Cells have evolved intricate systems to sense changes in internal and external conditions. These cues are relayed from sensors to actuators usually through post-translational modifications (PTMs) of proteins that allow cells to control cellular activities and generate an appropriate response. However, little is known about how these PTM regulatory networks change during evolution. In the past, comparative genomics has been crucial to study genome evolution and to identify functional DNA elements. In analogy to these studies, this project aims to advance our knowledge of cell signalling and evolution by performing a cross-species study of PTM regulation. Recent improvements in mass-spectrometry (MS) are unveiling a complex world of post-translational regulation with thousands of PTMs now routinely identified per study. PTMs have been shown to diverge quickly during evolution and it is not clear to what extent these changes have functional consequences. This rapid divergence underscores the importance of cross-species studies and urges the need to develop models of functionally relevant PTM regulatory networks. With this in mind, the main goals of this proposal are to: 1) experimentally determine PTMs for different species and predict the enzymes and binding domains that regulate them; 2) and develop and validate computational methods to predict the mechanistic functional role of PTMs. This work we will ultimately allow for a better understanding of how species adapt their regulatory networks during evolution and how genetic variability can generate diversity of PTM interactions that result in novel cell signaling responses.

Diabetes is a growing public health concern, with over 300 million type II diabetic patients worldwide. In the last decade, it has been shown that gastric bypass surgery leads to lasting diabetes remission in obese patients. Surprisingly, blood glucose level is normalized in a matter of days, before any significant weight loss. This clinical observation suggests that there are yet unknown mechanisms of diabetes remission evoked by the surgical procedure. Uncovering those mechanisms holds promise to identify new and less invasive treatments to diabetes, and gain new understanding on basic metabolic processes that come into play after this anatomical reconfiguration.
I will use a gastric bypass model in a diet induced obesity diabetic mice to comprehensively characterize of the dynamics of the metabolic shift following surgery. This will be carried by analyzing transcriptomics and histology data of the intestine, liver and pancreas, and metabolomics data of the portal vein connecting these tissues. As controls I will use sham operated obese and weight matched mice as well as healthy lean mice. The data will integrated to generate a wholistic view of metabolism, identify metabolic and signaling pathways key to diabetes remission and define the new metabolic state of the animal after surgery. In particular, I will study the changes in the endocrine of pancreas after surgery as a model for recovery from diabetes. Finally, I will use the gastric bypass model to study intestinal adaptation. This will be done by comparing gene expression and histology of the different intestinal limbs and using the intestinal crypt culture system.

Caveolae are cup-shaped plasma membrane invaginations and play a crucial role in mechanotransduction. Upon membrane stretch, caveolar integrity is lost and clustered lipids and proteins are released. Redistribution of the major caveolar component is implicated in the activation of multiple kinases, but the mechanism remains unclear. It is not known how caveolar disassembly is regulated and what determines the intensity and type of the stress response. Recent high-content imaging screens revealed that monoclonal cells display cell-to-cell variability in biochemical activities and cellular processes depending on their population context. I hypothesize that membrane tension and caveolae formation are heterogeneous in a cell population, and that mechanical stress results in different biochemical responses, undetectable by population-averaged studies. Thus, I will image caveolae formation and disassembly in large populations of cells and use computational image analysis to cluster individual cells into subpopulations with similar social context. I will monitor the stretch-mediated activation of diverse kinases with high spatiotemporal resolution at single cell level in a population context, and correlate their activation profiles with caveolar disassembly. Identified patterns might reveal novel regulatory networks of caveolar mechanotransduction, as well as mechanisms by which individual cells adapt caveolar integrity and dynamics to their social context. Together, this study will provide important insights into the underlying mechanisms of how cells adapt to membrane stress, and will advance our perception of the fundamental concept of population-determined heterogeneity.

Oxygen, that supports all aerobic life, is abundant in the atmosphere because of its constant regeneration by photosynthetic water oxidation by green plants, algae, and cyanobacteria. This reaction is catalyzed by a Mn4CaO5 cluster associated with photosystem II (PSII), a multi-subunit membrane protein complex. Given the role of PSII in maintaining life in the biosphere and the future vision of a renewable energy economy, understanding the mechanism of the photosynthetic water oxidation is considered to be one of science’s grand challenges. Although the structure of PSII and the chemistry at the catalytic site have been studied intensively, an understanding of the atomic-scale chemistry from light absorption to water-oxidation requires a new approach beyond the conventional steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure of the Mn4CaO5 cluster and PSII at ambient conditions, while overcoming the severe X-ray damage to the redox active catalytic center, is key for deriving the reaction mechanism. The intense and ultra-short femtosecond (fs) X-ray pulses of the LCLS (Linac Coherent Light Source) X-ray free electron laser provide an opportunity to overcome the current limitations of room temperature data collection for biological samples at regular X-ray sources. The fs X-ray pulses make it possible to acquire the signal before the sample is destroyed, which realizes the light-induced snap-shot study proposed here. The objective of this proposal is to study the protein structure and dynamics of PSII, as well as the chemical structure and dynamics of the Mn4CaO5 cluster (charge, spin, and covalency) during the light-driven process of PSII to elucidate the mechanism by which water is oxidized to dioxygen. We will design and apply a full suite of time-resolved X-ray diffraction and X-ray absorption/emission spectroscopy methods to follow the reaction at room temperature, that will provide an unprecedented combination of correlated data between the protein, the co-factors, and the Mn4CaO5 cluster, all of which are necessary for a complete understanding of photosynthetic water oxidation.

This project aims to artificially assemble a functional ‘switch complex’ of the bacterial flagellar motor (BFM) using DNA self-assembly to build a structural scaffold. The switch complex is a ~4 mega Dalton protein superstructure that converts a flux of ions across a membrane into mechanical rotation at speeds of up to 1700 Hz and consists of a reverse gear that switches rotation direction in a handful of milliseconds, making the BFM the most sophisticated nanoscale rotary motor known.
Building biology on a molecular scale is a fundamentally new approach to the Biological Sciences, which adopts a ‘top-down’ approach where the subcomponents are teased out of complete systems rather than pieced together from the bottom up. This suits the study of biological systems, which conveniently self assemble in vivo, but also limits our ability to manipulate systems to understand them. The BFM is one such example which can only be assembled and functionally characterised in vivo. As a result, elucidating a molecular picture of its structure and function is extremely difficult or impossible, and proposed molecular models remain controversial and not conclusively tested. However, the BFM may also present an important opportunity to study biology from the bottom-up. It is one of the best-characterised large protein complexes and decades of study have given us a blue print outlining the requirements for building the switch complex, which include a circular nanoscale structural scaffold. Recent advances in DNA self-assembly make it possible to build such a scaffold, allowing the programmed construction of nanoscale 3D objects at near-atomic precision.
We will design and build DNA nanostructures as structural scaffolds to artificially assemble the BFM switch complex ex vivo. By controlling and manipulating the assembly process, we will exploit remarkable new opportunities to address long-standing questions about the BFM and large, dynamic protein complexes in general, including the elucidation of a complete atomic-scale picture of this ~4 mega Dalton protein superstructure.

The epidermal growth factor receptor (EGFR), a prime target for cancer drugs, plays a key role in intercellular signal transduction. Activation of EGFR is modulated by several layers of regulation that is built into its structural components, a breakdown of which has been implicated in many cancers. It is therefore important to uncover the detailed molecular mechanism that regulates EGFR activity. Although the significance of the EGFR-plasma membrane interactions is realized, the molecular details of such interactions are poorly understood. The proposed project aims at gaining insights into receptor-membrane interactions and the effect of perturbation of such interactions on the activation of EGFR.
I will examine the effect of specific charge interactions, between the receptor and the plasma membrane, on the activation of EGFR. Quantitative response of EGFR to charge reversal mutations, leading to a breakdown of EGFR-membrane interactions, will be assayed in the cell based context. This will enable identification of a set of mutations that would lift the inhibitory interactions with the membrane and stimulate the full length receptor in the absence of ligand. Additionally, I will investigate the influence of membrane localization on the activity of EGFR. The other question aims to test the hypothesis that membrane attachment of the amphipathic helix in the juxtamembrane segments leads to EGFR autoinhibition. I will adopt experimental (single-cell microscopy assays) and theoretical (molecular dynamics simulations) approaches to address these questions.

This project aims to elucidate the principles of self-assembly of multiprotein complexes through statistical mechanics. Key questions are how self-assembly is constrained and how it can be optimized. Specifically, I aim to understand the observed necessity for "excess" numbers of copies of multiprotein complex constituents, and to examine the importance of cooperativity and nucleation barriers in self-assembly. I will initially focus on chemoreceptor arrays and flagellar motors in bacteria, two tractable and well-studied multiprotein complexes. My study will benefit from the long-term collaboration of Prof. Ned Wingreen at my host institution with the experimental group of Prof. Victor Sourjik at Heidelberg on chemotaxis. First, I plan to develop a coarse-grained model for extended multiprotein complexes, where self-assembly corresponds to a phase separation between a rich phase (e.g., rich in chemoreceptors) and a poor phase. I will study how the presence of a large variety and a large quantity of bystander proteins affects the efficiency and extent of phase separation. Second, I plan to investigate in more microscopic detail the interactions involved in self-assembly. Both direct protein-protein interactions and membrane-mediated interactions will be considered, with emphasis on their anisotropy, which yields specific assembly shapes. In particular, I will investigate the existence of an optimal strength of specific interactions, and I will study under what conditions a nucleation barrier exists, whether it is necessary for robust self-assembly, and how it constrains the range of possible self-assembled structures.

Photosynthesis is essential for all life on Earth and has enormous environmental and agricultural impacts. The most advanced photosynthetic machinery is that of higher-plant chloroplasts, where it is hosted by one of the most complex membranous systems found in cells. Notably, this massive and elaborate system forms essentially from scratch, commencing with undifferentiated plastids that contain no or very little photosynthetic proteins or internal membranes. Equally remarkable, upon aging of leaves, seasonal changes, or the plant’s transition to the reproductive phase, this system, along with other components of the chloroplast, breaks down and is recycled in a highly regulated process reminiscent of programmed cell death. In addition to being of fundamental scientific interest, these processes bear an immediate agricultural relevance as they have a direct impact on biomass production, plant longevity, flowering, fruiting and seed production. Despite their importance, the processes underlying the biogenesis and breakdown of the photosynthetic machinery in higher-plant chloroplasts are largely elusive. This is because their study necessitates an integrative approach that combines structural and biophysical studies with high-throughput transcriptomic, proteomic and metabolomic analyses, all of which must be performed along the entire developmental pathway of the plastids, from incipient chloroplast differentiation to senescence. Here we propose to undertake such an interdisciplinary approach to comprehensively characterize the buildup and disintegration of the photosynthetic machinery in the chloroplasts of dicots – the largest group of flowering plants, which includes many agriculturally important plant species as well as most trees. These studies, along with subsequent systems analysis and reverse genetic approaches, are expected to significantly advance our knowledge of the elementary processes that underlie differentiation and senescence of higher-plant chloroplasts and, in this way, the construction and dismantling of the most efficient energy conversion device that exists in nature. In addition, they will aid future in silico modeling approaches, as well as efforts to improve photosynthesis by breeding or genetic engineering.

This project takes a multidisciplinary quantitative approach to investigate the consequences of maximal growth rates. It draws on a range of cutting-edge approaches, from in vitro biochemistry to evolution and high-throughput characterization. It will also be guided by mathematics at every step. In addition to the insights into cell physiology, we expect to set a new world record in rapid growth of biomass.

Sophisticated models exist for the evolutionary pathways by which proteins have evolved in Nature over billions of years to form an impressive diversity of structure and to carry out many functions with unrivalled efficiency. Directed protein evolution in the test tube can emulate natural evolution, but is often limited by low hit rates and small improvements during evolution cycles. Burdened by their evolutionary history, proteins often show low evolvability, due to loss-of-function and/or structure or epistatic ratchets (ie deleterious or permissive mutations that only become beneficial upon occurrence of further mutations). Given the danger of getting lost or stuck in sequence space, the question arises how natural and forced evolution avoids dead ends. We will create a mathematical model to simulate the effects of mutational robustness on evolvability using parameters that can be adjusted to experimentally determined values for neutrality and accessibility of novel phenotypes. Based on experimental measurements of the dynamics of fitness landscapes of a protein population during evolution cycles we aim at insight into the fundamental as well as an effective basis for protein engineering by evolution. Investigations of duplicated specialized proteins (paralogs) and their ancestral precursors showed the latter often to be promiscuous (i.e. to have multiple functions) and more highly evolvable, suggesting that evolution may proceed by shifting the relative balance of two functions along paths of successive point mutations. We probe the evolution dynamics of promiscuous members of the metallo-ß-lactamase and alkaline phosphatase superfamilies. Ancestral sequences will be computed from alignments and databases as starting points for evolution to give a dynamic experimental picture of fitness of a protein population in response to the ancestor sequence and to selective pressure. Such distribution functions will be gathered by ultrahigh-throughput screening of >10^6 clones in picolitre droplet compartments during in vitro and in vivo evolution cycles, followed by systematic characterization of multiple fitness traits such as expression level, stability or reaction kinetics for multiple activities. These approaches will enable a dynamic picture of protein evolution at various levels from transition state recognition, protein biophysics to population genetics.

Our goal is to study physiologically important components within the voltage-gated sodium (Nav) channel signaling complex that may cause disorders when mutated, and to resolve their mechanisms of action at the molecular level. In particular, we will focus on vital members of the Nav channel signaling complex that do not contribute to the ion-conducting pore. Reflecting their medical importance, mutations found in several of these proteins have been implicated in various epilepsy syndromes, long-QT syndrome (LQTS), LQTS-associated Sudden Infant Death Syndrome, and Brugada syndrome, possibly through dysregulation of the Nav channel signaling complex in the brain and the heart, respectively. Little is known about the underlying machinery that governs the influence of these elements and their mutants on the functional properties of Nav channels. To elucidate the mechanisms by which these proteins influence Nav channel function, we will combine innovative biophysical techniques with molecular biology and animal toxin-based pharmacology, state-of-the-art biochemical approaches, mouse genetics, and X-ray crystallography. Successful completion of our aims will help define the function of the larger Nav channel signaling complex as found in humans, which is essential for developing new strategies that can correct for abnormal behaviors.

We address a key question in developmental biology: how does an organism reach its final size and shape, in the face of stochastic variation at the cellular level? Previous research has focused on mutants and conditions that affect the global size and shape of organs, enabling the discovery of a large number of regulators. However, most analyses have considered only average cell behaviors, overlooking local heterogeneity and stochastic variation. In this context, how the final size and shape of an organism are determined remains poorly understood.
Here we adopt an orthogonal strategy by taking advantage of the observation that many organisms have a remarkably consistent shape, yet at the cellular level, cell growth and shape can be highly variable. Our goal is to resolve this apparent contradiction between the cellular and organismal levels. We propose that cell-cell communication mechanisms coordinate cell stochastic variability so as to yield consistent organs. Since growth remains locally heterogeneous, these mechanisms may, counter-intuitively, maintain or even enhance local heterogeneity.
We will test this hypothesis in Arabidopsis thaliana, as it produces a large number of almost identical flowers with stereotyped floral organs. We choose to work on the sepal, which is accessible for live imaging and mechanical measurements. Using a combination of experimental and theoretical approaches, we will identify mutants with variable sepal size and shape. Because of their role in coordination, the corresponding genes are likely to include the hormonal/nutrient pathways and mechanical sensing and response. As plant growth is driven by the internal turgor pressure and restrained by cell walls, we will assess the activity of these growth regulators by quantifying the mechanics of cells in these mutants and in wild type to causally link gene activity and shape. We will use data-driven approach and information theory to extract the underlying patterns of correlations between cells, notably to qualify the most plausible coordinating mechanisms that regulate cell stochasticity. These hypotheses will be tested by combining a mechanical model of sepal growth that incorporates identified signals and local experimental perturbations, such as cell ablation, force application, and genetic mosaics.

Alternative splicing of pre-mRNAs allows the synthesis of multiple gene products from a single gene and therefore dramatically expands the eukaryotic proteome, especially in mammals. The task of splicing is achieved by the spliceosome, a highly complex and dynamic macromolecular machinery. The molecular mechanism of alternative splicing has been studied intensively, however a lack of information on kinetic data has hampered a comprehensive understanding of this dynamic process and its regulation.
Here I propose to study the kinetics of human spliceosome assembly and their regulation, which are the key to deepening our understanding of the molecular mechanism of alternative splicing. I specifically plan to establish colocalization single molecule spectroscopy (CoSMoS) in human nuclear extracts to follow single splicing events. CoSMoS will enable me to follow the comings and goings of components of individual spliceosomes in real time. I will also apply this technique to study how and at which steps spliceosome assembly is regulated during alternative splice site selection. For this, I will use a combination of CoSMoS and single molecule fluorescence resonance energy transfer (smFRET) to follow spliceosome assembly simultaneously on the different splice sites of an alternatively spliced pre-mRNA. Taken together, my project will yield insights into spliceosome assembly in mammals and the mechanism of alternative splicing.

The fundamental physical processes determining microscale transport of constituents to and from a coral’s ciliated surface are largely uncharted and their exploration will strongly benefit from an interdisciplinary effort, integrating quantitative modeling of transport and accurate microscale observations. I propose to focus on the origin, characterization and consequences of the ubiquitous ciliary flows recently discovered in my future host lab at MIT. I will undertake the first quantitative study of such flows, using digital holographic microscopy to acquire 3D, time-dependent data with micrometer precision. This will facilitate tracking bacterial pathogens as they target their coral host, yielding unprecedented access to investigate the physical mechanisms responsible for the emergence of the collective ciliary motion, the spatiotemporal nature of these flows, and their effect on mass transport and bacterial motility. I will integrate this experimental effort with the development of a mathematical model that incorporates ciliary flows into the prediction of mass transport and bacterial recruitment to the coral surface. Modeling and observation will be synergistic, providing an ideal approach for hypothesis testing. Specifically, I will determine the circumstances (e.g., times of day; regions on the coral surface; external flow conditions) in which the coral pathogen Vibrio coralliilyticus is most favored in locating and reaching the surface of its coral host, Pocillopora damicornis. This will provide valuable insight into the mechanistic processes underpinning coral health, and its generality will facilitate applicability to a wide range of cilia-driven flows.

Except for the nucleus, mitochondria are the only organelles in our cells that contain their own genome. Acquired genomic aberrations in mtDNA lead to mitochondrial dysfunction, a chief cause of neurological and aging diseases. mtDNA mutations, ranging from single-base substitutions to large-scale deletions, are also found in high frequency in many tumors, and recent experiments have established their role in driving metastasis. While the underlying basis for mtDNA deletions is unknown, two scenarios can explain their formation – infidelity of DNA replication machinery or errors in DNA double-strand break (DSB) repair.
The overall goal of our proposed study is to elucidate the molecular mechanism of mtDNA instability by deciphering both facets of mtDNA metabolism – replication and repair – and test if their deregulation causes deletions in the mitochondrial genome. Then, we will directly study the impact of the most common mtDNA deletion on cancer progression.
We will combine a single-molecule DNA combing approach with super-resolution microscopy to unravel the replication profile of mtDNA and highlight specific regions that might stall replication forks, causing replicative slippage and subsequent sequence loss. Biophysical analysis will be carried out to emphasize the structural nature of these deletion-prone regions. Using these molecular and biophysical tools, we will then address the genetic basis for misregulation of mtDNA replication in cancer cells.
The prevalence of short repetitive sequences at the junction of mtDNA deletions prompted us to ask if they are due the activity of alt-NHEJ, a recently identified pathway of DNA DSB repair that is predominantly driven by sequence-homology and often associated with significant sequence loss. To address this question we plan to manipulate the mitochondrial genome using TALE-technology (Transcription Activator-Like effector) both to incorporate reporter plasmids into its DNA and induce targeted DSBs. This innovative approach will allow us to investigate the DSB repair pathway(s) operating in the mitochondria and test if alt-NHEJ is the predominant pathway in deletion-bearing cells. Once the basis of the deletions is underscored, we will recapitulate the most common mtDNA deletion in human cells by coordinated cleavage with two TALE-nuclease and directly assess its contribution to tumor progression.
Despite the profound impact of aberrant mtDNA on cellular function, mitochondrial genomic instability remains largely an uncharted territory of cancer research. Our proposed study surpasses the abundance of descriptive literature to firmly establish the role of deletions in tumor progression. More importantly, it will highlight the underlying mechanism behind their formation, a critical step towards predicting and preventing their accumulation in cancer cells.

Cohesin complexes are required for holding together sister chromatids during DNA replication, repair of doubled-strand breaks, and chromosome segregation in cycling cells. More recently it has been shown that cohesin contributes to the control of gene expression in non-dividing cells. In mammalian cells, the study of the specific effects of cohesin deficiency in transcriptional control, independently of its role in the cell cycle, requires an approach which would allow temporally-controlled cohesin depletion and restoration in non-cycling cells. We propose the use of a proteolytically-degradable cohesin subunit, in combination with regulated TEV (tobacco etch virus) protease activity, Cre transgenes and conditional cohesin alleles to investigate: 1) if cohesin is required for the regulated expression of pluripotency genes in mouse embryonic stem cells, independently of its essential role in the cell cycle; 2) the developmental potential of T cells generated from non-cycling precursor thymocytes in the absence of cohesin; and 3) whether functional defects that arise in cohesin-depleted post-mitotic neurons can be reversed by restoring cohesin expression. In humans, cohesin deficiency causes Cornelia de Lange Syndrome, which is a rare multiple malformation disorder distinguished by growth and cognitive retardation, and abnormalities of several tissues and organs. Our study will help to gain insight into the molecular pathology of this disorder, which could lead to novel approaches for its management. Additionally, it should reveal unique aspects of cohesin function in gene regulation and development.

The contractile cortex is a thin actomyosin network that subtends the membrane of animal cells. The cortex is the main determinant of animal cell shape. As such, it plays a key role in processes such as cell division, where morphogenesis is driven by cortex remodelling in response to cell-cycle dependent signalling. In spite of its physiological importance, our knowledge of the cortex remains poor. In the first grant, we have established a multidisciplinary collaboration that allowed us to uncovered the proteic composition of the cortex and identify the two key proteins that nucleate actin in the cortex: the Arp2/3 complex and the formin Diaph1. This led to the proposal that the cortex is a composite network, which mechanics can be fine-tuned by regulating the relative contribution of the two main nucleators.
The aim of this project is to build on these findings to investigate the control of cortex mechanics during the cell cycle. Taking a mutiscale approach, we will explore how changes in the activity and abundance of cortical proteins during the cell cycle modulate the network architecture of the cortex, and how this, in turn, gives rise to whole cell mechanics. Building on the first grant, we propose to systematically examine how cell-cycle dependent signalling influences cortex composition and cortical protein activity, and ultimately cortex mechanical properties.
As the cortex is an intrinsically mechanical structure, its biological properties cannot be understood in isolation from its mechanics. In particular, our preliminary results indicate that the dramatic cell shape changes occurring during the cell cycle are driven by changes in cortical mechanics. Cell cycle-dependent modulation of the activity of actin nucleators provides a potential mechanism. Therefore, having identified the cortical actin nucleators, we will determine how changes in their activity affect cortex architecture and mechanics. Coupled biochemical and proteomic studies will search for signalling pathways altering nucleator activity. In vitro studies will examine how actin networks assembled by different nucleators interact and if tension can modulate nucleation activity. Electron microscopy and subdiffraction imaging techniques will enable us to characterise cortex architecture, while Atomic Force Microscopy will be used to characterise cortex mechanics. Furthermore, we will use proteomics to identify proteins whose abundance and activity vary most during the cell cycle. Using these results, we will determine key cortex regulators by systematically depleting/overactivating each in turn and measuring the resulting changes in cortex architecture and mechanics. Our integrated interdisciplinary approach will allow us to uncover how signalling regulates cell mechanics through modulating cortex architecture.

One of the most important unresolved questions in neuroscience is how memories are encoded and stored in the brain. Memory is a multi-step process, in which the acquisition phase begins as the animal forms a new memory. The nascent memory is then converted to a stable state through a process called consolidation. Later on, when the hardwired memory is reactivated, it transiently returns to a labile state and requires a second process of reconsolidation in order to persist. Therefore, retrieving a memory not only reactivates it, but also subjects it to further modifications such as extinction or formation of new memories. These intriguing observations give rise to questions regarding how neural circuits operate during these events. First, which neurons are part of the discrete network that is involved in encoding a given memory. Secondly, when the memory is retrieved, are the same neurons involved in the original training get reactivated? To address these questions, I will perform in vivo two-photon calcium imaging, using a genetically encoded calcium indicator, GCaMP5, in head-fixed mice during the performance of an auditory fear-conditioning task. Calcium imaging of hundreds of neurons simultaneously allows identification of precise neurons that are involved in encoding the given fear memory. In addition, I will track the same population of neurons over weeks to examine whether the same ensemble of neurons is being activated during memory retrieval. This research will close the gap between behavioral studies and underlying network plasticity, providing answers to some of the most fundamental questions of systems neurobiology.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with limited treatment options and dismal prognosis, urging the need to develop novel therapeutic strategies. Recent work from our group suggests that regulation of oxidative stress is crucial for pancreatic cancer progression, with Nrf2, a master transcriptional activator of cytoprotective genes, playing a central role. We have shown in an earlier publication that Nrf2 transcription is directly induced by oncogenic Kras to decrease levels of intracellular reactive oxygen species. The goal of this project is to delineate the molecular mechanisms by which Nrf2 promotes pancreatic cancer. We propose to generate mouse models in which the pattern and the level of Nrf2 expression can be manipulated in vivo, permitting evaluation of not only cell intrinsic versus non-cell autonomous functions of Nrf2, but also the dose and duration of activation that is relevant for disease progression. Complementary to this approach, we also propose to biochemically interrogate the molecular mechanisms by which Nrf2 promotes pancreatic cancer. This will involve identifying the most prominent Nrf2 transcriptional target genes in PDAC by performing ChIP-Seq on freshly isolated murine and human tumors. In addition, we also aim to identify proteins that carry out effector functions of Nrf2 by characterizing the global cysteine proteome of PDAC tumors. The less oxidized cytoplasmic environment following Nrf2 activation will reduce thiol groups on cysteine containing proteins, thereby activating such molecules, which will be likely candidates to carry out the effector functions of Nrf2 to promote pancreatic cancer.

Arabidopsis lateral root development is an excellent system to understand spatio-temporal regulation of stem cell fate. Oscillating-auxin-dependent priming predetermines stem cell fate of pericycle cells, although these primed cells initiate cell division only when they reach elongation zone of roots. Currently, it is unknown why this early marking of pericycle cell fate is required. Here, I propose that this early marking secures the transcriptional competence of primordium-specific genes against assembly of repressive chromatin marker. To confirm this idea, specific labeling, tracing, and isolation of primed pericycle cells as they develop into lateral root primordia for comparative analysis of epigenome and transcriptome is required. Currently, there are no primed-pericycle specific marker genes. By integrating microarray data, I will isolate marker genes that are expressed at early pericycle in phase with oscillating priming signal (auxin). Activation of ubiquitous GFP expression by primed-pericycle specific inducible recombinase allows tracing of recombinant pericycle cells. Following enrichment of recombinant cells by FACS at priming, founder cell, and primordium initiation stage, integration of 1) transcriptome, 2) histone 3 lysine 27 trimethylation (H3K27me3) and H3K4me3, 3) DNA methylation, and 4) H2A.Z profile in these cells would identify early epigenetic markers for the transcriptional competence of primordium-specific genes. This project should expend our knowledge on the epigenetic regulation of transcription competence during stem cell development that aid efficient engineering of stem cells.