The tiny nematode worm C.elegans has been instrumental for identifying genes that increase lifespan. Although most of the genes increasing lifespan in C.elegans are also present in humans, only very few have been associated with human longevity. This is mainly due to the fact that humans are more complex than worms. Instead of studying single genes we will investigate a multi-gene approach where we assume that lifespan in the worm can be predicted by the behaviour of all these genes together. We do this by viewing the worm genetic system as an ecological system. Similar to the fact that ecological systems display characteristic behaviors before they can collapse, we will test if the gene-regulatory network of worms displays characteristic behavior when approaching a collapse leading to death. This will provide insight in the mechanisms of aging and might help to translate the results to humans.

1. Astrocytes, classically considered as simply supportive cells for neurons, are emerging as relevant elements in brain information processing through their ability to regulate synaptic activity. Indeed, the tripartite synapse formed by pre- and post-synaptic neurons and surrounding astrocyte structures has been proposed as a functional unit of brain processes. These novel and important functions of astrocytes are under control of G protein signaling-dependent processes, which trigger astrocyte Ca2+ signals eventually leading to the release gliotransmitters and other mediators regulating synaptic functions.
2. Recent evidence indicates that the roles of mitochondria in the brain may go beyond the mere needs of energy supply for cell survival and maintenance, being possibly involved in the regulation of synaptic functions. It is conceivable that mitochondrial G protein signaling participates in these processes. Various subtypes of G protein-coupled receptors (GPCRs) and the associated signaling molecular elements are present within mitochondria, suggesting the existence of mitochondrial G (mtG) protein signaling pathways.

Thus, astroglial mtG signaling potentially plays an important role in the regulation of tripartite synapse and hence brain functions. However, no studies have addressed this issue so far.
The present project proposes to investigate the consequences of the activation of astroglial mtG signaling pathways in brain physiology, identifying the underlying mechanisms at cellular, network, behavioral and theoretical modeling levels. We propose to generate and develop novel pharmacogenetic tools (DREADDs specifically expressed by astroglial mitochondria, mtDREADDs) that will allow the specific control of astroglial mtG protein activity. We will experimentally and theoretically analyze the consequences of activation of different mtG proteins (via mtDREADDs) on neuronal, synaptic, and network activity as well as brain functions in living animals.
The expected results will reveal novel processes of cellular signaling in the CNS, and will identify new regulatory mechanisms mediated by astroglial cells in brain function.

One of the main challenges of this century is to better understand the function of the brain in order to solve its devastating disorders. We have learned a great deal about the basic components of the brain: the different types of neurons, each with its unique arborization, and the connections, termed synapses that enable neurons to communicate with each other. We also have a basic understanding of how information is transmitted through electrical and chemical signals. However, we know little about how individual neurons integrate the multiplicity of inputs, both excitatory and inhibitory, to compute a single output. Nor do we understand much about how individual neurons work in concert within a specific circuit to perform complicated computations. A major limitation has been our inability to monitor and control the electrical and synaptic activities of neurons inside the brain of a living animal. Overcoming this limitation requires the combination of different technologies to enable neuronal activity to be monitored with high sensitivity and fidelity while minimally interfering with brain function.
This grant exploits advances in optical methods, molecular biology, genetics, protein engineering, and electrophysiology and brings together tools developed in three laboratories, each with distinct expertise. These include a fiber-optic-based "micro-optrode" that can be inserted into the brain of a living animal in order to simultaneously record the electrical activity and biochemical signals. In particular, this instrument can detect and decode optical signals transmitted by probes that respond to the microenvironment inside the neuron. Such molecular probes, which are introduced into neurons through genetic manipulations, are designed to reveal how the neuron decodes synaptic messages that it receives from its connected partners in the brain of an animal receiving sensory stimulation. To dissect how signal processing within the various compartments and arbors of a neuron contributes to its function, we are exploiting a novel strategy to locate the molecular probes in specific neuronal microdomains. The combination of innovative approaches that we propose in this grant is expected to produce new knowledge about the contribution of specific neuronal subtypes and circuits to the complex processing of the brain in normal and pathological conditions.

Adolescence is marked by high onset and incidence of mental disorders and intense changes in specific brain areas. Prefrontal cortex (PFC) and basolateral amygdala (BLA) form a circuit involved in emotional learning believed to underlie many of these disorders. Anatomical and morphological changes occur in PFC, BLA and their connections during adolescence; nevertheless, how these changes affect PFC-BLA function and its consequences to sensitivity to stress and onset of mental illness is currently unknown. Aiming to achieve a more functional understanding of how the maturation of these sensitive areas might underlie normal and pathological brain function, we propose to investigate how their synaptic connectivity changes during adolescence. In aim 1 we will characterize the maturation of PFC-amygdala synaptic responses at different time points during adolescence and adulthood. In aim 2 we will examine whether developmental changes in plasticity underlie sensitivity to stress. Lastly, we will test whether selectively manipulating the activity of PFC projections can rescue the deleterious effects of stress in behaving animals. Understanding how these emotional regulatory circuits functionally mature will help identify the mechanisms responsible for the high incidence of mental disorders during adolescence and provide tools for earlier and more targeted interventions.

Although strong data from mouse models show that HMP deregulation causes AML development, its role in the human disease has not been demonstrated yet. Using the most advanced techniques to target gene expression in primary human cells as well as by taking advantage of the novel conservative culture method developed by the host laboratory for maintaining leukemia patient samples in vitro, this research proposal aims to investigate the role of HMP genes in the regulation of human LSC activity. More specifically, benefiting from the advanced expertise of the host laboratory in lentiviral overexpression and shRNA approaches in human primary cells, I will investigate the role of each one of the HMP genes in the regulation of patient LSC frequency, self-renewal activity and survival to chemotherapeutic treatment. The read-out of this study will rely on a state-of-the-art functional xeno-transplantation assay in immuno-compromised mice. This work will lead to a deeper understanding of the molecular biology of human AML, possibly revealing new options for therapeutic targeting of the disease in patients.

The functionalization of O2 within Fe containing enzymes will be investigated to obtain a detailed understanding of their geometric and electronic structure at intermediate stages throughout catalysis. Fe Intermediates are important in many human drug interactions and disease states but heme Fe (HFe) intermediates have not been accessible at an insightful level by more generally applied methods such as NRVS or VTVH MCD, while the experimental condition and energy range of L-edge XAS cause ligation damage. The development of resonant inelastic x-ray scattering (RIXS), a high resolution probe of 3d metal-ligand orbital covalence, will be used to investigate HFe and non heme Fe (NHFe) intermediates.
The research requires; (1) the development of the selection rules, drawing information content of RIXS relative to the established method of L edge XAS. (2) Following which RIXS measurements can be performed for heme model complexes where there is already K and L edge XAS data available. (3) RIXS measurements will then be performed on simple heme proteins where L edge XAS information is inaccessible. (4) This will be developed to the investigation of oxyhemoglobin on both the protein and the model complex. (5) With the acquired means of accurate interpretation of RIXS measurements (obtained from steps 1 to 4) the high valence Fe(IV) intermediates compound I & II and equivalent enzyme systems will be investigated and compared to RIXS results for equivalent non-heme complexes.

Transposons are genetic elements that can cut themselves out of the genome and hop to a new location. Remarkably, they comprise half the human genome, but their functions are still ambiguous. Because transposition can wreak havoc in the genome, causing severe diseases, many mechanisms have evolved across the tree of life to quell transposons. Astonishingly, recent research revealed that some transposons possess functions essential to their host. They contribute to regulation of gene expression, human development and neuronal diversification. These findings have led to a paradigm shift in biology: no longer are transposons mere parasites of the genome but can contribute to host function.
A class of single celled eukaryotes, called ciliates, exploit transposons in a particularly impressive way. They employ them to clean up their genomes by tossing out junk DNA, and this produces easier-to-maintain smaller chromosomes that express most genes in the cell. We have proposed that ciliates specifically recruited transposons for this ‘house-keeping’ job, but how they managed to tame these intrinsically harmful elements so that they are essential members of the cellular machinery is unknown. To test our hypothesis and to answer these questions, we will merge a unique set of state-of-the-art interdisciplinary expertise spanning chemistry, physics, computer science and biology. We aim to track the evolutionary pathway of transposon recruitment, to understand the chemistry and biophysics of these ‘good’ transposons and to investigate how they differ from canonical ‘bad’ transposons. We will visualize the structure of the molecular machines at a resolution that resolves individual atoms, and analyze how these beneficial transposons are programmed and directed to act only at the right time and place to defend the cell from their potential harmful effects. Our work will discover if and how such potentially harmful genomic parasites can be adapted for essential biological roles in ciliates and beyond, providing unmatched insight into a fundamental biological mechanism for the acquisition of novel genetic and physiologic functions.
Moreover, the elucidation of how these eukaryotes have managed to customize and tame transposons to function efficiently but safely will provide invaluable implications for engineering transposon-based tools for genetic research and gene therapy.

We propose to use DNA-based scaffolds to build lectins of pre-specified stoichiometry and precisely defined three-dimensional geometry to test mechanochemical requirements of plasma membrane invagination mediated by their membrane-resident glycosphingolipid (GSL) binding partners.
It is striking that numerous pathogenic GSL-binding lectins including the B-subunits of Shiga and cholera toxins and the VP1 protein of SV40 (i) consist of a pentameric fold (ii) bind GSLs with similar geometries (i.e., angles and distances between carbohydrate recognitions sites (CRDs)); and (iii) finally adopt a curvature-active state that enables their clathrin-independent uptake into cells despite the fact that they do not exhibit any sequence similarity. This suggests the exciting possibility that their clustering, membrane bending and invagination capacities are the result of parallel, convergent evolution.
Probing lectin functionality experimentally is infeasible using conventional approaches such as targeted mutagenesis because it is impossible to alter lectin oligomerization state and CRD geometry in a controlled manner. Therefore, we will engineer synthetic scaffolds composed of DNA to display GSL-binding CRDs in specific natural and non-natural geometries and stoichiometries. Such designer DNA-based lectins indeed offer the ability to address structural questions with unprecedented molecular precision. Innovative computational and chemical biological tools will be developed that should be of general interest to the fields of DNA devices and synthetic biology. Novel DNA-based GSL-binding scaffolds will be characterized as to their capacities to cluster on membranes, to adopt a curvature-active state (i.e. to induce or sense curvature), and to traffic into cells via defined pathways. Based on structural input, experiments will be modeled at length scales from 0.1 nm to 10 microns to provide a molecular understanding of underlying mechanistic aspects, and guide the design of new scaffolds. We expect to achieve an unprecedented understanding of GSL-based membrane mechanical processes, including the discovery of novel physical principles. Beyond the world of pathogens, our study bears relevance to the GSL-dependent functions of signaling receptors and the design of novel delivery tools for therapeutic intervention against GSL-expressing tumors.

Epigenetics is a complex, dynamic and largely mysterious layer of gene-expression regulation. Recent studies have shown that a large part of the breakdown in cellular control that is dysregulated in cancer is in vast changes in epigenetic regulation. In normal cells, each epigenetic marks define states of gene activation, transcription/RNA processing[1, 2], which in turn drive protein production to establish appropriate cellular functionality. Not only cancer cells use epigenetics as an engine of diversification, but part of my proposal’s driving assumption is that they become addicted to transient substates enabled by dysregulation. Cancers use dysregulation as a “search & evade” mechanism. They can search for states not normally accessible to healthy cells to “solve” an environmental insult or—as a corollary—they might rapidly be able to transition out of states that are susceptible to drug action, for instance. But this might come at a price. However, rather than viewing this as a robust trait that enables rapid evolution in the face of chemotherapy or immune detection, we see a fragile mechanism that –-at his heart-- can be the target of intervention.
The aim of this proposal is to apply CyTOF mass-cytometery and state-of-art RNA-sequencing protocols to (i) map the network of co-regulated epigenetic substates for specific cancer-cell populations on a single-cell level; (ii) correlate the transcriptomes of such populations to link epigenetic states to transcription modules to derive the control points that epigenetic dysregulation enables in cancer. A critical goal of this project will be the discovery of target networks against which drugs can be screened.

Polycomb group (PcG) proteins are important for silencing of key developmental genes in higher eukaryotes. Transcriptional polycomb repression is believed to involve changes in chromatin structure. Yet it remains controversial how PcG-dependent chromatin modifications serve as signal for chromatin compaction and epigenetic inheritance of gene silencing. To gain insight to the molecular mechanisms of polycomb gene silencing, I propose to investigate the temporal order and epigenetic inheritance of PcG-induced chromatin modifications. Traditional genetic and biochemical analyses have mostly provided a static picture of PcG activity and failed to generate an understanding of the functional consequences of chromatin modifications. I will employ conditional control of PcG activity using synthetic small molecule ligands to induce H3K27me3 and determine the sequence of events, kinetics and epigenetic memory of this histone mark. Rapid and reversible recruitment of PRC2 will enable kinetic measurement of H3K27me3 at high resolution and form the basis for mathematical modeling of complex histone modification dynamics and patterns. This will lead to a better understanding of the mechanisms involved in establishment and maintenance PcG-dependent gene repression. Furthermore, tight temporal control of PCR2 activity provides a powerful tool to identify and discern novel modulators of initiation, spreading and memory of PcG-dependent gene repression. These studies aim to unravel the crosstalk between polycomb function and cell plasticity and ultimately help to incorporate epigenetic regulation into current transcriptional regulatory models.

It is well known that the context modifies the perception of a stimulus but the underlying neural mechanisms are unknown. My proposal aims to elucidate the neuronal circuits for the contextual modulation of visually evoked activity in the neocortex. Surround suppression is a classical example of how visually evoked responses in the cortex are modulated by the context within which the stimulus is presented. I aim to understand the physiological role of a large class of inhibitory neurons, the Vasoactive-Intestinal-Peptide expressing interneurons (VIP-INs), on surround suppression.
By combining two-photon imaging with specific Cre mouse lines, I will establish the visual responses of three genetically defined neuronal populations in visual cortex: pyramidal cells (PCs), that experience surround suppression, somatostatin expressing interneurons (SOM-INs), which inhibit PCs causing surround suppression, and VIP-INs that selectively inhibit SOM-INs.
I will then perturb the activity of VIP-INs through pharmacogenetic or optogenetic approaches, while monitoring the activity of SOM-INs and PCs through optical imaging and targeted electrophysiological recordings. These experiments will reveal whether, by modulating the activity of SOM-INs, VIP-INs affect surround suppression.
Finally, I will develop a behavioral task in awake head-fixed mice to determine whether the modulation of surround suppression by VIP-INs affects perception.
In conclusion, by integrating these electrophysiological, imaging and behavioral strategies, I will reveal the impact of a large group of inhibitory neurons, the VIP-INs, in the contextual modulation of visually evoked activity in the cortex.

Human cancers are susceptible to genomic instability, resulting in chromosomal abnormalities, such as duplications, deletions and translocations. Different types of tumors typically exhibit distinct chromosomal aberrations, but how these tumor-specific abnormalities affect the cellular phenotype and responsiveness to drugs remains largely unknown. In my research project, I aim to characterize the effect of tumor-specific chromosomal aberrations on the cellular response to anticancer drugs. First, I intend to screen human cancer cell lines against a large library of anticancer drugs and analyze the interactions between the tumor type, its recurrent chromosomal aberrations and its cellular response to the drugs. Second, I intend to introduce tumor-specific aberrations into human embryonic stem cells using the CRISPR/Cas genome engineering system, differentiate the cells into the relevant cell types and screen them against the drug library. By applying cutting-edge genome-editing, high-throughput screening and gene expression profiling technologies to these two in-vitro model systems, I hope to identify unique vulnerabilities of characteristic chromosomal aberrations in various tumor types. I will then study the genes and the pathways behind the selective drug responses, and will analyze clinical data to correlate the selective sensitivities with differential clinical outcome in human patients. This work may have profound biological and clinical implications: it will shed light on the cellular and molecular mechanisms underlying common aberrations in human cancer; and it will allow the stratification of cancer patients based on the cytogenetic status of their tumors.

Macromolecular protein assemblies exhibit phenomenal functional versatility, and have recently been suggested to conduct long-range (micrometer scale and above) electrical signals that drive biological functions. To understand how electrical conductance emerges from structure it is crucial to distinguish between different transport mechanisms. Shared structural properties with conducting polymers motivate me to explore long range conductance by phonon-assisted hopping mechanism. This mechanism is valid in elongated aromatic, pi-conjugated, regions where electrons are delocalized. However, in analyzing natural protein assemblies it is difficult to eliminate interfering influences due to the proteins’ and protein assemblies’ complexity and susceptibility to environment influence, e.g. structure integrity in measurement setups.
Instead of analyzing native complexes, we will construct artificial “de novo” ones as a model system. We propose to establish this platform using novel computational design tools for proteins and protein assemblies, based on the Rosetta modeling software, and realize an electron delocalized region using natural, aromatic amino acids. Through a design of an “ideal” assembly, we expect structure-conductance relation to be less susceptible to external environmental influences, allowing us to gain clearer insights on the structural elements that underpin conductance in protein assemblies.
The results of this research will give new perspective on electrical conductance in protein assemblies and possibly lead to fresh reevaluation of known natural protein assemblies to uncover hitherto-unknown basic biological functionalities.

How does a nervous system adapt to the body plan of a growing animal from birth to adult? This question is fundamental to biology, yet is currently impossible to answer without turning to small model organisms, which provide an extraordinary level of access to the nervous system at the molecular, anatomical, and physiological levels. We will explore how a nematode C. elegans manages to drive undulatory locomotion at all life stages, with different motor circuit components, using powerful new tools in serial-section electron microscopy, optical neurophysiology in freely behaving animals, super resolution light microscopy, and biophysical modeling. This research program should provide a unique data set that illuminates developmental dynamics from neural activities to animal behaviors.

Oligomeric lectins from bacterial toxins and animal viruses induce glycosphingolipid (GSL) reorganization on target cells, leading to the clathrin-independent construction of endocytic pits. These lectins have no sequence similarity, but nevertheless bind GSLs with similar geometries. I will bioengineer innovative 3D DNA objects to address the structural code underlying this apparently convergent evolution for a shared membrane mechanical function. I will systematically vary GSL binding geometry, and then submit the corresponding synthetic lectins to functional testing on cell and model membranes. My experimental data will be scrutinized by theoretical and computer modeling, which should in return suggest new experiments. I expect to uncover novel clustering mechanisms by which a previously unrecognized highly organized exomembrane structure is obtained, made from regularly spaced infectious disease particles in which specific geometries are optimally exploited for membrane bending.

Chronic inflammation of the gastrointestinal tract results in pathological conditions known collectively as Inflammatory Bowel Disease (IBD), affecting ~0.5% of the world population. However, only ~20% of IBD incidences are currently accounted for by genetic studies. Genome wide association studies suggest that genetic variants in many loci may account for the risk of initiation of IBD. The gut has many lines of defense against invasion of luminal microbes, which are thought to be the major cause for IBD, including the epithelial barrier, and the innate and adaptive immune responses. The traditional view of gut function suggests that intestinal epithelial cells (IEC) serve simply as a physical barrier, while immune cells, residing in the lamina propria, orchestrate mucosal immune homeostasis, and thus play a major role in IBD. However, I hypothesize that IEC may also have innate immune properties that affect the response to or tolerance towards different insults. Dissecting the relative contribution of different epithelial cells, immune cells and microflora in gut homeostasis is challenging; therefore it is necessary to study each component independently. Here, I will use a systems biology approach – combining genomic profiling, computational modeling and biological experiments – to dissect the innate immune role of IEC in mouse models and human clinical samples. I will identify a gene expression signature of IEC immune-effectors, and will test their function in mucosal immunity in vitro and then in vivo in mouse models, as well as in human IBD samples. This work will help identify novel immune effectors in IEC, broaden our understanding of mucosal immunity and IBD, and will provide a paradigm for determining the circuits underlying related pathological conditions in complex tissues.

A vast number of intracellular processes have been shown to involve oscillating gene expression levels, including yeast metabolism, immune response mediated by NF-kappaB, tumor suppression by protein p53 and growth factor response via ERK. Synthetic biology has offered an efficient way of studying the design principles of the regulatory networks maintaining these rhythms, also known as biological clocks. However, investigating such functional units in isolation neglects mutual interactions with other components of the network. The proposed research aims at discovering the mechanisms that determine the emergent dynamics resulting from this interaction. Our first step is to experimentally explore the sensitivity of an already existing biological clock (a transformed Escherichia coli strain) to periodic input stimuli of different amplitudes and frequencies, delivered using micro-fluidic tools. By systematically varying experimental parameters, the free-running system can be tuned to show different dynamics with or without spontaneous oscillations, resulting in various possible responses to the stimulation, e.g., induction of oscillatory dynamics, entrainment of an existing oscillation or failure to do so. In a next step, a quantitative model constructed in prior work for the oscillatory regime of this gene network will be adapted to accurately capture the experimental results and thus will allow us to dissect the essential mechanisms that lead to tight coupling between stimulus and response and represent an efficient way of intracellular signal transmission. This information will suggest modifications of the genetic circuitry that will finally be experimentally tested.

The thyroid is a critical regulator of growth, development and metabolism in most tissues of the vertebrate body. While recent studies have identified some of the factors that regulate the migration, differentiation and growth of the thyroid gland, little is known about its regenerative capacity.
I propose to use genetic and chemical tools as complementary approaches in order to study the cellular and molecular mechanisms regulating thyroid regeneration. First, I intend to generate a zebrafish line in which thyroid follicle cells are conditionally targeted for ablation. To do so I will activate thyroid-specific expression of nitroreductase (NTR), an enzyme that converts metronidazole (MTZ) into a cytotoxic product. To examine thyroid cell survival and regeneration, I will use the photoconvertible property of the fluorescent protein Kaede to label thyroid cells before and after ablation. Upon successful setup of the ablation system I will conduct in-vivo chemical screens on the thyroid-ablated zebrafish to identify molecular pathways involved in thyroid regeneration. Simultaneously, I will investigate the possibility that cells from adjacent tissues contribute to thyroid regeneration using cell lineage tracing approaches, including those enabled by the Cre-ER system. Complementary to the chemical screens I will perform gene expression profiling of the suspected cells during thyroid regeneration. Combining these various genetic and chemical tools, I aim to identify molecular pathways and progenitor cells which contribute to thyroid regeneration.

Ribosomes stand amongst the most complicated structures in biology. Their assembly is a question of fundamental interest, but is still poorly understood in spite of efforts of excellent groups. We propose to address this challenging question using a quantitative approach, inspired by recent results in cell-free translation and in single-molecule biophysics. By applying a force to specially designed DNA/RNA duplexes, we can repetitively release and re-anneal RNA with full control over velocity and orientation of the strands. We can thus mimic the gradual appearance and folding of rRNA in co-transcriptional assembly of bacterial ribosomes, including varying transcription rate and pauses. Combined force and fluorescence experiments will allow us to follow in real time the binding of ribosomal (r-)proteins to the gradually released rRNA. We will focus with our single molecule measurements on the initial phase of the assembly of the large ribosomal subunit of E. coli. This involves the 23S rRNA, the five r-proteins that have been identified as essential and sufficient for the first assembly stage (L4, L13, L20, L22 and L24), and a selection of assembly helper proteins. The single-molecule studies will be complemented by coupled transcription-assembly experiments, in which the assembly will be followed in fluorescence experiments using Förster Resonance Energy Transfer between fluorescent labels on r-proteins adjacent in the ribosome and by testing the ribosomal capacity of protein synthesis. Using this combination of approaches, we will provide a dynamic view of the choreography of ribosome assembly, identify essential assembly factors among the about 15 known assembly helpers and identify their roles. Our interdisciplinary team combines two biophysics groups, well known for single-molecule studies on nucleic acids and associated proteins, and two biochemistry groups, well known in the fields of ribosome assembly and cell-free translation. Close collaboration between these four groups will bring together the expertise required to successfully perform the proposed experiments and will bring our understanding of ribosome assembly to a new level.

The ENCODE project and RNA deep-sequencing have revealed that the genome is pervasively transcribed, however, a considerable fraction of the RNA that is produced does not code for protein, but instead functions as a crucial layer of regulation over the messenger RNAs that do code for proteins. A major question in biology is how the activity of these regulatory non-coding RNAs (ncRNA) are controlled. Our 4 groups have now independently reported, through different fields of study, that the seemingly unruly network of ncRNAs are regulated by programmed interactions between the ncRNA themselves.
We propose to combine our expertise in RNA biology, computation, biotechnology, and oncology, to uncover the principles governing ncRNA interactions, with particular focus on ncRNA regulation of miRNAs, and to test the hypothesis that a major function of ncRNAs is to regulate other regulatory ncRNAs, particularly miRNAs by acting as competitive antagonists through cis-encoded sequences and recruitment of RNA binding proteins, and that ncRNA regulation of miRNA activity is important for controlling cancer relevant cell functions. To test our hypotheses, we will, (1) determine which ncRNAs have ceRNA/sponge activity through large-scale loss-of-function studies, (2) determine how the target site sequence encoded by an ncRNA affects the efficiency of ncRNA-mediated sponging, and (3) determine the relevance of linear and circular ncRNAs in cell proliferation and survival using a high-throughput functional approach.
These studies will assign functions to 100s of little studied non-protein coding genes, determine the mechanisms and principles of a novel form of gene regulation, and establish a new understanding of how a major part of the transcriptome, the ncRNAs, interact with each other, and potentially identify genes with relevance to cancer cell biology.