Browsing by Subject "Genes, Reporter"

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Open Access
A complex intronic enhancer regulates expression of the CFTR gene by direct interaction with the promoter.
(J Cell Mol Med, 2009-04) Ott, Christopher J; Suszko, Magdalena; Blackledge, Neil P; Wright, Jane E; Crawford, Gregory E; Harris, Ann
Genes can maintain spatiotemporal expression patterns by long-range interactions between cis-acting elements. The cystic fibrosis transmembrane conductance regulator gene (CFTR) is expressed primarily in epithelial cells. An element located within a DNase I-hypersensitive site (DHS) 10 kb into the first intron was previously shown to augment CFTR promoter activity in a tissue-specific manner. Here, we reveal the mechanism by which this element influences CFTR transcription. We employed a high-resolution method of mapping DHS using tiled microarrays to accurately locate the intron 1 DHS. Transfection of promoter-reporter constructs demonstrated that the element displays classical tissue-specific enhancer properties and can independently recruit factors necessary for transcription initiation. In vitro DNase I footprinting analysis identified a protected region that corresponds to a conserved, predicted binding site for hepatocyte nuclear factor 1 (HNF1). We demonstrate by electromobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) that HNF1 binds to this element both in vitro and in vivo. Moreover, using chromosome conformation capture (3C) analysis, we show that this element interacts with the CFTR promoter in CFTR-expressing cells. These data provide the first insight into the three- dimensional (3D) structure of the CFTR locus and confirm the contribution of intronic cis-acting elements to the regulation of CFTR gene expression.
Open Access
A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress.
(Science (New York, N.Y.), 2005-02) Boyce, Michael; Bryant, Kevin F; Jousse, Céline; Long, Kai; Harding, Heather P; Scheuner, Donalyn; Kaufman, Randal J; Ma, Dawei; Coen, Donald M; Ron, David; Yuan, Junying
Most protein phosphatases have little intrinsic substrate specificity, making selective pharmacological inhibition of specific dephosphorylation reactions a challenging problem. In a screen for small molecules that protect cells from endoplasmic reticulum (ER) stress, we identified salubrinal, a selective inhibitor of cellular complexes that dephosphorylate eukaryotic translation initiation factor 2 subunit alpha (eIF2alpha). Salubrinal also blocks eIF2alpha dephosphorylation mediated by a herpes simplex virus protein and inhibits viral replication. These results suggest that selective chemical inhibitors of eIF2alpha dephosphorylation may be useful in diseases involving ER stress or viral infection. More broadly, salubrinal demonstrates the feasibility of selective pharmacological targeting of cellular dephosphorylation events.
Open Access
Allo-Specific Humoral Responses: New Methods for Screening Donor-Specific Antibody and Characterization of HLA-Specific Memory B Cells.
(Frontiers in immunology, 2021-01) Song, Shengli; Manook, Miriam; Kwun, Jean; Jackson, Annette M; Knechtle, Stuart J; Kelsoe, Garnett
Antibody-mediated allograft rejection (AMR) causes more kidney transplant failure than any other single cause. AMR is mediated by antibodies recognizing antigens expressed by the graft, and antibodies generated against major histocompatibility complex (MHC) mismatches are especially problematic. Most research directed towards the management of clinical AMR has focused on identifying and characterizing circulating donor-specific HLA antibody (DSA) and optimizing therapies that reduce B-cell activation and/or block antibody secretion by inhibiting plasmacyte survival. Here we describe a novel set of reagents and techniques to allow more specific measurements of MHC sensitization across different animal transplant models. Additionally, we have used these approaches to isolate and clone individual HLA-specific B cells from patients sensitized by pregnancy or transplantation. We have identified and characterized the phenotypes of individual HLA-specific B cells, determined the V(D)J rearrangements of their paired H and L chains, and generated recombinant antibodies to determine affinity and specificity. Knowledge of the BCR genes of individual HLA-specific B cells will allow identification of clonally related B cells by high-throughput sequence analysis of peripheral blood mononuclear cells and permit us to re-construct the origins of HLA-specific B cells and follow their somatic evolution by mutation and selection.
Open Access
ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coat.
(J Cell Biol, 2002-10-14) Yang, JS; Lee, SY; Gao, M; Bourgoin, S; Randazzo, PA; Premont, RT; Hsu, VW
The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPgammaS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.
Open Access
Bioengineered human myobundles mimic clinical responses of skeletal muscle to drugs.
(Elife, 2015-01-09) Madden, Lauran; Juhas, Mark; Kraus, William E; Truskey, George A; Bursac, Nenad
Existing in vitro models of human skeletal muscle cannot recapitulate the organization and function of native muscle, limiting their use in physiological and pharmacological studies. Here, we demonstrate engineering of electrically and chemically responsive, contractile human muscle tissues ('myobundles') using primary myogenic cells. These biomimetic constructs exhibit aligned architecture, multinucleated and striated myofibers, and a Pax7(+) cell pool. They contract spontaneously and respond to electrical stimuli with twitch and tetanic contractions. Positive correlation between contractile force and GCaMP6-reported calcium responses enables non-invasive tracking of myobundle function and drug response. During culture, myobundles maintain functional acetylcholine receptors and structurally and functionally mature, evidenced by increased myofiber diameter and improved calcium handling and contractile strength. In response to diversely acting drugs, myobundles undergo dose-dependent hypertrophy or toxic myopathy similar to clinical outcomes. Human myobundles provide an enabling platform for predictive drug and toxicology screening and development of novel therapeutics for muscle-related disorders.
Open Access
Cytokinesis proteins Tum and Pav have a nuclear role in Wnt regulation.
(J Cell Sci, 2010-07-01) Jones, Whitney M; Chao, Anna T; Zavortink, Michael; Saint, Robert; Bejsovec, Amy
Wg/Wnt signals specify cell fates in both invertebrate and vertebrate embryos and maintain stem-cell populations in many adult tissues. Deregulation of the Wnt pathway can transform cells to a proliferative fate, leading to cancer. We have discovered that two Drosophila proteins that are crucial for cytokinesis have a second, largely independent, role in restricting activity of the Wnt pathway. The fly homolog of RacGAP1, Tumbleweed (Tum)/RacGAP50C, and its binding partner, the kinesin-like protein Pavarotti (Pav), negatively regulate Wnt activity in fly embryos and in cultured mammalian cells. Unlike many known regulators of the Wnt pathway, these molecules do not affect stabilization of Arm/beta-catenin (betacat), the principal effector molecule in Wnt signal transduction. Rather, they appear to act downstream of betacat stabilization to control target-gene transcription. Both Tum and Pav accumulate in the nuclei of interphase cells, a location that is spatially distinct from their cleavage-furrow localization during cytokinesis. We show that this nuclear localization is essential for their role in Wnt regulation. Thus, we have identified two modulators of the Wnt pathway that have shared functions in cell division, which hints at a possible link between cytokinesis and Wnt activity during tumorigenesis.
Open Access
Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells.
(Viruses, 2020-09-25) Lennemann, Nicholas J; Evans, Azia S; Coyne, Carolyn B
Enteroviruses manipulate host membranes to form replication organelles, which concentrate viral and host factors to allow for efficient replication. However, this process has not been well-studied in living cells throughout the course of infection. To define the dynamic process of enterovirus membrane remodeling of major secretory pathway organelles, we have developed plasmid-based reporter systems that utilize viral protease-dependent release of a nuclear-localized fluorescent protein from the endoplasmic reticulum (ER) membrane during infection, while retaining organelle-specific fluorescent protein markers such as the ER and Golgi. This system thus allows for the monitoring of organelle-specific changes induced by infection in real-time. Using long-term time-lapse imaging of living cells infected with coxsackievirus B3 (CVB), we detected reporter translocation to the nucleus beginning ~4 h post-infection, which correlated with a loss of Golgi integrity and a collapse of the peripheral ER. Lastly, we applied our system to study the effects of a calcium channel inhibitor, 2APB, on virus-induced manipulation of host membranes. We found that 2APB treatment had no effect on the kinetics of infection or the percentage of infected cells. However, we observed aberrant ER structures in CVB-infected cells treated with 2APB and a significant decrease in viral-dependent cell lysis, which corresponded with a decrease in extracellular virus titers. Thus, our system provides a tractable platform to monitor the effects of inhibitors, gene silencing, and/or gene editing on viral manipulation of host membranes, which can help determine the mechanism of action for antivirals.
Open Access
Intrastromal Gene Therapy Prevents and Reverses Advanced Corneal Clouding in a Canine Model of Mucopolysaccharidosis I.
(Molecular therapy : the journal of the American Society of Gene Therapy, 2020-06) Miyadera, Keiko; Conatser, Laura; Llanga, Telmo A; Carlin, Kendall; O'Donnell, Patricia; Bagel, Jessica; Song, Liujiang; Kurtzberg, Joanne; Samulski, R Jude; Gilger, Brian; Hirsch, Matthew L
Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disease characterized by severe phenotypes, including corneal clouding. MPS I is caused by mutations in alpha-l-iduronidase (IDUA), a ubiquitous enzyme that catalyzes the hydrolysis of glycosaminoglycans. Currently, no treatment exists to address MPS I corneal clouding other than corneal transplantation, which is complicated by a high risk for rejection. Investigation of an adeno-associated virus (AAV) IDUA gene addition strategy targeting the corneal stroma addresses this deficiency. In MPS I canines with early or advanced corneal disease, a single intrastromal AAV8G9-IDUA injection was well tolerated at all administered doses. The eyes with advanced disease demonstrated resolution of corneal clouding as early as 1 week post-injection, followed by sustained corneal transparency until the experimental endpoint of 25 weeks. AAV8G9-IDUA injection in the MPS I canine eye with early corneal disease prevented the development of advanced corneal changes while restoring clarity. Biodistribution studies demonstrated vector genomes in ocular compartments other than the cornea and in some systemic organs; however, a capsid antibody response was detected in only the highest dosed subject. Collectively, the results suggest that intrastromal AAV8G9-IDUA therapy prevents and reverses visual impairment associated with MPS I corneal clouding.
Open Access
Localization of DIR1 at the tissue, cellular and subcellular levels during Systemic Acquired Resistance in Arabidopsis using DIR1:GUS and DIR1:EGFP reporters.
(BMC plant biology, 2011-01) Champigny, Marc J; Shearer, Heather; Mohammad, Asif; Haines, Karen; Neumann, Melody; Thilmony, Roger; He, Sheng Yang; Fobert, Pierre; Dengler, Nancy; Cameron, Robin K
BACKGROUND: Systemic Acquired Resistance (SAR) is an induced resistance response to pathogens, characterized by the translocation of a long-distance signal from induced leaves to distant tissues to prime them for increased resistance to future infection. DEFECTIVE in INDUCED RESISTANCE 1 (DIR1) has been hypothesized to chaperone a small signaling molecule to distant tissues during SAR in Arabidopsis. RESULTS: DIR1 promoter:DIR1-GUS/dir1-1 lines were constructed to examine DIR1 expression. DIR1 is expressed in seedlings, flowers and ubiquitously in untreated or mock-inoculated mature leaf cells, including phloem sieve elements and companion cells. Inoculation of leaves with SAR-inducing avirulent or virulent Pseudomonas syringae pv tomato (Pst) resulted in Type III Secretion System-dependent suppression of DIR1 expression in leaf cells. Transient expression of fluorescent fusion proteins in tobacco and intercellular washing fluid experiments indicated that DIR1's ER signal sequence targets it for secretion to the cell wall. However, DIR1 expressed without a signal sequence rescued the dir1-1 SAR defect, suggesting that a cytosolic pool of DIR1 is important for the SAR response. CONCLUSIONS: Although expression of DIR1 decreases during SAR induction, the protein localizes to all living cell types of the vasculature, including companion cells and sieve elements, and therefore DIR1 is well situated to participate in long-distance signaling during SAR.
Open Access
Pairing of competitive and topologically distinct regulatory modules enhances patterned gene expression.
(Mol Syst Biol, 2008) Yanai, Itai; Baugh, L Ryan; Smith, Jessica J; Roehrig, Casey; Shen-Orr, Shai S; Claggett, Julia M; Hill, Andrew A; Slonim, Donna K; Hunter, Craig P
Biological networks are inherently modular, yet little is known about how modules are assembled to enable coordinated and complex functions. We used RNAi and time series, whole-genome microarray analyses to systematically perturb and characterize components of a Caenorhabditis elegans lineage-specific transcriptional regulatory network. These data are supported by selected reporter gene analyses and comprehensive yeast one-hybrid and promoter sequence analyses. Based on these results, we define and characterize two modules composed of muscle- and epidermal-specifying transcription factors that function together within a single cell lineage to robustly specify multiple cell types. The expression of these two modules, although positively regulated by a common factor, is reliably segregated among daughter cells. Our analyses indicate that these modules repress each other, and we propose that this cross-inhibition coupled with their relative time of induction function to enhance the initial asymmetry in their expression patterns, thus leading to the observed invariant gene expression patterns and cell lineage. The coupling of asynchronous and topologically distinct modules may be a general principle of module assembly that functions to potentiate genetic switches.
Open Access
Plasticity of Hopx(+) type I alveolar cells to regenerate type II cells in the lung.
(Nature communications, 2015-04-13) Jain, Rajan; Barkauskas, Christina E; Takeda, Norifumi; Bowie, Emily J; Aghajanian, Haig; Wang, Qiaohong; Padmanabhan, Arun; Manderfield, Lauren J; Gupta, Mudit; Li, Deqiang; Li, Li; Trivedi, Chinmay M; Hogan, Brigid LM; Epstein, Jonathan A
The plasticity of differentiated cells in adult tissues undergoing repair is an area of intense research. Pulmonary alveolar type II cells produce surfactant and function as progenitors in the adult, demonstrating both self-renewal and differentiation into gas exchanging type I cells. In vivo, type I cells are thought to be terminally differentiated and their ability to give rise to alternate lineages has not been reported. Here we show that Hopx becomes restricted to type I cells during development. However, unexpectedly, lineage-labelled Hopx(+) cells both proliferate and generate type II cells during adult alveolar regrowth following partial pneumonectomy. In clonal 3D culture, single Hopx(+) type I cells generate organoids composed of type I and type II cells, a process modulated by TGFβ signalling. These findings demonstrate unanticipated plasticity of type I cells and a bidirectional lineage relationship between distinct differentiated alveolar epithelial cell types in vivo and in single-cell culture.
Open Access
Ratiometric GPCR signaling enables directional sensing in yeast.
(PLoS biology, 2019-10-17) Henderson, Nicholas T; Pablo, Michael; Ghose, Debraj; Clark-Cotton, Manuella R; Zyla, Trevin R; Nolen, James; Elston, Timothy C; Lew, Daniel J
Accurate detection of extracellular chemical gradients is essential for many cellular behaviors. Gradient sensing is challenging for small cells, which can experience little difference in ligand concentrations on the up-gradient and down-gradient sides of the cell. Nevertheless, the tiny cells of the yeast Saccharomyces cerevisiae reliably decode gradients of extracellular pheromones to find their mates. By imaging the behavior of polarity factors and pheromone receptors, we quantified the accuracy of initial polarization during mating encounters. We found that cells bias the orientation of initial polarity up-gradient, even though they have unevenly distributed receptors. Uneven receptor density means that the gradient of ligand-bound receptors does not accurately reflect the external pheromone gradient. Nevertheless, yeast cells appear to avoid being misled by responding to the fraction of occupied receptors rather than simply the concentration of ligand-bound receptors. Such ratiometric sensing also serves to amplify the gradient of active G protein. However, this process is quite error-prone, and initial errors are corrected during a subsequent indecisive phase in which polarity clusters exhibit erratic mobile behavior.
Open Access
RNA-Seq and ChIP-Seq reveal SQSTM1/p62 as a key mediator of JunB suppression of NF-κB-dependent inflammation.
(J Invest Dermatol, 2015-04) Zhang, Xiaoling; Jin, Jane Y; Wu, Joseph; Qin, Xiaoxia; Streilein, Robert; Hall, Russell P; Zhang, Jennifer Y
Mice with epidermal deletion of JunB transcription factor displayed a psoriasis-like inflammation. The relevance of these findings to humans and the mechanisms mediating JunB function are not fully understood. Here we demonstrate that impaired JunB function via gene silencing or overexpression of a dominant negative mutant increased human keratinocyte cell proliferation but decreased cell barrier function. RNA-seq revealed over 500 genes affected by JunB loss of function, which included the upregulation of an array of proinflammatory molecules relevant to psoriasis. Among these were tumor necrosis factor α (TNFα), CCL2, CXCL10, IL6R, and SQSTM1, an adaptor protein involved in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Chromatin immunoprecipitation (ChIP)-Seq and gene reporter analyses showed that JunB directly suppressed SQSTM1 by binding to a consensus AP-1 cis element located around 2 kb upstream of SQSTM1-transcription start site. Similar to JunB loss of function, SQSTM1-overexpression induced TNFα, CCL2, and CXCL10. Conversely, NF-κB inhibition genetically with a mutant IκBα or pharmacologically with pyrrolidine dithiocarbamate (PDTC) prevented cytokine, but not IL6R, induction by JunB deficiency. Taken together, our findings indicate that JunB controls epidermal growth, barrier formation, and proinflammatory responses through direct and indirect mechanisms, pinpointing SQSTM1 as a key mediator of JunB suppression of NF-κB-dependent inflammation.
Open Access
Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling.
(J Vis Exp, 2015-09-14) Arriaga, Gustavo; Macopson, Joshua J; Jarvis, Erich D
Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)(1). PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site(2,3). Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)(4,5). This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3'-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites(6-11).
Open Access
Unmasking Proteolytic Activity for Adult Visual Cortex Plasticity by the Removal of Lynx1.
(The Journal of neuroscience : the official journal of the Society for Neuroscience, 2015-09) Bukhari, Noreen; Burman, Poromendro N; Hussein, Ayan; Demars, Michael P; Sadahiro, Masato; Brady, Daniel M; Tsirka, Stella E; Russo, Scott J; Morishita, Hirofumi
Unlabelled
Experience-dependent cortical plasticity declines with age. At the molecular level, experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain if mice are raised in standard cages. Understanding the mechanism for the loss of permissive proteolytic activity is therefore a key link for improving function in adult brains. Using the mouse primary visual cortex (V1) as a model, we demonstrate that tPA activity in V1 can be unmasked following 4 d of monocular deprivation when the mice older than 2 months are raised in standard cages by the genetic removal of Lynx1, a negative regulator of adult plasticity. This was also associated with the reduction of stubby and thin spine density and enhancement of ocular dominance shift in adult V1 of Lynx1 knock-out (KO) mice. These structural and functional changes were tPA-dependent because genetic removal of tPA in Lynx1 KO mice can block the monocular deprivation-dependent reduction of dendritic spine density, whereas both genetic and adult specific inhibition of tPA activity can ablate the ocular dominance shift in Lynx1 KO mice. Our work demonstrates that the adult brain has an intrinsic potential for experience-dependent elevation of proteolytic activity to express juvenile-like structural and functional changes but is effectively limited by Lynx1 if mice are raised in standard cages. Insights into the Lynx1-tPA plasticity mechanism may provide novel therapeutic targets for adult brain disorders.
Significance statement
Experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain in correlation with the decline in cortical plasticity when mice are raised in standard cages. We demonstrated that removal of Lynx1, one of negative regulators of plasticity, unmasks experience-dependent tPA elevation in visual cortex of adult mice reared in standard cages. This proteolytic elevation facilitated dendritic spine reduction and ocular dominance plasticity in adult visual cortex. This is the first demonstration of adult brain to retain the intrinsic capacity to elevate tPA in an experience-dependent manner but is effectively limited by Lynx1. tPA-Lynx1 may potentially be a new candidate mechanism for interventions that were shown to activate plasticity in adult brain.

Browsing by Subject "Genes, Reporter"

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