The gut is a dynamic and complex organ characterized by constant epithelium turnover and crosstalk among various cell types and the microbiota, the ecological community of commensal and, sometimes, pathogenic microorganisms. Changes in the composition of the gut microbiota have been connected to multiple aspects of host physiology and human disease, however relatively few microbe-host interactions have been defined at the level of molecular mechanisms. Here I propose to investigate the role of intestinal lymphatic vasculature in bacteria-derived metabolite transport and signaling. Intestine is richly supplied by lymphatic vessels. Known functions of lymphatic vessels of the gut include dietary fat absorption and transport of immune cells and dietary antigens to regulate tolerance or immune responses. To date, nothing is known about the microbiota-derived metabolites transported by intestinal lymph flow through lymphatic vessels and the role of those metabolites in body homeostasis. My main goal will be to use unbiased metabolomics and other systems biology approaches in combination with genetic animal models to characterize lymph bacteria-derived and host metabolomes and to establish an impact of extrinsic or intrinsic factors such as diet and lymphatic function on production and distribution of such metabolites in the body. I anticipate that the project will uncover previously unknown roles of intestinal lymphatic vasculature and bacterial metabolite signaling in specific target cell populations.

During the past decade, the tumor microenvironment and in particular its immune cell compartment have moved into the focus of cancer research, offering promising therapeutic opportunities. However, therapeutic approaches targeting the tumor microenvironment, including the highly promising field of immunotherapy, struggle with widespread tumor heterogeneity. To further our understanding of tumor biology and ultimately improve treatment, we need to assess the tumor microenvironment in its complexity and recognize all the different cellular and extra-cellular components as well as their interactions and spatial relations. To this end, I will connect two powerful single-cell technologies, DroNc-seq (providing genomic scale cellular resolution) and MERFISH (providing spatial tissue organization), to create high resolution, spatio-molecular maps of the tumor microenvironment in two tumor types with different etiologies (colon cancer & melanoma). These maps will allow the study of cellular, and by inference, extra-cellular components as well as their physical and effectual interactions, creating an integrated, quantitative image of the tumor microenvironment. Finally, I will apply machine-learning techniques to extract features predictive for different states of the immune cell compartment and establish their relevance through integration with clinical data.

Engineered mammalian cells have huge potential for next-generation medicine, but their use requires isolation and ex-vivo engineering of patients’ cells with sophisticated biotech instruments, as well as laborious quality checks. One possible approach to address this issue would be selective in situ cellular programming in vivo, but current modalities for gene delivery (e.g. viruses) involve risks due to insufficiently tight specificity and potential safety issues. The aim of my proposal is to apply a “focused discovery” approach to the basic biology of exosomes, which are highly biocompatible, cell-derived nanovesicles, using a range of synthetic/chemical biology tools to identify key control functions in exosomes and receiver cells. Then I will utilize this information to extract design principles for engineering polyvalent targeting layers to deliver exosome cargo to a cell type of interest with extremely high specificity without toxicities, thus enabling in situ cell programming. Specifically, I propose the following three sub-projects. 1. High-throughput exploration of effector proteins that efficiently direct exosomes to target cells in vivo. 2. Discovery of factors in exosome receiver cells that promote exosome uptake. 3. Creation of engineering/loading principles for cell-classifying exosome cargos. The combination of these different targeting layers (polyvalent targeting) should ultimately make it possible to use exosomes to program any desired type of target cells in vivo. This work should contribute to a better understanding of exosome biology, and provide a basis for developing new therapeutic strategies, e.g., for cancer and genetic/metabolic disorders.

Understanding the evolution and function of genetic variants affecting human fitness opens new avenues to investigate and explain health disparities among different human populations. The non-coding genome is arising as a promising target; however, it remains largely experimentally untested which and how genetic changes shape human molecular circuits, physiology, and disease.

This calls for novel approaches that experimentally test functionality and causality in a relevant testbed. My aim is to develop an interdisciplinary approach using Tibetan adaptation to the Himalayan Plateau (>4000 m) as a paradigm. Tibetans have genetically and physiologically adapted to live in a low oxygen environment by modulating the production of red blood cells (erythropoiesis). My aim is thus to 1) combine bioinformatics analysis of Tibetan and Han-Chinese genomes and integrate epigenetic data to identify candidate blood enhancers and regulatory variants associated with adaptation; 2) use high-throughput massively parallel reporter assay to experimentally test the activity of thousands of candidate blood enhancers and associated variants; and 3) use CRISPR-Cas base editing to stepwise dissect variants and determine enhancer-gene interactions that are causative and modify erythropoiesis. Together, my project will be the first of its kind to decipher the principles of non-coding genome adaptation, identifying new regulatory regions that control erythropoiesis and human fitness. If successful, my unique strategy and vision can be applied to many other rapidly evolving human traits such as immune regulation, metabolism and many more.

Aging is characterised as a progressive deterioration of physiological function leading to an increased vulnerability to death over time. This loss of viability starts at the age of thirty in humans. Evidences strongly suggest that aging is not an irremediable result of physical limitations, but instead a plastic, regulated and species-dependent process. However, despite the amount of experimental data gathered in the last decades, the dynamics governing longevity is still poorly understood. We propose a novel multidisciplinary method to model the aging of a tissue based on its cellular and molecular pathways. Current models focus on explicit simulations to create an organ from cells, but this approach is computationally very intensive and sensitive to initial conditions. We will investigate the age-related changes from an implicit approach, by using an “effective theory”, in order to simplify the parameters between the biological levels and predict the general evolution of the tissue. Such model will give us a much-needed overview of the multiscale dynamics of the aging process, help us identify new targets for therapeutic interventions and help us predict the effects of potential studies. This is especially valuable in the field of aging, since in vivo experiments are difficult on humans. Alternate approaches can make a difference to find ways to extend our healthy years of life and to counteract the burden of our aging population on society.

Adaptation to climate change, particularly to increasing temperatures, is vital to plant survival. Stomata are the gatekeepers of plant gas exchange, balancing CO2 uptake and water vapor release, and hence obvious targets for climate change adaptation. However, while stomata formation is well characterized on the molecular level, little is known about the variation of core stomata genes across climatic gradients, and variation through time has not been investigated at all.
The central goals of this proposal are to (i) characterize the last ~200 years of stomata variation, from the start of the industrial revolution to today, in preserved historical European Arabidopsis thaliana individuals from herbaria. Combined stomata imaging of historical specimens with herbarium genomics will reveal how populations changed genetically and whether the historic stomatal changes have a genetic basis. I will then (ii) determine the temporal genetic variation in the known core stomata genes, and identify coinciding genetic variation across temporal-climatic and spatial environmental-climatic clines. Finally, I will (iii) infer expression of core stomata pathway genes (including transcriptional regulators and intercellular signaling genes) across historical timescales and determine the role of changing gene expression in stomatal adaptation, using published transcriptome data.
This work will reveal how a wild, non-domesticated plant copes with anthropogenic climate change, from historical trends to genetic mechanisms. It will provide the basis to predict future trends not only in a model weed, but also other plant species.

Endoplasmic reticulum (ER) stress and unfolded protein response are energetically challenging under glucose deprivation due to a decrease in glycolytic rates and protein glycosylation. Under these conditions mitochondrial respiration and oxidative phosphorylation (OXPHOS) are necessary for survival, however the mechanisms whereby these energetic requirements are regulated are unknown. This mitochondrial bioenergetic response is impaired in human mitochondrial diseases. The existence of respiratory chain supercomplexes (SC) and their organization in cells has been subject of debate for decades arising the fluid and solid-state models. To date, the function, structure and organization of SC are poorly understood. Preliminary data show that the PERK-eIF2a axis promotes SC assembly and increases OXPHOS activity. However, the mechanisms that link PERK activation and SC assembly are not known. Thus, the major goal of this application is to identify the mechanisms and molecular interactions that allow SC assembly in cells under ER stress conditions through the PERK-eIF2a axis using different in-vitro and in-vivo approaches: 1. Identification of the components that control ER and nutrient-dependent stress SC assembly through the PERK-eIF2a axis. 2. Determination of cristae architecture on SC assembly during ER stress. 3. Bioenergetic rescue of PERK activation in complex I defective mice. Outcomes from this application will provide information on the mechanisms that allow ER and nutrient stress-dependent SC assembly and electron transport chain activity with implications for the development of therapies for mitochondrial disorders.

Animal mitochondrial DNA (mtDNA) has a higher substitution rate than nuclear DNA, with the accumulation of mtDNA mutations being one of the hallmarks of ageing. This discrepancy in the rates of evolution is partially due to the lack of mismatch repair activities in the mitochondria. Octocorals – a group of cnidarians – have a reduced rate of mitochondrial evolution and encode a MUTS-like protein (mt-MutS) in their mtDNA. Previous analyses suggested that this enzyme was acquired from a virus and has been universally retained among octocoral taxa. Its function, however, remains unknown. The project will combine comparative, structural, and experimental approaches to investigate the function of mt-MutS and to test whether mt-MutS expression results in lower mutation rates in mtDNA and improve heath in ageing. Comparative analysis of octocoral mtDNA will be used to identify distinct mutation patterns among its lineages and correlate them with the changes in mt-MutS. Partial mt-MutS sequences will be used to identify clades with unusual or accelerated rates of mtDNA evolution for additional sampling. Site- and taxa-specific evolutionary rates in mt-MutS will be analyzed to infer functional and structural constraints and to optimize the choice of the mt-MutS for transgenesis. Ancestral sequences of mt-MutS for the nodes of interest will also be reconstructed and analyzed. A structure-function approach will be utilized for in vitro dissection of mt-MutS functions. The full-length proteins from several species and a reconstructed ancestral sequence will be tested for stability and for amenable structure determination. Isolated domains will also be used for high-resolution structural analysis by diverse biophysical techniques to probe molecular details of binding and nuclease activity for understanding and improving function. Finally, we will generate transgenic mice that express a mitochondrially-targeted version of this optimized mt-MutS enzyme to test its effects on mtDNA mutation rate. The transgene construct will be knocked-in to mice by directed Easi-CRISPR template repair or BAC-transgenesis. mtDNA mutation rate analyses in wildtype and mice with enhanced mitochondrial mutation rates will be undertaken. An ageing study on mice expressing mt-MutS will determine if enhanced mtDNA fidelity can positively affect organismal lifespan and healthspan.

Genomic DNA is packaged by nucleosomes assembling the four core histone components (H3, H4, H2A and H2B). Histone modifications and DNA methylations regulate specific transcriptional states in the genome, and play important roles in various cellular processes of eukaryotes. The four core histones share characteristics with the common ancestor of eukaryotes and Archaea. In eukaryotes, the primitive histones diverged as histone variants that are associated with histone modifications and DNA methylations to form functional chromatin states. In eukaryotes, chromatin states have been studied only in model species from the later diverging branches of the evolutionary tree and we know very little of their evolutionary origins. Photosynthetic eukaryotes (Archaeplastida) are the result of a primary endosymbiotic event involving a cyanobacterium that occurred ca. 1.9 Bya. Chromatin has been well studied in land plants that, in evolutionary terms, have diverged more recently (ca. 500 Mya), whereas studies are wholly lacking from their sister lineages in the Archaeplastida (i.e., glaucophyte algae containing blue-green plastids and the red algae). To understand the origin of ancestral chromatin states in photosynthetic eukaryotes, this project will elucidate ancestral chromatin states in several species of glaucophyte and red algae. This study will trace the evolutionary trajectory of chromatin organization into functional units from its archaeal origin to photosynthetic eukaryotes. The models used to dissect the evolution of chromatin states will provide platforms to study other deep branching lineages within the eukaryotic tree of life.

Approximately 30% of mammalian genes code for transmembrane proteins, which comprise the majority of signal receptors and transducers. These functions are not solely encoded in protein structure, but are also regulated by the unique physicochemical environment of mammalian membranes. A key unmet challenge is to understand the interplay between the composition of membranes, their collective physical properties, and their resulting effect on protein function. The knowledge gap is especially apparent for mammalian neural tissue, whose membranes are highly enriched in omega-3 polyunsaturated fatty acids (PUFAs), which our bodies do not synthesize. This composition is central to neural function as evidenced by brain lipid alterations in numerous developmental, psychological, and neurodegenerative disorders; however the mechanistic relationships between the brain’s unique lipid composition and neurological functions are unknown. Major open questions are how neuronal function is influenced by the lipid content of the membranes that host neural signal transduction receptors, and how factors like diet and environment can influence those lipid compositions. Here, we assess the paradigm-shifting hypothesis that alterations of neuronal membrane lipid composition affect the signaling in the brain and contribute to the pathogenesis of neurological disorders. Particular emphasis is placed on the role of dietary lipids in modulating membrane composition, and the functional consequences thereof. Breakthroughs in understanding the central role of lipids will emerge from the project’s interdisciplinary crosstalk between detailed comprehensive lipidomics, molecular computer simulations, quantitative cellular biophysics, and molecular neurobiology. We focus on two parallel research streams: pattern-recognition receptors and G protein-coupled receptors, which are here used as representative systems to explore the regulation of neural receptors by lipids in a pipeline involving computational, synthetic, and natural model systems, as well as cultured cells and in vivo studies. As the influence of lipids on neuronal receptor function has so far been almost completely ignored, these studies will generate significant impact. Further, the modulation of membrane composition by diet may provide important translational insights and drug-free therapeutic strategies.

A primary goal in neuroscience is to understand how the nervous system interprets salient information from the external world and generate appropriate behaviors. Across the animal kingdom, animals respond to sensory cues emitted by conspecifics, preys or predators by initiating a repertoire of social behaviors, such as mating, fighting, prey capture, and predator avoidance. Although these behaviors are driven mainly by genetically pre-programmed instinctive circuits, they are also profoundly regulated by the animal’s internal states, suggesting an adaptive modulatory regulation of the underlying circuits. In rodents, the processing of chemical cues plays a crucial role in orchestrating essential instinctive social responses. The vomeronasal pathway, in particular, has been shown to generate sex-specific instinctive behavioral responses. Unraveling the neural mechanism underlying the processing of social cues through successive stages of the vomeronasal pathway will provide unique insights into the internal representation of social information in the brain. In primates, social behavior relies strongly upon visual and acoustic communication and, in contrast to rodents, depends minimal on chemical signaling. Studying social behaviors in primates will provide complementary insights into the neurobiology of social behaviors and emotional reactions more relate to that in humans. The marmoset, owing to its enriched social behaviors, is emerging as an ideal model for studying the neural substrates of primate social behaviors. In the long-term, we are interested in studying how the marmoset brain extracts social meaning from the environment and generate different social behaviors.

The gut microbiome plays a critical role in human health. For example, the commensal bacterium Akkermansia muciniphila is associated with health benefits against metabolic disorders and obesity. However, clear mechanistic understanding of host-microbe interactions is lacking due to the limitations of animal models and existing in vitro systems. In vivo measurements do not capture key physiological features such as bacterial colonization dynamics and spatial organization of mucosal biofilms. In addition, no method exists for reproducing human epithelial physiology for systematic visualization of the microbiota in vitro. I propose to address these challenges by developing a micro-physiological in vitro platform based on tube-shaped gut organoids generated from primary stem cells, and perform functional and dynamic studies of microbiota down to the single cell level. I will optimize the physicochemical conditions within these engineered mini-gut organoids to facilitate stable microbiota colonization to ultimately culture defined bacterial consortia and arbitrary human microbiota. Finally, I will investigate the molecular pathways of how A. muciniphila affects host physiology and pathogen infection using imaging, spatially-resolved transcriptomics and biophysical measurements. I expect that the mini-guts will fill a crucial technological gap in microbiome studies, contributing to our understanding of the principles and regulations of bacterial colonization and host-microbe interactions, and enabling potential future clinical intervention and manipulation.

Sense of balance is an equilibrium reflex that relies on feedback from sensory organs to perform body posture adjustments. Maintaining proper balance is vital for animal movement. However, the molecular and cellular mechanisms of the sensory inputs in the equilibrium reflex are not well understood. The Drosophila possesses a mechanosensitive balance organ, the haltere, which detects the angular velocity-dependent force (Coriolis force) generated when the body rotates during flight. The halteres provide sensory information about rotations in three dimensions to guide the adjustments of body positions, an attractive system to dissect the mechanism underlying sensory processing in the balance control. Taking advantage of the powerful genetic tools and behavioral assays in Drosophila, I aim to identify the molecular components essential for mechanotransduction in haltere and study their functional roles in the sense of balance. I will also genetically label subpopulations of sensory neurons in halteres and characterize their coding properties in response to body rotations in different dimensions. This study should provide a basis for a molecular and cellular dissection of how balance information is detected and encoded when animals move in three-dimensional space.

A major challenge of genomics has been to characterize all functional elements in the human noncoding genome, which generally lack appropriate methodologies to perturb them and measure the impact of the perturbation. Recently, CRISPR-based genome editing technologies opened new possibilities to probe noncoding regions of the genome. The easy programmability of CRISPR using short RNA guides enabled the development of high-throughput pooled CRISPR libraries for functional screens that can probe noncoding regions. However, the scale of these screens is not enough to target the entire human noncoding genome, which constitute more than 98% of the genome. Thus, a different approach is needed to engineer genome-scaled CRISPR screens over the entire noncoding genome.

We propose to develop an approach where entire chromosomes will be comprehensively screened for essential noncoding elements using tiled, overlapping deletions, generated by Cpf1 with a dual-guide delivery system. To pinpoint the genes and mechanisms through which the noncoding regions affect cell growth, we will develop a coupled deletion-single cell RNA sequencing approach which will link changes in gene expression with each deletion. This method will allow us to map essential noncoding elements at chromosome- or genome-scale and then, for these elements, map the genetic mechanisms they use to manipulate cell proliferation. Our whole genome functional noncoding screen is expected to identify many new regulatory genomic regions, with the long-term goal to create a truly genome-wide atlas of all noncoding elements that impact cell growth and to identify the genes that they modulate.

Vocal production control, i.e. the adjustments in the timing and the spectro-temporal features of vocalizations, is fundamental to acoustic communication, including human speech. Many species of bats demonstrate the capability for precise vocal production control, raising the intriguing possibility that this animal model can yield valuable insights to the mechanisms of human speech. In this project, I propose to study the neural circuits underlying echolocation call control in freely behaving bats (Hipposideros armiger), with an integrative approach which draws upon behavioral, neurophysiological, and modeling methods. At the subcortical level, we will make extracellular neural recordings from the midbrain inferior colliculus of the auditory pathway and the midbrain periaqueductal grey of the vocal-motor pathway. These experiments will be carried out using a pendulum setup which evokes dynamic adjustments in the bat’s call production features. At the cortical level, we will make extracellular neural recordings from the motor and prefrontal cortices when individual bats are trained to perform various vocal matching tasks from a hanging stand. For both behavioral setups, two types of the auditory feedback perturbations will be introduced to study the neural mechanisms for audio-vocal integration. The auditory feedback perturbations include a Lombard condition, in which the bat listens to signals embedded in white noise, and a frequency-shift condition, in which the bat listens to frequency-shifted copies of its echolocation calls. The proposed research will offer critical insights into the brain circuits mediating vocal production control in mammals, including humans.

Optical nanostructures are highly organized composites of materials with varying refractive indices (e.g. keratin, melanin and air) that produce some of the brightest colors found in nature through coherent light scattering. How these tissues organise themselves at the nanometer scale to produce colors is poorly understood, despite its fundamental significance to developmental and evolutionary biology and potential to spark advances in the biomimetic design and "green" commercial manufacture of self-assembling optical materials.
We thus propose to use both transcriptomic, laser diffraction and microscopy-based tools of developmental biology to elucidate the mechanisms by which these nanostructures self-assemble in a subsample of birds (Class Aves), a group with incredibly diverse structural colors and mechanisms. Our working hypothesis is that iridescent colors form through depletion-attraction, phase separation and other self-assembly mechanisms. Because most developmental biology is done at larger size scales, testing these hypotheses will require the use and development of methods such as wet cell TEM and in situ laser diffraction analysis to adequately resolve nanometer-scale changes in developing tissue. We will then test these proposed mechanisms using biomimetic approaches that replicate natural conditions as closely as possible (e.g. at room temperature,at biological pH) using natural or semi-natural materials. Use of optical techniques including angle-resolved spectrophotometry and microspectrophotometry will enable us to compare these properties between the natural and synthetic versions. This approach will enable us to not only experimentally test modes of development but also generate and test new materials and/or processes to produce them.
There are three highly innovative aspects to this proposal. First, it attempts to unlock the developmental pathways producing nanostructured tissues. This is a long-standing question with few answers thus far. Second, it uses biomimicry in novel ways to test developmental hypotheses and pushes the technical boundaries of developmental biology by focusing on nanometer-scale organisation of tissues. Finally, the use of biologically realistic chemistry in our biomimetic approaches is a huge leap forward in this field where most work is done at high temperature or with non-biocompatible materials. This work will therefore significantly advance both our fundamental understanding of these materials and the tools to study them and other nanoscale materials.

The recruitment of distinct neuronal populations to generate specific behavioral responses is a fundamental property of the central nervous system. However, understanding how defined neuronal sub-circuits encode different behavioral responses is still a major challenge of modern neuroscience. In this project I aim to elucidate how neuronal populations of the dorsomedial prefrontal cortex (dmPFC) encode the selection of active and passive fear responses. To achieve this goal, I will perform large-scale recordings of spiking and oscillatory activity in the dmPFC during conditions that will trigger either passive or active fear behavior, aiming to identify behavior-specific neuronal populations, assemblies and oscillations and the mechanisms mediating their interaction. Then, I aim to elucidate the projection patterns of the dmPFC neurons involved in active and passive fear and their targets in different brain regions. Finally, I will use optogenetics to manipulate specific neuronal populations in a phase specific manner aiming to reveal temporal encoding of fear behavior. Using these approaches, my goal is to demonstrate the role of specific coding mechanisms governing circuit selection during active and passive fear. This will largely expand our understanding of how particular neuronal circuits are selected based on environmental information and how specific fear coping strategies are coded. Anxiety disorders are a major health problem in western societies. It is estimated that 14% of the European adult population suffers anxiety disorders. Understanding the neural circuits involved in fear behavior will potentially lead to future therapies targeting anxiety disorders.

T cells are a type of lymphocyte that are able to recognize and destroy pathogens and tumor cells. The initial step in T cell activation is the binding of the major histocompatibility complex (MHC) presented antigen peptide to the T cell antigen receptor (TCR), which causes Lck mediated phosphorylation of its associated CD3 chains, including a CD3z/CD3z homodimer, a CD3g/CD3e heterodimer, and a CD3d/CD3e heterodimer. It is well established that CD3z/CD3z homodimer recruits ZAP70, a key cytosolic kinase, upon tyrosine phosphorylation. While much research has focused on CD3z, the roles of other CD3 chains are poorly understood. It is not clear why they are needed, what cytosolic proteins they recruit, and why they exist in a well-defined stoichiometry and geometry. To determine the role of the TCR architecture, I propose to assemble CD3 subunits on synthetic lipid bilayers with precisely controlled stoichiometry and inter-subunit distance using DNA nanotechnology. Using our recently developed fluorescence and microscopy readouts, I will dissect how the TCR architecture affects the recruitment of ZAP70 and other T cell signaling proteins. I will begin with a simple membrane reconstitution system to recapitulate each of the three types of CD3 dimerization, and determine their respective effect on signaling. I will then assemble the whole CD3 complex by precisely controlling the organization of CD3 dimers using a DNA scaffold. By exploring the potential geometry reported previously, this highly innovative approach may allow me to uncover the fundamental design principle of the TCR-CD3 complex and provide a mechanistic basis for designing better T cell-based immunotherapies.

Hox clusters are uniquely regulated, their collinear transcriptional patterns specifies an anterior-posterior axis of mammalian body plans reflecting their order on the genome. Spatio-temporal cues regulates the unique expression of these genes. Genetic disruption of the cluster uncovered multiple levels of cis-regulation. Two enhancer “archipelagos” in gene deserts surrounding the cluster control gene activation. Further, CTCF boundary elements separate the cluster in two TADs that target each archipelago to specific portions of the cluster.
Using transgenic constructs in drosophila embryos, it was shown that transcription occurs by series of bursts. The “static” view provided by classical approaches such as RNAseq or in situ hybridization limits our ability to dissect gene regulation. Thus, the specific contributions of each layer of gene regulation is still poorly understood.
Recently, part of the Duboule laboratory switched to the culture of "gastruloid" from mouse ES cells. These objects fully implement Hox collinear expression, providing a unique access to visualization of gastrulation-like patterning events that are normally obscured by implantation of the mammalian embryo.
I propose to analyse bursting using gastruloids and to follow Hox transcription through live imaging. First, we will assess how spatiotemporal cues regulate bursting at the cellular level. Then, we will dissect the functional consequences on bursting after disruption of the enhancers or loss of boundary elements.
This will provide us with a unique system to uncover mechanistic contributions of enhancer-promoter contact and boundary elements to the unique pattern of Hox gene expression.

Memory is a fundamental function in animals and humans. Memories can be classified into short-term memory that lasts for minutes to hours, and long-term memory that lasts for hours, days or even a lifetime. Different molecular mechanisms underlie these two types of memory: trafficking and modification of pre-existing proteins are sufficient for short-term memory, while synthesis of new proteins is required for long-term memory. Although the mechanism for short-term memory has been intensively studied, less is known about the mechanism for long-term memory. This is due to the lack of a good method to monitor and manipulate specific synthesized proteins in brain tissue. In this proposal, I aim to understand when, where and how protein synthesis mediates long-term memory in the mouse brain. Toward this aim, I will first develop a high-throughput method to image and control distinct synthesized proteins in brain tissue. For this, I will use my previously established genome editing methods (Cell 2016, Neuron 2017) as well as chemical labeling and optogenetic techniques. Using this method combined with two-photon imaging, electrophysiology and behavioral analysis, I will comprehensively understand the spatio-temporal dynamics and role of a number of newly synthesized proteins in long-lasting synaptic plasticity in vitro as a cellular model of long-term memory, and long-term memory in behaving mice in vivo. My proposed research will provide (1) a new platform to understand the molecular mechanism for various brain processes that depend on protein synthesis, and (2) new insight into the molecular mechanism for long-term memory from synaptic, cellular to systems levels.