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.

Photosynthesis is the major process used by plants, algae and cyanobacteria to generate energy from sunlight, being indispensable to the existence of all the advanced forms of life on Earth. In this process, with the consumption of the inorganic CO2, O2 is formed together with different organic compounds, mostly sugars and other carbohydrates. This maintains the level of O2 in the atmosphere, and also captures CO2 - with important ecological and environmental effects. These processes occur largely in light-harvesting complexes, which are groups of interacting subunit proteins that may be part of a larger supercomplex of a photosystem - the functional unit in photosynthesis. In this project we will investigate the coupling between the protein environment and the excitation states of the reaction center (RC) and the CP43 and CP47 “antennae” pigment subunits of the Photosystem II (PSII) complex. The main aim is to combine (i) the recent experimental data available on high-resolution PSII structures and energetics, with (ii) the latest developments in simulations and analysis of atomistic systems with both quantum and classical, atomistic levels of theory. In particular, we will use semi-classical (SC) and quantum mechanics / molecular mechanics (QM/MM) methods to probe and characterize quantitatively the role played at multiple scales by the specific molecular environment provided by PSII in mediating and controlling the excitation energy transfer between the different PSII subunits.
This work should lead to the identification of the most important molecular-mechanistics steps and factors that control the efficiency of energy transduction within PSII.

Determining how sensory information is detected and processed in the brain to produce adaptive behaviors is a fundamental issue in neuroscience. Although sensory inputs have been comprehensively-described in many systems, the central elements of sensory circuits are often much less well understood. The Drosophila gustatory system is an attractive model to investigate the neurobiology of sensory perception, because individual taste stimuli evoke robust and stereotyped behavioral responses under the control of neural circuitry that is amenable to molecular genetic perturbation, electrophysiological analysis and high-resolution imaging. Gustatory sensory neurons (GSNs) project from the peripheral taste organs to the primary gustatory center in the brain, the suboesophageal ganglion. Here, they are presumed to synapse with different types of interneurons, which integrate the information and send their projections to either higher brain regions and/or to the motor output. Although such interneurons have been well characterized in other brain regions of Drosophila, such as the olfactory system, almost nothing is known about gustatory interneurons. The goal of this project is to develop activity-dependent genetic reporters that will unequivocally label gustatory interneurons and to use these to characterize their morphology, neurochemistry and physiology to determine how these neurons transduce and transform taste evoked-sensory information.

To address the cellular and molecular requirements for Th17 differentiation during commensal colonization, I will focus SAA1 protein, which is strongly induced by SFB colonization. To monitor SFB colonization easily and surely, I propose to generate SAA1 luciferase mCherry knock in reporter mice and to use an ileal looping surgical approach that does not impair host circulation. Using this approach, we can understand when and where SAA1 is induced in response to SFB colonization. Although SFB may attach intestinal epithelial cells (IECs) and CX3CR1+ cells in intestine, which cells recognize SFB are not known. To determine the cellular requirement for SFB recognition, SAA induction and commensally driven Th17 differentiation, I will use specific cell linage depletion strategy that is well established. Using CX3CR1+ cells depleted mice, we can understand whether CX3CR1+ cells are necessary for SAA1 induction and Th17 differentiation or not.
To identify the genes which are required for Th17 differentiation in response to SFB colonization, I will try identifying the genes which are strongly induced by SFB. To eliminate secondary effects, I will purify IECs, DCs and CD4+ T cells from ileal loops of mice that are depleted CX3CR1+ cells and CD103+ DCs. Comparing these all gene profiles, we can understand the genes that are directly induced by SFB colonization in each cell linages. Thus, these genes are strong candidate that affect Th17 differentiation. In the last part of my study, I will elucidate whether the candidate genes are required for Th17 differentiation in response to SFB colonization by using conditional knock out mice.

Formation of beta-sheet-rich protein aggregates is linked with several functional and many pathological biological processes. In the latter case, the small early aggregates, rather than the fully-grown fibrils, have been implicated in cellular toxicity, but it is difficult to probe the pathway for aggregate formation in experiments due to the heterogeneity inherent in the aggregation process and the small concentration of such toxic oligomers present in the system at any given time. Yet, a good understanding of the factors that facilitate or inhibit to primary and secondary nucleation of protein aggregates is of key importance for controlling and inhibiting the formation of toxic species. Computer simulations can provide invaluable insights to that end since they allow input from experimental data relating to different length and timescales to be integrated. The aim of the proposed research is to explore the nucleation pathways of early oligomeric species, using a simple coarse-grained model that takes into account that aggregation prone peptides can occur in different states with different interaction properties. We will investigate how sensitive the kinetics of the fibril-growth process is to the interaction properties of the states that the peptide can acquire and the oligomeric species they produce. Furthermore, we will explore how the surface of existing fibrils can catalyze the formation of new oligomers, a process called secondary nucleation. Finally, we will investigate the effect of one particular external factor- mechanical stress- on the aggregation mechanism.

Tight regulation of cell size is essential for both multicellular and unicellular organisms to achieve an optimal size for specific physiological functions – for example, to balance nutrient uptake and cell growth. In principle, size regulation requires coordination of cell growth and division. In the budding yeast Saccharomyces cerevisiae, this occurs at a size check-point for cell cycle progression within the G1 phase of the cell division cycle prior to DNA replication. Although extensive studies have identified several regulatory proteins that critically affect size control, the molecular mechanism that allows cells to measure their own size remains elusive. One major candidate for a size sensor is the unstable G1-cyclin Cln3, which during G1 triggers irreversible commitment to cell division. Since the Cln3 concentration remains constant through the cell division cycle, we hypothesize that cells may titrate the number of Cln3 molecules, which increases proportionally with cell size, against their DNA, which remains constant in G1. Such a mechanism could result in an activity proportional to the number of Cln3 molecules rather than concentration. Here, we propose to directly test this hypothesis using a reconstituted, single-molecule imaging approach combining Total Internal Reflection Fluorescence (TIRF) microscopy and microfluidics to examine the interaction of single purified Cln3 molecules with its downstream target proteins on flow-stretched DNA. The observation of Cln3 binding non-specifically and sliding on DNA would provide strong support for the titration model of size control, thereby solving a longstanding fundamental problem in cell biology.

Transcriptional co-repressors are essential components of the highly complex gene regulatory system. Despite their importance in ensuring cell type and state-specific gene expression, we still have little understanding of their precise molecular mechanisms of action. The goal of this integrative genomics project is to provide a comprehensive understanding of co-repressor action using adipogenesis as a model system. Specifically, I aim to (1) determine the genome-wide DNA binding dynamics of the co-repressor KAP1, (2) integrate these data with available binding profiles of the co-repressors SMRT and NCor1; and (3) derive the regulatory logic of co-repressor complexes. This will be achieved through explorative computational investigation of the dynamic relationship between co-repressor complex formation, transcription factor DNA binding, gene expression, and chromatin structure during differentiation, and by experimentally validating emerging hypotheses through knock-down or over-expression. The choice of adipogenesis is motivated by the established importance of co-repressors in this system, its medical relevance, the availability of well-established in vitro models and the wealth of molecular data and tools available for downstream validation. The proposed approach will uniquely integrate complementary genome-wide datasets in a controlled differentiation set-up, aiming to unravel the dynamic gene regulatory properties of co-repressor complexes. A thorough understanding of co-repressor function may also lead to translational applications, including the development of new strategies to tackle fat mass excess-related health problems.

The symbiosis between the calcifying marine microalga Emiliana huxleyi (E. huxleyi) and the marine bacterium Phaeobacter gallaeciensis (P. gallaeciensis), is unique as it shifts from mutualism into parasitism. Although various data suggest that this symbiosis is highly prevailing and significant in oceanic habitats, little is known regarding this association and its affect on the single organism and the entire ecosystem.
By merging molecular and cell biological approaches, microscopy and geochemical techniques, I will characterize various aspects of the algal-bacterial interaction and address the following issues:
The colonization pattern of E. huxleyi by P. gallaeciensis: What algal and bacterial life modes are presented during colonization? Is a bacterial biofilm created during the process? Do algal-derived molecules, known to be utilized by bacteria, promote the establishment of symbiosis? Exploring these questions will allow me to portray the molecular and physiological profile, of both alga and bacterium, in the course of colonization.
The bacterial biosignature in algal calcified remnants: The calcified disks that are produced by E. huxleyi rely on uptake of seawater for ion supply, and thus record in their chemistry various characteristics of the algal environment and physiology. By exploiting experimental approaches commonly used in Earth sciences, I will examine how the algal-bacterial relation shapes the chemical signals encapsulated in the calcified remnants of algae, known to be of importance in the reconstruction of past environments.

Stem cells (SCs) hold promise for regenerative medicine. SCs were first cultured from skin, and are now used to treat burn patients. In addition, corneal blindness in one eye can often be fully cured by culturing corneal SCs from the good eye. I propose to address whether molecular distinctions between these two related adult SCs can be exploited to improve and broaden therapies for corneal blindness. Using mice as a model, I will: (1) Devise methods to purify corneal SCs, profile the mRNAs they express, and determine how they differ molecularly from well-characterized skin SCs; (2) Culture and compare long-term passaging potentials and retention of molecular differences of corneal and skin SCs. If differences are lost in vitro, do corneal SCs reacquire and sustain their identity when engrafted to common sites (cornea, backskin, kidney capsule) on a recipient mouse? This should reveal suitability of corneal SCs for long-term tissue regeneration. (3) Focus on ~100 genes highly and preferentially expressed in corneal SCs, and use in utero RNA interference to rapidly find which are critical for maintaining corneal SC identity in vivo. Promising candidates will be further tested by cellular, biochemical and classical genetic approaches. (4) Explore whether candidates from (3) can be used in gain of function experiments to see if I can reprogram skin SCs into corneal SCs. Pending promising results in vitro, I will engraft and test the efficacy of corneal-reprogrammed skin SCs to repair and regenerate damaged corneal tissue. Long-term, I hope to apply my findings to human SCs and small molecule-based reprogramming aimed at restoring corneal tissue in cases of total blindness.

A fundamental question in infection biology is what makes some diseases chronic. The disease agent, which may be a virus, bacterium or parasite, is cleared very slowly, or never cleared, by the immune system or therapy. Instead, infection is seemingly controlled but persists at low levels. Reasons for persistence may include mechanisms that involve new cycles of pathogen replication. In HIV infection, the mechanism of persistence in the face of the immune response relies on ongoing cycles of replication, and this may also contribute to persistence in the face of therapy. We previously observed that cell-to-cell spread, a directed form of HIV transmission to adjacent cells occurring in the immediate vicinity of a virus-producing cell and involving large viral particle numbers, dramatically decreases HIV infection sensitivity to antiretroviral drugs. Here we propose to use a probabilistic model of infection and mixtures of HIV particles, each expressing either CFP or YFP upon successful infection, to understand the persistence of ongoing replication in the face of antiretroviral drugs. We will quantify numbers of infecting viruses per cell in different infection modes: cell-free infection, where viral particles find a new cell to infect by diffusion in a non-directed manner, and cell-to-cell spread. We will determine whether large numbers of transmitted viral particles per cell increase the probability that at least one escapes drug action and infects the cell. Ongoing replication may lead to therapy failure and potentially contribute to viral persistence. Understanding it is of potential high interest to future curative strategies.

Sister chromatid cohesion is essential for proper chromosome segregation. Chromosome cohesion is mediated by cohesin complexes which form a ring-like structure that embraces replicated sister chromatids and hold them together until anaphase onset. Although cohesin binds to DNA dynamically before S phase, cohesion is only established during DNA replication, which requires acetylation of cohesin complexes and subsequent recruitment of a cohesin’s regulatory subunit called Sororin. Sororin additionally promotes cohesion maintenance from S phase until mitosis by antagonizing another cohesin’s regulatory subunit named Wapl. Maintenance of chromosome cohesion is extremely important in mammalian meiosis, because chromosome cohesion needs to be stably maintained for very long periods of time. In oocytes, cohesion establishment occurs during pre-meiotic S phase, which takes place before birth, and then has to be stably maintained until completion of meiosis, which only occurs several months or years later during ovulation. The mechanism responsible for meiotic cohesin stabilization for such long periods of time remains unclear. Importantly, the increase of trisomies and other birth defects with maternal age correlates with a gradual loss of sister chromosome cohesion during oocyte growth and maturation. Here, we intend to study how chromosome cohesion is established and regulated during meiosis, and to unravel the detailed mechanism responsible for maintenance of sister chromatid cohesion for very long periods of time. We will additionally study how the dynamics of meiotic cohesin complexes affect homologous pairing and meiotic recombination.

Humans, and other primates, are intensely social animals. We smoothly maneuver in our social environments by effortlessly understanding others’ interactions. Understanding others’ interactions has been found to engage several brain regions in humans [1]. In our monkey kin, though, homologous neural circuits remain unknown. This state of affairs raises fundamental questions about the biological and evolutionary foundations of this skill, which is essential for both humans and monkeys. Do monkeys rely on anatomical regions related to the human ones to read their peers’ interactions? And how is such a complex yet automatic social skill generated by the actions of single neurons? This project aims to answer these questions by using a multi-scale approach. First, those parts of the primate brain devoted to understanding others’ interactions will be charted. The use of functional magnetic resonance (fMRI) as an exploratory tool will enable me to compare monkey and human circuits. Then, the project will characterize the fMRI-identified areas, in order to understand how single neurons contribute to generating this skill. Electrophysiological and pharmacological techniques will enable fine-grained characterization of the features processed by individual neurons at a mechanistic level not achievable in human studies.