Browsing by Subject "DNA"
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Item AB1-42 Antibody Producing Plasma Cells in DNA AB42 Trimer Immunized Mice Reside Predominantly in the Bone Marrow(2013-01-22) Zacharias, Tresa; Langworthy, Suzanna; Fu, Min; Anderson, Larry; Stuve, Olaf; Rosenburg, Roger; Lambracht-Washington, DorisAlzheimer's disease (AD) is the most common form of age-related dementia and affects nearly 40 million people worldwide. Immunotherapy provides a possible avenue for prophylaxis of AD, but a clinical trial (AN1792) in which patients with early AD were immunized with Aβ1-42 peptide was halted after the occurrence of meningoencephalitis in 6% of the immunized people which was attributed to a T cell autoimmune response. DNA vaccination has been shown to have a polarized Th2 immune response that lacks many of the features responsible for inflammation seen in peptide immunizations. In this study, we show a new feature of the DNA Aβ42 trimer elicited B cell immune response and present data for the presence of a long lived plasma cell pool residing within the bone marrow in DNA immunized mice but not in peptide immunized mice. Two groups of mice were analyzed: one group of B6C3F1 mice (n=20) were studied 4 months after the last DNA vaccination, and a second group of BALB/c mice (n=14), which received DNA or peptide immunizations, were analyzed 10 days following the last immunization. The comparison of antibody producing cells in bone marrow and spleen for the DNA and peptide immunized mice with an Antibody Forming Cell (AFC) ELISPOT assay and subsequent ELISAs showed that bone marrow plasma cells from DNA immunized mice produced more anti-Aβ42 IgG producing cells and higher levels of secreted IgG antibodies. In peptide immunized mice, more IgG antibody producing cells were found to reside in the spleen. These data indicate that the bone marrow may be an important reservoir for B cells following DNA Aβ42 immunization and is in line with studies showing that the bone marrow represents an excellent niche for the survival of long lived plasma cells and a lifetime source for antibody producing B cells which are independent of continuous antigen specific stimulation. Further studies are needed to show whether it is possible to define additional phenotypic characteristics for the antigen specific B cell immune response in DNA Aβ42 trimer immunized mice or differences in the TH subsets directly involved in initial signaling events to B cells in the germinal center reactions.Item Characterization of Receptor Protein Tyrosine Phosphatase Epsilon (PTPRE) Gene Promoter(2015-01-26) Isaacs, Thomas; Shukla, Abhay A.; Amatruda, JamesBACKGROUND: Receptor protein tyrosine phosphatase epsilon (PTPRE) is a receptor bound phosphatase that has been shown to be downregulated in Wilms' tumors compared to normal tissue, and could potentially be a target for future therapy. Our objective is to identify and characterize the promoter of the PTPRE gene and define the critical role of Wt1 transcription factor (commonly downregulated in Wilm's tumor) in PTPRE gene expression and in Wilms' tumor progression. METHODS: Our first step involved cloning and sequence analysis of the upstream region of the human PTPRE gene followed by PCR primer design and PCR amplification. The amplified fragment was then cloned into a promoterless reporter vector (pGl3 Basic) and transfected in Hek293 cells. Promoter DNA was used for deletion analysis where multiple PCRs were performed using a single forward primer and multiple reverse primers with nucleotides sequentially deleted from the 3' end. The different size PCR products were then cloned into pGL3 Basic vector DNA, transfected into HEK cells and had reporter assay (luciferase assay) performed to calculate fold change in PTPRE expression over promoterless vector. After the critical transcription factor binding motif was identified, PCR was performed to amplify full length promoter lacking 76 bases. The role of the deleted nucleotides was confirmed via luciferase assay. The sequence of deleted nucleotides was then analyzed for the presence of transcription factor binding motifs. Next, a predicted Wt1 transcription factor binding motif was mutated using site directed mutagenesis. Mutated fragment was cloned into pGl3Basic vector. Both wild type and mutated vector were transfected and luciferase assay was performed to confirm role of Wt1 binding motif for PTPRE promoter activity. Chromatin immunoprecipitation assay was then performed for further evidence. Promoter activity was also compared in two cells lines having differential expression of Wt1. Western blot and semi-quantitative PCR are used to confirm the expression levels of Wt1. RESULTS: Promoter deletion analysis confirmed that the Wt1 binding motif present at -16 position is critical for PTPRE expression and mutation of this site results in 95% loss in promoter activity in Hek293 cells. PTPRE promoter activity was shown to be high in Hek293 cells and low in Hela cells (high and low WT1 expression respectively), suggesting WT1 driving promoter activity. ChiP using WT1 antibody confirmed WT1 binding of the critical transcription factor binding motif. CONCLUSION: These results shed light on why PTPRE expression is lower in Wilms' tumors and reveals potential future targeted therapy.Item The Development of an Animated Teaching Module Designed to Increase Understanding of the Basic Concepts of DNA, RNA, and Protein Synthesis Among Ninth Grade Biology Students(2011-12-14) Swensen, Jennie; Krumwiede, Kimberly HoggattCan a two-dimensional Flash animation be created to help teach about DNA? The goal of this thesis was to create a teaching module for DNA, RNA, and protein synthesis, designed for ninth grade students. The module contains animations created in Adobe Flash and quizzes after each section. It is to be used as a textbook and lecture supplement for high school students. Quantitative assessment showed an improvement in comprehension. Qualitative assessment showed positive feedback from both students and the teacher.Item Functional Significance of Extracellular Signal Regulated Kinase (ERK2) Phosphorylation States: Implications for DNA Binding(2013-11-04) McReynolds, Andrea Christine; Rice, Luke M.; Cobb, Melanie H.; Albanesi, Joseph P.; Yu, GangThe protein kinase extracellular signal-regulated kinase 2 (ERK2) has been well understood structurally for nearly twenty years. New insight is emerging about its structure and function. A novel autophosphorylation has recently been found to occur on a critical active site residue, threonine 188. Autophosphorylation of this residue has been suggested to occur as a result of the confluence of receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) signaling pathways. The possibility of autophosphorylation on threonine 188 seemed to be inconsistent with what was known about the structure and function of protein kinases generally and ERK2 in particular; T188 and the comparable residue in other protein kinases is required for catalytic activity. I found ERK2 phosphorylated on T188 in vitro in partially purified preparations of the recombinant protein purified following expression in bacteria. This suggests that phosphorylation of ERK2 on T188 can occur without the input of upstream RTK or GPCR signaling. Through mutagenesis experiments, I found mutation of T188 sharply reduced activity toward substrates in vitro. Protein fractions containing pT188 and purified ERK2 T188D and T188E mutants, that may act as phosphomimetics, appear to have an increased affinity for DNA binding. I examined critical residues in the activation loop important for phosphorylation and activation of ERK2 and found that perturbation of these residues influences DNA binding specificity. Although the MAPK pathway and role of ERK2 is well understood, our data suggest that previously unrecognized, higher-order signaling mechanisms that arise from additional phosphorylation events may be involved in less well characterized properties of ERK2. Second, our finding that T188 autophosphorylation can occur in recombinant ERK2 independently from upstream GPCR signaling illustrates the need for a reinvestigation of the regulation of ERK2 autophosphorylation. Finally, direct and specific DNA binding may be driven by differences in phosphorylation. The result of this work serves to redefine a most important signaling molecule in terms of its structural modifications and relationship to overall function.Item Innate Immune Sensing and Signaling of Cytosolic DNA(2015-04-10) Wu, Jiaxi; Mendell, Joshua T.; Chen, Zhijian J.; Olson, Eric N.; Cobb, Melanie H.In eukaryotic cells, DNA is normally confined within the nucleus and mitochondria. DNA exposed in the cytosol is a danger signal that warns the host of invading microbial pathogens and triggers innate immune responses including the production of type-I interferons (IFNs). Endogenous DNA that is inappropriately cleared can also accumulate in cytosol and drive pathological inflammation and autoimmune diseases such as systemic lupus erythematosus (SLE). It is well known that cytosolic DNA induces IFNs through the STING-TBK1-IRF3 axis. However, how DNA is sensed in the cytosol and how this sensing event leads to the activation of STING remains elusive. Using a cell-free complementation assay, we identified cyclic GMP-AMP (cGAMP), as a novel eukaryotic second messenger generated by DNA stimulated or DNA virus infected cells. cGAMP contains a unique phosphodiester linkage combination (both 2'-5' and 3'-5'), for which we referred to it as 2'3'-cGAMP. 2'3'-cGAMP bound to STING with nanomolar affinity and induced a dramatic conformational change that led to its activation. Through biochemical purification and quantitative mass spectrometry, we identified the enzyme that synthesizes cGAMP in a DNA dependent manner. This enzyme, which we named cyclic GMP-AMP synthase (cGAS), turned out to be the long sought-after cytosolic DNA sensor. Structural and functional studies revealed that cGAS is activated by DNA-induced dimerization. We further generated and characterized a cGAS knockout mouse strain, which failed to produce IFNs and other cytokines in response to DNA stimulation and was more vulnerable to lethal infection by DNA viruses. Together, these results not only elucidate the mechanism of cytosolic DNA sensing, but also uncover a novel second messenger-mediated signaling mechanism in innate immunity.Item Liquid-Liquid Phase Separations in Innate Immune DNA Sensing and NF-κB Signaling Pathways(August 2021) Du, Mingjian; Liu, Yi; Chen, Zhijian J.; Beutler, Bruce; O'Donnell, Kathryn A.The binding of DNA to cyclic GMP-AMP synthase (cGAS) leads to the production of the secondary messenger cyclic GMP-AMP (cGAMP), which activates innate immune responses. We have shown that DNA binding to cGAS robustly induced the formation of liquidlike droplets in which cGAS was activated. The disordered and positively charged cGAS N terminus enhanced cGAS-DNA phase separation by increasing the valencies of DNA binding. Long DNA was more efficient in promoting cGAS liquid phase separation and cGAS enzyme activity than short DNA. Moreover, free zinc ions enhanced cGAS enzyme activity both in vitro and in cells by promoting cGAS-DNA phase separation. These results demonstrated that the DNA-induced phase transition of cGAS promotes cGAMP production and innate immune signaling. Beyond cGAS-DNA phase separation, we sought to determine whether protein liquid-liquid phase separation is a ubiquitous mechanism across immune signaling pathways. NF-kappa-B essential modulator (NEMO), also known as IKBKG, is essential for the activation of IκB kinase (IKK) complex in NF-κB signaling, including Interleukin-1 (IL-1β), Tumor Necrosis Factor (TNFα) and Toll-like receptors (TLR) pathways. NEMO activates IKK complex by binding to polyubiquitin chains. Here we show that Lys63(K63)-linked or linear(M1)-linked polyubiquitin chains binding to NEMO robustly induced the formation of liquidlike droplets in which IKK was activated both in vitro and in cells. Both NEMO ubiquitin binding (NUB) domain and zinc finger (ZF) domain of NEMO contributed the multivalencies for binding to polyubiquitin chains. Long polyubiquitin chains were more efficient in promoting NEMO phase separation than short polyubiquitin chains. These results demonstrated that polyubiquitin chains induced phase transition of NEMO to promote IKK complex activation and NF-κB signaling.Item Measuring Activation of the Cytosolic DNA Sensing Pathway(2019-04-15) Varnado, Nicole L.; Tu, Benjamin; Beutler, Bruce; Cobb, Melanie H.; Chen, Zhijian J.In mammalian cells, DNA is normally sequestered within the confines of the nucleus or mitochondria. Entrance of DNA into the cytosol, whether foreign or self in origin, acts as a danger signal that triggers a host innate immune response. Cytosolically localized DNA is sensed by cyclic GMP-AMP synthase (cGAS), which synthesizes a novel second messenger known as cyclic GMP-AMP (2'3'-cGAMP). 2'3'-cGAMP, in turn, binds to and activates the ER resident adaptor Stimulator of Interferon Genes (STING), which triggers downstream signaling that culminates in the production of type-I interferons and other immune modulatory molecules. The pathway underlies the recognition of pathogenic DNA necessary to quell microbial infections, as well as the aberrant detection of self-DNA responsible for inducing certain autoimmune diseases. Such appreciation for the involvement of cGAS-cGAMP-STING signaling in numerous clinical phenotypes necessitates development of tools that can outline the extent of its contribution to various diseases. Additionally, numerous questions remain regarding the regulation of cGAS-cGAMP signaling. As 2'3'-cGAMP production is a hallmark of the pathway's activation, we sought to develop a robust method to monitor its formation in vivo, and quantify its levels in a wide variety of settings. Herein we present the development of an antibody of high sensitivity and specificity for this small molecule second messenger, capable of recognizing and quantifying 2'3'-cGAMP production in vivo. We show it can be adapted for use in a variety of techniques, to track and measure levels of 2'3'-cGAMP quantitatively, to visualize 2'3'-cGAMP produced in cells, and to quickly identify cGAMP-positive cell populations within live samples. We show this antibody to be an invaluable tool to elucidate outstanding questions in the field, and demonstrate its potential to detect patients with aberrant activation of the cGAS-STING pathway. We foresee a future in which the 2'3'-cGAMP antibody is used to quantify activation of the cGAS pathway in a variety of clinical and research settings.Item Mechanistic Link Between DNA Damage Response (DDR) Signaling & Immune Activation(2018-11-26) Bhattacharya, Souparno; Story, Michael; Shay, Jerry W.; Sadek, Hesham A.; Aroumougame, AsaithambyProper maintenance of an intact genome is crucial for cellular homeostasis. To combat threats posed by DNA damage, cells have evolved sophisticated mechanisms - collectively termed as the DNA-damage response (DDR) signaling -, which detect DNA lesions, signal their presence, and promote their repair. Contribution of proper DDR signaling in not just confined to prevention of genomic instability and carcinogenesis, as emerging evidence indicates crosstalk exists at different levels between DDR signaling machinery and our immune system. In my dissertation work, using innovative models and techniques, I deciphered how RAD51, a protein normally associated with repair and replication of DNA, regulates innate immune response. Besides detection and repair of damaged DNA, proper DDR signaling also enables checkpoint activation, which prevents cell cycle progression with unrepaired DNA lesions. In my thesis work, I have proved how failure to arrest cells in the G2-M boundary after genotoxic stress, leads to generation of micronuclei, present in the cytoplasm and subsequent immune activation. Work emanating from my thesis projects will add to the growing body of literature showing how different DDR factors' roles in modulating immune signaling are most often a consequence of their inherent ability to sense, repair and signal in response to DNA damage. Finally, our improving understanding of DDR has already provided new avenues for disease management (e.g. Use of PARP inhibitors in treating BRCA mutant tumors). A more precise understanding of mechanisms by which DDR factors are involved in regulation of cellular immunity can also be exploited to redirect the immune system for both preventing and treating variety of human pathologies including cancer, autoimmune diseases and age related disorders.Item The Role of the cGAS-Sting Pathway in DNA Vaccination and Autoimmune Disease(2017-07-10) Cheng, Philip R.; Hooper, Lora V.; Zinn, Andrew R.; Wakeland, Edward K.; Chen, Zhijian J.The innate immune system recognizes certain molecular patterns expressed by pathogens via pattern recognition receptors (PRR). As a PRR, cyclic GMP-AMP synthase (cGAS) functions as a cytosolic DNA sensor. Stimulator of Interferon Genes (STING) functions as the downstream adaptor protein. Activation of the cGAS-STING pathway results in proinflammatory cytokine production. Here I show that the cGAS-STING pathway plays dual roles in mediating DNA adjuvant activity and the use of 2'3'-cyclic GMP-AMP (cGAMP) as a vaccine adjuvant in mice, in addition to promoting autoantibody production and autoimmune inflammatory cell accumulation in lupus-prone mice. It is unclear which DNA sensor is responsible for mediating the adjuvant effects of plasmid DNA during the course of DNA vaccination. I show that the cGAS-STING pathway is required for generation of antigen-specific immune responses following DNA-adjuvanted vaccination. Mice vaccinated with influenza antigens co-administered with 2'3'-cGAMP develop robust neutralizing antibody titers, enhanced antigen-specific CD8+ T-cell responses, and are protected against lethal influenza virus challenge. The efficacy of 2'3'-cGAMP as a vaccine adjuvant can be enhanced by liposome-assisted delivery, the use of non-hydrolyzable analogs, or co-administration with CpG-C DNA. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease. The exact etiology of SLE is unclear, but work utilizing mouse models of SLE have shown that the PRRs of the innate immune system contribute to disease pathogenesis. My results show that in C57BL/6J-Faslpr/Faslpr mice, genetic ablation of cGAS or STING significantly decreases antinuclear autoantibody titers as well as a number of autoimmune inflammatory cell populations. These results are dependent on the genetic background of the mice, as genetic ablation of cGAS or STING in B6.MRL/Mp-Faslpr/Faslpr mice or B6.Sle1-Faslpr/Faslpr mice does not recapitulate the phenotype of cGAS-/- or STING gt/gt.C57BL/6J-Faslpr/Faslpr mice. My results provide more insight into the innate immune mechanisms involved in DNA vaccination and show that 2'3'-cGAMP promotes the enhanced development of protective immune responses, thereby demonstrating the potential utility of 2'3'-cGAMP as a molecular adjuvant for vaccines. Furthermore, my results demonstrate that the cGAS-STING pathway contributes to autoimmune disease development in C57BL/6J-Faslpr/Faslpr mice and implicates cGAS or STING as potential therapeutic targets for the treatment of SLE.Item Roles of Cyclic GMP-AMP Synthase in Immune Defense Against Retroviruses and Autoimmunity(2015-12-10) Gao, Daxing; Yan, Nan; Chen, Zhijian J.; Hooper, Lora V.; Pasare, ChandrashekharThe presence of DNA in the cytosol is known to trigger robust innate immunity. Cyclic GMP-AMP synthase (cGAS) is the sensor of cytosolic DNA and activation of cGAS initiates cytokine production. Here we show cGAS plays essential roles in immune defense against retroviruses as well as in autoimmune diseases caused by self-DNA. HIV infection abrogates adaptive immunity by the depletion of CD4 T cells. However, innate immune defense mechanisms against HIV are largely unknown. We show that pseudotyped HIV can infect human and mouse cell lines, leading to the production of interferons (IFN) and other antiviral cytokines. Activation of innate immunity by HIV requires viral cDNA synthesis but not cDNA integration. Furthermore, retrotranscribed HIV cDNA is sensed by the cytosolic DNA sensor cGAS, which then produces the second messenger 2'3'-cyclic GMP-AMP (cGAMP) to activate the adaptor STING. Importantly, wild-type HIV also triggers cGAMP production in human primary macrophages, underscoring the key role of cGAS in HIV sensing. Moreover, cytosolic sensing of other retroviruses such as murine leukemia virus and simian immunodeficiency virus also depends on cGAS. cGAS is important for the immune response against retroviruses, however, overactive cGAS causes autoimmunity. TREX1 is a cytosolic DNase which clears mislocalized DNA in the cytosol. Loss-of-function mutations in TREX1 cause the human disease Aicardi-Goutières syndrome (AGS). AGS manifests with abnormal type I IFN production and inflammation in multiple organs. Trex1-/- mice exhibit autoimmune and inflammatory phenotypes that are associated with elevated expression of IFN-induced genes (ISGs). Here we show that genetic ablation of cGAS in Trex1-/- mice eliminates all detectable pathological and molecular phenotypes, including ISG induction, autoantibody production, aberrant T-cell activation, and lethality. Similarly, deletion of cGAS in mice lacking DNaseII, a lysosomal enzyme that digests DNA, rescues the lethal autoimmune phenotype of the DNaseII-/- mice. Also, polyarthritis in DNaseII-/- Ifnar1-/- mice is dependent on cGAS. These results improve our understanding of immune detection of HIV and provide cGAMP as a new adjuvant for developing HIV vaccines. Moreover, our results suggest that inhibition of cGAS may lead to new treatments of some human autoimmune diseases caused by self-DNA.Item Sequencing the human genome: implications for clinical medicine(1989-09-28) Goldstein, Joseph L.Item Silencing Transcription: Promoter-Targeted Oligonucleotides Bind Chromosomal DNA Inside Cells(2009-01-08) Beane, Randall L.; Corey, David R.Aberrant gene expression can lead to multiple disease-states that can be difficult or impossible to treat using traditional small-molecule medications. An alternative approach to treating such diseases is oligonucleotide-based therapeutics, which are theoretically capable of treating or curing genetic diseases, infections, and abnormalities. Oligonucleotide-based molecules targeted to DNA are referred to as antigene agents. These molecules can silence or activate gene transcription of alleles and have many potential medical applications. However, the growth of antigene technologies has been slow despite broad therapeutic potential and unique molecular applications. Through the development of chemical modifications, oligonucleotide-based molecules are actively being improved and refined. Chemical modifications can alter the cellular uptake, toxicity, biodistribution, and plasma retention of oligonucleotides. My research goal was to further the field of synthetic antigene oligonucleotides. To do this, I targeted endogenous genes in human cancer cell lines with chemically-modified oligonucleotides, including MOEs, PNAs, ENAs, and LNAs. I established that LNAs were robust antigene agents capable of inhibiting transcription under multiple conditions. Specifically, I established that mixed-base antigene agents physically associate with the hPR-B promoter and decrease the occupancy of RNA polymerase II on the hAR and hPR genes inside human cells. Furthermore, my research indicates that antigene LNAs function in an orientation-dependent manner and that functional LNAs must target the template strand of DNA to have appreciable potency. This body of work comprised the first extensive study of a mixed-base antigene oligonucleotide in multiple human cell lines and provides the first evidence that mixed-base antigene agents can physically associate with chromosomal DNA and inhibit transcription of endogenous mammalian genes inside human cells. Collectively, my data suggest that antigene LNAs are a potent and general strategy for silencing gene expression, and that antigene LNAs also have potential therapeutic applications and possible utility in modern functional genomics.Item [Southwestern News](1996-11-25) Steeves, Susan A.Item [Southwestern News](1998-05-20) Stieglitz, HeatherItem [Southwestern News](1998-06-23) Steeves, Susan A.Item [Southwestern News](2004-07-08) Siegfried, AmandaItem [UT Southwestern Medical Center News](2007-05-31) Rian, RussellItem The Yeast Transcription Factor GAL4: A Model for Understanding Eukaryotic Transcription(2009-06-18) O'Neal, Melissa Ann; Kodadek, Thomas J.The 26S proteasome regulates numerous cellular pathways, including transcription, through proteolytic and non-proteolytic methods. The Kodadek and Johnston laboratories recently established a novel function for the proteasomal ATPases: the destabilization of activator-DNA complexes. This is independent of proteolysis but requires direct activator-ATPase interactions as well as ATP hydrolysis. The Gal4 mutant Gap71, which is hyper-sensitive to destabilization from a GAL promoter, was instrumental to this discovery. Gal4, but not Gap71, was mono-ubiquitylated in a HeLa nuclear extract and in vivo, suggesting that mono-ubiquitylation of an activator is critical to resisting destabilization by the proteasomal ATPases. To gain a better understanding of these events, the three amino acid substitutions in the Gap71 DNA-binding domain were individually cloned and analyzed for their contributions to the function of Gal4. The data showed that Serine 22 and Lysine 23 but not Lysine 25 were important for the efficiency of the activator. The charge at Lysine 23 was found to be important for Gal4-based transcription and subsequent in vitro work revealed that Gal4 was not only phosphorylated at Serine 22 but that this phosphorylation event was essential for the function of the activator. Many times a phosphorylation event precedes a mono-ubiquitylation event on an activator. Knowing the kinase and ligase machinery that modifies Gal4 would permit us to further test our model. As a result, I designed selection screens in an attempt to isolate the kinase and/or ligase machinery components that modify Gal4. While these particular enzymes were not identified, other novel genes were found to negatively affect the galactose utilization pathway, MSU1 and SPS1. Altogether, the data demonstrated that two post-translation events, phosphorylation and mono-ubiquitylation, prevent an activator-DNA complex from being disrupted, leading to an elegant model in which the proteasomal ATPases act as an important check point in transcription.