Breast milk is the optimal dietary source for infants, as it supplies all the nutritional requirements for the first six months of life. However, over 50% of mothers express concerns about insufficient milk volume or poor milk quality as pertinent to their motivation to wean their infants before six months of exclusive lactation. Human breast-milk contains macro- and micronutrients as well as numerous bioactive compounds and several different cell types, including epithelial, myoepithelial, stem cells and leukocytes. The human mammary gland is distinct from other mammals in many aspects, including milk composition, gene expression and architecture. This renders commonly used mammalian animal models non-representative systems for studying the human lactation process. During my post-doctoral fellowship, I aim to characterize the breast-milk cells transcriptome (single-cell RNA sequence), regulome (ATAC-seq and Cut&Run), and variome (genotyping arrays) and link them to create a novel and important genomic lactation dataset. I will use samples collected under both physiological (e.g., colostrum, mature milk, and involution) and pathological (e.g., low milk production) conditions, to study the mechanisms underlying different lactation pathologies and to characterize how genetic variation influences lactation outcomes and infant growth. Collectively, the results of this study will significantly impact mother and infant health worldwide and expand our toolbox to improve breastfeeding medicine and help mothers dealing with breastfeeding issues and related diseases.

In our lives, we are continuously generating a sequence of actions. What are the neural mechanisms that cause the selection of one action above all others? These mechanisms can provide key insights for designing artificial robotic controllers that are as flexible as their neurobiological counterparts. The dominant ‘feedforward’ model for action selection suggests that decisions are made in the brain, upon which a command signal is sent to downstream motor circuits. However, this model ignores the critical role that peripheral mechanosensory feedback signals likely play in action selection. Such a potentially paradigm-shifting ‘feedback-centric’ model of action selection, its dynamical mechanisms, and its neuronal instantiation have yet to be explored. As an HFSP Cross-Disciplinary Fellow in the laboratories of Pavan Ramdya (experimental neuroscience) and Auke Ijspeert (dynamical systems modelling), I will develop such a model through a combination of data-driven dynamical systems theory, optogenetics, and behavioral analysis in Drosophila melanogaster. I aim to discover how a variety of Drosophila limbed behaviors can be generated by a common modelling framework, whereby a set of neural oscillators are coupled through limb-mechanosensory feedback loops and driven by descending neurons. Using these models, I will predict and experimentally test how perturbing sensory feedback pathways affect action selection. Finally, I will cluster the distribution of model feedback topologies that explain different Drosophila behaviors to identify fundamental structure-function relationships and to reveal how one neural network can select and generate a remarkable diversity of actions.

Site-selective modifications of proteins at a predetermined location have been proven to be very useful to gather information and explore composite biological processes. In addition, these reactions give opportunities for researchers in academia and industry to prepare functional protein based materials for various applications in our daily life. Today, the chemical toolbox for site selective modifications is quite developed for targeting highly reactive amino acids residues such as cysteine and lysine, however targeting less reactive amino acids is much more challenging and therefore examples are rarer. Among the unreactive amino acids, histidine site selective modification is highly desirable. Although several reports exist for this modification there is no concrete general approach for selectively modifying histidine residues. This fact motivated us in this research proposal. Herein, we propose to develop a general strategy to selectively modify histidine residues on proteins without the need of complex genetic manipulations. We plan to leverage the exquisite functional and water group tolerance of Minisci type radical reactions to address this challenge. The bioorthogonality and feasibility of the developed reaction will be tested for drug development and fluorescent labelling of enzymes. We believe that the success of this selective histidine modification will have tremendous impact in the biomedical sciences.

Directed evolution is a technique to investigate evolutionary dynamics and evolve nucleic acids and proteins for research, diagnostic and therapeutic applications. It uses cycles of mutation and screening or selection to mimic Darwinian Evolution. The aim of these experiments is to screen or select large libraries of molecule variants with different sequences (‘genotypes’) and retain those sequences having the required properties (or required ‘phenotypes’). At the moment one essential limitation of Directed Evolution methods is that the properties of each variant in the library are assessed based on bulk samples, i.e. on the average activity of a large numbers of copies of the same molecule. While these copies are identical at the sequence level, they may still exhibit different phenotypes, for example due to the fact that the same sequence can assume multiple three dimensional structures. These ‘heterogeneous’ phenotypes can play important roles in evolutionary dynamics, but at the moment there is no convenient Directed Evolution tool to address them. Here we combine expertise from the fields of directed evolution and single-molecule microscopy to develop a novel approach that allows us to perform directed evolution while looking at the behaviour of single-molecules. The method can be used to perform accurate phenotypic characterization measuring multiple aspects of the phenotype in parallel, something that can be challenging with conventional approaches. For example it can be used to study how a molecule with a specific sequence can sometimes play a role in different chemical reactions, how to achieve ‘specialization’ and what the consequences of specialization are in terms of efficiency. As discussed above, the method can also reveal the existence of phenotypic heterogeneity in molecules with identical sequence. This approach will allow us to address several biological questions that cannot be addressed with conventional methods. These include, the evolution of heterogeneous phenotypes in populations of RNA molecules under selection for binding, the role of heterogeneity in defining the evolutionary potential of antibodies and the correlation between rate and accuracy in DNA polymerases. It will also provide a potent route to develop biomolecules for biomedical and industrial applications, notably in the field of single-molecule technologies.

Loss or imbalance of chromosomes can trigger various pathologies, threatening healthy cell proliferation. During the cell cycle, each chromosome is replicated, generating sister chromatids. Before cell division, duplicated chromosomes must be equally segregated and transmitted to daughter cells. Chromosome segregation is organized by cohesin, which topologically links chromatids from their synthesis onwards. Despite its vital importance, the molecular basis of sister chromatid cohesion establishment at the replication fork remains mostly elusive. A clear model is lacking of how cohesin transitions from interactions with one parental duplex DNA at the front of the replisome to two duplicated DNA strands behind the replisome. Detailed analysis of cohesin assembly at the fork is critical for the formation of a structural framework for cohesion establishment. This proposal aims to address two fundamental questions. First, I seek to describe the conformational changes that cohesin undergoes when it transitions from encircling one to two DNA molecules. Second, I aim to capture cohesin interacting with the replication fork. To tackle these questions, I will use in vitro reconstituted systems combined with cryo-EM imaging. Leveraging my expertise in chemistry and single-molecule techniques, I will develop a new DNA-affinity cryo-EM grid. This new tool will allow me to visualize cohesin and the replication machinery in a DNA-bound state, building on in vitro reconstituted systems available in the Costa and Uhlmann labs. With this multidisciplinary approach, I aim to characterize the elusive molecular intermediates of sister chromatid cohesion establishment.

Development is the critical link between genetic and morphological change. Thus, to understand how organisms come to differ in appearance we must understand how the developmental pathways that build them evolve. We know that changes in these pathways can drive the evolutionary loss or modification of traits. But we have so far failed to explain how new developmental pathways evolve. This is the greatest gap in our understanding of morphological diversification: innovations, such as wings, horns, and feathers, all need to evolve before they can be modified. This problem is further confounded in the most extreme parts of organismal design, sex-specific traits, which require not just the acquisition of a new developmental pathway but its limitation to a single sex. In this proposal, I develop a new analytical approach, which repurposes emerging biomedical methods, to identify for the first time the developmental genetic processes by which a novel trait, the recently-evolved Drosophila sex comb, has evolved. Combining time-series, single-cell RNA-sequencing with developmental genetic manipulations, I will use a comparative, multi-species approach to (a) reconstruct the developmental pathway that builds sex combs, (b) identify the developmental genetic changes that have driven morphological diversification, (c) elucidate how the novel pathway integrates sex-determination machinery, and (d) assess the evolvability of this innovation. Ultimately, this approach represents a new way of doing ‘evo-devo’ research – an approach that simultaneously charts trait evolution across levels of biological organisation, from gene to tissue, to understand the diversification of life.

The extracellular space (ECS) forms an important but understudied frontier in neuroscience. It consists of the narrow gaps that surround all brain cells, which are filled with interstitial fluid and extracellular matrix molecules, occupying around one fifth of the volume of the brain. Even though all extracellular signaling molecules and nutrients must transit through the ECS to reach their targets, we know very little about the shape and dynamics of this brain compartment, or its influence on brain function. Although the ECS has received much less attention than neuronal and glial networks, it plays a fundamental functional role in brain health and disease, serving as a reservoir of ions for electrical activity and providing an essential microenvironment for the well-being of cells and brain homeostasis. Based on pioneering theoretical and biophysical studies, we know the diffusivity and geometry of the ECS are major determinants of how molecules (endogenous substances or medical drugs) can spread around the brain or get cleared from it. However, mapping the biophysical landscape of the ECS with enough spatial resolution in live brain tissue has been impossible to accomplish until now for lack of appropriate tools. Bringing together a team of leading researchers, this multidisciplinary project will study key properties of the ECS, focusing on its dynamic organization, its role in material transport (in brain perivascular spaces and parenchyma) and its impact on brain function at the cellular and systems level. To this end, we will develop several innovative investigative tools that will allow us to image and manipulate key aspects of the ECS, and to study its structure and function in brain slices, in vivo and in silico. Specifically, we will (1) engineer novel chemical tools to label hyaluronan (HA) and to manipulate its biological activity, (2) develop super-resolution microscopy technology to study impact of ECS on synaptic function in brain slices and to enable ECS visualization in the intact brain in vivo, (3) make biophysical measurements to quantify diffusion of molecules through the ECS, (4) investigate ECS control of ‘glymphatic’ function in the sleep-wake cycle, (5) construct morphologically realistic mathematical models to simulate substance transport through the ECS. This combination of methodologies will enable us to better understand how this brain compartment influences brain physiology, focusing on single neuron function and sleep.

Sexual reproduction is an obligate stage in the complex life cycle of Plasmodium parasites, the causative agents of malaria. While cyclic asexual replication of the parasite in the vertebrate host is associated with all clinical symptoms of malaria, the transmission of the disease to a new host relies on the sexual reproduction of the parasite in the mosquito. Transmission and reproductive success depend on the ability of the parasite to produce fertile male and female gametes at an optimal ratio. The molecular mechanisms of sex determination and development, therefore, hold the key to new transmission-blocking interventions and to a broader understanding of how life cycle decisions in an important group of parasites can be regulated. Recent breakthroughs have identified a master regulator of sexual commitment, but how one transcription factor can give rise to the completely different gene expression programs of male and female gametocytes remains elusive. Genetic screens and transcriptomic analyses in a rodent model have now led to the identification of a small panel of genes which I propose here will unlock the question of how sex ratio is determined. By applying a combination of state-of-the-art biochemical, cell biological and genetic techniques, I aim to characterize the function of three strongly supported, male-determining candidate genes. The obtained results will provide insights into how sex, and thus transmission, of malaria parasites is regulated. By elucidating the fascinating biology of sex determination in an ancient eukaryote that lacks sex chromosomes, the proposed work can reveal potentially novel pathways involved in the origin of eukaryotic sex.

Uncontrolled loss of neurons is a hallmark of neurodegenerative diseases (NDs) such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS). In NDs, an abnormal assembly of proteins and mRNAs known as a biomolecular condensate (BC) is believed to contribute to neurotoxicity and ensuing neuronal death. Despite high mortality and poor quality of patients’ lives, as well as the enormous socio-economic burden of patient care, the exact molecular and cellular mechanisms underlying ND pathogenesis remain largely unknown. This roadblock in ND studies primarily originates from mutual dependency between “molecular assembly” of general ND proteins and “functionality” of individual ND proteins. Molecular assembly is a dynamic process that can take place locally, and changes actively in its physical property by transitioning among soluble phase, liquid-like droplets, hydrogels and insoluble aggregates. To our knowledge, there is currently no technique to disassemble ND-related BCs to distinguish these physical and biochemical characteristics, especially in a physiologically relevant context. By integrating expertise in neurophysiology, chemical biology, and soft-matter biophysics, we propose to develop a conceptually unique methodology termed “dePOLYMER”. Here, light illumination or chemical administration actuates a genetically-encoded molecular probe designed to polymerize actin to generate constrictive force against an intended object such as BCs in living cells. When force density exceeds the surface tension of target BCs, dePOLYMER is expected to trigger their dispersion. We will specifically target stress granules as a model for non-pathogenic BCs, as well as pathogenic ALS-related BCs. Using a model for ALS, we will next aim to demonstrate dispersion of BCs consisting of ALS proteins in a manner specific for constituent proteins and cell types. In summary, the goal of our study is to offer a novel technique to efficiently disperse BCs in a rapidly inducible manner. Implementation of dePOLYMER in ALS model systems may enable future identification of the molecular and cellular foundation of neurotoxicity, thus a potentially new treatment strategy for NDs. Due to the inherently modular design of dePOLYMER probes, the technique should be generalizable to virtually all BCs whose molecular components are known, let alone other ND-related BCs.

Dense commensal microbial communities are found within the gut that protect us against pathogens. Yet, a much-discussed gap in our understanding is how these communities organise within the intestine, and how this organisation links to health outcomes. One particularly dense area of colonisation is the gut epithelial surface and associated mucus, as the region with the most permanent and intimate association with the host. My key hypothesis is that microbes at the mucus interface are particularly important for protection against pathogens that must overcome this barrier to access the epithelium in order to cause disease. I will test this in three aims: 1) I will develop new imaging methods to map the spatial organisation of microbes at the gut mucus interface facing challenge from the important pathogen Salmonella enterica serovar Typhimurium, with unprecedented throughput and detail. I will pioneer new methods for label-free pathogen and commensal identification in the gut what will allow to overcome the limited throughput in imaging-based microbiome research. 2) Based on the new imaging data, I will use custom image analysis to identify those commensal species that associate preferentially with the epithelial surface and with the pathogen. 3) Finally, I will test whether the spatial ecology of gut bacteria is predictive for colonisation resistance by assembling defined communities with different spatial organisations in gnotobiotic mice. Overall, my project promises for the first time to show how the spatial ecology of the microbiota is critical for the balance between health and disease.

In a rapidly changing world, a key focus in evolutionary biology is to better understand and predict how populations will respond to alterations in their environment. Sexual selection is a pervasive force that can alter how populations respond to natural selection. But, how sexual selection and the broader environment interact to influence adaptation is not well understood. I will determine the influence of male-male competition on adaptation of an herbivorous insect to a novel host plant using the leaf-footed bug Acanthocephala femorata. Male leaf-footed bugs compete for territories on host plants, using enlarged hind femurs to grapple and push other males off the plant. Host plants differ in their architecture, which affects which weapons and fighting styles are most effective and are selected for, altering sexual selection on male weapon morphology. Male success at colonising a novel host plant is therefore influenced by both sexually selected traits (fighting behaviour and weapon shape) and the environment (the plant’s architecture). I will manipulate sexual selection directly by changing the sex ratio and indirectly with different host plants to test how sexual selection influences the ability of leaf-footed bugs to adapt to a novel host plant. I predict that the role of sexual selection in facilitating adaptation to a novel host plant depends on the architecture of the host plant. These results will be important for understanding how host range evolution can occur in species that experience sexual selection, especially when the environment indirectly impacts sexual selection.

Some coral reefs recover from climate change-driven disturbances relatively quickly, while others stay barren because coral populations fail to replenish themselves though larval recruitment. Here, we propose to study neuro-molecular mechanisms of settlement decision-making in coral larvae, to identify intrinsic processes that might be responsible for success or failure of coral reef recovery. Our first aim is to identify and map all types of neurons (connectome) in a coral larva at different stages of competency and commitment to settlement. It will be accomplished by performing single-cell RNA-sequencing to identify neuronal types and mapping their molecular markers onto the neural network using serial section electron microscopy, in situ hybridization and immunohistochemistry. Our second aim is to validate the role of neuron-specific candidate genes by perturbing their function (using CRISPR/Cas9 knock-downs and antibody blocking) and assessing the effect on larval settlement behavior, gene expression, and neuronal morphology. We will use Acropora millepora, which is a representative of the largest and the most ecologically important genus of reef-building corals.

One of the ultimate goals in neuroscience is to understand neural correlates of perception, sensation and behaviors. For the past several decades, a number of studies have successfully imaged neuronal activities in behaving animals and further showed the behavioral causality of specific neural circuits by using various optogenetic techniques. More recently, additional significant efforts have made in order to minimize the size of optical system, such that real-time imaging can be done even in freely moving animals. However, high-resolution imaging of deep brain structure in behaving animals still remains challenging. This limitation has hampered our understanding of neural mechanisms in subcortical areas at the resolution of individual cells or synapses. Here I propose to develop a new design of miniaturized microscope by implementing my expertise with optical coherence detection into two-photon (2P) microscopy. This system will allow us to image deep brain areas with synapse resolution in freely behaving animals. Furthermore, both aberration correction and holographic excitation will be implemented with wavefront shaping, which will enables simultaneous imaging and holographic optogenetic control of neural circuits. Using this newly developed microscope, I would like to define neural activities in Nucleus Accumbens (NAc) (4 mm deep from the surface) that are associated with goal-directed behaviors. I will further test direct behavioral causality by selectively activating behaviorally-relevant neuronal populations. In summary, the proposed study will provide new optical approaches that will uncover various neural mechanisms underlying animal’s behaviors or cognition.

Brain metastases (BrM) represent the most common and aggressive neurological complication of systemic cancers, typically arising from melanoma, lung and breast cancers. With most patients living just over one year following BrM diagnosis, it is essential to understand the multistage process of dissemination of cancer cells from the primary tumor to the CNS and the importance of the tumor microenvironment (TME) in which BrM develop, to uncover novel therapeutic targets. The brain TME comprises a diverse spectrum of resident and peripheral immune cells, glial, vascular, and cancer cells. As the CNS was previously believed to be devoid of a lymphatic system, the trafficking of these cells into the CNS was thought to occur only via blood vessels. However, we hypothesize that the recently identified CNS lymphatic system may represent a potential route for both cancer and immune cells to reach the CNS through the lymphatic vessels within the dura mater. For this reason, I aim to evaluate how the lymphatic network evolves during BrM establishment, to determine to what extent cancer cells and peripheral immune cells are being transported through the lymphatics into the CNS, and to identify which molecular cues are responsible for the recruitment of these cells to the dura mater. To address these goals, transcriptomic and histological analyses of the dural microenvironment, in vivo imaging of the dura mater, and manipulation of the lymphatic system will be performed at different stages of BrM development. This experimental strategy will allow me to determine the potential impact of the lymphatic system in BrM establishment, an intriguing link that has not yet been explored.

Cells respond to physical force-induced signals by converting the mechanical stimulus into a biochemical response. This process, termed mechanotransduction, is an integral part of cellular physiology and has a profound impact on many biological processes including cell development and differentiation. The project goal is to provide a better understanding of the molecular mechanisms of the initiating 'mechanosensing' and its connection to variations in gene expression by studying the dynamic features of this mechanism at a single molecule level. Force transmission is a highly dynamic process and its investigation requires single molecule techniques with high temporal resolution. Therefore, the immediate aim will be to take advantage of and improve a unique ultra-fast optical trap, recently developed by the supervisor's group at the University of Florence that will allow the investigation of protein-protein interactions in vitro. In particular, the researcher will study the interactions between actin and putative mechanosensitive proteins, under various conditions of mechanical load. In a parallel research line, the researcher will develop an experimental setup and a methodology to apply mechanical stimuli on living cells and monitor the changes in gene expression profiles, with sensitivity down to the single molecule. The aim will be to correlate external mechanical stimuli on specific membrane receptors to intracellular signaling pathways and gene expression profiles, thus creating a powerful tool to enhance our current understanding of the molecular mechanisms of mechanotransduction in vivo.

One of the great questions concerns fate vs. chance. Are events destined to occur, or are they the result of specific antecedent conditions, any of which, were they different, would have led to a different outcome? Anolis lizards are recognized as among the best groups to investigate this question, and detailed study of their evolution has suggested a strong role for determinism. These studies, however, have focused on the phenotype; the extent to which genome evolution is deterministic or historically contingent is an open question. Until recently, detailed investigation of genome evolution has been limited to model organisms, for which little data are available on their ecology and behavior in natural settings, precluding study of the factors that have shaped their evolution. Now, advances in genomics and gene editing methods allow evolution to be studied in non-model organisms, permitting holistic studies from the genome to selective pressures in nature. Anolis lizards are the ideal group to combine genomic and organismal studies at multiple levels to address the extent to which evolutionary diversification is deterministic or contingent. This project will proceed as follows: Genomics: We will test for evolutionary determinism by investigating whether convergently evolved phenotypes are the result of the same or different genetic changes. Functional Tests of Candidate Loci: We will take advantage of recent advances in gene editing to directly test the role of these genetic changes in producing convergent phenotypes. To do so, we will use CRISPR-cas9 gene editing to modify genes in the lizard Anolis sagrei. We will examine genetically modified offspring to test the hypothesis that sequence variants produce the putative phenotypes. Behavior and Physiology: We will identify genes and the underlying sequence changes associated with the evolution of these traits, introduce these specific sequence variants into brown anoles, and then test whether the introduced sequence alterations produce the hypothesized behavioral and physiological phenotypes. Natural Selection: Once we have identified genetic changes underlying putatively adaptive phenotypes, we will design field experiments to test the hypothesis that natural selection favors those genotypes/phenotypes under particular environmental circumstances.

Sleep is a reversible state of quiescence during which an animal loses awareness of the external world. Although sleep is important for many brain functions, the mechanisms that underpin sleep and its functions are still poorly understood. The fruit fly, Drosophila melanogaster, has become a popular model for sleep research, because of the ability to probe the molecules and circuits involved in sleep. In this study, we will investigate the neural mechanisms by which the brain suppresses perception during sleep, with a focus on the well-characterized Drosophila olfactory system. Firstly, we will establish a system in which we can image neural activity of olfactory circuits during sleep. Secondly, we will investigate how sleep alters odor representations. Thirdly, we will examine the neural mechanisms underlying this sleep-dependent olfactory processing. Finally, we will examine the functional impact of sleep on olfactory behaviour. Overall, this study aims to reveal fundamental principles of sleep functions in sensory perception.

Precise control of gene expression is required for virtually all cellular functions, ensuring correct maintenance of cell identity and genomic stability. Chromatin remodeling complexes regulate gene expression and reorganize chromatin architecture by sliding, ejecting or changing the composition of nucleosomes along DNA. The ATP-dependent multi-subunit BRG-/BRM-associated factor (BAF or mammalian SWI/SNF) remodeling complex is one of the most frequently disrupted macromolecular assemblies in human cancers, as well as in a range of autism and intellectual disability syndromes. While the molecular activities of BAF complexes and their role in driving disease mechanisms are intensively studied, the specific roles for post-translational modifications (PTMs) on individual BAF subunits in mediating overall BAF complex function has remained largely unexplored. Further, specific upstream signaling pathways that alter BAF remodeling activity via placement of subunit PTMs in both healthy and disease states have not been explored. To address these key questions, we aim to define the compendium of BAF subunit PTMs, assess their functional roles, and identify the corresponding PTM-mediating enzymes and pathways. These approaches will enable us to answer fundamental questions regarding whether and how BAF subunit PTMs control complex activity, targeting, and functions in mediating DNA accessibility lending new insights in to remodeler function in both healthy and disease states. Delineating the pathways which activate or repress BAF function may implicate novel strategies for therapeutic intervention in cancer and other diseases.

Advancing age is a major risk factor for many neurodegenerative diseases, but the underlying mechanisms are not understood. The Crowston laboratory has recently demonstrated age-related impairment of neuronal recovery and associated increased neuronal death in an established optic nerve injury model. The predominant age-related changes were localized to excitatory synapses that activate ganglion cells in the retina. Because cholesterol is known to be important for synaptogenesis and recent evidence linking mutations in cholesterol metabolism with numerous neurodegenerative diseases, we hypothesize that age-related changes in cholesterol metabolism could be responsible for the observed defect in neuronal recovery through loss of synaptic plasticity. To test this hypothesis, experiments would be divided into two stages, a discovery stage and a hypothesis-driven stage. The aim of the discovery stage is to compare central nervous system (CNS) cholesterol metabolism in young versus aged animals, either with or without injury. If the hypothesis is correct, aged animals would be expected to have altered CNS cholesterol metabolism more susceptible to injury challenge. The hypothesis-driven stage is to then test how manipulating CNS cholesterol metabolism impacts synaptogenesis and recovery of visual function. This project will be done in collaboration with the Silver lab which has expertise in lipid transport and metabolism. Results would further our understanding of cholesterol metabolism in the aging CNS and how it impacts neuronal recovery.

The majority of the human transcriptome encodes non-coding RNAs that are not translated into protein. Long ncRNAs (LncRNAs) are 200 nucleotides or longer, and are involved in many cellular processes, including embryogenesis and DNA damage repair. Xist is an evolutionarily conserved LncRNA that plays a key role in dosage compensation in eutherian females. It does so by recruiting protein complexes that deposit repressive marks on histones on one X chromosome (XC), thereby leading to heterochromatin formation. Recent computational and biophysical studies suggest that Xist, as well as other LncRNAs, adopt higher order tertiary structures similar to ribozymes and group-II introns. Nonetheless, no high-resolution 3-D structures of Xist and its associated protein complexes have been determined. Thus, the precise molecular mechanism of XC inactivation remains unclear. In this proposal, I aim to biochemically identify and reconstitute Xist ribonucleoprotein (RNP) complexes involved in spreading and silencing of the XC. I will then determine the 3-D structures of Xist-protein complexes using a combination of electron cryomicroscopy and X-ray crystallography. In combination with biochemical and biophysical assays, I will characterize the native proteome associated with Xist, as well as the contributions of Xist tertiary structure towards recruitment of protein machineries including the polycomb complexes. These studies aim to elucidate molecular mechanisms of how Xist carries out gene silencing of the XC.