Rockefeller University

NIH Research Projects · FY 2024 · 2024-09

Project Summary The recent completion of a Telomere-to-Telomere (T2T) human genome has demonstrated that, in principle, existing sequencing technologies allow gapless and nearly error-free, assembly of complex, human-sized genomes. Despite these technological advancements, genome assemblies that are currently being generated and released in public archives are still incomplete and contain a significant number of errors, which can dramatically impact downstream analyses. Algorithms that can generate T2T genomes are still in their infancy. The few that are available require extensive manual validation and curation and have so far worked on only a handful of model species. Dedicated algorithms and software tools are essential for achieving T2T assembly completeness and accuracy in all species. In particular, extensive evaluation and sophisticated manipulation of genome assembly graphs are required for T2T genome assembly. To this end, an efficient tool suite is missing. To bridge this gap, gfastar, a suite of algorithms and tools created for the evaluation and manipulation of assembly graphs will be further advanced and continuously maintained. Gfastar is under active development, and it is currently used by large-scale initiatives aimed at the generation of high-quality reference genomes such as the Vertebrate Genomes Project. Gfastar is powered by a dedicated C++ library, gfalibs. Gfalibs will be expanded to provide a comprehensive library dedicated to genome sequences and assembly graphs that can support multiple file formats commonly used by the genome assembly community (e.g. FASTA, FASTQ, GFA1/2, AGP, GAF, BAM, and FASTG), parallelized input/output (I/O) processing and many other general purpose functions and utilities. This library will be extensively used by the whole gfastar software ecosystem (rdeval, gfastats, gfalign, kcount, kreeq, teloscope, and gfase). Currently, several modules have already been implemented in gfastar. These existing modules will be expanded with additional functionalities and new tools will be developed. All these tools will synergistically contribute to the generation of T2T reference genomes at scale. As a whole, the gfastar tool suite will provide unparallelled algorithms and functionalities for assembly graph evaluation, manipulation and analysis, significantly supporting the genomic community by helping improve the completeness and accuracy of genomes.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Nutrient homeostasis in most living cells is mediated by membrane carrier proteins, which facilitate the translocation of small molecule metabolites across cellular membranes. Despite their clear roles in physiology and disease, many of the small molecule carriers in mammals are poorly studied owing to their hydrophobicity. Indeed, approximately 30% of these carriers still do not have known substrates or physiological functions. Among small molecule metabolites, choline is a vitamin-like metabolite that is indispensable for cellular and organismal viability. Choline is a dietary component that is critical for the structural integrity of cell membranes, one carbon metabolism, signaling, cholinergic neurotransmission, and lipid and cholesterol transport and metabolism. Most human cells need to import choline from their extracellular environment. Since serum choline concentration is ~10µM in mammals, choline uptake should occur almost exclusively through high affinity plasma membrane transporters. However, the identity of the high affinity choline transporter ubiquitously expressed across mammalian tissues remains to be discovered. To address this, in our preliminary work, we used a genome-wide association study (GWAS) of plasma metabolites from a cohort of Finnish individuals and linked biochemical pathways to uncharacterized membrane transporter genes. This analysis identified a ubiquitously expressed plasma membrane transporter, feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), as a genetic determinant of phosphocholine and phosphatidylcholine levels in human plasma. Biochemical characterization of cells lacking FLVCR1 revealed striking defects in choline metabolism. Additionally, FLVCR1 loss impairs proliferation of cells under choline limitation. Building upon this observation, in this proposal, we will test the hypothesis that FLVCR1 and its close paralog FLVCR2 are required for choline transport and homeostasis in mammals. To address this, we will first investigate how loss of FLVCR1-mediated choline import impacts mammalian cell metabolism and physiology. In the second aim, we propose to enhance our understanding of how FLVCR1 and FLVCR2 facilitate choline transport using biochemical and structural studies. Specifically, we will determine structures of FLVCR1/2 in a ligand-free condition to visualize the conformational changes associated with ligand-binding and release and use mutagenesis to probe the role of residues that directly coordinate choline and those associated with disease. Finally, we will determine the role of FLVCR1-mediated choline import in tissue physiology. In particular, given the role of choline in liver metabolism, we will focus on the impact of FLVCR1 loss in liver metabolism and non- alcoholic fatty liver disease (NAFLD).

NIH Research Projects · FY 2024 · 2024-09

Obesity directly contributes to comorbid diseases which account for the leading causes of morbidity and mortality in much of the world. Although functional brown adipose tissue (BAT) was only recently shown to be present in adult humans, studies in animal models and humans over the past fifteen years have shown that BAT can convey remarkable protection against obesity, type 2 diabetes, and associated disorders. However, we still have a very limited understanding of how BAT function in humans is regulated, and its genetic underpinnings have yet to be studied. At present there is no reliable biomarker or widely accessible diagnostic test to measure BAT function, necessitating creative approaches to study its genetic regulation. This proposal employs innovative strategies to leverage data from unique human cohorts to discover genetic variants explaining BAT function and linking BAT to metabolic traits and susceptibility to disease. We will utilize two complementary approaches: (1) Identify functional genetic variants in individuals with exceptionally high BAT activity and then cross-reference candidates with data from large human cohorts to ascertain whether these variants are associated with cardiometabolic phenotypes, and (2) Identify predicted functional variants in genes important for BAT function and perform phenome wide association study (PheWAS) and quantitative trait analyses focused on cardiometabolic traits. High confidence candidate variants identified through these approaches will then be investigated functionally in human brown adipocytes. This bedside-bench approach has the potential to provide a fundamentally new understanding of human BAT biology, a valuable new resource for the field, and to identify new mechanism-based targets for obesity, type 2 diabetes, and other metabolic diseases.

NIH Research Projects · FY 2026 · 2024-09

SUMMARY Laser scanning microscopy (LSM) – including optical coherence tomography (OCT), confocal and multiphoton microscopy (MPM) – allows imaging thick, living tissue at depth, and thus has been widely adopted for many biological contexts. To facilitate further research and discovery, increase in the total information capacity with larger fields-of-view, faster imaging, or deeper penetration is desired, but is ultimately limited by tissue heating. This project will employ distributed amplification in optical fibers – a technique pioneered and honed for telecommunications – in order to bypass this limitation. Two fiber-based amplifier and detection systems (F- BADS) will be constructed leveraging both the sensitivity of optical amplification, and the bandwidth and dynamic range of photodiode detectors in order to increase the signal-to-noise ratio (SNR) and acquisition speed of LSM by ~100-fold relative to conventional detection modalities. The first F-BADS will employ Raman amplification in multi-mode fibers to provide gain in the visible spectrum. The module will be installed into an existing two-photon microscope and characterize the benefits in SNR and speed afforded by this technique relative to photo- multiplier-tube-based detection. The second F-BADS will use four-wave mixing in single-mode fiber (SMF) to provide gain in the 13XX tissue transparency window. The SMF-based design of this amplifier will facilitate alignment-free integration into a fiber-based reflectance microscope. SNR, speed, and penetration depth achievable with this method will be characterized for both confocal microscopy and OCT. Both proposed F-BADS are drop-in compatible as add-on modules for any microscope, and can amplify spatially coherent or incoherent light across the visible and near-infrared spectrum, including broadband fluorescent signals. Therefore, these proof-of-principle demonstrations of the utility of nonlinear fiber optical amplification will encourage wide adoption for a broad range of optical imaging applications, as well as facilitate further scaling of LSM information capacity.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY The skin represents the first and outermost body’s line of defense, continuously dealing with potentially harmful environmental and physical stressors. To protect internal tissues, skin epithelial stem cells (SC) must exert diverse functions and form a barrier by proliferating, differentiating, secreting lipids and anti-microbial molecules. To allow for such a diverse functional output and specialization, SC rely on a high degree of heterogeneity within the skin. While this is mostly dictated at a developmental and anatomical level (such as the differences between the interfollicular and the hair follicle compartments), a considerable level of heterogeneity persists within the same compartment, as observed by recent single-cell RNA sequencing and clonal proliferation studies. However, how is this heterogeneity distributed across the tissue and what drives it remains unexplored. More importantly, what is the advantage and the consequence of establishing SC heterogeneity across the tissue remains an open question. In my preliminary studies, I observed that immune cells (and more specifically dendritic epidermal T cells, or DETC) in the skin fine tune and tailor their effector functions depending on the stem cells they are directly contacting (i.e. hair follicle of interfollicular epidermal SC). These findings provide a proof of concept that functional heterogeneity might arise from immune-SC crosstalk in the tissue. Therefore, here I plan to investigate whether the immune niche can drive SC heterogeneity within one SC unit (using the hair follicle as a model) and among distinct units across the tissue. More in detail, I aim to decipher: 1) how direct contact with DETC (which are uniformly distributed in every upper hair follicle) mediates functional SC specification within the hair follicle (Aim I); and 2) how proximity to lymphoid clusters (which are sparsely distributed across the skin and resemble tertiary lymphoid structures) dictates spatial and functional SC heterogeneity among different hair follicles across the tissue (Aim II). The results generated in this proposal will: 1) shed a new light in our understanding of SC-immune crosstalk in the skin and provide a novel perspective on the role of the immune system in the tissue and of SC heterogeneity; 2) allow to decode new molecular laws governing tissue homeostasis, which will be critical to comprehend what goes awry during disease; 3) establish a new paradigm and generate a toolkit to study cellular crosstalk within tissue microenvironments. Under the supervision of the world-renowned expert in stem cell biology, Dr. Elaine Fuchs, the co-mentorship of a leading immunologist and pioneer in genetic tools development for the study of cellular interaction, Dr. Gabriel Victora, I am ideally positioned to develop my technical skills, knowledge, and training. With my research background, training and career development, I will be able to establish a unique niche at the intersection of immunology and stem cell biology as an independent investigator.

NSF Awards · FY 2024 · 2024-08

The National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) is a highly competitive, federal fellowship program. GRFP helps ensure the vitality and diversity of the scientific and engineering workforce of the United States. The program recognizes and supports outstanding graduate students who are pursuing research-based master's and doctoral degrees in science, technology, engineering, and mathematics (STEM) and in STEM education. The GRFP provides three years of financial support for the graduate education of individuals who have demonstrated their potential for significant research achievements in STEM and STEM education. This award supports the NSF Graduate Fellows pursuing graduate education at this GRFP institution. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Choline is a vitamin-like metabolite that is indispensable for cellular and organismal viability which must be obtained through diet. Once imported into the cell, choline has multiple metabolic fates influencing diverse cellular processes ranging from membrane biosynthesis to epigenetics. Choline it is a key constituent of phospholipids, acetylcholine, and betaine which in turn impacts S-adenosylmethionine (SAM) and DNA methylation. Despite the influence of choline on diverse cellular processes, the identity of a high affinity choline transporter ubiquitously expressed across mammalian tissues was unknown. In our recently published preliminary data, we utilized genome-wide association studies (GWAS) of serum metabolites to identify a poorly characterized plasma membrane protein, feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), as the predominant choline transporter in mammals. In human cells and the developing mouse embryo, FLVCR1 loss severely impacts choline metabolism resulting in depletion of betaine and phosphatidylcholine (PC) – the predominant phospholipid species in cellular and organellar membranes. Mechanistically, FLVCR1 directly transports choline into cells and we have recently used CryoEM to identify the residue necessary for transport. In this effort, we also discovered that FLVCR1 can also transport ethanolamine, suggesting that it may also affect phosphatidylethanolamine (PE) synthesis, the second most abundant membrane phospholipid. Taken together, these data suggest that FLVCR1 is a crucial transporter for phospholipid metabolism. Broadly, this proposal seeks to investigate the influence of phospholipid metabolism on cellular and organismal physiology. Utilizing a conditional knockout mouse, in Aim 1, we will study the role of FLVCR1 in organismal physiology and metabolism and assess the efficacy of FLVCR1 as a target in metabolic disease. Our preliminary data suggest that mitochondrial stress and activation of the integrated stress response are defining features of cells and embryos lacking FLVCR1. In Aim 2 we will study how FLVCR1 loss and phospholipid metabolism impacts mitochondrial function and the subsequent cellular stress response. In Aim 3 we seek to understand how phospholipid homeostasis is maintained and regulated. Spanning basic biochemistry to mouse modeling, this application will address outstanding fundamental questions in cellular metabolism and seek to apply these findings to the possible treatment of human disease. The innovative studies proposed in this application in addition to the personalized training plan, will provide rigorous scientific training and professional development which will enable my transition to independence and start my own laboratory as a tenure-track professor.

NIH Research Projects · FY 2026 · 2024-08

Abstract Working memory is a form of short-term memory that is required for most daily activities, from reading, to driving, to conversing. As such, deficits in working memory are highly disabling and are prominent in learning disability, ADHD, and in Schizophrenia where it is the most important predictor of long-term prognosis. Currently, no treatments exist to improve working memory. Pioneering studies in humans and animal models established a central role for the prefrontal cortex in enabling working memory. Emerging work from our lab and others demonstrate important complimentary roles of thalamus in shaping thalamo-cortical synchrony underlying working memory. An important next step is to understand the molecular programs in thalamus that sustain such long-range thalamo-cortical neural activity on the time-scale of seconds to tens of seconds during working memory. This will provide new mechanisms for how memory is enabled on short time-scales, and also offer therapeutic targets for reversing working memory deficits. Towards this, we recently identified a novel orphan receptor that functions in thalamus as a potent modifier of short-term memory. In this proposal, we will delineate the chronic (steady-state) transcriptional and gene-regulatory programs downstream of Gpr12 that promote and maintain working memory. We will also identify acute (activity-dependent) recruitment of Gpr12 downstream signaling during working memory behavior. Finally, we will link Gpr12 molecular function with its role in maintaining sustained and synchronized thalamo-cortical activity during working memory. The overarching goal of this work is to provide new insight into molecular programs in thalamus that shape thalamo-cortical activity underlying working memory. Such discoveries will inform how our brains enable us to maintain task-relevant information on short time-scales. More broadly, this study will help work towards novel therapeutic strategies for reversing working memory deficits associated with schizophrenia, and related mental illnesses.

NIH Research Projects · FY 2025 · 2024-08

Abstract Working memory is a form of short-term memory that is required for most daily activities, from reading, to driving, to conversing. As such, deficits in working memory are highly disabling and are prominent in learning disability, ADHD, and in Schizophrenia where it is the most important predictor of long-term prognosis. Currently, no treatments exist to improve working memory. Pioneering studies in humans and animal models established a central role for the prefrontal cortex in enabling working memory. Emerging work from our lab and others demonstrate important complimentary roles of thalamus in shaping thalamo-cortical synchrony underlying working memory. An important next step is to understand the molecular programs in thalamus that sustain such long-range thalamo-cortical neural activity on the time-scale of seconds to tens of seconds during working memory. This will provide new mechanisms for how memory is enabled on short time-scales, and also offer therapeutic targets for reversing working memory deficits. Towards this, we recently identified a novel orphan receptor that functions in thalamus as a potent modifier of short-term memory. In this proposal, we will delineate the chronic (steady-state) transcriptional and gene-regulatory programs downstream of Gpr12 that promote and maintain working memory. We will also identify acute (activity-dependent) recruitment of Gpr12 downstream signaling during working memory behavior. Finally, we will link Gpr12 molecular function with its role in maintaining sustained and synchronized thalamo-cortical activity during working memory. The overarching goal of this work is to provide new insight into molecular programs in thalamus that shape thalamo-cortical activity underlying working memory. Such discoveries will inform how our brains enable us to maintain task-relevant information on short time-scales. More broadly, this study will help work towards novel therapeutic strategies for reversing working memory deficits associated with schizophrenia, and related mental illnesses.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY Germinal centers (GCs) are the site of affinity maturation, a prototypical Darwinian process that is required to generate the potent antibodies that protect against infectious disease. Over the past decades, work by several groups, including our own, has led to a comprehensive general understanding of the cellular and molecular mechanisms that drive evolution in the GC. However, this mechanistic understanding has yet to reach sufficient granularity to allow for accurate prediction of the outcomes of GC evolution and efficient guidance of GC B cells along predetermined mutational trajectories. Several population-level phenomena observed in GCs remain poorly understood, including the apparent stochasticity of “clonal bursts,” proliferative sprees in which the entire GC is taken over by the descendants of a single cell in a matter of a few days, the continuous presence of B cells with low affinities in GCs even at late stages of the reaction, and the apparent inability of GCs to find certain somatic mutations despite their benefit in terms of affinity. Greater understanding of these apparently aberrant evolutionary pathways may improve our ability to predict and potentially control the output of the GC reaction. In this application, we develop a full toolset consisting of a mouse monoclonal for Ig genes encoding an antibody to a classic protein antigen, a deep mutational scan (DMS) experiment in which the effects on affinity of every possible mutation in both chains of this Ig are measured experimentally, and a computational framework that allows us to assign affinities to any Ig sequence that we recover from these monoclonal GC B cells. We will use these tools to conduct replicated evolution experiments on hundreds of GCs, reconstructing the evolutionary paths and affinities of thousands of B cells in the course of GC maturation. We propose to use this approach to gain insight into how B cell evolution plays out within GCs in vivo, focusing on the interplay between reproducibility and stochasticity in evolution. Understanding such evolutionary aspects will be important for the field’s efforts to guide B cell clones through defined affinity maturation trajectories through vaccination.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Chronic hepatitis B virus (HBV) infection remains an enduring global public health challenge, affecting approximately 300 million individuals, despite prophylactic vaccines. The current FDA-approved anti-HBV drugs suppress viral replication but are unable to eliminate the viral covalently closed circular DNA (cccDNA) genome within infected hepatocytes. Restoring the immune response holds promise as a potential avenue toward a cure; however, T-cell exhaustion commonly observed in chronic HBV-infected patients impedes effective viral clearance. Regrettably, progress towards curative treatments has been stymied by the scarcity of appropriate immunocompetent animal models susceptible to HBV. HBV exhibits high species specificity, infecting only humans and chimpanzees. Mice, widely used for modeling human diseases due to their well-characterized immune system, high reproductive capability, and short gestation period, are not naturally susceptible to HBV infection, even after expressing human sodium taurocholate co-transporting polypeptide, the HBV entry receptor. The primary obstacle to HBV infection in murine hepatocytes is the inability to establish cccDNA, which is essential for HBV infection and persistence. Although HBV can infect mice xenotransplanted with human hepatocytes, these models are immunodeficient. Humanized mice engrafted with both human hepatocytes and human immune cells could surmount this deficiency, but their immune response tends to be weak and constrained. In this study, we aim to identify the host and viral determinants capable of breaching the species barrier for HBV infection in murine hepatocytes and thereby develop HBV-susceptible mouse models. In Aim 1, we will take a host-centric approach to determine if alterations in the murine hepatocyte cellular environment can lead to HBV cccDNA formation. Specifically, we will test whether genetic diversity across mouse strains or diversity induced by functional genomics approaches renders murine hepatocytes more permissive to HBV infection and replication. In Aim 2, we will take a virus-centric approach to determine the role of nucleocapsid uncoating in cccDNA formation within murine hepatocytes. We will also exploit the power of viral diversity to a degree never attempted with HBV to select viral variants capable of forming cccDNA in mouse hepatocytes. The proposed experiments have high reward potential. There is an inherent risk; however, the risks are more than justified because a simple mouse model would be accessible to scientists around the globe and accelerate research. We designed most experiments so that regardless of the outcome, the results will provide valuable novel insights into HBV host tropism and develop new technologies. We expect these efforts will ultimately lead to the creation of a fully HBV-susceptible immunocompetent mouse model that is suitable for developing therapeutic strategies, including immune perturbations, to promote a functional cure.

NIH Research Projects · FY 2026 · 2024-04

Project Summary Vision must transform patterns of light hitting the retina into an understanding of objects, their spatial relationships, and the surrounding scene context. This process of high-level information extraction is thought to occur in the ventral visual stream, a cortical pathway whose disruption is associated with face and object agnosias and visual memory loss. Both experimental neuroscience and theoretical work suggest that the traditional, purely feedforward model of the ventral stream fails to account for high-level vision in real-world scenarios. I hypothesize that visual cognitive information sent from the frontal cortex (FC) back to the ventral stream is critical for the remarkable robustness of primate vision to complex or ambiguous sensory inputs. The work proposed here examines interactions between the inferotemporal cortex (IT), the final station of the ventral stream, and two neighboring visually-responsive regions of FC: the frontal eye field (FEF), which controls eye movements and plays a role in visual spatial selection, and ventrolateral prefrontal cortex (PFC), which encodes abstract semantic object categories. This research plan leverages advantages of the marmoset, a small monkey with advanced visual behaviors and a lissencephalic brain allowing for application of experimental techniques that are intractable in the macaque. Aim 1 will use high-density electrophysiology probes and naturalistic stimuli to systematically characterize coding properties of visual FC. Aim 2 will use widefield calcium imaging, electrophysiological recordings, and electrical microsimulation to produce both a coarse map and a detailed cellular- and population-level account of how FC signals influence IT encoding. Finally, Aim 3 will use deep neural networks to model the influence of FEF and vlPFC on IT as a means for generating new hypotheses about biological vision. This project will expand understanding of the computational and cortical circuit bases of high-level vision, providing foundational knowledge of visual brain regions that are disrupted in neurological disorders such as frontotemporal dementia and autism. The training plan laid out in this proposal helps me acquire new experimental skills in optical imaging and multi- area recordings, theoretical skills in deep learning models, and scientific fluency in high-level vision. My sponsor, Dr. Elias Issa, will provide expertise in computational modeling and experimental work with the marmoset. My training will benefit from my co-sponsor, Dr. Michael Shadlen, who has decades of experience studying high-level associative cortex in the primate, and my collaborator on optical imaging, Dr. Aniruddha Das, an expert in mesoscale functional mapping. This work will be conducted at Columbia University’s Zuckerman Institute, a world-class neuroscience research and training institution whose faculty specialize in approaches ranging from molecular to systems to theoretical.

NIH Research Projects · FY 2026 · 2024-04

The gastrointestinal (GI) tract is the largest environmental interface of the mammalian body, which faces the challenge of maintaining tolerance to dietary and microbial antigens while protecting against pathogen invasion. In addition to hosting a large collection of immune cells, the GI tract contains diverse and numerous populations of glial cells and neurons, both intrinsic and extrinsic. Enteric neurons are responsible for controlling various physiological functions but can be targeted during enteric infections, resulting in functional gastrointestinal disorders after pathogen clearance. While some mechanisms underlying neuronal damage have been uncovered, the host and microbial factors that trigger enteric neuron loss and regeneration remain unclear. Over the past three years, we have uncovered new mechanisms of enteric neuronal cell death following infection or microbiota depletion and described the role of the gut microbiota and intestinal immune cells in regulating enteric neuron maintenance and function. In new preliminary data presented in the application, we provide evidence that enteric glial cells (EGC) experience a similar loss as enteric neurons following infection and antibiotic treatment. Additionally, our new data strongly suggest that EGC de-differentiation may play a role in neuronal recovery observed after microbiota transplantation. Finally, candidate metabolites involved in dysbiosis- associated neuronal death and neuronal recovery were identified. Based on these observations, we hypothesize that dysbiosis triggered by infection or antibiotic treatment generates ligands that trigger inflammasome- associated neuronal loss. Upon injury, glia-to-neuron dedifferentiation mediates neuronal recovery in a microbiota-dependent manner. The three complementary aims will delve deeper into these observations to reveal novel cellular and molecular mechanisms of microbiota-dependent neuronal death and regeneration, using novel imaging, state-of-the-art single-nuclei transcriptomics and chromatic accessibility, metabolomics, murine genetic fate-mapping, gain- and loss-of-function, and gnotobiotic approaches. In Aim 1, we will define the bacterial species and metabolites associated with specific glia and neuronal cell death after infection or antibiotic treatment. In Aim 2, we will assess the cellular dynamics of EGC and neuronal recovery after microbiota transplantation, followed by fate-mapping studies to define the possible role of glial cell de-differentiation in this process. Finally, Aim 3 will use complementary gnotobiotic and metabolomic approaches to identify bacterial signals and host pathways that induce gliogenesis and neurogenesis in different contexts. Overall, this proposal seeks to uncover novel mechanisms of microbiota-dependent neuronal death and regeneration with implications for the treatment of functional gastrointestinal disorders.

NIH Research Projects · FY 2025 · 2024-04

Project Summary Impaired social communication is a major feature of common neurodevelopmental disorders like autism spectrum disorder. Nevertheless, cellular mechanisms underlying the development of affected brain networks remain uncertain, despite the potential to inspire novel diagnostics and pharmacological interventions. Hence, this represents a key area of need in modern biomedical research. This career development proposal utilizes an emerging invertebrate model organism, the clonal raider ant, to investigate cellular mechanisms of neurodevelopment in olfactory brain circuits uniquely adapted for communication. Ants have evolved a remarkable capacity for chemical communication. Information is encoded by large arrays of pheromones exuded by dedicated exocrine glands and is received and processed by highly advanced olfactory systems. With approximately 500 olfactory glomeruli, the clonal raider ant antennal lobe (AL) is more complex than any other known insect and is evocative of the olfactory bulb in the brain of mammals (Drosophila have only ~50 AL glomeruli, for reference). Previous work in our lab suggests the evolution of sociality in ants may have coincided with unique neurodevelopmental logic in the AL supportive of this complexity. Over three aims, this project investigates early neuronal activity in pupal ant olfactory sensory neurons (OSNs) and its significance for the normal wiring of brain circuits in adults. First, Aim 1 utilizes GCaMP-expressing transgenic ants and in vivo two-photon microscopy to characterize spontaneous neuronal activity in the OSNs of clonal raider ant pupae. In Aim 2, OSN activity is manipulated throughout development using novel transgenic ant lines and the impact of these perturbations on olfactory circuit structure is investigated. In Aim 3, optogenetic tools are used to disrupt OSN activity during an isolated period of widespread synaptogenesis in mature ant pupae and the effect on neuropil volume is examined.

NIH Research Projects · FY 2026 · 2024-04

Project Summary/Abstract Modernization has been accompanied by a marked rise in disorders of allergic inflammation. These diseases are frequently borne by epithelial barrier tissues such as the skin, lung, and gut, which provide essential protection against the inflammatory stresses of the outside world. An emerging hallmark of barrier inflammation is the ability of these tissues to durably adapt to prior experiences, enhancing future responses against a broad range of stressors. The Fuchs Lab and others have revealed that long-lived epithelial stem cells (EpSCs), which replenish and repair their tissues throughout life, are key proprietors of these barrier adaptations in the context of type 17 inflammation, generally associated with extracellular pathogen responses. Conversely, how EpSCs adapt to allergic “type 2” inflammatory exposures remains a poorly understood and urgent unmet health need. To address this critical problem, I seek to use the prevalent epithelial alarmin thymic stromal lymphopoietin (TSLP) as a driver to uncover how type 2 skin inflammation impacts the long-term functional responsiveness and epigenetic state of EpSCs. My preliminary work reveals that transient TSLP-driven type 2 skin inflammation endows resident EpSCs with long-term increased stemness in vitro, and that inflammation- experienced skin retains a concurrently heightened ability to heal wounds in vivo. Surprisingly, I discovered that this lasting hyperresponsiveness extends beyond localized sites of inflammation, as even distal EpSCs and skin maintain comparably enhanced stemness and wound repair upon resolution, despite having never seen pathologically evident inflammation. With these data in hand, I will define the specific cell-intrinsic mechanisms that distinguish TSLP-driven inflammatory memory in both local and systemic contexts, and will interrogate the niche signaling that acts on EpSCs to establish cellular and tissue-level hyperresponsiveness in each. Lastly, given that my findings thus far could help explain the puzzling frequent interlinking of atopic inflammation in the skin and airway, I will determine whether TSLP-driven skin inflammation may have parallel cross-tissue effects, and alter the long-term functionality and epigenetic state of airway EpSCs. If successful, my findings will reveal important new insights into how allergic inflammation can shape long-term, organism-wide fitness beyond antigen-specific immune sensitization and memory, and add a molecular explanation for long-standing human pathologies that could advance treatment strategies.

NIH Research Projects · FY 2026 · 2024-04

Loss-of-function BRCA1/2 mutations lead to defective homologous recombination (HR) activity, a frontline DNA damage repair pathway that accurately addresses double-strand breaks. In the absence of sufficient HR activity, BRCA1/2-deficient cancers are suggested to activate a non-canonical DNA maintenance mechanism dependent on the oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A). CIP2A colocalizes with the genome stability factor TOPBP1 at sites of DNA damage during mitosis. This complex is suggested to physically tether fragmented chromosomes and centromere-bearing counterparts to ensure proper division and cancer cell survival. After mitosis, chromosome fragments can undergo chromothripsis, a large- scale error-prone genomic rearrangement. Chromothriptic fragments also rely on CIP2A for proper division in the next cell cycle to ultimately potentiate oncogenesis. CIP2A knockdown is lethal in BRCA1/2-deficient cancers but does not harm HR-proficient cells, indicating CIP2A to be a unique therapeutic vulnerability. However, a bonafide CIP2A inhibitor has not yet been produced via standard drug discovery efforts and may require innovative strategies. Recent chemoproteomic analyses identified nucleophilic cysteine residues in CIP2A that can be covalently labeled (“liganded”) with electrophiles. While lead fragments provide starting points for further development, these scaffolds also non-selectively engage other proteins. Furthermore, the functional consequences of labeling CIP2A are unknown. The proposal will therefore achieve chemical inhibition of CIP2A using covalent degraders (e.g., PROTAC) and focuses on the following aims: (1) Identify potent and selective covalent ligands for CIP2A. (2) Develop heterobifunctional probes and evaluate proximity-dependent degradation of CIP2A. (3) Analyze chemical inhibition of CIP2A in BRCA1/2-deficient cells. Encouragingly, preliminary data indicate the proposed ligands label CIP2A in both cellular and in vitro settings. Building on these results, the first aim will synthesize a focused probe collection based on computational guidance and then assess target engagement using a state-of-the-art chemoproteomic workflow. In the second aim, selective ligands will be converted into heterobifunctional degraders and characterized for CIP2A degradation in BRCA1/2-deficient cells. The third aim will then evaluate the cellular activity of covalent degraders by tracking phenotypes indicative of CIP2A inhibition and defective mitosis. This proposal will provide valuable chemical tools to probe CIP2A function and lay the foundation for therapeutics to treat BRCA1/2-deficient cancers. During this fellowship, I am grateful to be mentored by multiple professors from the Rockefeller University: Tarun Kapoor, my advisor, a master in chemical probe design and cell division biology; Jiankun Lyu, an expert in computational chemistry; Ekaterina Vinogradova, a leader in chemoproteomics. Their mentorship will be supplemented with additional training both within Rockefeller and in the broader academic community. This fellowship represents an essential step towards my goal of performing independent research at the interface of chemical and cancer biology.

NIH Research Projects · FY 2025 · 2024-03

Project Summary: Liver cancer is the third leading cause of cancer-related death and is associated with diverse range of oncogenic alterations. These driver mutations impact clinical outcomes, disease prognosis and response to therapy. Treatment options for liver cancer include a few multi-kinase inhibitors and surgical resection, providing only limited survival benefits. To this end, genotype-guided therapeutic approaches are urgently needed in clinics. Metabolism-focused approaches have recently gained interest to be explored as anti-cancer therapy. However, it is poorly understood how specific oncogenic alterations impact tumor metabolism and which tumors would benefit from metabolism-based therapies. Given the complex cellular and nutritional composition of tumors, studying tumor metabolism at cellular resolution is quite challenging. In this study, I propose a way to overcome this challenge by employing an organelle pull-down technology and profiling mitochondrial metabolites of cancer cells from highly heterogeneous tumor tissues. In my preliminary work, I focused on mitochondria, a central biosynthetic hub for cellular metabolism, applied the mitochondrial immunoprecipitation method (mito-IP) to capture cancer cell mitochondria from in vivo transformed liver tumors driven by different oncogenic alterations, including c-MYC; p53-/- and KrasG12D; p53-/-. This approach enabled mitochondrial metabolite profiling by LC/MS and identified metabolite changes specific to each oncogenic alteration. In particular, I found enrichment of creatine metabolism intermediates; guanidinoacetate, creatine (Cr) and phosphocreatine (P-Cr), specifically in KrasG12D tumors. Mitochondrial proteomics corroborated these findings, revealed upregulation of Gatm, the rate limiting enzyme in creatine biosynthesis, in KrasG12D tumors. Building upon these findings, in this proposal, I will test the central hypothesis that oncogenes impose distinct metabolic alterations in mitochondria, which can be exploited as targeted therapies. I will first test the essentiality of creatine metabolism for KrasG12D -mutant liver tumor growth. Then, I will determine the precise mechanisms by which creatine metabolism contributes to KrasG12D-driven tumorigenesis. Finally, given the success of my in vivo mito-IP approach, I will model frequent liver cancer mutations to map differentially regulated mitochondrial metabolites and proteins, and identify limiting metabolic reactions through unbiased genetic screens. By addressing the aims of this proposal, we will gain mechanistic insight into downstream metabolic effects of oncogenic alterations and potential of genotype- targeted metabolic therapies for targeting liver cancer. Collectively, these aims will provide foundations of my future research program committed to understanding how oncogenes (genetic determinants) alter cancer cell metabolism in vivo, with the goal of exploiting such alterations for genotype-targeted metabolic therapies.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY Innate behaviors are hard-wired and shared among most mammals, however, it is still not fully understood how these behaviors arise and what neuronal mechanisms control behavior selection. The goal of this proposal is to study feeding behavior as a typical innate behavior to determine the fundamentals of a circuit that connects sensing of the body’s energy state to motor centers for food consumption. This will be done using an integrative approach at behavior, circuit, neuron and synapse level that takes advantage of my existing and proposed training in neuroscience. Results will shed light on the neuronal computations that transform sensations of energy state into motor sequences of chewing and biting and will advance our understanding of behavior selection. Feeding behavior is elicited by changes in bodily energy state (fasted vs. fed) that result in jaw movements of chewing and biting. In contrast to other behaviors, the motor and premotor neurons for jaw muscles are located in the brain and not the spine. Thus, the arc from hunger sensation to motor control is fully brain-based. I previously identified a simple circuit for food consumption that connects neurons sensitive to signals of bodily energy state via only one intermediate node to premotor areas controlling biting and chewing. Building on this data, the mentored phase aims to delineate the role of premotor neurons and their activity patterns with respect to the hypothesis that premotor neurons are structured in a motor map for bite purposes. This aim aligns with my training plan to gain expertise in RNA profiling and single cell calcium imaging through my advisory committee and the high-quality scientific core and support opportunities at Rockefeller University. Additionally, I will also take advantage of the career development and lab management training opportunities offered locally and by the Tri-Institute area. Next, during the independent phase, I will use these skills to elucidate the computations that lead to the behavior selection of feeding and the effect of internal sensations of energy state change. Specifically, it will be determined how competing behaviors (social interactions, fear) can suppress feeding and how integration of food-derived sensations (sight, smell) and bodily signals causes food consumption. This phase is the logical extension of my extensive training in neuroscience and my career objectives to lead my own research group for which this proposal forms the basis of my research direction. Results from this study will shed light on the conceptual principles of how innate behaviors are encoded along a simple sensory to motor circuit. Additionally, results will also provide insight into the mechanisms of energy state coupling with food intake which has important implications for obesity and eating disorders. Finally, this proposal will equip me with the training and initial data needed to start an independent research program that is different from my mentors and aligned with my career goals of studying innate behavior in the context of interoception. .

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY/ABSTRACT The eukaryotic cell has evolved to compartmentalize DNA within the nucleus, surrounded by the barrier of the nuclear envelope (NE), providing protection to the DNA and a means to control information flow to and from the genome. This flow, namely the passage of small molecules, RNAs, and proteins across the NE, is mediated by large, 8-fold symmetrical structures known as nuclear pore complexes (NPCs). Arranged as a stack of rings, the NPC scaffold braces open pores within the NE, yet preserves its barrier function with a dense network of disordered protein domains, rich in repeating phenylalanine-glycine motifs (FG repeats), known as the NPC central transporter. The largest NPC cargoes, including pre-ribosomal subunits, mRNPs, and proteasome subcomplexes, approach the width of the ~55 nm NPC channel in size, yet are able to transit the NPC central transporter. That such large cargoes can traverse the NPC suggests that organized mechanisms and alternate structural states of the NPC are required to transport large cargoes through the FG repeat network of the central transporter. To address these questions, our multi-investigator, multi-site collaborative team recently determined structures of the affinity isolated S. cerevisiae budding yeast NPC at 8-11 Å resolution and the in situ yeast NPC at 30-40 Å resolution (Akey et al., 2022). Comparison of “ground state” (isolated) and “active state” (in situ) NPCs revealed that radial dilation of the NPC scaffold may accommodate changes in cargo flux or size. We now aim to investigate the dynamics of NPC transport function of model large cargoes that can be targeted for import into the nucleus and arrested during transport, allowing us to quantify cargo transport dynamics (function) and map the location of the transiting cargo within the NPC (structure). We will dissect NPC transport’s energy- dependence on nuclear Ran-GTP into separate studies of passive, Ran-insensitive cargo targeting to the NPC (Aim 1) and active, Ran-dependent cargo transport through the NPC (Aim 2). Using well-characterized NPC transport mutants, we will examine the behavior of actively transporting large cargoes by perturbing specific stages along the transport path (Aim 3). This level of control will allow us to map transport functions not only to specific Nups, but to specific regions of Nups. Quantitative fluorescence microscopy and cell fitness assays will screen for functionally important phenotypes to selectively pursue NPC-cargo interactomics by mass spectrometry and visualization of NPC structural changes by cryo-electron microscopy. Our functional and structural data will inform each other in a synergistic but non-dependent fashion to reveal how the NPC adopts distinct and discrete structural states to perform its transport functions for native (mRNPs, pre-ribosomes) and non-native (viral capsids, nanocarriers) large cargoes. This comprehensive research strategy will advance our understanding of (i) constitutive large cargo transport processes and malfunction of this process in disease states (ii) interactions between viral capsids and NPCs during infection and (iii) nuclear-targeting for nanocarriers in drug delivery and gene therapy.

NIH Research Projects · FY 2026 · 2024-02

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is an ongoing global health crisis. The WHO reports 10 million active cases and over 1.5 million deaths annually. RNA polymerase (RNAP), the enzyme responsible for all transcription in bacteria, is the target for rifampicin, a first-line treatment for TB. RNAP is thus a proven and attractive target for developing new drugs. These facts highlight the importance of our recent structural and functional characterization of Mtb RNAP, and the essential transcription factors required for full transcriptional activity and intrinsic antibiotic resistance. In addition, we have found that studying diverse clades of bacteria deepens our understanding of general principles of transcription. Therefore, we study Actinobacteria, as the biophysical and molecular mechanisms of transcription in this clade are relatively understudied and because it includes important pathogens. We determined high-resolution cryo-EM structures of several Mtb initiation transcription complexes with essential transcription factors and important antibiotics in the previous funding period. Here my vision is to complete the characterization of initiation regulation and expand our focus to the post-initiation steps throughout the transcription cycle. These steps include elongation, pausing (a regulatory step where the RNAP temporarily halts at specific sequences, permitting input from diverse signals), and termination. We will also continue characterizing new RNAP inhibitors. To complete our understanding of initiation, we will continue our studies of the WhiB factors, focusing on the essential factor WhiB1, which is required for Mtb viability and response to stress. To characterize post-initiation steps, we plan to study the mechanisms of mycobacterial NusA and NusG. NusG in Mycobacteria increases transcriptional pausing and termination, yet it suppresses both in the well-characterized E. coli. This difference in activity emphasizes the need to study these factors in other clades of bacteria. Most of these studies will involve single-particle cryo-EM, but we plan to extend our studies in situ by using cryo-electron tomography to investigate cellular processes and organizations, such as ribosomal coupling to RNAP. To rigorously study how transcription factors act genome-wide, we will finish developing cell-free genomics: using genomic DNA as a substrate for purified RNAP and transcription factors, and quantifying resultant transcripts with RNA-seq. This method is necessary to identify native DNA sequences regulated by essential factors WhiB1, NusA, and NusG, whose regulons have remained elusive due to pleiotropy upon perturbation in cells. In sum, I envision using a multidisciplinary approach that includes structural, genetic, biochemical, genomic, and in situ experiments to understand the roles and mechanisms of each step in the transcription cycle and how they are regulated by cis and trans-acting elements. The results from this proposal have the potential to elucidate the molecular and biological mechanisms of the entire transcription cycle.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY/ABSTRACT A major challenge in understanding how organs take shape is addressing how increases in morphological complexity arise. Over the past half-century, our understanding of such symmetry-breaking has been predominantly developed through molecular, genetic, and cellular frameworks. However, symmetry-breaking in developing organs occurs at scales far larger than their individual molecular and cellular constituents, prompting the question of how events at the molecular and cellular scale relate to the physical self-organization of organ structure. To address this question, my lab centers its studies on the behavior of cell collectives, an understudied nexus that serves as a mediator between subcellular processes and functionally relevant organ morphology. Our prior work in considering collective cell, or supracellular, material properties has led to the hypothesis that the role of key signals, known as morphogens, in sculpting organs is to influence cells and extracellular matrix such that tissue material properties or “phase” are modulated. Thus, a key functional role of morphogens may be to create diversity in supracellular mechanical behavior by fluidizing or solidifying tissue domains that result in the increase of complexity in organ form. Here we will use a unique set of supracellular behavioral assays to investigate new roles for signals in shaping emerging morphologies. We will first link where and when morphogens are expressed in the forming follicle to the transcriptional profiles and multicellular architectures that accompany morphogen activity (Aim 1). We will then leverage a constellation of novel collective cell mechanics platforms to probe the functional consequence of morphogen activity at the supracellular scale. (Aim 2). Finally, through the integration of theory and experiment, we will explore the hypothesis that interacting supracellular phases generate a mechanical instability in the skin that is sufficient to shape the organ (Aim 3). Together these studies stand to highlight the need to consider supracellular properties necessary for tissue symmetry breaking that are not reducible to the properties of individual cells. At the same time, our studies will identify the molecular and cellular component parts that enable the diversification of emergent supracellular material properties. Our investigations will pave the way for a generalizable paradigm for how morphogens can create adjacent material phases within a single tissue, thereby generating the potential for mechanical self- organization at the tissue scale. Finally, these studies will shed light on the multi-scale nature of organ formation and serve as a foundation for future work aimed at addressing congenital defects, engineering stromal tissues of specific material properties, and the rational channeling supracellular self-organizing potential in regenerative medicine.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT The fundamental unit of hierarchically organized eukaryotic chromatin is the nucleosome, which contains 147 base pairs of genomic DNA wrapped around an octamer of core histone proteins. Conventionally, nucleosomes have been viewed as DNA packaging units that inhibit gene expression by obstructing the accessibility of DNA to the transcriptional machinery. However, we and others have shown that nucleosomes also serve as potent hotspots which recruit, modulate, and stimulate the activity of various essential chromatin regulators, indicating a new role for nucleosomes in furnishing the genome with a multitude of physical features and interactions which direct protein function. As such, I hypothesize that the physical characteristics and topology of nucleosomes modulate the activity of chromatin regulators, constituting an underappreciated layer of physical parameters encoded within chromatin architecture that govern genomic transactions in the nucleus. These parameters include the shape and composition of the nucleosome core particle as well as the spacing and geometry of contiguous nucleosomes in an array. To test this hypothesis, I propose to use single-molecule fluorescence detection and force manipulation technologies established in my laboratory, which uniquely track real-time transient and heterogeneous molecular interactions, to investigate the physical characteristics of nucleosome topology that determine its capacity to tune the activity of several important classes of chromatin regulators at multiple scales. We will first investigate how the topology of individual nucleosomes directs the DNA targeting activity and cooperation of essential pioneer transcription factors (Aim 1). We will then probe how the geometry of local nucleosomes in an array modulates the engagement, recruitment, and propagation of chromatin- modifying enzymes on chromatin (Aim 2). Finally, we will investigate the biophysical basis of global nucleosome localization and functionalization by energy-consuming molecular machines (Aim 3). Together, these studies will zoom in on the topology of nucleosomes comprising a layer of biophysical parameters encoded within chromatin architecture that regulate genomic transactions in the nucleus. They will contribute evidence towards a new perspective that views nucleosomes as genomic regulators which harness their unique physical features to actively modulate, recruit, and stimulate the activity of chromatin-associated factors, rather than passive DNA packaging units. The proposed investigations will shed light on a nucleosome-focused angle for tackling several long-standing questions about the interplay between chromatin and its regulators and promise to mechanistically inform how disease-associated mutations perturb essential genomic activities, potentially revealing mutation- selective protein-chromatin interfaces that may be therapeutically exploited to treat human disease.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT Animals exhibit a remarkable array of creative, adaptive, and flexible behaviors. Birds and primates repurpose new materials to build nests and tools; rats efficiently construct navigational shortcuts, and humans generalize knowledge of one language to efficiently speak another. This ability to dynamically create novel behavior in one or a few trials often depends on compositional planning, or the ability to generate new combinations of a finite number of simple elements in a goal-directed manner. Despite its central importance for understanding cognition and its disorders, the neural mechanisms of compositionality remain unknown as there is a dearth of experimental frameworks for studying compositional planning. To address this critical need for new approaches, this proposal will elucidate neural mechanisms in a novel drawing task that I have developed in the Freiwald lab, in which macaques draw copies of never-before-seen visual figures. Subjects’ behavior exhibits a key signature of compositionality in the ability to construct novel combinations of previously learned elements to draw new images. I will investigate neural and computational mechanisms for compositional action planning by integrating this behavioral task two other innovations: (1) large-scale recordings in 12 frontal cortical areas, each implicated in cognition but never recorded simultaneously, which will allow me to discover how their distinct functions combine to support cognition (Aim 1), and (2) an integrative analysis framework building and comparing neural network (Aim 2) and symbolic (Aim 3) computational models of compositional planning with behavioral and neural data. I will test the main hypothesis that compositionality depends on neural dynamics implementing symbolic cognitive algorithms in hierarchically organized frontal cortical areas. These studies are expected to discover the first mechanisms, in neural substrates and dynamics, of compositional action planning. Further, because of these studies’ intersectional approach - testing neural network (Aim 2) and symbolic (Aim 3) modeling frameworks on the same data - they may unify these two influential approaches to cognition, which would be a foundational advance for the neuroscience of intelligence. Correspondingly, this study will contribute to understanding cognitive disorders, including frontal planning disorders, and to building brain-machine interfaces that decode cognitive plans from cortical activity. This award will also provide me with crucial training to prepare me for transitioning to independence. I will train in computational modeling - building, empirically testing, and interpreting these models - which will support my use of models to generate and test novel neural circuit and computational hypotheses. I will gain important career development skills in lab management and leadership, scientific communication, and grant writing, which will support my long term goal of establishing an independent research program on the neural substrates of intelligence and creative behavior.

NIH Research Projects · FY 2024 · 2023-09

Project Summary All eukaryotic cells, whether normal or cancerous, require the ability to sense changes in nutrients levels, ensuring their efficient use for survival and growth. Nutrient sensing mechanisms enable cells to rapidly adapt to environmental perturbation, a feature particularly essential for cancer cells to overcome diverse metabolic stresses along the metastatic cascade. Although many nutrient sensing mechanisms have been described, how metabolites are sensed in subcellular compartments remains a major open question. This question is particularly relevant for redox-active molecules such as NAD and glutathione, which display remarkably heterogenous distribution across subcellular compartments and have been shown to play key roles in cancer metastasis. Recent breakthroughs in deorphanizing mitochondrial metabolite transporters provided unprecedented opportunity to probe the dynamics and sensing mechanism of these metabolites at subcellular precision. In a recently published study, SLC25A39 has been identified as a key transporter for mitochondrial glutathione, a major antioxidant molecule implicated in cancer progression and metastasis. Remarkably, evidence suggests that SLC25A39 undergoes feedback regulation by mitochondrial glutathione and may be required for efficient metastatic colonization, implicating it in an adaptive mechanism for cancers to overcome metabolic stress during metastasis. This proposal seeks a deeper understanding of the implication of organellar glutathione metabolism in cancer. The Aim 1 of this proposal seeks to understand the role of mitochondrial glutathione homeostasis in tumor progression and metastasis and decipher the mechanism of its regulation. The Aim 2 of this proposal seeks to develop novel genetically encoded, single-cell RNAseq-compatible reporters for profiling intercellular heterogeneity in mitochondrial glutathione in tumors. Using a combination of biochemical analysis, unbiased CRISPR screens and novel animal models, this proposal aims to paint a multilayered picture of the dynamics, regulatory mechanisms and functional contribution of mitochondrial glutathione homeostasis in tumor progression and metastasis. Completion of the proposed studies will deepen our understanding on the role of compartmentalized metabolite pools in metabolic rewiring of cancers and shed light on novel therapeutic strategies to target metastasis.

NIH Research Projects · FY 2024 · 2023-09

Project Summary CRISPR-Cas are prokaryotic adaptive immune systems that protect bacteria and archaea from invading mobile genetic elements, such as phages and plasmids. CRISPR-Cas systems acquire immunological memories during infection by integrating short fragments from the invader’s genome into the CRISPR locus of the host. These fragments, called “spacers”, are later transcribed into CRISPR RNAs that are loaded on Cas nucleases and guide them to recognize and cleave infecting nucleic acids. Depending on their genetic composition, CRISPR- Cas systems are classified into six types (I-VI). While spacer acquisition has been extensively studied in type I and II systems, type III systems are just now starting to be explored. The overall goal of this application is to define the molecular mechanisms that govern spacer acquisition by the prevalent, yet less studied, type III-A CRISPR-Cas system, and understand its implications during CRISPR-Cas defense and tolerance. Preliminary work on the type III-A system of Staphylococcus epidermidis revealed that this system preferentially acquires new spacers by two independent modes. The first mode acquires spacers from some, but not all, highly transcribed genes, and spans their entire transcribed region. The first aim of this proposal is to elucidate how the acquisition machinery recognizes specific genes as substrates for preferential acquisition. This will be achieved by dissecting the DNA sequences that recruit the spacer-integrase complex to specific genes, finding host factors that mediate gene-specific spacer acquisition, and test for the physiological relevance of this process during the CRISPR-Cas immune response. The second mode of acquisition by the type III-A system is similar to the previously studied type I and II systems, where spacers are acquired from free dsDNA ends at the bacterial chromosomal terminus, in a manner that is dependent on the cell’s DNA-repair machinery. Such self-targeting spacers are expected to induce autoimmunity and be negatively selected, however we found them to be stably fixed in the bacterial population, suggesting the existence of unknown mechanisms that inhibit targeting by Cas nucleases at this site, thus preventing CRISPR autoimmunity. The second aim of this proposal will define the genomic context that allows self-targeting spacers to be tolerated, analyze the temporal dynamics of CRISPR- Cas immunity at free DNA ends, and explore the genetic components needed for CRISPR-tolerance and accumulation of self-targeting spacers. This proposed work will not only transform our conceptual understanding of the spacer acquisition process, but also could lead to CRISPR-based technological developments in molecular biology and diagnostics. To achieve these goals, I have assembled a team of experts in the fields of transcription, DNA repair, bioinformatics, biochemistry and biophysics. Their guidance, along with the continued mentorship of Prof. Luciano Marraffini and the scientific environment of the Rockefeller University, will allow me to perform the proposed research, as well as to develop writing, mentorship and communication skills, that will support my successful transition to an independent career.