such interaction maps together with gene expression signatures from ES cells, differentiated descendants and candidate lncRNA loss-of-function experiments hold the promise for a more comprehensive understanding of RNA-mediated genome regulation and ES cell differentiation.

Extracellular signals pattern tissues by instructing cells to acquire specific fates during embryogenesis. Multiple signaling pathways need to be precisely coordinated in space and time to pattern the body plan. While the intracellular pathways that mediate responses to extracellular signaling molecules have been well characterized, little is known about how these signals become distributed appropriately within the developing embryo such that cells receive the right amount of signal at the right time. In particular, how patterns proportionally scale with embryo size has been a mystery since the early days of experimental embryology. The spatial range of signals must adjust to the size of the embryo, because small and large embryos give rise to normally patterned adults, but it is currently unclear how this is achieved. I propose to address two major, long-standing questions in the field of developmental biology: (i) How are the appropriate spatial distributions of signaling molecules achieved? (ii) How is the spatial range of signaling molecules regulated to establish the correct tissue proportions and scale-invariant patterning? To address these questions, I will use quantitative experimental approaches in living zebrafish embryos combined with theoretical studies. By measuring the diffusivities and half-lives of signaling molecules and by using a systems biology approach encompassing genome-wide assays, mathematical modeling and functional interference, I expect to advance our understanding of how the biophysical properties of signaling networks give rise to robust patterning during vertebrate embryogenesis.

Although the multiple regulatory layers that result from modification of DNA and proteins have been extensively studied, RNA modifications still remain an uncharted territory. Recent studies have shown that RNA modification levels change under specific cellular conditions. Importantly, the increase of specific post-transcriptional modifications can affect the expression levels of specific subsets of genes, suggesting that specific modifications could be regulated and maintained in distinct cell types and physiological states in order to enhance the expression levels of those genes involved in a cellular response.

In this work we aim to characterize and decipher the functional roles of RNA modifications in the post-transcriptional regulation of human cells. By comparing the RNA modification levels of diverse human tissues under different stress conditions, we expect to correlate the diverse RNA modification profiles with the observed gene expression patterns found across different tissues and under different stress conditions.

Over the past decade, research in the field of DNA epigenetics has revealed that chemically modified bases are abundant components of the human genome, and its specific modifications have profound effects on their function both in health and disease. We expect that through the characterization of RNA modifications in human tissues, we may decipher novel regulation mechanisms through which defects in RNA modifications lead to disease.

Sexual dimorphism in various species does not only contribute to physical differences, but is also a major cause of the differences in behavior. At the molecular level, recent studies reveal extensive sex-biased gene expression changes in the nervous system. However, the mechanisms that regulate these differences are poorly understood. I propose to identify the underlying mechanism using C. elegans as an in vivo model. The recently reconstructed posterior nervous system of the male shows that many of the shared neurons between the male and the hermaphrodite are strongly sexually dimorphic in their wiring, but the mechanisms leading to these changes haven't been studied yet. First, I will define the sexual differences for patterned locomotory behavior. Although there are clear motility differences between C. elegans sexes, quantitative data have not been obtained yet. I will use automated tracking methods to determine the kinematic features that define male motility. By analyzing the locomotive features of laser-ablated candidate neurons and mutants, the circuits and neurons involved in generating dimorphic behaviors will be identified. Second, I will masculinize specific portions of the hermaphrodite nervous system using genetic manipulations and cell-specific promoters. Third, to identify transcripts that are selectively expressed in neurons from masculinized animals versus hermaphrodites I will combine genomic approaches. Whole-genome and cell-specific sequencing will be used for genome-wide and single-neuron gene expression profiling. Using these approaches, I will identify the molecular changes in the core nervous system responsible for sex-biased behaviors.

Sleep is a natural state that is characterized by a reversible decreased responsiveness to external stimuli. During this state, external information/stimulation cannot be processed because it hardly elicits evoked-activity in the corresponding cortical area, a process called sensory-gating. Sensory-gating is believed to be of paramount importance to protect the brain from external stimuli during sleep, and is mediated by the thalamus. The thalamus relays sensory inputs from sensory organs to the cortex and is responsible for filtering redundant stimuli. This function is achieved by oscillatory synchronous activity of thalamic neurons that can be influenced by many factors, some of which may originate from glial cells. Glial cells are non-neuronal cells that constitute the vast majority of brain volume but have been long ignored in understanding brain function. Very recently, they have been shown to detect and strongly impact neuronal activity through their capacity to release transmitters (gliotransmittion). In particular, they have been found to be crucial player in the mechanism of sleep through the release of ATP/adenosine. In the thalamus, ATP/adenosine is known to favor neuronal oscillatory activity. The purpose of this project is to demonstrate that glial cells are central to the process of sensory-gating by tightly controlling neuronal activity in the thalamus. We will use optogenetics and molecular genetics to promote and inhibit gliotransmission and test the hypothesis that release of ATP/adenosine by glial cells favors synchronous oscillation of thalamic neurons, filtering of sensory information, and ultimately favors sensory-gating.

Plants have evolved distinct tolerance mechanisms to abiotic environmental stresses such as drought. A plant hormone abscisic acid (ABA) is key to achieving abiotic stress resistance. Recent reports showed that ABA signal transduction can also interfere with plant immune responses upon biotic pathogen attack. Since plants are often exposed to biotic and abiotic stresses simultaneously, research on the interplay of signal transduction pathways of the two is essential to understand stress tolerance mechanisms. Furthermore, this may lead development of crop plants which optimize tolerance to both abiotic and biotic stresses and may achieve higher yields.

Recent chemical genetics research of Prof. Julian I. Schroeder identified reverse interference from biotic to abiotic stress tolerance signal transduction. They identified a new small molecule, DFPM, which rapidly interferes with the ABA signal transduction pathway by activating pathogen effector-triggered immune signaling. However, the molecular and cellular mechanisms for the cross interference remain unknown.

In this proposal, I aim to dissect mechanisms of DFPM-mediated biotic-to-ABA interference signaling and to identify and characterize genetic and cellular components important for the cross-talk. The research will use innovative techniques including chemical genetics approaches, next generation sequencing-based mapping and time-resolved cell biological signal transduction analyses. The HFSP fellowship would provide me with the opportunity for me to pursue my long-term goal in plant stress resistance and signaling which will be crucial to my future career as an independent scientist.

A hallmark of hominid duplications is their complex organization and their preponderance of interspersed duplications. While this organization promotes unequal crossovers associated with disease, it also harbors a potential for gene innovation and rapid adaptation, which I hypothesize may compensate for their cost as disease causing. To test this hypothesis, I propose to sequence hominid-specific duplicated gene families of which at least one gene is known to be expressed in the human brain. I will then apply phylogenetic analyses to infer selection forces and estimate the duplications timing and origin. I aim at detecting adaptation signals and find association of the genes to brain development by comparatively analyzing: (i) orthologous sequences from primates and mammals; (ii) paralogous sequences within the human species; (iii) variants within the human population; (iv) individuals with neurodevelopmental disease. In addition, I will conduct a spatiotemporal expression analysis of these genes during brain development. A major challenge in all of these analyses would be to distinguish between highly identical yet distinct duplicated genes that reside within highly complicated genomic regions.
I expect this study to shed light on the biological aspects of the human brain unique features, the evolution of our species, and human neurodevelopmental diseases.

In this project we will attempt to unravel the role of alternative polyadenylation (APA) on gene expression and protein production and its impact on cellular phenotypes relevant for cancer by applying four cutting-edge deep sequencing technologies. We propose to simultaneously measure APA (3’-seq) and translation efficiency (ribosome profiling), together with miRNA and mRNA expression levels (mRNA-seq and miRNA-seq) under different cellular conditions to create a comprehensive map of APA events that connects APA to gene expression and protein translation regulation by miRNAs and RNA-binding proteins. We will apply this approach to two different human cellular models under conditions of proliferation, transformation, arrest and senescence, to identify promising target effectors that may contribute to the cellular phenotype and be of relevance in cancer. Subsequently, selected targets subjected to APA regulation will be validated and studied in great detail using a vast repertoire of biochemical and molecular biology approaches to dissect their impact on cellular behavior and their potential implication on cancer processes. In addition, we will attempt to block APA events by using custom designed locked nucleic acid oligonucleotides, and the effectiveness of this strategy and its potential applicability for cancer treatment will be assayed using cellular and/or animal models.If successful, our innovative approaches will have the potential to uncover the gene expression and protein production changes caused by APA that are causally linked to cancer and may provide new tools to develop effective pharmacological treatments.

Organization of chromatin is regulated by post-translational modifications of histones to maintain specific patterns of gene expression. The Polycomb group (PcG) proteins play an important role in these modifications, acting as transcriptional repressors and regulating development and pluripotency. Recent reports suggest a role of these proteins also in genome stability. Loss of PcG proteins in mice results in phenotypes that mirror known disorders linked to genome instability. However, the mechanistic contribution of PcG proteins in the maintenance of genome stability is poorly understood. As deregulation of the DNA replication process is a major source of genome instability, we hypothesize that maintenance of certain chromatin marks during replication could be essential to assist genome stability during duplication. In this reserach proposal, we thus plan to investigate the involvement of PcG-dependent chromatin modifications in DNA replication stress and genome stability. The aims of this proposal are to uncover physical and functional interactions between PcG proteins and the DNA replication and repair machinery, using mouse as a model system. We also plan to map genome-wide the chromatin marks induced by PcG proteins during unperturbed and perturbed replication. The project combines biochemistry, mouse genetics, genomics and single molecule approaches and is designed to progressively focus specialized approaches on promising PcG candidate proteins. We are confident to gain significant insight into the mechanisms linking chromatin modifications and genome integrity, disruption of which results in a variety of human diseases, including developmental defects and cancer.