Now showing items 1-20 of 1215

    • Negative regulation of innate immunity by novel nuclear receptor NHR-42/NR1D1 with implications in infection survival, metabolism, and fitness

      Goswamy, Debanjan (2022-11-16)
      Detection of pathogenic signals leads to extensive changes in cellular transcriptional programs mediated by transcription factors. Positive and negative regulators of this host defense response work together to maintain immune homeostasis. Over the past two decades, several evolutionary conserved positive regulators of innate immunity have been identified in C. elegans. However, negative regulators remain unknown, and repression of the host defense response poorly understood. We previously discovered that HLH-30/TFEB is a positive regulator of immunity in C. elegans and murine macrophages after S. aureus infection, respectively. In this study, I identify nhr-42 as a negative regulator of immunity functioning downstream of HLH-30, with major implications for host survival, metabolism, and fitness. In nhr-42 mutants, several host defense genes, such as antimicrobial peptides, C-type lectins, and lysozymes, are upregulated constitutively. This enables nhr-42 mutants to have enhanced survival and lower pathogen burden after infection compared to wild type animals. I show that nhr-42 expression is induced in the pharynx and pharyngeal-intestinal valve after infection. Furthermore, I find that antimicrobial peptides abf-2 and cnc-2 are required for enhanced survival and lower pathogen burden in nhr-42 mutants. Moreover, induction of nhr-42 after infection leads to upregulation of lipid catabolism genes involved in beta-oxidation, driving lipid mobilization. These data show that nhr-42 functions to limit the host defense response to maintain immune homeostasis and reallocate energy resources through lipid mobilization towards other cellular processes. Additionally, I identify Nr1d1 (Rev-Erbα) as a functional homolog of nhr-42. I show that Nr1d1 functions downstream of TFEB to negatively regulate pro-inflammatory cytokines Il-6 and Il1b in macrophages, after S. aureus infection. These data open up new research avenues into mammalian nuclear receptor mediated regulation of immunity.
    • Prescribed Medications and Healthcare Resource Utilization in Reproductive-Age Women and Men with Rheumatic Disease

      Shridharmurthy, Divya (2022-11-15)
      Background: Axial spondyloarthritis (axSpA), rheumatoid arthritis (RA), and psoriatic arthritis (PsA) are the most prevalent forms of chronic immune-mediated inflammatory arthritis, affecting approximately 0.3-1.4% of adults in the US. These rheumatic diseases (RD) have an early age of onset and may have a significant impact on women and men of reproductive ages, particularly during pregnancy and the postpartum period. Methods: Using the IBM® MarketScan® Commercial Claims and Encounters Database (2013-2018), this dissertation first described sex differences in time to non-steroidal anti-inflammatory drugs (NSAIDs) or biologic disease modifying antirheumatic drugs (bDMARDs) initiation among patients with axSpA. We then evaluated co-management with rheumatology and bDMARD prescriptions filled during pregnancy among pregnant women with axSpA, RA, and PsA. Finally, we evaluated postpartum depression (PPD) rates among reproductive-age women with axSpA, RA or PsA compared to those without RD. Results: Overall, women experienced a delay in biologic treatment initiation compared with men. Dispensations for bDMARDs during pregnancy were low in the RA/PsA subgroup, and extremely uncommon among those with axSpA, and did not align with recommendations from clinical practice guidelines. While the receipt of rheumatologic care during and after pregnancy among RD patients was low, the prevalence of PPD in women with RD was higher compared to those without any RD. Conclusions: Findings from this dissertation emphasize the need for additional research to improve and prioritize care for reproductive-age women with rheumatic disease, particularly before, during, and after pregnancy. This could be accomplished through patient education, counseling, increased access, screening, and timely referrals to rheumatologists and mental health care specialists through enhanced care coordination of providers.
    • Introns Safeguard mRNA Expression in the C. elegans Germline against Multiple Surveillance Mechanisms

      Makeyeva, Yekaterina (2022-11-11)
      Organisms employ sophisticated systems for genome defense against foreign and potentially harmful elements, while leaving room for gene adaptation. In animals, conserved PIWI Argonautes use genomically encoded small RNA guides (called piRNAs) to detect and silence foreign nucleotide sequences, such as transposons. In Caenorhabditis elegans, the detection of foreign transcripts by PIWI triggers the production of a second class of antisense small RNAs (called 22G-RNAs), which guide worm-specific Argonautes (WAGOs) to direct transcriptional and posttranscriptional silencing. PIWI-piRNA complexes recognize targets via imperfect base-pairing, which could threaten the expression of endogenous host genes. Nevertheless, worms use yet a third small RNA pathway involving the Argonaute CSR-1 to license endogenous germline gene expression and prevent inappropriate silencing by the PIWI pathway. How and why certain genes are licensed remains unknown. Here I show that introns and, by inference, mRNA splicing protect messenger RNAs from germline silencing. Intronless reporters encounter 22G-RNA-dependent and -independent silencing mechanisms, which we collectively termed “intronless silencing.” Genetic studies revealed that primary Argonautes, e.g., PIWI, are not required for the 22G-RNA-dependent intronless silencing mechanism, suggesting that intronless reporters are silenced by default. Nuclear and cytoplasmic WAGOs enabled the transmission of silencing from an intronless allele to a homologous intron-containing allele. The 22G-RNA-independent mechanism not only reduced intronless reporter mRNA levels, compared to the homologous intron-containing genes, but also prevented polyadenylation and nuclear export. Cis-acting elements that promote export from the nucleus nevertheless failed to fully activate expression of intronless reporters, suggesting additional layers of regulation in the small RNA-independent mechanism of intronless silencing. These findings suggest that multiple germline surveillance systems monitor transcript splicing, reveal a protective role of splicing in transcript licensing, and provide evidence for a splicing-dependent, sequence-independent mode of Argonaute programming.
    • A Prognostic Model to Predict Survival in Children with Ebola Virus Disease

      Butler, Kelsey M. (2022-11-08)
      Repeated outbreaks of Ebola Virus Disease (EVD) in low-resource settings emphasize the importance of evidence-based guidelines to direct treatment. Previous research has shown that EVD causes high case fatality rates (CFRs) in young children, yet there are limited data focusing on pediatric patients. Here we present a prognostic model to predict mortality in children who are Ebola-positive using information available during the first 48 hours after admission to the treatment center. A logistic regression model was trained on triage data from the Ebola Data Platform, a repository of retrospective patient data compiled from actors that responded to the West African EVD outbreak from 2014-2016. Patients <18 years of age were included in the analysis (N=579) and the CFR was 40%. Overall 13% of data were missing, and multiple imputation was used to estimate missing values. Variable selection using elastic net regularization selected age, CT value, bleeding, breathlessness, bone or muscle pain, anorexia, swallowing problems, and diarrhea as predictors. Bootstrap validation yielded an optimism-corrected area under the curve (AUC) of 0.75 (95% CI: 0.71-0.79). The model was externally validated using data from the current EVD outbreak in the Democratic Republic of the Congo (DRC). While the model’s discriminative ability on the DRC data was similar (AUC=0.75, 95% CI: 0.63-0.87) to the training data, calibration was poor. We recalibrated the model by re-estimating the intercept and slope, and further improved model performance by including aspartate aminotransferase (AST) as a biomarker. The updated model with AST as an added predictor has an AUC of 0.90 (95% CI: 0.77-1). These preliminary results are encouraging but should be interpreted with caution because of limited availability of AST values in the validation data (n=25). The prognostic model described here has promising potential for use in a clinical setting and will continue to be validated as more data becomes available. Future efforts will focus on integrating the validated model into mHealth tools to aid clinicians in making informed, data-driven decisions about patient care.
    • Investigating Cell-Autonomous Mechanisms of Microglia Dysfunction in PFN1-ALS

      Funes, Salome (2022-10-19)
      Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by loss of motor neurons. Cumulative evidence shows that microglia contribute to disease progression, but the underlying mechanisms are unclear. Several ALS-related genes are highly expressed in microglia compared to neurons, including profilin-1 (PFN1). This raises the possibility that ALS-linked PFN1 mutations could induce microglia cell-autonomous dysfunction. Here, I sought to interrogate this possibility by differentiating human pluripotent stem cells (iPSCs) into microglia-like cells (iMGs). My work uncovered that ALS-PFN1 iMGs accumulate undegraded phagocytosed cargo in endo-lysosomal compartments which is recapitulated in vivo. ALS-PFN1 iMGs also exhibit dysregulation in the expression and cellular localization of crucial components of the endo-lysosomal pathway, impairments in the autophagy flux, and accumulation of lipid droplets. Intriguingly, rapamycin treatment ameliorates the accumulation of phagocytosed material in ALS-PFN1 iMGs and rescues the defects in the autophagy pathway, suggesting that an impaired autophagy flux contributes to ALS-PFN1-linked defects in microglial phagocytosis. In vitro experimentation uncovered that PFN1 interacts with phosphatidylinositol-3phosphate, a signaling molecule essential for autophagy and phagocytosis, and that this interaction is altered when PFN1 is mutated in ALS. Collectively, these findings implicate that ALS-PFN1 causes microglia dysfunction by hindering the autophagy flux, perturbing the endo-lysosomal pathway, and, in turn, causing delays in the degradation process during phagocytosis and inducing lipid dysmetabolism. These alterations may be partially driven by ALS-PFN1 distorted interactions with phosphoinositides. My work provides insight into PFN1 biology and opens new perspectives regarding microglia cell-autonomous defects in ALS that may contribute to neurodegeneration.
    • Investigating HNF4A in Intestinal Homeostasis and Inflammation

      Lei, Xuqiu (2022-10-19)
      Hepatocyte nuclear factor 4 alpha (HNF4A) is a highly conserved nuclear receptor that has been associated with ulcerative colitis. In mice, HNF4A is indispensable for the maintenance of intestinal homeostasis, yet the underlying mechanisms are poorly characterized. Here we demonstrate that the expression of HNF4A in intestinal epithelial cells (IECs) is required for the proper development and composition of the intraepithelial lymphocyte (IEL) compartment. HNF4A directly regulates expression of immune signaling molecules including butyrophilin-like (Btnl) 1, Btnl6, H2-T3, and Clec2e that control IEC-IEL crosstalk. HNF4A selectively enhances the expansion of natural IELs that are TCRγδ+ or TCRαβ+CD8αα+ to shape the composition of IEL compartment. In the small intestine, HNF4A cooperates with its paralog HNF4G, to drive expression of immune signaling molecules. Moreover, the HNF4A-BTNL regulatory axis is conserved in human IECs. Collectively, these findings underscore the importance of HNF4A as a conserved transcription factor controlling IEC-IEL crosstalk and suggest that HNF4A maintains intestinal homeostasis through regulation of the IEL compartment.
    • Leveraging Multi-Omic Data to Characterize Cis-Regulatory Elements and Investigate Their Roles in Gene Regulation

      Fan, Kaili (2022-10-11)
      Regulatory elements are non–coding genomic regions that interact with transcription factors to govern when, where, and how much of each gene is expressed. Understanding regulatory elements is essential to understanding mammalian gene regulation; however, our understanding of regulatory syntax is incomplete. Here, we have leveraged multi-omic data to characterize regulatory elements and investigate their roles in gene regulation. We examined a subset of CG-rich promoters exhibiting ubiquitous chromatin accessibility. While most promoters are cell-type specific, these promoters are enriched in cell-essential genes. To maintain universal transcription, they recruit distinct TFs. Furthermore, ubiquitous and cell-type specific promoters are enriched in different sets of Mendelian disease genes, suggesting different contributions to disease susceptibility. We investigated DNA methylation patterns among different categories of regulatory elements during mouse embryonic development and identified a novel class of highly conserved bivalent enhancers that are hypermethylated in cancers, suggesting a unique underlying mechanism involved in cancer. We developed a method to annotate transcription-initiating enhancers. We found that transcribed enhancers mark core enhancer regions, but flanking enhancers also contribute to overall activity. Next, we examined the evolutionary conservation pattern of regulatory elements across 240 mammal species. While conserved regulatory elements are associated with essential genes, recently evolving elements are involved in a species’ interaction with its surroundings. Finally, we performed genome-wide annotation of chromatin states across hundreds of samples, building a regulatory landscape in human and mouse genomes. Together, these works contribute to the characterization of regulatory elements and demonstrate distinct mechanisms of different regulatory subclasses.
    • The Role of Non-Coding Regulatory Elements in Complex Traits and Immune-Mediated Disease

      Pratt, Henry (2022-09-12)
      The completion of the Human Genome Project ushered in the age of genome-wide association studies (GWAS), which have associated thousands of single nucleotide polymorphisms (SNPs) and other sequence variants with complex traits and diseases. Despite this success, progress bridging these associations to pathophysiologic understanding and new therapeutic interventions has been limited. In large part, this owes to the fact that 90% of GWAS-identified variants are non-coding–they do not impact the structure or function of proteins. Unraveling the impacts of non-coding sequence variants is one of the most significant unsolved problems in biology. Non-coding GWAS variants are enriched within cis-regulatory elements (CREs), sequences of DNA which modulate the expression, rather than the function, of target genes. These include promoters, which are immediately adjacent to the gene they regulate; enhancers, which increase expression of distant genes; silencers, which reduce the expression of distant genes; and insulators, which divide chromatin into domains to regulate interactions between other CREs. The function of CREs is modulated in part by transcription factors (TFs), DNA binding proteins which recognize and bind short characteristic DNA sequences called motifs. TFs and CREs are tissue- and cell type-specific, frequently regulating gene expression in only a few of the thousands of distinct cell and tissue types comprising the human body. Here we present work leveraging deep sequencing data and evolutionary conservation to build comprehensive atlases of cis-regulatory elements and transcription factor binding sites in the human genome, along with work architecting visualization platforms to make these atlases more accessible to, and impactful for, the scientific community. We then illustrate a key role for the sites in our atlases, particularly those evolutionarily constrained throughout the mammalian lineage, in complex traits and diseases. We conclude by presenting two case studies utilizing these datasets: one to better understand the role of non-coding variants in primary sclerosing cholangitis, a rare immune-mediated liver disease, and a second to understand the sequence features underlying strong insulator elements in the human genome.
    • Functional Characterization of the 3’ Non-Seed Nucleotides of Conserved MicroRNA let-7a and Phenocritical Argonaute Residues in Caenorhabditis Elegans

      Duan, Ye (2022-09-08)
      MicroRNAs are endogenous non-coding RNAs important for post-transcriptional regulation of gene expression. miRNAs associate with Argonaute proteins to bind to the 3’ UTR of target genes, and post-transcriptionally regulate gene expression. It is well established that base pairing between the mRNA target and the miRNA seed region (g2-g8) is essential for targeting, but the function of the 3’ non-seed region (g9-g22) of microRNAs is less well understood. In this thesis, we systematically investigated the biological functions of 3’ non-seed nucleotides of C. elegans let-7a as an example of an evolutionarily conserved miRNA. We show that the 3’ non-seed sequence of let-7a determines target specificity among its family paralogs, and that the identity of each nucleotide at g11-g16 is essential for in vivo function. We confirmed that lin-41, as well as other heterochronic genes, are let-7 targets that require the 3’ pairing, in many cases to g11-g16 nucleotides. To investigate how 3’ pairing functionally interacts with seed pairing, we systematically re-configured the let-7a::lin-41 interactions and found that the 3’ pairing is critically required for full repression efficacy in the context of perfect seed pairing as well imperfect seed pairing. Certain de novo mutations on human Argonaute AGO1 have been reported to cause neuronal developmental defects with intellectual disability (ID). Many of these human ID mutations alter amino acids that are conserved between human AGO1 and its C. elegans homolog, ALG-1. We found that these mutations can be hypomorphic and antimorphic when modeled in C. elegans ALG-1. The human AGO1 mutations modeled in C. elegans ALG-1 can impact miRNA biogenesis, and lead to global perturbance in the translatome and transcriptome. We also show that the mutant ALG-1 can bind to microRNAs in C. elegans, and in so doing sequester microRNAs in nonfunctional miRISC, causing phenotypes stronger than alg-1(null). We show that the different ID mutations modeled in ALG-1 can exhibit distinct molecular phenotypes in terms of specific disruptions of miRNA biogenesis, and gene expression, suggesting that the mutated residues critically contribute to particular in vivo functions of Argonaute. Notably, different ID mutations affect distinct ALG-1 functions to varying degrees. These studies may provide insights into miRNA-mediated gene-regulatory mechanisms and advance the understanding of fundamental Argonaute functions.
    • In vitro Methods to Better Evaluate Drug Responses in Cancer

      Schwartz, Hannah (2022-09-08)
      Evaluating anti-cancer drugs in vitro is an important aspect of the drug development pipeline. When evaluating anti-cancer drugs, two different measurements are used: relative viability, which scores an amalgam of proliferative arrest and cell death, and fractional viability, which specifically scores the degree of cell killing. These two metrics are often used interchangeably despite measuring different aspects of a drug response. This study explored the relationship between drug-induced growth inhibition and cell death, and found that most drugs affect both proliferation and death, but in different proportions, and with different relative timing. This causes a non-uniform relationship between relative and fractional response measurements. To unify these measurements, I created a data visualization and analysis platform, called drug GRADE, which characterizes the degree to which death contributes to an observed drug response. GRADE captures drug- and genotype-specific responses, which are not captured using traditional pharmaco-metrics. Current in vitro anti-cancer drug evaluation practices measure drug responses with cancer cell lines in mono-culture. However, many cell types in the tumor microenvironment influence cancer’s drug response and disease progression. Therefore, current drug evaluation practices overlook complex cell-cell interactions that influence cancer’s drug response. In this study, I developed a high-throughput assay to study the effect of another cell type (cytotoxic T cells) on cancer viability in co-culture, in vitro. Further, I developed a reference framework to model the complex interaction between cancer cells and cytotoxic T cells, and to model how T cell-mediated cell death is modulated by anti-cancer drug treatment. Taken together, this study highlights two new methods which enable better in vitro evaluation of drug responses in cancer.
    • Insights into Nucleocytoplasmic Transport Decline in FUS-mediated ALS and Nanobody Biologics for SOD1-mediated ALS

      Kumar, Meenakshi Sundaram (2022-09-06)
      Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. The pathogenic mechanisms driving ALS are unclear, and no effective treatment is available. Herein, focusing on two ALS-linked proteins: Fused in Sarcoma (FUS) and Superoxide dismutase 1 (SOD1), I investigated the molecular bases of ALS pathophysiology and propose potential biologics for preclinical testing. Altered nucleocytoplasmic transport is an emerging, yet poorly understood disease pathway in FUS-mediated ALS and neurodegeneration. To understand the mechanism(s) underlying FUS-induced nucleocytoplasmic transport decline, I performed biochemical analyses of an interaction between FUS and the nucleoporin Nup62 that was recently uncovered by our lab. Phase separation of FUS and Phe/Gly-rich nucleoporins including Nup62 is implicated in their respective cellular functions. Using recombinant proteins, I found that FUS and Nup62 alter the phase separation properties of each other in a manner that is modulated by RNA. Our results implicate that the cytoplasmic versus nuclear environment influences FUS/Nup62 interaction, thereby supporting the notion that nucleocytoplasmic transport dysregulation is driven by ALS-mutation-induced mislocalization of FUS within the cytoplasm. Further, I found that the C-terminal domain of Nup62 plays an important role in the FUS/Nup62 interaction. Since this domain mediates Nup62 localization to the nuclear envelope, our results support a model whereby an interaction with cytoplasmic mutant FUS impairs Nup62 homeostasis in nucleocytoplasmic transport. As the first known genetic cause of ALS, SOD1 pathobiology has been studied longer than that of FUS. Notably, ALS-linked mutations induce toxic, misfolded SOD1 conformations which are potentially targetable for therapy. Herein, I characterized anti-SOD1 nanobodies that are capable of modulating the cellular properties of SOD1. Notably, a beneficial effect of anti-SOD1 nanobody expression was observed in human ALS-SOD1 neurons. Thus, this study supports the therapeutic potential of nanobody-based biologics for ALS-SOD1.
    • Cellular heterogeneity and gene regulatory network coordination during thymic epithelial cell development

      Magaletta, Margaret (2022-08-23)
      Thymic epithelial cells, derived from the pharyngeal endoderm, perform essential functions for establishing a self-tolerant immune system. Unsurprisingly, dysfunction of thymic epithelial cells resulting from maldevelopment of the pharyngeal endoderm causes immunodeficiency or autoimmunity syndromes, some of which cannot be fully explained according to known genetic errors. Despite the functional significance and disease-relevance of pharyngeal endoderm with respect to thymic epithelial cells, we lack a comprehensive understanding of the gene regulatory networks driving pharyngeal endoderm differentiation. To close this gap, we applied transcriptome and chromatin accessibility single cell profiling to generate a multi-omic developmental resource covering pharyngeal differentiation toward organ-specific epithelia in the mouse embryo. We identified cell-type specific gene regulation of developing organ domains and characterized the role of an immunodeficiency-associated forkhead box transcription factor, Foxn1, during early thymus development. Furthermore, analyses of the pharyngeal endoderm multi-omics atlas led us to discover a novel gene associated with thymus development, namely Grainyhead-like3 (Grhl3). We assessed the expression pattern and the functional importance of Grhl3 in the prenatal and postnatal thymus, uncovering a putative role in a specialized medullary thymic subtype. In conclusion, this dissertation provides insight on the molecular basis of pharyngeal endoderm differentiation and subsequent development of the thymus.
    • Regulated Gene Therapy Towards Glycosphingolipid Biosynthesis Deficiencies

      Yang, Huiya (2022-08-22)
      Glycosphingolipids (GSLs) are a group of amphipathic glycolipids essential for maintaining the normal ultrastructure and function of neural and oligodendrocyte cell membranes throughout the mammalian central nervous system (CNS). De novo GSL biosynthesis defects cause severe neurological diseases such as GM3 synthase deficiency (GM3SD) and hereditary sensory and autonomic neuropathy type 1A (HSAN1A), each lacking effective treatment. Here, we developed two distinct potential therapeutic approaches for these neurological diseases. For GM3SD that is caused by loss-of-function mutations in ST3GAL5, we employed a recombinant adeno-associated virus (rAAV)-mediated human ST3GAL5 gene replacement therapy. First, using ST3GAL5 mutant patient iPSC-derived neurons and St3gal5 knock-out mouse models, we have achieved ST3GAL5 gene normalization and restoration of the functional products, cerebral gangliosides. Importantly, we revealed the hepatic toxicity caused by ubiquitous expression of ST3GAL5 and optimized a CNS-restricted rAAV gene replacement therapy for the safe and efficacious rescue of the severe neurodevelopmental phenotypes and early lethality in disease mouse models, given by both intracerebroventricular and intravenous routes of administration. These results support for further clinical development of ST3GAL5 gene therapy. On the other hand, to target gain-of-function SPTLC1 mutation caused HSAN1A, we screened antisense oligonucleotides (ASOs) and achieved efficient reduction of mutant SPTLC1 transcripts and its toxic products in patient-fibroblasts. In summary, this thesis describes the potential of novel rAAV-mediated gene replacement therapy in GM3SD and allele-specific ASO silencing in HSAN1A, highlighting the significance of personalized gene therapy for monogenic neurological disorders.
    • Investigating Proteolytic Processing of Ataxin 2, a Neurodegenerative Disease Associated Protein

      Chitre, Monika (2022-08-08)
      Ataxin 2 (ATXN2) is a ubiquitously expressed mRNA binding protein involved in the development and progression of spinocerebellar ataxia 2 (SCA2) and amyotrophic lateral sclerosis (ALS). In the context of both neurodegenerative diseases, its N-terminal polyglutamine (polyQ) domain is mutated and expanded in length. Several other polyQ proteins, such as huntingtin (Htt), ataxin 3 (ATXN3), and ataxin 7 (ATXN7), undergo proteolytic processing that produces toxic fragments containing their polyQ domains. Investigating how ATXN2 is regulated by proteolysis is hindered by the lack of available molecular biological tools such as N-terminal ATXN2 antibodies to target and analyze the endogenous N-terminus of ATXN2. To circumvent this challenge, I developed a transient overexpression model of N-terminally tagged ATXN2 in HEK293E cells. Here, I demonstrate that both wild-type and mutant ATXN2 are targets of N-terminal proteolysis. I confirmed that ATXN2 produces an independent polyQ cleavage fragment like other polyQ proteins through basic molecular biology approaches such as Western blotting and immunoprecipitation. Additionally, I identified the specific region that is both necessary and sufficient for cleavage to occur via deletion mapping with multiple truncated ATXN2 mutants and reporter constructs. Further definition of ATXN2 as a target of proteolytic cleavage aligns it with other neurodegenerative polyQ proteins, and proteolysis is currently a less explored avenue of research for ATXN2-related disease development, progression, and therapeutic modalities. This work reveals a novel site that directs cleavage of ATXN2 and provides a potential avenue of investigation for how ATXN2 posttranslational modifications contribute to the progression of SCA2 and ALS.
    • High bacillary burden and the ESX-1 type VII secretion system promote MHC class I presentation to CD8 T-cells during Mycobacterium tuberculosis infection

      Mott, Daniel (2022-07-26)
      T-cell mediated immunity is required for optimal protection against Mycobacterium tuberculosis (Mtb) infection, but often fails to completely clear the pathogen. Mtb has evolved strategies to subvert host immunity so it can persist in host cells despite pressure from innate and adaptive immunity. While cytotoxic CD8 T-cells should recognize and clear infected host cells, eliminating Mtb’s intracellular niche, previous findings have demonstrated otherwise [1]. In fact, we have shown that CD8 T-cells specific to the immunodominant antigen TB10.4 poorly recognize Mtb infected macrophages in vitro. Here we extend our initial findings to show that class I MHC-restricted epitopes other than TB10.44-11 are inefficiently presented by Mtb-infected macrophages to CD8 T cells. The only exception we find is for heavily infected macrophages. During high burden infections, macrophages cross-present TB10.4 antigen to CD8 T-cells. These high burden infections result in considerable cell death, and we find that uninfected macrophages effectively scavenge dead cellular debris and cross-present this antigen to CD8 T cells. Furthermore, we find that cross-presentation by heavily infected cells is dependent on the ESX-1 type VII secretion system, suggesting that phagosomal membrane damage and host cell death are crucial for effective class I MHC cross-presentation during Mtb infection.
    • The Role of Age-Associated B Cells (ABC) in Combating Influenza Infection

      Kugler-Umana, Olivia (2022-07-12)
      With age, follicular helper T cell (TFH) dependent B cell responses erode, reducing B cell memory and long-term antibody responses. However, aged mice and humans develop an alternative B cell population, termed age-associated B cells (ABC), that may produce TFH independent antibodies. ABC lack CD21 and CD23 expression, and some express transcription factors and adhesion molecules indicative of antigen exposure. Some of these have been implicated in autoimmunity. We found a unique population of responding B cells following influenza A virus (IAV) infection, which was Fashi/GL7neg. We postulated that the CD21-CD23-ABC might be progenitors of these non-follicular B cells, that we called induced ABC (iABC). Using T-deficient RAG KO and TFH deficient SAP KO hosts, we found that the CD21-CD23-ABC can become iABC (FasHiGL7-) upon IAV infection. These iABC share the same phenotype of iABC found in infected aged mice and can become Ab-producing cells without T cell help. We showed that CD21-CD23-ABC can be separated into IgD+ (putative naïve B cells) vs. IgD- (memory-like B cells). We followed their ability to become iABC in SAP KO hosts. The IgD+ABC were most efficient at giving rise to iABC. Further transfer studies revealed that iABC generation from donor IgD+ABC requires extrinsic TLR signaling, and IgD+ABC can provide Ab-medicated protection. We concluded that upon T-independent stimulation, IgD+ABC (CD21-CD23-) become iABC (Fashi/GL7neg), some of which can secrete IAV-specific Ab and may provide protection against IAV.
    • Novel Genetic Pathways in Functional Regulation of Hematopoietic Stem Cells

      Desouza, Ngoc (2022-07-08)
      Hematopoietic stem cells (HSCs) are a rare population of bone marrow cells that have self-renewal and differentiating capabilities enabling them to produce all blood lineages during normal hematopoiesis. Many molecular pathways are involved in the regulation of HSCs, and the survival, maintenance and proliferation of these cells must be tightly controlled to avoid aberrant activities that can cause blood diseases, such as hematopoietic malignancies. Therefore, additional factors involved in the functional regulation of HSCs must be discovered to provide new therapeutic treatments for hematopoietic diseases. In chapter I, I briefly introduce the hematopoietic system and hierarchy through which HSCs can produce all mature blood cells in the lifespan. I also describe various methods to identify different hematopoietic cells, with a focus on using cell surface antigen markers. I additionally discuss an important method to study HSCs in mice by using bone marrow transplantation in which the donor cells are manipulated to examine the role of a gene of interest. I also briefly describe several signaling pathways important in HSCs, such as the Bmp, Wnt, Hedgehog and Notch signaling pathways. I provide known relevant information to my thesis work on c-Kit and Sca-1 receptors, as well as on LSK and LSK- cells. In chapter II, I describe my findings regarding a novel mechanism in which Ikzf3 plays a suppressive role in regulating HSC survival and maintenance. Ikzf3 suppresses the population of LSK (lineage-Sca-1+c-Kit+) cells that contains HSCs, and increases the LSK- (lineage-Sca-1+c-Kit-) population, which is highly apoptotic and derived from the LSK population. The DNA binding domain of Ikzf3 is required for its inhibition of HSCs, and Ikzf3 downregulates the expression of Bcl-2, Bcl-xL and c-Myc. Ikzf3 expression in HSCs is maintained at low levels by the c-Kit pathway. In chapter III, on the basis of data from microarray analysis previously performed to compare gene expression profiles between Hif1a knockout HSCs and wild type HSCs, the effects of both Hif1a and Notch1 deletion on HSC regulation are examined. Loss of both Hif1a and Notch1 induces the development of myelodysplastic/myeloproliferative diseases, which are clonal hematopoietic stem cell neoplasms characterized by abnormal regulation of the myeloid pathways for cellular proliferation, maturation and survival. Loss of both Hif1a and Notch1 also leads to a loss of HSC function. These findings indicate a mechanism through which the hypoxia pathway acts in coordination with the Notch pathway in HSCs. In chapter IV, I summarize the findings from chapter II and III and discuss the importance of these results in the field. I also provide the future directions that can answer more questions to expand our knowledge on these pathways. Together, the findings reveal two novel pathways involved in functional regulation of HSCs: (i) the Ikzf3 pathway, which involves c-Kit, Icsbp, Bcl-2, Bcl-xL and c-Myc, and suppresses normal HSCs to maintain homeostasis and (ii) the synergy of Hif1a and Notch1 in regulating HSCs. The loss of both genes can cause myelodysplastic/myeloproliferative-like diseases.
    • Gliotransmission Orchestrates Neuronal Type-specific Axon Regeneration

      Wang, Fei (2022-06-30)
      Why closely related neuronal types differ in their axon regenerative abilities remains elusive. Here, I demonstrate gliotransmission determines such a difference in Drosophila larval sensory neurons. Axotomy activates ensheathing glia, which signal to regenerative neurons through the gliotransmitter adenosine, to mount regenerative programs including neuronal activity and Ras. Surprisingly, ensheathing glia do not signal to non-regenerative neurons. Such neuronal type-specific responses to gliotransmission result from specific expression of adenosine receptors in regenerative neurons. Disrupting gliotransmission impedes regeneration of regenerative neurons. Strikingly, reconstitution of gliotransmission in non-regenerative neurons enables them to regenerate. Furthermore, activation of an adenosine receptor in adult mice promotes both regeneration and survival of retinal ganglion cells, uncovering a conserved pro-regenerative role of adenosine receptors. My studies demonstrate gliotransmission as a novel mechanism by which glia instruct axon regeneration, with neuronal type-specificity, and suggest targeting purinergic signaling as a new strategy for mammalian central nervous system repair.
    • Surveillance of Host and Pathogen Derived Metabolites Activates Intestinal Immunity

      Peterson, Nicholas D. (2022-06-30)
      Intestinal epithelial cells function, in part, to detect infection with pathogenic organisms and are key regulators of intestinal immune homeostasis. However, it is not fully understood how intestinal epithelial cells sense pathogen infection and coordinate the induction of protective immune defenses. Here, we define two new mechanisms of innate immune regulation in a metazoan host. First, we characterize the first bacterial pattern recognition receptor and its natural ligand in Caenorhabditis elegans. We show that the C. elegans nuclear hormone receptor NHR-86/HNF4 directly senses phenazine-1-carboxamide (PCN), a metabolite produced by pathogenic strains of Pseudomonas aeruginosa. PCN binds to the ligand-binding domain of NHR-86/HNF4, a ligand-gated transcription factor, and activates innate immunity in intestinal epithelial cells. In addition, we show that C. elegans NHR-86 senses PCN, and not other phenazine metabolites, as a marker of pathogen virulence to engage protective anti-pathogen defenses. Second, we show that a phase transition of the C. elegans Toll/interleukin-1 receptor domain protein (TIR-1) controls signaling by the C. elegans p38 PMK-1 MAPK pathway. Physiologic stress, both P. aeruginosa infection and sterol scarcity, induce multimerization of TIR-1 within intestinal epithelial cells. Like the mammalian homolog of TIR-1, SARM1, oligomerization and phase transition of C. elegans TIR-1 dramatically potentiate its NAD+ glycohydrolase activity. TIR-1/SARM1 multimerization and NAD+ glycohydrolase activity are required for activation of C. elegans p38 PMK-1 pathway signaling and pathogen resistance. These data uncover a mechanism by which nematodes interpret environmental conditions to prime innate immune defenses and promote survival in microbe rich environments. C. elegans animals augment these immune defenses by surveying for ligands specifically associated with toxigenic pathogens that are poised to cause disease. These findings define a new paradigm of intestinal immune control that informs the evolution of innate immunity in all metazoans.
    • Integrated Multi-omics Characterization of Human Disease Models

      Rodríguez, Tomás (2022-06-07)
      Animal and cell-based models of human disease offer simplified biological systems for studying the basis of more complex pathologies under well-controlled conditions. An ever-expanding suite of genomic and transcriptomic tools allows us to thoroughly characterize these models, highlighting disease-driving molecular features and exposing novel therapeutic targets. Here, we integrate diverse DNA- and RNA-sequencing strategies to describe the gene-regulatory chromatin landscape of models for hepatoblastoma and retrovirally-infected CD4+ T-cells. We first developed a conditional hepatoblastoma mouse model using doxycycline-inducible YAP1 overexpression and constitutive β-cateninDelN90. We found that YAP1 withdrawal alone is sufficient to trigger tumor regression and substantially increase survival. We reasoned that a thorough chromatin profile of this tumor model during YAP1 withdrawal could reveal YAP1-driven mechanisms of hepatoblastoma tumorigenesis. Our integrated approach revealed 31 novel YAP1-targeted cis-regulatory element-gene pairs. Subsequent validation confirmed that regulation of Jun-Dimerization Protein 2, among others, is both YAP1-dependent and functionally consequential for the hepatoblastoma phenotype in human cells and in hepatic malignancies. To expand our efforts to apply multi-omics technologies to disease models, we next engineered a fluorophore-containing murine leukemia virus (MLV-GFP) stably integrated into Jurkat CD4+ T-cells to report on defective transcriptional silencing by the retroelement-silencing complex, HUSH. A CRISPR knockout screen identified DHX29 as essential for HUSH-mediated silencing of newly-integrated retroviruses. Profiling genomic and transcriptomic features of MLV-GFP Jurkat cells after HUSH and DHX29 knockout revealed their epistatic roles in silencing, and revealed a suite of loci targeted by HUSH. Finally, we used site-specific proteomics and chromatin profiling to identify HUSH-associated factors at the newly integrated proviral reporter.