Journal articles on the topic 'In vivo inducible promoter'
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Dunstan, Sarah J., Cameron P. Simmons, and Richard A. Strugnell. "Use of In Vivo-Regulated Promoters To Deliver Antigens from Attenuated Salmonella enterica var. Typhimurium." Infection and Immunity 67, no. 10 (October 1, 1999): 5133–41. http://dx.doi.org/10.1128/iai.67.10.5133-5141.1999.
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ABSTRACT This study describes the construction and analysis of three in vivo-inducible promoter expression plasmids, containing pnirB, ppagC, and pkatG, for the delivery of foreign antigens in the ΔaroAD mutant ofSalmonella enterica var. Typhimurium (hereafter referred to as S. typhimurium). The reporter genes encoding β-galactosidase and firefly luciferase were used to assess the comparative levels of promoter activity in S. typhimuriumin vitro in response to different induction stimuli and in vivo in immunized mice. It was determined that the ppagC construct directed the expression of more β-galactosidase and luciferase inS. typhimurium than the pnirB and pkatG constructs, both in vitro and in vivo. The gene encoding the C fragment of tetanus toxin was expressed in thearoAD mutant of S. typhimurium (BRD509) under the control of the three promoters. Mice orally immunized with attenuated S. typhimurium expressing C fragment under control of the pagC promoter [BRD509(pKK/ppagC/C frag)] mounted the highest tetanus toxoid-specific serum antibody response. Levels of luciferase expression in vivo and C-fragment expression in vitro from thepagC promoter appeared to be equivalent to if not lower than the levels of expression detected with the constitutivetrc promoter. However, mice immunized with BRD509(pKK/ppagC/C frag) induced significantly higher levels of tetanus toxoid-specific antibody than BRD509(pKK/C frag)-immunized mice, suggesting that the specific location of foreign antigen expression may be important for immunogenicity. Mutagenesis of the ribosome binding sites (RBS) in the three promoter/C fragment expression plasmids was also performed. Despite optimization of the RBS in the three different promoter elements, the expression levels in vivo and overall immunogenicity of C fragment when delivered to mice by attenuated S. typhimurium were not affected. These studies suggest that in vivo-inducible promoters may give rise to enhanced immunogenicity and increase the efficacy of S. typhimuriumas a vaccine vector.
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Bateman, B. T., N. P. Donegan, T. M. Jarry, M. Palma, and A. L. Cheung. "Evaluation of a Tetracycline-Inducible Promoter inStaphylococcus aureus In Vitro and In Vivo and Its Application in Demonstrating the Role of sigB in Microcolony Formation." Infection and Immunity 69, no. 12 (December 1, 2001): 7851–57. http://dx.doi.org/10.1128/iai.69.12.7851-7857.2001.
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ABSTRACT An inducible promoter system provides a powerful tool for studying the genetic basis for virulence. A variety of inducible systems have been used in other organisms, including pXyl-xylR-inducible promoter, the pSpac-lacI system, and the arabinose-inducible PBAD promoter, but each of these systems has limitations in its application to Staphylococcus aureus. In this study, we demonstrated the efficacy of a tetracycline-inducible promoter system in inducing gene expression in S. aureus in vitro and inside epithelial cells as well as in an animal model of infection. Using the xyl/tetOpromoter::gfp uvr fusion carried on a shuttle plasmid, we demonstrated that dose-dependant tetracycline induction, as measured by bacterial fluorescence, occurred in each of the above environments while basal activation under noninduced conditions remained low. To ascertain how the system can be used to elucidate the genetic basis of a pathogenic phenotype, we cloned thesigB gene downstream of the inducible promoter. Induction of SigB expression led to dose-dependent attachment of the tested strain to polystyrene microtiter wells. Additionally, bacterial microcolony formation, an event preceding mature biofilm formation, also increased with tetracycline induction of SigB.
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LaPointe, Margot C., Xiao-Ping Yang, Oscar A. Carretero, and Quan He. "Left ventricular targeting of reporter gene expression in vivo by human BNP promoter in an adenoviral vector." American Journal of Physiology-Heart and Circulatory Physiology 283, no. 4 (October 1, 2002): H1439—H1445. http://dx.doi.org/10.1152/ajpheart.01090.2001.
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To selectively introduce genes into the mouse myocardium, we used a recombinant adenovirus encoding a transgene composed of a cardiac-specific promoter [the proximal human brain natriuretic peptide (hBNP) promoter] coupled to a luciferase reporter gene (Ad.hBNPLuc). Activity in vitro and in vivo was compared with Ad.CMVLuc, which contained the cytomegalovirus (CMV) enhancer/promoter. We tested cell-specific and inducible regulation of Ad.hBNPLuc in vitro. Expression was higher in neonatal cardiac myocytes than in a fibroblast cell line and was induced by interleukin-1β, phenylephrine, and isoproterenol in myocytes. For in vivo experiments, Ad.hBNPLuc, Ad.CMVLuc, or vehicle was injected into the left ventricular (LV) free wall of the mouse heart. In Ad.hBNPLuc-injected mice, luciferase activity was only detected in the heart. In contrast, Ad.CMVLuc-injected mice had detectable luciferase activity in all tissues examined. Our studies indicate that 1) the cardiac-specific hBNP promoter and direct cardiac injection limit expression of the transgene to the LV free wall; and 2) transgene expression in vitro is inducible and cardiac myocyte specific. Thus the use of the proximal hBNP promoter in recombinant adenoviral vectors may allow cardiac-specific and inducible expression of therapeutic genes in vivo and prevent some of the side effects of systemic adenovirus administration.
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Xiao, Yong, Takeo Kuwata, Tomoyuki Miura, Masanori Hayami, and Hisatoshi Shida. "Dox-Dependent SIVmac with Tetracycline-Inducible Promoter in the U3 Promoter Region." Virology 269, no. 2 (April 2000): 268–75. http://dx.doi.org/10.1006/viro.2000.0213.
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He, Quan, Ding Wang, Xiao-Ping Yang, Oscar A. Carretero, and Margot C. LaPointe. "Inducible regulation of human brain natriuretic peptide promoter in transgenic mice." American Journal of Physiology-Heart and Circulatory Physiology 280, no. 1 (January 1, 2001): H368—H376. http://dx.doi.org/10.1152/ajpheart.2001.280.1.h368.
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Studies have shown that brain natriuretic peptide (BNP) gene expression is rapidly induced in the infarcted heart and that plasma BNP levels reflect the degree of left ventricular dysfunction. Our previous in vitro work using transiently transfected neonatal rat cardiac myocytes has shown that the human BNP (hBNP) promoter, in particular a region extending from −127 to −40 relative to the start site of transcription, is more active in cardiac myocytes than in fibroblasts. To study tissue-specific and transcriptional regulation of the hBNP gene in vivo, we generated transgenic mice containing the proximal hBNP promoter (−408 to +100) coupled to a luciferase reporter gene. In four lines of transgenic mice, luciferase activity was ∼33- to 100-fold higher in the heart than in other tissues, including the whole brain. To test whether the transgene responded to a pathophysiological stimulus, we induced infarction by coronary artery ligation. Luciferase activity was fivefold higher in the infarcted region of the left ventricle at 48 h than in sham-operated animals and remained elevated for 4 wk. Endogenous BNP mRNA was similarly increased in the infarcted hearts of a separate group of mice. We conclude that 1) the proximal 408-bp region of the hBNP promoter confers cardiac-specific expression and 2) myocardial infarction activates the proximal hBNP promoter in vivo. These data suggest that we have a valid model for the study of basal and inducible regulation of the hBNP gene in vivo.
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Breton, Marc, Evelyne Sagné, Sybille Duret, Laure Béven, Christine Citti, and Joël Renaudin. "First report of a tetracycline-inducible gene expression system for mollicutes." Microbiology 156, no. 1 (January 1, 2010): 198–205. http://dx.doi.org/10.1099/mic.0.034074-0.
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Inducible promoter systems are powerful tools for studying gene function in prokaryotes but have never been shown to function in mollicutes. In this study we evaluated the efficacy of the tetracycline-inducible promoter Pxyl/tetO2 from Bacillus subtilis in controlling gene expression in two mollicutes, the plant pathogen Spiroplasma citri and the animal pathogen Mycoplasma agalactiae. An S. citri plasmid carrying the spiralin gene under the control of the xyl/tetO2 tetracycline-inducible promoter and the TetR repressor gene under the control of a constitutive spiroplasmal promoter was introduced into the spiralin-less S. citri mutant GII3-9a3. In the absence of tetracycline, expression of TetR almost completely abolished expression of spiralin from the xyl/tetO2 promoter. Adding tetracycline (>50 ng ml−1) to the medium induced high-level expression of spiralin. Interestingly, inducible expression of spiralin was also detected in vivo: in S. citri-infected leafhoppers fed on tetracycline-containing medium and in S. citri-infected plants watered with tetracycline. A similar construct was introduced into the M. agalactiae chromosome through transposition. Tetracycline-induced expression of spiralin proved the TetR-Pxyl/tetO2 system to be functional in the ruminant pathogen, suggesting that this tetracycline-inducible promoter system might be of general use in mollicutes.
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Charron, J., H. Richard-Foy, D. S. Berard, G. L. Hager, and J. Drouin. "Independent glucocorticoid induction and repression of two contiguous responsive genes." Molecular and Cellular Biology 9, no. 7 (July 1989): 3127–31. http://dx.doi.org/10.1128/mcb.9.7.3127.
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Specific DNA sequence elements which contain binding sites for the glucocorticoid receptor mediate the action of glucocorticoid hormones on gene transcription. In glucocorticoid-inducible genes, these glucocorticoid-responsive elements behave as hormone-inducible enhancers of transcription. We have taken advantage of the bovine papillomavirus (BPV) system to test the stringency of glucocorticoid regulation of transcription. BPV episomes were constructed to contain two hormone-regulated transcription units in close proximity; one transcription unit is under control of a glucocorticoid-inducible promoter (mouse mammary tumor virus) while the other is under control of a glucocorticoid-inhibited promoter (pro-opiomelanocortin). Glucocorticoids independently regulated transcription of the two physically linked transcription units, irrespective of their relative orientation and of their proximity on the BPV episomes. This result contrasts with the so-called position-independent activity of enhancers and suggests that the multicomponent organization of eucaryotic promoters restricts the action of hormone-responsive regulatory elements to a specific transcription unit, thus accounting for the stringency of hormonal regulation observed in vivo.
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Charron, J., H. Richard-Foy, D. S. Berard, G. L. Hager, and J. Drouin. "Independent glucocorticoid induction and repression of two contiguous responsive genes." Molecular and Cellular Biology 9, no. 7 (July 1989): 3127–31. http://dx.doi.org/10.1128/mcb.9.7.3127-3131.1989.
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Specific DNA sequence elements which contain binding sites for the glucocorticoid receptor mediate the action of glucocorticoid hormones on gene transcription. In glucocorticoid-inducible genes, these glucocorticoid-responsive elements behave as hormone-inducible enhancers of transcription. We have taken advantage of the bovine papillomavirus (BPV) system to test the stringency of glucocorticoid regulation of transcription. BPV episomes were constructed to contain two hormone-regulated transcription units in close proximity; one transcription unit is under control of a glucocorticoid-inducible promoter (mouse mammary tumor virus) while the other is under control of a glucocorticoid-inhibited promoter (pro-opiomelanocortin). Glucocorticoids independently regulated transcription of the two physically linked transcription units, irrespective of their relative orientation and of their proximity on the BPV episomes. This result contrasts with the so-called position-independent activity of enhancers and suggests that the multicomponent organization of eucaryotic promoters restricts the action of hormone-responsive regulatory elements to a specific transcription unit, thus accounting for the stringency of hormonal regulation observed in vivo.
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Piskurich, Janet F., Michael W. Linhoff, Ying Wang, and Jenny P. Y. Ting. "Two Distinct Gamma Interferon-Inducible Promoters of the Major Histocompatibility Complex Class II Transactivator Gene Are Differentially Regulated by STAT1, Interferon Regulatory Factor 1, and Transforming Growth Factor β." Molecular and Cellular Biology 19, no. 1 (January 1, 1999): 431–40. http://dx.doi.org/10.1128/mcb.19.1.431.
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ABSTRACT The major histocompatibility complex (MHC) class II transactivator (CIITA) is the master regulatory factor required for appropriate expression of class II MHC genes. Understanding the expression of CIITA is key to understanding the regulation of class II MHC genes. This report describes the independent regulation of two distinct CIITA promoters by cytokines with opposing functions, gamma interferon (IFN-γ) and transforming growth factor β (TGF-β). A functional analysis of deletion mutations of the upstream promoter (promoter III) identified an IFN-γ-responsive region located approximately 5 kb from the transcriptional start site. An in vivo DNase I hypersensitivity analysis detected a hypersensitive site in this area which supports the relevance of this region. When the downstream promoter (promoter IV) was studied by in vivo genomic footprinting, IFN-γ-induced changes at putative binding sites for STAT1, interferon regulatory factor 1 (IRF-1), and E-box proteins were seen. Gel shift and supershift analyses for IRF-1 confirmed the in vivo footprint results. The role of the IFN-γ-inducible transcription factor STAT1 was examined functionally. Although both promoters were controlled by STAT1, promoter-specific regulation was exhibited. The IFN-γ response of promoter III was completely dependent on STAT1 and not IRF-1, while promoter IV was partially activated by IRF-1 in the total absence of STAT1 expression. While both promoters were affected by TGF-β, activation of promoter III by IFN-γ was more severely diminished by TGF-β treatment. The differential control of CIITA promoters by TGF-β, IRF-1, and STAT1 may be important in refining regulation of class II MHC genes in different cell types and under different stimulatory conditions.
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Ko, H. P., S. T. Okino, Q. Ma, and J. P. Whitlock. "Transactivation domains facilitate promoter occupancy for the dioxin-inducible CYP1A1 gene in vivo." Molecular and Cellular Biology 17, no. 7 (July 1997): 3497–507. http://dx.doi.org/10.1128/mcb.17.7.3497.
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We have studied the transcriptional regulation of the dioxin-inducible mouse CYP1A1 gene in its native chromosomal setting. We analyzed the ability of aromatic hydrocarbon receptor (AhR) mutants and AhR chimeras to restore dioxin responsiveness to the CYP1A1 gene in AhR-defective mouse hepatoma cells. Our data reveal that transactivation domains in AhR's C-terminal half mediate occupancy of the nuclear factor 1 site and TATA box for the CYP1A1 promoter in vivo. Transactivation domains of VP16 and AhR nuclear translocator, but not Sp1, can substitute for AhR's C-terminal half in facilitating protein binding at the promoter. Our data also reveal an apparent linear relationship between promoter occupancy and CYP1A1 gene expression in chromatin. These findings provide new insights into the in vivo mechanism of transcriptional activation for an interesting mammalian gene.
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Szak, Suzanne T., Deborah Mays, and Jennifer A. Pietenpol. "Kinetics of p53 Binding to Promoter Sites In Vivo." Molecular and Cellular Biology 21, no. 10 (May 15, 2001): 3375–86. http://dx.doi.org/10.1128/mcb.21.10.3375-3386.2001.
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ABSTRACT Downstream target genes of p53 are thought to mediate its tumor-suppressive activity, but it is unknown whether differential transactivation of these genes is regulated at the level of p53 binding to their promoters. To address this issue, p53 binding in vivo to consensus sites in the p21Waf1, MDM2, and PIG3 promoters was investigated in cells exposed to adriamycin (ADR) or ionizing radiation as well as in an inducible p53 cell line. p53-DNA complexes were cross-linked in vivo by treating the cells with formaldehyde and processed by chromatin immunoprecipitation-PCR. This methodology allowed for the analysis of relevant p53-DNA complexes by preventing redistribution of cellular components upon collection of cell extracts. Increased p53 binding to the p21Waf1, MDM2, and PIG3 promoters occurred within 2 h after p53 activation; however, significant increases in PIG3 transcription did not occur until 15 h after p53 binding. Gel shift analyses indicated that p53 had lower affinity for the consensus binding site in the PIG3 promoters compared to its consensus sites in the p21 and MDM2 genes, which suggests that additional factors may be required to stabilize the interaction of p53 with the PIG3 promoter. Further, acetylated p53 (Lys382) was found in chemically cross-linked complexes at all promoter sites examined after treatment of cells with ADR. In summary, the kinetics of p53 binding in vivo to target gene regulatory regions does not uniformly correlate with target gene mRNA expression for the p53 target genes examined. Our results suggest that target genes with low-affinity p53 binding sites may require additional events and will have delayed kinetics of induction compared to those with high-affinity binding sites.
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Mellott, Jane K., Harry S. Nick, Michael F. Waters, Timothy R. Billiar, David A. Geller, and Sarah E. Chesrown. "Cytokine-induced changes in chromatin structure and in vivo footprints in the inducible NOS promoter." American Journal of Physiology-Lung Cellular and Molecular Physiology 280, no. 3 (March 1, 2001): L390—L399. http://dx.doi.org/10.1152/ajplung.2001.280.3.l390.
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Transcription of the human inducible nitric oxide synthase ( iNOS) gene is regulated by inflammatory cytokines in a tissue-specific manner. To determine whether differences in cytokine-induced mRNA levels between pulmonary epithelial cells (A549) and hepatic biliary epithelial cells (AKN-1) result from different protein or DNA regulatory mechanisms, we identified cytokine-induced changes in DNase I-hypersensitive (HS) sites in 13 kb of the iNOS 5′-flanking region. Data showed both constitutive and inducible HS sites in an overlapping yet cell type-specific pattern. Using in vivo footprinting and ligation-mediated PCR to detect potential DNA or protein interactions, we examined one promoter region near −5 kb containing both constitutive and cytokine-induced HS sites. In both cell types, three in vivo footprints were present in both control and cytokine-treated cells, and each mapped within a constitutive HS site. The remaining footprint appeared only in response to cytokine treatment and mapped to an inducible HS site. These studies, performed on chromatin in situ, identify a portion of the molecular mechanisms regulating transcription of the human iNOS gene in both lung- and liver-derived epithelial cells.
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Timmusk, T., U. Lendahl, H. Funakoshi, E. Arenas, H. Persson, and M. Metsis. "Identification of brain-derived neurotrophic factor promoter regions mediating tissue-specific, axotomy-, and neuronal activity-induced expression in transgenic mice." Journal of Cell Biology 128, no. 1 (January 1, 1995): 185–99. http://dx.doi.org/10.1083/jcb.128.1.185.
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The structure of rat brain-derived neurotrophic factor (BDNF) gene is complex; four 5' exons are linked to separate promoters and one 3' exon is encoding the BDNF protein. To analyze the relative importance of the regulatory regions in vivo, we have generated transgenic mice with six different promoter constructs of the BDNF gene fused to the chloramphenicol acetyl transferase reporter gene. High level and neuronal expression of the reporter gene, that in many respects recapitulated BDNF gene expression, was achieved by using 9 kb of genomic sequences covering the promoter regions that lie adjacent to each other in the genome (promoters I and II and promoters III and IV, respectively) and by including sequences of BDNF intron-exon splice junctions and 3' untranslated region in the constructs. The genomic regions responsible for the in vivo upregulation of BDNF expression in the axotomized sciatic nerve and in the brain after kainic acid-induced seizures and KCl-induced spreading depression were mapped. These data show that regulation of the different aspects of BDNF expression is controlled by different regions in vivo, and they suggest that these promoter constructs may be useful for targeted expression of heterologous genes to specific regions of the central and peripheral nervous systems in an inducible manner.
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Fortin, Pierre-Yves, Coralie Genevois, Mathilde Chapolard, Tomàs Santalucía, Anna M. Planas, and Franck Couillaud. "Dual-reporter in vivo imaging of transient and inducible heat-shock promoter activation." Biomedical Optics Express 5, no. 2 (January 13, 2014): 457. http://dx.doi.org/10.1364/boe.5.000457.
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Dubail, Iharilalao, Patrick Berche, and Alain Charbit. "Listeriolysin O as a Reporter To Identify Constitutive and In Vivo-Inducible Promoters in the Pathogen Listeria monocytogenes." Infection and Immunity 68, no. 6 (June 1, 2000): 3242–50. http://dx.doi.org/10.1128/iai.68.6.3242-3250.2000.
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ABSTRACT Listeria monocytogenes is a facultative intracellular gram-positive bacterium capable of growing in the cytoplasm of infected host cells. Bacterial escape from the phagosomal vacuole of infected cells is mainly mediated by the pore-forming hemolysin listeriolysin O (LLO) encoded by hly. LLO-negative mutants of L. monocytogenes are avirulent in the mouse model. We have developed a genetic system with hly as a reporter gene allowing the identification of both constitutive and in vivo-inducible promoters of this pathogen. Genomic libraries were created by randomly inserting L. monocytogenes chromosomal fragments upstream of the promoterless hly gene cloned into gram-positive and gram-negative shuttle vectors and expressed in an LLO-negative mutant strain. With this hly-based promoter trap system, combined with access to the L. monocytogenes genome database, we identified 20 in vitro-transcribed genes, including genes encoding (i) p60, a previously known virulence gene, (ii) a putative new hemolysin, and (iii) two proteins of the general protein secretion pathway. By using the hly-based system as an in vivo expression technology tool, nine in vivo-induced loci of L. monocytogenes were identified, including genes encoding (i) the previously known in vivo-inducible phosphatidylinositol phospholipase C and (ii) a putative N-acetylglucosamine epimerase, possibly involved in teichoic acid biosynthesis. The use of hly as a reporter is a simple and powerful alternative to classical methods for transcriptional analysis to monitor promoter activity in L. monocytogenes.
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Whitley, M. Z., D. Thanos, M. A. Read, T. Maniatis, and T. Collins. "A striking similarity in the organization of the E-selectin and beta interferon gene promoters." Molecular and Cellular Biology 14, no. 10 (October 1994): 6464–75. http://dx.doi.org/10.1128/mcb.14.10.6464.
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Transcription of the endothelial leukocyte adhesion molecule 1 (E-selectin or ELAM-1) gene is induced by the inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha (TNF-alpha). In this report, we identify four positive regulatory domains (PDI to PDIV) in the E-selectin promoter that are required for maximal levels of TNF-alpha induction in endothelial cells. In vitro DNA binding studies reveal that two of the domains contain novel adjacent binding sites for the transcription factor NF-kappa B (PDIII and PDIV), a third corresponds to a recently described CRE/ATF site (PDII), and a fourth is a consensus NF-kappa B site (PDI). Mutations that decrease the binding of NF-kappa B to any one of the NF-kappa B binding sites in vitro abolished cytokine-induced E-selectin gene expression in vivo. Previous studies demonstrated a similar correlation between ATF binding to PDII and E-selectin gene expression. Here we show that the high-mobility-group protein I(Y) [HMG I(Y)] also binds specifically to the E-selectin promoter and thereby enhances the binding of both ATF-2 and NF-kappa B to the E-selectin promoter in vitro. Moreover, mutations that interfere with HMG I(Y) binding decrease the level of cytokine-induced E-selectin expression. The organization of the TNF-alpha-inducible element of the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human beta interferon gene in that both promoters require NF-kappa B, ATF-2, and HMG I(Y). We propose that HMG I(Y) functions as a key architectural component in the assembly of inducible transcription activation complexes on both promoters.
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Whitley, M. Z., D. Thanos, M. A. Read, T. Maniatis, and T. Collins. "A striking similarity in the organization of the E-selectin and beta interferon gene promoters." Molecular and Cellular Biology 14, no. 10 (October 1994): 6464–75. http://dx.doi.org/10.1128/mcb.14.10.6464-6475.1994.
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Transcription of the endothelial leukocyte adhesion molecule 1 (E-selectin or ELAM-1) gene is induced by the inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha (TNF-alpha). In this report, we identify four positive regulatory domains (PDI to PDIV) in the E-selectin promoter that are required for maximal levels of TNF-alpha induction in endothelial cells. In vitro DNA binding studies reveal that two of the domains contain novel adjacent binding sites for the transcription factor NF-kappa B (PDIII and PDIV), a third corresponds to a recently described CRE/ATF site (PDII), and a fourth is a consensus NF-kappa B site (PDI). Mutations that decrease the binding of NF-kappa B to any one of the NF-kappa B binding sites in vitro abolished cytokine-induced E-selectin gene expression in vivo. Previous studies demonstrated a similar correlation between ATF binding to PDII and E-selectin gene expression. Here we show that the high-mobility-group protein I(Y) [HMG I(Y)] also binds specifically to the E-selectin promoter and thereby enhances the binding of both ATF-2 and NF-kappa B to the E-selectin promoter in vitro. Moreover, mutations that interfere with HMG I(Y) binding decrease the level of cytokine-induced E-selectin expression. The organization of the TNF-alpha-inducible element of the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human beta interferon gene in that both promoters require NF-kappa B, ATF-2, and HMG I(Y). We propose that HMG I(Y) functions as a key architectural component in the assembly of inducible transcription activation complexes on both promoters.
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Streker, Karin, Tina Schäfer, Christoph Freiberg, Heike Brötz-Oesterhelt, Jörg Hacker, Harald Labischinski, and Knut Ohlsen. "In Vitro and In Vivo Validation of ligA and tarI as Essential Targets in Staphylococcus aureus." Antimicrobial Agents and Chemotherapy 52, no. 12 (September 22, 2008): 4470–74. http://dx.doi.org/10.1128/aac.00548-07.
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ABSTRACT A conditional expression system has been developed using the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible Pspac promoter to validate essential genes of Staphylococcus aureus in vivo. The system has been applied to prove the essentiality of ligA and to evaluate the function of tarI, which was found to be essential in vitro but not in vivo.
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Cameron, S., DS Taylor, EC TePas, NA Speck, and B. Mathey-Prevot. "Identification of a critical regulatory site in the human interleukin-3 promoter by in vivo footprinting." Blood 83, no. 10 (May 15, 1994): 2851–59. http://dx.doi.org/10.1182/blood.v83.10.2851.2851.
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Abstract Interleukin-3 (IL-3) is involved in proliferation and differentiation of hematopoietic progenitor cells. Its expression is subject to precise, tissue-specific regulation, and has been studied extensively in the gibbon T-cell line MLA 144 by a combination of functional assays and DNA binding experiments. To extend these studies, the gibbon IL-3 promoter was cloned and in vivo footprinting of the gibbon and human IL- 3 proximal promoters was performed. The gibbon IL-3 promoter was found to be highly homologous to its human counterpart and both promoters yielded identical in vivo footprints after induction of IL-3 synthesis. In particular, we observed specific protection of three guanines over a core sequence TGTGGTTT (IF-1IL3) that had not been recognized in previous studies. IF-1IL3 is not found in other cytokine promoters, but it is conserved in the IL-3 promoter of several species and is similar to a recurring motif in viral and T-cell-specific cellular enhancers. IF-1IL3 binds a specific complex in MLA 144 and Jurkat nuclear extracts in vitro, which shares the same specificity as the complex bound by the polyoma virus and T-cell receptor delta enhancers. Mutation of the three guanines in IF-1IL3 core sequence disrupts binding in vitro and abrogates the ability of the IL-3 promoter to mediate inducible expression in T cells. Although IF-1IL3 is necessary for IL-3 expression, it is not sufficient: a truncated IL-3 promoter with an intact IF-1IL3 site but no other activator sites is transcriptionally silent. These studies describe a new regulatory element within the IL-3 promoter that is essential for expression and conserved between species.
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Chao, C. C. K., W. C. Yam, L. K. Chen, and S. Lin-Chao. "Cloning of a functional Burkitt's lymphoma polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) gene promoter by the polymerase chain reaction, and its interaction with inducible cellular factors." Biochemical Journal 286, no. 2 (September 1, 1992): 555–59. http://dx.doi.org/10.1042/bj2860555.
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The promoter of the human gene encoding the stress-responsive protein polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) was isolated from Burkitt's lymphoma cells by PCR. This promoter DNA segment (termed BiP670) or one of its 5′ deletion derivatives was fused to the bacterial chloramphenicol acetyltransferase gene and introduced into HeLa cells for transient expression. BiP670 retained transcriptional activity at both the basal and Ca2+ ionophore A23187-inducible levels. However, there was no significant increase in promoter activity following a 5 h induction with 7 microM-A23187, and less than 5-fold induction at 15 h. In contrast, the steady-state mRNA level was induced by 18-fold at 5 h. The in vivo transactivation assays with BiP670 5′ deletion derivatives indicate that the putative A23187-inducible element is located within a 70 bp DNA segment (i.e. spanning -39 to -107 bp upstream of the transcriptional initiation site). Using an in vitro gel mobility shift assay, A23187-inducible nuclear factors were identified from HeLa cell extracts. DNA-binding competition experiments also suggest that the 70 bp DNA segment contains a potential sequence motif for the binding of the A23187-inducible nuclear factors.
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Dehm, Scott M., Traci L. Hilton, Edith H. Wang, and Keith Bonham. "SRC Proximal and Core Promoter Elements Dictate TAF1 Dependence and Transcriptional Repression by Histone Deacetylase Inhibitors." Molecular and Cellular Biology 24, no. 6 (March 15, 2004): 2296–307. http://dx.doi.org/10.1128/mcb.24.6.2296-2307.2004.
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ABSTRACT Histone deacetylase inhibitors (HDIs) induce cell cycle arrest, differentiation, or apoptosis in numerous cancer cell types both in vivo and in vitro. These dramatic effects are the result of a specific reprogramming of gene expression. However, the mechanism by which these agents activate the transcription of some genes, such as p21 WAF1 , but repress others, such as cyclin D1, is currently unknown. We have been studying the human SRC gene as a model for HDI-mediated transcriptional repression. We found previously that both the tissue-specific and housekeeping SRC promoters were equally repressed by HDIs. Here we show that, despite an overt dissimilarity, both SRC promoters do share similar core promoter elements and transcription is TAF1 dependent. Detailed analysis of the SRC promoters suggested that both core and proximal promoter elements were responsible for HDI-mediated repression. This was confirmed in a series of promoter-swapping experiments with the HDI-inducible, TAF1-independent p21 WAF1 promoter. Remarkably, all the SRC-p21 WAF1 chimeric promoter constructs were not only repressed by HDIs but also dependent on TAF1. Together these experiments suggest that the overall promoter architecture, rather than discrete response elements, is responsible for HDI-mediated repression, and they implicate core promoter elements in particular as potential mediators of this response.
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Matic, Jake N., Tamsin D. Terry, David Van Bockel, Tracy Maddocks, David Tinworth, Michael P. Jennings, Steven P. Djordjevic, and Mark J. Walker. "Development of Non-Antibiotic-Resistant, Chromosomally Based, Constitutive and Inducible Expression Systems for aroA-Attenuated Salmonella enterica Serovar Typhimurium." Infection and Immunity 77, no. 5 (February 17, 2009): 1817–26. http://dx.doi.org/10.1128/iai.01301-08.
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ABSTRACT Live-vaccine delivery systems expressing two model antigens from Mycoplasma hyopneumoniae, F2P97 (Adh) and NrdF, were constructed using Salmonella enterica serovar Typhimurium aroA (STM-1), and immunogenicity in mice was evaluated. Recombinant plasmid-based expression (PBE) and chromosomally based expression (CBE) systems were constructed. The PBE system was formed by cloning both antigen genes into pJLA507 to create an operon downstream of temperature-inducible promoters. Constitutive CBE was achieved using a promoter-trapping technique whereby the promoterless operon was stably integrated into the chromosome of STM-1, and the expression of antigens was assessed. The chromosomal position of the operon was mapped in four clones. Inducible CBE was obtained by using the in vivo-induced sspA promoter and recombining the expression construct into aroD. Dual expression of the antigens was detected in all systems, with PBE producing much larger quantities of both antigens. The stability of antigen expression after in vivo passage was 100% for all CBE strains recovered. PBE and CBE strains were selected for comparison in a vaccination trial. The vaccine strains were delivered orally into mice, and significant systemic immunoglobulin M (IgM) and IgG responses against both antigens were detected among all CBE groups. No significant immune response was detected using PBE strains. Expression of recombinant antigens in S. enterica serovar Typhimurium aroA from chromosomally located strong promoters without the use of antibiotic resistance markers is a reliable and effective method of inducing a significant immune response.
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Saviola, Beatrice, Samuel C. Woolwine, and William R. Bishai. "Isolation of Acid-Inducible Genes of Mycobacterium tuberculosis with the Use of Recombinase-Based In Vivo Expression Technology." Infection and Immunity 71, no. 3 (March 2003): 1379–88. http://dx.doi.org/10.1128/iai.71.3.1379-1388.2003.
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ABSTRACT A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon γδ is active in Mycobacterium smegmatis and Mycobacterium tuberculosis. We developed a method to perform sequential double selection with mycobacteria by using RIVET, with a kanamycin preselection and a sucrose postselection. A library of M. tuberculosis DNA inserted upstream of tnpR was created, and using the double selection, we identified two promoters which are upregulated at low pH. The promoter regions drive the expression of a gene encoding a putative lipase, lipF (Rv3487c), as well as a PE-PGRS gene, Rv0834c, in a pH-dependent manner in both M. smegmatis and M. tuberculosis. The acid inducibility of lipF and Rv0834c was independent of the stress response sigma factor, SigF, as acid induction of the two genes in an M. tuberculosis sigF mutant strain was similar to that in the wild-type strain. No induction of lipF or Rv0834c was observed during infection of J774 murine macrophages, an observation which is in agreement with previous reports on the failure of phagosomes containing M. tuberculosis to acidify.
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Girbal, Laurence, Isabelle Mortier-Barrière, Frédéric Raynaud, Céline Rouanet, Christian Croux, and Philippe Soucaille. "Development of a Sensitive Gene Expression Reporter System and an Inducible Promoter-Repressor System for Clostridium acetobutylicum." Applied and Environmental Microbiology 69, no. 8 (August 2003): 4985–88. http://dx.doi.org/10.1128/aem.69.8.4985-4988.2003.
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ABSTRACT A sensitive gene expression reporter system was developed for Clostridium acetobutylicum ATCC 824 by using a customized gusA expression cassette. In discontinuous cultures, time course profiles of β-glucuronidase specific activity reflected adequately in vivo dynamic up- and down-regulation of acidogenesis- and/or solventogenesis-associated promoter expression in C. acetobutylicum. Furthermore, a new inducible gene expression system was developed in C. acetobutylicum, based on the Staphylococcus xylosus xylose operon promoter-repressor regulatory system.
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Anchisi, Stéphanie, Jessica Guerra, Geneviève Mottet-Osman, and Dominique Garcin. "Mismatches in the Influenza A Virus RNA Panhandle Prevent Retinoic Acid-Inducible Gene I (RIG-I) Sensing by Impairing RNA/RIG-I Complex Formation." Journal of Virology 90, no. 1 (October 7, 2015): 586–90. http://dx.doi.org/10.1128/jvi.01671-15.
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Influenza virus RNA (vRNA) promoter panhandle structures are believed to be sensed by retinoic acid-inducible gene I (RIG-I). The occurrence of mismatches in this double-stranded RNA structure raises questions about their effect on innate sensing. Our results suggest that mismatches in vRNA promoters decrease binding to RIG-Iin vivo, affecting RNA/RIG-I complex formation and preventing RIG-I activation. These results can be inferred to apply to other viruses and suggest that mismatches may represent a general viral strategy to escape RIG-I sensing.
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Jiang, J. G., and R. Zarnegar. "A novel transcriptional regulatory region within the core promoter of the hepatocyte growth factor gene is responsible for its inducibility by cytokines via the C/EBP family of transcription factors." Molecular and Cellular Biology 17, no. 10 (October 1997): 5758–70. http://dx.doi.org/10.1128/mcb.17.10.5758.
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Hepatocyte growth factor (HGF) is an inducible cytokine that is essential for the normal growth and development of various tissues, such as the liver. To decipher the molecular mechanisms that regulate HGF gene induction at the transcriptional level, we carried out in vitro and in vivo studies on the mouse HGF gene promoter. We have identified a novel regulatory element, located between -6 and +7 bp (from the transcription start site) in the HGF basal promoter region, which binds to inducible transcription factors and dictates responsiveness to extracellular stimuli that activate this gene. The core binding sequence for the inducible cis-acting factors was determined to be TTTGCAA (-4 to +3 bp) within the HGF promoter. Competition and gel mobility supershift assays showed that these binding complexes are composed of C/EBPbeta (CCAAT/enhancer-binding protein beta) and C/EBPdelta. DNA binding analysis also revealed that the binding site for the C/EBP family of transcription factors in the HGF promoter region overlaps that of another binding protein (complex C1), which binds specifically to a novel sequence with a core binding site of ACCGGT located adjacent to the C/EBP site (-9 to -4 bp). C1 binds to this region of the promoter and represses the inducible upregulation by C/EBP through direct competition for their individual binding sites. Partial hepatectomy, which is known to activate HGF gene expression in the liver, increased C/EBP (especially C/EBPbeta) binding activity to this region of the HGF promoter. Thus, our present results provide a mechanistic explanation for the transcriptional induction of the HGF gene by extracellular signals (i.e., cytokines) that induce tissue growth and regeneration.
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Wu, Chuanfeng, So Gun Hong, Alexander Jares, Vicky Guo, Thomas Winkler, and Cynthia E. Dunbar. "Development of an Inducible Caspase-9 Safety Switch for Pluripotent Stem Cell Based Therapies." Blood 120, no. 21 (November 16, 2012): 2047. http://dx.doi.org/10.1182/blood.v120.21.2047.2047.
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Abstract Abstract 2047 Induced pluripotent stem cell (iPSC) based therapies offer a promising path for patient-specific regenerative medicine. However, residual undifferentiated iPSCs within the graft present a potential risk of tumor development. Including a suicide gene system in the transplanted cells would allow the elimination of the transplant in case of an adverse event. For this purpose we propose the use of an inducible caspase 9 (iCasp9) suicide gene system (Di Stasi et al., 2011), consisting of human caspase 9 fused to a modified human FK-binding protein. After conditional iCasp9 dimerization by a synthetic small molecule chemical inducer (AP1903) the marked cells undergo rapid apoptosis (Clackson et al., 1998). To explore the system in the context of iPSC, we studied in vitro and in vivo purging strategies as well as therapeutically modalities using lentiviral constructs expressing iCasp9 either from a constitutional promoters or a pluripotency-specific promoter in a teratoma mouse model. We designed lentiviral vectors consisting of the iCasp9 gene linked via a 2A peptide to human CD19, driven by either CMV, EF1α, or embryonic stem cell-specific EOS (3+) promoters (Hotta et al., 2009). The iCasp9 lentiviral constructs were introduced into either into mouse iPSCs (C57Bl/6) or rhesus monkey fibroblasts that were subsequently used to derive monkey iCasp9-iPSCs for future nonhuman primate model. In mature rhesus monkey fibroblasts, cells expressing iCasp9/CD19 driven by either CMV or EF1α promoters underwent rapid and complete apoptosis with exposure to the AP1903 dimerizer (0.5μM to 10 μM), whereas the EOS (3+) promoter drove minimal iCasp9-CD19 expression in fibroblasts, as expected. Murine iPSCs transduced with the CMV-iCasp9 construct demonstrated silencing of the transgenes and no significant apoptosis induction with exposure to AP1903 (10 μM). In contrast, both the EF1α and the EOS (3+) promoters highly expressed iCasp9, allowing for successful induction of apoptosis in iPSCs following AP1903 treatment, with dosages ranging from 0.5μM to 10 μM in vitro, cell apoptosis were analyzed by Annexin V/7AAD using flow cytometry. We next tested the iCasp9 suicide gene system in an in vivo NSG mouse teratoma assay. For this we chose three different approaches: 1) In vitro treatment with AP1903(10 μM) of the iPSCs 4 hours prior to cells injection to NSG mouse (in vitro purging iCasp9-CD19 expressing cells); 2) Immediate I.V. injection of AP1903 (1mg/kg, single does) after injection of the iPSCs into NSG mouse (in vivo purging) ; and 3) Treatment with AP1903(1mg/kg) one week after iPSC injection (in vivo purging after initial teratoma formation). A non-iCasp9 mouse iPSC clone was used as a control in the assay. The results showed that mice injected with iPSCs expressing EOS (3+) or EF1α iCasp9 that underwent in vitro purging with 10 μM AP1903 did not show teratoma formation until 2 months, whereas the non-iCasp9 mouse iPSCs control mice reached the end point size of the teratoma after two weeks(1.494±0.29 g, mean±SD). Mice treated with a single dose of 1mg/kg AP1903 post iPSC cell injection demonstrated markedly delayed teratoma formation. Mice treated one week post iPSC injection with a daily AP1903 regimen (1mg/kg) for four days demonstrated inhibition of further tumor growth, but did not fully ablate existing tumors. In conclusion, we have shown that the iCasp9/AP1903 system is effective in eliminating iPSCs in vitro and in vivo. Furthermore, we show that efficient transgene expression and subsequent elimination of pluripotent cells depends on the internal promoter of the viral construct. Currently, we are investigating why the induction of the suicide gene failed to ablate existing tumors efficiently (insufficient dosage, silencing of transgenes in vivo). Disclosures: No relevant conflicts of interest to declare.
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Ohnuma, Mio, Nobuyuki Fujita, Akira Ishihama, Kan Tanaka, and Hideo Takahashi. "A Carboxy-Terminal 16-Amino-Acid Region of ς38 ofEscherichia coli Is Important for Transcription under High-Salt Conditions and Sigma Activities In Vivo." Journal of Bacteriology 182, no. 16 (August 15, 2000): 4628–31. http://dx.doi.org/10.1128/jb.182.16.4628-4631.2000.
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ABSTRACT ς38 (or ςS, the rpoS gene product) is a sigma subunit of RNA polymerase in Escherichia coli and directs transcription from a number of stationary-phase promoters as well as osmotically inducible promoters. In this study, we analyzed the function of the carboxy-terminal 16-amino-acid region of ς38 (residues 315 to 330), which is well conserved among the rpoS gene products of enteric bacterial species. Truncation of this region was shown to result in the loss of sigma activity in vivo using promoter-lacZ fusion constructs, but the mutant ς38 retained the binding activity in vivo to the core enzyme. The in vitro transcription analysis revealed that the transcription activity of ς38 holoenzyme under high potassium glutamate concentrations was significantly decreased by the truncation of the carboxy-terminal tail element.
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Mainwaring, Oliver, Holger Weishaupt, Sonja Hutter, Miao Zhao, Gabriela Rosén, Laura Breinschmid, Annemieke Verbaan, et al. "EMBR-07. MYC BUT NOT MYCN GENERATES AGGRESSIVE GROUP 3 MEDULLOBLASTOMA BY ARF PATHWAY SUPPRESSION." Neuro-Oncology 23, Supplement_1 (June 1, 2021): i7. http://dx.doi.org/10.1093/neuonc/noab090.025.
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Abstract Medulloblastoma (MB), the most common malignant pediatric brain tumor, often harbor MYC amplifications and arise in the presence of a functional p53 suppressor protein. To elucidate the mechanism behind this inexplicable tumor development we generated an inducible, immunocompetent transgenic mouse model of MYC-driven MB. Tumors driven from the glutamate transporter promoter molecularly resembled aggressive Group 3 MB driven by an enriched photoreceptor program. They developed embryonically in a monoclonal fashion in the presence of a functional unmutated p53 gene. Compared to MYCN-expressing MB driven from the same promoter, we discovered pronounced silencing of the ARF suppressor upstream of p53. We similarly found significant methylation of the ARF promoter in MYC-amplified as compared to MYCN-amplified human MB samples. While MYCN-driven tumor malignancy was more sensitive to ARF depletion, it dramatically increased metastatic spread of MYC-driven tumors. DNMT inhibition could restore ARF levels in MYC-expressing tumors but did not show any therapeutic advantage in tumors in vivo. Computational modeling suggested the HSP90 protein to act as a more specific target and ARF could indeed be restored by the HSP90 inhibitor onalespib that promoted increased survival in our inducible animal model suggesting that HSP90 inhibition could be potentially used in patients affected by MYC-driven ARF-silenced cancer.
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Algarté, Michèle, Hakju Kwon, Pierre Génin, and John Hiscott. "Identification by In Vivo Genomic Footprinting of a Transcriptional Switch Containing NF-κB and Sp1 That Regulates the IκBα Promoter." Molecular and Cellular Biology 19, no. 9 (September 1, 1999): 6140–53. http://dx.doi.org/10.1128/mcb.19.9.6140.
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ABSTRACT In unstimulated cells, NF-κB transcription factors are retained in the cytoplasm by inhibitory IκB proteins. Upon stimulation by multiple inducers including cytokines or viruses, IκBα is rapidly phosphorylated and degraded, resulting in the release of NF-κB and the subsequent increase in NF-κB-regulated gene expression. IκBα gene expression is also regulated by an NF-κB autoregulatory mechanism, via NF-κB binding sites in the IκBα promoter. In previous studies, tetracycline-inducible expression of transdominant repressors of IκBα (TD-IκBα) progressively decreased endogenous IκBα protein levels. In the present study, we demonstrate that expression of TD-IκBα blocked phorbol myristate acetate-phytohemagglutinin or tumor necrosis factor alpha-induced IκBα gene transcription and abolished NF-κB DNA binding activity, due to the continued cytoplasmic sequestration of RelA(p65) by TD-IκBα. In vivo genomic footprinting revealed stimulus-responsive protein-DNA binding not only to the −63 to −53 κB1 site but also to the adjacent −44 to −36 Sp1 site of the IκBα promoter. In vivo protection of both sites was inhibited by tetracycline-inducible TD-IκBα expression. Prolonged NF-κB binding and a temporal switch in the composition of NF-κB complexes bound to the −63 to −53 κB1 site of the IκBα promoter were also observed; with time after induction, decreased levels of transcriptionally active p50-p65 and increased p50–c-Rel heterodimers were detected at the κB1 site. Mutation of either the κB1 site or the Sp1 site abolished transcription factor binding to the respective sites and the inducibility of the IκBα promoter in transient transfection studies. These observations provide the first in vivo characterization of a promoter proximal transcriptional switch involving NF-κB and Sp1 that is essential for autoregulation of the IκBα promoter.
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Carroll, Paul, D. G. Niranjala Muttucumaru, and Tanya Parish. "Use of a Tetracycline-Inducible System for Conditional Expression in Mycobacterium tuberculosis and Mycobacterium smegmatis." Applied and Environmental Microbiology 71, no. 6 (June 2005): 3077–84. http://dx.doi.org/10.1128/aem.71.6.3077-3084.2005.
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ABSTRACT A number of essential genes have been identified in mycobacteria, but methods to study these genes have not been developed, leaving us unable to determine the function or biology of the genes. We investigated the use of a tetracycline-inducible expression system in Mycobacterium tuberculosis and Mycobacterium smegmatis. Using a reporter gene which encodes an unstable variant of GFP, we showed that tetracycline-inducible expression occurred in M. smegmatis and that expression levels were titratable to some extent by varying the concentration of tetracycline. The removal of tetracycline led to cessation of GFP expression, and we showed that this was a controllable on/off switch for fluorescence upon addition and removal of the antibiotic inducer. The system also functioned in M. tuberculosis, giving inducible expression of the reporter gene. We used homologous recombination to construct a strain of M. tuberculosis that expressed the only copy of the tryptophan biosynthetic enzyme, TrpD, from the tetracycline-inducible promoter. This strain was conditionally auxotrophic, showing auxotrophy only in the absence of tetracycline, confirming that trpD was tightly controlled by the foreign promoter. This is the first demonstration of the use of an inducible promoter to generate a conditional auxotroph of M. tuberculosis. The ability to tightly regulate genes now gives us the possibility to define the functions of essential genes by switching them off under defined conditions and paves the way for in vivo studies.
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He, Quan, and Xiao-Ping Yang. "Inducible Regulation of the Human Brain Natriuretic Peptide Promoter in Transgenic Mice." Hypertension 36, suppl_1 (October 2000): 727. http://dx.doi.org/10.1161/hyp.36.suppl_1.727-a.
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P187 Brain natriuretic peptide (BNP), a cardiac hormone, is rapidly elevated in the infarcted heart, and plasma BNP levels reflect the degree of left ventricular dysfunction. Our previous work indicated that the proximal promoter of the hBNP gene was active when injected into adult rat hearts. To study tissue-specific and inducible regulation of the hBNP gene in vivo, we generated transgenic mice containing the hBNP promoter (-408 to +100) coupled to a luciferase reporter gene. Cardiac function was measured by 2D echocardiography and transgene expression was measured by luciferase assay and tissue section immunostaining. Luciferase activity was measured in the heart, brain, kidney, liver, lung and skeletal muscle of 4 lines of mice. High levels of luciferase activity were only detected in the heart, with ventricular expression higher than atrial expression (LV=RV>RA>>LA). Immunostaining with a luciferase antibody showed staining in myocytes (line 203). There were no differences in the blood pressure, heart rate, heart size and function of transgenic mice compared with controls (line 203). To test whether the transgene responded to a pathophysiological stimulus, mice had coronary artery ligation to produce infarction. Luciferase activity was measured after 48 hr and 1, 3 and 4 weeks. Luciferase activity driven by the hBNP promoter was 4 to 41-fold higher in the infarcted left ventricle vs sham-operated animals (lines 203, 83 and 86) at 48hr, 3.1-fold higher at 1 week (line 203), and 2.5-fold at 3 and 4 weeks (line 203). There was a clear increase in heart weight to body weight ratio and a decrease in cardiac output and ejection fraction 4 weeks post-infarction. Endogenous BNP mRNA was increased in infarcted hearts from a separate group of non-transgenic mice at 48 hr. We concluded that: 1) the hBNP promoter is preferentially expressed in the heart; 2) 408hBNPLuc transgenic mice have normal blood pressure and cardiac function; and 3) the hBNP promoter is stimulated by ischemic injury following infarction, as is the endogenous mouse BNP gene. These data suggest that we have a valid model for the study of both basal and inducible regulation of the hBNP gene in vivo.
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Ko, H. P., S. T. Okino, Q. Ma, and J. P. Whitlock. "Dioxin-induced CYP1A1 transcription in vivo: the aromatic hydrocarbon receptor mediates transactivation, enhancer-promoter communication, and changes in chromatin structure." Molecular and Cellular Biology 16, no. 1 (January 1996): 430–36. http://dx.doi.org/10.1128/mcb.16.1.430.
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We have analyzed the dioxin-inducible transcriptional control mechanism for the mouse CYP1A1 gene in its native chromosomal context. Our genetic and biochemical studies indicate that a C-terminal segment of the aromatic hydrocarbon receptor (AhR) contains latent transactivation capability and communicates the induction signal from enhancer to promoter. Thus, transactivation and enhancer-promoter communication may be congruent functions of AhR. Both functions require heterodimerization between AhR and the AhR nuclear translocator (Arnt). Our findings also indicate that heterodimerization activates AhR's latent transactivation function and silences that of Arnt. Furthermore, removal of Arnt's transactivation domain does not affect dioxin-induced CYP1A1 transcription in vivo. In addition, our studies demonstrate that dioxin-induced changes in chromatin structure occur by different mechanisms at the CYP1A1 enhancer and promoter and that events at an enhancer can be experimentally dissociated from events at the cognate promoter during mechanistic analyses of mammalian transcription in vivo.
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Narumi, Ko, Akira Kojima, and Ronald G. Crystal. "Adenovirus Vector-Mediated Perforin Expression Driven by a Glucocorticoid-Inducible Promoter Inhibits Tumor GrowthIn Vivo." American Journal of Respiratory Cell and Molecular Biology 19, no. 6 (December 1998): 936–41. http://dx.doi.org/10.1165/ajrcmb.19.6.3289.
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Nguyen, Trinh Thi, Nam Hoai Nguyen, Yeonhee Kim, Jung Rae Kim, and Sunghoon Park. "In vivo Characterization of the Inducible Promoter System of 3-hydroxypropionic Dehydrogenase in Pseudomonas denitrificans." Biotechnology and Bioprocess Engineering 26, no. 4 (August 2021): 612–20. http://dx.doi.org/10.1007/s12257-020-0291-3.
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Jiang, Jie, Hui Yu, Yan Shou, Geoffrey Neale, Sheng Zhou, Taihe Lu, and Brian P. Sorrentino. "Hemgn is a direct transcriptional target of HOXB4 and induces expansion of murine myeloid progenitor cells." Blood 116, no. 5 (August 5, 2010): 711–19. http://dx.doi.org/10.1182/blood-2009-07-235341.
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HOXB4, a member of the Homeobox transcription factor family, promotes expansion of hematopoietic stem cells and hematopoietic progenitor cells in vivo and ex vivo when overexpressed. However, the molecular mechanisms underlying this effect are not well understood. To identify direct target genes of HOXB4 in primary murine hematopoietic progenitor cells, we induced HOXB4 function in lineage-negative murine bone marrow cells, using a tamoxifen-inducible HOXB4-ERT2 fusion protein. Using expression microarrays, 77 probe sets were identified with differentially changed expression in early response to HOXB4 induction. Among them, we show that Hemogen (Hemgn), encoding a hematopoietic-specific nuclear protein of unknown function, is a direct transcriptional target of HOXB4. We show that HOXB4 binds to the promoter region of Hemgn both ex vivo and in vivo. When we overexpressed Hemgn in bone marrow cells, we observed that Hemgn promoted cellular expansion in liquid cultures and increased self-renewal of myeloid colony-forming units in culture, partially recapitulating the effect of HOXB4 overexpression. Furthermore, down-regulation of Hemgn using an shRNA strategy proved that Hemgn contributes to HOXB4-mediated expansion in our myeloid progenitor assays. Our results identify a functionally relevant, direct transcriptional target of HOXB4 and identify other target genes that may also participate in the HOXB4 genetic network.
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Todd, M. D., M. J. Grusby, J. A. Lederer, E. Lacy, A. H. Lichtman, and L. H. Glimcher. "Transcription of the interleukin 4 gene is regulated by multiple promoter elements." Journal of Experimental Medicine 177, no. 6 (June 1, 1993): 1663–74. http://dx.doi.org/10.1084/jem.177.6.1663.
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Activation of T helper cell 1 (Th1) and Th2 results in transcription of the interleukin 2 (IL-2) and IL-4 cytokine genes, respectively. Whereas many of the regulatory elements and factors responsible for IL-2 transcription in T cells are well defined, little is known about parallel mechanisms that drive transcription of the IL-4 gene. Here we have analyzed the murine IL-4 promoter, both in vivo and in a Th2 clone. 3 kb of IL-4 upstream sequence is shown to be sufficient to achieve tissue-specific and inducible expression of a thymidine kinase reporter gene in vivo in a manner that mirrors the expression of endogenous IL-4. Tissue-specific and inducible expression is also demonstrated in a Th2 clone, but not in a B cell line. Deletional and mutational analysis of the IL-4 promoter demonstrated that sequences from -100 to -28 were necessary for a transcriptional response to Concanavalin A or anti-CD3 monoclonal antibody. An overlapping, yet smaller region, spanning the sequences from -60 to -28 bp was shown to be required for the response to ionomycin. Mutation of an 8-bp region from -43 to -35 of the IL-4 promoter completely abrogated IL-4 gene transcription in response to all stimuli tested. In addition, our results show that the effects of the immunosuppressive agent Cyclosporin A map to the same DNA sequences as the positive control elements. These results identify DNA sequences that are functionally important for the control of IL-4 gene transcription both in vivo and in vitro. Although these sequences are highly conserved in the human and murine IL-4 genes, they are largely not present in the IL-2 enhancer complex. Thus, cytokine-specific cis-acting elements may be one mechanism by which these two cytokine genes are differentially regulated.
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Gupta, Ashish K., and Bruce C. Kone. "USF-1 and USF-2 trans-repress IL-1β-induced iNOS transcription in mesangial cells." American Journal of Physiology-Cell Physiology 283, no. 4 (October 1, 2002): C1065—C1072. http://dx.doi.org/10.1152/ajpcell.00100.2002.
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Transcriptional activation of the inducible nitric oxide synthase (iNOS) gene requires multiple interactions of cis elements and trans-acting factors. Previous in vivo footprinting studies (Goldring CE, Reveneau S, Algarte M, and Jeannin JF. Nucleic Acids Res 24: 1682–1687, 1996) of the murine iNOS gene demonstrated lipopolysaccharide-inducible protection of guanines in the region −904/−883, which includes an E-box motif. In this report, by using site-directed mutagenesis of the −893/−888 E-box and correlating functional assays of the mutated iNOS promoter with upstream stimulatory factor (USF) DNA-binding activities, we demonstrate that the −893/−888 E-box motif is functionally required for iNOS regulation in murine mesangial cells and that USFs are in vivo components of the iNOS transcriptional response complex. Mutation of the E-box sequence augmented the iNOS response to interleukin-1β (IL-1β) in transiently transfected mesangial cells. Gel mobility shift assays demonstrated that USFs cannot bind to the −893/−888 E-box promoter region when the E-box is mutated. Cotransfection of USF-1 and USF-2 expression vectors with iNOS promoter-luciferase reporter constructs suppressed IL-1β-simulated iNOS promoter activity. Cotransfection of dominant-negative USF-2 mutants lacking the DNA binding domain or cis-element decoys containing concatamers of the −904/−883 region augmented IL-1β stimulation of iNOS promoter activity. Gel mobility shift assays showed that only USF-1 and USF-2 supershifted the USF protein-DNA complexes. These results demonstrated that USF binding to the E-box at −893/−888 serves to trans-repress basal expression and IL-1β induction of the iNOS promoter.
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Qian, Feng, and Weiqing Pan. "Construction of a tetR-Integrated Salmonella enterica Serovar Typhi CVD908 Strain That Tightly Controls Expression of the Major Merozoite Surface Protein of Plasmodium falciparum for Applications in Human Vaccine Production." Infection and Immunity 70, no. 4 (April 2002): 2029–38. http://dx.doi.org/10.1128/iai.70.4.2029-2038.2002.
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ABSTRACT Attenuated Salmonella strains are an attractive live vector for delivery of a foreign antigen to the human immune system. However, the problem with this vector lies with plasmid segregation and the low level of expression of the foreign gene in vivo when constitutive expression is employed, leading to a diminished immune response. We have established inducible expressions of foreign genes in the Salmonella enterica serovar Typhi CVD908 vaccine strain using the tetracycline response regulatory promoter. To set up this system, a tetracycline repressor (tetR) was integrated into a defined ΔaroC locus of the chromosome via suicide plasmid pJG12/tetR-neo. To remove the neo gene conferring kanamycin resistance from the locus, a cre expression vector under the control of the tetracycline response promoter was transformed into the clone; expression of the Cre recombinase excised the neo gene and generated the end strain CVD908-tetR. Expression of the luciferase reporter gene in this strain is dependent on the presence of tetracycline in the medium and can be regulated up to 4,773-fold. Moreover, the tightly controlled expression of major merozoite surface protein 1 (MSP1) and parts of Plasmodium falciparum was achieved, and the product yield was increased when the inducible expression system was employed. Inoculation of bacteria harboring plasmid pZE11/MSP142 in mice produced the protein in liver and spleen controlled by the inducer. The persistence of the plasmid-carrying bacteria in mice was determined. Peak colonization of both liver and spleen was detected on the third day postinoculation and was followed by a decline in growth curves. After 14 days postinfection, the majority of the bacteria (>90%) recovered from the liver and spleen of the mice retained the plasmid when expression was induced; this clearly indicated that stability of the expression vector in vivo was improved by inducible expression. Establishment of the regulatory system in the vaccine strain may broaden the range of its use by enhancing plasmid stability and expression levels in vivo. Moreover, the availability of the vaccine strain inducibly expressing the entire MSP1 provides possibilities for examining its immunogenicity, particularly the cellular response in animal models.
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Hull, M. W., J. Erickson, M. Johnston, and D. R. Engelke. "tRNA genes as transcriptional repressor elements." Molecular and Cellular Biology 14, no. 2 (February 1994): 1266–77. http://dx.doi.org/10.1128/mcb.14.2.1266.
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Eukaryotic genomes frequently contain large numbers of repetitive RNA polymerase III (pol III) promoter elements interspersed between and within RNA pol II transcription units, and in several instances a regulatory relationship between the two types of promoter has been postulated. In the budding yeast Saccharomyces cerevisiae, tRNA genes are the only known interspersed pol III promoter-containing repetitive elements, and we find that they strongly inhibit transcription from adjacent pol II promoters in vivo. This inhibition requires active transcription of the upstream tRNA gene but is independent of its orientation and appears not to involve simple steric blockage of the pol II upstream activator sites. Evidence is presented that different pol II promoters can be repressed by different tRNA genes placed upstream at varied distances in both orientations. To test whether this phenomenon functions in naturally occurring instances in which tRNA genes and pol II promoters are juxtaposed, we examined the sigma and Ty3 elements. This class of retrotransposons is always found integrated immediately upstream of different tRNA genes. Weakening tRNA gene transcription by means of a temperature-sensitive mutation in RNA pol III increases the pheromone-inducible expression of sigma and Ty3 elements up to 60-fold.
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Hull, M. W., J. Erickson, M. Johnston, and D. R. Engelke. "tRNA genes as transcriptional repressor elements." Molecular and Cellular Biology 14, no. 2 (February 1994): 1266–77. http://dx.doi.org/10.1128/mcb.14.2.1266-1277.1994.
Full textAbstract:
Eukaryotic genomes frequently contain large numbers of repetitive RNA polymerase III (pol III) promoter elements interspersed between and within RNA pol II transcription units, and in several instances a regulatory relationship between the two types of promoter has been postulated. In the budding yeast Saccharomyces cerevisiae, tRNA genes are the only known interspersed pol III promoter-containing repetitive elements, and we find that they strongly inhibit transcription from adjacent pol II promoters in vivo. This inhibition requires active transcription of the upstream tRNA gene but is independent of its orientation and appears not to involve simple steric blockage of the pol II upstream activator sites. Evidence is presented that different pol II promoters can be repressed by different tRNA genes placed upstream at varied distances in both orientations. To test whether this phenomenon functions in naturally occurring instances in which tRNA genes and pol II promoters are juxtaposed, we examined the sigma and Ty3 elements. This class of retrotransposons is always found integrated immediately upstream of different tRNA genes. Weakening tRNA gene transcription by means of a temperature-sensitive mutation in RNA pol III increases the pheromone-inducible expression of sigma and Ty3 elements up to 60-fold.
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McCall, G. E., D. L. Allen, F. Haddad, and K. M. Baldwin. "Transcriptional regulation of IGF-I expression in skeletal muscle." American Journal of Physiology-Cell Physiology 285, no. 4 (October 2003): C831—C839. http://dx.doi.org/10.1152/ajpcell.00047.2003.
Full textAbstract:
The present study investigated the role of transcription in the regulation of insulin-like growth factor (IGF)-I expression in skeletal muscle. RT-PCR was used to determine endogenous expression of IGF-I pre-mRNA and mRNA in control (Con) and functionally overloaded (FO) rat plantaris. The transcriptional activities of five different-length IGF-I promoter fragments controlling transcription of a firefly luciferase (FLuc) reporter gene were tested in vitro by transfection of myoblasts or in vivo during FO by direct gene transfer into the plantaris. Increased endogenous IGF-I gene transcription during 7 days of plantaris FO was evidenced by an ∼140-160% increase ( P < 0.0001) in IGF-I pre-mRNA (a transcriptional marker). IGF-I mRNA expression also increased by ∼90% ( P < 0.0001), and it was correlated ( R = 0.93; P < 0.0001) with the pre-mRNA increases. The three longest IGF-I exon 1 promoters induced reporter gene expression in proliferating C2C12 and L6E9 myoblasts. In differentiated L6E9 myotubes, promoter activity increased approximately two- to threefold over myoblasts. Overexpression of calcineurin and MyoD increased the activity of the -852/+192 promoter in C2C12 myotubes by ∼5- and ∼18-fold, respectively. However, FO did not induce these exogenous promoter fragments. Nevertheless, the present findings are consistent with the hypothesis that the IGF-I gene is transcriptionally regulated during muscle hypertrophy in vivo as evidenced by the induction of the endogenous IGF-I pre-mRNA during plantaris FO. The exon 1 promoter region of the IGF-I gene is sufficient to direct inducible expression in vitro; however, an in vivo response to FO may require elements outside the -852/+346 region of the exon 1 IGF-I promoter or features inherent to the endogenous IGF-I gene.
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Suzuki, M., C. Kuroda, E. Oda, S. Tsunoda, T. Nakamura, T. Nakajima, and K. Oda. "G10BP, an E1A-inducible negative regulator of Sp1, represses transcription of the rat fibronectin gene." Molecular and Cellular Biology 15, no. 10 (October 1995): 5423–33. http://dx.doi.org/10.1128/mcb.15.10.5423.
Full textAbstract:
Downregulation of the fibronectin (FN) gene in a rat 3Y1 derivative cell line, XhoC, transformed by the adenovirus E1A and E1B genes seems to be caused by the induction of a negative regulator, G10BP, which binds to three G-rich sequences in the promoter (T. Nakamura, T. Nakajima, S. Tsunoda, S. Nakada, K. Oda, H. Tsurui, and A. Wada, J. Virol. 66:6436-6450, 1992). These are the G10 stretch and two GC boxes consisting of the G10 stretch with one internal C residue insertion. The recognition sequences of G10BP and Sp1 (GGGCGG) overlap in these GC boxes. To analyze the mechanism of the downregulation, G10BP was purified by DNA affinity chromatography, and its molecular mass was estimated to be about 30 kDa. The promoter was modified by substituting the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, and by replacing the Sp1 motif by the T stretch. Transcription of FN promoter-chloramphenicol acetyltransferase fusion genes carrying the base substitution in one or more of these G-rich sequences both in vivo and in vitro revealed that the base substitution in any G-rich sequence results in reduction of promoter activity, although the downstream GC box (GCd) plays a primary role. The addition of G10BP severely inhibited the activities of the FN promoters carrying the wild-type GCd in vitro, while the promoters carrying the mutant GCd were unaffected. The binding affinity of G10BP and Sp1 to each of the G-rich sequences, analyzed by gel shift assays, indicated that G10BP binds strongly to the GCd, moderately to the G10 stretch, and weakly to GCu, while Sp1 binds strongly to GCu, moderately to GCd, and weakly to the G10 stretch. Sp1 binding to GCd and the G10 stretch was inhibited by G10BP, while binding to GCu was unaffected. These results indicate that FN gene transcription is inhibited in XhoC cells primarily by exclusion of Sp1 binding to GCd by G10BP and that G10BP is a new class of Sp1 negative regulator.
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Yu, Zhiyuan, Xuefeng Xia, and Bruce C. Kone. "Expression profile of a human inducible nitric oxide synthase promoter reporter in transgenic mice during endotoxemia." American Journal of Physiology-Renal Physiology 288, no. 1 (January 2005): F214—F220. http://dx.doi.org/10.1152/ajprenal.00258.2004.
Full textAbstract:
Inducible nitric oxide synthase (iNOS) is involved in many physiological and pathophysiological processes, including septic shock and acute kidney failure. Little is known about transcriptional regulation of the human iNOS gene in vivo under basal conditions or in sepsis. Accordingly, we developed transgenic mice carrying an insertional human iNOS promoter-reporter gene construct. In these mice, the proximal 8.3 kb of the human iNOS 5′-flanking region controls expression of the reporter gene of enhanced green fluorescent protein (EGFP). Patterns of human iNOS promoter/EGFP transgene expression in tissues were examined by fluorescence microscopy and immunoblotting. Endogenous murine iNOS was basally undetectable in kidney, intestine, spleen, heart, lung, liver, stomach, or brain. In contrast, EGFP from the transgene was basally expressed in kidney, brain, and spleen, but not the other tissues of the transgenic mice. Bacterial lipopolysaccharide induced endogenous iNOS expression in kidney, intestine, spleen, lung, liver, stomach, and heart, but not brain. In contrast, human iNOS promoter/EGFP transgene expression was induced above basal levels only in intestine, spleen, brain, stomach, and lung. Within kidney, human iNOS promoter/EGFP fluorescence was detected most prominently in proximal tubules of the outer cortex and collecting ducts and colocalized with endogenous mouse iNOS. Within the collecting duct, both endogenous iNOS and the human iNOS promoter/EGFP transgene were expressed in cells lacking aquaporin-2 immunoreactivity, consistent with expression in intercalated cells. Although it remains possible that essential regulatory elements reside in remote locations of the gene, our data concerning this 8.3-kb region provide the first in vivo evidence suggesting differential transcriptional control of the human iNOS gene in these organs and marked differences in transcriptional regulatory regions between the murine and human genes.
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Sužiede˙liene˙, Edita, Ke˛stutis Sužiede˙lis, Vaida Garbenčiute˙, and Staffan Normark. "The Acid-Inducible asr Gene inEscherichia coli: Transcriptional Control by thephoBR Operon." Journal of Bacteriology 181, no. 7 (April 1, 1999): 2084–93. http://dx.doi.org/10.1128/jb.181.7.2084-2093.1999.
Full textAbstract:
ABSTRACT Escherichia coli responds to external acidification (pH 4.0 to 5.0) by synthesizing a newly identified, ∼450-nucleotide RNA component. At maximal levels of induction it is one of the most abundant small RNAs in the cell and is relatively stable bacterial RNA. The acid-inducible RNA was purified, and the gene encoding it, designated asr (for acid shock RNA), mapped at 35.98 min on the E. coli chromosome. Analysis of the asr DNA sequence revealed an open reading frame coding for a 111-amino-acid polypeptide with a deduced molecular mass of approximately 11.6 kDa. According to computer-assisted analysis, the predicted polypeptide contains a typical signal sequence of 30 amino acids and might represent either a periplasmic or an outer membrane protein. Theasr gene cloned downstream from a T7 promoter was translated in vivo after transcription using a T7 RNA polymerase transcription system. Expression of a plasmid-encodedasr::lacZ fusion under a nativeasr promoter was reduced ∼15-fold in a complex medium, such as Luria-Bertani medium, versus the minimal medium. Transcription of the chromosomal asr was abolished in the presence of aphoB-phoR (a two-component regulatory system, controlling the pho regulon inducible by phosphate starvation) deletion mutant. Acid-mediated induction of the asr gene in the Δ(phoB-phoR) mutant strain was restored by introduction of the plasmid with cloned phoB-phoR genes. Primer extension analysis of the asr transcript revealed a region similar to the Pho box (the consensus sequence found in promoters transcriptionally activated by the PhoB protein) upstream from the determined transcription start. The asr promoter DNA region was demonstrated to bind PhoB protein in vitro. We discuss our results in terms of how bacteria might employ the phoB-phoRregulatory system to sense an external acidity and regulate transcription of the asr gene.
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Liang, Lu, Zhiwei Yue, Wei Du, Yang Li, Hongyan Tao, Di Wang, Ran Wang, et al. "Molecular Imaging of Inducible VEGF Expression and Tumor Progression in a Breast Cancer Model." Cellular Physiology and Biochemistry 42, no. 1 (2017): 407–15. http://dx.doi.org/10.1159/000477485.
Full textAbstract:
Background: Tumor derived vascular endothelial growth factor (VEGF) can stimulate proliferation and migration of endothelial cells and recruit endothelial progenitor cells into tumors for vascular formation via a paracrine manner. Now increasing evidence suggests that VEGF also serves as an autocrine factor promoting cell survival and tumor angiogenesis. Real time visualization of VEGF activity in the early stages of tumor formation using molecular imaging will provide unprecedented insight into the biological processes of cancer. Methods: The mouse breast cancer cell line 4T1 was transfected with an inducible, bidirectional tetracycline (Bi-Tet) promoter driving VEGF and renilla luciferase (Rluc). This was used to quantitatively image conditional switching of VEGF by bioluminescence imaging (BLI) under the control of systemic administration of doxycycline. Simultaneously, 4T1 cells were labelled with the double fusion reporter gene (Fluc-eGFP) to establish a breast cancer model. Results: We found that inducible VEGF could promote proliferation and attenuate apoptosis due to oxidative stress in an autocrine manner in vitro. In vivo studies revealed that induction of VEGF expression during early tumor development not only dramatically enhanced tumor growth but also increased tumor angiogenesis as visualized by BLI. Finally, immunohistochemistry staining confirmed that inducing VEGF expression promoted cell survival and tumor neovascularization. Conclusion: Together the inducible bidirectional tetracycline (Bi-Tet) co-expression system combined with the dual bioluminescence imaging (BLI) system provides a platform to investigate a target gene’s role in the pathologic process of cancer and facilitates noninvasive monitoring of biological responses in real time.
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Pedchenko, Tetyana V., Gye Young Park, Myungsoo Joo, Timothy S. Blackwell, and John W. Christman. "Inducible binding of PU.1 and interacting proteins to the Toll-like receptor 4 promoter during endotoxemia." American Journal of Physiology-Lung Cellular and Molecular Physiology 289, no. 3 (September 2005): L429—L437. http://dx.doi.org/10.1152/ajplung.00046.2005.
Full textAbstract:
We hypothesized that PU.1 and PU.1 interacting proteins (PIP) binding to the Toll-like receptor 4 (TLR4) promoter is involved in endotoxin-induced upregulation of TLR4 gene expression. Our results employing chromatin immunoprecipitation assays indicate that PU.1 binds to the murine TLR4 promoter both in macrophage cells and, most importantly, in whole lung tissue. Treatment of RAW 264.7 cells with endotoxin induced the association of PU.1 and the TLR4 promoter in a time-dependent manner, and this was closely tied to interactions between the TLR4 promoter and the PIP interferon regulatory factors (IRF)4 and IRF8. PU.1 binding was related to increases in steady-state TLR4 mRNA and total TLR4 protein in RAW cells. Endotoxemia in animals caused the similar inducible interaction between PU.1 and IRF4 and the TLR4 promoter in lung tissue of mice that was treated with a single intraperitoneal injection of endotoxin. PU.1 binding to the TLR4 promoter was not enhanced in the lung tissue of endotoxin-resistant C3H/HeJ mice in response to endotoxemia. Transient transfection studies in RAW cells indicate that inducible binding of PU.1 to the TLR4 promoter is abrogated by a Ser148 to Ala mutation in PU.1. These data suggest that induction of PU.1/PIP binding to the TLR4 promoter is involved in endotoxin response in vivo and may mediate transcriptional changes in TLR4 gene expression.
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Funnell, Alister P. W., Christopher A. Maloney, Lucinda J. Thompson, Janelle Keys, Michael Tallack, Andrew C. Perkins, and Merlin Crossley. "Erythroid Krüppel-Like Factor Directly Activates the Basic Krüppel-Like Factor Gene in Erythroid Cells." Molecular and Cellular Biology 27, no. 7 (February 5, 2007): 2777–90. http://dx.doi.org/10.1128/mcb.01658-06.
Full textAbstract:
ABSTRACT The Sp/Krüppel-like factor (Sp/Klf) family is comprised of around 25 zinc finger transcription factors that recognize CACCC boxes and GC-rich elements. We have investigated basic Krüppel-like factor (Bklf/Klf3) and show that in erythroid tissues its expression is highly dependent on another family member, erythroid Krüppel-like factor (Eklf/Klf1). We observe that Bklf mRNA is significantly reduced in erythroid tissues from Eklf-null murine embryos. We find that Bklf is driven primarily by two promoters, a ubiquitously active GC-rich upstream promoter, 1a, and an erythroid downstream promoter, 1b. Transcripts from the two promoters encode identical proteins. Interestingly, both the ubiquitous and the erythroid promoter are dependent on Eklf in erythroid cells. Eklf also activates both promoters in transient assays. Experiments utilizing an inducible form of Eklf demonstrate activation of the endogenous Bklf gene in the presence of an inhibitor of protein synthesis. The kinetics of activation are also consistent with Bklf being a direct Eklf target. Chromatin immunoprecipitation assays confirm that Eklf associates with both Bklf promoters. Eklf is typically an activator of transcription, whereas Bklf is noted as a repressor. Our results support the hypothesis that feedback cross-regulation occurs within the Sp/Klf family in vivo.
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Chowdhury, Rakhi Pait, Surbhi Gupta, and Dipankar Chatterji. "Identification and Characterization of the dps Promoter of Mycobacterium smegmatis: Promoter Recognition by Stress-Specific Extracytoplasmic Function Sigma Factors σH and σF." Journal of Bacteriology 189, no. 24 (October 5, 2007): 8973–81. http://dx.doi.org/10.1128/jb.01222-07.
Full textAbstract:
ABSTRACT The survival of a bacterium with a depleted oxygen or nutrient supply is important for its long-term persistence inside the host under stressful conditions. We studied a gene, dps, from Mycobacterium smegmatis, encoding a protein, Dps (for DNA binding protein from starved cells), which is overexpressed under oxidative and nutritional stresses and provides bimodal protection to the bacterial DNA. Characterization of the dps promoter in vivo is therefore important. We cloned a 1-kb putative promoter region of the dps gene of M. smegmatis in an Escherichia coli-Mycobacterium shuttle vector, pSD5B, immediately upstream of the lacZ gene. Promoter activities were assayed in vivo both in solid medium and in liquid cultures by quantitative β-galactosidase activity measurements. To characterize the minimal promoter region, a 200-bp fragment from the whole 1-kb sequence was further cloned in the same vector, and in a similar way, β-galactosidase activity was quantitated. Primer extension analysis was performed to determine the +1 transcription start site of the gene. Point mutations were inserted in the putative promoter sequences in the −10 and −20 regions, and the promoter sequence was confirmed. The promoter was not recognized by purified M. smegmatis core RNA polymerase reconstituted with purified Mycobacterium tuberculosis σA or σB during multiple- and single-round in vitro transcription assays. Promoter-specific in vivo pull-down assays with an immobilized 1-kb DNA fragment containing the dps promoter established that extracellular function sigma factors were associated with this starvation-inducible promoter. Single-round transcription at the dps promoter further supported the idea that only core RNA polymerase reconstituted with σF or σH can generate proper transcripts.
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Bhoopathi, Praveen, Anjan K. Pradhan, Amit Kumar, Santanu Maji, Padmanabhan Mannangatti, Xiaoyan Deng, Dipankar Bandyopadhyay, et al. "Conversion of a Non-Cancer-Selective Promoter into a Cancer-Selective Promoter." Cancers 14, no. 6 (March 15, 2022): 1497. http://dx.doi.org/10.3390/cancers14061497.
Full textAbstract:
Progression-elevated gene-3 (PEG-3) and rat growth arrest and DNA damage-inducible gene-34 (GADD34) display significant sequence homology with regulation predominantly transcriptional. The rat full-length (FL) and minimal (min) PEG-3 promoter display cancer-selective expression in rodent and human tumors, allowing for cancer-directed regulation of transgenes, viral replication and in vivo imaging of tumors and metastases in animals, whereas the FL- and min-GADD34-Prom lack cancer specificity. Min-PEG-Prom and min-GADD34-Prom have identical sequences except for two single-point mutation differences (at −260 bp and +159 bp). Engineering double mutations in the min-GADD34-Prom produce the GAPE-Prom. Changing one base pair (+159) or both point mutations in the min-GADD34-Prom, but not the FL-GADD34-Prom, results in cancer-selective transgene expression in diverse cancer cells (including prostate, breast, pancreatic and neuroblastoma) vs. normal counterparts. Additionally, we identified a GATA2 transcription factor binding site, promoting cancer specificity when both min-PEG-Prom mutations are present in the GAPE-Prom. Taken together, introducing specific point mutations in a rat min-GADD34-Prom converts this non-cancer-specific promoter into a cancer-selective promoter, and the addition of GATA2 with existing AP1 and PEA3 transcription factors enhances further cancer-selective activity of the GAPE-Prom. The GAPE-Prom provides a genetic tool to specifically regulate transgene expression in cancer cells.