Grants

NINDS - National Institute of Neurological Disorders and Stroke

ABSTRACT During brain development, neurons undergo dramatic growth and branching to establish the intricate morphology required for neural network connectivity and function. This cellular expansion involves a substantial and rapid increase in neuronal surface area and therefore requires extensive insertion of material into the plasma membrane. Defects in neuronal morphogenesis can result in improper synaptic connectivity, neurodevelopmental disorders, and psychiatric syndromes. Insertion of membrane material is facilitated by SNARE-mediated exocytosis. Two SNARE proteins, Vesicle-associated membrane protein (VAMP) 2 and VAMP7, are enriched in the embryonic brain and are associated with distinct vesicle populations even prior to synapse formation. While knockout studies in mice have shown that both VAMP2 and VAMP7-mediated vesicle fusion are required for proper neuronal morphogenesis, the specific mechanisms regulating the trafficking and fusion of these vesicles during development is not known. My preliminary data reveal that VAMP2 and VAMP7 mediate non-synaptic exocytic events that cluster in different areas of the developing neuron, suggesting the existence of distinct regulatory pathways governing their distribution and fusion. Additionally, my preliminary data indicate that VAMP2-mediated exocytosis is sensitive to Ca2+ chelation, whereas VAMP7-mediated exocytosis is not, mirroring the differential Ca2+ sensitivity of VAMP2 and VAMP7 vesicle pools observed at the synapse of mature neurons. This suggests a potential role for Ca2+ signaling in the regulation of VAMP2 and VAMP7- mediated exocytosis during neuronal morphogenesis. Endoplasmic reticulum (ER)-PM membrane contact sites, key regulators of Ca2+ signaling, may also be involved in this regulatory process. The goal of this proposal is to investigate the regulation of VAMP2 and VAMP7-mediated exocytosis during neuronal morphogenesis using hypothesis driven science coupled with unbiased proteomic analysis. I will utilize total internal reflection fluorescence (TIRF) microscopy and advanced image analysis techniques to quantitatively define the interplay between SNARE-mediated exocytosis, Ca2+ signaling, and ER-PM membrane contact sites during neurite outgrowth in stage 2 developing neurons. Using a proximity biotinylation approach I will define the interactome of VAMP2 and VAMP7 during neuronal morphogenesis, providing insights into the molecular machinery regulating their trafficking and fusion. These findings will advance our understanding of the complex regulation of exocytic membrane trafficking during neuronal development. Successful completion of this proposed research will provide advanced training in live cell microscopy, the development of advanced image analysis pipelines, proteomics, and bioinformatics.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY: The ability to tolerate the complete loss of water is a rare but conserved trait observed across the diversity of life, including in many bacterial species. While desiccation is a common stress experienced by bacteria that primarily inhabit the natural environment, there are rare examples of host-associated bacteria that can tolerate extended periods of water loss. Acinetobacter baumannii is an emerging opportunistic bacterial pathogen that frequently colonizes mammalian hosts after contact with contaminated surfaces. The long-term persistence of A. baumannii on abiotic surfaces is well-documented and has been attributed to the extreme desiccation tolerance of this organism. Moreover, the ability of A. baumannii to readily transition between environmental and host-associated niches implies the evolution of sophisticated regulatory mechanisms that sense and transduce environmental stimuli, including water loss. However, the precise regulatory mechanisms A. baumannii employs to sense and respond to desiccation stress are not well understand. Previously, we uncovered two central regulatory proteins that interface to control desiccation tolerance in A. baumannii, the two-component transcriptional regulator BfmR, and the conserved protease Lon. We discovered that the regulatory processes controlled by these proteins are tightly linked, suggesting a mechanism whereby A. baumannii coordinates the regulated turnover of protein content with a global transcriptional response as a mechanism to program its long- term survival on abiotic surfaces. Over the next five years, we build on these recent discoveries to further delineate the precise regulatory networks and signal transduction pathways controlled by Lon and BfmR to facilitate adaptation to water loss. First, we will explore how regulated turnover of protein cargo by a newly discovered adaptor of Lon protease influences adaptation to desiccation. Second, we will elucidate the signal transduction mechanisms governing transcriptional control of desiccation tolerance by the BfmRS two- component system. Finally, we will define the mechanisms by which transcriptional and post-translational regulatory processes crosstalk to control desiccation tolerance in this organism. Together, this work will reveal novel factors mediating the extreme desiccation tolerance of A. baumannii and will create a framework for better understanding how bacteria induce multi-faceted regulatory processes to transition between host and environmental reservoirs.

NIH

Posttraumatic stress disorder (PTSD) is a highly prevalent and often chronically disabling disorder among Veterans. Sleep impairment with frightening trauma combat nightmares and distressed awakenings accompanied by both increased peripheral and brain noradrenergic signaling are common features of PTSD in Veterans. In addition to the adverse of impacts of PTSD on Veterans’ sense of well-being and ability to function, epidemiologic studies demonstrate that PTSD is a risk factor for development of all cause dementia and Alzheimer’s disease (AD). Another disorder highly prevalent among Veterans and particularly among Veterans with PTSD is obstructive sleep apnea (OSA). Epidemiologic studies demonstrate that OSA also is a risk factor for dementia and AD. Thus, Veterans with both PTSD and OSA are likely at particularly high risk for later life dementia and AD. A potential mechanism by which both PTSD and OSA increase the risk for AD is impairment of the brain glymphatic system. The glymphatic system clears the brain of metabolic waste products and neurotoxic proteins, including amyloid beta Abeta42 and tau, the pathological hallmarks of AD. The brain glymphatic system is active during normal sleep and relatively quiescent during wakefulness. In addition, the glymphatic system is suppressed by noradrenergic signaling. Impaired sleep and elevated noradrenergic signaling (particularly at night) are important features of both PTSD and OSA. We hypothesize that PTSD and OSA impair glymphatic function and this impairment is greatest in Veterans with both conditions. We also hypothesize that plasma biomarker signature of AD will be greatest in Veterans with both PTSD and OSA. Our preliminary findings support these hypotheses. In a cohort of Veterans of Operations Enduring Freedom/Iraqi Freedom/New Dawn (OEF/OIF/OND) who met diagnostic criteria for PTSD, 80% reported a moderate-to-high burden of trauma nightmares and sleep disturbance. In these Veterans, poor sleep was associated with both increased cerebrospinal fluid (CSF) norepinephrine (NE) -- consistent with increased brain noradrenergic signaling -- and lower CSF amyloid-beta42 (Abeta42) -- an early biomarker for AD. We will explore this hypothesis through a mechanistic translational study. Participants will be 100 middle- aged OEF/OIF/OND-deployed Veterans: 70 with a diagnosis of PTSD with and without OSA and 30 deployed control Veterans with neither PTSD nor OSA. Because of our preliminary data establishing a relationship among impaired sleep, increased brain noradrenergic signaling, and CSF Abeta42 in OEF/OIF/OND Veterans, we will select what we consider to be the “noradrenergic subtype” of PTSD, characterized by moderate-to- extreme distressing dreams and sleep disruption assessed by the Clinician-Administered PTSD Scale For DSM-5. Recent improvements in biofluid biomarkers have allowed measurement of AD biomarkers in plasma rather than CSF. Plasma Abeta42, Abeta40, and phosphorylated tau (ptau) 217 will be measured on the state- of-the-art Lumipulse 1200G platform. Specific Aim 1. Define the effect of PTSD and OSA on glymphatic function in Veterans. Specific Aim 2. Define the effect of PTSD and OSA on AD-related biomarkers and cognitive function. Exploratory Aim. To determine if CPAP therapy improves glymphatic function and plasma AD biomarkers in Veterans with PTSD and OSA. The Research Project as well as the Mentorship and Career Plans in this application will prepare the candidate, Dr. Cho, a sleep medicine physician, to pursue an independent research career in the VA focusing on the effects of PTSD and OSA on Veterans’ health and pursuing novel strategies to reduce risk of AD.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY The complex communication mechanisms involved in bladder function are not fully understood. Due to this lack of physiological understanding, bladder diseases have remained challenging to treat, resulting in severe detriment to patient quality of life, particularly in the case of interstitial cystitis/bladder pain syndrome (IC/BPS). To address this, I aim to contribute to the ongoing investigation of the role of renin-angiotensin signaling in the bladder to inform future therapeutic strategies. Angiotensin II (Ang II) is the primary effector molecule of the renin-angiotensin system, and action on either of its receptors, Ang II type 1 receptor (AT1R) and Ang II type 2 receptor (AT2R), has been shown to elicit major effects in other organ systems. While Ang II signaling research in the bladder has primarily focused on defining the role of AT1R in mediating contractions and pathological effects on bladder tissue, AT2R should also be considered for its potential protective effects. Despite the understanding that AT2R counteracts the pathological impact of AT1R in other organ systems, the role of AT2R in the bladder remains largely unknown. Preliminary investigations have demonstrated that AT2R is expressed in the bladder, and one investigation showed that blocking AT2R in mice worsened the pathological effects in a cystitis model. However, there has not been a comprehensive investigation solely regarding the presence and role of AT2R in the bladder. Given these preliminary findings, I hypothesize that AT2R has a functional role in the bladder, and its activation can alleviate hyperalgesia and bladder dysfunction associated with cystitis conditions. This work will be conducted with two main aims: Aim 1 will provide a physiological evaluation by using naïve mouse bladder tissue to characterize the presence and location of AT2R mRNA expression and define the role of AT2R in ex vivo contractile responses through pharmacological manipulation of AT2R activity; Aim 2 will explore the role of AT2R in a pathophysiological bladder condition in vivo through chronic administration of an AT2R agonist, C21, in a cystitis mouse model. The effect of AT2R activation will be evaluated through voiding function, abdominal hypersensitivity, molecular changes, and ex vivo contractile responses. This work begins to uncover the role of AT2R in bladder physiology and pathology and is the first to broach the potential of AT2R as a therapeutic target for bladder diseases such as IC/BPS. Expanding the knowledge of angiotensin signaling in bladder dysfunction provides a foundation for pharmacological and non-pharmacological interventions that could improve patient outcomes and quality of life. The proposed research will help me develop as an independent scientist, providing me the opportunity to lead an experiment and gain the technical skills needed for a career in science. As a PhD student in the Joint Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University under mentorship from Dr. Aaron Mickle and Dr. Justin Grobe, I have all the tools necessary to complete this fellowship effectively and obtain preparation for my career.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Most influenza cases resolve without complications, but some progress to serious conditions like pneumonia, acute respiratory distress syndrome, or encephalitis. Genetic predispositions, such as inborn errors of immunity, are known factors that contribute to these severe outcomes, yet the underlying mechanisms in otherwise healthy individuals, particularly children, remain unclear. This proposal focuses on ANKEF1, a ciliary protein identified as having a loss-of-function mutation in a child who succumbed to influenza-associated rhombencephalitis—a rare and severe complication characterized by inflammation of the brainstem that can occur in some severe cases of influenza-associated encephalitis (IAE). Our preliminary findings indicate that ANKEF1 is critical for ciliary motility in airway epithelial cells, which is essential for effective mucociliary clearance (MCC). In mouse models, ANKEF1 deficiency leads to impaired MCC, reducing the ability to clear pathogens from the respiratory tract and leading to increased susceptibility to influenza infection, higher morbidity, and greater mortality. We hypothesize that persistent viral presence in the airways due to defective MCC triggers an exaggerated inflammatory response, which elevates the risk of IAE through increased cytokine production and disruption of brain barrier integrity In this proposal, we will leverage novel mouse models, human iPSC-derived airway epithelial cultures, and advanced multi-omics technologies to define how ANKEF1 regulates ciliary function and host defense. Aim 1 will investigate how ANKEF1 deficiency alters ciliary beat frequency, coordination, and motility in the airways and brain, and assess the resulting impact on viral replication and immune responses ex vivo. Aim 2 will characterize disease progression in ANKEF1-deficient mice by measuring viral titers, immune responses, and tissue damage in both the respiratory system and brain. Additionally, we will use single-cell RNA sequencing and spatial transcriptomics to identify key pathways associated with increased disease severity. Aim 3 will elucidate the molecular mechanisms by which ANKEF1 modulates ciliary functions and identify key interactions and regulatory networks that contribute to host defense against IAV infection. This research will provide new insights into the role of ciliary function in airway and brain defenses, potentially revealing therapeutic targets for severe influenza and other respiratory diseases.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary Chronic liver diseases are a collection of disorders that result in significant morbidity and mortality worldwide. The overwhelming burden of these diseases results in the liver being the second most transplanted organ. As a result of a dramatic rise in the incidence of obesity and alcohol consumption, the rate of liver transplantation continues to climb world-wide. Despite the significant impact of chronic liver disease on society, there are no effective therapies to ameliorate disease outside of lifestyle modification and liver transplantation. Thus, a more rigorous understanding of the mechanisms that drive the progression of chronic liver disease is required to identify novel targets for therapeutic intervention. Our previous publication demonstrated a significant accumulation of T lymphocytes in the liver of individuals with Metabolic Dysfunction-Associated Steatohepatitis (MASH)-induced cirrhosis. However, the precise role of T cells in the progression of MASH has yet to be firmly established. The preliminary studies presented in this application have identified a yet to be appreciated role for T cell activation and clonal expansion during MASH. In this application we demonstrate that T cell clonal expansion is a common event that correlates with fibrosis stage in both humans and mice with MASH. These are the first studies to identify T cell clonal expansion in the liver of humans and/or mice with MASH and link T cell activation and function with MASH pathology. However, the exact antigenic cause of T cell activation or if these T cells are inducing or suppressing disease progression is unknown. The studies in this application aim to answer these two fundamental questions by defining (1) the antigens recognized by clonally expanded T cells, (2) if there are shared stimulatory antigens between human and mouse disease, (3) the antigen presenting cell (APC) that drives T cell clonal expansion, and (4) if eliminating T cell clonal expansion through depletion of either clonally expanded T cells or the APCs presenting antigens to these T cells results in prevention and/or resolution of disease. Completion of these studies will provide a paradigm shift in our understanding of the pathogenesis of MASH and will generate the tools necessary to define the role of T cells in the progression of other chronic liver diseases.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that results from the breakdown of tolerance mechanisms in self-reactive T cells, leading to immune infiltration of the pancreas and the destruction of insulin- producing b cells. Patients with T1D require lifelong exogenous insulin treatment and often develop multiorgan dysfunction, emphasizing the need for effective treatment strategies. β cell-specific CD8 T cells are the primary pathogenic population that eliminate insulin-producing b cells in the pancreatic islets, yet intriguingly, MHC class II haplotypes confer the greatest genetic risk for T1D development suggesting a critical role of CD4 T cells in disease initiation and progression. However, many aspects of autoimmune β cell-specific CD4 T cell differentiation and function remain enigmatic, including where and how autoimmune CD4 T cell populations arise and are maintained and their exact function in driving T1D. Our lab previously identified a stem-like CD8 T cell population which self-renews and gives rise to differentiated progenies which migrate to the pancreas and eliminate b cells; the stem-CD8 T cell pool is absolutely required for sustained b cell destruction. The goal of this application is to generate a deep understanding of the phenotypic and molecular features and functional role of β cell-specific CD4 T cell populations in driving T1D and specifically CD8 T cell stemness, differentiation and function. We will leverage the non-obese diabetic (NOD) mouse model, a clinically relevant model of T1D, which shares many features with human disease to (i) assess heterogeneity and polarization/differentiation states of CD4 T cells, (ii) determine the importance of key transcription factors in regulating CD4 T cell function, and (iii) employ innovative spatial sequencing technologies to identify cell-cell interactions and signals driving β cell destruction. Our proposed studies will provide important insights into autoimmune b cell-specific T cell programming and function which could yield novel therapeutic targets for the prevention or treatment of T1D and other T cell-mediated autoimmune diseases.

NIA - National Institute on Aging

Project Summary: The overarching goal of this application is to investigate cell mechanics-mediated regulation of hair follicle stem cells (HFSCs) during homeostasis and aging. We propose to study microRNA-205- mediated regulation of extracellular matrix (ECM) and actin cytoskeleton for HFSC quiescence and activation and leverage the ability of microRNA-205 (miR-205) to stimulate HFSC activation to enhance HFSC aging. MicroRNA (miRNA) is a class of small noncoding, regulatory RNAs that play important roles in mammalian development, stem cells, diseases and aging. In our preliminary studies, we have determined mechanical properties of HFSCs during homeostasis and aging. We have revealed that bulge HFSCs reside in a stiff microenvironment with high actomyosin contraction forces. In contrast, hair germ progenitors are relatively soft and undergo periodic enlargement and contraction. Notably, induction of miR-205, one of the most highly expressed miRNAs in HFSCs, downregulates many bona fide targets, which are enriched in the function of ECM, actomyosin cytoskeleton and mechanosensing. And this leads to rapid activation of HFSC cell division and promotes hair regeneration in both young and aged mice. Mechanistically, we have identified Piezo1 as a novel target of miR-205, which functions downstream of miR-205 and translates mechanical cues into a gene expression program to reinforce the mechanical properties and maintain cellular states of quiescent HFSCs. To examine the role of PIEZO1-mediated calcium influx in HFSCs, we have further developed a high-resolution intravital imaging system to accurately record calcium influx in HFSCs over an extended period of time during quiescence and activation. This allows us to quantify cumulative calcium levels and further identify transcription factors, NFATC1 and JUN (AP1), which function downstream of PIEZO1-mediated calcium influx to promote the expression of the ECM and actin cytoskeleton genes. Based on these exciting findings and promising preliminary data, we propose to further elucidate the mechanism of miR-205-mediated HFSC activation and aging through the regulation of ECM and actomyosin contraction forces (Aim 1), determine the regulation of PIEZO1-mediated mechanosensing by miR-205 (Aim 2), and leverage miR-205-induced HFSC activation to improve HFSC functions and hair growth during aging (Aim 3). Together, this application will provide new insights into the mechanisms orchestrating the mechanical properties and stem cell functions of HFSCs. By harnessing the powerful combination of live imaging, cell biology, mouse genetics, and single-cell genomics, we will establish a new paradigm for studying tissue architecture, cell mechanics and underlying mechanisms. These results will lay the foundation for leveraging noncoding, regulatory RNAs to enhance HFSC functions during aging.

NIA - National Institute on Aging

Project Summary Collagen XII is known to bind to collagen I fibrils to create inter-fibrillar bridges that influence collagen fibril assembly and organization. Collagen XII also localizes at the cell surface in the pericellular matrix to form inter-cellular bridges that are critical for establishing cell organization in bone and tendon during development. Clinically, collagen XII deficiency presents with both features of connective tissue disorders and myopathy, and similar functional deficits have been recapitulated in a murine model for collagen XII deficiency. Recent work has established the role of collagen XII in development and injury regeneration in various tissues. However, despite myopathy appearing in middle-age collagen XII-deficient patients and worsening with age, no work has been done on aged (post-skeletal maturity) collagen XII-deficient animals to determine what is driving this clinical pattern. Thus, the mechanisms by which collagen XII regulates muscle-tendon structure and mechanical function during aging remain unknown. Therefore, our overall goal is to determine the differential role of collagen XII in the murine muscle-tendon unit in progressing age-associated changes to overall tissue function and regulating overall tissue function after skeletal maturity. Our global hypothesis is that collagen XII bridging is necessary for gap junction formation between cells throughout the muscle-tendon unit after development and that collagen XII mediates matrix interactions that maintain muscle-tendon unit composition, structure, and function during aging. Using powerful genetic mouse models that allow us to alter the expression of collagen XII, we will study the following aims: Aim 1: Define the role of collagen XII in cell-cell communication and ECM remodeling across the murine muscle-tendon unit during aging; Aim 2: Elucidate the differential roles of collagen XII in determining changes to structure, composition, and function across the murine muscle-tendon unit during aging. Rigorous, carefully chosen, and well-established structural, functional, compositional, and biological assays will allow us to define the differential role of collagen XII in musculoskeletal tissues in the contexts of aging and disease. The activities described by this proposal provide a strong foundation for scientific inquiry in the fields of musculoskeletal biology and biomechanics, preparing me to be valuable contributor to these fields as an independent investigator.

NIA - National Institute on Aging

PROJECT SUMMARY The incidence of diastolic dysfunction increases dramatically in women after menopause, which has long been attributed to loss of protection from female sex hormones, specifically estradiol. The mechanisms underlying estradiol-mediated cardioprotection are still unknown. A better understanding of biochemical pathways that are modulated in the female heart in response to estradiol has the potential to improve how we treat post-menopausal women with diastolic dysfunction. We have found that sarcomeric relaxation is significantly prolonged in healthy women after menopause compared to women who are pre-menopausal. We hypothesize that changes in sarcomeric PTMs due to decreased estradiol may contribute to this prolongation in relaxation. In addition to prolonged relaxation, we have also found that crotonylation of proteins is upregulated in old female hearts. In an unbiased screen of aged human hearts using mass spectrometry, we found a specific enzyme, short-chain enoyl-CoA hydratase (ECHS1), is significantly down regulated in the hearts of females over 60 years of age. Lower levels of ECHS1 increase crotonyl-CoA. This increase in crotonyl-CoA, in turn, leads to higher protein crotonylation, which is a novel PTM that occurs on lysine residues. The enzymes that facilitate this PTM include p300 and the enzymes that remove crotonylation from proteins are class I histone deacetylases (HDACs). While it has been shown that sarcomeric proteins are crotonylated, the role that estradiol plays in crotonylation and the functional effects of crotonylation are completely unknown. The objective of this proposal is to determine the role of estradiol in regulating cardiomyocyte crotonyl-CoA thereby modifying sarcomeric crotonylation. In this proposal, we will establish if lower ECHS1 in aged female cardiomyocytes is due to decreased estradiol (Aim 1) and determine the functional role of sarcomeric crotonylation (Aim 2). We hypothesize that reduced estradiol levels in aged female hearts lead to decreased ECHS1 in cardiomyocytes, increasing cellular crotonyl- CoA, sarcomeric protein crotonylation, thereby contributing to prolongation of sarcomeric relaxation. This R21 exploratory proposal will identify a completely novel mechanism underlying changes in cardiac diastolic function in response to estradiol. There are no data examining the role estradiol plays in regulating crotonylation nor data about how crotonylation of sarcomeric proteins modulate function. Completion of the proposed experiments will provide critical insights into specific modifications that occur in female hearts after menopause as well as contributing critical data necessary to elucidate our understanding of how an unexplored post-translational modification impacts cardiac function.

NHLBI - National Heart Lung and Blood Institute

Project Summary The lymphatic system plays an important role in maintaining tissue-fluid homeostasis, the absorption of dietary fats, and the transport of immune cells throughout the body to combat infection. Central conducting lymphatic anomaly (CCLA) is a disorder resulting from lymphatic network conduction abnormalities which can result in the accumulation of fluid in tissues. Despite recent advances in patient sample collection and more thorough genetic testing, only about 40% of patients with CCLA have a defined genetic cause. Identification of the underlying disease mechanism in these patients has resulted in the targeted trial of specific pathway inhibitors including MEK, ERK, and PI3K. Elucidating novel genetic causes of CCLA will inform the development of new custom therapies. Exome sequencing in a cohort of five families with children who presented with non-immune fetal hydrops revealed FZD6 variants. FZD6 has therefore emerged as a potential novel cause of CCLA. FZD6 is a WNT receptor and a core member of the planar cell polarity (PCP) signaling pathway. In humans, loss of FZD6 function causes nail dysplasia, which is also present in the identified cohort. The core PCP genes Celsr1 and Vangl2 are shown to be involved in lymphatic valve formation in mice and defects in PCP signaling due to pathogenic variants in CELSR1 have been implicated in lymphatic malformations in humans. However, the role of FZD6 in lymphatic network development and function is unstudied. We hypothesize that FZD6 is important for lymphatic system development, and the identified variants result in CCLA by disrupting PCP signaling. Further, in addition to the role PCP plays during valve formation, we propose PCP has an earlier role in shaping the lymphatic network by regulating lymphatic endothelial cell dynamics. I will test these hypotheses using PCP loss of function mouse models. I will assess the morphology of the developing lymphatic vessels and valves in PCP mutant embryos compared to littermate controls in the dermis and mesentery (Aim 1). I will then assess the molecular function of FZD6 variants identified in patients. Subsequently, I will determine how the loss of PCP function affects lymphatic network uptake and conductance capabilities by injecting a tracer molecule (Aim 2). Finally, I will determine which lymphatic endothelial cellular behaviors are influenced by PCP signaling through a live-imaging approach (Aim 3). Completing these aims will identify a novel genetic driver of CCLA and inform targeted treatments to rescue the effects of FZD6 loss. Further, the results of this study will fill the gaps in our understanding of the role of PCP signaling in lymphatic network formation during development.

NCI - National Cancer Institute

Project Summary/Abstract Urothelial carcinoma (UC) involves the urothelial cells that line the bladder, kidney and ureters and is a major cause of morbidity and mortality in the US, especially in men. Bladder UC can be clinically separated into nonmuscle invasive (NMIBC) and muscle invasive (MIBC). MIBC accounts for the vast majority of metastasis and mortality, having only a ~50% cure rate. Patients with treated NMIBC are at risk of recurrence or progression to MIBC at prior or de novo sites. Over half of urothelial cancer, regardless of site of origin, harbor loss of function mutations in the histone demethylase KDM6A (UTX) and in two highly homologous histone methyltransferases KMT2C (MLL3) and KMT2D (MLL4). These proteins form the MLL3/4-COMPASS (COMplex of Proteins ASsociated with Set1)-like complex that regulate enhancer function, partly through methylation of histones at enhancers. Enhancers are regions of DNA that regulate lineage specific transcriptional programs. Recent studies have shown that patients with two urothelial carcinomas in far away sites (ureter and bladder) harbor the same COMPASS-like mutation. Further sequencing of histologically benign urothelium identify frequent mutations in the complex at expand over time. Our hypothesis is that these mutations under “field-cancerization” of the urothelium. Our lab has generated a genetically engineered mouse model with deletion of Kmt2c, Kmt2d, or the combination in the urothelium. The urothelium of these mice exhibit no histologic abnormalities. However, transcriptome analysis shows the urothelium exhibit increased stemness and functional studies show they exhibit increased organoid forming abilities. When crossed into the Pten conditional deletion mouse, there was robust cooperativity in tumorigenesis. The overall objective of this proposal is to utilize our recently generated mouse models of urothelial this COMPASS-like complex loss to mechanistically understand its role in tumor urothelial suppression. Specifically, in Aim 1, we seek to determine the stemness, clonal dynamics, oncogene and carcinogen susceptibility of urothelium harboring mutations in this COMPASS-like complex, using lineage tracing, organoid culture, and single-cell RNA-sequencing. In Aim 2, we seek to determine the functional interplay between MLL3/4-COMPASS dysfunction and oncogene activation. In Aim 3, we will seek to define how loss of Kmt2c and Kmt2d in urothelial cells affect enhancer and promoter function. Active enhancers are genomic regions of open chromatin with transcription factor binding, divergent transcription of enhancer RNA, and looping to promoters. We will study each step by global mapping of histone marks, chromatin accessibility, mRNA transcription of associated gene and looping to promoters using state-of the art epigenetics techniques.

NIA - National Institute on Aging

PROJECT SUMMARY Down syndrome (DS), caused by an extra copy of chromosome 21 (Hsa21), is the most common genetic cause of intellectual disability and is also strongly linked to dementia. About 75% of DS individuals eventually develop Alzheimer’s disease (AD)-like symptoms, with amyloid-beta (Aβ) plaques emerging decades earlier than in typical AD patients. APP, an Hsa21 gene almost universally overexpressed in DS, has long been considered a key driver of these pathological changes. However, dementia onset varies widely, with some individuals developing symptoms in their 40s, while others remain unaffected into their 60s or beyond. Given that Hsa21 contains over 200 protein-coding genes, this variability suggests that genetic factors beyond APP may contribute to dementia risk and neurodegeneration progression in DS. Identifying these additional factors is crucial for understanding DS-AD pathogenesis and developing early interventions. While some Hsa21 genes have been linked to DS traits, a systematic mapping of most Hsa21 genes to DS phenotypes, especially aging- related, is lacking. Investigating numerous Hsa21 genes in mammalian models is particularly challenging due to time and cost constraints. To overcome this, we will leverage Caenorhabditis elegans as an efficient and genetically tractable in vivo system to study aging. Our preliminary investigation, based on existing genetic and functional data, identified that C. elegans have orthologs for over 50 Hsa21 genes, many of which are highly conserved in protein sequence and biological function. However, for most, their roles in neuronal health post- development remain unclear. This project will investigate how overexpression of Hsa21 orthologs affects neurodegeneration specifically during aging, using an integrated approach combining genetics, cell biology, biochemistry, and behavioral analysis. Overexpression will be induced via traditional transgenic approaches as well as CRISPR-based gene activation, the latter enabling controlled, physiologically relevant expression increases (~50%), similar to those seen in DS. Aim 1 will identify individual Hsa21 orthologs that accelerate or exacerbate neuronal aging using our well-characterized neurodegeneration models. We will also examine potential synergistic interactions among genes with highly correlated expression patterns in DS individuals, addressing whether combinatorial effects contribute to neurodegeneration progression. Recognizing that Aβ accumulation also occurs in healthy aging, Aim 2 will determine whether specific Hsa21 orthologs interact with Aβ to mediate neurotoxicity, using established C. elegans Aβ models. Successful completion of this project will identify key Hsa21 genes that contribute to neurodegeneration and generate a comprehensive set of relevant Hsa21 overexpression strains and reagents as a widely accessible resource for the research community. These findings and resources will facilitate future mechanistic studies and validation in mammalian systems, ultimately informing the development of preventive and therapeutic strategies to improve care for DS individuals, while also uncovering novel genetic factors potentially relevant to sporadic AD.

NIGMS - National Institute of General Medical Sciences

The extracellular matrix (ECM) provides essential tissue infrastructure and mechanical cues that regulates cell metabolism. Mechanotransduction is the conversion of mechanical forces from the ECM to intracellular chemical signals and plays a vital role in health and disease through cell-matrix interactions. While evidence supports a ‘mechano-metabolic link’ between mechanotransduction and metabolism, the precise pathway connecting ECM mechanical states to cellular metabolism remains poorly understood due to the ECM’s complexity, including its viscoelastic properties, which exhibit both strain-independent (linear) and strain-dependent (nonlinear) regimes. All tissues exhibit both linear and nonlinear viscoelasticity, typically reported as stiffness and strain-stiffening, respectively; however, a fundamental gap remains in understanding how these distinct mechanical properties counterbalance each other to regulate cell metabolism. Key open questions include how cells engage with nonlinear viscoelastic environments, how mechanotransduction scales with cell and tissue maturity, and how nonlinear viscoelasticity influences cellular uptake and consumption of metabolic biomolecules. To address these gaps, this proposal uses primitive and differentiated induced pluripotent stem cells (iPSCs), both as single-cell and organoid cultures, in a 3D in vitro polymeric hydrogel systems to independently present cell-accessible and cell-inaccessible nonlinear viscoelastic regimes. We combine this with material- and omics-based modeling approaches to define viscoelastic and metabolic signaling regimes. In Project 1, we will use ECM ligand-binding motifs and both primary cells and iPSCs to investigate integrin-mediated cell-matrix interactions across viscoelastic regimes, cell maturity, and tissue complexity. In Project 2, we will examine how nonlinear viscoelasticity influences cellular uptake of metabolic precursors such as lipids and apply model-based approaches to define characteristic metabolic and proteomic signatures associated with linear and nonlinear regimes. The outcomes of this proposal will advance our understanding of mechanosignaling in nonlinear viscoelastic environments and establish a mechanistic model of the mechanical regulation of cellular metabolism. The long-term goal of the lab is to develop complex in vitro models to investigate how physiological processes such as aging and pregnancy induce systemic tissue alterations that drive changes in cell-matrix interactions and mechanosignaling, ultimately influencing cell and tissue function. By uncovering a direct mechano-metabolic connection, this work will have broad implications for fundamental biology while also developing critical tools and workflows for studying cell-matrix interactions. Importantly, while this proposal focuses on fundamental biological processes, the methods and tools developed will be broadly applicable across various cell, tissue, and disease states.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY To ensure a robust immune response to pathogens without risking immunopathology, the kinetics and amplitude of inflammatory gene expression in macrophages need to be exquisitely well-controlled. To mount balanced inflammatory responses, macrophages need to be able to coordinate the transcription and processing of thousands of genes that are synthesized de novo following pathogen sensing. Yet, we have an incomplete understanding of the molecular mechanisms employed by macrophages to properly regulate the timing and amplitude of inflammatory gene expression. Key to organization and compartmentalization of the nucleus are biomolecular condensates. Condensates, which include structures like the nucleolus, Cajal body, and nuclear speckle, are known to concentrate functionally related nucleic acids and proteins. We have become interested in one such biomolecular condensate called the nuclear paraspeckle, which forms on a long lncRNA called Neat1. Studies of model cell types (e.g. HeLa and HEK293T) have shown that paraspeckles can aggregate in response to a variety of cellular stresses (e.g. osmotic stress, hypoxia, sodium arsenite), but how paraspeckle dynamics change in response to more physiological stresses—and how they may help regulate the cell’s response to stress—is not well-understood. My project aims to define the role of the paraspeckle in macrophage activation. Our lab recently discovered that macrophage paraspeckles are dynamically regulated over an early time-course of macrophage activation via lipopolysaccharide (LPS). We also found that in response to LPS treatment, Neat1 KO macrophages fail to properly express a large cohort of proinflammatory cytokines, chemokines, and antimicrobial mediators and consequently, cannot control replication of Salmonella enterica or vesicular stomatitis virus (VSV). I hypothesize that macrophage paraspeckles regulate innate immune gene expression by dynamically sequestering and/or releasing nuclear proteins/RNA binding proteins, thus impacting their concentration/bioavailability for participation in innate immune gene expression. Here, I will investigate how different stimuli impact paraspeckle dynamics in macrophages, how paraspeckle composition changes in response to LPS, and how differential sequestration of nuclear proteins in paraspeckles controls innate immune gene expression. In Aim 1, I will define the dynamics of macrophage paraspeckles in response to various cellular stresses, carrying out a high-throughput screen using macrophage cell lines that express GFP-tagged paraspeckles. In Aim 2, I will apply a cutting-edge biotin proximity labeling approach called O- MAP to compositionally define the paraspeckle over a time course of LPS and follow up on how novel paraspeckle-associated proteins contribute to macrophage antimicrobial responses. Together, these experiments will provide important insights into how cellular stresses are transmitted to the nucleus to control paraspeckle dynamics and how reorganization of proteins in and out of paraspeckles controls innate immune gene expression in macrophages.

NCI - National Cancer Institute

PROJECT SUMMARY Over the past decade, non-genetic mechanisms have emerged as powerful drivers of melanoma initiation and progression. These mechanisms include epigenetic, transcriptional, and post-transcriptional alterations that lead to dynamic and reversible changes in gene expression programs. Among these, the deregulation of transcription factors plays particularly potent roles in melanomagenesis, and we have identified the transcription factor MAFG as a critical player in melanoma development. MAFG exhibits increased expression levels in human melanoma and promotes the oncogenic behavior of melanoma cells. Additionally, overexpression of MAFG in a genetic mouse model of melanoma dramatically accelerated melanomagenesis. Conversely, genetic deletion of MAFG in a genetic mouse model of aggressive melanoma completely prevented the transition from nevi to frank melanomas, indicating MAFG’s critical role during tumor initiation. The oncogenic function of MAFG may, in part, be mediated by a phenotype switch and an interaction with the lineage transcription factor MITF. We hypothesize that MAFG acts as both a potent driver and a specific vulnerability in melanoma, making it a promising target for therapeutic intervention. However, critical questions remain about the cell biological and molecular influence of MAFG on melanomagenesis, which we will address in this proposal. Aim 1 will uncover the role of MAFG in melanocyte transformation and melanomagenesis. Using our ESC-GEMM platform, we will comprehensively evaluate MAFG’s role in melanocyte transformation, melanoma initiation, malignant progression, and metastasis across various genetic contexts. This Aim seeks to establish the foundational impact of MAFG in melanoma development. Aim 2 will decipher the oncogenic mechanisms of MAFG through its binding partners. We will investigate how MAFG cooperates with MITF to drive melanomagenesis and determine if MITF is required for the oncogenic effects of MAFG. Moreover, we will determine if the canonical binding partner BACH2 cooperates with MAFG to drive melanoma. This Aim will elucidate the molecular pathways by which MAFG exerts its oncogenic effects, focusing on its interaction with key transcriptional regulators. Aim 3 will define the vulnerability of melanoma to MAFG depletion. We will assess the molecular consequences of acute MAFG depletion in melanoma and perform a CRISPR screen to identify actionable upstream regulators of MAFG. This Aim will uncover potential therapeutic targets by highlighting the dependency of melanoma cells on MAFG. By addressing these Aims, our studies will provide critical insights into MAFG’s role in melanoma biology and pave the way for novel therapeutic strategies targeting MAFG and its downstream pathways. This work holds significant potential for improving outcomes for patients with metastatic melanoma, a currently intractable malignancy.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Affecting over 12 million people worldwide, Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common inherited kidney diseases and is caused primarily by mutations in two genes—PKD1 and PKD2. In the 30 years since the identification of these genes, extensive effort has been made to understand how their protein products function under normal conditions and further, how naturally occurring mutations alter this function in ADPKD. While significant progress has been made to characterize PKD2 as a Ca2+-permeable nonselective ion channel, the function of PKD1 has proven more elusive. PKD1 has been proposed to act as an atypical G protein-coupled receptor (GPCR), but studies to define this function are limited by the absence of a known activating stimulus and lack of a typical 7-transmembrane (7-TM) domain signature present in all known GPCRs. We have discovered secreted WNT proteins as the activating ligands of the PKD1/PKD2 complex, defining this complex as a distinct class of WNT receptors. We asked the question of whether PKD1 functions as a WNT-activated GPCR. Using super-resolution live cell imaging and bioluminescence resonance energy transfer-based G protein biosensors, we find that PKD1 is directly coupled to specific subsets of heterotrimeric G proteins, such as Gαi1-3 and Gαq, but not Gαs or Gα12/13 in a WNT ligand-dependent manner. Specifically, we show that WNT ligands induce: 1) PKD1-mediated dissociation of Gα from Gβγ subunits, 2) time-dependent recruitment of Gα subunits to PKD1, and 3) Gα-GDP to -GTP transition via PKD1. The WNT-induced coupling of PKD1 to Gαi1-3 inhibits basal and forskolin-induced cAMP accumulation. PKD2 has an essential role in enabling PKD1 to function as a GPCR by chaperoning it to the plasma membrane. Naturally occurring mutations in PKD1 or PKD2 that compromise cell surface expression, assembly of the PKD1/PKD2 complex, or disrupt G protein recruitment diminish WNT-induced PKD1-mediated GPCR signaling. These data, which form the premise of this application, define the molecular function of the Polycystin complex and provide a direct mechanistic link of the complex to cAMP metabolism. We propose complementary in vitro and in vivo genetic approaches to take these findings to the next level to determine whether the direct coupling of PKD1 to Gαi and cAMP signaling is critical for the initiation of cystogenesis. Successful completion of the proposed studies will remove previously impossible technical and conceptual roadblocks to determine how naturally occurring mutations alter the function of these complex molecules and lead towards the development of new and more effective treatments for ADPKD.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Coronary artery disease (CAD) remains a rising cause of morbidity and mortality in the United States. Human genetics suggest that a significant portion of the inheritable risk of CAD is related to fibroblast-specific regions of the genome. Using single cell transcriptomics, our group has identified a transcriptionally distinct adventitial fibroblast (AdvFib) population, which undergoes early phenotypic modulation during atherosclerosis development. Specific ablation of AdvFib significantly reduces plaque burden, suggesting their ability to influence atherosclerosis. To identify regulators of AdvFib, we characterized regions of altered chromatin accessibility upon AdvFib activation, revealing the transcription factor Tcf21. Tcf21 is predominantly expressed in AdvFib, and its expression declines upon AdvFib phenotypic activation. Tcf21 knockout in murine AdvFib increases vascular calcifications and upregulation of pro-atherogenic inflammatory cytokines. Given the restricted expression of Tcf21 to AdvFib and phenotypic activation of AdvFib upon Tcf21 knockdown, the central hypothesis underlying this proposal is that overexpression of Tcf21 locks AdvFib in a quiescent state, which then prevents the downstream activation of non-cell-autonomous signals that trigger plaque formation. Using an AdvFib-specific Tcf21 overexpression mouse model, I will characterize histological changes in plaque and alterations in non-AdvFib plaque cells, such as smooth muscle cells and inflammatory cells. I will then use single-cell RNA sequencing to characterize transcriptional changes of AdvFib upon Tcf21 overexpression and downstream transcriptional changes in non-AdvFib plaque cells. Given the putative effect of Tcf21 on the activation/recruitment of inflammatory cells, my goal is to characterize the precise epigenetic regions of Tcf21 binding to understand how Tcf21 regulates inflammatory cytokine production. Overall, this study will elucidate the role of AdvFib in atherosclerosis and characterize how Tcf21 regulates AdvFib.

NEI - National Eye Institute

Project Summary: Neurodevelopmental disorders, including syndromic disorders of retinal and brain development, are a major cause of morbidity in children. A subset of these disorders results from abnormal vascular development, and microcephaly and chorioretinopathy (MCCRP) is a recently identified disease that may belong in this category. It is characterized by small head circumference, brain anomalies, developmental delay, and vision loss due to chorioretinopathy and abnormal retinal vasculature. Autosomal recessive MCCRP results from defects in TUBGCP4 or TUBGCP6, which encode components of the gamma-tubulin ring complex (γ-TuRC), a ubiquitous structure necessary for microtubule nucleation and spindle formation in cells. However, γ-TuRC has not previously been implicated in vascular development and it is unknown why defects in γ-TuRC lead to blindness and microcephaly. We demonstrated that Tubgcp4 and Tubgcp6 expression is highly upregulated in murine vascular endothelial cells (EC) from the retina and brain (relative to EC from other tissues), and that murine EC deficiency of TUBGCP4 results in embryonic lethality, indicating a critical role for TUBGCP4 in EC. Our long-term goal is to identify the role of the γ-TuRC in retinal and brain development. The objective of this application is to define the pathophysiology of TUBGCP4 and TUBGCP6-associated MCCRP. We will test the hypothesis that TUBGCP4 and TUBGCP6 serve critical, EC-specific roles in the retina and brain, and that deficiency of these proteins results in MCCRP due to a primary vascular developmental defect. Since retinal and cerebral vascular development is not complete until several weeks after birth, we will use novel conditional knockout mouse models of Tubgcp4 and Tubgcp6 and a tamoxifen-inducible EC-specific Cre recombinase to eliminate expression of these genes in EC postnatally. Ophthalmic studies will demonstrate the necessity of EC-specific TUBGCP4 and TUBGCP6 in retina and retinal vascular development, including optical coherence tomography (OCT), OCT-angiography, electroretinogram, optokinetic response, and retinal histology (Aim 1). Neurologic studies in mice and/or embryos lacking TUBGCP4 or TUBGCP6 in EC will demonstrate the necessity of these proteins in cerebral and neurovascular development, including MRI brain imaging, neurobehavioral studies, and brain immunohistochemistry (Aim 2). Elucidating the pathophysiology of MCCRP will improve our understanding of the genetic mechanisms controlling retinal and brain vascular development, and may reveal new therapeutic targets for more common blinding retinal vascular diseases. The career development objective of this proposal is to develop the mentorship and expertise needed to become a productive independent clinician-scientist and international leader working at the intersection of inherited retinal diseases (IRD) and disorders of vascular development. OHSU is a center of renowned expertise in IRDs, in vivo retinal vascular imaging, and the neurosciences; it provides state of the art resources and world-class faculty to support Dr. Everett’s scientific and career development goals for this proposal.

NCI - National Cancer Institute

ABSTRACT Neuroendocrine prostate cancer (NEPC) is an aggressive and lethal subtype of prostate cancer (PC). While NEPC can develop as de novo disease, more commonly it develops as a resistance mechanism to Androgen Receptor (AR) targeted therapies in castrate resistance prostate cancer (CRPC) patients. NEPC patients often present with high metastatic burden. The prognosis of NEPC is poor with a 5-year survival rate of less than 20% with limited treatment options. Thus, new tractable molecular targets to combat NEPC are urgently needed. In our preliminary studies we have identified Tyrosine Threonine Kinase (TTK) as a new high value NEPC molecular target. TTK, also referred to as Monopolar Spindle 1 (Mps1), is a dual specificity protein kinase that plays a significant role in mitosis and the spindle assembly checkpoint (SAC). TTK expression is undetectable in normal cells and upregulated in transformed cells, making it an ideal therapeutic target. Moreover, TTK is a highly translational target, which as five small molecule inhibitors in clinical development. TTK expression progressively increases throughout PC disease progression and is highest in NEPC. The TTK expression trend holds in PC cell lines and patient-derived xenograft (PDX) models, wherein TTK expression is highest in cell lines and PDXs of NEPC origin. Genetically and pharmacologically targeting TTK slows NEPC cell growth in vitro and in vivo. These data support that TTK is an actionable target that may impede NEPC tumor growth. The objective of our proposal is to test the potential of TTK as a target for NEPC and define TTK mechanisms of action. For the latter, we conducted high resolution differential phospho-proteomic analysis in TTK targeted NEPC cells. We have prioritized Rab-Like Protein 6 (RABL6) as a candidate TTK NEPC phospho-target. In specific Aim 1, we will evaluate TTK phospho-target RABL6 and test the role of RABL6 in NEPC. In Aim 2, we will utilize a combination of NEPC cell line, patient derived xenografts and syngeneic NEPC models to rigorously test the effects of targeting TTK in vivo. Along with genetic approaches we will utilize clinical grade TTK inhibitors and preclinical TTK proteolysis targeting chimeras (PROTAC). In Aim 3 we will evaluate the therapeutic efficacy of combining leading TTK clinical inhibitor with first line NEPC chemotherapies. Transcriptomic analysis and immune cell profiling of tumors will be conducted to assess therapeutic response and identify biomarkers. Successful completion of these studies will confidently determine if TTK is a high value target which may ultimately help to lessen the morbidity of NEPC.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Non-typhoidal Salmonella enterica (NTS) is a leading cause of bacterial foodborne gastroenteritis and leading cause of death amongst foodborne pathogens. NTS elicit neutrophilic inflammation both during gut colonization and in systemic infection. Neutrophils produce toxic products to kill invading pathogens. One toxic product produced by activated neutrophils during severe bacterial infection is sulfite. Sulfites are reactive sulfur species that are toxic to all life forms. In addition to their presence during severe bacterial infection, sulfites are endogenously produced during metabolism of sulfated amino acids and are ingested in food products. Sulfites are also added to food products as antimicrobial agents. Therefore, enteric pathogens such as NTS must resist sulfite toxicity in several stages of infection. Our preliminary data link yeiH with sulfite stress resistance in Salmonella Typhimurium. We show a ∆yeiH mutant is defective for growth in sulfite stress conditions and yeiH expression is specifically activated by sulfite. yeiH encodes a highly conserved putative inner membrane protein with no documented function. The purpose of the proposed work is to establish the role of YeiH in sulfite stress resistance. We hypothesize that YeiH exports sulfites to allow Salmonella to resist neutrophil-derived sulfite stress during infection. We will test our hypothesis in two aims. In Aim 1, we will establish the role of yeiH in infection. We will use animal models of colitis and sepsis to establish whether yeiH is needed for Salmonella fitness during infection. Using a yeiH transcriptional reporter as a novel sulfite biosensor, we will determine the tissues in which yeiH is expressed to establish the spatial organization of sulfite stress during enteric and systemic salmonellosis. In Aim 2, we will determine how YeiH contributes to sulfite stress resistance. We will determine whether the proton motive force drives YeiH function and establish the amino acid residues important for YeiH role in sulfite stress resistance. We will also establish whether YeiH function is to export sulfite from the cell. YeiH orthologs are present in many bacteria, including in different pathogens with importance to human health. Successful completion of the proposed experiments will allow us to establish how YeiH contributes to Salmonella sulfite stress resistance in vitro and in vivo. This work will lead to future studies to define the mechanism of YeiH function and establish how sulfites contribute to human health and resistance to bacterial infections.

NIAID - National Institute of Allergy and Infectious Diseases

Project summary Enterovirus D70 (EVD70) is an understudied human enterovirus that causes acute hemorrhagic conjunctivitis, a highly contagious and painful ocular disease and that can, in rare instances, progress to polio-like paralysis. The molecular mechanisms underlying EVD70 infection, including 1) the host factors required for the viral life cycle and the 2) cellular tropism within the eye, remain poorly understood, due in part to a lack of eye-specific models. EVD70 attaches to ocular cells, at least in 2D cell culture, via sialic acids. There is, however, limited evidence suggesting the existence of an as-yet-unidentified proteinaceous receptor for EVD70 that triggers entry and uncoating in ocular cells. Through a genome-wide genetic knockout screen, I have identified host factors necessary for EVD70 infection in vitro, including the orphan receptor Jumping translocation breakpoint (JTB), which has limited demonstrated cellular functions. Notably, genetic knockout of JTB confers resistance to EVD70. I hypothesize that JTB serves as a receptor critical for EVD70 entry and uncoating in ocular cells and that JTB expression contributes to cellular tropism for corneal and conjunctival epithelial cells in the eye. The objectives of this proposal are to 1) determine the mechanism by which JTB is required for EVD70 infection and 2) define the cell-specific tropism and host immune responses to EVD70 in corneal organoids, which recapitulate the cellular diversity and organization of the ocular surface. Aim 1 will systematically evaluate if JTB is an entry and uncoating receptor for EVD70. Aim 2 will define the tropism of EVD70 and investigate host responses, such as interferon signaling, in corneal organoids using unbiased single cell RNA- sequencing and multiplexed cytokine analysis. Together, these aims will provide critical new insights into EVD70 biology and its interaction with ocular cells. Completing the proposed aims will advance our understanding of EVD70 molecular biology and pathogenesis and provide training to better equip me to progress towards independence as a scientist.

NIGMS - National Institute of General Medical Sciences

DNA binding to chromosomal DNA is essential for many fundamental biological processes including: transcriptional regulation, DNA replication and repair, recombination, and chromosome segregation. There is a fundamental gap in understanding how transcription factors (TF) bind regulatory regions located in compacted, high-order chromatin. Therefore, there is a fundamental need to determine the mechanistic rules defining TF binding to chromatin. Our long-term goal is to define the biological rules dictating TF binding to chromosomal DNA in a cell. All TFs, even pioneer factors, are not able to bind all their targets throughout the genome, indicating that there are binding constraints imposed on all binding events. Multiple factors appear to impact how TFs identify TF binding sites (TFBSs) including: TFBS positioning within a nucleosome, TFBS variants, sequence of the nucleosome, cooperativity, histone modifications, DNA methylation, and chromatin remodeling. To accomplish our goal, we will combine in vitro with in-cell assays to allow the systematic dissection of factors required for initial and sustained/functional TF binding to chromatin. At the nucleosome level, we will use our in vitro Pioneer-seq assay, which allows the direct comparison of TFBSs located in all possible nucleosome positions, with differing nucleosome sequences, across many TFBS variants, with modified histones. At the regulatory region level, we will ectopically express pioneer TFs in comprehensively characterized cell lines, with cell models of disease, to uncover the chromatin characteristics, chromatin remodelers, or cooperativity required for TF binding. With this MIRA our lab will close these knowledge gaps by answering three fundamental questions: 1) How do TFs recognize their binding sites within a nucleosome? 2) How do histone modifications and nucleosome positioning regulate TF binding? 3) How does chromatin organization regulate TF binding? Our results are expected to have an important positive impact on our mechanistic understanding of how all TFs regulate transcription and direct gene expression during normal development and disease. Our findings will likely be transformative by providing quantitative models for how proteins bind to their targets within or near nucleosomes.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract While the advent of antiretroviral therapy (ART) has improved the lives of many people with HIV (PWH), HIV infection remains persistent for the lifetime of most individuals. ART interruption leads to viral rebound and renewed disease progression, thus highlighting the existence of the latent HIV which exists even when viral RNA is undetectable in plasma during ART. To date, there is no broadly accessible cure for HIV. This requires most PWH to stay virally suppressed with a constant ART regimen. As such, there remains an urgent need to find functional cures of HIV and alleviate the need for a lifetime of costly ART and its associated long-term side effects. However, the complex nature of the HIV reservoir has impeded our collective efforts to understand the reservoir for elimination strategies. Viral transcription decreases over time on ART and can be nonexistent in many HIV+ cells, thus leading to evasion of immunosurveillance. The leading concept in the field for eliminating the reservoir is to reverse latency using pharmacological agents so that viral antigen is produced to render HIV+ cells permissive for elimination by an interventional therapy. However, current attempts at latency reversal are neither efficient nor complete as only a fraction of HIV+ cells are reactivated. This suggests that transcriptional regulation of the various proviruses (i.e. the integrated viral DNA) is highly heterogeneous. Evidence further suggests that proviral epigenetics, defined as the regulation of transcription at the chromatin level independent of sequence, is critical in understanding how the latent HIV reservoir is established, regulated, and disrupted. Some proviruses are situated in more condensed (heterochromatic) DNA without transcription, while others are in more accessible (euchromatin) DNA that is more conducive to transcription. This spectrum of reactivation potential means that reservoir elimination will first require a single-cell investigation to precisely define these proviral epigenetic states and assess their contribution to latency reversal. Therefore, the primary objective for this project is to define the ex vivo proviral epigenetic landscape at single-cell resolution and to develop a pioneering strategy to selectively reverse latency. The underlying central hypothesis is that HIV+ cells with heterochromatin-associated proviruses are more resistant to reservoir disruption and latency reversal. The central hypothesis will be tested through two specific aims: 1) define the proviral epigenetic landscape and its susceptibility to reservoir disruption from ART-treated PWH; and 2) determine the efficacy of mRNA-LNP delivery of HIV-specific transcription activator-like effectors (TALEs) for latency reversal. This proposal is highly innovative because it represents a major shift in how the field studies the HIV reservoir by using new single-cell methodology to circumvent the challenges inherent to studying the HIV reservoir. The outcomes will uniquely define the identities of the latent HIV reservoir and will offer additional opportunities to target and eliminate the HIV reservoir.