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Sample records for acid mediated transcriptional

  1. What makes ribosome-mediated transcriptional attenuation sensitive to amino acid limitation?

    Directory of Open Access Journals (Sweden)

    Johan Elf

    2005-06-01

    Full Text Available Ribosome-mediated transcriptional attenuation mechanisms are commonly used to control amino acid biosynthetic operons in bacteria. The mRNA leader of such an operon contains an open reading frame with "regulatory" codons, cognate to the amino acid that is synthesized by the enzymes encoded by the operon. When the amino acid is in short supply, translation of the regulatory codons is slow, which allows transcription to continue into the structural genes of the operon. When amino acid supply is in excess, translation of regulatory codons is rapid, which leads to termination of transcription. We use a discrete master equation approach to formulate a probabilistic model for the positioning of the RNA polymerase and the ribosome in the attenuator leader sequence. The model describes how the current rate of amino acid supply compared to the demand in protein synthesis (signal determines the expression of the amino acid biosynthetic operon (response. The focus of our analysis is on the sensitivity of operon expression to a change in the amino acid supply. We show that attenuation of transcription can be hyper-sensitive for two main reasons. The first is that its response depends on the outcome of a race between two multi-step mechanisms with synchronized starts: transcription of the leader of the operon, and translation of its regulatory codons. The relative change in the probability that transcription is aborted (attenuated can therefore be much larger than the relative change in the time it takes for the ribosome to read a regulatory codon. The second is that the general usage frequencies of codons of the type used in attenuation control are small. A small percentage decrease in the rate of supply of the controlled amino acid can therefore lead to a much larger percentage decrease in the rate of reading a regulatory codon. We show that high sensitivity further requires a particular choice of regulatory codon among several synonymous codons for the

  2. Abscisic acid promotes proteasome-mediated degradation of the transcription coactivator NPR1 in Arabidopsis thaliana.

    Science.gov (United States)

    Ding, Yezhang; Dommel, Matthew; Mou, Zhonglin

    2016-04-01

    Proteasome-mediated turnover of the transcription coactivator NPR1 is pivotal for efficient activation of the broad-spectrum plant immune responses known as localized acquired resistance (LAR) and systemic acquired resistance (SAR) in adjacent and systemic tissues, respectively, and requires the CUL3-based E3 ligase and its adaptor proteins, NPR3 and NPR4, which are receptors for the signaling molecule salicylic acid (SA). It has been shown that SA prevents NPR1 turnover under non-inducing and LAR/SAR-inducing conditions, but how cellular NPR1 homeostasis is maintained remains unclear. Here, we show that the phytohormone abscisic acid (ABA) and SA antagonistically influence cellular NPR1 protein levels. ABA promotes NPR1 degradation via the CUL3(NPR) (3/) (NPR) (4) complex-mediated proteasome pathway, whereas SA may protect NPR1 from ABA-promoted degradation through phosphorylation. Furthermore, we demonstrate that the timing and strength of SA and ABA signaling are critical in modulating NPR1 accumulation and target gene expression. Perturbing ABA or SA signaling in adjacent tissues alters the temporal dynamic pattern of NPR1 accumulation and target gene transcription. Finally, we show that sequential SA and ABA treatment leads to dynamic changes in NPR1 protein levels and target gene expression. Our results revealed a tight correlation between sequential SA and ABA signaling and dynamic changes in NPR1 protein levels and NPR1-dependent transcription in plant immune responses. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  3. Mediator complex cooperatively regulates transcription of retinoic acid target genes with Polycomb Repressive Complex 2 during neuronal differentiation.

    Science.gov (United States)

    Fukasawa, Rikiya; Iida, Satoshi; Tsutsui, Taiki; Hirose, Yutaka; Ohkuma, Yoshiaki

    2015-11-01

    The Mediator complex (Mediator) plays key roles in transcription and functions as the nexus for integration of various transcriptional signals. Previously, we screened for Mediator cyclin-dependent kinase (CDK)-interacting factors and identified three proteins related to chromatin regulation. One of them, SUZ12 is required for both stability and activity of Polycomb Repressive Complex 2 (PRC2). PRC2 primarily suppresses gene expression through histone H3 lysine 27 trimethylation, resulting in stem cell maintenance and differentiation; perturbation of this process leads to oncogenesis. Recent work showed that Mediator contributes to the embryonic stem cell state through DNA loop formation, which is strongly associated with chromatin architecture; however, it remains unclear how Mediator regulates gene expression in cooperation with chromatin regulators (i.e. writers, readers and remodelers). We found that Mediator CDKs interact directly with the PRC2 subunit EZH2, as well as SUZ12. Known PRC2 target genes were deregulated by Mediator CDK knockdown during neuronal differentiation, and both Mediator and PRC2 complexes co-occupied the promoters of developmental genes regulated by retinoic acid. Our results provide a mechanistic link between Mediator and PRC2 during neuronal differentiation. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  4. Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

    International Nuclear Information System (INIS)

    Song, Jun; Ren, Pingping; Zhang, Lin; Wang, Xing Li; Chen, Li; Shen, Ying H.

    2010-01-01

    Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

  5. Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

    Energy Technology Data Exchange (ETDEWEB)

    Song, Jun [Qilu Hospital, Shandong University, Jinan, Shandong (China); Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Ren, Pingping; Zhang, Lin [Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Wang, Xing Li [Qilu Hospital, Shandong University, Jinan, Shandong (China); Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Chen, Li [Qilu Hospital, Shandong University, Jinan, Shandong (China); Shen, Ying H., E-mail: hyshen@bcm.edu [Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States)

    2010-02-26

    Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

  6. Assessing the Role of ETHYLENE RESPONSE FACTOR Transcriptional Repressors in Salicylic Acid-Mediated Suppression of Jasmonic Acid-Responsive Genes.

    Science.gov (United States)

    Caarls, Lotte; Van der Does, Dieuwertje; Hickman, Richard; Jansen, Wouter; Verk, Marcel C Van; Proietti, Silvia; Lorenzo, Oscar; Solano, Roberto; Pieterse, Corné M J; Van Wees, Saskia C M

    2017-02-01

    Salicylic acid (SA) and jasmonic acid (JA) cross-communicate in the plant immune signaling network to finely regulate induced defenses. In Arabidopsis, SA antagonizes many JA-responsive genes, partly by targeting the ETHYLENE RESPONSE FACTOR (ERF)-type transcriptional activator ORA59. Members of the ERF transcription factor family typically bind to GCC-box motifs in the promoters of JA- and ethylene-responsive genes, thereby positively or negatively regulating their expression. The GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Here, we investigated whether SA-induced ERF-type transcriptional repressors, which may compete with JA-induced ERF-type activators for binding at the GCC-box, play a role in SA/JA antagonism. We selected ERFs that are transcriptionally induced by SA and/or possess an EAR transcriptional repressor motif. Several of the 16 ERFs tested suppressed JA-dependent gene expression, as revealed by enhanced JA-induced PDF1.2 or VSP2 expression levels in the corresponding erf mutants, while others were involved in activation of these genes. However, SA could antagonize JA-induced PDF1.2 or VSP2 in all erf mutants, suggesting that the tested ERF transcriptional repressors are not required for SA/JA cross-talk. Moreover, a mutant in the co-repressor TOPLESS, that showed reduction in repression of JA signaling, still displayed SA-mediated antagonism of PDF1.2 and VSP2. Collectively, these results suggest that SA-regulated ERF transcriptional repressors are not essential for antagonism of JA-responsive gene expression by SA. We further show that de novo SA-induced protein synthesis is required for suppression of JA-induced PDF1.2, pointing to SA-stimulated production of an as yet unknown protein that suppresses JA-induced transcription. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  7. Abscisic Acid Antagonizes Ethylene Production through the ABI4-Mediated Transcriptional Repression of ACS4 and ACS8 in Arabidopsis.

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    Dong, Zhijun; Yu, Yanwen; Li, Shenghui; Wang, Juan; Tang, Saijun; Huang, Rongfeng

    2016-01-04

    Increasing evidence has revealed that abscisic acid (ABA) negatively modulates ethylene biosynthesis, although the underlying mechanism remains unclear. To identify the factors involved, we conducted a screen for ABA-insensitive mutants with altered ethylene production in Arabidopsis. A dominant allele of ABI4, abi4-152, which produces a putative protein with a 16-amino-acid truncation at the C-terminus of ABI4, reduces ethylene production. By contrast, two recessive knockout alleles of ABI4, abi4-102 and abi4-103, result in increased ethylene evolution, indicating that ABI4 negatively regulates ethylene production. Further analyses showed that expression of the ethylene biosynthesis genes ACS4, ACS8, and ACO2 was significantly decreased in abi4-152 but increased in the knockout mutants, with partial dependence on ABA. Chromatin immunoprecipitation-quantitative PCR assays showed that ABI4 directly binds the promoters of these ethylene biosynthesis genes and that ABA enhances this interaction. A fusion protein containing the truncated ABI4-152 peptide accumulated to higher levels than its full-length counterpart in transgenic plants, suggesting that ABI4 is destabilized by its C terminus. Therefore, our results demonstrate that ABA negatively regulates ethylene production through ABI4-mediated transcriptional repression of the ethylene biosynthesis genes ACS4 and ACS8 in Arabidopsis. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  8. Amino Acids of Epstein-Barr Virus Nuclear Antigen 3A Essential for Repression of Jκ-Mediated Transcription and Their Evolutionary Conservation

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    Dalbiès-Tran, Rozenn; Stigger-Rosser, Evelyn; Dotson, Travis; Sample, Clare E.

    2001-01-01

    Epstein-Barr virus (EBV) nuclear antigen 3A (EBNA-3A) is essential for virus-mediated immortalization of B lymphocytes in vitro and is believed to regulate transcription of cellular and/or viral genes. One known mechanism of regulation is through its interaction with the cellular transcription factor Jκ. This interaction downregulates transcription mediated by EBNA-2 and Jκ. To identify the amino acids that play a role in this interaction, we have generated mutant EBNA-3A proteins. A mutant EBNA-3A protein in which alanine residues were substituted for amino acids 199, 200, and 202 no longer downregulated transcription. Surprisingly, this mutant protein remained able to coimmunoprecipitate with Jκ. Using a reporter gene assay based on the recruitment of Jκ by various regions spanning EBNA-3A, we have shown that this mutation abolished binding of Jκ to the N-proximal region (amino acids 125 to 222) and that no other region of EBNA-3A alone was sufficient to mediate an association with Jκ. To determine the biological significance of the interaction of EBNA-3A with Jκ, we have studied its conservation in the simian lymphocryptovirus herpesvirus papio (HVP) by cloning HVP-3A, the homolog of EBNA-3A encoded by this virus. This 903-amino-acid protein exhibited 37% identity with its EBV counterpart, mainly within the amino-terminal half. HVP-3A also interacted with Jκ through a region located between amino acids 127 and 223 and also repressed transcription mediated through EBNA-2 and Jκ. The evolutionary conservation of this function, in proteins that have otherwise significantly diverged, argues strongly for an important biological role in virus-mediated immortalization of B lymphocytes. PMID:11119577

  9. Gallic Acid Inhibited Matrix Invasion and AP-1/ETS-1-Mediated MMP-1 Transcription in Human Nasopharyngeal Carcinoma Cells.

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    Pang, Jong-Hwei S; Yen, Jia-Hau; Wu, Hsiao-Ting; Huang, Sheng-Teng

    2017-06-24

    Gallic acid is a trihydroxybenzoic acid found in natural herbal plants. Gallic acid has been reported to inhibit the migration and invasive capability of various cancers. Little is known about the underlying mechanisms of invasion responsible for cancer metastasis via gallic acid. The present study was intended to investigate the anti-invasive effect of gallic acid on human nasopharyngeal carcinoma cells (NPC-BM1) and its related mechanism. Gallic acid inhibited the invasion of NPC-BM1 cells dose- and time-dependently without significant cytotoxic effect. Affymetrix oligonucleotide microarray analysis revealed matrix metalloproteinase-1 (MMP-1) as the most down-regulated gene in NPC-BM1 cells by gallic acid. The cytosolic and secreted MMP-1 levels were both found to be inhibited by gallic acid as demonstrated by western blot analysis and ELISA respectively. The mRNA expression and transcription of MMP-1 gene was also down-regulated as determined by RT/real-time PCR and promoter activity assay. The expression of two major transcription binding factors in the MMP-1 promoter, AP-1 and ETS-1, were demonstrated to be reduced by gallic acid in NPC-BM1 cells. The effect of gallic acid was associated with the inhibition of p38 MAPK signaling pathway. In addition, gallic acid enhanced the gene expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) which further suppressed the MMP-1 activity. These findings may be useful to develop a novel chemotherapeutic agent to inhibit the metastasis of nasopharyngeal cancer.

  10. The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation

    International Nuclear Information System (INIS)

    Parl, Angelika; Mitchell, Sabrina L.; Clay, Hayley B.; Reiss, Sara; Li, Zhen; Murdock, Deborah G.

    2013-01-01

    Highlights: •The function of the mitochondria fatty acid synthesis pathway is partially unknown. •Overexpression of the pathway causes transcriptional activation through PPARs. •Knock down of the pathway attenuates that activation. •The last enzyme in the pathway regulates its own transcription. •Products of the mtFASII pathway are able to drive nuclear transcription. -- Abstract: Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease

  11. The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation

    Energy Technology Data Exchange (ETDEWEB)

    Parl, Angelika; Mitchell, Sabrina L.; Clay, Hayley B.; Reiss, Sara; Li, Zhen; Murdock, Deborah G., E-mail: deborah.murdock@vanderbilt.edu

    2013-11-15

    Highlights: •The function of the mitochondria fatty acid synthesis pathway is partially unknown. •Overexpression of the pathway causes transcriptional activation through PPARs. •Knock down of the pathway attenuates that activation. •The last enzyme in the pathway regulates its own transcription. •Products of the mtFASII pathway are able to drive nuclear transcription. -- Abstract: Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease.

  12. Assessing the Role of ETHYLENE RESPONSE FACTOR Transcriptional Repressors in Salicylic Acid-Mediated Suppression of Jasmonic Acid-Responsive Genes

    NARCIS (Netherlands)

    Caarls, Lotte; van der Does, Adriana; Hickman, Richard; Jansen, Wouter; van Verk, Marcel; Proietti, Silvia; Lorenzo, Oscar; Solano, Roberto; Pieterse, Corné M J; Van Wees, Saskia C M

    2017-01-01

    Salicylic acid (SA) and jasmonic acid (JA) cross-communicate in the plant immune signaling network to finely regulate induced defenses. In Arabidopsis, SA antagonizes many JA-responsive genes, partly by targeting the ETHYLENE RESPONSE FACTOR (ERF)-type transcriptional activator ORA59. Members of the

  13. Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae

    KAUST Repository

    Aouida, Mustapha; Li, Lixin; Mahjoub, Ali; Alshareef, Sahar; Ali, Zahir; Piatek, Agnieszka Anna; Mahfouz, Magdy M.

    2015-01-01

    Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S.cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S.cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. © 2015 The Society for Biotechnology, Japan.

  14. Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae

    KAUST Repository

    Aouida, Mustapha

    2015-04-01

    Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S.cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S.cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. © 2015 The Society for Biotechnology, Japan.

  15. Transcription regulation by the Mediator complex.

    Science.gov (United States)

    Soutourina, Julie

    2018-04-01

    Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.

  16. How salicylic acid takes transcriptional control over jasmonic acid signaling

    Directory of Open Access Journals (Sweden)

    Lotte eCaarls

    2015-03-01

    Full Text Available Transcriptional regulation is a central process in plant immunity. The induction or repression of defense genes is orchestrated by signaling networks that are directed by plant hormones of which salicylic acid (SA and jasmonic acid (JA are the major players. Extensive cross-communication between the hormone signaling pathways allows for fine tuning of transcriptional programs, determining resistance to invaders and trade-offs with plant development. Here, we give an overview of how SA can control transcriptional reprogramming of JA-induced genes in Arabidopsis thaliana. SA can influence activity and/or localization of transcriptional regulators by post-translational modifications of transcription factors and co-regulators. SA-induced redox changes, mediated by thioredoxins and glutaredoxins, modify transcriptional regulators that are involved in suppression of JA-dependent genes, such as NPR1 and TGA transcription factors, which affects their localization or DNA binding activity. Furthermore, SA can mediate sequestering of JA-responsive transcription factors away from their target genes by stalling them in the cytosol or in complexes with repressor proteins in the nucleus. SA also affects JA-induced transcription by inducing degradation of transcription factors with an activating role in JA signaling, as was shown for the ERF transcription factor ORA59. Additionally, SA can induce negative regulators, among which WRKY transcription factors, that can directly or indirectly inhibit JA-responsive gene expression. Finally, at the DNA level, modification of histones by SA-dependent factors can result in repression of JA-responsive genes. These diverse and complex regulatory mechanisms affect important signaling hubs in the integration of hormone signaling networks. Some pathogens have evolved effectors that highjack hormone crosstalk mechanisms for their own good, which are described in this review as well.

  17. Comparative transcriptional profiling of Gracilariopsis lemaneiformis in response to salicylic acid- and methyl jasmonate-mediated heat resistance.

    Directory of Open Access Journals (Sweden)

    Fangjun Wang

    Full Text Available Culturing the economically important macroalga Gracilariopsis lemaneiformis (Rhodophyta is limited due to the high temperatures in the summertime on the southern Chinese coast. Previous studies have demonstrated that two phytohormones, salicylic acid (SA and methyl jasmonate (MJ, can alleviate the adverse effects of high-temperature stress on Gp. lemaneiformis. To elucidate the molecular mechanisms underlying SA- and MJ-mediated heat tolerance, we performed comprehensive analyses of transcriptome-wide gene expression profiles using RNA sequencing (RNA-seq technology. A total of 14,644 unigenes were assembled, and 10,501 unigenes (71.71% were annotated to the reference databases. In the SA, MJ and SA/MJ treatment groups, 519, 830, and 974 differentially expressed unigenes were detected, respectively. Unigenes related to photosynthesis and glycometabolism were enriched by SA, while unigenes associated with glycometabolism, protein synthesis, heat shock and signal transduction were increased by MJ. A crosstalk analysis revealed that 216 genes were synergistically regulated, while 18 genes were antagonistically regulated by SA and MJ. The results indicated that the two phytohormones could mitigate the adverse effects of heat on multiple pathways, and they predominantly acted synergistically to resist heat stress. These results will provide new insights into how SA and MJ modulate the molecular mechanisms that counteract heat stress in algae.

  18. Signal Transducer and Activator of Transcription 3 (STAT3) Mediates Amino Acid Inhibition of Insulin Signaling through Serine 727 Phosphorylation*

    OpenAIRE

    Kim, Jeong-Ho; Yoon, Mee-Sup; Chen, Jie

    2009-01-01

    Nutrient overload is associated with the development of obesity, insulin resistance, and type II diabetes. High plasma concentrations of amino acids have been found to correlate with insulin resistance. At the cellular level, excess amino acids impair insulin signaling, the mechanisms of which are not fully understood. Here, we report that STAT3 plays a key role in amino acid dampening of insulin signaling in hepatic cells. Excess amino acids inhibited insulin-stimulated Akt phosphorylation a...

  19. The Mediator complex and transcription regulation

    Science.gov (United States)

    Poss, Zachary C.; Ebmeier, Christopher C.

    2013-01-01

    The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module. PMID:24088064

  20. Auxin-dependent compositional change in Mediator in ARF7- and ARF19-mediated transcription.

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    Ito, Jun; Fukaki, Hidehiro; Onoda, Makoto; Li, Lin; Li, Chuanyou; Tasaka, Masao; Furutani, Masahiko

    2016-06-07

    Mediator is a multiprotein complex that integrates the signals from transcription factors binding to the promoter and transmits them to achieve gene transcription. The subunits of Mediator complex reside in four modules: the head, middle, tail, and dissociable CDK8 kinase module (CKM). The head, middle, and tail modules form the core Mediator complex, and the association of CKM can modify the function of Mediator in transcription. Here, we show genetic and biochemical evidence that CKM-associated Mediator transmits auxin-dependent transcriptional repression in lateral root (LR) formation. The AUXIN/INDOLE 3-ACETIC ACID 14 (Aux/IAA14) transcriptional repressor inhibits the transcriptional activity of its binding partners AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 by making a complex with the CKM-associated Mediator. In addition, TOPLESS (TPL), a transcriptional corepressor, forms a bridge between IAA14 and the CKM component MED13 through the physical interaction. ChIP assays show that auxin induces the dissociation of MED13 but not the tail module component MED25 from the ARF7 binding region upstream of its target gene. These findings indicate that auxin-induced degradation of IAA14 changes the module composition of Mediator interacting with ARF7 and ARF19 in the upstream region of their target genes involved in LR formation. We suggest that this regulation leads to a quick switch of signal transmission from ARFs to target gene expression in response to auxin.

  1. Transcription, Signaling Receptor Activity, Oxidative Phosphorylation, and Fatty Acid Metabolism Mediate the Presence of Closely Related Species in Distinct Intertidal and Cold-Seep Habitats.

    Science.gov (United States)

    Van Campenhout, Jelle; Vanreusel, Ann; Van Belleghem, Steven; Derycke, Sofie

    2015-12-03

    Bathyal cold seeps are isolated extreme deep-sea environments characterized by low species diversity while biomass can be high. The Håkon Mosby mud volcano (Barents Sea, 1,280 m) is a rather stable chemosynthetic driven habitat characterized by prominent surface bacterial mats with high sulfide concentrations and low oxygen levels. Here, the nematode Halomonhystera hermesi thrives in high abundances (11,000 individuals 10 cm(-2)). Halomonhystera hermesi is a member of the intertidal Halomonhystera disjuncta species complex that includes five cryptic species (GD1-5). GD1-5's common habitat is characterized by strong environmental fluctuations. Here, we compared the transcriptomes of H. hermesi and GD1, H. hermesi's closest relative. Genes encoding proteins involved in oxidative phosphorylation are more strongly expressed in H. hermesi than in GD1, and many genes were only observed in H. hermesi while being completely absent in GD1. Both observations could in part be attributed to high sulfide concentrations and low oxygen levels. Additionally, fatty acid elongation was also prominent in H. hermesi confirming the importance of highly unsaturated fatty acids in this species. Significant higher amounts of transcription factors and genes involved in signaling receptor activity were observed in GD1 (many of which were completely absent in H. hermesi), allowing fast signaling and transcriptional reprogramming which can mediate survival in dynamic intertidal environments. GC content was approximately 8% higher in H. hermesi coding unigenes resulting in differential codon usage between both species and a higher proportion of amino acids with GC-rich codons in H. hermesi. In general our results showed that most pathways were active in both environments and that only three genes are under natural selection. This indicates that also plasticity should be taken in consideration in the evolutionary history of Halomonhystera species. Such plasticity, as well as possible

  2. Ursodeoxycholic acid protects cardiomyocytes against cobalt chloride induced hypoxia by regulating transcriptional mediator of cells stress hypoxia inducible factor 1α and p53 protein.

    Science.gov (United States)

    Mohamed, Anis Syamimi; Hanafi, Noorul Izzati; Sheikh Abdul Kadir, Siti Hamimah; Md Noor, Julina; Abdul Hamid Hasani, Narimah; Ab Rahim, Sharaniza; Siran, Rosfaiizah

    2017-10-01

    In hepatocytes, ursodeoxycholic acid (UDCA) activates cell signalling pathways such as p53, intracellular calcium ([Ca 2+ ] i ), and sphingosine-1-phosphate (S1P)-receptor via Gα i -coupled-receptor. Recently, UDCA has been shown to protect the heart against hypoxia-reoxygenation injury. However, it is not clear whether UDCA cardioprotection against hypoxia acts through a transcriptional mediator of cells stress, HIF-1α and p53. Therefore, in here, we aimed to investigate whether UDCA could protect cardiomyocytes (CMs) against hypoxia by regulating expression of HIF-1α, p53, [Ca 2+ ] i , and S1P-Gα i -coupled-receptor. Cardiomyocytes were isolated from newborn rats (0-2 days), and hypoxia was induced by using cobalt chloride (CoCl 2 ). Cardiomyocytes were treated with UDCA and cotreated with either FTY720 (S1P-receptor agonist) or pertussis toxin (PTX; Gα i inhibitor). Cells were subjected for proliferation assay, beating frequency, QuantiGene Plex assay, western blot, immunofluorescence, and calcium imaging. Our findings showed that UDCA counteracted the effects of CoCl 2 on cell viability, beating frequency, HIF-1α, and p53 protein expression. We found that these cardioprotection effects of UDCA were similar to FTY720, S1P agonist. Furthermore, we observed that UDCA protects CMs against CoCl 2 -induced [Ca 2+ ] i dynamic alteration. Pharmacological inhibition of the Gα i -sensitive receptor did not abolish the cardioprotection of UDCA against CoCl 2 detrimental effects, except for cell viability and [Ca 2+ ] i . Pertussis toxin is partially effective in inhibiting UDCA protection against CoCl 2 effects on CM cell viability. Interestingly, PTX fully inhibits UDCA cardioprotection on CoCl 2 -induced [Ca 2+ ] i dynamic changes. We conclude that UDCA cardioprotection against CoCl 2 -induced hypoxia is similar to FTY720, and its actions are not fully mediated by the Gα i -coupled protein sensitive pathways. Ursodeoxycholic acid is the most hydrophilic bile

  3. A retinoic acid response element that overlaps an estrogen response element mediates multihormonal sensitivity in transcriptional activation of the lactoferrin gene.

    Science.gov (United States)

    Lee, M O; Liu, Y; Zhang, X K

    1995-08-01

    The lactoferrin gene is highly expressed in many different tissues, and its expression is controlled by different regulators. In this report, we have defined a retinoic acid response element (RARE) in the 5'-flanking region of the lactoferrin gene promoter. The lactoferrin-RARE is composed of two AGGTCA-like motifs arranged as a direct repeat with 1-bp spacing (DR-1). A gel retardation assay demonstrated that it bound strongly with retinoid X receptor (RXR) homodimers and RXR-retinoic acid receptor (RAR) heterodimers as well as chicken ovalbumin upstream promoter transcription factor (COUP-TF) orphan receptor. In CV-1 cells, the lactoferrin-RARE linked with a heterologous thymidine kinase promoter was strongly activated by RXR homodimers in response to 9-cis-retinoic acid (9-cis-RA) but not to all-trans-RA. When the COUP-TF orphan receptor was cotransfected, the 9-cis-RA-induced RXR homodimer activity was strongly repressed. A unique feature of the lactoferrin-RARE is that it has an AGGTCA-like motif in common with an estrogen-responsive element (ERE). The composite RARE/ERE contributes to the functional interaction between retinoid receptors and the estrogen receptor (ER) and their ligands. In CV-1 cells, cotransfection of the retinoid and estrogen receptors led to mutual inhibition of the other's activity, while an RA-dependent inhibition of ER activity was observed in breast cancer cells. Furthermore, the lactoferrin-RARE/ERE showed differential transactivation activity in different cell types. RAs could activate the lactoferrin-RARE/ERE in human leukemia HL-60 cells and U937 cells but not in human breast cancer cells. By gel retardation analyses, we demonstrated that strong binding of the endogenous COUP-TF in breast cancer cells to the composite element contributed to diminished RA response in these cells. Thus, the lactoferrin-RARE/ERE functions as a signaling switch module that mediates multihormonal responsiveness in the regulation of lactoferrin gene

  4. Salacia oblonga root improves cardiac lipid metabolism in Zucker diabetic fatty rats: Modulation of cardiac PPAR-α-mediated transcription of fatty acid metabolic genes

    International Nuclear Information System (INIS)

    Huang, Tom H.-W.; Yang Qinglin; Harada, Masaki; Uberai, Jasna; Radford, Jane; Li, George Q.; Yamahara, Johji; Roufogalis, Basil D.; Li Yuhao

    2006-01-01

    Excess cardiac triglyceride accumulation in diabetes and obesity induces lipotoxicity, which predisposes the myocytes to death. On the other hand, increased cardiac fatty acid (FA) oxidation plays a role in the development of myocardial dysfunction in diabetes. PPAR-α plays an important role in maintaining homeostasis of lipid metabolism. We have previously demonstrated that the extract from Salacia oblonga root (SOE), an Ayurvedic anti-diabetic and anti-obesity medicine, improves hyperlipidemia in Zucker diabetic fatty (ZDF) rats (a genetic model of type 2 diabetes and obesity) and possesses PPAR-α activating properties. Here we demonstrate that chronic oral administration of SOE reduces cardiac triglyceride and FA contents and decreases the Oil red O-stained area in the myocardium of ZDF rats, which parallels the effects on plasma triglyceride and FA levels. Furthermore, the treatment suppressed cardiac overexpression of both FA transporter protein-1 mRNA and protein in ZDF rats, suggesting inhibition of increased cardiac FA uptake as the basis for decreased cardiac FA levels. Additionally, the treatment also inhibited overexpression in ZDF rat heart of PPAR-α mRNA and protein and carnitine palmitoyltransferase-1, acyl-CoA oxidase and 5'-AMP-activated protein kinase mRNAs and restored the downregulated acetyl-CoA carboxylase mRNA. These results suggest that SOE inhibits cardiac FA oxidation in ZDF rats. Thus, our findings suggest that improvement by SOE of excess cardiac lipid accumulation and increased cardiac FA oxidation in diabetes and obesity occurs by reduction of cardiac FA uptake, thereby modulating cardiac PPAR-α-mediated FA metabolic gene transcription

  5. WRKY Transcription Factors: Key Components in Abscisic Acid Signaling

    Science.gov (United States)

    2011-01-01

    networks that take inputs from numerous stimuli and that they are involved in mediating responses to numerous phytohormones including salicylic acid ... jasmonic acid , ABA and GA. These roles in multiple signalling pathways may in turn partly explain the pleiotropic effects commonly seen when TF genes are...Review article WRKY transcription factors: key components in abscisic acid signalling Deena L. Rushton1, Prateek Tripathi1, Roel C. Rabara1, Jun Lin1

  6. Proteins mediating DNA loops effectively block transcription.

    Science.gov (United States)

    Vörös, Zsuzsanna; Yan, Yan; Kovari, Daniel T; Finzi, Laura; Dunlap, David

    2017-07-01

    Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  7. Adenovirus DNA binding protein inhibits SrCap-activated CBP and CREB-mediated transcription

    International Nuclear Information System (INIS)

    Xu Xiequn; Tarakanova, Vera; Chrivia, John; Yaciuk, Peter

    2003-01-01

    The SNF2-related CBP activator protein (SrCap) is a potent activator of transcription mediated by CBP and CREB. We have previously demonstrated that the Adenovirus 2 DNA Binding Protein (DBP) binds to SrCap and inhibits the transcription mediated by the carboxyl-terminal region of SrCap (amino acids 1275-2971). We report here that DBP inhibits the ability of full-length SrCap (1-2971) to activate transcription mediated by Gal-CREB and Gal-CBP. In addition, DBP also inhibits the ability of SrCap to enhance Protein Kinase A (PKA) activated transcription of the enkaphalin promoter. DBP was found to dramatically inhibit transcription of a mammalian two-hybrid system that was dependent on the interaction of SrCap and CBP binding domains. We also found that DBP has no effect on transcription mediated by a transcriptional activator that is not related to SrCap, indicating that our reported transcriptional inhibition is specific for SrCap and not due to nonspecific effects of DBP's DNA binding activity on the CAT reporter plasmid. Taken together, these results suggest a model in which DBP inhibits cellular transcription mediated by the interaction between SrCap and CBP

  8. How salicylic acid takes transcriptional control over jasmonic acid signaling

    NARCIS (Netherlands)

    Caarls, Lotte|info:eu-repo/dai/nl/371746213; Pieterse, Corné M J|info:eu-repo/dai/nl/113115113; van Wees, Saskia C M|info:eu-repo/dai/nl/185445373

    2015-01-01

    Transcriptional regulation is a central process in plant immunity. The induction or repression of defense genes is orchestrated by signaling networks that are directed by plant hormones of which salicylic acid (SA) and jasmonic acid (JA) are the major players. Extensive cross-communication between

  9. Plant Mediator complex and its critical functions in transcription regulation.

    Science.gov (United States)

    Yang, Yan; Li, Ling; Qu, Li-Jia

    2016-02-01

    The Mediator complex is an important component of the eukaryotic transcriptional machinery. As an essential link between transcription factors and RNA polymerase II, the Mediator complex transduces diverse signals to genes involved in different pathways. The plant Mediator complex was recently purified and comprises conserved and specific subunits. It functions in concert with transcription factors to modulate various responses. In this review, we summarize the recent advances in understanding the plant Mediator complex and its diverse roles in plant growth, development, defense, non-coding RNA production, response to abiotic stresses, flowering, genomic stability and metabolic homeostasis. In addition, the transcription factors interacting with the Mediator complex are also highlighted. © 2015 Institute of Botany, Chinese Academy of Sciences.

  10. The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor.

    Science.gov (United States)

    Kim, Mi Jung; Jang, In-Cheol; Chua, Nam-Hai

    2016-07-01

    The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15 However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

  11. Suppression of Thyroid Hormone Receptor-Mediated Transcription ...

    African Journals Online (AJOL)

    TH)-induced TR-mediated transcription. We further examined the effects of methamidophos on TR-thyroid hormone response element (TRE) binding using the liquid chemiluminescent DNA pull-down assay (LCDPA), and found no dissociation of ...

  12. Fungal mediator tail subunits contain classical transcriptional activation domains.

    Science.gov (United States)

    Liu, Zhongle; Myers, Lawrence C

    2015-04-01

    Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. A retinoic acid response element that overlaps an estrogen response element mediates multihormonal sensitivity in transcriptional activation of the lactoferrin gene.

    OpenAIRE

    Lee, M O; Liu, Y; Zhang, X K

    1995-01-01

    The lactoferrin gene is highly expressed in many different tissues, and its expression is controlled by different regulators. In this report, we have defined a retinoic acid response element (RARE) in the 5'-flanking region of the lactoferrin gene promoter. The lactoferrin-RARE is composed of two AGGTCA-like motifs arranged as a direct repeat with 1-bp spacing (DR-1). A gel retardation assay demonstrated that it bound strongly with retinoid X receptor (RXR) homodimers and RXR-retinoic acid re...

  14. The Mediator complex: a central integrator of transcription

    Science.gov (United States)

    Allen, Benjamin L.; Taatjes, Dylan J.

    2016-01-01

    The RNA polymerase II (pol II) enzyme transcribes all protein-coding and most non-coding RNA genes and is globally regulated by Mediator, a large, conformationally flexible protein complex with variable subunit composition (for example, a four-subunit CDK8 module can reversibly associate). These biochemical characteristics are fundamentally important for Mediator's ability to control various processes important for transcription, including organization of chromatin architecture and regulation of pol II pre-initiation, initiation, re-initiation, pausing, and elongation. Although Mediator exists in all eukaryotes, a variety of Mediator functions appear to be specific to metazoans, indicative of more diverse regulatory requirements. PMID:25693131

  15. TALE-mediated modulation of transcriptional enhancers in vivo.

    Science.gov (United States)

    Crocker, Justin; Stern, David L

    2013-08-01

    We tested whether transcription activator-like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.

  16. Mediator Undergoes a Compositional Change during Transcriptional Activation.

    Science.gov (United States)

    Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

    2016-11-03

    Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Malleable machines in transcription regulation: the mediator complex.

    Directory of Open Access Journals (Sweden)

    Agnes Tóth-Petróczy

    2008-12-01

    Full Text Available The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II. The complex has a modular architecture (Head, Middle, and Tail and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs distinguished by a high protein-protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the

  18. Nucleic Acid Analogue Induced Transcription of Double Stranded DNA

    DEFF Research Database (Denmark)

    1998-01-01

    RNA is transcribed from a double stranded DNA template by forming a complex by hybridizing to the template at a desired transcription initiation site one or more oligonucleic acid analogues of the PNA type capable of forming a transcription initiation site with the DNA and exposing the complex...... to the action of a DNA dependant RNA polymerase in the presence of nucleoside triphosphates. Equal length transcripts may be obtained by placing a block to transcription downstream from the initiation site or by cutting the template at such a selected location. The initiation site is formed by displacement...... of one strand of the DNA locally by the PNA hybridization....

  19. The Mediator Complex: At the Nexus of RNA Polymerase II Transcription.

    Science.gov (United States)

    Jeronimo, Célia; Robert, François

    2017-10-01

    Mediator is an essential, large, multisubunit, transcriptional co-activator highly conserved across eukaryotes. Mediator interacts with gene-specific transcription factors at enhancers as well as with the RNA polymerase II (RNAPII) transcription machinery bound at promoters. It also interacts with several other factors involved in various aspects of transcription, chromatin regulation, and mRNA processing. Hence, Mediator is at the nexus of RNAPII transcription, regulating its many steps and connecting transcription with co-transcriptional events. To achieve this flexible role, Mediator, which is divided into several functional modules, reorganizes its conformation and composition while making transient contacts with other components. Here, we review the mechanisms of action of Mediator and propose a unifying model for its function. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. The Mediator complex of Caenorhabditis elegans: insights into the developmental and physiological roles of a conserved transcriptional coregulator.

    Science.gov (United States)

    Grants, Jennifer M; Goh, Grace Y S; Taubert, Stefan

    2015-02-27

    The Mediator multiprotein complex ('Mediator') is an important transcriptional coregulator that is evolutionarily conserved throughout eukaryotes. Although some Mediator subunits are essential for the transcription of all protein-coding genes, others influence the expression of only subsets of genes and participate selectively in cellular signaling pathways. Here, we review the current knowledge of Mediator subunit function in the nematode Caenorhabditis elegans, a metazoan in which established and emerging genetic technologies facilitate the study of developmental and physiological regulation in vivo. In this nematode, unbiased genetic screens have revealed critical roles for Mediator components in core developmental pathways such as epidermal growth factor (EGF) and Wnt/β-catenin signaling. More recently, important roles for C. elegans Mediator subunits have emerged in the regulation of lipid metabolism and of systemic stress responses, engaging conserved transcription factors such as nuclear hormone receptors (NHRs). We emphasize instances where similar functions for individual Mediator subunits exist in mammals, highlighting parallels between Mediator subunit action in nematode development and in human cancer biology. We also discuss a parallel between the association of the Mediator subunit MED12 with several human disorders and the role of its C. elegans ortholog mdt-12 as a regulatory hub that interacts with numerous signaling pathways. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Environmental phthalate monoesters activate pregnane X receptor-mediated transcription

    International Nuclear Information System (INIS)

    Hurst, Christopher H.; Waxman, David J.

    2004-01-01

    Phthalate esters, widely used as plasticizers in the manufacture of products made of polyvinyl chloride, induce reproductive and developmental toxicities in rodents. The mechanism that underlies these effects of phthalate exposure, including the potential role of members of the nuclear receptor superfamily, is not known. The present study investigates the effects of phthalates on the pregnane X receptor (PXR), which mediates the induction of enzymes involved in steroid metabolism and xenobiotic detoxification. The ability of phthalate monoesters to activate PXR-mediated transcription was assayed in a HepG2 cell reporter assay following transfection with mouse PXR (mPXR), human PXR (hPXR), or the hPXR allelic variants V140M, D163G, and A370T. Mono-2-ethylhexyl phthalate (MEHP) increased the transcriptional activity of both mPXR and hPXR (5- and 15-fold, respectively) with EC 50 values of 7-8 μM. mPXR and hPXR were also activated by monobenzyl phthalate (MBzP, up to 5- to 6-fold) but were unresponsive to monomethyl phthalate and mono-n-butyl phthalate (M(n)BP) at the highest concentrations tested (300 μM). hPXR-V140M and hPXR-A370T exhibited patterns of phthalate responses similar to the wild-type receptor. By contrast, hPXR-D163G was unresponsive to all phthalate monoesters tested. Further studies revealed that hPXR-D163G did respond to rifampicin, but required approximately 40-fold higher concentrations than wild-type receptor, suggesting that the ligand-binding domain D163G variant has impaired ligand-binding activity. The responsiveness of PXR to activation by phthalate monoesters demonstrated here suggests that these ubiquitous environmental chemicals may, in part, exhibit their endocrine disruptor activities by altering PXR-regulated steroid hormone metabolism with potential adverse health effects in exposed individuals

  2. Effect of ARA9 on dioxin receptor mediated transcription

    International Nuclear Information System (INIS)

    Lees, M.J.; Whitelaw, M.L.

    2002-01-01

    The dioxin (Aryl hydrocarbon) receptor (DR) is a unique bHLH transcription factor which is activated by binding of planar aromatic hydrocarbons typified by dioxin (TCDD). The active receptor is key to metabolism of aryl hydrocarbon xenobiotics by being a potent inducer of CYP1A1 gene activity. Chlorinated dioxins are inert to metabolism and initiate multifarious toxicities, including potent tumour promotion. These ill-effects are mediated by the activated DR and we are studying the mechanisms by which the ligand binding domain of the DR controls activity of the protein. The DR ligand binding domain resides within a PAS (Per/Arnt/Sim homology) region which is contiguous with the bHLH. The latent bHLH/PAS dioxin receptor (DR) is found in the cytoplasm of most mammalian cell types in a complex with heat shock protein 90, a novel immunophilin like protein termed ARA9/XAP2/AIP, and the co-chaperone p23. Here we use antisense ARA9 constructs to reveal that in the absence of ARA9, the DR is unable to form a transcriptionally active complex. Co-expression of antisense ARA9 with a form of the DR which is constitutively targeted to the nucleus leads to dramatically decreased levels of the nuclear DR protein, implying that ARA9 may function beyond its currently proposed role in cytoplasmic retention of the latent DR

  3. Modulation of organic acids and sugar content in tomato fruits by an abscisic acid-regulated transcription factor.

    Science.gov (United States)

    Bastías, Adriana; López-Climent, María; Valcárcel, Mercedes; Rosello, Salvador; Gómez-Cadenas, Aurelio; Casaretto, José A

    2011-03-01

    Growing evidence suggests that the phytohormone abscisic acid (ABA) plays a role in fruit development. ABA signaling components of developmental programs and responses to stress conditions include the group of basic leucine zipper transcriptional activators known as ABA-response element binding factors (AREBs/ABFs). AREB transcription factors mediate ABA-regulated gene expression involved in desiccation tolerance and are expressed mainly in seeds and in vegetative tissues under stress; however, they are also expressed in some fruits such as tomato. In order to get an insight into the role of ABA signaling in fruit development, the expression of two AREB-like factors were investigated during different developmental stages. In addition, tomato transgenic lines that overexpress and downregulate one AREB-like transcription factor, SlAREB1, were used to determine its effect on the levels of some metabolites determining fruit quality. Higher levels of citric acid, malic acid, glutamic acid, glucose and fructose were observed in SlAREB1-overexpressing lines compared with those in antisense suppression lines in red mature fruit pericarp. The higher hexose concentration correlated with increased expression of genes encoding a vacuolar invertase (EC 3.2.1.26) and a sucrose synthase (EC 2.4.1.13). No significant changes were found in ethylene content which agrees with the normal ripening phenotype observed in transgenic fruits. These results suggest that an AREB-mediated ABA signal affects the metabolism of these compounds during the fruit developmental program. Copyright © Physiologia Plantarum 2010.

  4. Subunit architecture and functional modular rearrangements of the transcriptional mediator complex.

    Science.gov (United States)

    Tsai, Kuang-Lei; Tomomori-Sato, Chieri; Sato, Shigeo; Conaway, Ronald C; Conaway, Joan W; Asturias, Francisco J

    2014-06-05

    The multisubunit Mediator, comprising ∼30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA-binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. WRKY transcription factors: key components in abscisic acid signalling.

    Science.gov (United States)

    Rushton, Deena L; Tripathi, Prateek; Rabara, Roel C; Lin, Jun; Ringler, Patricia; Boken, Ashley K; Langum, Tanner J; Smidt, Lucas; Boomsma, Darius D; Emme, Nicholas J; Chen, Xianfeng; Finer, John J; Shen, Qingxi J; Rushton, Paul J

    2012-01-01

    WRKY transcription factors (TFs) are key regulators of many plant processes, including the responses to biotic and abiotic stresses, senescence, seed dormancy and seed germination. For over 15 years, limited evidence has been available suggesting that WRKY TFs may play roles in regulating plant responses to the phytohormone abscisic acid (ABA), notably some WRKY TFs are ABA-inducible repressors of seed germination. However, the roles of WRKY TFs in other aspects of ABA signalling, and the mechanisms involved, have remained unclear. Recent significant progress in ABA research has now placed specific WRKY TFs firmly in ABA-responsive signalling pathways, where they act at multiple levels. In Arabidopsis, WRKY TFs appear to act downstream of at least two ABA receptors: the cytoplasmic PYR/PYL/RCAR-protein phosphatase 2C-ABA complex and the chloroplast envelope-located ABAR-ABA complex. In vivo and in vitro promoter-binding studies show that the target genes for WRKY TFs that are involved in ABA signalling include well-known ABA-responsive genes such as ABF2, ABF4, ABI4, ABI5, MYB2, DREB1a, DREB2a and RAB18. Additional well-characterized stress-inducible genes such as RD29A and COR47 are also found in signalling pathways downstream of WRKY TFs. These new insights also reveal that some WRKY TFs are positive regulators of ABA-mediated stomatal closure and hence drought responses. Conversely, many WRKY TFs are negative regulators of seed germination, and controlling seed germination appears a common function of a subset of WRKY TFs in flowering plants. Taken together, these new data demonstrate that WRKY TFs are key nodes in ABA-responsive signalling networks. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  6. Selective activation of human heat shock gene transcription by nitrosourea antitumor drugs mediated by isocyanate-induced damage and activation of heat shock transcription factor.

    Science.gov (United States)

    Kroes, R A; Abravaya, K; Seidenfeld, J; Morimoto, R I

    1991-01-01

    Treatment of cultured human tumor cells with the chloroethylnitrosourea antitumor drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) selectively induces transcription and protein synthesis of a subset of the human heat shock or stress-induced genes (HSP90 and HSP70) with little effect on other stress genes or on expression of the c-fos, c-myc, or beta-actin genes. The active component of BCNU and related compounds appears to be the isocyanate moiety that causes carbamoylation of proteins and nucleic acids. Transcriptional activation of the human HSP70 gene by BCNU is dependent on the heat shock element and correlates with the level of heat shock transcription factor and its binding to the heat shock element in vivo. Unlike activation by heat or heavy metals, BCNU-mediated activation is strongly dependent upon new protein synthesis. This suggests that BCNU-induced, isocyanate-mediated damage to newly synthesized protein(s) may be responsible for activation of the heat shock transcription factor and increased transcription of the HSP90 and HSP70 genes. Images PMID:2052560

  7. Mediator binding to UASs is broadly uncoupled from transcription and cooperative with TFIID recruitment to promoters.

    Science.gov (United States)

    Grünberg, Sebastian; Henikoff, Steven; Hahn, Steven; Zentner, Gabriel E

    2016-11-15

    Mediator is a conserved, essential transcriptional coactivator complex, but its in vivo functions have remained unclear due to conflicting data regarding its genome-wide binding pattern obtained by genome-wide ChIP Here, we used ChEC-seq, a method orthogonal to ChIP, to generate a high-resolution map of Mediator binding to the yeast genome. We find that Mediator associates with upstream activating sequences (UASs) rather than the core promoter or gene body under all conditions tested. Mediator occupancy is surprisingly correlated with transcription levels at only a small fraction of genes. Using the same approach to map TFIID, we find that TFIID is associated with both TFIID- and SAGA-dependent genes and that TFIID and Mediator occupancy is cooperative. Our results clarify Mediator recruitment and binding to the genome, showing that Mediator binding to UASs is widespread, partially uncoupled from transcription, and mediated in part by TFIID. © 2016 The Authors.

  8. Thyroid hormone and retinoic acid nuclear receptors: specific ligand-activated transcription factors

    International Nuclear Information System (INIS)

    Brtko, J.

    1998-01-01

    Transcriptional regulation by both the thyroid hormone and the vitamin A-derived 'retinoid hormones' is a critical component in controlling many aspects of higher vertebrate development and metabolism. Their functions are mediated by nuclear receptors, which comprise a large super-family of ligand-inducible transcription factors. Both the thyroid hormone and the retinoids are involved in a complex arrangement of physiological and development responses in many tissues of higher vertebrates. The functions of 3,5,3'-triiodothyronine (T 3 ), the thyromimetically active metabolite of thyroxine as well as all-trans retinoic acid, the biologically active vitamin A metabolite are mediated by nuclear receptor proteins that are members of the steroid/thyroid/retinoid hormone receptor family. The functions of all members of the receptor super family are discussed. (authors)

  9. Perfluorooctanoic acid stimulated mitochondrial biogenesis and gene transcription in rats

    International Nuclear Information System (INIS)

    Walters, M.W.; Bjork, J.A.; Wallace, K.B.

    2009-01-01

    Perfluorooctanoic acid (PFOA), used in the production of non-stick surface compounds, exhibits a worldwide distribution in the serum of humans and wildlife. In rodents PFOA transactivates PPARα and PPARγ nuclear receptors and increases mitochondrial DNA (mtDNA) copy number, which may be critical to the altered metabolic state of affected animals. A key regulator of mitochondrial biogenesis and transcription of mitochondrial genes is the PPARγ coactivator-1α (Pgc-1α) protein. The purpose of this study was to determine if Pgc-1α is implicated in the stimulation of mitochondrial biogenesis that occurs following the treatment of rats with PFOA. Livers from adult male Sprague-Dawley rats that received a 30 mg/kg daily oral dose of PFOA for 28 days were used for all experiments. Analysis of mitochondrial replication and transcription was performed by real time PCR, and proteins were detected using western blotting. PFOA treatment caused a transcriptional activation of the mitochondrial biogenesis pathway leading to a doubling of mtDNA copy number. Further, transcription of OXPHOS genes encoded by mtDNA was 3-4 times greater than that of nuclear encoded genes, suggestive of a preferential induction of mtDNA transcription. Western blot analysis revealed an increase in Pgc-1α, unchanged Tfam and decreased Cox II and Cox IV subunit protein expression. We conclude that PFOA treatment in rats induces mitochondrial biogenesis at the transcriptional level with a preferential stimulation of mtDNA transcription and that this occurs by way of activation of the Pgc-1α pathway. Implication of the Pgc-1α pathway is consistent with PPARγ transactivation by PFOA and reveals new understanding and possibly new critical targets for assessing or averting the associated metabolic disease.

  10. HSF1 transcriptional activity mediates alcohol induction of Vamp2 expression and GABA release

    Directory of Open Access Journals (Sweden)

    Florence P. Varodayan

    2013-12-01

    Full Text Available Many central synapses are highly sensitive to alcohol, and it is now accepted that short-term alterations in synaptic function may lead to longer term changes in circuit function. The regulation of postsynaptic receptors by alcohol has been well studied, but the mechanisms underlying the effects of alcohol on the presynaptic terminal are relatively unexplored. To identify a pathway by which alcohol regulates neurotransmitter release, we recently investigated the mechanism by which ethanol induces the Vamp2 gene, but not Vamp1, in mouse primary cortical cultures. These two genes encode isoforms of synaptobrevin, a vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE protein required for synaptic vesicle fusion. We found that alcohol activates the transcription factor heat shock factor 1 (HSF1 to induce Vamp2 gene expression, while Vamp1 mRNA levels remain unaffected. As the Vamp2 gene encodes a SNARE protein, we then investigated whether ethanol exposure and HSF1 transcriptional activity alter neurotransmitter release using electrophysiology. We found that alcohol increased the frequency of γ-aminobutyric acid (GABA-mediated miniature IPSCs via HSF1, but had no effect on mEPSCs. Overall, these data indicate that alcohol induces HSF1 transcriptional activity to trigger a specific coordinated adaptation in GABAergic presynaptic terminals. This mechanism could explain some of the changes in synaptic function that occur soon after alcohol exposure, and may underlie some of the more enduring effects of chronic alcohol intake on local circuit function.

  11. Role of arachidonic acid metabolism in transcriptional induction of tumor necrosis factor gene expression by phorbol ester

    Energy Technology Data Exchange (ETDEWEB)

    Horiguchi, J.; Spriggs, D.; Imamura, K.; Stone, R.; Luebbers, R.; Kufe, D.

    1989-01-01

    The treatment of human HL-60 promyelocytic leukemia cells with 12-0 tetradecanoylphorbol-13-acetate (TPA) is associated with induction of tumor necrosis factor (TNF) transcripts. The study reported here has examined TPA-induced signaling mechanisms responsible for the regulation of TNF gene expression in these cells. Run-on assays demonstrated that TPA increases TNS mRNA levels by transcriptional activation of this gene. The induction of TNF transcripts by TPA was inhibited by the isoquinolinesulfonamide derivative H7 but not by HA1004, suggesting that this effect of TPA is mediated by activation of protein kinase C. TPA treatment also resulted in increased arachidonic acid release. Moreover, inhibitors of phospholipase, A/sub 2/ blocked both the increase in arachidonic acid release and the induction of TNF transcripts. These findings suggest that TPA induces TNF gene expression through the formation of arachidonic acid metabolites. Although indomethacin had no detectable effect on this induction of TNF transcripts, ketoconazole, an inhibitor of 5-lipoxygenase, blocked TPA-induced increases in TNF mRNA levels. Moreover, TNF mRNA levels were increased by the 5-lipoxygenase metabolite leukotriene B/sub 4/. In contrast, the cyclooxygenase metabolite prostaglandin E/sub 2/ inhibited the induction of TNF transcripts by TPA. Taken together, these results suggest that TPA induces TNF gene expression through the arachidonic acid cascade and that the level of TNF transcripts is regulated by metabolites of the pathway, leukotriene B/sub 4/ and prostaglandin E/sub 2/.

  12. Azadirachtin interacts with retinoic acid receptors and inhibits retinoic acid-mediated biological responses.

    Science.gov (United States)

    Thoh, Maikho; Babajan, Banaganapalli; Raghavendra, Pongali B; Sureshkumar, Chitta; Manna, Sunil K

    2011-02-11

    Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies.

  13. Azadirachtin Interacts with Retinoic Acid Receptors and Inhibits Retinoic Acid-mediated Biological Responses*

    Science.gov (United States)

    Thoh, Maikho; Babajan, Banaganapalli; Raghavendra, Pongali B.; Sureshkumar, Chitta; Manna, Sunil K.

    2011-01-01

    Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies. PMID:21127062

  14. DNA damage mediated transcription arrest: Step back to go forward.

    Science.gov (United States)

    Mullenders, Leon

    2015-12-01

    The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Mediator phosphorylation prevents stress response transcription during non-stress conditions.

    Science.gov (United States)

    Miller, Christian; Matic, Ivan; Maier, Kerstin C; Schwalb, Björn; Roether, Susanne; Strässer, Katja; Tresch, Achim; Mann, Matthias; Cramer, Patrick

    2012-12-28

    The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an end point of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here, we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast Saccharomyces cerevisiae is phosphorylated at multiple sites of 17 of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.

  16. Nanoparticle-mediated transcriptional modification enhances neuronal differentiation of human neural stem cells following transplantation in rat brain.

    Science.gov (United States)

    Li, Xiaowei; Tzeng, Stephany Y; Liu, Xiaoyan; Tammia, Markus; Cheng, Yu-Hao; Rolfe, Andrew; Sun, Dong; Zhang, Ning; Green, Jordan J; Wen, Xuejun; Mao, Hai-Quan

    2016-04-01

    Strategies to enhance survival and direct the differentiation of stem cells in vivo following transplantation in tissue repair site are critical to realizing the potential of stem cell-based therapies. Here we demonstrated an effective approach to promote neuronal differentiation and maturation of human fetal tissue-derived neural stem cells (hNSCs) in a brain lesion site of a rat traumatic brain injury model using biodegradable nanoparticle-mediated transfection method to deliver key transcriptional factor neurogenin-2 to hNSCs when transplanted with a tailored hyaluronic acid (HA) hydrogel, generating larger number of more mature neurons engrafted to the host brain tissue than non-transfected cells. The nanoparticle-mediated transcription activation method together with an HA hydrogel delivery matrix provides a translatable approach for stem cell-based regenerative therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Essential fatty acids and lipid mediators. Endocannabinoids

    Directory of Open Access Journals (Sweden)

    G. Caramia

    2012-03-01

    Full Text Available In 1929 Burr and Burr discovered the essential fatty acids omega-6 and omega-3. Since then, researchers have shown a growing interest in polyunsaturated fatty acids (PUFA as precursors of “lipid mediator” molecules, often with opposing effects, prostaglandins, prostacyclins, thromboxanes, leukotrienes, lipossines, resolvines, protectines, maresins that regulate immunity, platelet aggregation, inflammation, etc. They showed that the balance between omega-3 and omega-6 acids has a profound influence on all the body’s inflammatory responses and a raised level of PUFA omega-3 in tissue correlate with a reduced incidence of degenerative cardiovascular disease, some mental illnesses such as depression, and neuro-degenerative diseases such as Alzheimer’s. The CYP-catalyzed epoxidation and hydroxylation of arachidonic acid (AA were established recently as the so-called third branch of AGE cascade. Cytochrome P450 (CYP epoxygenases convert AA to four epoxyeicosatrienoic acid (EET regioisomers, that produce vascular relaxation anti-inflammatory effects on blood vessels and in the kidney, promote angiogenesis, and protect ischemic myocardium and brain. Eicosapentaenoic acid (EPA and docosahexaenoic acid (DHA are accessible to CYP enzymes in the same way as AA. Metabolites derived from EPA include epoxyeicosatetraenoic acids (EETR and hydroxyeicosapentaenoic acids (19- and 20-HEPE, whereas DHA include epoxydocosapentaenoic acids (EDPs hydroxydocosahexaenoic acids (21- and 22-HDoHE. For many of the CYP isoforms, the n-3 PUFAs are the preferred substrates and the available data suggest that some of the vasculo- and cardioprotective effects attributed to dietary n-3 PUFAs may be mediated by CYP-dependent metabolites of EPA and DHA. From AA derives also endocannabinoids like anandamide (N-arachidonoylethanolamine and 2-arachidonoylglycerol, capable of mimicking the pharmacological actions of the active principle of Cannabis sativa preparations such as

  18. APETALA 2-domain-containing transcription factors: focusing on abscisic acid and gibberellins antagonism.

    Science.gov (United States)

    Shu, Kai; Zhou, Wenguan; Yang, Wenyu

    2018-02-01

    The phytohormones abscisic acid (ABA) and gibberellin (GA) antagonistically mediate diverse plant developmental processes including seed dormancy and germination, root development, and flowering time control, and thus the optimal balance between ABA and GA is essential for plant growth and development. Although more than a half and one century have passed since the initial discoveries of ABA and GA, respectively, the precise mechanisms underlying ABA-GA antagonism still need further investigation. Emerging evidence indicates that two APETALA 2 (AP2)-domain-containing transcription factors (ATFs), ABI4 in Arabidopsis and OsAP2-39 in rice, play key roles in ABA and GA antagonism. These two transcription factors precisely regulate the transcription pattern of ABA and GA biosynthesis or inactivation genes, mediating ABA and GA levels. In this Viewpoint article, we try to shed light on the effects of ATFs on ABA-GA antagonism, and summarize the overlapping but distinct biological functions of these ATFs in the antagonism between ABA and GA. Finally, we strongly propose that further research is needed into the detailed roles of additional numerous ATFs in ABA and GA crosstalk, which will improve our understanding of the antagonism between these two phytohormones. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  19. Polycomb group protein-mediated repression of transcription

    DEFF Research Database (Denmark)

    Morey, Lluís; Helin, Kristian

    2010-01-01

    The polycomb group (PcG) proteins are essential for the normal development of multicellular organisms. They form multi-protein complexes that work as transcriptional repressors of several thousand genes controlling differentiation pathways during development. How the PcG proteins work as transcri......The polycomb group (PcG) proteins are essential for the normal development of multicellular organisms. They form multi-protein complexes that work as transcriptional repressors of several thousand genes controlling differentiation pathways during development. How the PcG proteins work...... as transcriptional repressors is incompletely understood, but involves post-translational modifications of histones by two major PcG protein complexes: polycomb repressive complex 1 and polycomb repressive complex 2....

  20. The Mediator subunit SFR6/MED16 controls defence gene expression mediated by salicylic acid and jasmonate responsive pathways.

    Science.gov (United States)

    Wathugala, Deepthi L; Hemsley, Piers A; Moffat, Caroline S; Cremelie, Pieter; Knight, Marc R; Knight, Heather

    2012-07-01

    • Arabidopsis SENSITIVE TO FREEZING6 (SFR6) controls cold- and drought-inducible gene expression and freezing- and osmotic-stress tolerance. Its identification as a component of the MEDIATOR transcriptional co-activator complex led us to address its involvement in other transcriptional responses. • Gene expression responses to Pseudomonas syringae, ultraviolet-C (UV-C) irradiation, salicylic acid (SA) and jasmonic acid (JA) were investigated in three sfr6 mutant alleles by quantitative real-time PCR and susceptibility to UV-C irradiation and Pseudomonas infection were assessed. • sfr6 mutants were more susceptible to both Pseudomonas syringae infection and UV-C irradiation. They exhibited correspondingly weaker PR (pathogenesis-related) gene expression than wild-type Arabidopsis following these treatments or after direct application of SA, involved in response to both UV-C and Pseudomonas infection. Other genes, however, were induced normally in the mutants by these treatments. sfr6 mutants were severely defective in expression of plant defensin genes in response to JA; ectopic expression of defensin genes was provoked in wild-type but not sfr6 by overexpression of ERF5. • SFR6/MED16 controls both SA- and JA-mediated defence gene expression and is necessary for tolerance of Pseudomonas syringae infection and UV-C irradiation. It is not, however, a universal regulator of stress gene transcription and is likely to mediate transcriptional activation of specific regulons only. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  1. VirF-Independent Regulation of Shigella virB Transcription is Mediated by the Small RNA RyhB

    Science.gov (United States)

    Broach, William H.; Egan, Nicholas; Wing, Helen J.; Payne, Shelley M.; Murphy, Erin R.

    2012-01-01

    Infection of the human host by Shigella species requires the coordinated production of specific Shigella virulence factors, a process mediated largely by the VirF/VirB regulatory cascade. VirF promotes the transcription of virB, a gene encoding the transcriptional activator of several virulence-associated genes. This study reveals that transcription of virB is also regulated by the small RNA RyhB, and importantly, that this regulation is not achieved indirectly via modulation of VirF activity. These data are the first to demonstrate that the regulation of virB transcription can be uncoupled from the master regulator VirF. It is also established that efficient RyhB-dependent regulation of transcription is facilitated by specific nucleic acid sequences within virB. This study not only reveals RyhB-dependent regulation of virB transcription as a novel point of control in the central regulatory circuit modulating Shigella virulence, but also highlights the versatility of RyhB in controlling bacterial gene expression. PMID:22701677

  2. The transcription factor DREAM represses A20 and mediates inflammation

    OpenAIRE

    Tiruppathi, Chinnaswamy; Soni, Dheeraj; Wang, Dong-Mei; Xue, Jiaping; Singh, Vandana; Thippegowda, Prabhakar B.; Cheppudira, Bopaiah P.; Mishra, Rakesh K.; DebRoy, Auditi; Qian, Zhijian; Bachmaier, Kurt; Zhao, Youyang; Christman, John W.; Vogel, Stephen M.; Ma, Averil

    2014-01-01

    Here we show that the transcription-repressor DREAM binds to the A20 promoter to repress the expression of A20, the deubiquitinase suppressing inflammatory NF-κB signaling. DREAM-deficient (Dream−/− ) mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, USF1 binding to the DRE-associated E-box domain activated A20 expression in response to inf...

  3. Histone acetyltransferase (HAT) activity of p300 modulates human T lymphotropic virus type 1 p30II-mediated repression of LTR transcriptional activity

    International Nuclear Information System (INIS)

    Michael, Bindhu; Nair, Amrithraj M.; Datta, Antara; Hiraragi, Hajime; Ratner, Lee; Lairmore, Michael D.

    2006-01-01

    Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T cell leukemia/lymphoma, and is implicated in a variety of lymphocyte-mediated inflammatory disorders. HTLV-1 provirus has regulatory and accessory genes in four pX open reading frames. HTLV-1 pX ORF-II encodes two proteins, p13 II and p30 II , which are incompletely defined in virus replication or pathogenesis. We have demonstrated that pX ORF-II mutations block virus replication in vivo and that ORF-II encoded p30 II , a nuclear-localizing protein that binds with CREB-binding protein (CBP)/p300, represses CREB and Tax responsive element (TRE)-mediated transcription. Herein, we have identified p30 II motifs important for p300 binding and in regulating TRE-mediated transcription in the absence and presence of HTLV-1 provirus. Within amino acids 100-179 of p30 II , a region important for repression of LTR-mediated transcription, we identified a single lysine residue at amino acid 106 (K3) that significantly modulates the ability of p30 II to repress TRE-mediated transcription. Exogenous p300, in a dose-responsive manner, reverses p30 II -dependent repression of TRE-mediated transcription, in the absence or presence of the provirus, In contrast to wild type p300, p300 HAT mutants (defective in histone acetyltransferase activity) only partially rescued p30 II -mediated LTR repression. Deacetylation by histone deacetylase-1 (HDAC-1) enhanced p30 II -mediated LTR repression, while inhibition of deacetylation by trichostatin A decreases p30 II -mediated LTR repression. Collectively, our data indicate that HTLV-1 p30 II modulates viral gene expression in a cooperative manner with p300-mediated acetylation

  4. Cross-talk between an activator of nuclear receptors-mediated transcription and the D1 dopamine receptor signaling pathway.

    Science.gov (United States)

    Schmidt, Azriel; Vogel, Robert; Rutledge, Su Jane; Opas, Evan E; Rodan, Gideon A; Friedman, Eitan

    2005-03-01

    Nuclear receptors are transcription factors that usually interact, in a ligand-dependent manner, with specific DNA sequences located within promoters of target genes. The nuclear receptors can also be controlled in a ligand-independent manner via the action of membrane receptors and cellular signaling pathways. 5-Tetradecyloxy-2-furancarboxylic acid (TOFA) was shown to stimulate transcription from the MMTV promoter via chimeric receptors that consist of the DNA binding domain of GR and the ligand binding regions of the PPARbeta or LXRbeta nuclear receptors (GR/PPARbeta and GR/LXRbeta). TOFA and hydroxycholesterols also modulate transcription from NF-kappaB- and AP-1-controlled reporter genes and induce neurite differentiation in PC12 cells. In CV-1 cells that express D(1) dopamine receptors, D(1) dopamine receptor stimulation was found to inhibit TOFA-stimulated transcription from the MMTV promoter that is under the control of chimeric GR/PPARbeta and GR/LXRbeta receptors. Treatment with the D(1) dopamine receptor antagonist, SCH23390, prevented dopamine-mediated suppression of transcription, and by itself increased transcription controlled by GR/LXRbeta. Furthermore, combined treatment of CV-1 cells with TOFA and SCH23390 increased transcription controlled by the GR/LXRbeta chimeric receptor synergistically. The significance of this in vitro synergy was demonstrated in vivo, by the observation that SCH23390 (but not haloperidol)-mediated catalepsy in rats was potentiated by TOFA, thus showing that an agent that mimics the in vitro activities of compounds that activate members of the LXR and PPAR receptor families can influence D1 dopamine receptor elicited responses.

  5. A recombinase-mediated transcriptional induction system in transgenic plants

    DEFF Research Database (Denmark)

    Hoff, T; Schnorr, K M; Mundy, J

    2001-01-01

    We constructed and tested a Cre-loxP recombination-mediated vector system termed pCrox for use in transgenic plants. In this system, treatment of Arabidopsis under inducing conditions mediates an excision event that removes an intervening piece of DNA between a promoter and the gene to be expressed......-mediated GUS activation. Induction was shown to be possible at essentially any stage of plant growth. This single vector system circumvents the need for genetic crosses required by other, dual recombinase vector systems. The pCrox system may prove particularly useful in instances where transgene over...

  6. The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation

    OpenAIRE

    Malik, Sohail; Roeder, Robert G.

    2010-01-01

    The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action ...

  7. The Mediator complex: a master coordinator of transcription and cell lineage development.

    Science.gov (United States)

    Yin, Jing-wen; Wang, Gang

    2014-03-01

    Mediator is a multiprotein complex that is required for gene transcription by RNA polymerase II. Multiple subunits of the complex show specificity in relaying information from signals and transcription factors to the RNA polymerase II machinery, thus enabling control of the expression of specific genes. Recent studies have also provided novel mechanistic insights into the roles of Mediator in epigenetic regulation, transcriptional elongation, termination, mRNA processing, noncoding RNA activation and super enhancer formation. Based on these specific roles in gene regulation, Mediator has emerged as a master coordinator of development and cell lineage determination. Here, we describe the most recent advances in understanding the mechanisms of Mediator function, with an emphasis on its role during development and disease.

  8. Investigating the Regulation of Estrogen Receptor-Mediated Transcription

    National Research Council Canada - National Science Library

    Thackray, Varykina

    2002-01-01

    ...-mediated regulation of specific target genes are still lacking. We have developed an estrogen responsive system in the fruit fly, Drosophila melanogaster in order to explore the functional interactions between ER and other cellular proteins...

  9. Investigating the Regulation of Estrogen Receptor-Mediated Transcription

    National Research Council Canada - National Science Library

    Thackray, Varykina

    2001-01-01

    ...-mediated regulation of specific target genes are still lacking. We have developed an estrogen responsive system in the fruit fly, Drosophila melanogaster in order to explore the functional interactions between ER and other cellular proteins...

  10. Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice.

    Science.gov (United States)

    De Domenico, Ivana; Zhang, Tian Y; Koening, Curry L; Branch, Ryan W; London, Nyall; Lo, Eric; Daynes, Raymond A; Kushner, James P; Li, Dean; Ward, Diane M; Kaplan, Jerry

    2010-07-01

    Hepcidin is a peptide hormone that regulates iron homeostasis and acts as an antimicrobial peptide. It is expressed and secreted by a variety of cell types in response to iron loading and inflammation. Hepcidin mediates iron homeostasis by binding to the iron exporter ferroportin, inducing its internalization and degradation via activation of the protein kinase Jak2 and the subsequent phosphorylation of ferroportin. Here we have shown that hepcidin-activated Jak2 also phosphorylates the transcription factor Stat3, resulting in a transcriptional response. Hepcidin treatment of ferroportin-expressing mouse macrophages showed changes in mRNA expression levels of a wide variety of genes. The changes in transcript levels for half of these genes were a direct effect of hepcidin, as shown by cycloheximide insensitivity, and dependent on the presence of Stat3. Hepcidin-mediated transcriptional changes modulated LPS-induced transcription in both cultured macrophages and in vivo mouse models, as demonstrated by suppression of IL-6 and TNF-alpha transcript and secreted protein. Hepcidin-mediated transcription in mice also suppressed toxicity and morbidity due to single doses of LPS, poly(I:C), and turpentine, which is used to model chronic inflammatory disease. Most notably, we demonstrated that hepcidin pretreatment protected mice from a lethal dose of LPS and that hepcidin-knockout mice could be rescued from LPS toxicity by injection of hepcidin. The results of our study suggest a new function for hepcidin in modulating acute inflammatory responses.

  11. Human GW182 Paralogs Are the Central Organizers for RNA-Mediated Control of Transcription.

    Science.gov (United States)

    Hicks, Jessica A; Li, Liande; Matsui, Masayuki; Chu, Yongjun; Volkov, Oleg; Johnson, Krystal C; Corey, David R

    2017-08-15

    In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Mediator MED23 regulates basal transcription in vivo via an interaction with P-TEFb.

    Science.gov (United States)

    Wang, Wei; Yao, Xiao; Huang, Yan; Hu, Xiangming; Liu, Runzhong; Hou, Dongming; Chen, Ruichuan; Wang, Gang

    2013-01-01

    The Mediator is a multi-subunit complex that transduces regulatory information from transcription regulators to the RNA polymerase II apparatus. Growing evidence suggests that Mediator plays roles in multiple stages of eukaryotic transcription, including elongation. However, the detailed mechanism by which Mediator regulates elongation remains elusive. In this study, we demonstrate that Mediator MED23 subunit controls a basal level of transcription by recruiting elongation factor P-TEFb, via an interaction with its CDK9 subunit. The mRNA level of Egr1, a MED23-controlled model gene, is reduced 4-5 fold in Med23 (-/-) ES cells under an unstimulated condition, but Med23-deficiency does not alter the occupancies of RNAP II, GTFs, Mediator complex, or activator ELK1 at the Egr1 promoter. Instead, Med23 depletion results in a significant decrease in P-TEFb and RNAP II (Ser2P) binding at the coding region, but no changes for several other elongation regulators, such as DSIF and NELF. ChIP-seq revealed that Med23-deficiency partially reduced the P-TEFb occupancy at a set of MED23-regulated gene promoters. Further, we demonstrate that MED23 interacts with CDK9 in vivo and in vitro. Collectively, these results provide the mechanistic insight into how Mediator promotes RNAP II into transcription elongation.

  13. Comprehensive analysis of the transcriptional profile of the Mediator complex across human cancer types.

    Science.gov (United States)

    Syring, Isabella; Klümper, Niklas; Offermann, Anne; Braun, Martin; Deng, Mario; Boehm, Diana; Queisser, Angela; von Mässenhausen, Anne; Brägelmann, Johannes; Vogel, Wenzel; Schmidt, Doris; Majores, Michael; Schindler, Anne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg; Shaikhibrahim, Zaki; Perner, Sven

    2016-04-26

    The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking.We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

  14. Nuclear Matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling

    International Nuclear Information System (INIS)

    Sreenath, Kadreppa; Pavithra, Lakshminarasimhan; Singh, Sandeep; Sinha, Surajit; Dash, Prasanta K.; Siddappa, Nagadenahalli B.; Ranga, Udaykumar; Mitra, Debashis; Chattopadhyay, Samit

    2010-01-01

    Nuclear Matrix and MARs have been implicated in the transcriptional regulation of host as well as viral genes but their precise role in HIV-1 transcription remains unclear. Here, we show that > 98% of HIV sequences contain consensus MAR element in their promoter. We show that SMAR1 binds to the LTR MAR and reinforces transcriptional silencing by tethering the LTR MAR to nuclear matrix. SMAR1 associated HDAC1-mSin3 corepressor complex is dislodged from the LTR upon cellular activation by PMA/TNFα leading to an increase in the acetylation and a reduction in the trimethylation of histones, associated with the recruitment of RNA Polymerase II on the LTR. Overexpression of SMAR1 lead to reduction in LTR mediated transcription, both in a Tat dependent and independent manner, resulting in a decreased virion production. These results demonstrate the role of SMAR1 in regulating viral transcription by alternative compartmentalization of LTR between the nuclear matrix and chromatin.

  15. Nucleosome mediated crosstalk between transcription factors at eukaryotic enhancers

    International Nuclear Information System (INIS)

    Teif, Vladimir B; Rippe, Karsten

    2011-01-01

    A recent study of transcription regulation in Drosophila embryonic development revealed a complex non-monotonic dependence of gene expression on the distance between binding sites of repressor and activator proteins at the corresponding enhancer cis-regulatory modules (Fakhouri et al 2010 Mol. Syst. Biol. 6 341). The repressor efficiency was high at small separations, low around 30 bp, reached a maximum at 50–60 bp, and decreased at larger distances to the activator binding sites. Here, we propose a straightforward explanation for the distance dependence of repressor activity by considering the effect of the presence of a nucleosome. Using a method that considers partial unwrapping of nucleosomal DNA from the histone octamer core, we calculated the dependence of activator binding on the repressor–activator distance and found a quantitative agreement with the distance dependence reported for the Drosophila enhancer element. In addition, the proposed model offers explanations for other distance-dependent effects at eukaryotic enhancers. (communication)

  16. A chemical perspective on transcriptional fidelity dominant contributions of sugar integrity revealed by unlocked nucleic acids

    DEFF Research Database (Denmark)

    Xu, Liang; Plouffe, Steven W; Chong, Jenny

    2013-01-01

    Transcription unlocked: A synthetic chemical biology approach involving unlocked nucleic acids was used to dissect the contribution of sugar backbone integrity to the RNA Polymerase II (Pol II) transcription process. An unexpected dominant role for sugar-ring integrity in Pol II transcriptional...

  17. Transcriptional profile of sweet orange in response to chitosan and salicylic acid.

    Science.gov (United States)

    Coqueiro, Danila Souza Oliveira; de Souza, Alessandra Alves; Takita, Marco Aurélio; Rodrigues, Carolina Munari; Kishi, Luciano Takeshi; Machado, Marcos Antonio

    2015-04-12

    Resistance inducers have been used in annual crops as an alternative for disease control. Wood perennial fruit trees, such as those of the citrus species, are candidates for treatment with resistance inducers, such as salicylic acid (SA) and chitosan (CHI). However, the involved mechanisms in resistance induced by elicitors in citrus are currently few known. In the present manuscript, we report information regarding the transcriptional changes observed in sweet orange in response to exogenous applications of SA and CHI using RNA-seq technology. More genes were induced by SA treatment than by CHI treatment. In total, 1,425 differentially expressed genes (DEGs) were identified following treatment with SA, including the important genes WRKY50, PR2, and PR9, which are known to participate in the salicylic acid signaling pathway, and genes involved in ethylene/Jasmonic acid biosynthesis (ACS12, AP2 domain-containing transcription factor, and OPR3). In addition, SA treatment promoted the induction of a subset of genes involved in several metabolic processes, such as redox states and secondary metabolism, which are associated with biotic stress. For CHI treatment, there were 640 DEGs, many of them involved in secondary metabolism. For both SA and CHI treatments, the auxin pathway genes were repressed, but SA treatment promoted induction in the ethylene and jasmonate acid pathway genes, in addition to repressing the abscisic acid pathway genes. Chitosan treatment altered some hormone metabolism pathways. The DEGs were validated by quantitative Real-Time PCR (qRT-PCR), and the results were consistent with the RNA-seq data, with a high correlation between the two analyses. We expanded the available information regarding induced defense by elicitors in a species of Citrus that is susceptible to various diseases and identified the molecular mechanisms by which this defense might be mediated.

  18. The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation.

    Science.gov (United States)

    Malik, Sohail; Roeder, Robert G

    2010-11-01

    The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action of numerous other co-activators and co-repressors, including those acting at the level of chromatin. These interactions ultimately allow the Mediator to deliver outputs that range from maximal activation of genes to modulation of basal transcription to long-term epigenetic silencing.

  19. Ursolic acid mediates photosensitization by initiating mitochondrial-dependent apoptosis

    Science.gov (United States)

    Lee, Yuan-Hao; Wang, Exing; Kumar, Neeru; Glickman, Randolph D.

    2013-02-01

    The signaling pathways PI3K/Akt and MAPK play key roles in transcription, translation and carcinogenesis, and may be activated by light exposure. These pathways may be modulated or inhibited by naturally-occurring compounds, such as the triterpenoid, ursolic acid (UA). Previously, the transcription factors p53 and NF-kB, which transactivate mitochondrial apoptosis-related genes, were shown to be differentially modulated by UA. Our current work indicates that UA causes these effects via the mTOR and insulin-mediated pathways. UA-modulated apoptosis, following exposure to UV radiation, is observed to correspond to differential levels of oxidative stress in retinal pigment epithelial (RPE) and skin melanoma (SM) cells. Flow cytometry analysis, DHE (dihydroethidium) staining and membrane permeability assay showed that UA pretreatment potentiated cell cycle arrest and radiation-induced apoptosis selectively on SM cells while DNA photo-oxidative damage (i.e. strand breakage) was reduced, presumably by some antioxidant activity of UA in RPE cells. The UA-mediated NF-κB activation in SM cells was reduced by rapamycin pretreatment, which indicates that these agents exert inter-antagonistic effects in the PI3K/Akt/mTOR pathway. In contrast, the antagonistic effect of UA on the PI3K/Akt pathway was reversed by insulin leading to greater NF-κB and p53 activation in RPE cells. MitoTracker, a mitochondrial functional assay, indicated that mitochondria in RPE cells experienced reduced oxidative stress while those in SM cells exhibited increased oxidative stress upon UA pretreatment. When rapamycin administration was followed by UA, mitochondrial oxidative stress was increased in RPE cells but decreased in SM cells. These results indicate that UA modulates p53 and NF-κB, initiating a mitogenic response to radiation that triggers mitochondria-dependent apoptosis.

  20. Mediator can regulate mitotic entry and direct periodic transcription in fission yeast.

    Science.gov (United States)

    Banyai, Gabor; Lopez, Marcela Davila; Szilagyi, Zsolt; Gustafsson, Claes M

    2014-11-01

    Cdk8 is required for correct timing of mitotic progression in fission yeast. How the activity of Cdk8 is regulated is unclear, since the kinase is not activated by T-loop phosphorylation and its partner, CycC, does not oscillate. Cdk8 is, however, a component of the multiprotein Mediator complex, a conserved coregulator of eukaryotic transcription that is connected to a number of intracellular signaling pathways. We demonstrate here that other Mediator components regulate the activity of Cdk8 in vivo and thereby direct the timing of mitotic entry. Deletion of Mediator components Med12 and Med13 leads to higher cellular Cdk8 protein levels, premature phosphorylation of the Cdk8 target Fkh2, and earlier entry into mitosis. We also demonstrate that Mediator is recruited to clusters of mitotic genes in a periodic fashion and that the complex is required for the transcription of these genes. We suggest that Mediator functions as a hub for coordinated regulation of mitotic progression and cell cycle-dependent transcription. The many signaling pathways and activator proteins shown to function via Mediator may influence the timing of these cell cycle events. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  1. Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment.

    Science.gov (United States)

    Eyboulet, Fanny; Cibot, Camille; Eychenne, Thomas; Neil, Helen; Alibert, Olivier; Werner, Michel; Soutourina, Julie

    2013-12-01

    Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3' endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes.

  2. Transcription regulator TRIP-Br2 mediates ER stress-induced brown adipocytes dysfunction.

    Science.gov (United States)

    Qiang, Guifen; Whang Kong, Hyerim; Gil, Victoria; Liew, Chong Wee

    2017-01-09

    In contrast to white adipose tissue, brown adipose tissue (BAT) is known to play critical roles for both basal and inducible energy expenditure. Obesity is associated with reduction of BAT function; however, it is not well understood how obesity promotes BAT dysfunction, especially at the molecular level. Here we show that the transcription regulator TRIP-Br2 mediates ER stress-induced inhibition of lipolysis and thermogenesis in BAT. Using in vitro, ex vivo, and in vivo approaches, we demonstrate that obesity-induced inflammation upregulates brown adipocytes TRIP-Br2 expression via the ER stress pathway and amelioration of ER stress in mice completely abolishes high fat diet-induced upregulation of TRIP-Br2 in BAT. We find that increased TRIP-Br2 significantly inhibits brown adipocytes thermogenesis. Finally, we show that ablation of TRIP-Br2 ameliorates ER stress-induced inhibition on lipolysis, fatty acid oxidation, oxidative metabolism, and thermogenesis in brown adipocytes. Taken together, our current study demonstrates a role for TRIP-Br2 in ER stress-induced BAT dysfunction, and inhibiting TRIP-Br2 could be a potential approach for counteracting obesity-induced BAT dysfunction.

  3. Novel isoforms of the TFIID subunit TAF4 modulate nuclear receptor-mediated transcriptional activity

    International Nuclear Information System (INIS)

    Brunkhorst, Adrian; Neuman, Toomas; Hall, Anita; Arenas, Ernest; Bartfai, Tamas; Hermanson, Ola; Metsis, Madis

    2004-01-01

    The transcription factor TFIID consists of TATA-binding protein (TBP) and TBP-associated factors (TAFs). TAFs are essential for modulation of transcriptional activity but the regulation of TAFs is complex and many important aspects remain unclear. In this study, we have identified and characterized five novel truncated forms of the TFIID subunit TAF4 (TAF II 135). Analysis of the mouse gene structure revealed that all truncations were the results of alternative splicing and resulted in the loss of domains or parts of domains implicated in TAF4 functional interactions. Results from transcriptional assays showed that several of the TAF4 isoforms exerted dominant negative effects on TAF4 activity in nuclear receptor-mediated transcriptional activation. In addition, alternative TAF4 isoforms could be detected in specific cell types. Our results indicate an additional level of complexity in TAF4-mediated regulation of transcription and suggest context-specific roles for these new TAF4 isoforms in transcriptional regulation in vivo

  4. Milk: an epigenetic amplifier of FTO-mediated transcription? Implications for Western diseases.

    Science.gov (United States)

    Melnik, Bodo C

    2015-12-21

    Single-nucleotide polymorphisms within intron 1 of the FTO (fat mass and obesity-associated) gene are associated with enhanced FTO expression, increased body weight, obesity and type 2 diabetes mellitus (T2DM). The N (6) -methyladenosine (m(6)A) demethylase FTO plays a pivotal regulatory role for postnatal growth and energy expenditure. The purpose of this review is to provide translational evidence that links milk signaling with FTO-activated transcription of the milk recipient. FTO-dependent demethylation of m(6)A regulates mRNA splicing required for adipogenesis, increases the stability of mRNAs, and affects microRNA (miRNA) expression and miRNA biosynthesis. FTO senses branched-chain amino acids (BCAAs) and activates the nutrient sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1), which plays a key role in translation. Milk provides abundant BCAAs and glutamine, critical components increasing FTO expression. CpG hypomethylation in the first intron of FTO has recently been associated with T2DM. CpG methylation is generally associated with gene silencing. In contrast, CpG demethylation generally increases transcription. DNA de novo methylation of CpG sites is facilitated by DNA methyltransferases (DNMT) 3A and 3B, whereas DNA maintenance methylation is controlled by DNMT1. MiRNA-29s target all DNMTs and thus reduce DNA CpG methylation. Cow´s milk provides substantial amounts of exosomal miRNA-29s that reach the systemic circulation and target mRNAs of the milk recipient. Via DNMT suppression, milk exosomal miRNA-29s may reduce the magnitude of FTO methylation, thereby epigenetically increasing FTO expression in the milk consumer. High lactation performance with increased milk yield has recently been associated with excessive miRNA-29 expression of dairy cow mammary epithelial cells (DCMECs). Notably, the galactopoietic hormone prolactin upregulates the transcription factor STAT3, which induces miRNA-29 expression. In a retrovirus-like manner

  5. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    International Nuclear Information System (INIS)

    Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-01

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

  6. Core Mediator structure at 3.4 Å extends model of transcription initiation complex.

    Science.gov (United States)

    Nozawa, Kayo; Schneider, Thomas R; Cramer, Patrick

    2017-05-11

    Mediator is a multiprotein co-activator that binds the transcription pre-initiation complex (PIC) and regulates RNA polymerase (Pol) II. The Mediator head and middle modules form the essential core Mediator (cMed), whereas the tail and kinase modules play regulatory roles. The architecture of Mediator and its position on the PIC are known, but atomic details are limited to Mediator subcomplexes. Here we report the crystal structure of the 15-subunit cMed from Schizosaccharomyces pombe at 3.4 Å resolution. The structure shows an unaltered head module, and reveals the intricate middle module, which we show is globally required for transcription. Sites of known Mediator mutations cluster at the interface between the head and middle modules, and in terminal regions of the head subunits Med6 (ref. 16) and Med17 (ref. 17) that tether the middle module. The structure led to a model for Saccharomyces cerevisiae cMed that could be combined with the 3.6 Å cryo-electron microscopy structure of the core PIC (cPIC). The resulting atomic model of the cPIC-cMed complex informs on interactions of the submodules forming the middle module, called beam, knob, plank, connector, and hook. The hook is flexibly linked to Mediator by a conserved hinge and contacts the transcription initiation factor IIH (TFIIH) kinase that phosphorylates the carboxy (C)-terminal domain (CTD) of Pol II and was recently positioned on the PIC. The hook also contains residues that crosslink to the CTD and reside in a previously described cradle. These results provide a framework for understanding Mediator function, including its role in stimulating CTD phosphorylation by TFIIH.

  7. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription.

    Science.gov (United States)

    Lai, Fan; Orom, Ulf A; Cesaroni, Matteo; Beringer, Malte; Taatjes, Dylan J; Blobel, Gerd A; Shiekhattar, Ramin

    2013-02-28

    Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions. We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism. To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease- displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

  8. A Direct, Biomass-Based Synthesis of Benzoic Acid: Formic Acid-Mediated Deoxygenation of the Glucose-Derived Materials Quinic Acid and Shikimic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Arceo, Elena; Ellman, Jonathan; Bergman, Robert

    2010-05-03

    An alternative biomass-based route to benzoic acid from the renewable starting materials quinic acid and shikimic acid is described. Benzoic acid is obtained selectively using a highly efficient, one-step formic acid-mediated deoxygenation method.

  9. Role of the hinge region of glucocorticoid receptor for HEXIM1-mediated transcriptional repression

    International Nuclear Information System (INIS)

    Yoshikawa, Noritada; Shimizu, Noriaki; Sano, Motoaki; Ohnuma, Kei; Iwata, Satoshi; Hosono, Osamu; Fukuda, Keiichi; Morimoto, Chikao

    2008-01-01

    We previously reported that HEXIM1 (hexamethylene bisacetamide-inducible protein 1), which suppresses transcription elongation via sequestration of positive transcription elongation factor b (P-TEFb) using 7SK RNA as a scaffold, directly associates with glucocorticoid receptor (GR) to suppress glucocorticoid-inducible gene activation. Here, we revealed that the hinge region of GR is essential for its interaction with HEXIM1, and that oxosteroid receptors including GR show sequence homology in their hinge region and interact with HEXIM1, whereas the other members of nuclear receptors do not. We also showed that HEXIM1 suppresses GR-mediated transcription in two ways: sequestration of P-TEFb by HEXIM1 and direct interaction between GR and HEXIM1. In contrast, peroxisome proliferator-activated receptor γ-dependent gene expression is negatively modulated by HEXIM1 solely via sequestration of P-TEFb. We, therefore, conclude that HEXIM1 may act as a gene-selective transcriptional regulator via direct interaction with certain transcriptional regulators including GR and contribute to fine-tuning of, for example, glucocorticoid-mediated biological responses

  10. Structures of transcription pre-initiation complex with TFIIH and Mediator.

    Science.gov (United States)

    Schilbach, S; Hantsche, M; Tegunov, D; Dienemann, C; Wigge, C; Urlaub, H; Cramer, P

    2017-11-09

    For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC-core Mediator complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the 'E-bridge' helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC-core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II.

  11. TRIM32 promotes retinoic acid receptor α-mediated differentiation in human promyelogenous leukemic cell line HL60

    International Nuclear Information System (INIS)

    Sato, Tomonobu; Okumura, Fumihiko; Iguchi, Akihiro; Ariga, Tadashi; Hatakeyama, Shigetsugu

    2012-01-01

    Highlights: ► TRIM32 enhanced RARα-mediated transcriptional activity even in the absence of RA. ► TRIM32 stabilized RARα in the human promyelogenous leukemic cell line HL60. ► Overexpression of TRIM32 in HL60 cells induced granulocytic differentiation. ► TRIM32 may function as a coactivator for RARα-mediated transcription in APL cells. -- Abstract: Ubiquitination, one of the posttranslational modifications, appears to be involved in the transcriptional activity of nuclear receptors including retinoic acid receptor α (RARα). We previously reported that an E3 ubiquitin ligase, TRIM32, interacts with several important proteins including RARα and enhances transcriptional activity of RARα in mouse neuroblastoma cells and embryonal carcinoma cells. Retinoic acid (RA), which acts as a ligand to nuclear receptors including RARα, plays crucial roles in development, differentiation, cell cycles and apoptosis. In this study, we found that TRIM32 enhances RARα-mediated transcriptional activity even in the absence of RA and stabilizes RARα in the human promyelogenous leukemic cell line HL60. Moreover, we found that overexpression of TRIM32 in HL60 cells suppresses cellular proliferation and induces granulocytic differentiation even in the absence of RA. These findings suggest that TRIM32 functions as one of the coactivators for RARα-mediated transcription in acute promyelogenous leukemia (APL) cells, and thus TRIM32 may become a potentially therapeutic target for APL.

  12. Overexpression of the transcription factor NF-YC9 confers abscisic acid hypersensitivity in Arabidopsis.

    Science.gov (United States)

    Bi, Chao; Ma, Yu; Wang, Xiao-Fang; Zhang, Da-Peng

    2017-11-01

    Nuclear factor Y (NF-Y) family proteins are involved in many developmental processes and responses to environmental cues in plants, but whether and how they regulate phytohormone abscisic acid (ABA) signaling need further studies. In the present study, we showed that over-expression of the NF-YC9 gene confers ABA hypersensitivity in both the early seedling growth and stomatal response, while down-regulation of NF-YC9 does not affect ABA response in these processes. We also showed that over-expression of the NF-YC9 gene confers salt and osmotic hypersensitivity in early seedling growth, which is likely to be directly associated with the ABA hypersensitivity. Further, we observed that NF-YC9 physically interacts with the ABA-responsive bZIP transcription factor ABA-INSENSITIVE5 (ABI5), and facilitates the function of ABI5 to bind and activate the promoter of a target gene EM6. Additionally, NF-YC9 up-regulates expression of the ABI5 gene in response to ABA. These findings show that NF-YC9 may be involved in ABA signaling as a positive regulator and likely functions redundantly together with other NF-YC members, and support the model that the NF-YC9 mediates ABA signaling via targeting to and aiding the ABA-responsive transcription factors such as ABI5.

  13. Post-transcriptional regulation of the arginine transporter Cat-1 by amino acid availability

    NARCIS (Netherlands)

    Aulak, K. S.; Mishra, R.; Zhou, L.; Hyatt, S. L.; de Jonge, W.; Lamers, W.; Snider, M.; Hatzoglou, M.

    1999-01-01

    The regulation of the high affinity cationic amino acid transporter (Cat-1) by amino acid availability has been studied. In C6 glioma and NRK kidney cells, cat-1 mRNA levels increased 3.8-18-fold following 2 h of amino acid starvation. The transcription rate of the cat-1 gene remained unchanged

  14. Ultraviolet Radiation and the Slug Transcription Factor Induce Proinflammatory and Immunomodulatory Mediator Expression in Melanocytes

    Directory of Open Access Journals (Sweden)

    Stephanie H. Shirley

    2012-01-01

    Full Text Available Despite extensive investigation, the precise contribution of the ultraviolet radiation (UVR component of sunlight to melanoma etiology remains unclear. UVR induces keratinocytes to secrete proinflammatory and immunomodulatory mediators that promote inflammation and skin tumor development; expression of the slug transcription factor in keratinocytes is required for maximal production of these mediators. In the present studies we examined the possibility that UVR-exposed melanocytes also produce proinflammatory mediators and that Slug is important in this process. Microarray studies revealed that both UVR exposure and Slug overexpression altered transcription of a variety of proinflammatory mediators by normal human melanocytes; some of these mediators are also known to stimulate melanocyte growth and migration. There was little overlap in the spectra of cytokines produced by the two stimuli. However IL-20 was similarly induced by both stimuli and the NFκB pathway appeared to be important in both circumstances. Further exploration of UVR-induced and Slug-dependent pathways of cytokine induction in melanocytes may reveal novel targets for melanoma therapy.

  15. Ultraviolet Radiation and the Slug Transcription Factor Induce Pro inflammatory and Immunomodulatory Mediator Expression in Melanocytes

    International Nuclear Information System (INIS)

    Shirley, S. H.; Kusewitt, D. F.; Grimm, E. A.

    2012-01-01

    Despite extensive investigation, the precise contribution of the ultraviolet radiation (UVR) component of sunlight to melanoma etiology remains unclear. UVR induces keratinocytes to secrete pro inflammatory and immunomodulatory mediators that promote inflammation and skin tumor development; expression of the slug transcription factor in keratinocytes is required for maximal production of these mediators. In the present studies we examined the possibility that UVR-exposed melanocytes also produce pro inflammatory mediators and that Slug is important in this process. Micro array studies revealed that both UVR exposure and Slug overexpression altered transcription of a variety of pro inflammatory mediators by normal human melanocytes; some of these mediators are also known to stimulate melanocyte growth and migration. There was little overlap in the spectra of cytokines produced by the two stimuli. However IL-20 was similarly induced by both stimuli and the NFκB pathway appeared to be important in both circumstances. Further exploration of UVR-induced and Slug-dependent pathways of cytokine induction in melanocytes may reveal novel targets for melanoma therapy.

  16. Nfatc1 Is a Functional Transcriptional Factor Mediating Nell-1-Induced Runx3 Upregulation in Chondrocytes

    Directory of Open Access Journals (Sweden)

    Chenshuang Li

    2018-01-01

    Full Text Available Neural EGFL like 1 (Nell-1 is essential for chondrogenic differentiation, maturation, and regeneration. Our previous studies have demonstrated that Nell-1’s pro-chondrogenic activities are predominantly reliant upon runt-related transcription factor 3 (Runx3-mediated Indian hedgehog (Ihh signaling. Here, we identify the nuclear factor of activated T-cells 1 (Nfatc1 as the key transcriptional factor mediating the Nell-1 → Runx3 signal transduction in chondrocytes. Using chromatin immunoprecipitation assay, we were able to determine that Nfatc1 binds to the −833–−810 region of the Runx3-promoter in response to Nell-1 treatment. By revealing the Nell-1 → Nfatc1 → Runx3 → Ihh cascade, we demonstrate the involvement of Nfatc1, a nuclear factor of activated T-cells, in chondrogenesis, while providing innovative insights into developing a novel therapeutic strategy for cartilage regeneration and other chondrogenesis-related conditions.

  17. Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma.

    Science.gov (United States)

    Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias

    2016-02-01

    Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma. Copyright © 2015 Federation of European Biochemical Societies

  18. Inhibition of CRM1-mediated nuclear export of transcription factors by leukemogenic NUP98 fusion proteins.

    Science.gov (United States)

    Takeda, Akiko; Sarma, Nayan J; Abdul-Nabi, Anmaar M; Yaseen, Nabeel R

    2010-05-21

    NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

  19. NGF-mediated transcriptional targets of p53 in PC12 neuronal differentiation

    Directory of Open Access Journals (Sweden)

    Labhart Paul

    2007-05-01

    Full Text Available Abstract Background p53 is recognized as a critical regulator of the cell cycle and apoptosis. Mounting evidence also suggests a role for p53 in differentiation of cells including neuronal precursors. We studied the transcriptional role of p53 during nerve growth factor-induced differentiation of the PC12 line into neuron-like cells. We hypothesized that p53 contributed to PC12 differentiation through the regulation of gene targets distinct from its known transcriptional targets for apoptosis or DNA repair. Results Using a genome-wide chromatin immunoprecipitation cloning technique, we identified and validated 14 novel p53-regulated genes following NGF treatment. The data show p53 protein was transcriptionally activated and contributed to NGF-mediated neurite outgrowth during differentiation of PC12 cells. Furthermore, we describe stimulus-specific regulation of a subset of these target genes by p53. The most salient differentiation-relevant target genes included wnt7b involved in dendritic extension and the tfcp2l4/grhl3 grainyhead homolog implicated in ectodermal development. Additional targets included brk, sdk2, sesn3, txnl2, dusp5, pon3, lect1, pkcbpb15 and other genes. Conclusion Within the PC12 neuronal context, putative p53-occupied genomic loci spanned the entire Rattus norvegicus genome upon NGF treatment. We conclude that receptor-mediated p53 transcriptional activity is involved in PC12 differentiation and may suggest a contributory role for p53 in neuronal development.

  20. Identification of a Transcription Factor Controlling pH-Dependent Organic Acid Response in Aspergillus niger

    DEFF Research Database (Denmark)

    Poulsen, Lars; Andersen, Mikael Rørdam; Lantz, Anna Eliasson

    2012-01-01

    exhibiting an oxalate overproducing phenotype were identified. The yield of oxalate was increased up to 158% compared to the wild type and the corresponding transcription factor was therefore entitled Oxalic Acid repression Factor, OafA. Detailed physiological characterization of one of the ΔoafA mutants......, compared to the wild type, showed that both strains produced substantial amounts of gluconic acid, but the mutant strain was more efficient in re-uptake of gluconic acid and converting it to oxalic acid, particularly at high pH (pH 5.0). Transcriptional profiles showed that 241 genes were differentially......Acid formation in Aspergillus niger is known to be subjected to tight regulation, and the acid production profiles are fine-tuned to respond to the ambient pH. Based on transcriptome data, putative trans-acting pH responding transcription factors were listed and through knock out studies, mutants...

  1. Set1/COMPASS and Mediator are repurposed to promote epigenetic transcriptional memory.

    Science.gov (United States)

    D'Urso, Agustina; Takahashi, Yoh-Hei; Xiong, Bin; Marone, Jessica; Coukos, Robert; Randise-Hinchliff, Carlo; Wang, Ji-Ping; Shilatifard, Ali; Brickner, Jason H

    2016-06-23

    In yeast and humans, previous experiences can lead to epigenetic transcriptional memory: repressed genes that exhibit mitotically heritable changes in chromatin structure and promoter recruitment of poised RNA polymerase II preinitiation complex (RNAPII PIC), which enhances future reactivation. Here, we show that INO1 memory in yeast is initiated by binding of the Sfl1 transcription factor to the cis-acting Memory Recruitment Sequence, targeting INO1 to the nuclear periphery. Memory requires a remodeled form of the Set1/COMPASS methyltransferase lacking Spp1, which dimethylates histone H3 lysine 4 (H3K4me2). H3K4me2 recruits the SET3C complex, which plays an essential role in maintaining this mark. Finally, while active INO1 is associated with Cdk8(-) Mediator, during memory, Cdk8(+) Mediator recruits poised RNAPII PIC lacking the Kin28 CTD kinase. Aspects of this mechanism are generalizable to yeast and conserved in human cells. Thus, COMPASS and Mediator are repurposed to promote epigenetic transcriptional poising by a highly conserved mechanism.

  2. Disrupting SUMOylation enhances transcriptional function and ameliorates polyglutamine androgen receptor–mediated disease

    Science.gov (United States)

    Chua, Jason P.; Reddy, Satya L.; Yu, Zhigang; Giorgetti, Elisa; Montie, Heather L.; Mukherjee, Sarmistha; Higgins, Jake; McEachin, Richard C.; Robins, Diane M.; Merry, Diane E.; Iñiguez-Lluhí, Jorge A.; Lieberman, Andrew P.

    2015-01-01

    Expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR) causes neuromuscular degeneration in individuals with spinobulbar muscular atrophy (SBMA). PolyQ AR has diminished transcriptional function and exhibits ligand-dependent proteotoxicity, features that have both been implicated in SBMA; however, the extent to which altered AR transcriptional function contributes to pathogenesis remains controversial. Here, we sought to dissociate effects of diminished AR function from polyQ-mediated proteotoxicity by enhancing the transcriptional activity of polyQ AR. To accomplish this, we bypassed the inhibitory effect of AR SUMOylation (where SUMO indicates small ubiquitin-like modifier) by mutating conserved lysines in the polyQ AR that are sites of SUMOylation. We determined that replacement of these residues by arginine enhances polyQ AR activity as a hormone-dependent transcriptional regulator. In a murine model, disruption of polyQ AR SUMOylation rescued exercise endurance and type I muscle fiber atrophy; it also prolonged survival. These changes occurred without overt alterations in polyQ AR expression or aggregation, revealing the favorable trophic support exerted by the ligand-activated receptor. Our findings demonstrate beneficial effects of enhancing the transcriptional function of the ligand-activated polyQ AR and indicate that the SUMOylation pathway may be a potential target for therapeutic intervention in SBMA. PMID:25607844

  3. Transcriptional Regulation of Arabidopsis MIR168a and ARGONAUTE1 Homeostasis in Abscisic Acid and Abiotic Stress Responses1[W

    Science.gov (United States)

    Li, Wei; Cui, Xiao; Meng, Zhaolu; Huang, Xiahe; Xie, Qi; Wu, Heng; Jin, Hailing; Zhang, Dabing; Liang, Wanqi

    2012-01-01

    The accumulation of a number of small RNAs in plants is affected by abscisic acid (ABA) and abiotic stresses, but the underlying mechanisms are poorly understood. The miR168-mediated feedback regulatory loop regulates ARGONAUTE1 (AGO1) homeostasis, which is crucial for gene expression modulation and plant development. Here, we reveal a transcriptional regulatory mechanism by which MIR168 controls AGO1 homeostasis during ABA treatment and abiotic stress responses in Arabidopsis (Arabidopsis thaliana). Plants overexpressing MIR168a and the AGO1 loss-of-function mutant ago1-27 display ABA hypersensitivity and drought tolerance, while the mir168a-2 mutant shows ABA hyposensitivity and drought hypersensitivity. Both the precursor and mature miR168 were induced under ABA and several abiotic stress treatments, but no obvious decrease for the target of miR168, AGO1, was shown under the same conditions. However, promoter activity analysis indicated that AGO1 transcription activity was increased under ABA and drought treatments, suggesting that transcriptional elevation of MIR168a is required for maintaining a stable AGO1 transcript level during the stress response. Furthermore, we showed both in vitro and in vivo that the transcription of MIR168a is directly regulated by four abscisic acid-responsive element (ABRE) binding factors, which bind to the ABRE cis-element within the MIR168a promoter. This ABRE motif is also found in the promoter of MIR168a homologs in diverse plant species. Our findings suggest that transcriptional regulation of miR168 and posttranscriptional control of AGO1 homeostasis may play an important and conserved role in stress response and signal transduction in plants. PMID:22247272

  4. Transcriptional regulation of Arabidopsis MIR168a and argonaute1 homeostasis in abscisic acid and abiotic stress responses.

    Science.gov (United States)

    Li, Wei; Cui, Xiao; Meng, Zhaolu; Huang, Xiahe; Xie, Qi; Wu, Heng; Jin, Hailing; Zhang, Dabing; Liang, Wanqi

    2012-03-01

    The accumulation of a number of small RNAs in plants is affected by abscisic acid (ABA) and abiotic stresses, but the underlying mechanisms are poorly understood. The miR168-mediated feedback regulatory loop regulates ARGONAUTE1 (AGO1) homeostasis, which is crucial for gene expression modulation and plant development. Here, we reveal a transcriptional regulatory mechanism by which MIR168 controls AGO1 homeostasis during ABA treatment and abiotic stress responses in Arabidopsis (Arabidopsis thaliana). Plants overexpressing MIR168a and the AGO1 loss-of-function mutant ago1-27 display ABA hypersensitivity and drought tolerance, while the mir168a-2 mutant shows ABA hyposensitivity and drought hypersensitivity. Both the precursor and mature miR168 were induced under ABA and several abiotic stress treatments, but no obvious decrease for the target of miR168, AGO1, was shown under the same conditions. However, promoter activity analysis indicated that AGO1 transcription activity was increased under ABA and drought treatments, suggesting that transcriptional elevation of MIR168a is required for maintaining a stable AGO1 transcript level during the stress response. Furthermore, we showed both in vitro and in vivo that the transcription of MIR168a is directly regulated by four abscisic acid-responsive element (ABRE) binding factors, which bind to the ABRE cis-element within the MIR168a promoter. This ABRE motif is also found in the promoter of MIR168a homologs in diverse plant species. Our findings suggest that transcriptional regulation of miR168 and posttranscriptional control of AGO1 homeostasis may play an important and conserved role in stress response and signal transduction in plants.

  5. Systematic identification of core transcription factors mediating dysregulated links bridging inflammatory bowel diseases and colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Yun Xiao

    Full Text Available Accumulating evidence shows a tight link between inflammation and cancer. However, comprehensive identification of pivotal transcription factors (i.e., core TFs mediating the dysregulated links remains challenging, mainly due to a lack of samples that can effectively reflect the connections between inflammation and tumorigenesis. Here, we constructed a series of TF-mediated regulatory networks from a large compendium of expression profiling of normal colonic tissues, inflammatory bowel diseases (IBDs and colorectal cancer (CRC, which contains 1201 samples in total, and then proposed a network-based approach to characterize potential links bridging inflammation and cancer. For this purpose, we computed significantly dysregulated relationships between inflammation and their linked cancer networks, and then 24 core TFs with their dysregulated genes were identified. Collectively, our approach provides us with quite important insight into inflammation-associated tumorigenesis in colorectal cancer, which could also be applied to identify functionally dysregulated relationships mediating the links between other different disease phenotypes.

  6. Acetylation-mediated suppression of transcription-independent memory: bidirectional modulation of memory by acetylation.

    Directory of Open Access Journals (Sweden)

    Katja Merschbaecher

    Full Text Available Learning induced changes in protein acetylation, mediated by histone acetyl transferases (HATs, and the antagonistic histone deacetylases (HDACs play a critical role in memory formation. The status of histone acetylation affects the interaction between the transcription-complex and DNA and thus regulates transcription-dependent processes required for long-term memory (LTM. While the majority of studies report on the role of elevated acetylation in memory facilitation, we address the impact of both, increased and decreased acetylation on formation of appetitive olfactory memory in honeybees. We show that learning-induced changes in the acetylation of histone H3 at aminoacid-positions H3K9 and H3K18 exhibit distinct and different dynamics depending on the training strength. A strong training that induces LTM leads to an immediate increase in acetylation at H3K18 that stays elevated for hours. A weak training, not sufficient to trigger LTM, causes an initial increase in acetylation at H3K18, followed by a strong reduction in acetylation at H3K18 below the control group level. Acetylation at position H3K9 is not affected by associative conditioning, indicating specific learning-induced actions on the acetylation machinery. Elevating acetylation levels by blocking HDACs after conditioning leads to an improved memory. While memory after strong training is enhanced for at least 2 days, the enhancement after weak training is restricted to 1 day. Reducing acetylation levels by blocking HAT activity after strong training leads to a suppression of transcription-dependent LTM. The memory suppression is also observed in case of weak training, which does not require transcription processes. Thus, our findings demonstrate that acetylation-mediated processes act as bidirectional regulators of memory formation that facilitate or suppress memory independent of its transcription-requirement.

  7. Histone H4 Lys 20 methyltransferase SET8 promotes androgen receptor-mediated transcription activation in prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Lushuai [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Yanyan; Du, Fengxia [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Han, Xiao [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Xiaohua [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Niu, Yuanjie [Chawnshang Chang Sex Hormone Research Center, Tianjin Institute of Urology, Tianjin Medical University, Tianjin 300070 (China); Ren, Shancheng, E-mail: renshancheng@gmail.com [Department of Urology, Shanghai Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Sun, Yingli, E-mail: sunyl@big.ac.cn [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

    Highlights: • Dihydrotestosterone stimulates H4K20me1 enrichment at the PSA promoter. • SET8 promotes AR-mediated transcription activation. • SET8 interacts with AR and promotes cell proliferation. - Abstract: Histone methylation status in different lysine residues has an important role in transcription regulation. The effect of H4K20 monomethylation (H4K20me1) on androgen receptor (AR)-mediated gene transcription remains unclear. Here we show that AR agonist stimulates the enrichment of H4K20me1 and SET8 at the promoter of AR target gene PSA in an AR dependent manner. Furthermore, SET8 is crucial for the transcription activation of PSA. Co-immunoprecipitation analyses demonstrate that SET8 interacts with AR. Therefore, we conclude that SET8 is involved in AR-mediated transcription activation, possibly through its interaction with AR and H4K20me1 modification.

  8. CHD8, A Novel Beta-Catenin Associated Chromatin Remodeling Enzyme, Regulates Androgen Receptor Mediated Gene Transcription

    National Research Council Canada - National Science Library

    Bochar, Daniel A

    2008-01-01

    .... To better understand the function of beta-catenin in AR mediated transcription, we have identified a novel chromatin remodeling enzyme, CHD8, that can associate with beta-catenin and functions in AR...

  9. Structural basis of transcriptional gene silencing mediated by Arabidopsis MOM1.

    Directory of Open Access Journals (Sweden)

    Taisuke Nishimura

    2012-02-01

    Full Text Available Shifts between epigenetic states of transcriptional activity are typically correlated with changes in epigenetic marks. However, exceptions to this rule suggest the existence of additional, as yet uncharacterized, layers of epigenetic regulation. MOM1, a protein of 2,001 amino acids that acts as a transcriptional silencer, represents such an exception. Here we define the 82 amino acid domain called CMM2 (Conserved MOM1 Motif 2 as a minimal MOM1 fragment capable of transcriptional regulation. As determined by X-ray crystallography, this motif folds into an unusual hendecad-based coiled-coil. Structure-based mutagenesis followed by transgenic complementation tests in plants demonstrate that CMM2 and its dimerization are effective for transcriptional suppression at chromosomal loci co-regulated by MOM1 and the siRNA pathway but not at loci controlled by MOM1 in an siRNA-independent fashion. These results reveal a surprising separation of epigenetic activities that enable the single, large MOM1 protein to coordinate cooperating mechanisms of epigenetic regulation.

  10. Structural basis of transcriptional gene silencing mediated by Arabidopsis MOM1.

    Science.gov (United States)

    Nishimura, Taisuke; Molinard, Guillaume; Petty, Tom J; Broger, Larissa; Gabus, Caroline; Halazonetis, Thanos D; Thore, Stéphane; Paszkowski, Jerzy

    2012-02-01

    Shifts between epigenetic states of transcriptional activity are typically correlated with changes in epigenetic marks. However, exceptions to this rule suggest the existence of additional, as yet uncharacterized, layers of epigenetic regulation. MOM1, a protein of 2,001 amino acids that acts as a transcriptional silencer, represents such an exception. Here we define the 82 amino acid domain called CMM2 (Conserved MOM1 Motif 2) as a minimal MOM1 fragment capable of transcriptional regulation. As determined by X-ray crystallography, this motif folds into an unusual hendecad-based coiled-coil. Structure-based mutagenesis followed by transgenic complementation tests in plants demonstrate that CMM2 and its dimerization are effective for transcriptional suppression at chromosomal loci co-regulated by MOM1 and the siRNA pathway but not at loci controlled by MOM1 in an siRNA-independent fashion. These results reveal a surprising separation of epigenetic activities that enable the single, large MOM1 protein to coordinate cooperating mechanisms of epigenetic regulation.

  11. TRIM45 negatively regulates NF-κB-mediated transcription and suppresses cell proliferation

    International Nuclear Information System (INIS)

    Shibata, Mio; Sato, Tomonobu; Nukiwa, Ryota; Ariga, Tadashi; Hatakeyama, Shigetsugu

    2012-01-01

    Highlights: ► NF-κB plays an important role in cell survival and carcinogenesis. ► TRIM45 negatively regulates TNFα-induced NF-κB-mediated transcription. ► TRIM45 overexpression suppresses cell growth. ► TRIM45 acts as a repressor for the NF-κB signal and regulates cell growth. -- Abstract: The NF-κB signaling pathway plays an important role in cell survival, immunity, inflammation, carcinogenesis, and organogenesis. Activation of NF-κB is regulated by several posttranslational modifications including phosphorylation, neddylation and ubiquitination. The NF-κB signaling pathway is activated by two distinct signaling mechanisms and is strictly modulated by the ubiquitin–proteasome system. It has been reported that overexpression of TRIM45, one of the TRIM family ubiquitin ligases, suppresses transcriptional activities of Elk-1 and AP-1, which are targets of the MAPK signaling pathway. In this study, we showed that TRIM45 also negatively regulates TNFα-induced NF-κB-mediated transcription by a luciferase reporter assay and that TRIM45 lacking a RING domain also has an activity to inhibit the NF-κB signal. Moreover, we found that TRIM45 overexpression suppresses cell growth. These findings suggest that TRIM45 acts as a repressor for the NF-κB signal and regulates cell growth.

  12. Estrogen promotes megakaryocyte polyploidization via estrogen receptor beta-mediated transcription of GATA1.

    Science.gov (United States)

    Du, C; Xu, Y; Yang, K; Chen, S; Wang, X; Wang, S; Wang, C; Shen, M; Chen, F; Chen, M; Zeng, D; Li, F; Wang, T; Wang, F; Zhao, J; Ai, G; Cheng, T; Su, Y; Wang, J

    2017-04-01

    Estrogen is reported to be involved in thrombopoiesis and the disruption of its signaling may cause myeloproliferative disease, yet the underlying mechanisms remain largely unknown. GATA-binding factor 1 (GATA1) is a key regulator of megakaryocyte (MK) differentiation and its deficiency will lead to megakaryoblastic leukemia. Here we show that estrogen can dose-dependently promote MK polyploidization and maturation via activation of estrogen receptor beta (ERβ), accompanied by a significant upregulation of GATA1. Chromatin immunoprecipitation and a dual luciferase assay demonstrate that ERβ can directly bind the promoter region of GATA1 and activate its transcription. Steroid receptor coactivator 3 (SRC3) is involved in ERβ-mediated GATA1 transcription. The deficiency of ERβ or SRC3, similar to the inhibition of GATA1, leads to the impediment of estrogen-induced MK polyploidization and platelet production. Further investigations reveal that signal transducer and activator of transcription 1 signaling pathway downstream of GATA1 has a crucial role in estrogen-induced MK polyploidization, and ERβ-mediated GATA1 upregulation subsequently enhances nuclear factor erythroid-derived 2 expression, thereby promoting proplatelet formation and platelet release. Our study provides a deep insight into the molecular mechanisms of estrogen signaling in regulating thrombopoiesis and the pathogenesis of ER deficiency-related leukemia.

  13. The transcription factor MEF2C mediates cardiomyocyte hypertrophy induced by IGF-1 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Munoz, Juan Pablo; Collao, Andres; Chiong, Mario; Maldonado, Carola; Adasme, Tatiana; Carrasco, Loreto; Ocaranza, Paula; Bravo, Roberto; Gonzalez, Leticia; Diaz-Araya, Guillermo [Centro FONDAP Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Facultad de Ciencias Quimicas y Farmaceuticas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Hidalgo, Cecilia [Centro FONDAP Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Lavandero, Sergio, E-mail: slavander@uchile.cl [Centro FONDAP Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Facultad de Ciencias Quimicas y Farmaceuticas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile)

    2009-10-09

    Myocyte enhancer factor 2C (MEF2C) plays an important role in cardiovascular development and is a key transcription factor for cardiac hypertrophy. Here, we describe MEF2C regulation by insulin-like growth factor-1 (IGF-1) and its role in IGF-1-induced cardiac hypertrophy. We found that IGF-1 addition to cultured rat cardiomyocytes activated MEF2C, as evidenced by its increased nuclear localization and DNA binding activity. IGF-1 stimulated MEF2 dependent-gene transcription in a time-dependent manner, as indicated by increased MEF2 promoter-driven reporter gene activity; IGF-1 also induced p38-MAPK phosphorylation, while an inhibitor of p38-MAPK decreased both effects. Additionally, inhibitors of phosphatidylinositol 3-kinase and calcineurin prevented IGF-1-induced MEF2 transcriptional activity. Via MEF2C-dependent signaling, IGF-1 also stimulated transcription of atrial natriuretic factor and skeletal {alpha}-actin but not of fos-lux reporter genes. These novel data suggest that MEF2C activation by IGF-1 mediates the pro-hypertrophic effects of IGF-1 on cardiac gene expression.

  14. The transcription factor MEF2C mediates cardiomyocyte hypertrophy induced by IGF-1 signaling

    International Nuclear Information System (INIS)

    Munoz, Juan Pablo; Collao, Andres; Chiong, Mario; Maldonado, Carola; Adasme, Tatiana; Carrasco, Loreto; Ocaranza, Paula; Bravo, Roberto; Gonzalez, Leticia; Diaz-Araya, Guillermo; Hidalgo, Cecilia; Lavandero, Sergio

    2009-01-01

    Myocyte enhancer factor 2C (MEF2C) plays an important role in cardiovascular development and is a key transcription factor for cardiac hypertrophy. Here, we describe MEF2C regulation by insulin-like growth factor-1 (IGF-1) and its role in IGF-1-induced cardiac hypertrophy. We found that IGF-1 addition to cultured rat cardiomyocytes activated MEF2C, as evidenced by its increased nuclear localization and DNA binding activity. IGF-1 stimulated MEF2 dependent-gene transcription in a time-dependent manner, as indicated by increased MEF2 promoter-driven reporter gene activity; IGF-1 also induced p38-MAPK phosphorylation, while an inhibitor of p38-MAPK decreased both effects. Additionally, inhibitors of phosphatidylinositol 3-kinase and calcineurin prevented IGF-1-induced MEF2 transcriptional activity. Via MEF2C-dependent signaling, IGF-1 also stimulated transcription of atrial natriuretic factor and skeletal α-actin but not of fos-lux reporter genes. These novel data suggest that MEF2C activation by IGF-1 mediates the pro-hypertrophic effects of IGF-1 on cardiac gene expression.

  15. Chk2 regulates transcription-independent p53-mediated apoptosis in response to DNA damage

    International Nuclear Information System (INIS)

    Chen Chen; Shimizu, Shigeomi; Tsujimoto, Yoshihide; Motoyama, Noboru

    2005-01-01

    The tumor suppressor protein p53 plays a central role in the induction of apoptosis in response to genotoxic stress. The protein kinase Chk2 is an important regulator of p53 function in mammalian cells exposed to ionizing radiation (IR). Cells derived from Chk2-deficient mice are resistant to the induction of apoptosis by IR, and this resistance has been thought to be a result of the defective transcriptional activation of p53 target genes. It was recently shown, however, that p53 itself and histone H1.2 translocate to mitochondria and thereby induces apoptosis in a transcription-independent manner in response to IR. We have now examined whether Chk2 also regulates the transcription-independent induction of apoptosis by p53 and histone H1.2. The reduced ability of IR to induce p53 stabilization in Chk2-deficient thymocytes was associated with a marked impairment of p53 and histone H1 translocation to mitochondria. These results suggest that Chk2 regulates the transcription-independent mechanism of p53-mediated apoptosis by inducing stabilization of p53 in response to IR

  16. Strategies to regulate transcription factor-mediated gene positioning and interchromosomal clustering at the nuclear periphery.

    Science.gov (United States)

    Randise-Hinchliff, Carlo; Coukos, Robert; Sood, Varun; Sumner, Michael Chas; Zdraljevic, Stefan; Meldi Sholl, Lauren; Garvey Brickner, Donna; Ahmed, Sara; Watchmaker, Lauren; Brickner, Jason H

    2016-03-14

    In budding yeast, targeting of active genes to the nuclear pore complex (NPC) and interchromosomal clustering is mediated by transcription factor (TF) binding sites in the gene promoters. For example, the binding sites for the TFs Put3, Ste12, and Gcn4 are necessary and sufficient to promote positioning at the nuclear periphery and interchromosomal clustering. However, in all three cases, gene positioning and interchromosomal clustering are regulated. Under uninducing conditions, local recruitment of the Rpd3(L) histone deacetylase by transcriptional repressors blocks Put3 DNA binding. This is a general function of yeast repressors: 16 of 21 repressors blocked Put3-mediated subnuclear positioning; 11 of these required Rpd3. In contrast, Ste12-mediated gene positioning is regulated independently of DNA binding by mitogen-activated protein kinase phosphorylation of the Dig2 inhibitor, and Gcn4-dependent targeting is up-regulated by increasing Gcn4 protein levels. These different regulatory strategies provide either qualitative switch-like control or quantitative control of gene positioning over different time scales. © 2016 Randise-Hinchliff et al.

  17. Malondialdehyde inhibits an AMPK-mediated nuclear translocation and repression activity of ALDH2 in transcription

    International Nuclear Information System (INIS)

    Choi, Ji-Woong; Kim, Jae-Hwan; Cho, Sung-Chun; Ha, Moon-Kyung; Song, Kye-Yong; Youn, Hong-Duk; Park, Sang Chul

    2011-01-01

    Research highlights: → ALDH2 is an MDA-modified protein in old rat kidney tissues. → AMPK associates with ALDH2 and triggers the nuclear localization of ALDH2. → ALDH2 serves as a general transcriptional repressor by associating with HDACs. → MDA inhibits the AMPK-mediated translocation of ALDH2 and its repression activity. -- Abstract: Aging process results from deleterious damages by reactive oxygen species, in particular, various metabolic aldehydes. Aldehyde dehydrogenase 2 (ALDH2) is one of metabolic enzymes detoxifying various aldehydes under oxidative conditions. AMP-activated protein kinase (AMPK) plays a key role in controlling metabolic process. However, little was known about the relationship of ALDH2 with AMPK under oxidative conditions. Here, we, by using MDA-specific monoclonal antibody, screened the tissues of young and old rats for MDA-modified proteins and identified an ALDH2 as a prominent MDA-modified protein band in the old rat kidney tissue. ALDH2 associates with AMPK and is phosphorylated by AMPK. In addition, AICAR, an activator of AMP-activated protein kinase, induces the nuclear translocation of ALDH2. ALDH2 in nucleus is involved in general transcription repression by association with histone deacetylases. Furthermore, MDA modification inhibited the translocation of ALDH2 and the association with AMPK, and ultimately led to de-repression of transcription in the reporter system analysis. In this study, we have demonstrated that ALDH2 acts as a transcriptional repressor in response to AMPK activation, and MDA modifies ALDH2 and inhibits repressive activity of ALDH2 in general transcription. We thus suggest that increasing amount of MDA during aging process may interrupt the nuclear function of ALDH2, modulated by AMPK.

  18. Malondialdehyde inhibits an AMPK-mediated nuclear translocation and repression activity of ALDH2 in transcription

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Ji-Woong [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Aging and Apoptosis Research Center (AARC), Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799, (Korea, Republic of); Kim, Jae-Hwan [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Cho, Sung-Chun; Ha, Moon-Kyung [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Aging and Apoptosis Research Center (AARC), Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799, (Korea, Republic of); Song, Kye-Yong [Department of Pathology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Youn, Hong-Duk, E-mail: hdyoun@snu.ac.kr [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Park, Sang Chul, E-mail: scpark@snu.ac.kr [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Aging and Apoptosis Research Center (AARC), Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799, (Korea, Republic of)

    2011-01-07

    Research highlights: {yields} ALDH2 is an MDA-modified protein in old rat kidney tissues. {yields} AMPK associates with ALDH2 and triggers the nuclear localization of ALDH2. {yields} ALDH2 serves as a general transcriptional repressor by associating with HDACs. {yields} MDA inhibits the AMPK-mediated translocation of ALDH2 and its repression activity. -- Abstract: Aging process results from deleterious damages by reactive oxygen species, in particular, various metabolic aldehydes. Aldehyde dehydrogenase 2 (ALDH2) is one of metabolic enzymes detoxifying various aldehydes under oxidative conditions. AMP-activated protein kinase (AMPK) plays a key role in controlling metabolic process. However, little was known about the relationship of ALDH2 with AMPK under oxidative conditions. Here, we, by using MDA-specific monoclonal antibody, screened the tissues of young and old rats for MDA-modified proteins and identified an ALDH2 as a prominent MDA-modified protein band in the old rat kidney tissue. ALDH2 associates with AMPK and is phosphorylated by AMPK. In addition, AICAR, an activator of AMP-activated protein kinase, induces the nuclear translocation of ALDH2. ALDH2 in nucleus is involved in general transcription repression by association with histone deacetylases. Furthermore, MDA modification inhibited the translocation of ALDH2 and the association with AMPK, and ultimately led to de-repression of transcription in the reporter system analysis. In this study, we have demonstrated that ALDH2 acts as a transcriptional repressor in response to AMPK activation, and MDA modifies ALDH2 and inhibits repressive activity of ALDH2 in general transcription. We thus suggest that increasing amount of MDA during aging process may interrupt the nuclear function of ALDH2, modulated by AMPK.

  19. Involvement of WRKY Transcription Factors in Abscisic-Acid-Induced Cold Tolerance of Banana Fruit.

    Science.gov (United States)

    Luo, Dong-Lan; Ba, Liang-Jie; Shan, Wei; Kuang, Jian-Fei; Lu, Wang-Jin; Chen, Jian-Ye

    2017-05-10

    Phytohormone abscisic acid (ABA) and plant-specific WRKY transcription factors (TFs) have been implicated to play important roles in various stress responses. The involvement of WRKY TFs in ABA-mediated cold tolerance of economical fruits, such as banana fruit, however remains largely unknown. Here, we reported that ABA application could induce expressions of ABA biosynthesis-related genes MaNCED1 and MaNCED2, increase endogenous ABA contents, and thereby enhance cold tolerance in banana fruit. Four banana fruit WRKY TFs, designated as MaWRKY31, MaWRKY33, MaWRKY60, and MaWRKY71, were identified and characterized. All four of these MaWRKYs were nuclear-localized and displayed transactivation activities. Their expressions were induced by ABA treatment during cold storage. More importantly, the gel mobility shift assay and transient expression analysis revealed that MaWRKY31, MaWRKY33, MaWRKY60, and MaWRKY71 directly bound to the W-box elements in MaNCED1 and MaNCED2 promoters and activated their expressions. Taken together, our findings demonstrate that banana fruit WRKY TFs are involved in ABA-induced cold tolerance by, at least in part, increasing ABA levels via directly activating NECD expressions.

  20. Exosome proteomics reveals transcriptional regulator proteins with potential to mediate downstream pathways.

    Science.gov (United States)

    Ung, Timothy H; Madsen, Helen J; Hellwinkel, Justin E; Lencioni, Alex M; Graner, Michael W

    2014-11-01

    Exosomes are virus-sized, membrane-enclosed vesicles with origins in the cellular endosomal system, but are released extracellularly. As a population, these tiny vesicles carry relatively enormous amounts of information in their protein, lipid and nucleic acid content, and the vesicles can have profound impacts on recipient cells. This review employs publically-available data combined with gene ontology applications to propose a novel concept, that exosomes transport transcriptional and translational machinery that may have direct impacts on gene expression in recipient cells. Here, we examine the previously published proteomic contents of medulloblastoma-derived exosomes, focusing on transcriptional regulators; we found that there are numerous proteins that may have potential roles in transcriptional and translational regulation with putative influence on downstream, cancer-related pathways. We expanded this search to all of the proteins in the Vesiclepedia database; using gene ontology approaches, we see that these regulatory factors are implicated in many of the processes involved in cancer initiation and progression. This information suggests that some of the effects of exosomes on recipient cells may be due to the delivery of protein factors that can directly and fundamentally change the transcriptional landscape of the cells. Within a tumor environment, this has potential to tilt the advantage towards the cancer. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  1. E2F1-Mediated Induction of NFYB Attenuates Apoptosis via Joint Regulation of a Pro-Survival Transcriptional Program.

    Directory of Open Access Journals (Sweden)

    Xiaolei Jiang

    Full Text Available The E2F1 transcription factor regulates cell proliferation and apoptosis through the control of a considerable variety of target genes. Previous work has detailed the role of other transcription factors in mediating the specificity of E2F function. Here we identify the NF-YB transcription factor as a novel direct E2F1 target. Genome-wide expression analysis of the effects of NFYB knockdown on E2F1-mediated transcription identified a large group of genes that are co-regulated by E2F1 and NFYB. We also provide evidence that knockdown of NFYB enhances E2F1-induced apoptosis, suggesting a pro-survival function of the NFYB/E2F1 joint transcriptional program. Bioinformatic analysis suggests that deregulation of these NFY-dependent E2F1 target genes might play a role in sarcomagenesis as well as drug resistance.

  2. Arabidopsis MYC Transcription Factors Are the Target of Hormonal Salicylic Acid/Jasmonic Acid Cross Talk in Response to Pieris brassicae Egg Extract.

    Science.gov (United States)

    Schmiesing, André; Emonet, Aurélia; Gouhier-Darimont, Caroline; Reymond, Philippe

    2016-04-01

    Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence, expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Cross talk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating this phenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of the JA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domain transcriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negative regulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYC protein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JA cross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation of SA/JA antagonism depending on the biological context in which the SA pathway is activated. © 2016 American Society of Plant Biologists. All Rights Reserved.

  3. Arabidopsis MYC Transcription Factors Are the Target of Hormonal Salicylic Acid/Jasmonic Acid Cross Talk in Response to Pieris brassicae Egg Extract1[OPEN

    Science.gov (United States)

    Schmiesing, André; Gouhier-Darimont, Caroline

    2016-01-01

    Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence, expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Cross talk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating this phenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of the JA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domain transcriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negative regulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYC protein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JA cross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation of SA/JA antagonism depending on the biological context in which the SA pathway is activated. PMID:26884488

  4. EWS/FLI mediates transcriptional repression via NKX2.2 during oncogenic transformation in Ewing's sarcoma.

    Directory of Open Access Journals (Sweden)

    Leah A Owen

    2008-04-01

    Full Text Available EWS/FLI is a master regulator of Ewing's sarcoma formation. Gene expression studies in A673 Ewing's sarcoma cells have demonstrated that EWS/FLI downregulates more genes than it upregulates, suggesting that EWS/FLI, and/or its targets, function as transcriptional repressors. One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature. We now report that microarray analysis demonstrated that the transcriptional profile of NKX2.2 consists solely of downregulated genes, and overlaps with the EWS/FLI downregulated signature, suggesting that NKX2.2 mediates oncogenic transformation via transcriptional repression. Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable. Furthermore, blockade of TLE or HDAC function, two protein families thought to mediate the repressive function of NKX2.2, inhibited the transformed phenotype and reversed the NKX2.2 transcriptional profile in Ewing's sarcoma cells. Whole genome localization studies (ChIP-chip revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding. These data demonstrate that the transcriptional repressive function of NKX2.2 is necessary, and sufficient, for the oncogenic phenotype of Ewing's sarcoma, and suggest a therapeutic approach to this disease.

  5. Abscisic acid represses the transcription of chloroplast genes

    Czech Academy of Sciences Publication Activity Database

    Yamburenko, M.V.; Zubo, Y.O.; Vaňková, Radomíra; Kusnetsov, V.; Kulaeva, O.N.; Borner, T.

    2013-01-01

    Roč. 64, č. 14 (2013), s. 4491-4502 ISSN 0022-0957 R&D Projects: GA ČR GA522/09/2058 Institutional research plan: CEZ:AV0Z50380511 Keywords : Abscisic acid (ABA) * chloroplast * cytokinin Subject RIV: ED - Physiology Impact factor: 5.794, year: 2013

  6. Analysis of the Varicella-Zoster Virus IE62 N-Terminal Acidic Transactivating Domain and Its Interaction with the Human Mediator Complex▿

    OpenAIRE

    Yamamoto, Shinobu; Eletsky, Alexander; Szyperski, Thomas; Hay, John; Ruyechan, William T.

    2009-01-01

    The varicella-zoster virus major transactivator, IE62, contains a potent N-terminal acidic transcriptional activation domain (TAD). Our experiments revealed that the minimal IE62 TAD encompasses amino acids (aa) 19 to 67. We showed that the minimal TAD interacts with the human Mediator complex. Site-specific mutations revealed residues throughout the minimal TAD that are important for both activation and Mediator interaction. The TAD interacts directly with aa 402 to 590 of the MED25 subunit,...

  7. Abscisic-acid-dependent basic leucine zipper (bZIP) transcription factors in plant abiotic stress.

    Science.gov (United States)

    Banerjee, Aditya; Roychoudhury, Aryadeep

    2017-01-01

    One of the major causes of significant crop loss throughout the world is the myriad of environmental stresses including drought, salinity, cold, heavy metal toxicity, and ultraviolet-B (UV-B) rays. Plants as sessile organisms have evolved various effective mechanism which enable them to withstand this plethora of stresses. Most of such regulatory mechanisms usually follow the abscisic-acid (ABA)-dependent pathway. In this review, we have primarily focussed on the basic leucine zipper (bZIP) transcription factors (TFs) activated by the ABA-mediated signalosome. Upon perception of ABA by specialized receptors, the signal is transduced via various groups of Ser/Thr kinases, which phosphorylate the bZIP TFs. Following such post-translational modification of TFs, they are activated so that they bind to specific cis-acting sequences called abscisic-acid-responsive elements (ABREs) or GC-rich coupling elements (CE), thereby influencing the expression of their target downstream genes. Several in silico techniques have been adopted so far to predict the structural features, recognize the regulatory modification sites, undergo phylogenetic analyses, and facilitate genome-wide survey of TF under multiple stresses. Current investigations on the epigenetic regulation that controls greater accessibility of the inducible regions of DNA of the target gene to the bZIP TFs exclusively under stress situations, along with the evolved stress memory responses via genomic imprinting mechanism, have been highlighted. The potentiality of overexpression of bZIP TFs, either in a homologous or in a heterologous background, in generating transgenic plants tolerant to various abiotic stressors have also been addressed by various groups. The present review will provide a coherent documentation on the functional characterization and regulation of bZIP TFs under multiple environmental stresses, with the major goal of generating multiple-stress-tolerant plant cultivars in near future.

  8. RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Smialowska, Agata; Djupedal, Ingela; Wang, Jingwen; Kylsten, Per; Swoboda, Peter; Ekwall, Karl

    2014-01-01

    Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe

  9. Transcription factor CREB is involved in CaSR-mediated cytoskeleton gene expression.

    Science.gov (United States)

    Huang, Shuaishuai; Ren, Yu; Wang, Ping; Li, Yanyuan; Wang, Xue; Zhuang, Haihui; Fang, Rong; Wang, Yuduo; Liu, Ningsheng; Hehir, Michael; Zhou, Jeff X

    2015-03-01

    Our previous studies illustrated that a steady increase of intracellular calcium concentration ([Ca2+]i) was important for maintaining microtubules (MTs) rearrangement in apoptotic cells. However, little is known about the effect of calcium sensing receptor (CaSR)-mediated increase in [Ca2+]i on cytoskeleton gene expression. We examined the impact of taxol or CaSR agonist/antagonist on the regulation of [Ca2+]i concentration, cytoskeleton arrangement, phosphorylated CREB and cytoskeleton gene expressions in HeLa cells with dominant negative plasmid of CREB (PM). This study demonstrated that Gdcl3 (a specific CaSR agonist) evoked a rapid increase of [Ca2+]i, formed a rigid bundle of MTs which surrounded the nucleus and decreased the cytoskeleton gene expressions in HeLa cells. These effects were rescued by addition of NPS2390 (a specific CaSR antagonist). Moreover, CaSR activity affected cytoskeleton gene expression through transcription factor CREB. Histoscores of pCREB immunoreactivity in tissues of cervical adenocarcinoma, renal clear cell carcinoma, and diffuse large B-cell lymphoma were markedly increased compared with non malignant tissue. These data demonstrate, for the first time, that CaSR-mediated increase in [Ca2+]i probably modulate cytoskeleton organization and gene expression via transcription factor. © 2014 Wiley Periodicals, Inc.

  10. Ultradian hormone stimulation induces glucocorticoid receptor-mediated pulses of gene transcription.

    Science.gov (United States)

    Stavreva, Diana A; Wiench, Malgorzata; John, Sam; Conway-Campbell, Becky L; McKenna, Mervyn A; Pooley, John R; Johnson, Thomas A; Voss, Ty C; Lightman, Stafford L; Hager, Gordon L

    2009-09-01

    Studies on glucocorticoid receptor (GR) action typically assess gene responses by long-term stimulation with synthetic hormones. As corticosteroids are released from adrenal glands in a circadian and high-frequency (ultradian) mode, such treatments may not provide an accurate assessment of physiological hormone action. Here we demonstrate that ultradian hormone stimulation induces cyclic GR-mediated transcriptional regulation, or gene pulsing, both in cultured cells and in animal models. Equilibrium receptor-occupancy of regulatory elements precisely tracks the ligand pulses. Nascent RNA transcripts from GR-regulated genes are released in distinct quanta, demonstrating a profound difference between the transcriptional programs induced by ultradian and constant stimulation. Gene pulsing is driven by rapid GR exchange with response elements and by GR recycling through the chaperone machinery, which promotes GR activation and reactivation in response to the ultradian hormone release, thus coupling promoter activity to the naturally occurring fluctuations in hormone levels. The GR signalling pathway has been optimized for a prompt and timely response to fluctuations in hormone levels, indicating that biologically accurate regulation of gene targets by GR requires an ultradian mode of hormone stimulation.

  11. Repression of HNF1α-mediated transcription by amino-terminal enhancer of split (AES)

    Energy Technology Data Exchange (ETDEWEB)

    Han, Eun Hee [Section of Structural Biology, Hormel Institute, University of Minnesota, Austin, MN 55912 (United States); Gorman, Amanda A. [Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536 (United States); Singh, Puja [Section of Structural Biology, Hormel Institute, University of Minnesota, Austin, MN 55912 (United States); Chi, Young-In, E-mail: ychi@hi.umn.edu [Section of Structural Biology, Hormel Institute, University of Minnesota, Austin, MN 55912 (United States)

    2015-12-04

    HNF1α (Hepatocyte Nuclear Factor 1α) is one of the master regulators in pancreatic beta-cell development and function, and the mutations in Hnf1α are the most common monogenic causes of diabetes mellitus. As a member of the POU transcription factor family, HNF1α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge in their functional complex formation. In this study, we identified the Groucho protein AES (Amino-terminal Enhancer of Split) as a HNF1α-specific physical binding partner and functional repressor of HNF1α-mediated transcription, which has a direct link to glucose-stimulated insulin secretion in beta-cells that is impaired in the HNF1α mutation-driven diabetes. - Highlights: • We identified AES as a transcriptional repressor for HNF1α in pancreatic beta-cell. • AES's repressive activity was HNF1α-specific and was not observed with HNF1β. • AES interacts with the transactivation domain of HNF1α. • Small molecules can be designed or discovered to disrupt this interaction and improve insulin secretion and glucose homeostasis.

  12. Repression of HNF1α-mediated transcription by amino-terminal enhancer of split (AES)

    International Nuclear Information System (INIS)

    Han, Eun Hee; Gorman, Amanda A.; Singh, Puja; Chi, Young-In

    2015-01-01

    HNF1α (Hepatocyte Nuclear Factor 1α) is one of the master regulators in pancreatic beta-cell development and function, and the mutations in Hnf1α are the most common monogenic causes of diabetes mellitus. As a member of the POU transcription factor family, HNF1α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge in their functional complex formation. In this study, we identified the Groucho protein AES (Amino-terminal Enhancer of Split) as a HNF1α-specific physical binding partner and functional repressor of HNF1α-mediated transcription, which has a direct link to glucose-stimulated insulin secretion in beta-cells that is impaired in the HNF1α mutation-driven diabetes. - Highlights: • We identified AES as a transcriptional repressor for HNF1α in pancreatic beta-cell. • AES's repressive activity was HNF1α-specific and was not observed with HNF1β. • AES interacts with the transactivation domain of HNF1α. • Small molecules can be designed or discovered to disrupt this interaction and improve insulin secretion and glucose homeostasis.

  13. Snail recruits Ring1B to mediate transcriptional repression and cell migration in pancreatic cancer cells.

    Science.gov (United States)

    Chen, Jiangzhi; Xu, Hong; Zou, Xiuqun; Wang, Jiamin; Zhu, Yi; Chen, Hao; Shen, Baiyong; Deng, Xiaxing; Zhou, Aiwu; Chin, Y Eugene; Rauscher, Frank J; Peng, Chenghong; Hou, Zhaoyuan

    2014-08-15

    Transcriptional repressor Snail is a master regulator of epithelial-mesenchymal transition (EMT), yet the epigenetic mechanism governing Snail to induce EMT is not well understood. Here, we report that in pancreatic ductal adenocarcinoma (PDAC), elevated levels of the ubiquitin E3 ligase Ring1B and Snail, along with elevated monoubiquitination of H2A at K119 (H2AK119Ub1), are highly correlated with poor survival. Mechanistic investigations identified Ring1B as a Snail-interacting protein and showed that the carboxyl zinc fingers of Snail recruit Ring1B and its paralog Ring1A to repress its target promoters. Simultaneous depletion of Ring1A and Ring1B in pancreatic cancer cells decreased Snail binding to the target chromatin, abolished H2AK119Ub1 modification, and thereby compromised Snail-mediated transcriptional repression and cell migration. We found that Ring1B and the SNAG-associated chromatin modifier EZH2 formed distinct protein complexes with Snail and that EZH2 was required for Snail-Ring1A/B recruitment to the target promoter. Collectively, our results unravel an epigenetic mechanism underlying transcriptional repression by Snail, suggest Ring1A/B as a candidate therapeutic target, and identify H2AK119Ub1 as a potential biomarker for PDAC diagnosis and prognosis. ©2014 American Association for Cancer Research.

  14. Identification of a transcription factor controlling pH-dependent organic acid response in Aspergillus niger.

    Directory of Open Access Journals (Sweden)

    Lars Poulsen

    Full Text Available Acid formation in Aspergillus niger is known to be subjected to tight regulation, and the acid production profiles are fine-tuned to respond to the ambient pH. Based on transcriptome data, putative trans-acting pH responding transcription factors were listed and through knock out studies, mutants exhibiting an oxalate overproducing phenotype were identified. The yield of oxalate was increased up to 158% compared to the wild type and the corresponding transcription factor was therefore entitled Oxalic Acid repression Factor, OafA. Detailed physiological characterization of one of the ΔoafA mutants, compared to the wild type, showed that both strains produced substantial amounts of gluconic acid, but the mutant strain was more efficient in re-uptake of gluconic acid and converting it to oxalic acid, particularly at high pH (pH 5.0. Transcriptional profiles showed that 241 genes were differentially expressed due to the deletion of oafA and this supported the argument of OafA being a trans-acting transcription factor. Furthermore, expression of two phosphoketolases was down-regulated in the ΔoafA mutant, one of which has not previously been described in fungi. It was argued that the observed oxalate overproducing phenotype was a consequence of the efficient re-uptake of gluconic acid and thereby a higher flux through glycolysis. This results in a lower flux through the pentose phosphate pathway, demonstrated by the down-regulation of the phosphoketolases. Finally, the physiological data, in terms of the specific oxygen consumption, indicated a connection between the oxidative phosphorylation and oxalate production and this was further substantiated through transcription analysis.

  15. Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation.

    Science.gov (United States)

    Banik, Mitali; Duguid, Scott; Cloutier, Sylvie

    2011-06-01

    Three genes encoding fatty acid desaturase 3 (fad3a, fad3b, and a novel fad3c) were cloned from four flax genotypes varying in linolenic acid content. Real-time PCR was used to quantify expression levels of the three fad3 genes during seed development. High amounts of both fad3a and fad3b transcripts were observed and reached their peak levels at 20 days after anthesis, except for fad3a from SP2047 where only low level expression was observed throughout seed development. Transcript accumulation of the novel fad3c gene was at similar background levels. The fatty acid composition was analysed for all genotypes and stages of development and compared with the fad3 gene expression patterns. α-Linolenic acid gradually accumulated during seed development, while linoleic acid was transient and decreased in M5791, UGG5-5, and AC McDuff. In contrast, the linolenic acid present in the early stages of development nearly completely disappeared in SP2047, while linoleic acid steadily accumulated. fad3a of the low linolenic acid line SP2047 encoded a truncated protein caused by a premature stop codon resulting from a single point mutation, and the low level of transcript accumulation in this genotype is likely due to nonsense-mediated mRNA decay caused by the premature termination of translation as a result of this early stop codon. Although substantial amounts of transcript accumulation occurred with fad3b of SP2047 genotype, cloning of the gene revealed a mutation in the first histidine box causing an amino acid change. Heterologous expression in yeast of the SP2047 and UGG5-5 fad3b genes showed that the mutation in the histidine box in SP2047 caused the enzyme inactivity. Taken together, these results showed that fad3a and fad3b are responsible for linolenic acid accumulation in flax seeds but did not support a major role for the novel fad3c. These observations were further supported by phenotypic and genotypic assessment of a doubled haploid population. Expression patterns

  16. Salicylic Acid Suppresses Jasmonic Acid Signaling Downstream of SCFCOI1-JAZ by Targeting GCC Promoter Motifs via Transcription Factor ORA59[C][W][OA

    Science.gov (United States)

    Van der Does, Dieuwertje; Leon-Reyes, Antonio; Koornneef, Annemart; Van Verk, Marcel C.; Rodenburg, Nicole; Pauwels, Laurens; Goossens, Alain; Körbes, Ana P.; Memelink, Johan; Ritsema, Tita; Van Wees, Saskia C.M.; Pieterse, Corné M.J.

    2013-01-01

    Antagonism between the defense hormones salicylic acid (SA) and jasmonic acid (JA) plays a central role in the modulation of the plant immune signaling network, but the molecular mechanisms underlying this phenomenon are largely unknown. Here, we demonstrate that suppression of the JA pathway by SA functions downstream of the E3 ubiquitin-ligase Skip-Cullin-F-box complex SCFCOI1, which targets JASMONATE ZIM-domain transcriptional repressor proteins (JAZs) for proteasome-mediated degradation. In addition, neither the stability nor the JA-induced degradation of JAZs was affected by SA. In silico promoter analysis of the SA/JA crosstalk transcriptome revealed that the 1-kb promoter regions of JA-responsive genes that are suppressed by SA are significantly enriched in the JA-responsive GCC-box motifs. Using GCC:GUS lines carrying four copies of the GCC-box fused to the β-glucuronidase reporter gene, we showed that the GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Using plants overexpressing the GCC-box binding APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors ERF1 or ORA59, we found that SA strongly reduces the accumulation of ORA59 but not that of ERF1. Collectively, these data indicate that the SA pathway inhibits JA signaling downstream of the SCFCOI1-JAZ complex by targeting GCC-box motifs in JA-responsive promoters via a negative effect on the transcriptional activator ORA59. PMID:23435661

  17. Salicylic acid suppresses jasmonic acid signaling downstream of SCFCOI1-JAZ by targeting GCC promoter motifs via transcription factor ORA59.

    Science.gov (United States)

    Van der Does, Dieuwertje; Leon-Reyes, Antonio; Koornneef, Annemart; Van Verk, Marcel C; Rodenburg, Nicole; Pauwels, Laurens; Goossens, Alain; Körbes, Ana P; Memelink, Johan; Ritsema, Tita; Van Wees, Saskia C M; Pieterse, Corné M J

    2013-02-01

    Antagonism between the defense hormones salicylic acid (SA) and jasmonic acid (JA) plays a central role in the modulation of the plant immune signaling network, but the molecular mechanisms underlying this phenomenon are largely unknown. Here, we demonstrate that suppression of the JA pathway by SA functions downstream of the E3 ubiquitin-ligase Skip-Cullin-F-box complex SCF(COI1), which targets JASMONATE ZIM-domain transcriptional repressor proteins (JAZs) for proteasome-mediated degradation. In addition, neither the stability nor the JA-induced degradation of JAZs was affected by SA. In silico promoter analysis of the SA/JA crosstalk transcriptome revealed that the 1-kb promoter regions of JA-responsive genes that are suppressed by SA are significantly enriched in the JA-responsive GCC-box motifs. Using GCC:GUS lines carrying four copies of the GCC-box fused to the β-glucuronidase reporter gene, we showed that the GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Using plants overexpressing the GCC-box binding APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors ERF1 or ORA59, we found that SA strongly reduces the accumulation of ORA59 but not that of ERF1. Collectively, these data indicate that the SA pathway inhibits JA signaling downstream of the SCF(COI1)-JAZ complex by targeting GCC-box motifs in JA-responsive promoters via a negative effect on the transcriptional activator ORA59.

  18. Mediator MED23 Links Pigmentation and DNA Repair through the Transcription Factor MITF.

    Science.gov (United States)

    Xia, Min; Chen, Kun; Yao, Xiao; Xu, Yichi; Yao, Jiaying; Yan, Jun; Shao, Zhen; Wang, Gang

    2017-08-22

    DNA repair is related to many physiological and pathological processes, including pigmentation. Little is known about the role of the transcriptional cofactor Mediator complex in DNA repair and pigmentation. Here, we demonstrate that Mediator MED23 plays an important role in coupling UV-induced DNA repair to pigmentation. The loss of Med23 specifically impairs the pigmentation process in melanocyte-lineage cells and in zebrafish. Med23 deficiency leads to enhanced nucleotide excision repair (NER) and less DNA damage following UV radiation because of the enhanced expression and recruitment of NER factors to chromatin for genomic stability. Integrative analyses of melanoma cells reveal that MED23 controls the expression of a melanocyte master regulator, Mitf, by modulating its distal enhancer activity, leading to opposing effects on pigmentation and DNA repair. Collectively, the Mediator MED23/MITF axis connects DNA repair to pigmentation, thus providing molecular insights into the DNA damage response and skin-related diseases. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. Basic aspects of tumor cell fatty acid-regulated signaling and transcription factors.

    Science.gov (United States)

    Comba, Andrea; Lin, Yi-Hui; Eynard, Aldo Renato; Valentich, Mirta Ana; Fernandez-Zapico, Martín Ernesto; Pasqualini, Marìa Eugenia

    2011-12-01

    This article reviews the current knowledge and experimental research about the mechanisms by which fatty acids and their derivatives control specific gene expression involved during carcinogenesis. Changes in dietary fatty acids, specifically the polyunsaturated fatty acids of the ω-3 and ω-6 families and some derived eicosanoids from lipoxygenases, cyclooxygenases, and cytochrome P-450, seem to control the activity of transcription factor families involved in cancer cell proliferation or cell death. Their regulation may be carried out either through direct binding to DNA as peroxisome proliferator-activated receptors or via modulation in an indirect manner of signaling pathway molecules (e.g., protein kinase C) and other transcription factors (nuclear factor kappa B and sterol regulatory element binding protein). Knowledge of the mechanisms by which fatty acids control specific gene expression may identify important risk factors for cancer and provide insight into the development of new therapeutic strategies for a better management of whole body lipid metabolism.

  20. Nuclear import of transcription factor BR-C is mediated by its interaction with RACK1.

    Science.gov (United States)

    Cheng, Daojun; Qian, Wenliang; Wang, Yonghu; Meng, Meng; Wei, Ling; Li, Zhiqing; Kang, Lixia; Peng, Jian; Xia, Qingyou

    2014-01-01

    The transcription factor Broad Complex (BR-C) is an early ecdysone response gene in insects and contains two types of domains: two zinc finger domains for the activation of gene transcription and a Bric-a-brac/Tramtrack/Broad complex (BTB) domain for protein-protein interaction. Although the mechanism of zinc finger-mediated gene transcription is well studied, the partners interacting with the BTB domain of BR-C has not been elucidated until now. Here, we performed a yeast two-hybrid screen using the BTB domain of silkworm BR-C as bait and identified the receptor for activated C-kinase 1 (RACK1), a scaffolding/anchoring protein, as the novel partner capable of interacting with BR-C. The interaction between BR-C and RACK1 was further confirmed by far-western blotting and pull-down assays. Importantly, the disruption of this interaction, via RNAi against the endogenous RACK1 gene or deletion of the BTB domain, abolished the nuclear import of BR-C in BmN4 cells. In addition, RNAi against the endogenous PKC gene as well as phosphorylation-deficient mutation of the predicted PKC phosphorylation sites at either Ser373 or Thr406 in BR-C phenocopied RACK1 RNAi and altered the nuclear localization of BR-C. However, when BTB domain was deleted, phosphorylation mimics of either Ser373 or Thr406 had no effect on the nuclear import of BR-C. Moreover, mutating the PKC phosphorylation sites at Ser373 and Thr406 or deleting the BTB domain significantly decreased the transcriptional activation of a BR-C target gene. Given that RACK1 is necessary for recruiting PKC to close and phosphorylate target proteins, we suggest that the PKC-mediated phosphorylation and nuclear import of BR-C is determined by its interaction with RACK1. This novel finding will be helpful for further deciphering the mechanism underlying the role of BR-C proteins during insect development.

  1. Nuclear import of transcription factor BR-C is mediated by its interaction with RACK1.

    Directory of Open Access Journals (Sweden)

    Daojun Cheng

    Full Text Available The transcription factor Broad Complex (BR-C is an early ecdysone response gene in insects and contains two types of domains: two zinc finger domains for the activation of gene transcription and a Bric-a-brac/Tramtrack/Broad complex (BTB domain for protein-protein interaction. Although the mechanism of zinc finger-mediated gene transcription is well studied, the partners interacting with the BTB domain of BR-C has not been elucidated until now. Here, we performed a yeast two-hybrid screen using the BTB domain of silkworm BR-C as bait and identified the receptor for activated C-kinase 1 (RACK1, a scaffolding/anchoring protein, as the novel partner capable of interacting with BR-C. The interaction between BR-C and RACK1 was further confirmed by far-western blotting and pull-down assays. Importantly, the disruption of this interaction, via RNAi against the endogenous RACK1 gene or deletion of the BTB domain, abolished the nuclear import of BR-C in BmN4 cells. In addition, RNAi against the endogenous PKC gene as well as phosphorylation-deficient mutation of the predicted PKC phosphorylation sites at either Ser373 or Thr406 in BR-C phenocopied RACK1 RNAi and altered the nuclear localization of BR-C. However, when BTB domain was deleted, phosphorylation mimics of either Ser373 or Thr406 had no effect on the nuclear import of BR-C. Moreover, mutating the PKC phosphorylation sites at Ser373 and Thr406 or deleting the BTB domain significantly decreased the transcriptional activation of a BR-C target gene. Given that RACK1 is necessary for recruiting PKC to close and phosphorylate target proteins, we suggest that the PKC-mediated phosphorylation and nuclear import of BR-C is determined by its interaction with RACK1. This novel finding will be helpful for further deciphering the mechanism underlying the role of BR-C proteins during insect development.

  2. Fatty Acids, Lipid Mediators, and T-Cell Function

    Science.gov (United States)

    de Jong, Anja J.; Kloppenburg, Margreet; Toes, René E. M.; Ioan-Facsinay, Andreea

    2014-01-01

    Research toward the mechanisms underlying obesity-linked complications has intensified during the last years. As a consequence, it has become clear that metabolism and immunity are intimately linked. Free fatty acids and other lipids acquired in excess by current feeding patterns have been proposed to mediate this link due to their immune modulatory capacity. The functional differences between saturated and unsaturated fatty acids, in combination with their dietary intake are believed to modulate the outcome of immune responses. Moreover, unsaturated fatty acids can be oxidized in a tightly regulated and specific manner to generate either potent pro-inflammatory or pro-resolving lipid mediators. These oxidative derivatives of fatty acids have received detailed attention during the last years, as they have proven to have strong immune modulatory capacity, even in pM ranges. Both fatty acids and oxidized fatty acids have been studied especially in relation to macrophage and T-cells functions. In this review, we propose to focus on the effect of fatty acids and their oxidative derivatives on T-cells, as it is an active area of research during the past 5 years. The effect of fatty acids and their derivatives on activation and proliferation of T-cells, as well as the delicate balance between stimulation and lipotoxicity will be discussed. Moreover, the receptors involved in the interaction between free fatty acids and their derivatives with T-cells will be summarized. Finally, the mechanisms involved in modulation of T-cells by fatty acids will be addressed, including cellular signaling and metabolism of T-cells. The in vitro results will be placed in context of in vivo studies both in humans and mice. In this review, we summarize the latest findings on the immune modulatory function of lipids on T-cells and will point out novel directions for future research. PMID:25352844

  3. Novel Hematopoietic Target Genes in the NRF2-Mediated Transcriptional Pathway

    Directory of Open Access Journals (Sweden)

    Michelle R. Campbell

    2013-01-01

    Full Text Available Nuclear factor- (erythroid-derived 2 like 2 (NFE2L2, NRF2 is a key transcriptional activator of the antioxidant response pathway and is closely related to erythroid transcription factor NFE2. Under oxidative stress, NRF2 heterodimerizes with small Maf proteins and binds cis-acting enhancer sequences found near oxidative stress response genes. Using the dietary isothiocyanate sulforaphane (SFN to activate NRF2, chromatin immunoprecipitation sequencing (ChIP-seq identified several hundred novel NRF2-mediated targets beyond its role in oxidative stress. Activated NRF2 bound the antioxidant response element (ARE in promoters of several known and novel target genes involved in iron homeostasis and heme metabolism, including known targets FTL and FTH1, as well as novel binding in the globin locus control region. Five novel NRF2 target genes were chosen for followup: AMBP, ABCB6, FECH, HRG-1 (SLC48A1, and TBXAS1. SFN-induced gene expression in erythroid K562 and lymphoid cells were compared for each target gene. NRF2 silencing showed reduced expression in lymphoid, lung, and hepatic cells. Furthermore, stable knockdown of NRF2 negative regulator KEAP1 in K562 cells resulted in increased NQO1, AMBP, and TBXAS1 expression. NFE2 binding sites in K562 cells revealed similar binding profiles as lymphoid NRF2 sites in all potential NRF2 candidates supporting a role for NRF2 in heme metabolism and erythropoiesis.

  4. DNA replication factor C1 mediates genomic stability and transcriptional gene silencing in Arabidopsis

    KAUST Repository

    Liu, Qian; Wang, Junguo; Miki, Daisuke; Xia, Ran; Yu, Wenxiang; He, Junna; Zheng, Zhimin; Zhu, Jian-Kang; Gonga, Zhizhong

    2010-01-01

    Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylationindependent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana. © 2010 American Society of Plant Biologists.

  5. DNA replication factor C1 mediates genomic stability and transcriptional gene silencing in Arabidopsis

    KAUST Repository

    Liu, Qian

    2010-07-01

    Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylationindependent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana. © 2010 American Society of Plant Biologists.

  6. Role of cocaine- and amphetamine-regulated transcript in estradiol-mediated neuroprotection

    Science.gov (United States)

    Xu, Yun; Zhang, Wenri; Klaus, Judith; Young, Jennifer; Koerner, Ines; Sheldahl, Laird C.; Hurn, Patricia D.; Martínez-Murillo, Francisco; Alkayed, Nabil J.

    2006-09-01

    Estrogen reduces brain injury after experimental cerebral ischemia in part through a genomic mechanism of action. Using DNA microarrays, we analyzed the genomic response of the brain to estradiol, and we identified a transcript, cocaine- and amphetamine-regulated transcript (CART), that is highly induced in the cerebral cortex by estradiol under ischemic conditions. Using in vitro and in vivo models of neural injury, we confirmed and characterized CART mRNA and protein up-regulation by estradiol in surviving neurons, and we demonstrated that i.v. administration of a rat CART peptide is protective against ischemic brain injury in vivo. We further demonstrated binding of cAMP response element (CRE)-binding protein to a CART promoter CRE site in ischemic brain and rapid activation by CART of ERK in primary cultured cortical neurons. The findings suggest that CART is an important player in estrogen-mediated neuroprotection and a potential therapeutic agent for stroke and other neurodegenerative diseases. ischemia | stroke | estrogen

  7. Physiological and transcriptional responses and cross protection of Lactobacillus plantarum ZDY2013 under acid stress.

    Science.gov (United States)

    Huang, Renhui; Pan, Mingfang; Wan, Cuixiang; Shah, Nagendra P; Tao, Xueying; Wei, Hua

    2016-02-01

    Acid tolerance responses (ATR) in Lactobacillus plantarum ZDY2013 were investigated at physiological and molecular levels. A comparison of composition of cell membrane fatty acids (CMFA) between acid-challenged and unchallenged cells showed that acid adaptation evoked a significantly higher percentage of saturated fatty acids and cyclopropane fatty acids in acid-challenged than in unchallenged cells. In addition, reverse transcription-quantitative PCR analysis in acid-adapted cells at different pH values (ranging from 3.0 to 4.0) indicated that several genes were differently regulated, including those related to proton pumps, amino acid metabolism, sugar metabolism, and class I and class III stress response pathways. Expression of genes involved in fatty acid synthesis and production of alkali was significantly upregulated. Upon exposure to pH 4.5 for 2 h, a higher survival rate (higher viable cell count) of Lactobacillus plantarum ZDY2013 was achieved following an additional challenge to 40 mM hydrogen peroxide for 60 min, but no difference in survival rate of cells was found with further challenge to heat, ethanol, or salt. Therefore, we concluded that the physiological and metabolic changes of acid-treated cells of Lactobacillus plantarum ZDY2013 help the cells resist damage caused by acid, and further initiated global response signals to bring the whole cell into a state of defense to other stress factors, especially hydrogen peroxide. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  8. E-cadherin is transcriptionally activated via suppression of ZEB1 transcriptional repressor by small RNA-mediated gene silencing.

    Directory of Open Access Journals (Sweden)

    Minami Mazda

    Full Text Available RNA activation has been reported to be induced by small interfering RNAs (siRNAs that act on the promoters of several genes containing E-cadherin. In this study, we present an alternative mechanism of E-cadherin activation in human PC-3 cells by siRNAs previously reported to possess perfect-complementary sequences to E-cadherin promoter. We found that activation of E-cadherin can be also induced via suppression of ZEB1, which is a transcriptional repressor of E-cadherin, by seed-dependent silencing mechanism of these siRNAs. The functional seed-complementary sites of the siRNAs were found in the coding region in addition to the 3' untranslated region of ZEB1 mRNA. Promoter analyses indicated that E-boxes, which are ZEB1-binding sites, in the upstream promoter region are indispensable for E-cadherin transcription by the siRNAs. Thus, the results caution against ignoring siRNA seed-dependent silencing effects in genome-wide transcriptional regulation. In addition, members of miR-302/372/373/520 family, which have the same seed sequences with one of the siRNAs containing perfect-complementarity to E-cadherin promoter, are also found to activate E-cadherin transcription. Thus, E-cadherin could be upregulated by the suppression of ZEB1 transcriptional repressor by miRNAs in vivo.

  9. TALEN-mediated targeted mutagenesis of fatty acid desaturase 2 (FAD2) in peanut (Arachis hypogaea L.) promotes the accumulation of oleic acid.

    Science.gov (United States)

    Wen, Shijie; Liu, Hao; Li, Xingyu; Chen, Xiaoping; Hong, Yanbin; Li, Haifen; Lu, Qing; Liang, Xuanqiang

    2018-05-01

    A first creation of high oleic acid peanut varieties by using transcription activator-like effecter nucleases (TALENs) mediated targeted mutagenesis of Fatty Acid Desaturase 2 (FAD2). Transcription activator like effector nucleases (TALENs), which allow the precise editing of DNA, have already been developed and applied for genome engineering in diverse organisms. However, they are scarcely used in higher plant study and crop improvement, especially in allopolyploid plants. In the present study, we aimed to create targeted mutagenesis by TALENs in peanut. Targeted mutations in the conserved coding sequence of Arachis hypogaea fatty acid desaturase 2 (AhFAD2) were created by TALENs. Genetic stability of AhFAD2 mutations was identified by DNA sequencing in up to 9.52 and 4.11% of the regeneration plants at two different targeted sites, respectively. Mutation frequencies among AhFAD2 mutant lines were significantly correlated to oleic acid accumulation. Genetically, stable individuals of positive mutant lines displayed a 0.5-2 fold increase in the oleic acid content compared with non-transgenic controls. This finding suggested that TALEN-mediated targeted mutagenesis could increase the oleic acid content in edible peanut oil. Furthermore, this was the first report on peanut genome editing event, and the obtained high oleic mutants could serve for peanut breeding project.

  10. Endoplasmic reticulum stress-responsive transcription factor ATF6α directs recruitment of the Mediator of RNA polymerase II transcription and multiple histone acetyltransferase complexes.

    Science.gov (United States)

    Sela, Dotan; Chen, Lu; Martin-Brown, Skylar; Washburn, Michael P; Florens, Laurence; Conaway, Joan Weliky; Conaway, Ronald C

    2012-06-29

    The basic leucine zipper transcription factor ATF6α functions as a master regulator of endoplasmic reticulum (ER) stress response genes. Previous studies have established that, in response to ER stress, ATF6α translocates to the nucleus and activates transcription of ER stress response genes upon binding sequence specifically to ER stress response enhancer elements in their promoters. In this study, we investigate the biochemical mechanism by which ATF6α activates transcription. By exploiting a combination of biochemical and multidimensional protein identification technology-based mass spectrometry approaches, we have obtained evidence that ATF6α functions at least in part by recruiting to the ER stress response enhancer elements of ER stress response genes a collection of RNA polymerase II coregulatory complexes, including the Mediator and multiple histone acetyltransferase complexes, among which are the Spt-Ada-Gcn5 acetyltransferase (SAGA) and Ada-Two-A-containing (ATAC) complexes. Our findings shed new light on the mechanism of action of ATF6α, and they outline a straightforward strategy for applying multidimensional protein identification technology mass spectrometry to determine which RNA polymerase II transcription factors and coregulators are recruited to promoters and other regulatory elements to control transcription.

  11. Upregulation of the coagulation factor VII gene during glucose deprivation is mediated by activating transcription factor 4.

    Directory of Open Access Journals (Sweden)

    Katherine R Cronin

    Full Text Available BACKGROUND: Constitutive production of blood coagulation proteins by hepatocytes is necessary for hemostasis. Stressful conditions trigger adaptive cellular responses and delay processing of most proteins, potentially affecting plasma levels of proteins secreted exclusively by hepatocytes. We examined the effect of glucose deprivation on expression of coagulation proteins by the human hepatoma cell line, HepG2. METHODOLOGY/PRINCIPAL FINDINGS: Expression of coagulation factor VII, which is required for initiation of blood coagulation, was elevated by glucose deprivation, while expression of other coagulation proteins decreased. Realtime PCR and ELISA demonstrated that the relative percentage expression +/- SD of steady-state F7 mRNA and secreted factor VII antigen were significantly increased (from 100+/-15% to 188+/-27% and 100+/-8.8% to 176.3+/-17.3% respectively, p<0.001 at 24 hr of treatment. The integrated stress response was induced, as indicated by upregulation of transcription factor ATF4 and of additional stress-responsive genes. Small interfering RNAs directed against ATF4 potently reduced basal F7 expression, and prevented F7 upregulation by glucose deprivation. The response of the endogenous F7 gene was replicated in reporter gene assays, which further indicated that ATF4 effects were mediated via interaction with an amino acid response element in the F7 promoter. CONCLUSIONS/SIGNIFICANCE: Our data indicated that glucose deprivation enhanced F7 expression in a mechanism reliant on prior ATF4 upregulation primarily due to increased transcription from the ATF4 gene. Of five coagulation protein genes examined, only F7 was upregulated, suggesting that its functions may be important in a systemic response to glucose deprivation stress.

  12. Detection of Enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification

    OpenAIRE

    D Wang; X Wang; Y Geng; C An

    2014-01-01

    Purpose : The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD) for an early treatment by using loop-mediated isothermal amplification (LAMP) technique. Materials and Methods : A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for detecting EV71 virus was developed, the specificity and sensitivity of RT-LAMP was tested, and the clinical specimens was assayed by the RT-LAMP comparing with conven...

  13. Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo.

    Science.gov (United States)

    Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

    2017-07-12

    The Mediator complex has been described as a general transcription factor, but it is unclear if it is essential for Pol II transcription and/or is a required component of the preinitiation complex (PIC) in vivo. Here, we show that depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in transcription. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in transcription. Depletion of head or middle subunits, but not tail subunits, causes a downstream shift in the Pol II occupancy profile, suggesting that Mediator at the core promoter inhibits promoter escape. Interestingly, a functional PIC and Pol II transcription can occur when Mediator is not detected at core promoters. These results provide strong evidence that Mediator is essential for Pol II transcription and stimulates PIC formation, but it is not a required component of the PIC in vivo.

  14. Transcription factor YY1 can control AID-mediated mutagenesis in mice.

    Science.gov (United States)

    Zaprazna, Kristina; Basu, Arindam; Tom, Nikola; Jha, Vibha; Hodawadekar, Suchita; Radova, Lenka; Malcikova, Jitka; Tichy, Boris; Pospisilova, Sarka; Atchison, Michael L

    2018-02-01

    Activation-induced cytidine deminase (AID) is crucial for controlling the immunoglobulin (Ig) diversification processes of somatic hypermutation (SHM) and class switch recombination (CSR). AID initiates these processes by deamination of cytosine, ultimately resulting in mutations or double strand DNA breaks needed for SHM and CSR. Levels of AID control mutation rates, and off-target non-Ig gene mutations can contribute to lymphomagenesis. Therefore, factors that control AID levels in the nucleus can regulate SHM and CSR, and may contribute to disease. We previously showed that transcription factor YY1 can regulate the level of AID in the nucleus and Ig CSR. Therefore, we hypothesized that conditional knock-out of YY1 would lead to reduction in AID localization at the Ig locus, and reduced AID-mediated mutations. Using mice that overexpress AID (IgκAID yy1 f/f ) or that express normal AID levels (yy1 f/f ), we found that conditional knock-out of YY1 results in reduced AID nuclear levels, reduced localization of AID to the Sμ switch region, and reduced AID-mediated mutations. We find that the mechanism of YY1 control of AID nuclear accumulation is likely due to YY1-AID physical interaction which blocks AID ubiquitination. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. HosA, a MarR Family Transcriptional Regulator, Represses Nonoxidative Hydroxyarylic Acid Decarboxylase Operon and Is Modulated by 4-Hydroxybenzoic Acid.

    Science.gov (United States)

    Roy, Ajit; Ranjan, Akash

    2016-02-23

    Members of the Multiple antibiotic resistance Regulator (MarR) family of DNA binding proteins regulate transcription of a wide array of genes required for virulence and pathogenicity of bacteria. The present study reports the molecular characterization of HosA (Homologue of SlyA), a MarR protein, with respect to its target gene, DNA recognition motif, and nature of its ligand. Through a comparative genomics approach, we demonstrate that hosA is in synteny with nonoxidative hydroxyarylic acid decarboxylase (HAD) operon and is present exclusively within the mutS-rpoS polymorphic region in nine different genera of Enterobacteriaceae family. Using molecular biology and biochemical approach, we demonstrate that HosA binds to a palindromic sequence downstream to the transcription start site of divergently transcribed nonoxidative HAD operon and represses its expression. Furthermore, in silico analysis showed that the recognition motif for HosA is highly conserved in the upstream region of divergently transcribed operon in different genera of Enterobacteriaceae family. A systematic chemical search for the physiological ligand revealed that 4-hydroxybenzoic acid (4-HBA) interacts with HosA and derepresses HosA mediated repression of the nonoxidative HAD operon. Based on our study, we propose a model for molecular mechanism underlying the regulation of nonoxidative HAD operon by HosA in Enterobacteriaceae family.

  16. CFLAP1 and CFLAP2 Are Two bHLH Transcription Factors Participating in Synergistic Regulation of AtCFL1-Mediated Cuticle Development in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Shibai Li

    2016-01-01

    Full Text Available The cuticle is a hydrophobic lipid layer covering the epidermal cells of terrestrial plants. Although many genes involved in Arabidopsis cuticle development have been identified, the transcriptional regulation of these genes is largely unknown. Previously, we demonstrated that AtCFL1 negatively regulates cuticle development by interacting with the HD-ZIP IV transcription factor HDG1. Here, we report that two bHLH transcription factors, AtCFL1 associated protein 1 (CFLAP1 and CFLAP2, are also involved in AtCFL1-mediated regulation of cuticle development. CFLAP1 and CFLAP2 interact with AtCFL1 both in vitro and in vivo. Overexpression of either CFLAP1 or CFLAP2 led to expressional changes of genes involved in fatty acids, cutin and wax biosynthesis pathways and caused multiple cuticle defective phenotypes such as organ fusion, breakage of the cuticle layer and decreased epicuticular wax crystal loading. Functional inactivation of CFLAP1 and CFLAP2 by chimeric repression technology caused opposite phenotypes to the CFLAP1 overexpressor plants. Interestingly, we find that, similar to the transcription factor HDG1, the function of CFLAP1 in cuticle development is dependent on the presence of AtCFL1. Furthermore, both HDG1 and CFLAP1/2 interact with the same C-terminal C4 zinc finger domain of AtCFL1, a domain that is essential for AtCFL1 function. These results suggest that AtCFL1 may serve as a master regulator in the transcriptional regulation of cuticle development, and that CFLAP1 and CFLAP2 are involved in the AtCFL1-mediated regulation pathway, probably through competing with HDG1 to bind to AtCFL1.

  17. Inhibition of hepatitis B virus (HBV) by LNA-mediated nuclear interference with HBV DNA transcription

    International Nuclear Information System (INIS)

    Sun, Zhen; Xiang, Wenqing; Guo, Yajuan; Chen, Zhi; Liu, Wei; Lu, Daru

    2011-01-01

    Highlights: → LNA-modified oligonucleotides can pass through the plasma membrane of cultured cells even without using transfection machinery. → LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. → LNA-oligonucleotide designed to target nuclear HBV DNA efficiently suppresses HBV replication and transcription in cultured hepatic cells. -- Abstract: Silencing target genes with small regulatory RNAs is widely used to investigate gene function and therapeutic drug development. Recently, triplex-based approaches have provided another attractive means to achieve targeted gene regulation and gene manipulation at the molecular and cellular levels. Nuclear entry of oligonucleotides and enhancement of their affinity to the DNA targets are key points of such approaches. In this study, we developed lipid-based transport of a locked-nucleic-acid (LNA)-modified oligonucleotide for hepatitis B virus (HBV) DNA interference in human hepatocytes expressing HBV genomic DNA. In these cells, the LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. The oligonucleotide specifically targeting HBV DNA clearly interfered with HBV DNA transcription as shown by a block in pregenomic RNA (pgRNA) production. The HBV DNA-targeted oligonucleotide suppressed HBV DNA replication and HBV protein production more efficiently than small interfering RNAs directed to the pgRNA. These results demonstrate that fusion with lipid can carry LNA-modified oligonucleotides to the nucleus where they regulate gene expression. Interfering with HBV DNA transcription by LNA-modified oligonucleotides has strong potential as a new strategy for HBV inhibition.

  18. Inhibition of hepatitis B virus (HBV) by LNA-mediated nuclear interference with HBV DNA transcription

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Zhen [The State Key Laboratory of Genetic Engineering and The MOE Key Laboratory of Contemporary Anthropology, School of Life Science, Fudan University, Shanghai 200433 (China); Department of Biochemistry and Molecular Biology, Program in Molecular Cell Biology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058 (China); Xiang, Wenqing; Guo, Yajuan [Department of Biochemistry and Molecular Biology, Program in Molecular Cell Biology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058 (China); Chen, Zhi [The State Key Laboratory for Infectious Disease, Institute of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003 (China); Liu, Wei, E-mail: liuwei666@zju.edu.cn [Department of Biochemistry and Molecular Biology, Program in Molecular Cell Biology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058 (China); Lu, Daru, E-mail: drlu@fudan.edu.cn [The State Key Laboratory of Genetic Engineering and The MOE Key Laboratory of Contemporary Anthropology, School of Life Science, Fudan University, Shanghai 200433 (China)

    2011-06-10

    Highlights: {yields} LNA-modified oligonucleotides can pass through the plasma membrane of cultured cells even without using transfection machinery. {yields} LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. {yields} LNA-oligonucleotide designed to target nuclear HBV DNA efficiently suppresses HBV replication and transcription in cultured hepatic cells. -- Abstract: Silencing target genes with small regulatory RNAs is widely used to investigate gene function and therapeutic drug development. Recently, triplex-based approaches have provided another attractive means to achieve targeted gene regulation and gene manipulation at the molecular and cellular levels. Nuclear entry of oligonucleotides and enhancement of their affinity to the DNA targets are key points of such approaches. In this study, we developed lipid-based transport of a locked-nucleic-acid (LNA)-modified oligonucleotide for hepatitis B virus (HBV) DNA interference in human hepatocytes expressing HBV genomic DNA. In these cells, the LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. The oligonucleotide specifically targeting HBV DNA clearly interfered with HBV DNA transcription as shown by a block in pregenomic RNA (pgRNA) production. The HBV DNA-targeted oligonucleotide suppressed HBV DNA replication and HBV protein production more efficiently than small interfering RNAs directed to the pgRNA. These results demonstrate that fusion with lipid can carry LNA-modified oligonucleotides to the nucleus where they regulate gene expression. Interfering with HBV DNA transcription by LNA-modified oligonucleotides has strong potential as a new strategy for HBV inhibition.

  19. Epigenetic mediated transcriptional activation of WNT5A participates in arsenical-associated malignant transformation

    International Nuclear Information System (INIS)

    Jensen, Taylor J.; Wozniak, Ryan J.; Eblin, Kylee E.; Wnek, Sean M.; Gandolfi, A. Jay; Futscher, Bernard W.

    2009-01-01

    Arsenic is a human carcinogen with exposure associated with cancer of the lung, skin, and bladder. Many potential mechanisms have been implicated as playing a role in the process of arsenical-induced malignancy including the perturbation of signaling pathways and aberrant epigenetic regulation. We initiated studies to examine the role of a member of the non-canonical WNT signaling pathway, WNT5A, in UROtsa cells and arsenite [URO-ASSC] and monomethylarsonous acid [URO-MSC] malignantly transformed variants. We present data herein that suggest that WNT5A is transcriptionally activated during arsenical-induced malignant transformation. This WNT5A transcriptional activation is correlated with the enrichment of permissive histone modifications and the reduction of repressive modifications in the WNT5A promoter region. The epigenetic activation of WNT5A expression and acetylation of its promoter remain after the removal of the arsenical, consistent with the maintenance of an anchorage independent growth phenotype in these cells. Additionally, treatment with epigenetic modifying drugs supports a functional role for these epigenetic marks in controlling gene expression. Reduction of WNT5A using lentiviral shRNA greatly attenuated the ability of these cells to grow in an anchorage independent fashion. Extension of our model into human bladder cancer cell lines indicates that each of the cell lines examined also express WNT5A. Taken together, these data suggest that the epigenetic remodeling of the WNT5A promoter is correlated with its transcriptional activation and this upregulation likely participates in arsenical-induced malignant transformation

  20. Transcriptional and metabolomic analysis of Ascophyllum nodosum mediated freezing tolerance in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Nair Prasanth

    2012-11-01

    Full Text Available Abstract Background We have previously shown that lipophilic components (LPC of the brown seaweed Ascophyllum nodosum (ANE improved freezing tolerance in Arabidopsis thaliana. However, the mechanism(s of this induced freezing stress tolerance is largely unknown. Here, we investigated LPC induced changes in the transcriptome and metabolome of A. thaliana undergoing freezing stress. Results Gene expression studies revealed that the accumulation of proline was mediated by an increase in the expression of the proline synthesis genes P5CS1 and P5CS2 and a marginal reduction in the expression of the proline dehydrogenase (ProDH gene. Moreover, LPC application significantly increased the concentration of total soluble sugars in the cytosol in response to freezing stress. Arabidopsis sfr4 mutant plants, defective in the accumulation of free sugars, treated with LPC, exhibited freezing sensitivity similar to that of untreated controls. The 1H NMR metabolite profile of LPC-treated Arabidopsis plants exposed to freezing stress revealed a spectrum dominated by chemical shifts (δ representing soluble sugars, sugar alcohols, organic acids and lipophilic components like fatty acids, as compared to control plants. Additionally, 2D NMR spectra suggested an increase in the degree of unsaturation of fatty acids in LPC treated plants under freezing stress. These results were supported by global transcriptome analysis. Transcriptome analysis revealed that LPC treatment altered the expression of 1113 genes (5% in comparison with untreated plants. A total of 463 genes (2% were up regulated while 650 genes (3% were down regulated. Conclusion Taken together, the results of the experiments presented in this paper provide evidence to support LPC mediated freezing tolerance enhancement through a combination of the priming of plants for the increased accumulation of osmoprotectants and alteration of cellular fatty acid composition.

  1. Pnrc2 regulates 3'UTR-mediated decay of segmentation clock-associated transcripts during zebrafish segmentation.

    Science.gov (United States)

    Gallagher, Thomas L; Tietz, Kiel T; Morrow, Zachary T; McCammon, Jasmine M; Goldrich, Michael L; Derr, Nicolas L; Amacher, Sharon L

    2017-09-01

    Vertebrate segmentation is controlled by the segmentation clock, a molecular oscillator that regulates gene expression and cycles rapidly. The expression of many genes oscillates during segmentation, including hairy/Enhancer of split-related (her or Hes) genes, which encode transcriptional repressors that auto-inhibit their own expression, and deltaC (dlc), which encodes a Notch ligand. We previously identified the tortuga (tor) locus in a zebrafish forward genetic screen for genes involved in cyclic transcript regulation and showed that cyclic transcripts accumulate post-splicing in tor mutants. Here we show that cyclic mRNA accumulation in tor mutants is due to loss of pnrc2, which encodes a proline-rich nuclear receptor co-activator implicated in mRNA decay. Using an inducible in vivo reporter system to analyze transcript stability, we find that the her1 3'UTR confers Pnrc2-dependent instability to a heterologous transcript. her1 mRNA decay is Dicer-independent and likely employs a Pnrc2-Upf1-containing mRNA decay complex. Surprisingly, despite accumulation of cyclic transcripts in pnrc2-deficient embryos, we find that cyclic protein is expressed normally. Overall, we show that Pnrc2 promotes 3'UTR-mediated decay of developmentally-regulated segmentation clock transcripts and we uncover an additional post-transcriptional regulatory layer that ensures oscillatory protein expression in the absence of cyclic mRNA decay. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Upregulation of the coagulation factor VII gene during glucose deprivation is mediated by activating transcription factor 4.

    Science.gov (United States)

    Cronin, Katherine R; Mangan, Thomas P; Carew, Josephine A

    2012-01-01

    Constitutive production of blood coagulation proteins by hepatocytes is necessary for hemostasis. Stressful conditions trigger adaptive cellular responses and delay processing of most proteins, potentially affecting plasma levels of proteins secreted exclusively by hepatocytes. We examined the effect of glucose deprivation on expression of coagulation proteins by the human hepatoma cell line, HepG2. Expression of coagulation factor VII, which is required for initiation of blood coagulation, was elevated by glucose deprivation, while expression of other coagulation proteins decreased. Realtime PCR and ELISA demonstrated that the relative percentage expression +/- SD of steady-state F7 mRNA and secreted factor VII antigen were significantly increased (from 100+/-15% to 188+/-27% and 100+/-8.8% to 176.3+/-17.3% respectively, pfactor ATF4 and of additional stress-responsive genes. Small interfering RNAs directed against ATF4 potently reduced basal F7 expression, and prevented F7 upregulation by glucose deprivation. The response of the endogenous F7 gene was replicated in reporter gene assays, which further indicated that ATF4 effects were mediated via interaction with an amino acid response element in the F7 promoter. Our data indicated that glucose deprivation enhanced F7 expression in a mechanism reliant on prior ATF4 upregulation primarily due to increased transcription from the ATF4 gene. Of five coagulation protein genes examined, only F7 was upregulated, suggesting that its functions may be important in a systemic response to glucose deprivation stress.

  3. HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Inae; Hur, Jung; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

    2015-01-30

    Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells.

  4. A pp32-retinoblastoma protein complex modulates androgen receptor-mediated transcription and associates with components of the splicing machinery

    International Nuclear Information System (INIS)

    Adegbola, Onikepe; Pasternack, Gary R.

    2005-01-01

    We have previously shown pp32 and the retinoblastoma protein interact. pp32 and the retinoblastoma protein are nuclear receptor transcriptional coregulators: the retinoblastoma protein is a coactivator for androgen receptor, the major regulator of prostate cancer growth, while pp32, which is highly expressed in prostate cancer, is a corepressor of the estrogen receptor. We now show pp32 increases androgen receptor-mediated transcription and the retinoblastoma protein modulates this activity. Using affinity purification and mass spectrometry, we identify members of the pp32-retinoblastoma protein complex as PSF and nonO/p54nrb, proteins implicated in coordinate regulation of nuclear receptor-mediated transcription and splicing. We show that the pp32-retinoblastoma protein complex is modulated during TPA-induced K562 differentiation. Present evidence suggests that nuclear receptors assemble multiprotein complexes to coordinately regulate transcription and mRNA processing. Our results suggest that pp32 and the retinoblastoma protein may be part of a multiprotein complex that coordinately regulates nuclear receptor-mediated transcription and mRNA processing

  5. HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

    International Nuclear Information System (INIS)

    Kim, Inae; Hur, Jung; Jeong, Sunjoo

    2015-01-01

    Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells

  6. Wnt-mediated down-regulation of Sp1 target genes by a transcriptional repressor Sp5

    Czech Academy of Sciences Publication Activity Database

    Fujimura, Naoko; Vacík, Tomáš; Machoň, Ondřej; Vlček, Čestmír; Scalabrin, S.; Speth, M.; Diep, D.; Krauss, S.; Kozmik, Zbyněk

    2007-01-01

    Roč. 282, č. 2 (2007), s. 1225-1237 ISSN 0021-9258 Institutional research plan: CEZ:AV0Z50520514 Keywords : Wnt -mediated signaling * Sp5 transcription factor * Sp1 target genes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.581, year: 2007

  7. Functional Genomic investigation of Peroxisome Proliferator-Activated Receptor Gamma (PPARG mediated transcription response in gastric cancer

    Directory of Open Access Journals (Sweden)

    Karthikeyan Selvarasu

    2017-10-01

    Full Text Available Cancer is a complex and progressive multi-step disorder that results from the transformation of normal cells to malignant derivatives. Several oncogenic signaling pathways are involved in this transformation. PPARG (Peroxisome proliferator-activated receptor gamma mediated transcription and signaling is involved in few cancers. We have investigated the PPARG in gastric tumors. The objective of the present study was to investigate the PPARG mediated transcriptional response in gastric tumors. Gene-set based and pathway focused gene-set enrichment analysis of available PPARG signatures in gastric tumor mRNA profiles shows that PPARG mediated transcription is highly activated in intestinal sub-type of gastric tumors. Further, we have derived the PPARG associated genes in gastric cancer and their expression was identified for the association with the better survival of the patients. Analysis of the PPARG associated genes reveals their involvement in mitotic cell cycle process, chromosome organization and nuclear division. Towards identifying the association with other oncogenic signaling process, E2F regulated genes were found associated with PPARG mediated transcription. The current results reveal the possible stratification of gastric tumors based on the PPARG gene expression and the possible development of PPARG targeted gastric cancer therapeutics. The identified PPARG regulated genes were identified to be targetable by pioglitazone and rosiglitazone. The identification of PPARG genes also in the normal stomach tissues reveal the possible involvement of these genes in the normal physiology of stomach and needs to be investigated.

  8. Mediator, SWI/SNF and SAGA complexes regulate Yap8-dependent transcriptional activation of ACR2 in response to arsenate.

    Science.gov (United States)

    Menezes, Regina Andrade; Pimentel, Catarina; Silva, Ana Rita Courelas; Amaral, Catarina; Merhej, Jawad; Devaux, Frédéric; Rodrigues-Pousada, Claudina

    2017-04-01

    Response to arsenic stress in Saccharomyces cerevisiae is orchestrated by the regulatory protein Yap8, which mediates transcriptional activation of ACR2 and ACR3. This study contributes to the state of art knowledge of the molecular mechanisms underlying yeast stress response to arsenate as it provides the genetic and biochemical evidences that Yap8, through cysteine residues 132, 137, and 274, is the sensor of presence of arsenate in the cytosol. Moreover, it is here reported for the first time the essential role of the Mediator complex in the transcriptional activation of ACR2 by Yap8. Based on our data, we propose an order-of-function map to recapitulate the sequence of events taking place in cells injured with arsenate. Modification of the sulfhydryl state of these cysteines converts Yap8 in its activated form, triggering the recruitment of the Mediator complex to the ACR2/ACR3 promoter, through the interaction with the tail subunit Med2. The Mediator complex then transfers the regulatory signals conveyed by Yap8 to the core transcriptional machinery, which culminates with TBP occupancy, ACR2 upregulation and cell adaptation to arsenate stress. Additional co-factors are required for the transcriptional activation of ACR2 by Yap8, particularly the nucleosome remodeling activity of SWI/SNF and SAGA complexes. Copyright © 2017. Published by Elsevier B.V.

  9. Transcription Profiles Reveal Sugar and Hormone Signaling Pathways Mediating Flower Induction in Apple (Malus domestica Borkh.).

    Science.gov (United States)

    Xing, Li-Bo; Zhang, Dong; Li, You-Mei; Shen, Ya-Wen; Zhao, Cai-Ping; Ma, Juan-Juan; An, Na; Han, Ming-Yu

    2015-10-01

    Flower induction in apple (Malus domestica Borkh.) is regulated by complex gene networks that involve multiple signal pathways to ensure flower bud formation in the next year, but the molecular determinants of apple flower induction are still unknown. In this research, transcriptomic profiles from differentiating buds allowed us to identify genes potentially involved in signaling pathways that mediate the regulatory mechanisms of flower induction. A hypothetical model for this regulatory mechanism was obtained by analysis of the available transcriptomic data, suggesting that sugar-, hormone- and flowering-related genes, as well as those involved in cell-cycle induction, participated in the apple flower induction process. Sugar levels and metabolism-related gene expression profiles revealed that sucrose is the initiation signal in flower induction. Complex hormone regulatory networks involved in cytokinin (CK), abscisic acid (ABA) and gibberellic acid pathways also induce apple flower formation. CK plays a key role in the regulation of cell formation and differentiation, and in affecting flowering-related gene expression levels during these processes. Meanwhile, ABA levels and ABA-related gene expression levels gradually increased, as did those of sugar metabolism-related genes, in developing buds, indicating that ABA signals regulate apple flower induction by participating in the sugar-mediated flowering pathway. Furthermore, changes in sugar and starch deposition levels in buds can be affected by ABA content and the expression of the genes involved in the ABA signaling pathway. Thus, multiple pathways, which are mainly mediated by crosstalk between sugar and hormone signals, regulate the molecular network involved in bud growth and flower induction in apple trees. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  10. Transcriptional Response to Lactic Acid Stress in the Hybrid Yeast Zygosaccharomyces parabailii.

    Science.gov (United States)

    Ortiz-Merino, Raúl A; Kuanyshev, Nurzhan; Byrne, Kevin P; Varela, Javier A; Morrissey, John P; Porro, Danilo; Wolfe, Kenneth H; Branduardi, Paola

    2018-03-01

    Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH and a fermentative metabolism with a high growth rate. Here we used mRNA sequencing (RNA-seq) to analyze Z. parabailii 's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than under control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/Aft2, previously described as lactic acid responsive in Saccharomyces cerevisiae Furthermore, formate dehydrogenase ( FDH ) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii in comparison to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production. IMPORTANCE Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression under conditions of interest and relating gene expression patterns

  11. Gibberellic acid and cGMP-dependent transcriptional regulation in arabidopsis thaliana

    KAUST Repository

    Bastian, René

    2010-03-01

    An ever increasing amount of transcriptomic data and analysis tools provide novel insight into complex responses of biological systems. Given these resources we have undertaken to review aspects of transcriptional regulation in response to the plant hormone gibberellic acid (GA) and its second messenger guanosine 3\\',5\\'-cyclic monophosphate (cGMP) in Arabidopsis thaliana, both wild type and selected mutants. Evidence suggests enrichment of GA-responsive (GARE) elements in promoters of genes that are transcriptionally upregulated in response to cGMP but downregulated in a GA insensitive mutant (ga1-3). In contrast, in the genes upregulated in the mutant, no enrichment in the GARE is observed suggesting that GARE motifs are diagnostic for GA-induced and cGMP-dependent transcriptional upregulation. Further, we review how expression studies of GA-dependent transcription factors and transcriptional networks based on common promoter signatures derived from ab initio analyses can contribute to our understanding of plant responses at the systems level. © 2010 Landes Bioscience.

  12. Valproic acid disrupts the oscillatory expression of core circadian rhythm transcription factors.

    Science.gov (United States)

    Griggs, Chanel A; Malm, Scott W; Jaime-Frias, Rosa; Smith, Catharine L

    2018-01-15

    Valproic acid (VPA) is a well-established therapeutic used in treatment of seizure and mood disorders as well as migraines and a known hepatotoxicant. About 50% of VPA users experience metabolic disruptions, including weight gain, hyperlipidemia, and hyperinsulinemia, among others. Several of these metabolic abnormalities are similar to the effects of circadian rhythm disruption. In the current study, we examine the effect of VPA exposure on the expression of core circadian transcription factors that drive the circadian clock via a transcription-translation feedback loop. In cells with an unsynchronized clock, VPA simultaneously upregulated the expression of genes encoding core circadian transcription factors that regulate the positive and negative limbs of the feedback loop. Using low dose glucocorticoid, we synchronized cultured fibroblast cells to a circadian oscillatory pattern. Whether VPA was added at the time of synchronization or 12h later at CT12, we found that VPA disrupted the oscillatory expression of multiple genes encoding essential transcription factors that regulate circadian rhythm. Therefore, we conclude that VPA has a potent effect on the circadian rhythm transcription-translation feedback loop that may be linked to negative VPA side effects in humans. Furthermore, our study suggests potential chronopharmacology implications of VPA usage. Copyright © 2017. Published by Elsevier Inc.

  13. Mechanisms of bile acid mediated inflammation in the liver.

    Science.gov (United States)

    Li, Man; Cai, Shi-Ying; Boyer, James L

    2017-08-01

    Bile acids are synthesized in the liver and are the major component in bile. Impaired bile flow leads to cholestasis that is characterized by elevated levels of bile acid in the liver and serum, followed by hepatocyte and biliary injury. Although the causes of cholestasis have been extensively studied, the molecular mechanisms as to how bile acids initiate liver injury remain controversial. In this chapter, we summarize recent advances in the pathogenesis of bile acid induced liver injury. These include bile acid signaling pathways in hepatocytes as well as the response of cholangiocytes and innate immune cells in the liver in both patients with cholestasis and cholestatic animal models. We focus on how bile acids trigger the production of molecular mediators of neutrophil recruitment and the role of the inflammatory response in this pathological process. These advances point to a number of novel targets where drugs might be judged to be effective therapies for cholestatic liver injury. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system.

    Science.gov (United States)

    D'Souza, Alicia D; Belotserkovskii, Boris P; Hanawalt, Philip C

    2018-02-01

    The selective inhibition of transcription of a chosen gene by an artificial agent has numerous applications. Usually, these agents are designed to bind a specific nucleotide sequence in the promoter or within the transcribed region of the chosen gene. However, since optimal binding sites might not exist within the gene, it is of interest to explore the possibility of transcription inhibition when the agent is designed to bind at other locations. One of these possibilities arises when an additional transcription initiation site (e.g. secondary promoter) is present upstream from the primary promoter of the target gene. In this case, transcription inhibition might be achieved by inducing the formation of an RNA-DNA hybrid (R-loop) upon transcription from the secondary promoter. The R-loop could extend into the region of the primary promoter, to interfere with promoter recognition by RNA polymerase and thereby inhibit transcription. As a sequence-specific R-loop-inducing agent, a peptide nucleic acid (PNA) could be designed to facilitate R-loop formation by sequestering the non-template DNA strand. To investigate this mode for transcription inhibition, we have employed a model system in which a PNA binding site is localized between the T3 and T7 phage RNA polymerase promoters, which respectively assume the roles of primary and secondary promoters. In accord with our model, we have demonstrated that with PNA-bound DNA substrates, transcription from the T7 promoter reduces transcription from the T3 promoter by 30-fold, while in the absence of PNA binding there is no significant effect of T7 transcription upon T3 transcription. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.

    Directory of Open Access Journals (Sweden)

    Zach Hensel

    Full Text Available DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The "genetic switch" of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences O(R and O(L (separated by 2.3 kb, mediated by the λ repressor CI (accession number P03034, play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms.

  16. Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1

    OpenAIRE

    Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2006-01-01

    bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid subs...

  17. Transcriptional profiling of PBMCs unravels B cell mediated immunopathogenic imprints of HCV vasculitis.

    Science.gov (United States)

    Comstock, Emily; Kim, Cheol-Woo; Murphy, Alison; Emmanuel, Benjamin; Zhang, Xi; Sneller, Michael; Poonia, Bhawna; Kottilil, Shyamasundaran

    2017-01-01

    B cell depletion therapy using rituximab has been shown to be effective in achieving remission in patients with HCV-mixed cryoglobulinemic (MC) vasculitis. Previously, we have demonstrated abnormalities in peripheral immune cells involving neutrophils, chemotaxis, and innate immune activation among patients with HCV-MC vasculitis when compared to HCV patients without vasculitis. In this study, we evaluated the effect of B cell depletion therapy on transcriptional profiles of peripheral blood mononuclear cells before and after riruximab therapy, in order to unravel the pathogenic mechanism involved in HCV-MC vasculitis induced by abnormal B cell proliferation. DNA microarray analysis was performed using RNA from PBMCs from seven patients with HCV-MC vasculitis and seven normal volunteers. DNA was hybridized to Affymetrix U133A chips. After normalization, differentially expressed gene list with treatment was generated using partitional clustering. RT-PCR, flow cytometry, and enzyme immunoassay (EIA) was used to validate DNA microarray findings. Differentially expressed genes included B cells and non-B cell genes. Validation of genes using purified cell subsets demonstrated distinct effect of B cell depletion therapy on non-B cells, such as monocytes, T cells, and NK cells. Notably, B lymphocyte stimulator (BLyS) levels were persistently elevated in patients who subsequently relapsed. In conclusion, pathogenesis of HCV-MC vasculitis is mediated by abnormal proliferation of B cells, driven by BLyS, leading to significant effects on non-B cells in mediating symptomatology. Future therapeutics using a combination approach of B cell depletion and proliferation may be desired to achieve long-term remission.

  18. Nucleus accumbens cocaine-amphetamine regulated transcript mediates food intake during novelty conflict

    Science.gov (United States)

    Burghardt, PR; Krolewski, DM; Dykhuis, KE; Ching, J; Pinawin, AM; Britton, SL; Koch, LG; Watson, SJ; Akil, H.

    2016-01-01

    Obesity is a persistent and pervasive problem, particularly in industrialized nations. It has come to be appreciated that the metabolic health of an individual can influence brain function and subsequent behavioral patterns. To examine the relationship between metabolic phenotype and central systems that regulate behavior, we tested rats with divergent metabolic phenotypes (Low Capacity Runner: LCR vs. High Capacity Runner: HCR) for behavioral responses to the conflict between hunger and environmental novelty using the novelty suppressed feeding (NSF) paradigm. Additionally, we measured expression of mRNA, for peptides involved in energy management, in response to fasting. Following a 24-h fast, LCR rats showed lower latencies to begin eating in a novel environment compared to HCR rats. A 48-h fast equilibrated the latency to begin eating in the novel environment. A 24-h fast differentially affected expression of cocaine-amphetamine regulated transcript (CART) mRNA in the nucleus accumbens (NAc), where 24-h of fasting reduced CART mRNA in LCR rats. Bilateral microinjections of CART 55–102 peptide into the NAc increased the latency to begin eating in the NSF paradigm following a 24-h fast in LCR rats. These results indicate that metabolic phenotype influences how animals cope with the conflict between hunger and novelty, and that these differences are at least partially mediated by CART signaling in the NAc. For individuals with poor metabolic health who have to navigate food-rich and stressful environments, changes in central systems that mediate conflicting drives may feed into the rates of obesity and exacerbate the difficulty individuals have in maintaining weight loss. PMID:26926827

  19. A GntR-type transcriptional repressor controls sialic acid utilization in Bifidobacterium breve UCC2003.

    Science.gov (United States)

    Egan, Muireann; O'Connell Motherway, Mary; van Sinderen, Douwe

    2015-02-01

    Bifidobacterium breve strains are numerically prevalent among the gut microbiota of healthy, breast-fed infants. The metabolism of sialic acid, a ubiquitous monosaccharide in the infant and adult gut, by B. breve UCC2003 is dependent on a large gene cluster, designated the nan/nag cluster. This study describes the transcriptional regulation of the nan/nag cluster and thus sialic acid metabolism in B. breve UCC2003. Insertion mutagenesis and transcriptome analysis revealed that the nan/nag cluster is regulated by a GntR family transcriptional repressor, designated NanR. Crude cell extract of Escherichia coli EC101 in which the nanR gene had been cloned and overexpressed was shown to bind to two promoter regions within this cluster, each of which containing an imperfect inverted repeat that is believed to act as the NanR operator sequence. Formation of the DNA-NanR complex is prevented in the presence of sialic acid, which we had previously shown to induce transcription of this gene cluster. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells.

    Science.gov (United States)

    Zhang, Liyuan; Gu, Lingkun; Ringler, Patricia; Smith, Stanley; Rushton, Paul J; Shen, Qingxi J

    2015-07-01

    Members of the WRKY transcription factor superfamily are essential for the regulation of many plant pathways. Functional redundancy due to duplications of WRKY transcription factors, however, complicates genetic analysis by allowing single-mutant plants to maintain wild-type phenotypes. Our analyses indicate that three group I WRKY genes, OsWRKY24, -53, and -70, act in a partially redundant manner. All three showed characteristics of typical WRKY transcription factors: each localized to nuclei and yeast one-hybrid assays indicated that they all bind to W-boxes, including those present in their own promoters. Quantitative real time-PCR (qRT-PCR) analyses indicated that the expression levels of the three WRKY genes varied in the different tissues tested. Particle bombardment-mediated transient expression analyses indicated that all three genes repress the GA and ABA signaling in a dosage-dependent manner. Combination of all three WRKY genes showed additive antagonism of ABA and GA signaling. These results suggest that these WRKY proteins function as negative transcriptional regulators of GA and ABA signaling. However, different combinations of these WRKY genes can lead to varied strengths in suppression of their targets. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. The human tartrate-resistant acid phosphatase (TRAP): involvement of the hemin responsive elements (HRE) in transcriptional regulation.

    Science.gov (United States)

    Fleckenstein, E C; Dirks, W G; Drexler, H G

    2000-02-01

    The biochemical properties and protein structure of the tartrate-resistant acid phosphatase (TRAP), an iron-containing lysosomal glycoprotein in cells of the mononuclear phagocyte system, are well known. In contrast, little is known about the physiology and genic structure of this unique enzyme. In some diseases, like hairy cell leukemia, Gaucher's disease and osteoclastoma, cytochemically detected TRAP expression is used as a disease-associated marker. In order to begin to elucidate the regulation of this gene we generated different deletion constructs of the TRAP 5'-flanking region, placed them upstream of the luciferase reporter gene and assayed them for their ability to direct luciferase expression in human 293 cells. Treatment of these cells with the iron-modulating reagents transferrin and hemin causes opposite effects on the TRAP promoter activity. Two regulatory GAGGC tandem repeat sequences (the hemin responsive elements, HRE) within the 5'-flanking region of the human TRAP gene were identified. Studies with specific HRE-deletion constructs of the human TRAP 5'-flanking region upstream of the luciferase reporter gene document the functionality of these HRE-sequences which are apparently responsible for mediating transcriptional inhibition upon exposure to hemin. In addition to the previously published functional characterization of the murine TRAP HRE motifs, these results provide the first description of a new iron/hemin-responsive transcriptional regulation in the human TRAP gene.

  2. Trim24 (Tif1 alpha): an essential 'brake' for retinoic acid-induced transcription to prevent liver cancer.

    Science.gov (United States)

    Khetchoumian, Konstantin; Teletin, Marius; Tisserand, Johan; Herquel, Benjamin; Ouararhni, Khalid; Losson, Régine

    2008-12-01

    Retinoic acid (RA), the active derivative of vitamin A, is an important signaling molecule that controls various developmental processes and influence the proliferation and differentiation of a variety of cell types. RA exerts its biological functions primarily through binding to and activating nuclear RA receptors (RARs, which include the RAR alpha, beta and gamma isotypes RARA, RARB and RARC). Aberrant expression or impaired function of these nuclear receptors has been linked to diverse types of cancer. RARs are RA-dependent transcription factors that regulate gene expression through the recruitment of different co-regulators (co-activators and co-repressors). TRIM24 (formerly known as TIF1 alpha) was among the first co-regulators identified as interacting with RARs in a ligand-dependent fashion, and it was recently shown to function in mice as a potent liver-specific tumor suppressor by attenuating Rara-mediated transcription. The fact that Trim24(-/-), but not Trim24(-/-)Rara(+/-), mutant mice are highly predisposed to the development of hepatocellular carcinoma (HCC) has significant implications in cancer research. This result, along with the observation that in response to pharmacological inhibition of the RA signaling, hepatocytes lacking Trim24 loose their ability to proliferate, strongly implicates Rara as a proto-oncogene in hepatocytes and demonstrates that overactivated RA signaling is deleterious to liver homeostasis.

  3. Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination.

    Science.gov (United States)

    Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P; Raviram, Ramya; Schiavo, Ebe; Heyer, Vincent; Bonneau, Richard; Luo, Vincent M; Reddy, Janardan K; Borggrefe, Tilman; Skok, Jane A; Reina-San-Martin, Bernardo

    2016-03-07

    Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation. © 2016 Thomas-Claudepierre et al.

  4. Hierarchical mechanisms for transcription factor-mediated reprogramming of fibroblasts to neurons

    Science.gov (United States)

    Wapinski, Orly L.; Vierbuchen, Thomas; Qu, Kun; Lee, Qian Yi; Chanda, Soham; Fuentes, Daniel R.; Giresi, Paul G.; Ng, Yi Han; Marro, Samuele; Neff, Norma F.; Drechsel, Daniela; Martynoga, Ben; Castro, Diogo S.; Webb, Ashley E.; Brunet, Anne; Guillemot, Francois; Chang, Howard Y.; Wernig, Marius

    2013-01-01

    SUMMARY Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine with poorly understood mechanisms. Here we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an “on target” pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead Ascl1 recruits Brn2 to Ascl1 sites genome-wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, precise match between pioneer factor and the chromatin context at key target genes is determinative for trans-differentiation to neurons and likely other cell types. PMID:24243019

  5. Regulation of FOXO1-mediated transcription and cell proliferation by PARP-1

    Energy Technology Data Exchange (ETDEWEB)

    Sakamaki, Jun-ichi; Daitoku, Hiroaki; Yoshimochi, Kenji [Center for Tsukuba Advanced Research Alliance, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Miwa, Masanao [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829 (Japan); Fukamizu, Akiyoshi, E-mail: akif@tara.tsukuba.ac.jp [Center for Tsukuba Advanced Research Alliance, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan)

    2009-05-08

    Forkhead box O (FOXO) transcription factors play an important role in a wide range of biological processes, including cell cycle control, apoptosis, detoxification of reactive oxygen species, and gluconeogenesis through regulation of gene expression. In this study, we demonstrated that PARP-1 functions as a negative regulator of FOXO1. We showed that PARP-1 directly binds to and poly(ADP-ribosyl)ates FOXO1 protein. PARP-1 represses FOXO1-mediated expression of cell cycle inhibitor p27{sup Kip1} gene. Notably, poly(ADP-ribosyl)ation activity was not required for the repressive effect of PARP-1 on FOXO1 function. Furthermore, knockdown of PARP-1 led to a decrease in cell proliferation in a manner dependent on FOXO1 function. Chromatin immunoprecipitation experiments confirmed that PARP-1 is recruited to the p27{sup Kip1} gene promoter through a binding to FOXO1. These results suggest that PARP-1 acts as a corepressor for FOXO1, which could play an important role in proper cell proliferation by regulating p27{sup Kip1} gene expression.

  6. Novel RNA Duplex Locks HIV-1 in a Latent State via Chromatin-mediated Transcriptional Silencing

    Directory of Open Access Journals (Sweden)

    Chantelle Ahlenstiel

    2015-01-01

    Full Text Available Transcriptional gene silencing (TGS of mammalian genes can be induced by short interfering RNA (siRNA targeting promoter regions. We previously reported potent TGS of HIV-1 by siRNA (PromA, which targets tandem NF-κB motifs within the viral 5′LTR. In this study, we screened a siRNA panel with the aim of identifying novel 5′LTR targets, to provide multiplexing potential with enhanced viral silencing and application toward developing alternate therapeutic strategies. Systematic examination identified a novel siRNA target, si143, confirmed to induce TGS as the silencing mechanism. TGS was prolonged with virus suppression >12 days, despite a limited ability to induce post- TGS. Epigenetic changes associated with silencing were suggested by partial reversal by histone deacetylase inhibitors and confirmed by chromatin immunoprecipitation analyses, which showed induction of H3K27me3 and H3K9me3, reduction in H3K9Ac, and recruitment of argonaute-1, all characteristic marks of heterochromatin and TGS. Together, these epigenetic changes mimic those associated with HIV-1 latency. Further, robust resistance to reactivation was observed in the J-Lat 9.2 cell latency model, when transduced with shPromA and/or sh143. These data support si/shRNA-mediated TGS approaches to HIV-1 and provide alternate targets to pursue a functional cure, whereby the viral reservoir is locked in latency following antiretroviral therapy cessation.

  7. A downy mildew effector attenuates salicylic acid-triggered immunity in Arabidopsis by interacting with the host mediator complex.

    Directory of Open Access Journals (Sweden)

    Marie-Cécile Caillaud

    2013-12-01

    Full Text Available Plants are continually exposed to pathogen attack but usually remain healthy because they can activate defences upon perception of microbes. However, pathogens have evolved to overcome plant immunity by delivering effectors into the plant cell to attenuate defence, resulting in disease. Recent studies suggest that some effectors may manipulate host transcription, but the specific mechanisms by which such effectors promote susceptibility remain unclear. We study the oomycete downy mildew pathogen of Arabidopsis, Hyaloperonospora arabidopsidis (Hpa, and show here that the nuclear-localized effector HaRxL44 interacts with Mediator subunit 19a (MED19a, resulting in the degradation of MED19a in a proteasome-dependent manner. The Mediator complex of ∼25 proteins is broadly conserved in eukaryotes and mediates the interaction between transcriptional regulators and RNA polymerase II. We found MED19a to be a positive regulator of immunity against Hpa. Expression profiling experiments reveal transcriptional changes resembling jasmonic acid/ethylene (JA/ET signalling in the presence of HaRxL44, and also 3 d after infection with Hpa. Elevated JA/ET signalling is associated with a decrease in salicylic acid (SA-triggered immunity (SATI in Arabidopsis plants expressing HaRxL44 and in med19a loss-of-function mutants, whereas SATI is elevated in plants overexpressing MED19a. Using a PR1::GUS reporter, we discovered that Hpa suppresses PR1 expression specifically in cells containing haustoria, into which RxLR effectors are delivered, but not in nonhaustoriated adjacent cells, which show high PR1::GUS expression levels. Thus, HaRxL44 interferes with Mediator function by degrading MED19, shifting the balance of defence transcription from SA-responsive defence to JA/ET-signalling, and enhancing susceptibility to biotrophs by attenuating SA-dependent gene expression.

  8. Copper(I) mediated cross-coupling of amino acid derived organozinc reagents with acid chlorides

    DEFF Research Database (Denmark)

    Hjelmgaard, Thomas; Tanner, David Ackland

    2006-01-01

    This paper describes the development of a straightforward experimental protocol for copper-mediated cross-coupling of amino acid derived beta-amido-alkylzinc iodides 1 and 3 with a range of acid chlorides. The present method uses CuCN center dot 2LiCl as the copper source and for organozinc reagent...... 1 the methodology appears to be limited to reaction with more stable acid chlorides, providing the desired products in moderate yields. When applied to organozinc reagent 3, however, the protocol is more general and provides the products in good yields in all but one of the cases tested....

  9. Transcriptomic profiling-based mutant screen reveals three new transcription factors mediating menadione resistance in Neurospora crassa.

    Science.gov (United States)

    Zhu, Jufen; Yu, Xinxu; Xie, Baogui; Gu, Xiaokui; Zhang, Zhenying; Li, Shaojie

    2013-06-01

    To gain insight into the regulatory mechanisms of oxidative stress responses in filamentous fungi, the genome-wide transcriptional response of Neurospora crassa to menadione was analysed by digital gene expression (DGE) profiling, which identified 779 upregulated genes and 576 downregulated genes. Knockout mutants affecting 130 highly-upregulated genes were tested for menadione sensitivity, which revealed that loss of the transcription factor siderophore regulation (SRE) (a transcriptional repressor for siderophore biosynthesis), catatase-3, cytochrome c peroxidase or superoxide dismutase 1 copper chaperone causes hypersensitivity to menadione. Deletion of sre dramatically increased transcription of the siderophore biosynthesis gene ono and the siderophore iron transporter gene sit during menadione stress, suggesting that SRE is required for repression of iron uptake under oxidative stress conditions. Contrary to its phenotype, the sre deletion mutant showed higher transcriptional levels of genes encoding reactive oxygen species (ROS) scavengers than wild type during menadione stress, which implies that the mutant suffers a higher level of oxidative stress than wild type. Uncontrolled iron uptake in the sre mutant might exacerbate cellular oxidative stress. This is the first report of a negative regulator of iron assimilation participating in the fungal oxidative stress response. In addition to SRE, eight other transcription factor genes were also menadione-responsive but their single gene knockout mutants showed wild-type menadione sensitivity. Two of them, named as mit-2 (menadione induced transcription factor-2) and mit-4 (menadione induced transcription factor-4), were selected for double mutant analysis. The double mutant was hypersensitive to menadione. Similarly, the double mutation of mit-2 and sre also had additive effects on menadione sensitivity, suggesting multiple transcription factors mediate oxidative stress resistance in an additive manner

  10. Regulation of wheat seed dormancy by after-ripening is mediated by specific transcriptional switches that induce changes in seed hormone metabolism and signaling.

    Directory of Open Access Journals (Sweden)

    Aihua Liu

    Full Text Available Treatments that promote dormancy release are often correlated with changes in seed hormone content and/or sensitivity. To understand the molecular mechanisms underlying the role of after-ripening (seed dry storage in triggering hormone related changes and dormancy decay in wheat (Triticum aestivum, temporal expression patterns of genes related to abscisic acid (ABA, gibberellin (GA, jasmonate and indole acetic acid (IAA metabolism and signaling, and levels of the respective hormones were examined in dormant and after-ripened seeds in both dry and imbibed states. After-ripening mediated developmental switch from dormancy to germination appears to be associated with declines in seed sensitivity to ABA and IAA, which are mediated by transcriptional repressions of PROTEIN PHOSPHATASE 2C, SNF1-RELATED PROTEIN KINASE2, ABA INSENSITIVE5 and LIPID PHOSPHATE PHOSPHTASE2, and AUXIN RESPONSE FACTOR and RELATED TO UBIQUITIN1 genes. Transcriptomic analysis of wheat seed responsiveness to ABA suggests that ABA inhibits the germination of wheat seeds partly by repressing the transcription of genes related to chromatin assembly and cell wall modification, and activating that of GA catabolic genes. After-ripening induced seed dormancy decay in wheat is also associated with the modulation of seed IAA and jasmonate contents. Transcriptional control of members of the ALLENE OXIDE SYNTHASE, 3-KETOACYL COENZYME A THIOLASE, LIPOXYGENASE and 12-OXOPHYTODIENOATE REDUCTASE gene families appears to regulate seed jasmonate levels. Changes in the expression of GA biosynthesis genes, GA 20-OXIDASE and GA 3-OXIDASE, in response to after-ripening implicate this hormone in enhancing dormancy release and germination. These findings have important implications in the dissection of molecular mechanisms underlying regulation of seed dormancy in cereals.

  11. The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro.

    Science.gov (United States)

    Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

    2008-06-01

    The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44-61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.

  12. The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

    Science.gov (United States)

    Reilman, Ewoud; Mars, Ruben A T; van Dijl, Jan Maarten; Denham, Emma L

    2014-10-01

    Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Transcriptional regulatory programs underlying barley germination and regulatory functions of Gibberellin and abscisic acid

    Science.gov (United States)

    2011-01-01

    Background Seed germination is a complex multi-stage developmental process, and mainly accomplished through concerted activities of many gene products and biological pathways that are often subjected to strict developmental regulation. Gibberellins (GA) and abscisic acid (ABA) are two key phytohormones regulating seed germination and seedling growth. However, transcriptional regulatory networks underlying seed germination and its associated biological pathways are largely unknown. Results The studies examined transcriptomes of barley representing six distinct and well characterized germination stages and revealed that the transcriptional regulatory program underlying barley germination was composed of early, late, and post-germination phases. Each phase was accompanied with transcriptional up-regulation of distinct biological pathways. Cell wall synthesis and regulatory components including transcription factors, signaling and post-translational modification components were specifically and transiently up-regulated in early germination phase while histone families and many metabolic pathways were up-regulated in late germination phase. Photosynthesis and seed reserve mobilization pathways were up-regulated in post-germination phase. However, stress related pathways and seed storage proteins were suppressed through the entire course of germination. A set of genes were transiently up-regulated within three hours of imbibition, and might play roles in initiating biological pathways involved in seed germination. However, highly abundant transcripts in dry barley and Arabidopsis seeds were significantly conserved. Comparison with transcriptomes of barley aleurone in response to GA and ABA identified three sets of germination responsive genes that were regulated coordinately by GA, antagonistically by ABA, and coordinately by GA but antagonistically by ABA. Major CHO metabolism, cell wall degradation and protein degradation pathways were up-regulated by both GA and seed

  14. Transcriptional regulatory programs underlying barley germination and regulatory functions of Gibberellin and abscisic acid

    Directory of Open Access Journals (Sweden)

    Lin Li

    2011-06-01

    Full Text Available Abstract Background Seed germination is a complex multi-stage developmental process, and mainly accomplished through concerted activities of many gene products and biological pathways that are often subjected to strict developmental regulation. Gibberellins (GA and abscisic acid (ABA are two key phytohormones regulating seed germination and seedling growth. However, transcriptional regulatory networks underlying seed germination and its associated biological pathways are largely unknown. Results The studies examined transcriptomes of barley representing six distinct and well characterized germination stages and revealed that the transcriptional regulatory program underlying barley germination was composed of early, late, and post-germination phases. Each phase was accompanied with transcriptional up-regulation of distinct biological pathways. Cell wall synthesis and regulatory components including transcription factors, signaling and post-translational modification components were specifically and transiently up-regulated in early germination phase while histone families and many metabolic pathways were up-regulated in late germination phase. Photosynthesis and seed reserve mobilization pathways were up-regulated in post-germination phase. However, stress related pathways and seed storage proteins were suppressed through the entire course of germination. A set of genes were transiently up-regulated within three hours of imbibition, and might play roles in initiating biological pathways involved in seed germination. However, highly abundant transcripts in dry barley and Arabidopsis seeds were significantly conserved. Comparison with transcriptomes of barley aleurone in response to GA and ABA identified three sets of germination responsive genes that were regulated coordinately by GA, antagonistically by ABA, and coordinately by GA but antagonistically by ABA. Major CHO metabolism, cell wall degradation and protein degradation pathways were up

  15. GPBAR1/TGR5 mediates bile acid-induced cytokine expression in murine Kupffer cells.

    Directory of Open Access Journals (Sweden)

    Guiyu Lou

    Full Text Available GPBAR1/TGR5 is a novel plasma membrane-bound G protein-coupled bile acid (BA receptor. BAs are known to induce the expression of inflammatory cytokines in the liver with unknown mechanism. Here we show that without other external stimuli, TGR5 activation alone induced the expression of interleukin 1β (IL-1β and tumor necrosis factor-α (TNF-α in murine macrophage cell line RAW264.7 or murine Kupffer cells. The TGR5-mediated increase of pro-inflammatory cytokine expression was suppressed by JNK inhibition. Moreover, the induced pro-inflammatory cytokine expression in mouse liver by 1% cholic acid (CA diet was blunted in JNK-/- mice. TGR5 activation by its ligands enhanced the phosphorylation levels, DNA-binding and trans-activities of c-Jun and ATF2 transcription factors. Finally, the induced pro-inflammatory cytokine expression in Kupffer cells by TGR5 activation correlated with the suppression of Cholesterol 7α-hydroxylase (Cyp7a1 expression in murine hepatocytes. These results suggest that TGR5 mediates the BA-induced pro-inflammatory cytokine production in murine Kupffer cells through JNK-dependent pathway. This novel role of TGR5 may correlate to the suppression of Cyp7a1 expression in hepatocytes and contribute to the delicate BA feedback regulation.

  16. Acid-Mediated Tumor Proteolysis: Contribution of Cysteine Cathepsins

    Directory of Open Access Journals (Sweden)

    Jennifer M Rothberg

    2013-10-01

    Full Text Available One of the noncellular microenvironmental factors that contribute to malignancy of solid tumors is acidic peritumoral pH. We have previously demonstrated that extracellular acidosis leads to localization of the cysteine pro-tease cathepsin B on the tumor cell membrane and its secretion. The objective of the present study was to determine if an acidic extracellular pH such as that observed in vivo (i.e., pHe 6.8 affects the activity of proteases, e.g., cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional (3D cultures. For these studies, we used 1 3D reconstituted basement membrane overlay cultures of human carcinomas, 2 live cell imaging assays to assess proteolysis, and 3 in vivo imaging of active tumor proteases. At pHe 6.8, there were increases in pericellular active cysteine cathepsins and in degradation of dye-quenched collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of bound probe decreased in tumors in bicarbonate-treated mice, a treatment previously shown to increase peritumoral pHe and reduce local invasion of the tumors. Our results are consistent with the acid-mediated invasion hypothesis and with a role for cathepsin B in promoting degradation of a basement membrane protein substrate, i.e., type IV collagen, in an acidic peritumoral environment.

  17. Rapid detection of Prunus necrotic ringspot virus using magnetic nanoparticle-assisted reverse transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Zong, Xiaojuan; Wang, Wenwen; Wei, Hairong; Wang, Jiawei; Chen, Xin; Xu, Li; Zhu, Dongzi; Tan, Yue; Liu, Qingzhong

    2014-11-01

    Prunus necrotic ringspot virus (PNRSV) has seriously reduced the yield of Prunus species worldwide. In this study, a highly efficient and specific two-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect PNRSV. Total RNA was extracted from sweet cherry leaf samples using a commercial kit based on a magnetic nanoparticle technique. Transcripts were used as the templates for the assay. The results of this assay can be detected using agarose gel electrophoresis or by assessing in-tube fluorescence after adding SYBR Green I. The assay is highly specific for PNRSV, and it is more sensitive than reverse-transcription polymerase chain reaction (RT-PCR). Restriction enzyme digestion verified further the reliability of this RT-LAMP assay. To our knowledge, this is the first report of the application of RT-LAMP to PNRSV detection in Prunus species. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Lysophosphatidic acid as a lipid mediator with multiple biological actions.

    Science.gov (United States)

    Aikawa, Shizu; Hashimoto, Takafumi; Kano, Kuniyuki; Aoki, Junken

    2015-02-01

    Lysophosphatidic acid (LPA) is one of the simplest glycerophospholipids with one fatty acid chain and a phosphate group as a polar head. Although LPA had been viewed just as a metabolic intermediate in de novo lipid synthetic pathways, it has recently been paid much attention as a lipid mediator. LPA exerts many kinds of cellular processes, such as cell proliferation and smooth muscle contraction, through cognate G protein-coupled receptors. Because lipids are not coded by the genome directly, it is difficult to know their patho- and physiological roles. However, recent studies have identified several key factors mediating the biological roles of LPA, such as receptors and producing enzymes. In addition, studies of transgenic and gene knockout animals for these LPA-related genes, have revealed the biological significance of LPA. In this review we will summarize recent advances in the studies of LPA production and its roles in both physiological and pathological conditions. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  19. Hyperammonemia in cirrhosis induces transcriptional regulation of myostatin by an NF-κB–mediated mechanism

    Science.gov (United States)

    Qiu, Jia; Thapaliya, Samjhana; Runkana, Ashok; Yang, Yu; Tsien, Cynthia; Mohan, Maradumane L.; Narayanan, Arvind; Eghtesad, Bijan; Mozdziak, Paul E.; McDonald, Christine; Stark, George R.; Welle, Stephen; Naga Prasad, Sathyamangla V.; Dasarathy, Srinivasan

    2013-01-01

    Loss of muscle mass, or sarcopenia, is nearly universal in cirrhosis and adversely affects patient outcome. The underlying cross-talk between the liver and skeletal muscle mediating sarcopenia is not well understood. Hyperammonemia is a consistent abnormality in cirrhosis due to impaired hepatic detoxification to urea. We observed elevated levels of ammonia in both plasma samples and skeletal muscle biopsies from cirrhotic patients compared with healthy controls. Furthermore, skeletal muscle from cirrhotics had increased expression of myostatin, a known inhibitor of skeletal muscle accretion and growth. In vivo studies in mice showed that hyperammonemia reduced muscle mass and strength and increased myostatin expression in wild-type compared with postdevelopmental myostatin knockout mice. We postulated that hyperammonemia is an underlying link between hepatic dysfunction in cirrhosis and skeletal muscle loss. Therefore, murine C2C12 myotubes were treated with ammonium acetate resulting in intracellular concentrations similar to those in cirrhotic muscle. In this system, we demonstrate that hyperammonemia stimulated myostatin expression in a NF-κB–dependent manner. This finding was also observed in primary murine muscle cell cultures. Hyperammonemia triggered activation of IκB kinase, NF-κB nuclear translocation, binding of the NF-κB p65 subunit to specific sites within the myostatin promoter, and stimulation of myostatin gene transcription. Pharmacologic inhibition or gene silencing of NF-κB abolished myostatin up-regulation under conditions of hyperammonemia. Our work provides unique insights into hyperammonemia-induced myostatin expression and suggests a mechanism by which sarcopenia develops in cirrhotic patients. PMID:24145431

  20. Stat1-Vitamin D Receptor Interactions Antagonize 1,25-Dihydroxyvitamin D Transcriptional Activity and Enhance Stat1-Mediated Transcription

    Science.gov (United States)

    Vidal, Marcos; Ramana, Chilakamarti V.; Dusso, Adriana S.

    2002-01-01

    The cytokine gamma interferon (IFN-γ) and the calcitropic steroid hormone 1,25-dihydroxyvitamin D (1,25D) are activators of macrophage immune function. In sarcoidosis, tuberculosis, and several granulomatoses, IFN-γ induces 1,25D synthesis by macrophages and inhibits 1,25D induction of 24-hydroxylase, a key enzyme in 1,25D inactivation, causing high levels of 1,25D in serum and hypercalcemia. This study delineates IFN-γ-1,25D cross talk in human monocytes-macrophages. Nuclear accumulation of Stat1 and vitamin D receptor (VDR) by IFN-γ and 1,25D promotes protein-protein interactions between Stat1 and the DNA binding domain of the VDR. This prevents VDR-retinoid X receptor (RXR) binding to the vitamin D-responsive element, thus diverting the VDR from its normal genomic target on the 24-hydroxylase promoter and antagonizing 1,25D-VDR transactivation of this gene. In contrast, 1,25D enhances IFN-γ action. Stat1-VDR interactions, by preventing Stat1 deactivation by tyrosine dephosphorylation, cooperate with IFN-γ/Stat1-induced transcription. This novel 1,25D-IFN-γ cross talk explains the pathogenesis of abnormal 1,25D homeostasis in granulomatous processes and provides new insights into 1,25D immunomodulatory properties. PMID:11909970

  1. UV-C-Induced alleviation of transcriptional gene silencing through plant–plant communication: Key roles of jasmonic acid and salicylic acid pathways

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Wei; Wang, Ting [Key Laboratory of Ion Beam Bio-engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, P.O. Box 1138, Hefei, Anhui, 230031 (China); Xu, Shaoxin [School of physics and materials science, Anhui University, Hefei, Anhui, 230601 (China); Li, Fanghua; Deng, Chenguang; Wu, Lijun; Wu, Yuejin [Key Laboratory of Ion Beam Bio-engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, P.O. Box 1138, Hefei, Anhui, 230031 (China); Bian, Po, E-mail: bianpo@ipp.ac.cn [Key Laboratory of Ion Beam Bio-engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, P.O. Box 1138, Hefei, Anhui, 230031 (China)

    2016-08-15

    Highlights: • Transcriptional gene silencing (TGS) in plants can be epigenetically alleviated by volatile signals from UV-C- irradiated neighboring plants. • Alleviation of TGS can be induced by UV-C irradiation through plant–plant–plant communication. • JA and SA signals take part in interplant communication for alleviation of TGS. - Abstract: Plant stress responses at the epigenetic level are expected to allow more permanent changes of gene expression and potentially long-term adaptation. While it has been reported that plants subjected to adverse environments initiate various stress responses in their neighboring plants, little is known regarding epigenetic responses to external stresses mediated by plant–plant communication. In this study, we show that DNA repetitive elements of Arabidopsis thaliana, whose expression is inhibited epigenetically by transcriptional gene silencing (TGS) mechanism, are activated by UV-C irradiation through airborne plant–plant and plant–plant–plant communications, accompanied by DNA demethylation at CHH sites. Moreover, the TGS is alleviated by direct treatments with exogenous methyl jasmonate (MeJA) and methyl salicylate (MeSA). Further, the plant–plant and plant–plant–plant communications are blocked by mutations in the biosynthesis or signaling of jasmonic acid (JA) or salicylic acid (SA), indicating that JA and SA pathways are involved in the interplant communication for epigenetic responses. For the plant–plant–plant communication, stress cues are relayed to the last set of receiver plants by promoting the production of JA and SA signals in relaying plants, which exhibit upregulated expression of genes for JA and SA biosynthesis and enhanced emanation of MeJA and MeSA.

  2. UV-C-Induced alleviation of transcriptional gene silencing through plant–plant communication: Key roles of jasmonic acid and salicylic acid pathways

    International Nuclear Information System (INIS)

    Xu, Wei; Wang, Ting; Xu, Shaoxin; Li, Fanghua; Deng, Chenguang; Wu, Lijun; Wu, Yuejin; Bian, Po

    2016-01-01

    Highlights: • Transcriptional gene silencing (TGS) in plants can be epigenetically alleviated by volatile signals from UV-C- irradiated neighboring plants. • Alleviation of TGS can be induced by UV-C irradiation through plant–plant–plant communication. • JA and SA signals take part in interplant communication for alleviation of TGS. - Abstract: Plant stress responses at the epigenetic level are expected to allow more permanent changes of gene expression and potentially long-term adaptation. While it has been reported that plants subjected to adverse environments initiate various stress responses in their neighboring plants, little is known regarding epigenetic responses to external stresses mediated by plant–plant communication. In this study, we show that DNA repetitive elements of Arabidopsis thaliana, whose expression is inhibited epigenetically by transcriptional gene silencing (TGS) mechanism, are activated by UV-C irradiation through airborne plant–plant and plant–plant–plant communications, accompanied by DNA demethylation at CHH sites. Moreover, the TGS is alleviated by direct treatments with exogenous methyl jasmonate (MeJA) and methyl salicylate (MeSA). Further, the plant–plant and plant–plant–plant communications are blocked by mutations in the biosynthesis or signaling of jasmonic acid (JA) or salicylic acid (SA), indicating that JA and SA pathways are involved in the interplant communication for epigenetic responses. For the plant–plant–plant communication, stress cues are relayed to the last set of receiver plants by promoting the production of JA and SA signals in relaying plants, which exhibit upregulated expression of genes for JA and SA biosynthesis and enhanced emanation of MeJA and MeSA.

  3. Polyunsaturated fatty acid regulation of gene transcription: a molecular mechanism to improve the metabolic syndrome.

    Science.gov (United States)

    Clarke, S D

    2001-04-01

    This review addresses the hypothesis that polyunsaturated fatty acids (PUFA), particularly those of the (n-3) family, play pivotal roles as "fuel partitioners" in that they direct fatty acids away from triglyceride storage and toward oxidation, and that they enhance glucose flux to glycogen. In doing this, PUFA may protect against the adverse symptoms of the metabolic syndrome and reduce the risk of heart disease. PUFA exert their beneficial effects by up-regulating the expression of genes encoding proteins involved in fatty acid oxidation while simultaneously down-regulating genes encoding proteins of lipid synthesis. PUFA govern oxidative gene expression by activating the transcription factor peroxisome proliferator-activated receptor alpha. PUFA suppress lipogenic gene expression by reducing the nuclear abundance and DNA-binding affinity of transcription factors responsible for imparting insulin and carbohydrate control to lipogenic and glycolytic genes. In particular, PUFA suppress the nuclear abundance and expression of sterol regulatory element binding protein-1 and reduce the DNA-binding activities of nuclear factor Y, Sp1 and possibly hepatic nuclear factor-4. Collectively, the studies discussed suggest that the fuel "repartitioning" and gene expression actions of PUFA should be considered among criteria used in defining the dietary needs of (n-6) and (n-3) and in establishing the dietary ratio of (n-6) to (n-3) needed for optimum health benefit.

  4. Transcription factor Sox4 is required for PUMA-mediated apoptosis induced by histone deacetylase inhibitor, TSA.

    Science.gov (United States)

    Jang, Sang-Min; Kang, Eun-Jin; Kim, Jung-Woong; Kim, Chul-Hong; An, Joo-Hee; Choi, Kyung-Hee

    2013-08-23

    PUMA is a crucial regulator of apoptotic cell death mediated by p53-dependent and p53-independent mechanisms. In many cancer cells, PUMA expression is induced in response to DNA-damaging reagent in a p53-dependent manner. However, few studies have investigated transcription factors that lead to the induction of PUMA expression via p53-independent apoptotic signaling. In this study, we found that the transcription factor Sox4 increased PUMA expression in response to trichostatin A (TSA), a histone deacetylase inhibitor in the p53-null human lung cancer cell line H1299. Ectopic expression of Sox4 led to the induction of PUMA expression at the mRNA and protein levels, and TSA-mediated up-regulation of PUMA transcription was repressed by the knockdown of Sox4. Using luciferase assays and chromatin immunoprecipitation, we also determined that Sox4 recruits p300 on the PUMA promoter region and increases PUMA gene expression in response to TSA treatment. Taken together, these results suggest that Sox4 is required for p53-independent apoptotic cell death mediated by PUMA induction via TSA treatment. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

  5. The Mediator subunit MED23 couples H2B mono-ubiquitination to transcriptional control and cell fate determination.

    Science.gov (United States)

    Yao, Xiao; Tang, Zhanyun; Fu, Xing; Yin, Jingwen; Liang, Yan; Li, Chonghui; Li, Huayun; Tian, Qing; Roeder, Robert G; Wang, Gang

    2015-12-02

    The Mediator complex orchestrates multiple transcription factors with the Pol II apparatus for precise transcriptional control. However, its interplay with the surrounding chromatin remains poorly understood. Here, we analyze differential histone modifications between WT and MED23(-/-) (KO) cells and identify H2B mono-ubiquitination at lysine 120 (H2Bub) as a MED23-dependent histone modification. Using tandem affinity purification and mass spectrometry, we find that MED23 associates with the RNF20/40 complex, the enzyme for H2Bub, and show that this association is critical for the recruitment of RNF20/40 to chromatin. In a cell-free system, Mediator directly and substantially increases H2Bub on recombinant chromatin through its cooperation with RNF20/40 and the PAF complex. Integrative genome-wide analyses show that MED23 depletion specifically reduces H2Bub on a subset of MED23-controlled genes. Importantly, MED23-coupled H2Bub levels are oppositely regulated during myogenesis and lung carcinogenesis. In sum, these results establish a mechanistic link between the Mediator complex and a critical chromatin modification in coordinating transcription with cell growth and differentiation. © 2015 The Authors.

  6. CAR-mediated repression of Foxo1 transcriptional activity regulates the cell cycle inhibitor p21 in mouse livers

    International Nuclear Information System (INIS)

    Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Pustylnyak, Vladimir O.

    2014-01-01

    Highlights: • CAR activation decreased the level of Foxo1 in mouse livers. • CAR activation decreased the level of p21 in mouse livers. • CAR activation inhibited Foxo1 transcriptional activity in mouse livers. - Abstract: 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor (CAR), is a well-known strong primary chemical mitogen for the mouse liver. Despite extensive investigation of the role of CAR in the regulation of cell proliferation, our knowledge of the intricate mediating mechanism is incomplete. In this study, we demonstrated that long-term CAR activation by TCPOBOP increased liver-to-body weight ratio and decreased tumour suppressor Foxo1 expression and transcriptional activity, which were correlated with reduced expression of genes regulated by Foxo1, including the cell-cycle inhibitor Cdkn1a(p21), and upregulation of the cell-cycle regulator Cyclin D1. Moreover, we demonstrated the negative regulatory effect of TCPOBOP-activated CAR on the association of Foxo1 with the target Foxo1 itself and Cdkn1a(p21) promoters. Thus, we identified CAR-mediated repression of cell cycle inhibitor p21, as mediated by repression of FOXO1 expression and transcriptional activity. CAR-FOXO1 cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments

  7. Understanding large multiprotein complexes: applying a multiple allosteric networks model to explain the function of the Mediator transcription complex.

    Science.gov (United States)

    Lewis, Brian A

    2010-01-15

    The regulation of transcription and of many other cellular processes involves large multi-subunit protein complexes. In the context of transcription, it is known that these complexes serve as regulatory platforms that connect activator DNA-binding proteins to a target promoter. However, there is still a lack of understanding regarding the function of these complexes. Why do multi-subunit complexes exist? What is the molecular basis of the function of their constituent subunits, and how are these subunits organized within a complex? What is the reason for physical connections between certain subunits and not others? In this article, I address these issues through a model of network allostery and its application to the eukaryotic RNA polymerase II Mediator transcription complex. The multiple allosteric networks model (MANM) suggests that protein complexes such as Mediator exist not only as physical but also as functional networks of interconnected proteins through which information is transferred from subunit to subunit by the propagation of an allosteric state known as conformational spread. Additionally, there are multiple distinct sub-networks within the Mediator complex that can be defined by their connections to different subunits; these sub-networks have discrete functions that are activated when specific subunits interact with other activator proteins.

  8. Enhanced B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation contributes to ABCC1-mediated chemoresistance and glutathione-mediated survival in acquired topoisomerase II poison-resistant cancer cells.

    Science.gov (United States)

    Chen, Huang-Hui; Chang, Hsin-Huei; Chang, Jang-Yang; Tang, Ya-Chu; Cheng, Yung-Chi; Lin, Li-Mei; Cheng, Shu-Ying; Huang, Chih-Hsiang; Sun, Man-Wu; Chen, Chiung-Tong; Kuo, Ching-Chuan

    2017-12-01

    Nuclear factor erythroid-2-related factor 2 (NRF2) mainly regulates transcriptional activation through antioxidant-responsive elements (AREs) present in the promoters of NRF2 target genes. Recently, we found that NRF2 was overexpressed in a KB-derived drug-resistant cancer cell panel. In this panel, KB-7D cells, which show acquired resistance to topoisomerase II (Top II) poisons, exhibited the highest NRF2 activation. To investigate whether NRF2 directly contributed to acquired resistance against Top II poisons, we manipulated NRF2 by genetic and pharmacological approaches. The result demonstrated that silencing of NRF2 by RNA interference increased the sensitivity and treatment with NRF2 activator decreased the sensitivity of KB and KB-7D cells toward Top II poisons. Further, increased B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation activated NRF2 signaling in KB-7D cells. Moreover, increased binding of NRF2 to an ARE in the promoter of ATP-binding cassette subfamily C member 1 (ABCC1) directly contributed to Top II poison resistance. In addition, activation of NRF2 increased glutathione level and antioxidant capacity in KB-7D cells compared with that in KB cells; moreover, high glutathione level provided survival advantage to KB-7D cells. Our study is the first to show that aberrant NRF2 activation is via increased B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation, which increases the acquired resistance and promote the survival of Top II poison-resistant cancer cells. Importantly, NRF2 downstream effectors ABCC1 and glutathione directly contribute to acquired resistance and survival, respectively. These results suggest that blockade of NRF2 signaling may enhance therapeutic efficacy and reduce the survival of Top II poison-refractory tumors in clinical. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Transcriptional and post-transcriptional upregulation of p27 mediates growth inhibition of isorhapontigenin (ISO) on human bladder cancer cells.

    Science.gov (United States)

    Jiang, Guosong; Huang, Chao; Li, Jingxia; Huang, Haishan; Wang, Jingjing; Li, Yawei; Xie, Fei; Jin, Honglei; Zhu, Junlan; Huang, Chuanshu

    2018-03-08

    There are few approved drugs available for the treatment of muscle-invasive bladder cancer (MIBC). Recently, we have demonstrated that isorhapontigenin (ISO), a new derivative isolated from the Chinese herb Gnetum cleistostachyum, effectively induces cell-cycle arrest at the G0/G1 phase and inhibits anchorage-independent cell growth through the miR-137/Sp1/cyclin D1 axis in human MIBC cells. Herein, we found that treatment of bladder cancer (BC) cells with ISO resulted in a significant upregulation of p27, which was also observed in ISO-treated mouse BCs that were induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Importantly, knockdown of p27 caused a decline in the ISO-induced G0-G1 growth arrest and reversed ISO suppression of anchorage-independent growth in BC cells. Mechanistic studies revealed that ISO promoted p27 expression at mRNA transcription level through increasing direct binding of forkhead box class O1 (FOXO1) to its promoter, while knockdown of FOXO1 attenuated ISO inhibition of BC cell growth. On the other hand, ISO upregulated the 3'-untranslated region (3'-UTR) activity of p27, which was accompanied by a reduction of miR-182 expression. In line with these observations, ectopic expression of miR-182 significantly blocked p27 3'-UTR activity, whereas mutation of the miR-182-binding site at p27 mRNA 3'-UTR effectively reversed this inhibition. Accordingly, ectopic expression of miR-182 also attenuated ISO upregulation of p27 expression and impaired ISO inhibition of BC cell growth. Our results not only provide novel insight into understanding of the underlying mechanism related to regulation of MIBC cell growth but also identify new roles and mechanisms underlying ISO inhibition of BC cell growth.

  10. Antioxidative Activities of Both Oleic Acid and Camellia tenuifolia Seed Oil Are Regulated by the Transcription Factor DAF-16/FOXO in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Chia-Cheng Wei

    Full Text Available Tea seed oil is a high quality edible oil, yet lacking sufficient scientific evidences to support the nutritional and medical purposes. We identified major and minor components in Camellia tenuifolia seed oil and investigated the antioxidative activity and its underlying mechanisms in Caenorhabditis elegans.The results showed that the major constitutes in C. tenuifolia seed oil were unsaturated fatty acids (~78.4%. Moreover, two minor compounds, β-amyrin and β-sitosterol, were identified and their antioxidative activity was examined. We found that oleic acid was the major constitute in C. tenuifolia seed oil and plays a key role in the antioxidative activity of C. tenuifolia seed oil in C. elegans.This study found evidences that the transcription factor DAF-16/FOXO was involved in both oleic acid- and C. tenuifolia seed oil-mediated oxidative stress resistance in C. elegans. This study suggests the potential of C. tenuifolia seed oil as nutrient or functional foods.

  11. UV-C-Induced alleviation of transcriptional gene silencing through plant-plant communication: Key roles of jasmonic acid and salicylic acid pathways.

    Science.gov (United States)

    Xu, Wei; Wang, Ting; Xu, Shaoxin; Li, Fanghua; Deng, Chenguang; Wu, Lijun; Wu, Yuejin; Bian, Po

    2016-08-01

    Plant stress responses at the epigenetic level are expected to allow more permanent changes of gene expression and potentially long-term adaptation. While it has been reported that plants subjected to adverse environments initiate various stress responses in their neighboring plants, little is known regarding epigenetic responses to external stresses mediated by plant-plant communication. In this study, we show that DNA repetitive elements of Arabidopsis thaliana, whose expression is inhibited epigenetically by transcriptional gene silencing (TGS) mechanism, are activated by UV-C irradiation through airborne plant-plant and plant-plant-plant communications, accompanied by DNA demethylation at CHH sites. Moreover, the TGS is alleviated by direct treatments with exogenous methyl jasmonate (MeJA) and methyl salicylate (MeSA). Further, the plant-plant and plant-plant-plant communications are blocked by mutations in the biosynthesis or signaling of jasmonic acid (JA) or salicylic acid (SA), indicating that JA and SA pathways are involved in the interplant communication for epigenetic responses. For the plant-plant-plant communication, stress cues are relayed to the last set of receiver plants by promoting the production of JA and SA signals in relaying plants, which exhibit upregulated expression of genes for JA and SA biosynthesis and enhanced emanation of MeJA and MeSA. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Jensen, Michael Krogh; Lindemose, Søren; De Masi, Federico

    2013-01-01

    ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin-immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT[A,C,G...... abscisic acid (ABA) phytohormone biosynthetic gene NCED3. ChIP-qPCR and expression analysis showed that ATAF1 binding to the NCED3 promoter correlated with increased NCED3 expression and ABA hormone levels. These results indicate that ATAF1 regulates ABA biosynthesis....

  13. Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA)

    DEFF Research Database (Denmark)

    Robaczewska, Magdalena; Narayan, Ramamurthy; Seigneres, Beatrice

    2005-01-01

    BACKGROUND/AIMS: Peptide nucleic acids (PNAs) appear as promising new antisense agents, that have not yet been examined as hepatitis B virus (HBV) inhibitors. Our aim was to study the ability of PNAs targeting the duck HBV (DHBV) encapsidation signal epsilon to inhibit reverse transcription (RT...... in primary duck hepatocytes (PDH). RESULTS: Both PNAs reproducibly inhibited DHBV RT in a dose-dependent manner with IC(50) of 10nM, whereas up to 600-fold higher concentration of S-ODNs was required for similar inhibition. The PNA targeting the bulge and upper stem of epsilon appeared as more efficient RT...

  14. Stk1-mediated phosphorylation stimulates the DNA-binding properties of the Staphylococcus aureus SpoVG transcriptional factor.

    Science.gov (United States)

    Bischoff, Markus; Brelle, Solène; Minatelli, Sabrina; Molle, Virginie

    2016-05-13

    The stage V sporulation protein G (SpoVG) homolog of Staphylococcus aureus is a modulator of virulence factor synthesis and antibiotic resistance in this clinically important gram-positive pathogen. Here we demonstrate that SpoVG can be phosphorylated by the staphylococcal Ser/Thr protein kinase Stk1 and that phosphorylation positively affects its DNA-binding properties. Mass spectrometric analyses and site directed mutagenesis identified Thr4, Thr13, Thr24 and Ser41 as phospho-acceptors. Stk1-mediated phosphorylation markedly enhanced the DNA binding activity of SpoVG towards the promoter regions of target genes such as capA, lip, and nuc1. Similarly, trans-complementation of the S. aureus ΔyabJ-spoVG mutant SM148 with a SpoVG derivative that mimics constitutive phosphorylation, SpoVG_Asp, exhibited capA, lip, and nuc1 transcript levels that were comparable to the levels seen with the wild-type, whereas trans-complementation with a phosphoablative variant of SpoVG (SpoVG_Ala) produced transcript levels similar to the ones seen in SM148. Our data suggest that the expression/activity of this transcription factor is tightly controlled in S. aureus by transcriptional, post-transcriptional and post-translational mechanisms. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Inference of Transcription Regulatory Network in Low Phytic Acid Soybean Seeds

    Directory of Open Access Journals (Sweden)

    Neelam Redekar

    2017-11-01

    Full Text Available A dominant loss of function mutation in myo-inositol phosphate synthase (MIPS gene and recessive loss of function mutations in two multidrug resistant protein type-ABC transporter genes not only reduce the seed phytic acid levels in soybean, but also affect the pathways associated with seed development, ultimately resulting in low emergence. To understand the regulatory mechanisms and identify key genes that intervene in the seed development process in low phytic acid crops, we performed computational inference of gene regulatory networks in low and normal phytic acid soybeans using a time course transcriptomic data and multiple network inference algorithms. We identified a set of putative candidate transcription factors and their regulatory interactions with genes that have functions in myo-inositol biosynthesis, auxin-ABA signaling, and seed dormancy. We evaluated the performance of our unsupervised network inference method by comparing the predicted regulatory network with published regulatory interactions in Arabidopsis. Some contrasting regulatory interactions were observed in low phytic acid mutants compared to non-mutant lines. These findings provide important hypotheses on expression regulation of myo-inositol metabolism and phytohormone signaling in developing low phytic acid soybeans. The computational pipeline used for unsupervised network learning in this study is provided as open source software and is freely available at https://lilabatvt.github.io/LPANetwork/.

  16. The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities.

    Science.gov (United States)

    Valdés, Ana Elisa; Overnäs, Elin; Johansson, Henrik; Rada-Iglesias, Alvaro; Engström, Peter

    2012-11-01

    Plants perceiving drought activate multiple responses to improve survival, including large-scale alterations in gene expression. This article reports on the roles in the drought response of two Arabidopsis thaliana homeodomain-leucine zipper class I genes; ATHB7 and ATHB12, both strongly induced by water-deficit and abscisic acid (ABA). ABA-mediated transcriptional regulation of both genes is shown to depend on the activity of protein phosphatases type 2C (PP2C). ATHB7 and ATHB12 are, thus, targets of the ABA signalling mechanism defined by the PP2Cs and the PYR/PYL family of ABA receptors, with which the PP2C proteins interact. Our results from chromatin immunoprecipitation and gene expression analyses demonstrate that ATHB7 and ATHB12 act as positive transcriptional regulators of PP2C genes, and thereby as negative regulators of abscisic acid signalling. In support of this notion, our results also show that ATHB7 and ATHB12 act to repress the transcription of genes encoding the ABA receptors PYL5 and PYL8 in response to an ABA stimulus. In summary, we demonstrate that ATHB7 and ATHB12 have essential functions in the primary response to drought, as mediators of a negative feedback effect on ABA signalling in the plant response to water deficit.

  17. Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

    Science.gov (United States)

    Thanarajoo, Sathis Sri; Kong, Lih Ling; Kadir, Jugah; Lau, Wei Hongi; Vadamalai, Ganesan

    2014-06-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Abscisic acid and sucrose regulate tomato and strawberry fruit ripening through the abscisic acid-stress-ripening transcription factor.

    Science.gov (United States)

    Jia, Haifeng; Jiu, Songtao; Zhang, Cheng; Wang, Chen; Tariq, Pervaiz; Liu, Zhongjie; Wang, Baoju; Cui, Liwen; Fang, Jinggui

    2016-10-01

    Although great progress has been made towards understanding the role of abscisic acid (ABA) and sucrose in fruit ripening, the mechanisms underlying the ABA and sucrose signalling pathways remain elusive. In this study, transcription factor ABA-stress-ripening (ASR), which is involved in the transduction of ABA and sucrose signalling pathways, was isolated and analysed in the nonclimacteric fruit, strawberry and the climacteric fruit, tomato. We have identified four ASR isoforms in tomato and one in strawberry. All ASR sequences contained the ABA stress- and ripening-induced proteins and water-deficit stress-induced proteins (ABA/WDS) domain and all ASR transcripts showed increased expression during fruit development. The expression of the ASR gene was influenced not only by sucrose and ABA, but also by jasmonic acid (JA) and indole-3-acetic acid (IAA), and these four factors were correlated with each other during fruit development. ASR bound the hexose transporter (HT) promoter, which contained a sugar box that activated downstream gene expression. Overexpression of the ASR gene promoted fruit softening and ripening, whereas RNA interference delayed fruit ripening, as well as affected fruit physiological changes. Change in ASR gene expression influenced the expression of several ripening-related genes such as CHS, CHI, F3H, DFR, ANS, UFGT, PG, PL, EXP1/2, XET16, Cel1/2 and PME. Taken together, this study may provide new evidence on the important role of ASR in cross-signalling between ABA and sucrose to regulate tomato and strawberry fruit ripening. The findings of this study also provide new insights into the regulatory mechanism underlying fruit development. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  19. Suppression of sterol 27-hydroxylase mRNA and transcriptional activity by bile acids in cultured rat hepatocytes

    NARCIS (Netherlands)

    Twisk, J.; Wit, E.C.M. de; Princen, H.M.G.

    1995-01-01

    In previous work we have demonstrated suppression of cholesterol 7α-hydroxylase by bile acids at the level of mRNA and transcription, resulting in a similar decline in bile acid synthesis in cultured rat hepatocytes. In view of the substantial contribution of the 'alternative' or '27-hydroxylase'

  20. Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.

    Science.gov (United States)

    Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2006-02-07

    bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.

  1. Lysophosphatidic acid mediates pleiotropic responses in skeletal muscle cells

    International Nuclear Information System (INIS)

    Jean-Baptiste, Gael; Yang Zhao; Khoury, Chamel; Greenwood, Michael T.

    2005-01-01

    Lysophosphatidic acid (LPA) is a potent modulator of growth, cell survival, and apoptosis. Although all four LPA receptors are expressed in skeletal muscle, very little is known regarding the role they play in this tissue. We used RT-PCR to demonstrate that cultured skeletal muscle C2C12 cells endogenously express multiple LPA receptor subtypes. The demonstration that LPA mediates the activation of ERK1/2 MAP kinase and Akt/PKB in C2C12 cells is consistent with the widely observed mitogenic properties of LPA. In spite of these observations, LPA did not induce proliferation in C2C12 cells. Paradoxically, we found that prolonged treatment of C2C12 cells with LPA led to caspase 3 and PARP cleavage as well as the activation of stress-associated MAP kinases JNK and p38. In spite of these typically pro-apoptotic responses, LPA did not induce cell death. Blocking ERK1/2 and Akt/PKB activation with specific pharmacological inhibitors, nevertheless, stimulated LPA-mediated apoptosis. Taken together, these results suggest that both mitogenic and apoptotic responses serve to counterbalance the effects of LPA in cultured C2C12 cells

  2. Transcriptional up-regulation of restin by all-trans retinoic acid through STAT1 in cancer cell differentiation process

    International Nuclear Information System (INIS)

    Fu Haiyan; Yang Guodong; Lu Fan; Wang Ruihua; Yao Libo; Lu Zifan

    2006-01-01

    RESTIN, a member of the melanoma-associated antigen superfamily, is a nuclear protein induced by atRA (all-trans retinoic acid) in HL60 cells. HeLa cells stably transfected with restin results in G1 cell cycle arrest. How this gene is regulated by atRA in the cell differentiation process is still unclear. In this study, we observed that up-regulation of restin was present during the atRA-induced HL60 cell differentiation process, suggesting the functional relevance between RESTIN and atRA-induced cellular effects. In order to further define the transcriptional regulation of restin by atRA, we analyzed the promoter region of restin. About 2.1 kb 5' flanking sequence of this gene was cloned into vector pGL3 and its core promoter region was identified through systemic deletions. Interestingly, restin promoter containing several potential consensus-binding sites of STAT-1α was activated by atRA in ER + MCF-7 cells but not in ER - MDA-MB-231 cells, over-expression of STAT-1α in latter rescued the activation effect of restin promoter in response to atRA and IFNγ. Our evidence supported that STAT-1α plays an important role in the atRA-induced transcriptional up-regulation of restin, which was associated with the atRA-induced HL60 cell differentiation and potentially mediated the downstream effects of atRA signal pathway via STAT-1α in some cancer cells

  3. Rf8-Mediated T-urf13 Transcript Accumulation Coincides with a Pentatricopeptide Repeat Cluster on Maize Chromosome 2L

    Directory of Open Access Journals (Sweden)

    Julie Meyer

    2011-11-01

    Full Text Available Cytoplasmic male sterility (CMS is a maternally inherited inability to produce functional pollen. In Texas (T-cytoplasm maize ( L., CMS results from the action of the URF13 mitochondrial pore-forming protein encoded by the unique T- mitochondrial gene. Full or partial restoration of fertility to T-cytoplasm maize is mediated by the nuclear gene in combination with one of three other genes: , , or *. encodes a mitochondrial aldehyde dehydrogenase whereas , , and * are associated with the accumulation of distinctive T- mitochondrial transcripts. -associated RNA processing activity was mapped to a 4.55-Mbp region on chromosome 2L that contains 10 pentatricopeptide repeat (PPR encoding genes in the B73 5b.60 genome assembly. Genetic linkage analysis also indicated that * is positioned within this PPR cluster as well as , which restores USDA (S-cytoplasm maize. Partially male-fertile plants segregated for the presence or absence of the -associated T- 1.42- and 0.42-kbp transcripts, indicating that the RNA processing event associated with these transcripts is not necessary for anther exsertion. In addition, a statistically significant delay in flowering was observed between partially male-fertile and mostly male-fertile plants. Taken together, these new results indicate that -mediated male fertility is under the control of more than one nuclear locus.

  4. NikR mediates nickel-responsive transcriptional induction of urease expression in Helicobacter pylori

    NARCIS (Netherlands)

    A.H.M. van Vliet (Arnoud); S.W. Poppelaars (Sophie); B.J. Davies; J. Stoof (Jeroen); S. Bereswill (Stefan); M. Kist (Manfred); C.W. Penn (Charles); E.J. Kuipers (Ernst); J.G. Kusters (Johannes)

    2002-01-01

    textabstractThe important human pathogen Helicobacter pylori requires the abundant expression and activity of its urease enzyme for colonization of the gastric mucosa. The transcription, expression, and activity of H. pylori urease were previously demonstrated to be induced by

  5. Differential regulation of hepatic transcription factors in the Wistar rat offspring born to dams fed folic acid, vitamin B12 deficient diets and supplemented with omega-3 fatty acids.

    Directory of Open Access Journals (Sweden)

    Akshaya Meher

    Full Text Available Nutritional status of the mother is known to influence various metabolic adaptations required for optimal fetal development. These may be mediated by transcription factors like peroxisome proliferator activated receptors (PPARs, which are activated by long chain polyunsaturated fatty acids. The objective of the current study was to examine the expression of different hepatic transcription factors and the levels of global methylation in the liver of the offspring born to dams fed micronutrient deficient (folic acid and vitamin B12 diets and supplemented with omega-3 fatty acids. Female rats were divided into five groups (n = 8/group as follows; control, folic acid deficient (FD, vitamin B12 deficient (BD and omega-3 fatty acid supplemented groups (FDO and BDO. Diets were given starting from pre-conception and continued throughout pregnancy and lactation. Pups were dissected at the end of lactation. Liver tissues were removed; snap frozen and stored at -80°C. Maternal micronutrients deficiency resulted in lower (p<0.05 levels of pup liver docosahexaenoic acid (DHA and arachidonic acid (ARA as compared to the control group. Pup liver PPARα and PPARγ expression was lower (p<0.05 in the BD group although there were no differences in the expression of SREBP-1c, LXRα and RXRα expression. Omega-3 fatty acids supplementation to this group normalized (p<0.05 levels of both PPARα and PPARγ but reduced (p<0.05 SREBP-1c, LXRα and RXRα expression. There was no change in any of the transcription factors in the pup liver in the FD group. Omega-3 fatty acids supplementation to this group reduced (p<0.05 PPARα, SREBP-1c and RXRα expression. Pup liver global methylation levels were higher (p<0.01 in both the micronutrients deficient groups and could be normalized (p<0.05 by omega-3 fatty acid supplementation. Our novel findings suggest a role for omega-3 fatty acids in the one carbon cycle in influencing the hepatic expression of transcription factors

  6. Genome-wide Analysis of RARβ Transcriptional Targets in Mouse Striatum Links Retinoic Acid Signaling with Huntington's Disease and Other Neurodegenerative Disorders.

    Science.gov (United States)

    Niewiadomska-Cimicka, Anna; Krzyżosiak, Agnieszka; Ye, Tao; Podleśny-Drabiniok, Anna; Dembélé, Doulaye; Dollé, Pascal; Krężel, Wojciech

    2017-07-01

    Retinoic acid (RA) signaling through retinoic acid receptors (RARs), known for its multiple developmental functions, emerged more recently as an important regulator of adult brain physiology. How RAR-mediated regulation is achieved is poorly known, partly due to the paucity of information on critical target genes in the brain. Also, it is not clear how reduced RA signaling may contribute to pathophysiology of diverse neuropsychiatric disorders. We report the first genome-wide analysis of RAR transcriptional targets in the brain. Using chromatin immunoprecipitation followed by high-throughput sequencing and transcriptomic analysis of RARβ-null mutant mice, we identified genomic targets of RARβ in the striatum. Characterization of RARβ transcriptional targets in the mouse striatum points to mechanisms through which RAR may control brain functions and display neuroprotective activity. Namely, our data indicate with statistical significance (FDR 0.1) a strong contribution of RARβ in controlling neurotransmission, energy metabolism, and transcription, with a particular involvement of G-protein coupled receptor (p = 5.0e -5 ), cAMP (p = 4.5e -4 ), and calcium signaling (p = 3.4e -3 ). Many identified RARβ target genes related to these pathways have been implicated in Alzheimer's, Parkinson's, and Huntington's disease (HD), raising the possibility that compromised RA signaling in the striatum may be a mechanistic link explaining the similar affective and cognitive symptoms in these diseases. The RARβ transcriptional targets were particularly enriched for transcripts affected in HD. Using the R6/2 transgenic mouse model of HD, we show that partial sequestration of RARβ in huntingtin protein aggregates may account for reduced RA signaling reported in HD.

  7. After-ripening induced transcriptional changes of hormonal genes in wheat seeds: the cases of brassinosteroids, ethylene, cytokinin and salicylic acid.

    Directory of Open Access Journals (Sweden)

    Vijaya R Chitnis

    Full Text Available Maintenance and release of seed dormancy is regulated by plant hormones; their levels and seed sensitivity being the critical factors. This study reports transcriptional regulation of brassinosteroids (BR, ethylene (ET, cytokinin (CK and salicylic acid (SA related wheat genes by after-ripening, a period of dry storage that decays dormancy. Changes in the expression of hormonal genes due to seed after-ripening did not occur in the anhydrobiotic state but rather in the hydrated state. After-ripening induced dormancy decay appears to be associated with imbibition mediated increase in the synthesis and signalling of BR, via transcriptional activation of de-etiolated2, dwarf4 and brassinosteroid signaling kinase, and repression of brassinosteroid insensitive 2. Our analysis is also suggestive of the significance of increased ET production, as reflected by enhanced transcription of 1-aminocyclopropane-1-carboxylic acid oxidase in after-ripened seeds, and tight regulation of seed response to ET in regulating dormancy decay. Differential transcriptions of lonely guy, zeatin O-glucosyltransferases and cytokinin oxidases, and pseudo-response regulator between dormant and after-ripened seeds implicate CK in the regulation of seed dormancy in wheat. Our analysis also reflects the association of dormancy decay in wheat with seed SA level and NPR independent SA signaling that appear to be regulated transcriptionally by phenylalanine ammonia lyase, and whirly and suppressor of npr1 inducible1 genes, respectively. Co-expression clustering of the hormonal genes implies the significance of synergistic and antagonistic interaction between the different plant hormones in regulating wheat seed dormancy. These results contribute to further our understanding of the molecular features controlling seed dormancy in wheat.

  8. Reviewing the Tannic Acid Mediated Synthesis of Metal Nanoparticles

    International Nuclear Information System (INIS)

    Ahmad, T.

    2014-01-01

    Metal nanoparticles harbour numerous exceptional physiochemical properties absolutely different from those of bulk metal as a function of their extremely small size and large superficial area to volume. Naked metal nanoparticles are synthesized by various physical and chemical methods. Chemical methods involving metal salt reduction in solution enjoy an extra edge over other protocols owing to their relative facileness and capability of controlling particle size along with the attribute of surface tailoring. Although chemical methods are the easiest, they are marred by the use of hazardous chemicals such as borohydrides. This has led to inclination of scientific community towards eco-friendly agents for the reduction of metal salts to form nanoparticles. Tannic acid, a plant derived polyphenolic compound, is one such agent which embodies characteristics of being harmless and environmentally friendly combined with being a good reducing and stabilizing agent. In this review, first various methods used to prepare metal nanoparticles are highlighted and further tannic acid mediated synthesis of metal nanoparticles is emphasized. This review brings forth the most recent findings on this issue.

  9. Reviewing the Tannic Acid Mediated Synthesis of Metal Nanoparticles

    Directory of Open Access Journals (Sweden)

    Tufail Ahmad

    2014-01-01

    Full Text Available Metal nanoparticles harbour numerous exceptional physiochemical properties absolutely different from those of bulk metal as a function of their extremely small size and large superficial area to volume. Naked metal nanoparticles are synthesized by various physical and chemical methods. Chemical methods involving metal salt reduction in solution enjoy an extra edge over other protocols owing to their relative facileness and capability of controlling particle size along with the attribute of surface tailoring. Although chemical methods are the easiest, they are marred by the use of hazardous chemicals such as borohydrides. This has led to inclination of scientific community towards eco-friendly agents for the reduction of metal salts to form nanoparticles. Tannic acid, a plant derived polyphenolic compound, is one such agent which embodies characteristics of being harmless and environmentally friendly combined with being a good reducing and stabilizing agent. In this review, first various methods used to prepare metal nanoparticles are highlighted and further tannic acid mediated synthesis of metal nanoparticles is emphasized. This review brings forth the most recent findings on this issue.

  10. AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Changho Eun

    Full Text Available RNA-directed DNA methylation (RdDM is a small interfering RNA (siRNA-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points.

  11. SAF-A forms a complex with BRG1 and both components are required for RNA polymerase II mediated transcription.

    Directory of Open Access Journals (Sweden)

    Dzeneta Vizlin-Hodzic

    Full Text Available BACKGROUND: Scaffold attachment factor A (SAF-A participates in the regulation of gene expression by organizing chromatin into transcriptionally active domains and by interacting directly with RNA polymerase II. METHODOLOGY: Here we use co-localization, co-immunoprecipitation (co-IP and in situ proximity ligation assay (PLA to identify Brahma Related Gene 1 (BRG1, the ATP-driven motor of the human SWI-SNF chromatin remodeling complex, as another SAF-A interaction partner in mouse embryonic stem (mES cells. We also employ RNA interference to investigate functional aspects of the SAF-A/BRG1 interaction. PRINCIPAL FINDINGS: We find that endogenous SAF-A protein interacts with endogenous BRG1 protein in mES cells, and that the interaction does not solely depend on the presence of mRNA. Moreover the interaction remains intact when cells are induced to differentiate. Functional analyses reveal that dual depletion of SAF-A and BRG1 abolishes global transcription by RNA polymerase II, while the nucleolar RNA polymerase I transcription machinery remains unaffected. CONCLUSIONS: We demonstrate that SAF-A interacts with BRG1 and that both components are required for RNA Polymerase II Mediated Transcription.

  12. Protection from cyanide-induced brain injury by the Nrf2 transcriptional activator carnosic acid.

    Science.gov (United States)

    Zhang, Dongxian; Lee, Brian; Nutter, Anthony; Song, Paul; Dolatabadi, Nima; Parker, James; Sanz-Blasco, Sara; Newmeyer, Traci; Ambasudhan, Rajesh; McKercher, Scott R; Masliah, Eliezer; Lipton, Stuart A

    2015-06-01

    Cyanide is a life-threatening, bioterrorist agent, preventing cellular respiration by inhibiting cytochrome c oxidase, resulting in cardiopulmonary failure, hypoxic brain injury, and death within minutes. However, even after treatment with various antidotes to protect cytochrome oxidase, cyanide intoxication in humans can induce a delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Additional mechanisms are thought to underlie cyanide-induced neuronal damage, including generation of reactive oxygen species. This may account for the fact that antioxidants prevent some aspects of cyanide-induced neuronal damage. Here, as a potential preemptive countermeasure against a bioterrorist attack with cyanide, we tested the CNS protective effect of carnosic acid (CA), a pro-electrophilic compound found in the herb rosemary. CA crosses the blood-brain barrier to up-regulate endogenous antioxidant enzymes via activation of the Nrf2 transcriptional pathway. We demonstrate that CA exerts neuroprotective effects on cyanide-induced brain damage in cultured rodent and human-induced pluripotent stem cell-derived neurons in vitro, and in vivo in various brain areas of a non-Swiss albino mouse model of cyanide poisoning that simulates damage observed in the human brain. Cyanide, a potential bioterrorist agent, can produce a chronic delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Here, cyanide poisoning treated with the proelectrophillic compound carnosic acid, results in reduced neuronal cell death in both in vitro and in vivo models through activation of the Nrf2/ARE transcriptional pathway. Carnosic acid is therefore a potential treatment for the toxic central nervous system (CNS) effects of cyanide poisoning. ARE, antioxidant responsive element; Nrf2 (NFE2L2, Nuclear factor (erythroid-derived 2)-like 2). © 2015 International Society for Neurochemistry.

  13. Activating transcription factor 4 underlies the pathogenesis of arsenic trioxide-mediated impairment of macrophage innate immune functions

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, Ritesh K.; Li, Changzhao [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Wang, Yong [Department of Medicine, University of Alabama at Birmingham, Birmingham, AL (United States); Weng, Zhiping; Elmets, Craig A. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Harrod, Kevin S. [Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham, Birmingham, AL (United States); Deshane, Jessy S., E-mail: treena@uab.edu [Department of Medicine, University of Alabama at Birmingham, Birmingham, AL (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States)

    2016-10-01

    Chronic arsenic exposure to humans is considered immunosuppressive with augmented susceptibility to several infectious diseases. The exact molecular mechanisms, however, remain unknown. Earlier, we showed the involvement of unfolded protein response (UPR) signaling in arsenic-mediated impairment of macrophage functions. Here, we show that activating transcription factor 4 (ATF4), a UPR transcription factor, regulates arsenic trioxide (ATO)-mediated dysregulation of macrophage functions. In ATO-treated ATF4{sup +/+} wild-type mice, a significant down-regulation of CD11b expression was associated with the reduced phagocytic functions of peritoneal and lung macrophages. This severe immuno-toxicity phenotype was not observed in ATO-treated ATF4{sup +/−} heterozygous mice. To confirm these observations, we demonstrated in Raw 264.7 cells that ATF4 knock-down rescues ATO-mediated impairment of macrophage functions including cytokine production, bacterial engulfment and clearance of engulfed bacteria. Sustained activation of ATF4 by ATO in macrophages induces apoptosis, while diminution of ATF4 expression protects against ATO-induced apoptotic cell death. Raw 264.7 cells treated with ATO also manifest dysregulated Ca{sup ++} homeostasis. ATO induces Ca{sup ++}-dependent calpain-1 and caspase-12 expression which together regulated macrophage apoptosis. Additionally, apoptosis was also induced by mitochondria-regulated pathway. Restoring ATO-impaired Ca{sup ++} homeostasis in ER/mitochondria by treatments with the inhibitors of inositol 1,4,5-trisphosphate receptor (IP3R) and voltage-dependent anion channel (VDAC) attenuate innate immune functions of macrophages. These studies identify a novel role for ATF4 in underlying pathogenesis of macrophage dysregulation and immuno-toxicity of arsenic. - Highlights: • ATF4 regulates arsenic-mediated impairment in macrophage functions. • Arsenic-mediated alterations in pulmonary macrophage are diminished in ATF4{sup +/−} mice

  14. Activating transcription factor 4 underlies the pathogenesis of arsenic trioxide-mediated impairment of macrophage innate immune functions

    International Nuclear Information System (INIS)

    Srivastava, Ritesh K.; Li, Changzhao; Wang, Yong; Weng, Zhiping; Elmets, Craig A.; Harrod, Kevin S.; Deshane, Jessy S.; Athar, Mohammad

    2016-01-01

    Chronic arsenic exposure to humans is considered immunosuppressive with augmented susceptibility to several infectious diseases. The exact molecular mechanisms, however, remain unknown. Earlier, we showed the involvement of unfolded protein response (UPR) signaling in arsenic-mediated impairment of macrophage functions. Here, we show that activating transcription factor 4 (ATF4), a UPR transcription factor, regulates arsenic trioxide (ATO)-mediated dysregulation of macrophage functions. In ATO-treated ATF4 +/+ wild-type mice, a significant down-regulation of CD11b expression was associated with the reduced phagocytic functions of peritoneal and lung macrophages. This severe immuno-toxicity phenotype was not observed in ATO-treated ATF4 +/− heterozygous mice. To confirm these observations, we demonstrated in Raw 264.7 cells that ATF4 knock-down rescues ATO-mediated impairment of macrophage functions including cytokine production, bacterial engulfment and clearance of engulfed bacteria. Sustained activation of ATF4 by ATO in macrophages induces apoptosis, while diminution of ATF4 expression protects against ATO-induced apoptotic cell death. Raw 264.7 cells treated with ATO also manifest dysregulated Ca ++ homeostasis. ATO induces Ca ++ -dependent calpain-1 and caspase-12 expression which together regulated macrophage apoptosis. Additionally, apoptosis was also induced by mitochondria-regulated pathway. Restoring ATO-impaired Ca ++ homeostasis in ER/mitochondria by treatments with the inhibitors of inositol 1,4,5-trisphosphate receptor (IP3R) and voltage-dependent anion channel (VDAC) attenuate innate immune functions of macrophages. These studies identify a novel role for ATF4 in underlying pathogenesis of macrophage dysregulation and immuno-toxicity of arsenic. - Highlights: • ATF4 regulates arsenic-mediated impairment in macrophage functions. • Arsenic-mediated alterations in pulmonary macrophage are diminished in ATF4 +/− mice. • Changes in macrophage

  15. CDK11{sup p58} represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    Energy Technology Data Exchange (ETDEWEB)

    Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China); Gu, Jianxin, E-mail: jxgu@shmu.edu.cn [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China)

    2009-08-28

    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11{sup p58} as a novel protein involved in the regulation of VDR. CDK11{sup p58}, a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11{sup p58} interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11{sup p58} decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11{sup p58} is involved in the negative regulation of VDR.

  16. CDK11p58 represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    International Nuclear Information System (INIS)

    Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing; Gu, Jianxin

    2009-01-01

    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11 p58 as a novel protein involved in the regulation of VDR. CDK11 p58 , a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11 p58 interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11 p58 decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11 p58 is involved in the negative regulation of VDR.

  17. Using network component analysis to dissect regulatory networks mediated by transcription factors in yeast.

    Directory of Open Access Journals (Sweden)

    Chun Ye

    2009-03-01

    Full Text Available Understanding the relationship between genetic variation and gene expression is a central question in genetics. With the availability of data from high-throughput technologies such as ChIP-Chip, expression, and genotyping arrays, we can begin to not only identify associations but to understand how genetic variations perturb the underlying transcription regulatory networks to induce differential gene expression. In this study, we describe a simple model of transcription regulation where the expression of a gene is completely characterized by two properties: the concentrations and promoter affinities of active transcription factors. We devise a method that extends Network Component Analysis (NCA to determine how genetic variations in the form of single nucleotide polymorphisms (SNPs perturb these two properties. Applying our method to a segregating population of Saccharomyces cerevisiae, we found statistically significant examples of trans-acting SNPs located in regulatory hotspots that perturb transcription factor concentrations and affinities for target promoters to cause global differential expression and cis-acting genetic variations that perturb the promoter affinities of transcription factors on a single gene to cause local differential expression. Although many genetic variations linked to gene expressions have been identified, it is not clear how they perturb the underlying regulatory networks that govern gene expression. Our work begins to fill this void by showing that many genetic variations affect the concentrations of active transcription factors in a cell and their affinities for target promoters. Understanding the effects of these perturbations can help us to paint a more complete picture of the complex landscape of transcription regulation. The software package implementing the algorithms discussed in this work is available as a MATLAB package upon request.

  18. The transcription factors CREB and c-Fos play key roles in NCAM-mediated neuritogenesis in PC12-E2 cells

    DEFF Research Database (Denmark)

    Jessen, U; Novitskaya, V; Pedersen, N

    2001-01-01

    The neural cell adhesion molecule (NCAM) stimulates axonal outgrowth by activation of the Ras-mitogen activated protein kinase (MAPK) pathway and by generation of arachidonic acid. We investigated whether the transcription factors, cyclic-AMP response-element binding protein (CREB) and c-Fos play...... roles in this process by estimating NCAM-dependent neurite outgrowth from PC12-E2 cells grown in co-culture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with expression plasmids encoding wild-type or dominant negative forms of CREB and c-Fos or an activated...... form of the MAPK kinase, MEK2. Alternatively, PC12-E2 cells were treated with arachidonic acid, the cAMP analogue dBcAMP, or protein kinase A (PKA) inhibitors. The negative forms of CREB and c-Fos inhibited neurite outgrowth mediated by NCAM, arachidonic acid, dBcAMP, or MEK2. Neither CREB nor c...

  19. Repression of class I transcription by cadmium is mediated by the protein phosphatase 2A

    Science.gov (United States)

    Zhou, Lei; Le Roux, Gwenaëlle; Ducrot, Cécile; Chédin, Stéphane; Labarre, Jean; Riva, Michel; Carles, Christophe

    2013-01-01

    Toxic metals are part of our environment, and undue exposure to them leads to a variety of pathologies. In response, most organisms adapt their metabolism and have evolved systems to limit this toxicity and to acquire tolerance. Ribosome biosynthesis being central for protein synthesis, we analyzed in yeast the effects of a moderate concentration of cadmium (Cd2+) on Pol I transcription that represents >60% of the transcriptional activity of the cells. We show that Cd2+ rapidly and drastically shuts down the expression of the 35S rRNA. Repression does not result from a poisoning of any of the components of the class I transcriptional machinery by Cd2+, but rather involves a protein phosphatase 2A (PP2A)-dependent cellular signaling pathway that targets the formation/dissociation of the Pol I–Rrn3 complex. We also show that Pol I transcription is repressed by other toxic metals, such as Ag+ and Hg2+, which likewise perturb the Pol I–Rrn3 complex, but through PP2A-independent mechanisms. Taken together, our results point to a central role for the Pol I–Rrn3 complex as molecular switch for regulating Pol I transcription in response to toxic metals. PMID:23640330

  20. Inhibition of transcription of abscisic acid in relation to the binding with DNA

    International Nuclear Information System (INIS)

    Basak, Sukla; Basu, P.S.; Biswas, B.B.

    1976-01-01

    Abscisic acid (ABA), a plant substance inhibits RNA synthesis in vivo and vitro. In vitro inhibition by ABA has been demonstrated in isolated RNA polymerase system from coconut endosperm chromatin. This inhibition can be partly reversible with indole acetic acid-receptor protein complex if added in the system. To find the mechanism of inhibition of transcription by ABA, it has been found that ABA (10 -4 -10 -5 M) can bind with DNA and can prevent strand separation. This binding increases the Tm value. ABA binds with DNA but not with RNA. Moreover, ABA can equally bind and prevent denaturation of calfthymus DNA and E. coli DNA. pH optimum for this binding is 8.0. The bound complex is resistant to alkali and alcohol but susceptible to acid below pH 5.0. It has further been demonstrated that free aBA at this pH is changed to another component which has tentatively been identified as lactone form of ABA. (author)

  1. Rapid detection of genetically diverse tomato black ring virus isolates using reverse transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Hasiów-Jaroszewska, Beata; Budzyńska, Daria; Borodynko, Natasza; Pospieszny, Henryk

    2015-12-01

    A reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) has been developed for detection of tomato black ring virus (TBRV) isolates collected from different hosts. One-step RT-LAMP was performed with a set of four primers, the design of which was based on the coat protein gene. Results of RT-LAMP were visualized by direct staining of products with fluorescent dyes, agarose gel electrophoresis, and analysis of amplification curves. The sensitivity of RT-LAMP was 100-fold greater than that of RT-PCR. The RT-LAMP assay developed here is a useful and practical method for diagnosis of TBRV.

  2. Meta-analysis of global transcriptomics reveals conserved genetic pathways of Quercetin and Tannic acid mediated longevity in C. elegans

    Directory of Open Access Journals (Sweden)

    Kerstin ePietsch

    2012-04-01

    Full Text Available Recent research has highlighted that the polyphenols Quercetin and Tannic acid are capable of extending the lifespan of C. elegans. To gain a deep understanding of the underlying molecular genetics, we analyzed the global transcriptional patterns of nematodes exposed to Quercetin or Tannic acid concentrations that are non-effective (in lifespan extension, lifespan extending or toxic. By means of an intricate meta-analysis it was possible to compare the transcriptomes of polyphenol exposure to recently published data sets derived from i longevity mutants or ii infection. This detailed comparative in silico analysis facilitated the identification of compound specific and overlapping transcriptional profiles and allowed the formulation of mechanistic models of Quercetin and Tannic acid mediated longevity. Lifespan extension due to Quercetin was predominantly driven by the metabolome, TGF-beta signaling, Insulin-like signaling and the p38 MAPK pathway and Tannic acid’s impact involved, in part, the amino acid metabolism and was modulated by the TGF-beta and the p38 MAPK pathways. DAF-12, which integrates TGF-beta and Insulin-like downstream signaling, therefore seems to be a crucial regulator for both polyphenols.

  3. A transcription activator-like effector (TALE) induction system mediated by proteolysis.

    Science.gov (United States)

    Copeland, Matthew F; Politz, Mark C; Johnson, Charles B; Markley, Andrew L; Pfleger, Brian F

    2016-04-01

    Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic.

  4. Transcriptional regulator-mediated activation of adaptation genes triggers CRISPR de novo spacer acquisition

    DEFF Research Database (Denmark)

    Liu, Tao; Li, Yingjun; Wang, Xiaodi

    2015-01-01

    Acquisition of de novo spacer sequences confers CRISPR-Cas with a memory to defend against invading genetic elements. However, the mechanism of regulation of CRISPR spacer acquisition remains unknown. Here we examine the transcriptional regulation of the conserved spacer acquisition genes in Type I......, it was demonstrated that the transcription level of csa1, cas1, cas2 and cas4 was significantly enhanced in a csa3a-overexpression strain and, moreover, the Csa1 and Cas1 protein levels were increased in this strain. Furthermore, we demonstrated the hyperactive uptake of unique spacers within both CRISPR loci...... in the presence of the csa3a overexpression vector. The spacer acquisition process is dependent on the CCN PAM sequence and protospacer selection is random and non-directional. These results suggested a regulation mechanism of CRISPR spacer acquisition where a single transcriptional regulator senses the presence...

  5. Visual Detection of Potato leafroll virus by One-step Reverse Transcription Loop-Mediated Isothermal Amplification of DNA with Hydroxynaphthol Blue Dye

    NARCIS (Netherlands)

    Ahmadi, S.; Almasi, A.M.; Fatehi, F.; Struik, P.C.; Moradi, A.

    2013-01-01

    Loop-mediated isothermal amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. We applied reverse transcription loop-mediated isothermal amplification (RT-LAMP) to visually detect Potato leafroll

  6. Exploring transcriptional signalling mediated by OsWRKY13, a potential regulator of multiple physiological processes in rice

    Directory of Open Access Journals (Sweden)

    Li Xianghua

    2009-06-01

    Full Text Available Abstract Background Rice transcription regulator OsWRKY13 influences the functioning of more than 500 genes in multiple signalling pathways, with roles in disease resistance, redox homeostasis, abiotic stress responses, and development. Results To determine the putative transcriptional regulation mechanism of OsWRKY13, the putative cis-acting elements of OsWRKY13-influenced genes were analyzed using the whole genome expression profiling of OsWRKY13-activated plants generated with the Affymetrix GeneChip Rice Genome Array. At least 39 transcription factor genes were influenced by OsWRKY13, and 30 of them were downregulated. The promoters of OsWRKY13-upregulated genes were overrepresented with W-boxes for WRKY protein binding, whereas the promoters of OsWRKY13-downregulated genes were enriched with cis-elements putatively for binding of MYB and AP2/EREBP types of transcription factors. Consistent with the distinctive distribution of these cis-elements in up- and downregulated genes, nine WRKY genes were influenced by OsWRKY13 and the promoters of five of them were bound by OsWRKY13 in vitro; all seven differentially expressed AP2/EREBP genes and six of the seven differentially expressed MYB genes were suppressed by in OsWRKY13-activated plants. A subset of OsWRKY13-influenced WRKY genes were involved in host-pathogen interactions. Conclusion These results suggest that OsWRKY13-mediated signalling pathways are partitioned by different transcription factors. WRKY proteins may play important roles in the monitoring of OsWRKY13-upregulated genes and genes involved in pathogen-induced defence responses, whereas MYB and AP2/EREBP proteins may contribute most to the control of OsWRKY13-downregulated genes.

  7. Modulation of the nitrate reductase transcript by cytokinin and abscisic acid in etiolated barley seedlings

    International Nuclear Information System (INIS)

    Lu, Jia-ling; Enl, J.R.; Chen, Chong-maw

    1989-01-01

    To investigate the molecular mechanism of the hormonal modulation of nitrate reductase (NR) activity, the influence of benzyladenine (BA) and/or abscisic acid (ABA) on the level of NR poly(A)RNA was studied in etiolated barley seedlings using a 32 P-labelled NR cDNA as a probe. Enhancement of NR activity by 2 x 10 -5 M BA was measurable only after 60 minutes of exposure of the seedlings to light, while a significant stimulatory effect on the transcript level could by clearly detected within 15 minutes. Northern blot analyses of the levels of NR poly(A)RNA indicate that the amount present is proportional to the concentration of BA applied to the seedlings. The stimulatory effects seen for BA were nullified by ABA. The counteractive effects of ABA on BA were dose-responsive, with greater inhibition at higher concentrations of ABA. Evidence suggests that the interaction of BA and ABA on NR activity is at the transcriptional level, however, is also possible that interactions occur at the postranscriptional level as well

  8. Transcription factor TLX1 controls retinoic acid signaling to ensure spleen development

    Science.gov (United States)

    Lenti, Elisa; Farinello, Diego; Penkov, Dmitry; Castagnaro, Laura; Lavorgna, Giovanni; Wuputra, Kenly; Tjaden, Naomi E. Butler; Bernassola, Francesca; Caridi, Nicoletta; Wagner, Michael; Kozinc, Katja; Niederreither, Karen; Blasi, Francesco; Pasini, Diego; Trainor, Paul A.

    2016-01-01

    The molecular mechanisms that underlie spleen development and congenital asplenia, a condition linked to increased risk of overwhelming infections, remain largely unknown. The transcription factor TLX1 controls cell fate specification and organ expansion during spleen development, and Tlx1 deletion causes asplenia in mice. Deregulation of TLX1 expression has recently been proposed in the pathogenesis of congenital asplenia in patients carrying mutations of the gene-encoding transcription factor SF-1. Herein, we have shown that TLX1-dependent regulation of retinoic acid (RA) metabolism is critical for spleen organogenesis. In a murine model, loss of Tlx1 during formation of the splenic anlage increased RA signaling by regulating several genes involved in RA metabolism. Uncontrolled RA activity resulted in premature differentiation of mesenchymal cells and reduced vasculogenesis of the splenic primordium. Pharmacological inhibition of RA signaling in Tlx1-deficient animals partially rescued the spleen defect. Finally, spleen growth was impaired in mice lacking either cytochrome P450 26B1 (Cyp26b1), which results in excess RA, or retinol dehydrogenase 10 (Rdh10), which results in RA deficiency. Together, these findings establish TLX1 as a critical regulator of RA metabolism and provide mechanistic insights into the molecular determinants of human congenital asplenia. PMID:27214556

  9. Activation of Nrf2-mediated oxidative stress response in macrophages by hypochlorous acid

    International Nuclear Information System (INIS)

    Pi Jingbo; Zhang Qiang; Woods, Courtney G.; Wong, Victoria; Collins, Sheila; Andersen, Melvin E.

    2008-01-01

    Hypochlorous acid (HOCl), a potent oxidant generated when chlorine gas reacts with water, is important in the pathogenesis of many disorders. Transcription factor Nrf2-mediated antioxidant response represents a critical cellular defense mechanism that serves to maintain intracellular redox homeostasis and limit oxidative damage. In the present study, the effect of HOCl on Nrf2 activation was investigated in macrophages, one of the target cells of chlorine gas exposure. Exposure of RAW 264.7 macrophages to HOCl resulted in increased protein levels of Nrf2 in nuclear extractions, as well as a time- and dose-dependent increase in the expression of Nrf2 target genes, including heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 (NQO-1), glutamate cysteine ligase catalytic subunit (GCLC), and glutathione synthetase (GS). Additionally, intracellular glutathione (GSH), which is the prime scavenger for HOCl in cells, decreased within the first hour of HOCl exposure. The decline was followed by a GSH rebound that surpassed the initial basal levels by up to 4-fold. This reversal in GSH levels closely correlated with the gene expression profile of GCLC and GS. To study the mechanisms of Nrf2 activation in response to HOCl exposure, we examined the effects of several antioxidants on Nrf2-mediated response. Pretreatment with cell-permeable catalase, N-acetyl-L-cysteine or GSH-monoethyl ester markedly reduced expression of NQO-1 and GCLC under HOCl challenge conditions, suggesting intracellular ROS-scavenging capacity affects HOCl-induced Nrf2 activation. Importantly, pre-activation of Nrf2 with low concentrations of pro-oxidants protected the cells against HOCl-induced cell damage. Taken together, we provide direct evidence that HOCl activates Nrf2-mediated antioxidant response, which protects cells from oxidative damage

  10. The role of abscisic acid in regulating cucumber fruit development and ripening and its transcriptional regulation.

    Science.gov (United States)

    Wang, Yanping; Wang, Ya; Ji, Kai; Dai, Shengjie; Hu, Ying; Sun, Liang; Li, Qian; Chen, Pei; Sun, Yufei; Duan, Chaorui; Wu, Yan; Luo, Hao; Zhang, Dian; Guo, Yangdong; Leng, Ping

    2013-03-01

    Cucumber (Cucumis sativus L.), a kind of fruit usually harvested at the immature green stage, belongs to non-climacteric fruit. To investigate the contribution of abscisic acid (ABA) to cucumber fruit development and ripening, variation in ABA level was investigated and a peak in ABA level was found in pulp before fruit get fully ripe. To clarify this point further, exogenous ABA was applied to cucumber fruits at two different development stages. Results showed that ABA application at the turning stage promotes cucumber fruit ripening, while application at the immature green stage had inconspicuous effects. In addition, with the purpose of understanding the transcriptional regulation of ABA, two partial cDNAs of CsNCED1 and CsNCED2 encoding 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in ABA biosynthetic pathway; one partial cDNA of CsCYP707A1 for 8'-hydroxylase, a key enzyme in the oxidative catabolism of ABA and two partial cDNAs of CsBG1 and CsBG2 for β-glucosidase (BG) that hydrolyzes ABA glucose ester (ABA-GE) to release active ABA were cloned from cucumber. The DNA and deduced amino acid sequences of these obtained genes respectively showed high similarities to their homologous genes in other plants. Real-time PCR analysis revealed that ABA content may be regulated by its biosynthesis (CsNCEDs), catabolism (CsCYP707A1) and reactivation genes (CsBGs) at the transcriptional level during cucumber fruit development and ripening, in response to ABA application, dehydration and pollination, among which CsNCED1, CsCYP707A1 and CsBG1 were highly expressed in pulp and may play more important roles in regulating ABA metabolism. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  11. Altered cultivar resistance of kimchi cabbage seedlings mediated by salicylic Acid, jasmonic Acid and ethylene.

    Science.gov (United States)

    Lee, Young Hee; Kim, Sang Hee; Yun, Byung-Wook; Hong, Jeum Kyu

    2014-09-01

    Two cultivars Buram-3-ho (susceptible) and CR-Hagwang (moderate resistant) of kimchi cabbage seedlings showed differential defense responses to anthracnose (Colletotrichum higginsianum), black spot (Alternaria brassicicola) and black rot (Xanthomonas campestris pv. campestris, Xcc) diseases in our previous study. Defense-related hormones salicylic acid (SA), jasmonic acid (JA) and ethylene led to different transcriptional regulation of pathogenesis-related (PR) gene expression in both cultivars. In this study, exogenous application of SA suppressed basal defenses to C. higginsianum in the 1st leaves of the susceptible cultivar and cultivar resistance of the 2nd leaves of the resistant cultivar. SA also enhanced susceptibility of the susceptible cultivar to A. brassicicola. By contrast, SA elevated disease resistance to Xcc in the resistant cultivar, but not in the susceptible cultivar. Methyl jasmonate (MJ) treatment did not affect the disease resistance to C. higginsianum and Xcc in either cultivar, but it compromised the disease resistance to A. brassicicola in the resistant cultivar. Treatment with 1-aminocyclopropane-1-carboxylic acid (ACC) ethylene precursor did not change resistance of the either cultivar to C. higginsianum and Xcc. Effect of ACC pretreatment on the resistance to A. brassicicola was not distinguished between susceptible and resistant cultivars, because cultivar resistance of the resistant cultivar was lost by prolonged moist dark conditions. Taken together, exogenously applied SA, JA and ethylene altered defense signaling crosstalk to three diseases of anthracnose, black spot and black rot in a cultivar-dependent manner.

  12. Altered Cultivar Resistance of Kimchi Cabbage Seedlings Mediated by Salicylic Acid, Jasmonic Acid and Ethylene

    Directory of Open Access Journals (Sweden)

    Young Hee Lee

    2014-09-01

    Full Text Available Two cultivars Buram-3-ho (susceptible and CR-Hagwang (moderate resistant of kimchi cabbage seedlings showed differential defense responses to anthracnose (Colletotrichum higginsianum, black spot (Alternaria brassicicola and black rot (Xanthomonas campestris pv. campestris, Xcc diseases in our previous study. Defense-related hormones salicylic acid (SA, jasmonic acid (JA and ethylene led to different transcriptional regulation of pathogenesis-related (PR gene expression in both cultivars. In this study, exogenous application of SA suppressed basal defenses to C. higginsianum in the 1st leaves of the susceptible cultivar and cultivar resistance of the 2nd leaves of the resistant cultivar. SA also enhanced susceptibility of the susceptible cultivar to A. brassicicola. By contrast, SA elevated disease resistance to Xcc in the resistant cultivar, but not in the susceptible cultivar. Methyl jasmonate (MJ treatment did not affect the disease resistance to C. higginsianum and Xcc in either cultivar, but it compromised the disease resistance to A. brassicicola in the resistant cultivar. Treatment with 1-aminocyclopropane-1-carboxylic acid (ACC ethylene precursor did not change resistance of the either cultivar to C. higginsianum and Xcc. Effect of ACC pretreatment on the resistance to A. brassicicola was not distinguished between susceptible and resistant cultivars, because cultivar resistance of the resistant cultivar was lost by prolonged moist dark conditions. Taken together, exogenously applied SA, JA and ethylene altered defense signaling crosstalk to three diseases of anthracnose, black spot and black rot in a cultivar-dependent manner.

  13. CRISPR-Cas9-Mediated Genome Editing and Transcriptional Control in Yarrowia lipolytica.

    Science.gov (United States)

    Schwartz, Cory; Wheeldon, Ian

    2018-01-01

    The discovery and adaptation of RNA-guided nucleases has resulted in the rapid development of efficient, scalable, and easily accessible synthetic biology tools for targeted genome editing and transcriptional control. In these systems, for example CRISPR-Cas9 from Streptococcus pyogenes, a protein with nuclease activity is targeted to a specific nucleotide sequence by a short RNA molecule, whereupon binding it cleaves the targeted nucleotide strand. To extend this genome-editing ability to the industrially important oleaginous yeast Yarrowia lipolytica, we developed a set of easily usable and effective CRISPR-Cas9 episomal vectors. In this protocols chapter, we first present a method by which arbitrary protein-coding genes can be disrupted via indel formation after CRISPR-Cas9 targeting. A second method demonstrates how the same CRISPR-Cas9 system can be used to induce markerless gene cassette integration into the genome by inducing homologous recombination after DNA cleavage by Cas9. Finally, we describe how a catalytically inactive form of Cas9 fused to a transcriptional repressor can be used to control transcription of native genes in Y. lipolytica. The CRISPR-Cas9 tools and strategies described here greatly increase the types of genome editing and transcriptional control that can be achieved in Y. lipolytica, and promise to facilitate more advanced engineering of this important oleaginous host.

  14. Sox4 mediates Tbx3 transcriptional regulation of the gap junction protein Cx43

    NARCIS (Netherlands)

    Boogerd, C.J.; Wong, L.Y.; van den Boogaard, M.; Bakker, M.A.J.; Tessadori, F.; Bakkers, J.; 't Hoen, P.A.C.; Moorman, A.F.; Christoffels, V.M.; Barnett, P.

    2011-01-01

    Tbx3, a T-box transcription factor, regulates key steps in development of the heart and other organ systems. Here, we identify Sox4 as an interacting partner of Tbx3. Pull-down and nuclear retention assays verify this interaction and in situ hybridization reveals Tbx3 and Sox4 to co-localize

  15. Transcriptional response of bronchial epithelial cells to Pseudomonas aeruginosa: identification of early mediators of host defense.

    NARCIS (Netherlands)

    Vos, J.B.; Sterkenburg, M.A. van; Rabe, K.F.; Schalkwijk, J.; Hiemstra, P.S.; Datson, N.A.

    2005-01-01

    The airway epithelium responds to microbial exposure by altering expression of a variety of genes to increase innate host defense. We aimed to delineate the early transcriptional response in human primary bronchial epithelial cells exposed for 6 h to a mixture of IL-1beta and TNF-alpha or

  16. Putrescine biosynthesis in Lactococcus lactis is transcriptionally activated at acidic pH and counteracts acidification of the cytosol.

    Science.gov (United States)

    Del Rio, Beatriz; Linares, Daniel; Ladero, Victor; Redruello, Begoña; Fernandez, Maria; Martin, Maria Cruz; Alvarez, Miguel A

    2016-11-07

    Lactococcus lactis subsp. cremoris CECT 8666 is a lactic acid bacterium that synthesizes the biogenic amine putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The AGDI genes cluster includes aguR. This encodes a transmembrane protein that functions as a one-component signal transduction system, the job of which is to sense the agmatine concentration of the medium and accordingly regulate the transcription of the catabolic operon aguBDAC. The latter encodes the proteins necessary for agmatine uptake and its conversion into putrescine. This work reports the effect of extracellular pH on putrescine biosynthesis and on the genetic regulation of the AGDI pathway. Increased putrescine biosynthesis was detected at acidic pH (pH5) compared to neutral pH. Acidic pH induced the transcription of the catabolic operon via the activation of the aguBDAC promoter PaguB. However, the external pH had no significant effect on the activity of the aguR promoter PaguR, or on the transcription of the aguR gene. The transcriptional activation of the AGDI pathway was also found to require a lower agmatine concentration at pH5 than at neutral pH. Finally, the following of the AGDI pathway counteracted the acidification of the cytoplasm under acidic external conditions, suggesting it to provide protection against acid stress. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Ornithine Decarboxylase-Mediated Production of Putrescine Influences Ganoderic Acid Biosynthesis by Regulating Reactive Oxygen Species in Ganoderma lucidum.

    Science.gov (United States)

    Wu, Chen-Gao; Tian, Jia-Long; Liu, Rui; Cao, Peng-Fei; Zhang, Tian-Jun; Ren, Ang; Shi, Liang; Zhao, Ming-Wen

    2017-10-15

    Putrescine is an important polyamine that participates in a variety of stress responses. Ornithine decarboxylase (ODC) is a key enzyme that catalyzes the biosynthesis of putrescine. A homolog of the gene encoding ODC was cloned from Ganoderma lucidum In the ODC -silenced strains, the transcript levels of the ODC gene and the putrescine content were significantly decreased. The ODC -silenced strains were more sensitive to oxidative stress. The content of ganoderic acid was increased by approximately 43 to 46% in the ODC -silenced strains. The content of ganoderic acid could be recovered after the addition of exogenous putrescine. Additionally, the content of reactive oxygen species (ROS) was significantly increased by approximately 1.3-fold in the ODC -silenced strains. The ROS content was significantly reduced after the addition of exogenous putrescine. The gene transcript levels and the activities of four major antioxidant enzymes were measured to further explore the effect of putrescine on the intracellular ROS levels. Further studies showed that the effect of the ODC-mediated production of putrescine on ROS might be a factor influencing the biosynthesis of ganoderic acid. Our study reports the role of putrescine in large basidiomycetes, providing a basis for future studies of the physiological functions of putrescine in microbes. IMPORTANCE It is well known that ODC and the ODC-mediated production of putrescine play an important role in resisting various environmental stresses, but there are few reports regarding the mechanisms underlying the effect of putrescine on secondary metabolism in microorganisms, particularly in fungi. G. lucidum is gradually becoming a model organism for studying environmental regulation and metabolism. In this study, a homolog of the gene encoding ODC was cloned in Ganoderma lucidum We found that the transcript level of the ODC gene and the content of putrescine were significantly decreased in the ODC -silenced strains. The content of

  18. Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling.

    Science.gov (United States)

    Trusov, Yuri; Sewelam, Nasser; Rookes, James Edward; Kunkel, Matt; Nowak, Ekaterina; Schenk, Peer Martin; Botella, José Ramón

    2009-04-01

    Heterotrimeric G proteins are involved in the defense response against necrotrophic fungi in Arabidopsis. In order to elucidate the resistance mechanisms involving heterotrimeric G proteins, we analyzed the effects of the Gβ (subunit deficiency in the mutant agb1-2 on pathogenesis-related gene expression, as well as the genetic interaction between agb1-2 and a number of mutants of established defense pathways. Gβ-mediated signaling suppresses the induction of salicylic acid (SA)-, jasmonic acid (JA)-, ethylene (ET)- and abscisic acid (ABA)-dependent genes during the initial phase of the infection with Fusarium oxysporum (up to 48 h after inoculation). However, at a later phase it enhances JA/ET-dependent genes such as PDF1.2 and PR4. Quantification of the Fusarium wilt symptoms revealed that Gβ- and SA-deficient mutants were more susceptible than wild-type plants, whereas JA- and ET-insensitive and ABA-deficient mutants demonstrated various levels of resistance. Analysis of the double mutants showed that the Gβ-mediated resistance to F. oxysporum and Alternaria brassicicola was mostly independent of all of the previously mentioned pathways. However, the progressive decay of agb1-2 mutants was compensated by coi1-21 and jin1-9 mutations, suggesting that at this stage of F. oxysporum infection Gβ acts upstream of COI1 and ATMYC2 in JA signaling. © 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.

  19. Metabolic and Transcriptional Analysis of Acid Stress in Lactococcus lactis, with a Focus on the Kinetics of Lactic Acid Pools

    Science.gov (United States)

    Carvalho, Ana Lúcia; Turner, David L.; Fonseca, Luís L.; Solopova, Ana; Catarino, Teresa; Kuipers, Oscar P.; Voit, Eberhard O.; Neves, Ana Rute; Santos, Helena

    2013-01-01

    The effect of pH on the glucose metabolism of non-growing cells of L. lactis MG1363 was studied by in vivo NMR in the range 4.8 to 6.5. Immediate pH effects on glucose transporters and/or enzyme activities were distinguished from transcriptional/translational effects by using cells grown at the optimal pH of 6.5 or pre-adjusted to low pH by growth at 5.1. In cells grown at pH 5.1, glucose metabolism proceeds at a rate 35% higher than in non-adjusted cells at the same pH. Besides the upregulation of stress-related genes (such as dnaK and groEL), cells adjusted to low pH overexpressed H+-ATPase subunits as well as glycolytic genes. At sub-optimal pHs, the total intracellular pool of lactic acid reached approximately 500 mM in cells grown at optimal pH and about 700 mM in cells grown at pH 5.1. These high levels, together with good pH homeostasis (internal pH always above 6), imply intracellular accumulation of the ionized form of lactic acid (lactate anion), and the concomitant export of the equivalent protons. The average number, n, of protons exported with each lactate anion was determined directly from the kinetics of accumulation of intra- and extracellular lactic acid as monitored online by 13C-NMR. In cells non-adjusted to low pH, n varies between 2 and 1 during glucose consumption, suggesting an inhibitory effect of intracellular lactate on proton export. We confirmed that extracellular lactate did not affect the lactate: proton stoichiometry. In adjusted cells, n was lower and varied less, indicating a different mix of lactic acid exporters less affected by the high level of intracellular lactate. A qualitative model for pH effects and acid stress adaptation is proposed on the basis of these results. PMID:23844205

  20. Silencing of the Rice Gene LRR1 Compromises Rice Xa21 Transcript Accumulation and XA21-Mediated Immunity.

    Science.gov (United States)

    Caddell, Daniel F; Park, Chang-Jin; Thomas, Nicholas C; Canlas, Patrick E; Ronald, Pamela C

    2017-12-01

    The rice immune receptor XA21 confers resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. We previously demonstrated that an auxilin-like protein, XA21 BINDING PROTEIN 21 (XB21), positively regulates resistance to Xoo. To further investigate the function of XB21, we performed a yeast two-hybrid screen. We identified 22 unique XB21 interacting proteins, including LEUCINE-RICH REPEAT PROTEIN 1 (LRR1), which we selected for further analysis. Silencing of LRR1 in the XA21 genetic background (XA21-LRR1Ri) compromises resistance to Xoo compared with control XA21 plants. XA21-LRR1Ri plants have reduced Xa21 transcript levels and reduced expression of genes that serve as markers of XA21-mediated activation. Overexpression of LRR1 is insufficient to alter resistance to Xoo in rice lines lacking XA21. Taken together, our results indicate that LRR1 is required for wild-type Xa21 transcript expression and XA21-mediated immunity.

  1. Detection of enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Wang, D; Wang, X; Geng, Y; An, C

    2014-01-01

    The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD) for an early treatment by using loop-mediated isothermal amplification (LAMP) technique. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for detecting EV71 virus was developed, the specificity and sensitivity of RT-LAMP was tested, and the clinical specimens was assayed by the RT-LAMP comparing with conventional reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR. A total of 116 clinical specimens from the suspected HFMD individual were detected with the RT-LAMP. The detection rate for EV71 was 56.89% by RT-LAMP, 41.38% by real-time PCR and 34.48% by RT-PCR. The minimum detection limit of RT-LAMP was 0.01 PFU, both of RT-PCR and real-time PCR was 0.1PFU. Non-cross-reactive amplification with other enteroviruses was detected in the survey reports. The effectiveness of RT-LAMP is higher than RT-PCR and real-time PCR. The protocol is easy to operate and time saving. It was not an expensive instrument, which was needed; it is an applicable method for rapid diagnosis of the disease, especially in resource-poor countries or in developing countries.

  2. Detection of Enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    D Wang

    2014-01-01

    Full Text Available Purpose : The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD for an early treatment by using loop-mediated isothermal amplification (LAMP technique. Materials and Methods : A reverse-transcription loop-mediated isothermal amplification (RT-LAMP for detecting EV71 virus was developed, the specificity and sensitivity of RT-LAMP was tested, and the clinical specimens was assayed by the RT-LAMP comparing with conventional reverse-transcription polymerase chain reaction (RT-PCR and real-time PCR. Results : A total of 116 clinical specimens from the suspected HFMD individual were detected with the RT-LAMP. The detection rate for EV71 was 56.89% by RT-LAMP, 41.38% by real-time PCR and 34.48% by RT-PCR. The minimum detection limit of RT-LAMP was 0.01 PFU, both of RT-PCR and real-time PCR was 0.1PFU. Non-cross-reactive amplification with other enteroviruses was detected in the survey reports. Conclusions : The effectiveness of RT-LAMP is higher than RT-PCR and real-time PCR. The protocol is easy to operate and time saving. It was not an expensive instrument, which was needed; it is an applicable method for rapid diagnosis of the disease, especially in resource-poor countries or in developing countries.

  3. Development of mRNA-based body fluid identification using reverse transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Satoh, Tetsuya; Kouroki, Seiya; Ogawa, Keita; Tanaka, Yorika; Matsumura, Kazutoshi; Iwase, Susumu

    2018-04-25

    Identifying body fluids from forensic samples can provide valuable evidence for criminal investigations. Messenger RNA (mRNA)-based body fluid identification was recently developed, and highly sensitive parallel identification using reverse transcription polymerase chain reaction (RT-PCR) has been described. In this study, we developed reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a simple, rapid assay for identifying three common forensic body fluids, namely blood, semen, and saliva, and evaluated its specificity and sensitivity. Hemoglobin beta (HBB), transglutaminase 4 (TGM4), and statherin (STATH) were selected as marker genes for blood, semen, and saliva, respectively. RT-LAMP could be performed in a single step including both reverse transcription and DNA amplification under an isothermal condition within 60 min, and detection could be conveniently performed via visual fluorescence. Marker-specific amplification was performed in each assay, and no cross-reaction was observed among five representative forensically relevant body fluids. The detection limits of the assays were 0.3 nL, 30 nL, and 0.3 μL for blood, semen, and saliva, respectively, and their sensitivities were comparable with those of RT-PCR. Furthermore, RT-LAMP assays were applicable to forensic casework samples. It is considered that RT-LAMP is useful for body fluid identification.

  4. Photochemical decomposition of perfluorooctanoic acid mediated by iron in strongly acidic conditions

    Energy Technology Data Exchange (ETDEWEB)

    Ohno, Masaki, E-mail: mohno@hiroshima-u.ac.jp [Environmental Research and Management Center, Hiroshima University, 1-5-3 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8513 (Japan); Ito, Masataka; Ohkura, Ryouichi [Faculty of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1, Higashijima, Akiha-ku, Niigata 956-8603 (Japan); Mino A, Esteban R. [Environmental Research and Management Center, Hiroshima University, 1-5-3 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8513 (Japan); Kose, Tomohiro [Faculty of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1, Higashijima, Akiha-ku, Niigata 956-8603 (Japan); Okuda, Tetsuji [Environmental Research and Management Center, Hiroshima University, 1-5-3 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8513 (Japan); Nakai, Satoshi [Department of Chemical Engineering, Graduate School of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8527 (Japan); Kawata, Kuniaki [Faculty of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1, Higashijima, Akiha-ku, Niigata 956-8603 (Japan); Nishijima, Wataru [Environmental Research and Management Center, Hiroshima University, 1-5-3 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8513 (Japan)

    2014-03-01

    Highlights: • Perfluorooctanoic acid (PFOA) was decomposed based on ferric ion performance. • Complete decomposition of PFOA was confirmed in strongly acidic conditions. • Fe{sup 2+} changed to Fe{sup 3+} to restore chemical equilibrium in this condition. • Fe{sup 3+} was only produced from Fe{sup 2+} by hydroxyl radical in weakly acidic conditions. • The Fe{sup 3+} regeneration mechanisms resulted in the performance of Fe{sup 3+} for PFOA. - Abstract: The performance of a ferric ion mediated photochemical process for perfluorooctanoic acid (PFOA) decomposition in strongly acidic conditions of pH 2.0 was evaluated in comparison with those in weakly acidic conditions, pH 3.7 or pH 5.0, based on iron species composition and ferric ion regeneration. Complete decomposition of PFOA under UV irradiation was confirmed at pH 2.0, whereas perfluoroheptanoic acid (PFHpA) and other intermediates were accumulated in weakly acidic conditions. Iron states at each pH were evaluated using a chemical equilibrium model, Visual MINTEQ. The main iron species at pH 2.0 is Fe{sup 3+} ion. Although Fe{sup 3+} ion is consumed and is transformed to Fe{sup 2+} ion by photochemical decomposition of PFOA and its intermediates, the produced Fe{sup 2+} ion will change to Fe{sup 3+} ion to restore chemical equilibrium. Continuous decomposition will occur at pH 2.0. However, half of the iron cannot be dissolved at pH 3.7. The main species of dissolved iron is Fe(OH){sup 2+}. At pH 3.7 or higher pH, Fe{sup 3+} ion will only be produced from the oxidation of Fe{sup 2+} ion by hydroxyl radical produced by Fe(OH){sup 2+} under UV irradiation. These different mechanisms of Fe{sup 3+} regeneration that prevail in strongly and weakly acidic conditions will engender different performances of the ferric ion.

  5. Unique CCT repeats mediate transcription of the TWIST1 gene in mesenchymal cell lines

    International Nuclear Information System (INIS)

    Ohkuma, Mizue; Funato, Noriko; Higashihori, Norihisa; Murakami, Masanori; Ohyama, Kimie; Nakamura, Masataka

    2007-01-01

    TWIST1, a basic helix-loop-helix transcription factor, plays critical roles in embryo development, cancer metastasis and mesenchymal progenitor differentiation. Little is known about transcriptional regulation of TWIST1 expression. Here we identified DNA sequences responsible for TWIST1 expression in mesenchymal lineage cell lines. Reporter assays with TWIST1 promoter mutants defined the -102 to -74 sequences that are essential for TWIST1 expression in human and mouse mesenchymal cell lines. Tandem repeats of CCT, but not putative CREB and NF-κB sites in the sequences substantially supported activity of the TWIST1 promoter. Electrophoretic mobility shift assay demonstrated that the DNA sequences with the CCT repeats formed complexes with nuclear factors, containing, at least, Sp1 and Sp3. These results suggest critical implication of the CCT repeats in association with Sp1 and Sp3 factors in sustaining expression of the TWIST1 gene in mesenchymal cells

  6. Fucose-Mediated Transcriptional Activation of the fcs Operon by FcsR in Streptococcus pneumoniae.

    Science.gov (United States)

    Manzoor, Irfan; Shafeeq, Sulman; Afzal, Muhammad; Kuipers, Oscar P

    2015-01-01

    In this study, we explore the impact of fucose on the transcriptome of S. pneumoniae D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (fcs operon) was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the fcs operon, as a transcriptional activator of the fcs operon. We also predict a 19-bp putative FcsR regulatory site (5'-ATTTGAACATTATTCAAGT-3') in the promoter region of the fcs operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the fcs operon. © 2015 S. Karger AG, Basel.

  7. The Role of Estrogen Related Receptor in Modulating Estrogen Receptor Mediated Transcription in Breast Cancer Cells

    Science.gov (United States)

    2005-04-01

    tumors correlates with an unfavorable prognosis (Ariazi 2002; Lu 2001; Suzuki 2004; Vanacker 1999). The transcriptional activity of ERRa is not inhibited...SA. 101:6570-5. Needham, M ., S. Raines, J. McPheat, C. Stacey, J. Ellston, S. Hoare, and M . Parker. 2000. Differential interaction of steroid hormone...R. Graves, M . Wright, and B.M. Spiegelman. 1998. A cold- inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell. 92:829- 39

  8. Enhanced oxygen consumption in Herbaspirillum seropedicae fnr mutants leads to increased NifA mediated transcriptional activation.

    Science.gov (United States)

    Batista, Marcelo Bueno; Wassem, Roseli; Pedrosa, Fábio de Oliveira; de Souza, Emanuel Maltempi; Dixon, Ray; Monteiro, Rose Adele

    2015-05-07

    Orthologous proteins of the Crp/Fnr family have been previously implicated in controlling expression and/or activity of the NifA transcriptional activator in some diazotrophs. This study aimed to address the role of three Fnr-like proteins from H. seropedicae SmR1 in controlling NifA activity and consequent NifA-mediated transcription activation. The activity of NifA-dependent transcriptional fusions (nifA::lacZ and nifB::lacZ) was analysed in a series of H. seropedicae fnr deletion mutant backgrounds. We found that combined deletions in both the fnr1 and fnr3 genes lead to higher expression of both the nifA and nifB genes and also an increased level of nifH transcripts. Expression profiles of nifB under different oxygen concentrations, together with oxygen consumption measurements suggest that the triple fnr mutant has higher respiratory activity when compared to the wild type, which we believe to be responsible for greater stability of the oxygen sensitive NifA protein. This conclusion was further substantiated by measuring the levels of NifA protein and its activity in fnr deletion strains in comparison with the wild-type. Fnr proteins are indirectly involved in controlling the activity of NifA in H. seropedicae, probably as a consequence of their influence on respiratory activity in relation to oxygen availability. Additionally we can suggest that there is some redundancy in the physiological function of the three Fnr paralogs in this organism, since altered respiration and effects on NifA activity are only observed in deletion strains lacking both fnr1 and fnr3.

  9. Tumour–stromal interactions in acid-mediated invasion: A mathematical model

    KAUST Repository

    Martin, Natasha K.; Gaffney, Eamonn A.; Gatenby, Robert A.; Maini, Philip K.

    2010-01-01

    constraint. In particular, the production of acid by tumour cells and the subsequent creation of a low extracellular pH environment has been explored in several 'acid-mediated tumour invasion' models where the acidic environment facilitates normal cell death

  10. Human Galectin-9 Is a Potent Mediator of HIV Transcription and Reactivation.

    Directory of Open Access Journals (Sweden)

    Mohamed Abdel-Mohsen

    2016-06-01

    Full Text Available Identifying host immune determinants governing HIV transcription, latency and infectivity in vivo is critical to developing an HIV cure. Based on our recent finding that the host factor p21 regulates HIV transcription during antiretroviral therapy (ART, and published data demonstrating that the human carbohydrate-binding immunomodulatory protein galectin-9 regulates p21, we hypothesized that galectin-9 modulates HIV transcription. We report that the administration of a recombinant, stable form of galectin-9 (rGal-9 potently reverses HIV latency in vitro in the J-Lat HIV latency model. Furthermore, rGal-9 reverses HIV latency ex vivo in primary CD4+ T cells from HIV-infected, ART-suppressed individuals (p = 0.002, more potently than vorinostat (p = 0.02. rGal-9 co-administration with the latency reversal agent "JQ1", a bromodomain inhibitor, exhibits synergistic activity (p<0.05. rGal-9 signals through N-linked oligosaccharides and O-linked hexasaccharides on the T cell surface, modulating the gene expression levels of key transcription initiation, promoter proximal-pausing, and chromatin remodeling factors that regulate HIV latency. Beyond latent viral reactivation, rGal-9 induces robust expression of the host antiviral deaminase APOBEC3G in vitro and ex vivo (FDR<0.006 and significantly reduces infectivity of progeny virus, decreasing the probability that the HIV reservoir will be replenished when latency is reversed therapeutically. Lastly, endogenous levels of soluble galectin-9 in the plasma of 72 HIV-infected ART-suppressed individuals were associated with levels of HIV RNA in CD4+ T cells (p<0.02 and with the quantity and binding avidity of circulating anti-HIV antibodies (p<0.009, suggesting a role of galectin-9 in regulating HIV transcription and viral production in vivo during therapy. Our data suggest that galectin-9 and the host glycosylation machinery should be explored as foundations for novel HIV cure strategies.

  11. The Signal Transducer and Activator of Transcription 1 (STAT1 Inhibits Mitochondrial Biogenesis in Liver and Fatty Acid Oxidation in Adipocytes.

    Directory of Open Access Journals (Sweden)

    Jennifer D Sisler

    Full Text Available The transcription factor STAT1 plays a central role in orchestrating responses to various pathogens by activating the transcription of nuclear-encoded genes that mediate the antiviral, the antigrowth, and immune surveillance effects of interferons and other cytokines. In addition to regulating gene expression, we report that STAT1-/- mice display increased energy expenditure and paradoxically decreased release of triglycerides from white adipose tissue (WAT. Liver mitochondria from STAT1-/- mice show both defects in coupling of the electron transport chain (ETC and increased numbers of mitochondria. Consistent with elevated numbers of mitochondria, STAT1-/- mice expressed increased amounts of PGC1α, a master regulator of mitochondrial biogenesis. STAT1 binds to the PGC1α promoter in fed mice but not in fasted animals, suggesting that STAT1 inhibited transcription of PGC1α. Since STAT1-/- mice utilized more lipids we examined white adipose tissue (WAT stores. Contrary to expectations, fasted STAT1-/- mice did not lose lipid from WAT. β-adrenergic stimulation of glycerol release from isolated STAT1-/- WAT was decreased, while activation of hormone sensitive lipase was not changed. These findings suggest that STAT1-/- adipose tissue does not release glycerol and that free fatty acids (FFA re-esterify back to triglycerides, thus maintaining fat mass in fasted STAT1-/- mice.

  12. Untangling the Effect of Fatty Acid Addition at Species Level Revealed Different Transcriptional Responses of the Biogas Microbial Community Members

    DEFF Research Database (Denmark)

    Treu, Laura; Campanaro, Stefano; Kougias, Panagiotis

    2016-01-01

    In the present study, RNA-sequencing was used to elucidate the change of anaerobic digestion metatranscriptome after long chain fatty acids (oleate) exposure. To explore the general transcriptional behavior of the microbiome, the analysis was first performed on shotgun reads without considering...

  13. IRF-4-mediated CIITA transcription is blocked by KSHV encoded LANA to inhibit MHC II presentation.

    Directory of Open Access Journals (Sweden)

    Qiliang Cai

    2013-10-01

    Full Text Available Peptides presentation to T cells by MHC class II molecules is of importance in initiation of immune response to a pathogen. The level of MHC II expression directly influences T lymphocyte activation and is often targeted by various viruses. Kaposi's sarcoma-associated herpesvirus (KSHV encoded LANA is known to evade MHC class I peptide processing, however, the effect of LANA on MHC class II remains unclear. Here, we report that LANA down-regulates MHC II expression and presentation by inhibiting the transcription of MHC II transactivator (CIITA promoter pIII and pIV in a dose-dependent manner. Strikingly, although LANA knockdown efficiently disrupts the inhibition of CIITA transcripts from its pIII and pIV promoter region, the expression of HLA-DQβ but no other MHC II molecules was significantly restored. Moreover, we revealed that the presentation of HLA-DQβ enhanced by LANA knockdown did not help LANA-specific CD4+ T cell recognition of PEL cells, and the inhibition of CIITA by LANA is independent of IL-4 or IFN-γ signaling but dependent on the direct interaction of LANA with IRF-4 (an activator of both the pIII and pIV CIITA promoters. This interaction dramatically blocked the DNA-binding ability of IRF-4 on both pIII and pIV promoters. Thus, our data implies that LANA can evade MHC II presentation and suppress CIITA transcription to provide a unique strategy of KSHV escape from immune surveillance by cytotoxic T cells.

  14. Karrikins delay soybean seed germination by mediating abscisic acid and gibberellin biogenesis under shaded conditions

    Science.gov (United States)

    Meng, Yongjie; Chen, Feng; Shuai, Haiwei; Luo, Xiaofeng; Ding, Jun; Tang, Shengwen; Xu, Shuanshuan; Liu, Jianwei; Liu, Weiguo; Du, Junbo; Liu, Jiang; Yang, Feng; Sun, Xin; Yong, Taiwen; Wang, Xiaochun; Feng, Yuqi; Shu, Kai; Yang, Wenyu

    2016-01-01

    Karrikins (KAR) are a class of signal compounds, discovered in wildfire smoke, which affect seed germination. Currently, numerous studies have focused on the model plant Arabidopsis in the KAR research field, rather than on crops. Thus the regulatory mechanisms underlying KAR regulation of crop seed germination are largely unknown. Here, we report that KAR delayed soybean seed germination through enhancing abscisic acid (ABA) biosynthesis, while impairing gibberellin (GA) biogenesis. Interestingly, KAR only retarded soybean seed germination under shaded conditions, rather than under dark and white light conditions, which differs from in Arabidopsis. Phytohormone quantification showed that KAR enhanced ABA biogenesis while impairing GA biosynthesis during the seed imbibition process, and subsequently, the ratio of active GA4 to ABA was significantly reduced. Further qRT-PCR analysis showed that the transcription pattern of genes involved in ABA and GA metabolic pathways are consistent with the hormonal measurements. Finally, fluridone, an ABA biogenesis inhibitor, remarkably rescued the delayed-germination phenotype of KAR-treatment; and paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Taken together, these evidences suggest that KAR inhibit soybean seed germination by mediating the ratio between GA and ABA biogenesis. PMID:26902640

  15. Karrikins delay soybean seed germination by mediating abscisic acid and gibberellin biogenesis under shaded conditions.

    Science.gov (United States)

    Meng, Yongjie; Chen, Feng; Shuai, Haiwei; Luo, Xiaofeng; Ding, Jun; Tang, Shengwen; Xu, Shuanshuan; Liu, Jianwei; Liu, Weiguo; Du, Junbo; Liu, Jiang; Yang, Feng; Sun, Xin; Yong, Taiwen; Wang, Xiaochun; Feng, Yuqi; Shu, Kai; Yang, Wenyu

    2016-02-23

    Karrikins (KAR) are a class of signal compounds, discovered in wildfire smoke, which affect seed germination. Currently, numerous studies have focused on the model plant Arabidopsis in the KAR research field, rather than on crops. Thus the regulatory mechanisms underlying KAR regulation of crop seed germination are largely unknown. Here, we report that KAR delayed soybean seed germination through enhancing abscisic acid (ABA) biosynthesis, while impairing gibberellin (GA) biogenesis. Interestingly, KAR only retarded soybean seed germination under shaded conditions, rather than under dark and white light conditions, which differs from in Arabidopsis. Phytohormone quantification showed that KAR enhanced ABA biogenesis while impairing GA biosynthesis during the seed imbibition process, and subsequently, the ratio of active GA4 to ABA was significantly reduced. Further qRT-PCR analysis showed that the transcription pattern of genes involved in ABA and GA metabolic pathways are consistent with the hormonal measurements. Finally, fluridone, an ABA biogenesis inhibitor, remarkably rescued the delayed-germination phenotype of KAR-treatment; and paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Taken together, these evidences suggest that KAR inhibit soybean seed germination by mediating the ratio between GA and ABA biogenesis.

  16. Malaria infectivity of xanthurenic acid-deficient anopheline mosquitoes produced by TALEN-mediated targeted mutagenesis.

    Science.gov (United States)

    Yamamoto, Daisuke S; Sumitani, Megumi; Hatakeyama, Masatsugu; Matsuoka, Hiroyuki

    2018-02-01

    Anopheline mosquitoes are major vectors of malaria parasites. When the gametocytes of the malaria parasite are transferred from a vertebrate to mosquitoes, they differentiate into gametes, and are fertilized in the midguts of mosquitoes. Xanthurenic acid (XA), a waste product of the ommochrome synthesis pathway, has been shown to induce exflagellation during microgametogenesis in vitro; however, it currently remains unclear whether endogenous XA affects the infectivity of anopheline mosquitoes to malaria parasites in vivo due to the lack of appropriate experimental systems such as a XA-deficient line. In the present study, we produced a XA-deficient line in Anopheles stephensi using transcription activator-like effector nuclease (TALEN)-mediated gene targeting (knockout) of the kynurenine 3-monooxygenase (kmo) gene, which encodes an enzyme that participates in the ommochrome synthesis pathway. The knockout of kmo resulted in the absence of XA, and oocyst formation was inhibited in the midguts of these XA-deficient mosquitoes, which, in turn, reduced sporozoite numbers in their salivary glands. These results suggest that endogenous XA stimulates exflagellation, and enhances the infectivity of anopheline mosquitoes to malaria parasites in vivo. The XA-deficient line of the anopheline mosquito provides a useful system for analyzing and understanding the associated factors of malaria gametogenesis in the mosquito midgut.

  17. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription

    OpenAIRE

    Lai, Fan; Orom, Ulf A; Cesaroni, Matteo; Beringer, Malte; Taatjes, Dylan J; Blobel, Gerd A.; Shiekhattar, Ramin

    2013-01-01

    Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms 1-8 . While the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X inactivation and imprinting, different classes of lncRNAs may have varied biological functions 8-13 . We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their n...

  18. Paramutation of tobacco transgenes by small RNA-mediated transcriptional gene silencing

    Czech Academy of Sciences Publication Activity Database

    Crhák Khaitová, Lucie; Fojtová, M.; Křížová, Kateřina; Lunerová Bedřichová, Jana; Fulneček, Jaroslav; Depicker, A.; Kovařík, Aleš

    2011-01-01

    Roč. 6, č. 5 (2011), s. 650-660 ISSN 1559-2294 R&D Projects: GA ČR(CZ) GD204/09/H002; GA MŠk(CZ) LC06004 Grant - others:GA ČR(CZ) GPP501/11/P667 Program:GP Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : transcriptional gene silencing * transgene epialleles * DNA methylation Subject RIV: BO - Biophysics Impact factor: 4.318, year: 2011

  19. ATM-mediated transcriptional and developmental responses to gamma-rays in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Lilian Ricaud

    Full Text Available ATM (Ataxia Telangiectasia Mutated is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT. To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT and homozygous ATM-deficient mutants challenged with a dose of gamma-rays (IR that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post-IR were compiled in a single table, which was used to import gene information and extract gene sets. Sequence and function homology searches; import of spatio-temporal, cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes in all functional classes. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA replication, repair, and recombination; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints. Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of

  20. The transcription factor Krüppel homolog 1 is linked to hormone mediated social organization in bees

    Directory of Open Access Journals (Sweden)

    Fan Yongliang

    2010-04-01

    Full Text Available Abstract Background Regulation of worker behavior by dominant queens or workers is a hallmark of insect societies, but the underlying molecular mechanisms and their evolutionary conservation are not well understood. Honey bee and bumble bee colonies consist of a single reproductive queen and facultatively sterile workers. The queens' influences on the workers are mediated largely via inhibition of juvenile hormone titers, which affect division of labor in honey bees and worker reproduction in bumble bees. Studies in honey bees identified a transcription factor, Krüppel-homolog 1 (Kr-h1, whose expression in worker brains is significantly downregulated in the presence of a queen or queen pheromone and higher in forager bees, making this gene an ideal candidate for examining the evolutionary conservation of socially regulated pathways in Hymenoptera. Results In contrast to honey bees, bumble bees foragers do not have higher Kr-h1 levels relative to nurses: in one of three colonies levels were similar in nurses and foragers, and in two colonies levels were higher in nurses. Similarly to honey bees, brain Kr-h1 levels were significantly downregulated in the presence versus absence of a queen. Furthermore, in small queenless groups, Kr-h1 levels were downregulated in subordinate workers with undeveloped ovaries relative to dominant individuals with active ovaries. Brain Kr-h1 levels were upregulated by juvenile hormone treatment relative to a vehicle control. Finally, phylogenetic analysis indicates that KR-H1 orthologs are presence across insect orders. Though this protein is highly conserved between honey bees and bumble bees, there are significant differences between orthologs of insects from different orders. Conclusions Our results suggest that Kr-h1 is associated with juvenile hormone mediated regulation of reproduction in bumble bees. The expression of this transcription factor is inhibited by the queen and associated with endocrine mediated

  1. Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression.

    Science.gov (United States)

    Lomniczi, Alejandro; Wright, Hollis; Castellano, Juan Manuel; Matagne, Valerie; Toro, Carlos A; Ramaswamy, Suresh; Plant, Tony M; Ojeda, Sergio R

    2015-12-16

    In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty.

  2. Ultraviolet-B effects on transcript levels for photosynthetic genes are not mediated through carbohydrate metabolism

    International Nuclear Information System (INIS)

    Mackerness, S.A.H.; Surplus, S.L.; Jordan, B.R.; Thomas, B.

    1997-01-01

    The relationship between UV-B-induced changes in gene expression and carbohydrate levels in pea seedlings has been investigated. The effect of supplementary UV-B radiation on the transcript abundance for two photosynthetic genes, photosynthesis, respiration and the levels of carbohydrates was determined in fully expanded leaves of 17-d-old pea seedlings under high (HL: 350 μmol m −2 s −1 ) and low (LL: 150 μmol m −2 s 1 ) light. Supplementary UV-B caused down regulation of the photosynthetic genes in green leaves under LL and to a lesser extent under HL. In contrast to previous studies, UV-B radiation resulted in a decrease in glucose levels rather than an increase under LL. Sucrose and starch levels were not affected until longer exposure. Effects of UV-B on carbohydrate levels were, however, minimal under HL. The effects on transcript levels were most marked under LL and therefore could not be attributed to elevated carbohydrate levels. Comparison of UV-B effects on carbohydrates in source (leaf) and sink (green buds) organs indicated that changes in carbohydrates in response to UV-B are probably indirect and arise from effects of UV-B on photosynthesis in source organs. (author)

  3. Development of novel metabolite-responsive transcription factors via transposon-mediated protein fusion.

    Science.gov (United States)

    Younger, Andrew K D; Su, Peter Y; Shepard, Andrea J; Udani, Shreya V; Cybulski, Thaddeus R; Tyo, Keith E J; Leonard, Joshua N

    2018-02-01

    Naturally evolved metabolite-responsive biosensors enable applications in metabolic engineering, ranging from screening large genetic libraries to dynamically regulating biosynthetic pathways. However, there are many metabolites for which a natural biosensor does not exist. To address this need, we developed a general method for converting metabolite-binding proteins into metabolite-responsive transcription factors-Biosensor Engineering by Random Domain Insertion (BERDI). This approach takes advantage of an in vitro transposon insertion reaction to generate all possible insertions of a DNA-binding domain into a metabolite-binding protein, followed by fluorescence activated cell sorting to isolate functional biosensors. To develop and evaluate the BERDI method, we generated a library of candidate biosensors in which a zinc finger DNA-binding domain was inserted into maltose binding protein, which served as a model well-studied metabolite-binding protein. Library diversity was characterized by several methods, a selection scheme was deployed, and ultimately several distinct and functional maltose-responsive transcriptional biosensors were identified. We hypothesize that the BERDI method comprises a generalizable strategy that may ultimately be applied to convert a wide range of metabolite-binding proteins into novel biosensors for applications in metabolic engineering and synthetic biology. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. MED25 is a mediator component of HNF4α-driven transcription leading to insulin secretion in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Eun Hee Han

    Full Text Available Unique nuclear receptor Hepatocyte Nuclear Factor 4α (HNF4α is an essential transcriptional regulator for early development and proper function of pancreatic ß-cells, and its mutations are monogenic causes of a dominant inherited form of diabetes referred to as Maturity Onset Diabetes of the Young 1 (MODY1. As a gene-specific transcription factor, HNF4α exerts its function through various molecular interactions, but its protein recruiting network has not been fully characterized. Here we report the identification of MED25 as one of the HNF4α binding partners in pancreatic ß-cells leading to insulin secretion which is impaired in MODY patients. MED25 is one of the subunits of the Mediator complex that is required for induction of RNA polymerase II transcription by various transcription factors including nuclear receptors. This HNF4α-MED25 interaction was initially identified by a yeast-two-hybrid method, confirmed by in vivo and in vitro analyses, and proven to be mediated through the MED25-LXXLL motif in a ligand-independent manner. Reporter-gene based transcription assays and siRNA/shRNA-based gene silencing approaches revealed that this interaction is crucial for full activation of HNF4α-mediated transcription, especially expression of target genes implicated in glucose-stimulated insulin secretion. Selected MODY mutations at the LXXLL motif binding pocket disrupt these interactions and cause impaired insulin secretion through a 'loss-of-function' mechanism.

  5. Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Dukes, J.P.; King, D.P.; Alexandersen, Søren

    2006-01-01

    Speed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD virus (FMDV) to be detected in under an hour...... in a single tube without thermal cycling. A fragment of the 3D RNA polymerase gene of the virus is amplified at 65 degrees C in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase. Compared with real-time RT-PCR, RT-LAMP was consistently faster, and ten copies of FMDV...... vesicular diseases and from that of genetically related picornaviruses. Diagnostic sensitivity was validated by the amplification of reference FMDV strains and archival material from field cases of FMD. In comparison with the performance of the established diagnostic TaqMan (R) assay, RT-LAMP appears...

  6. Detection of foot-and-mouth disease virus rna by reverse transcription loop-mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    Chen Hao-tai

    2011-11-01

    Full Text Available Abstract A reverse transcription loop-mediated isothermal amplification (RT-LAMP assay was developed for foot-and-mouth disease virus (FMDV RNA. The amplification was able to finish in 45 min under isothermal condition at 64°C by employing a set of four primers targeting FMDV 2B. The assay showed higher sensitivity than RT-PCR. No cross reactivity was observed from other RNA viruses including classical swine fever virus, swine vesicular disease, porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus. Furthermore, the assay correctly detected 84 FMDV positive samples but not 65 FMDV negative specimens. The result indicated the potential usefulness of the technique as a simple and rapid procedure for the detection of FMDV infection.

  7. Transcriptional Elongation Factor Elongin A Regulates Retinoic Acid-Induced Gene Expression during Neuronal Differentiation

    Directory of Open Access Journals (Sweden)

    Takashi Yasukawa

    2012-11-01

    Full Text Available Elongin A increases the rate of RNA polymerase II (pol II transcript elongation by suppressing transient pausing by the enzyme. Elongin A also acts as a component of a cullin-RING ligase that can target stalled pol II for ubiquitylation and proteasome-dependent degradation. It is not known whether these activities of Elongin A are functionally interdependent in vivo. Here, we demonstrate that Elongin A-deficient (Elongin A−/− embryos exhibit abnormalities in the formation of both cranial and spinal nerves and that Elongin A−/− embryonic stem cells (ESCs show a markedly decreased capacity to differentiate into neurons. Moreover, we identify Elongin A mutations that selectively inactivate one or the other of the aforementioned activities and show that mutants that retain the elongation stimulatory, but not pol II ubiquitylation, activity of Elongin A rescue neuronal differentiation and support retinoic acid-induced upregulation of a subset of neurogenesis-related genes in Elongin A−/− ESCs.

  8. Genetic interaction of two abscisic acid signaling regulators, HY5 and FIERY1, in mediating lateral root formation

    KAUST Repository

    Chen, Hao

    2011-01-01

    Root architecture is continuously shaped in a manner that helps plants to better adapt to the environment. Gene regulation at the transcriptional or post-transcriptional levels largely controls this environmental response. Recently, RNA silencing has emerged as an important player in gene regulation and is involved in many aspects of plant development, including lateral root formation. In a recent study, we found that FIERY1, a bifunctional abiotic stress and abscisic acid (ABA) signaling regulator and an endogenous RNA silencing suppressor, mediates auxin response during lateral root formation in Arabidopsis. We proposed that FRY1 regulates lateral root development through its activity on adenosine 3,5-bisphosphate (PAP), a strong inhibitor of exoribonucleases (XRNs). Interestingly, some of the phenotypes of fry1, such as enhanced response to light in repressing hypocotyl elongation and hypersensitivity to ABA in lateral root growth, are opposite to those of another light- and ABA-signaling mutant, hy5. Here we analyzed the hy5 fry1 double mutant for root and hypocotyl growth. We found that the hy5 mutation can suppress the enhanced light sensitivity in fry1 hypocotyl elongation and restore the lateral root formation. The genetic interaction between HY5 and FRY1 indicates that HY5 and FRY1 may act in overlapping pathways that mediate light signaling and lateral root development. © 2011 Landes Bioscience.

  9. Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Murano, Tatsuro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Okamoto, Ryuichi, E-mail: rokamoto.gast@tmd.ac.jp [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Department of Advanced GI Therapeutics, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Ito, Go; Nakata, Toru; Hibiya, Shuji; Shimizu, Hiromichi; Fujii, Satoru; Kano, Yoshihito; Mizutani, Tomohiro; Yui, Shiro; Akiyama-Morio, Junko; Nemoto, Yasuhiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Tsuchiya, Kiichiro; Nakamura, Tetsuya [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Department of Advanced GI Therapeutics, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan)

    2014-01-17

    Highlights: •Hes1 enhances IL-22-STAT3 signaling in human intestinal epithelial cells. •Hes1 enhances REG family gene induction by IL-22-STAT3 signaling. •Protein level of Hes1 restricts the response to IL-22. •Present regulation of a cytokine signal represents a new mode of Hes1 function. -- Abstract: Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.

  10. Global identification of the full-length transcripts and alternative splicing related to phenolic acid biosynthetic genes in Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Zhichao eXu

    2016-02-01

    Full Text Available Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and 4 alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that 6 candidate cytochrome P450s and 5 candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza.

  11. Development of a Rapid Detection Method for Potato virus X by Reverse Transcription Loop-Mediated Isothermal Amplification

    Directory of Open Access Journals (Sweden)

    Joojin Jeong

    2015-09-01

    Full Text Available The primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP has been used to detect viral RNA molecules because of its simplicity and high sensitivity for a number of viruses. RT-LAMP for the detection of Potato virus X (PVX was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR to demonstrate its advantages over RT-PCR. RT-LAMP reactions were conducted with or without a set of loop primers since one out of six primers showed PVX specificity. Based on real-time monitoring, RT-LAMP detected PVX around 30 min, compared to 120 min for RT-PCR. By adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. RT-LAMP was conducted using simple inexpensive instruments and a regular incubator to evaluate whether RNA could be amplified at a constant temperature instead of using an expensive thermal cycler. This study shows the potential of RT-LAMP for the diagnosis of viral diseases and PVX epidemiology because of its simplicity and rapidness compared to RT-PCR.

  12. Drosophila brakeless interacts with atrophin and is required for tailless-mediated transcriptional repression in early embryos.

    Science.gov (United States)

    Haecker, Achim; Qi, Dai; Lilja, Tobias; Moussian, Bernard; Andrioli, Luiz Paulo; Luschnig, Stefan; Mannervik, Mattias

    2007-06-01

    Complex gene expression patterns in animal development are generated by the interplay of transcriptional activators and repressors at cis-regulatory DNA modules (CRMs). How repressors work is not well understood, but often involves interactions with co-repressors. We isolated mutations in the brakeless gene in a screen for maternal factors affecting segmentation of the Drosophila embryo. Brakeless, also known as Scribbler, or Master of thickveins, is a nuclear protein of unknown function. In brakeless embryos, we noted an expanded expression pattern of the Krüppel (Kr) and knirps (kni) genes. We found that Tailless-mediated repression of kni expression is impaired in brakeless mutants. Tailless and Brakeless bind each other in vitro and interact genetically. Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA. This suggests that Brakeless is a novel co-repressor. Orphan nuclear receptors of the Tailless type also interact with Atrophin co-repressors. We show that both Drosophila and human Brakeless and Atrophin interact in vitro, and propose that they act together as a co-repressor complex in many developmental contexts. We discuss the possibility that human Brakeless homologs may influence the toxicity of polyglutamine-expanded Atrophin-1, which causes the human neurodegenerative disease dentatorubral-pallidoluysian atrophy (DRPLA).

  13. Icaritin enhances mESC self-renewal through upregulating core pluripotency transcription factors mediated by ERα.

    Science.gov (United States)

    Tsang, Wing Pui; Zhang, Fengjie; He, Qiling; Cai, Waijiao; Huang, Jianhua; Chan, Wai Yee; Shen, Ziyin; Wan, Chao

    2017-01-16

    Utilization of small molecules in modulation of stem cell self-renewal is a promising approach to expand stem cells for regenerative therapy. Here, we identify Icaritin, a phytoestrogen molecule enhances self-renewal of mouse embryonic stem cells (mESCs). Icaritin increases mESCs proliferation while maintains their self-renewal capacity in vitro and pluripotency in vivo. This coincides with upregulation of key pluripotency transcription factors OCT4, NANOG, KLF4 and SOX2. The enhancement of mESCs self-renewal is characterized by increased population in S-phase of cell cycle, elevation of Cylin E and Cyclin-dependent kinase 2 (CDK2) and downregulation of p21, p27 and p57. PCR array screening reveals that caudal-related homeobox 2 (Cdx2) and Rbl2/p130 are remarkably suppressed in mESCs treated with Icaritin. siRNA knockdown of Cdx2 or Rbl2/p130 upregulates the expression of Cyclin E, OCT4 and SOX2, and subsequently increases cell proliferation and colony forming efficiency of mESCs. We then demonstrate that Icaritin co-localizes with estrogen receptor alpha (ERα) and activates its nuclear translocation in mESCs. The promotive effect of Icaritin on cell cycle and pluripotency regulators are eliminated by siRNA knockdown of ERα in mESCs. The results suggest that Icaritin enhances mESCs self-renewal by regulating cell cycle machinery and core pluripotency transcription factors mediated by ERα.

  14. Copper Sensing Function of Drosophila Metal-Responsive Transcription Factor-1 Is Mediated By a Tetranuclear Cu(I) Cluster

    Energy Technology Data Exchange (ETDEWEB)

    Chen, X.; Hua, H.; Balamurugan, K.; Kong, X.; Zhang, L.; George, G.N.; Georgiev, O.; Schaffner, W.; Giedroc, D.P.

    2009-05-12

    Drosophila melanogaster MTF-1 (dMTF-1) is a copper-responsive transcriptional activator that mediates resistance to Cu, as well as Zn and Cd. Here, we characterize a novel cysteine-rich domain which is crucial for sensing excess intracellular copper by dMTF-1. Transgenic flies expressing mutant dMTF-1 containing alanine substitutions of two, four or six cysteine residues within the sequence {sup 547}CNCTNCKCDQTKSCHGGDC{sup 565} are significantly or completely impaired in their ability to protect flies from copper toxicity and fail to up-regulate MtnA (metallothionein) expression in response to excess Cu. In contrast, these flies exhibit wild-type survival in response to copper deprivation thus revealing that the cysteine cluster domain is required only for sensing Cu load by dMTF-1. Parallel studies show that the isolated cysteine cluster domain is required to protect a copper-sensitive S. cerevisiae ace1 strain from copper toxicity. Cu(I) ligation by a Cys-rich domain peptide fragment drives the cooperative assembly of a polydentate [Cu{sub 4}-S{sub 6}] cage structure, characterized by a core of trigonally S{sub 3} coordinated Cu(I) ions bound by bridging thiolate ligands. While reminiscent of Cu{sub 4}-L{sub 6} (L = ligand) tetranuclear clusters in copper regulatory transcription factors of yeast, the absence of significant sequence homology is consistent with convergent evolution of a sensing strategy particularly well suited for Cu(I).

  15. Transcription activator-like effector-mediated regulation of gene expression based on the inducible packaging and delivery via designed extracellular vesicles

    International Nuclear Information System (INIS)

    Lainšček, Duško; Lebar, Tina; Jerala, Roman

    2017-01-01

    Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators. - Highlights: • Inducible dimerization enriched cargo proteins within extracellular vesicles (EV). • Farnesylation surpassed LAMP-1 fusion proteins for the EV packing. • Extracellular vesicles were able to deliver TALE regulators to mammalian cells. • TALE mediated transcriptional activation was achieved by designed EV.

  16. Chemotaxis to cyclic AMP and folic acid is mediated by different G proteins in Dictyostelium discoideum

    NARCIS (Netherlands)

    Kesbeke, Fanja; Haastert, Peter J.M. van; Wit, René J.W. de; Snaar-Jagalska, B. Ewa

    1990-01-01

    Mutant Frigid A (fgdA) of Dictyostelium discoideum is defective in a functional Gα2 subunit of a G protein and is characterized by a complete blockade of the cyclic AMP-mediated sensory transduction steps, including cyclic AMP relay, chemotaxis and the cyclic GMP response. Folic acid-mediated

  17. Agrobacterium-mediated transformation of tomato with the ICE1 transcription factor gene.

    Science.gov (United States)

    Juan, J X; Yu, X H; Jiang, X M; Gao, Z; Zhang, Y; Li, W; Duan, Y D; Yang, G

    2015-01-30

    ICE1 genes play a very important role in plants in cold conditions. To improve the cold resistance of tomato, the ICE1 gene of Arabidopsis thaliana was used to construct the plant expression vector p3301-ICE1, and was overexpressed in tomato through Agrobacterium-mediated transformation. Five strains of resistant plants were obtained. PCR and half-quantitative results showed that the ICE1 gene was transferred to tomato; three strains tested positive. After low-temperature stress treatment, praline content and peroxide and catalase activities in the transgenic tomato plants were higher compared with non-transgenic controls, while malondialdehyde content was clearly lower.

  18. E2F1-mediated transcriptional inhibition of the plasminogen activator inhibitor type 1 gene

    DEFF Research Database (Denmark)

    Koziczak, M; Müller, H; Helin, K

    2001-01-01

    but independent of binding to pocket-binding proteins, suggesting a novel mechanism for E2F-mediated negative gene regulation [Koziczak, M., Krek, W. & Nagamine, Y. (2000) Mol. Cell. Biol. 20, 2014-2022]. However, it remains to be seen whether endogenous E2F can exert a similar effect. We report here that down....... These results all indicate that endogenous E2F can directly repress the PAI-1 gene. DNase I hypersensitive-site analysis of the PAI-1 promoter suggested the involvement of conformation changes in chromatin structure of the PAI-1 promoter. 5' deletion analysis of the PAI-1 promoter showed that multiple sites...

  19. Effects of environmental estrogenic chemicals on AP1 mediated transcription with estrogen receptors alpha and beta.

    Science.gov (United States)

    Fujimoto, Nariaki; Honda, Hiroaki; Kitamura, Shigeyuki

    2004-01-01

    There has been much discussion concerning endocrine disrupting chemicals suspected of exerting adverse effects in both wildlife and humans. Since the majority of these compounds are estrogenic, a large number of in vitro tests for estrogenic characteristics have been developed for screening purpose. One reliable and widely used method is the reporter gene assay employing estrogen receptors (ERs) and a reporter gene with a cis-acting estrogen responsive element (ERE). Other elements such as AP1 also mediate estrogenic signals and the manner of response could be quite different from that of ERE. Since this has yet to be explored, the ER mediated AP1 activity in response to a series of environmental estrogens was investigated in comparison with ERE findings. All the compounds exhibited estrogenic properties with ERE-luc and their AP1 responses were quite similar. These was one exception, however, p,p'-DDT (1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane) did not exert any AP1-luc activity, while it appeared to be estrogenic at 10(-7) to 10(-5)M with the ERE action. None of the compounds demonstrated ER beta:AP1 activity. These data suggest that significant differences can occur in responses through the two estrogen pathways depending on environmental chemicals.

  20. Analysis of the combined effects of lanthanum and acid rain, and their mechanisms, on nitrate reductase transcription in plants.

    Science.gov (United States)

    Xia, Binxin; Sun, Zhaoguo; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

    2017-04-01

    Rare earth element (REE) pollution and acid rain are major global environmental concerns, and their spatial distributions overlap. Thus, both forms of pollution combine to act on plants. Nitrogen is important for plant growth, and nitrate reductase (NR) is a key plant enzyme that catalyzes nitrogen assimilation. Studying the combined effects of REEs and acid rain on plant nitrogen-based nutrients has important environmental significance. Here, soybean (Glycine max) plants, commonly used for toxicological studies, were exposed to lanthanum (La), a REE, and acid rain to study the NR activities and NR transcriptional levels in the roots. To explain how the pollution affected the NR transcriptional level, we simultaneously observed the contents of intracellular La and nutrient elements, protoplast morphology, membrane lipid peroxidation and intracellular pH. A combined treatment of 0.08mmol/L La and pH 4.5 acid rain increased the NR activity, decreased the NR transcriptional level, increased the intracellular nutrient elements' contents and caused deformations in membrane structures. Other combined treatments significantly decreased the aforementioned parameters and caused serious damage to the membrane structures. The variation in the amplitudes of combined treatments was greater than those of individual treatments. Compared with the control and individual treatments, combined treatments increased membrane permeability, the malondialdehyde content, and intracellular H + and La contents, and with an increasing La concentration or acid strength, the change in amplitude increased. Thus, the combined effects on NR gene transcription in soybean seedling roots were related to the intracellular nutrient elements' contents, protoplast morphology, membranous lipid peroxidation, intracellular pH and La content. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. LncRNA HOTAIR Enhances the Androgen-Receptor-Mediated Transcriptional Program and Drives Castration-Resistant Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Ali Zhang

    2015-10-01

    Full Text Available Understanding the mechanisms of androgen receptor (AR activation in the milieu of low androgen is critical to effective treatment of castration-resistant prostate cancer (CRPC. Here, we report HOTAIR as an androgen-repressed lncRNA, and, as such, it is markedly upregulated following androgen deprivation therapies and in CRPC. We further demonstrate a distinct mode of lncRNA-mediated gene regulation, wherein HOTAIR binds to the AR protein to block its interaction with the E3 ubiquitin ligase MDM2, thereby preventing AR ubiquitination and protein degradation. Consequently, HOTAIR expression is sufficient to induce androgen-independent AR activation and drive the AR-mediated transcriptional program in the absence of androgen. Functionally, HOTAIR overexpression increases, whereas HOTAIR knockdown decreases, prostate cancer cell growth and invasion. Taken together, our results provide compelling evidence of lncRNAs as drivers of androgen-independent AR activity and CRPC progression, and they support the potential of lncRNAs as therapeutic targets.

  2. Cohesin Rad21 Mediates Loss of Heterozygosity and Is Upregulated via Wnt Promoting Transcriptional Dysregulation in Gastrointestinal Tumors

    Directory of Open Access Journals (Sweden)

    Huiling Xu

    2014-12-01

    Full Text Available Summary: Loss of heterozygosity (LOH of the adenomatous polyposis coli (APC gene triggers a series of molecular events leading to intestinal adenomagenesis. Haploinsufficiency of the cohesin Rad21 influences multiple initiating events in colorectal cancer (CRC. We identify Rad21 as a gatekeeper of LOH and a β-catenin target gene and provide evidence that Wnt pathway activation drives RAD21 expression in human CRC. Genome-wide analyses identified Rad21 as a key transcriptional regulator of critical CRC genes and long interspersed element (LINE-1 or L1 retrotransposons. Elevated RAD21 expression tracks with reactivation of L1 expression in human sporadic CRC, implicating cohesin-mediated L1 expression in global genomic instability and gene dysregulation in cancer. : Rad21 holds the cohesin complex together as part of its role in chromosome partitioning and DNA repair. Xu et al. identify Rad21 as a key mediator of Apc gene heterozygous loss, the event initiating intestinal tumorigenesis. The subsequent activation of the Wnt pathway further induces Rad21, global gene dysregulation, chromosome instability, and pervasive retrotransposon activation.

  3. Forkhead box O transcription factors as possible mediators in the development of major depression.

    Science.gov (United States)

    Wang, Haitao; Quirion, Rémi; Little, Peter J; Cheng, Yufang; Feng, Zhong-Ping; Sun, Hong-Shuo; Xu, Jiangping; Zheng, Wenhua

    2015-12-01

    Forkhead box O (FoxO) transcription factors play important roles in cellular physiology and biology. Recent findings indicate that FoxOs are also involved in the development of major depressive disorder. Alterations in the upstream molecules of FoxOs, such as brain derived neurotrophic factor or protein kinase B, have been linked to depression. Antidepressants, such as imipramine and venlafaxine, modify the FoxOs phosphorylation. Furthermore, FoxOs could be regulated by serotonin and norepinephrine receptor signaling as well as the hypothalamic-pituitary-adrenal axis, all of which are involved in the pathogenesis of depression. FoxOs also regulate neuronal morphology, synaptogenesis and adult hippocampal neurogenesis, which are viewed as candidate mechanisms for the etiology of depression. In this review, we emphasize the possible roles of FoxOs during the development of depression and make some strategic recommendations for future research. We propose that FoxOs and its signaling pathways may constitute potential therapeutic targets in the treatment of depression. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Eosinophil Resistance to Glucocorticoid-Induced Apoptosis is Mediated by the Transcription Factor NFIL3

    Science.gov (United States)

    Pazdrak, Konrad; Moon, Young; Straub, Christof; Stafford, Susan; Kurosky, Alexander

    2016-01-01

    The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs' effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired prop-aptoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils' response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apoptotic response regardless of IL-5 stimulation. Proteomic analysis revealed that both Nuclear Factor IL-3 (NFIL3) and Map Kinase Phosphatase 1 (MKP1) were inducible by IL-5 and enhanced by Dex; however, CpdA had no effect on NFIL3 and MKP1 expression. We found that inhibiting NFIL3 with specific siRNA or by blocking the IL-5-inducible Pim-1 kinase abrogated the protective effect of IL-5 on Dex-induced apoptosis, indicating crosstalk between IL-5 anti-apoptotic pathways and GR-mediated TA signaling occurring via the NFIL3 molecule. Collectively, these results indicate that 1) GCs' TA pathway may support eosinophil viability in IL-5-stimulated cells through synergistic upregulation of NFIL3; and 2) functional inhibition of IL-5 signaling (anti-Pim1) or the use of selective GR agonists that don't upregulate NFIL3 may be effective strategies for the restoring pro-apoptotic effect of GCs on IL-5-activated eosinophils. PMID:26880402

  5. DNA-mediated cooperativity facilitates the co-selection of cryptic enhancer sequences by SOX2 and PAX6 transcription factors.

    Science.gov (United States)

    Narasimhan, Kamesh; Pillay, Shubhadra; Huang, Yong-Heng; Jayabal, Sriram; Udayasuryan, Barath; Veerapandian, Veeramohan; Kolatkar, Prasanna; Cojocaru, Vlad; Pervushin, Konstantin; Jauch, Ralf

    2015-02-18

    Sox2 and Pax6 are transcription factors that direct cell fate decision during neurogenesis, yet the mechanism behind how they cooperate on enhancer DNA elements and regulate gene expression is unclear. By systematically interrogating Sox2 and Pax6 interaction on minimal enhancer elements, we found that cooperative DNA recognition relies on combinatorial nucleotide switches and precisely spaced, but cryptic composite DNA motifs. Surprisingly, all tested Sox and Pax paralogs have the capacity to cooperate on such enhancer elements. NMR and molecular modeling reveal very few direct protein-protein interactions between Sox2 and Pax6, suggesting that cooperative binding is mediated by allosteric interactions propagating through DNA structure. Furthermore, we detected and validated several novel sites in the human genome targeted cooperatively by Sox2 and Pax6. Collectively, we demonstrate that Sox-Pax partnerships have the potential to substantially alter DNA target specificities and likely enable the pleiotropic and context-specific action of these cell-lineage specifiers. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Phenolic acids potentiate colistin-mediated killing of Acinetobacter baumannii by inducing redox imbalance.

    Science.gov (United States)

    Ajiboye, Taofeek O; Skiebe, Evelyn; Wilharm, Gottfried

    2018-05-01

    Phenolic acids with catechol groups are good prooxidants because of their low redox potential. In this study, we provided data showing that phenolic acids, caffeic acid, gallic acid and protocatechuic acid, enhanced colistin-mediated bacterial death by inducing redox imbalance. The minimum inhibitory concentrations of these phenolic acids against Acinetobacter baumannii AB5075 were considerably lowered for ΔsodB and ΔkatG mutants. Checkerboard assay shows synergistic interactions between colistin and phenolic acids. The phenolic acids exacerbated colistin-induced oxidative stress in A. baumannii AB5075 through increased superoxide anion generation, NAD + /NADH and ADP/ATP ratio. In parallel, the level of reduced glutathione was significantly lowered. We conclude that phenolic acids potentiate colistin-induced oxidative stress in A. baumannii AB5075 by increasing ROS generation, energy metabolism and electron transport chain activity with a concomitant decrease in glutathione. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  7. Upregulation of CREB-mediated transcription enhances both short- and long-term memory.

    Science.gov (United States)

    Suzuki, Akinobu; Fukushima, Hotaka; Mukawa, Takuya; Toyoda, Hiroki; Wu, Long-Jun; Zhao, Ming-Gao; Xu, Hui; Shang, Yuze; Endoh, Kengo; Iwamoto, Taku; Mamiya, Nori; Okano, Emiko; Hasegawa, Shunsuke; Mercaldo, Valentina; Zhang, Yue; Maeda, Ryouta; Ohta, Miho; Josselyn, Sheena A; Zhuo, Min; Kida, Satoshi

    2011-06-15

    Unraveling the mechanisms by which the molecular manipulation of genes of interest enhances cognitive function is important to establish genetic therapies for cognitive disorders. Although CREB is thought to positively regulate formation of long-term memory (LTM), gain-of-function effects of CREB remain poorly understood, especially at the behavioral level. To address this, we generated four lines of transgenic mice expressing dominant active CREB mutants (CREB-Y134F or CREB-DIEDML) in the forebrain that exhibited moderate upregulation of CREB activity. These transgenic lines improved not only LTM but also long-lasting long-term potentiation in the CA1 area in the hippocampus. However, we also observed enhanced short-term memory (STM) in contextual fear-conditioning and social recognition tasks. Enhanced LTM and STM could be dissociated behaviorally in these four lines of transgenic mice, suggesting that the underlying mechanism for enhanced STM and LTM are distinct. LTM enhancement seems to be attributable to the improvement of memory consolidation by the upregulation of CREB transcriptional activity, whereas higher basal levels of BDNF, a CREB target gene, predicted enhanced shorter-term memory. The importance of BDNF in STM was verified by microinfusing BDNF or BDNF inhibitors into the hippocampus of wild-type or transgenic mice. Additionally, increasing BDNF further enhanced LTM in one of the lines of transgenic mice that displayed a normal BDNF level but enhanced LTM, suggesting that upregulation of BDNF and CREB activity cooperatively enhances LTM formation. Our findings suggest that CREB positively regulates memory consolidation and affects memory performance by regulating BDNF expression.

  8. The transcriptional activator LdtR from 'Candidatus Liberibacter asiaticus' mediates osmotic stress tolerance.

    Directory of Open Access Journals (Sweden)

    Fernando A Pagliai

    2014-04-01

    Full Text Available The causal agent of Huanglongbing disease, 'Candidatus Liberibacter asiaticus', is a non-culturable, gram negative, phloem-limited α-proteobacterium. Current methods to control the spread of this disease are still limited to the removal and destruction of infected trees. In this study, we identified and characterized a regulon from 'Ca. L. asiaticus' involved in cell wall remodeling, that contains a member of the MarR family of transcriptional regulators (ldtR, and a predicted L,D-transpeptidase (ldtP. In Sinorhizobium meliloti, mutation of ldtR resulted in morphological changes (shortened rod-type phenotype and reduced tolerance to osmotic stress. A biochemical approach was taken to identify small molecules that modulate LdtR activity. The LdtR ligands identified by thermal shift assays were validated using DNA binding methods. The biological impact of LdtR inactivation by the small molecules was then examined in Sinorhizobium meliloti and Liberibacter crescens, where a shortened-rod phenotype was induced by growth in presence of the ligands. A new method was also developed to examine the effects of small molecules on the viability of 'Ca. Liberibacter asiaticus', using shoots from HLB-infected orange trees. Decreased expression of ldtRLas and ldtPLas was observed in samples taken from HLB-infected shoots after 6 h of incubation with the LdtR ligands. These results provide strong proof of concept for the use of small molecules that target LdtR, as a potential treatment option for Huanglongbing disease.

  9. A trans-acting enhancer modulates estrogen-mediated transcription of reporter genes in osteoblasts.

    Science.gov (United States)

    Sasaki-Iwaoka, H; Maruyama, K; Endoh, H; Komori, T; Kato, S; Kawashima, H

    1999-02-01

    The presence of bone-specific estrogen agonists and discovery of the osteoblast-specific transcription factor (TF), Cbfa1, together with the discovery of synergism between a TF Pit-1 and estrogen receptor alpha (ERalpha) on rat prolactin gene, led to investigation of Cbfa1 in the modulation of osteoblast-specific actions of estrogen. Reverse transcribed-polymerase chain reaction demonstrated expression of Cbfa1 in the osteoblastic cell lines, MG63, ROS17/2.8, and MC3T3E1, but not in nonosteoblastic cell lines, MCF7, C3H10T1/2, and HeLa. An ER expression vector and a series of luciferase (Luc) reporter plasmids harboring the Cbfa1 binding site OSE2 (the osteoblast-specific cis element in the osteocalcin promoter) and palindromic estrogen response elements (EREs) were cotransfected into both osteoblastic and nonosteoblastic cells. OSE2 worked as a cis- acting element in osteoblastic cells but not nonosteoblastic cells, whereas EREs were cis- acting in all cell lines. Synergistic transactivation was observed in osteoblastic cells only when both ERE and OSE2 were placed in juxtaposition to the promoter. Forced expression of Cbfa1 in C3H10T1/2 cells also induced synergism. Tamoxifen, a partial agonist/antagonist of estrogen, acted as an osteoblast-specific agonist in cells transfected with a promoter containing ERE and acted synergistically with a promoter containing the ERE-OSE2 enhancer combination. These results support the idea that bone-specific TFs modulate the actions of estrogen in a tissue-specific manner.

  10. ER-tethered Transcription Factor CREBH Regulates Hepatic Lipogenesis, Fatty Acid Oxidation, and Lipolysis upon Metabolic Stress

    OpenAIRE

    Zhang, Chunbin; Wang, Guohui; Zheng, Ze; Maddipati, Krishna Rao; Zhang, Xuebao; Dyson, Gregory; Williams, Paul; Duncan, Stephen A.; Kaufman, Randal J.; Zhang, Kezhong

    2012-01-01

    CREBH is a liver-specific transcription factor that is localized in the endoplasmic reticulum (ER) membrane. Our previous work demonstrated that CREBH is activated by ER stress or inflammatory stimuli to induce an acute-phase hepatic inflammation. Here we demonstrate that CREBH is a key metabolic regulator of hepatic lipogenesis, fatty acid (FA) oxidation, and lipolysis under metabolic stress. Saturated FA, insulin signals, or an atherogenic high-fat diet can induce CREBH activation in the li...

  11. Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic acid-responsive transcription factor gaaR.

    Science.gov (United States)

    Alazi, Ebru; Knetsch, Tim; Di Falco, Marcos; Reid, Ian D; Arentshorst, Mark; Visser, Jaap; Tsang, Adrian; Ram, Arthur F J

    2018-03-01

    The transcription factor GaaR is needed for the expression of genes required for pectin degradation and transport and catabolism of the main degradation product, D-galacturonic acid (GA) in Aspergillus niger. In this study, we used the strong constitutive gpdA promoter of Aspergillus nidulans to overexpress gaaR in A. niger. Overexpression of gaaR resulted in an increased transcription of the genes encoding pectinases, (putative) GA transporters, and catabolic pathway enzymes even under non-inducing conditions, i.e., in the absence of GA. Exoproteome analysis of a strain overexpressing gaaR showed that this strain secretes highly elevated levels of pectinases when grown in fructose. The genes encoding exo-polygalacturonases were found to be subjected to CreA-mediated carbon catabolite repression, even in the presence of fructose. Deletion of creA in the strain overexpressing gaaR resulted in a further increase in pectinase production in fructose. We showed that GaaR localizes mainly in the nucleus regardless of the presence of an inducer, and that overexpression of gaaR leads to an increased concentration of GaaR in the nucleus.

  12. Enhancement of Chlorogenic Acid Production in Hairy Roots of Platycodon grandiflorum by Over-Expression of An Arabidopsis thaliana Transcription Factor AtPAP1

    Directory of Open Access Journals (Sweden)

    Pham Anh Tuan

    2014-08-01

    Full Text Available To improve the production of chlorogenic acid (CGA in hairy roots of Platycodon grandiflorum, we induced over-expression of Arabidopsis thaliana transcription factor production of anthocyanin pigment (AtPAP1 using an Agrobacterium rhizogenes-mediated transformation system. Twelve hairy root lines showing over-expression of AtPAP1 were generated. In order to investigate the regulation of AtPAP1 on the activities of CGA biosynthetic genes, the expression levels of seven P. grandiflorum CGA biosynthetic genes were analyzed in the hairy root line that had the greatest accumulation of AtPAP1 transcript, OxPAP1-1. The introduction of AtPAP1 increased the mRNA levels of all examined CGA biosynthetic genes and resulted in a 900% up-regulation of CGA accumulation in OxPAP1-1 hairy roots relative to controls. This suggests that P. grandiflorum hairy roots that over-express the AtPAP1 gene are a potential alternative source of roots for the production of CGA.

  13. Nucleic acid and nucleotide-mediated synthesis of inorganic nanoparticles

    Science.gov (United States)

    Berti, Lorenzo; Burley, Glenn A.

    2008-02-01

    Since the advent of practical methods for achieving DNA metallization, the use of nucleic acids as templates for the synthesis of inorganic nanoparticles (NPs) has become an active area of study. It is now widely recognized that nucleic acids have the ability to control the growth and morphology of inorganic NPs. These biopolymers are particularly appealing as templating agents as their ease of synthesis in conjunction with the possibility of screening nucleotide composition, sequence and length, provides the means to modulate the physico-chemical properties of the resulting NPs. Several synthetic procedures leading to NPs with interesting photophysical properties as well as studies aimed at rationalizing the mechanism of nucleic acid-templated NP synthesis are now being reported. This progress article will outline the current understanding of the nucleic acid-templated process and provides an up to date reference in this nascent field.

  14. Lateral Root Development in Potato Is Mediated by Stu-mi164 Regulation of NAC Transcription Factor

    Directory of Open Access Journals (Sweden)

    Li Zhang

    2018-03-01

    Full Text Available The NAC designation is derived from petunia (Petunia hybrida gene NO APICAL MERISTEM (NAM and Arabidopsis genes ATAF1/ATAF2 and CUP-SHAPED COTYLEDON2 (CUC2, which belongs to the family of plant-specific transcription factors (TFs, and plays important role in plant development processes, such as response to biotic and abiotic stress, and hormone signaling. MicroRNAs (miRNAs are a class of small, non-coding endogenous RNAs which play versatile and significant role in plant stress response and development via negatively affecting gene expression at a post-transcriptional level. Here, we showed that Stu-mi164 had a complementary sequence in the CDS sequence of potato NAC TFs, and that NAC expression exhibited significant differences under osmotic stress. We measured expression levels of the Stu-mi164 target gene StNAC262 between control and PEG-treated plants using real-time PCR, and the results demonstrated that they had inverse relationship. We suggested that Stu-miR164 might drive overexpression of NAC gene under osmotic stress in potato. To confirm the regulation of NAC TFs by Stu-mi164, we developed transgenic plants, using Agrobacterium tumefaciens–mediated transformation, of the potato cultivars “Gannongshu 2” and “Kexin 3” overexpressing the Stu-mi164 or the TF StNAC262. Real-time PCR analysis of transgenic potato plants under osmotic (PEG stress, showed that potato plants overexpressing Stu-mi164 had reduced expression of StNAC262 and their osmotic resistance decreased. Furthermore, these plants had low number of lateral roots although the same length as the control. Our findings support the regulatory role of Stu-miRNAs in controlling plant response to osmotic stress via StNAC262.

  15. Transcription factor RBP-J-mediated signalling regulates basophil immunoregulatory function in mouse asthma model.

    Science.gov (United States)

    Qu, Shuo-Yao; He, Ya-Long; Zhang, Jian; Wu, Chang-Gui

    2017-09-01

    Basophils (BA) play an important role in the promotion of aberrant T helper type 2 (Th2) immune responses in asthma. It is not only the effective cell, but also modulates the initiation of Th2 immune responses. We earlier demonstrated that Notch signalling regulates the biological function of BAin vitro. However, whether this pathway plays the same role in vivo is not clear. The purpose of the present study was to investigate the effect of Notch signalling on BA function in the regulation of allergic airway inflammation in a murine model of asthma. Bone marrow BA were prepared by bone marrow cell culture in the presence of recombinant interleukin-3 (rIL-3; 300 pg/ml) for 7 days, followed by isolation of the CD49b + microbeads. The recombination signal binding protein J (RBP-J -/- ) BA were co-cultured with T cells, and the supernatant and the T-cell subtypes were examined. The results indicated disruption of the capacity of BA for antigen presentation alongside an up-regulation of the immunoregulatory function. This was possibly due to the low expression of OX40L in the RBP-J -/- BA. Basophils were adoptively transferred to ovalbumin-sensitized recipient mice, to establish an asthma model. Lung pathology, cytokine profiles of brobchoalveolar fluid, airway hyperactivity and the absolute number of Th1/Th2 cells in lungs were determined. Overall, our results indicate that the RBP-J-mediated Notch signalling is critical for BA-dependent immunoregulation. Deficiency of RBP-J influences the immunoregulatory functions of BA, which include activation of T cells and their differentiation into T helper cell subtypes. The Notch signalling pathway is a potential therapeutic target for BA-based immunotherapy against asthma. © 2017 John Wiley & Sons Ltd.

  16. The CaM Kinase CMK-1 Mediates a Negative Feedback Mechanism Coupling the C. elegans Glutamate Receptor GLR-1 with Its Own Transcription.

    Directory of Open Access Journals (Sweden)

    Benjamin J Moss

    2016-07-01

    Full Text Available Regulation of synaptic AMPA receptor levels is a major mechanism underlying homeostatic synaptic scaling. While in vitro studies have implicated several molecules in synaptic scaling, the in vivo mechanisms linking chronic changes in synaptic activity to alterations in AMPA receptor expression are not well understood. Here we use a genetic approach in C. elegans to dissect a negative feedback pathway coupling levels of the AMPA receptor GLR-1 with its own transcription. GLR-1 trafficking mutants with decreased synaptic receptors in the ventral nerve cord (VNC exhibit compensatory increases in glr-1 mRNA, which can be attributed to increased glr-1 transcription. Glutamatergic transmission mutants lacking presynaptic eat-4/VGLUT or postsynaptic glr-1, exhibit compensatory increases in glr-1 transcription, suggesting that loss of GLR-1 activity is sufficient to trigger the feedback pathway. Direct and specific inhibition of GLR-1-expressing neurons using a chemical genetic silencing approach also results in increased glr-1 transcription. Conversely, expression of a constitutively active version of GLR-1 results in decreased glr-1 transcription, suggesting that bidirectional changes in GLR-1 signaling results in reciprocal alterations in glr-1 transcription. We identify the CMK-1/CaMK signaling axis as a mediator of the glr-1 transcriptional feedback mechanism. Loss-of-function mutations in the upstream kinase ckk-1/CaMKK, the CaM kinase cmk-1/CaMK, or a downstream transcription factor crh-1/CREB, result in increased glr-1 transcription, suggesting that the CMK-1 signaling pathway functions to repress glr-1 transcription. Genetic double mutant analyses suggest that CMK-1 signaling is required for the glr-1 transcriptional feedback pathway. Furthermore, alterations in GLR-1 signaling that trigger the feedback mechanism also regulate the nucleocytoplasmic distribution of CMK-1, and activated, nuclear-localized CMK-1 blocks the feedback pathway. We

  17. Higher transcription levels in ascorbic acid biosynthetic and recycling genes were associated with higher ascorbic acid accumulation in blueberry.

    Science.gov (United States)

    Liu, Fenghong; Wang, Lei; Gu, Liang; Zhao, Wei; Su, Hongyan; Cheng, Xianhao

    2015-12-01

    In our preliminary study, the ripe fruits of two highbush blueberry (Vaccinium corymbosum L.) cultivars, cv 'Berkeley' and cv 'Bluecrop', were found to contain different levels of ascorbic acid. However, factors responsible for these differences are still unknown. In the present study, ascorbic acid content in fruits was compared with expression profiles of ascorbic acid biosynthetic and recycling genes between 'Bluecrop' and 'Berkeley' cultivars. The results indicated that the l-galactose pathway was the predominant route of ascorbic acid biosynthesis in blueberry fruits. Moreover, higher expression levels of the ascorbic acid biosynthetic genes GME, GGP, and GLDH, as well as the recycling genes MDHAR and DHAR, were associated with higher ascorbic acid content in 'Bluecrop' compared with 'Berkeley', which indicated that a higher efficiency ascorbic acid biosynthesis and regeneration was likely to be responsible for the higher ascorbic acid accumulation in 'Bluecrop'. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions.

    Science.gov (United States)

    Uno, Y; Furihata, T; Abe, H; Yoshida, R; Shinozaki, K; Yamaguchi-Shinozaki, K

    2000-10-10

    The induction of the dehydration-responsive Arabidopsis gene, rd29B, is mediated mainly by abscisic acid (ABA). Promoter analysis of rd29B indicated that two ABA-responsive elements (ABREs) are required for the dehydration-responsive expression of rd29B as cis-acting elements. Three cDNAs encoding basic leucine zipper (bZIP)-type ABRE-binding proteins were isolated by using the yeast one-hybrid system and were designated AREB1, AREB2, and AREB3 (ABA-responsive element binding protein). Transcription of the AREB1 and AREB2 genes is up-regulated by drought, NaCl, and ABA treatment in vegetative tissues. In a transient transactivation experiment using Arabidopsis leaf protoplasts, both the AREB1 and AREB2 proteins activated transcription of a reporter gene driven by ABRE. AREB1 and AREB2 required ABA for their activation, because their transactivation activities were repressed in aba2 and abi1 mutants and enhanced in an era1 mutant. Activation of AREBs by ABA was suppressed by protein kinase inhibitors. These results suggest that both AREB1 and AREB2 function as transcriptional activators in the ABA-inducible expression of rd29B, and further that ABA-dependent posttranscriptional activation of AREB1 and AREB2, probably by phosphorylation, is necessary for their maximum activation by ABA. Using cultured Arabidopsis cells, we demonstrated that a specific ABA-activated protein kinase of 42-kDa phosphorylated conserved N-terminal regions in the AREB proteins.

  19. Jasmonic acid-mediated defense suppresses brassinosteroid-mediated susceptibility to Rice black streaked dwarf virus infection in rice.

    Science.gov (United States)

    He, Yuqing; Zhang, Hehong; Sun, Zongtao; Li, Junmin; Hong, Gaojie; Zhu, Qisong; Zhou, Xuebiao; MacFarlane, Stuart; Yan, Fei; Chen, Jianping

    2017-04-01

    Plant hormones play a vital role in plant immune responses. However, in contrast to the relative wealth of information on hormone-mediated immunity in dicot plants, little information is available on monocot-virus defense systems. We used a high-throughput-sequencing approach to compare the global gene expression of Rice black-streaked dwarf virus (RBSDV)-infected rice plants with that of healthy plants. Exogenous hormone applications and transgenic rice were used to test RBSDV infectivity and pathogenicity. Our results revealed that the jasmonic acid (JA) pathway was induced while the brassinosteroid (BR) pathway was suppressed in infected plants. Foliar application of methyl jasmonate (MeJA) or brassinazole (BRZ) resulted in a significant reduction in RBSDV incidence, while epibrassinolide (BL) treatment increased RBSDV infection. Infection studies using coi1-13 and Go mutants demonstrated JA-mediated resistance and BR-mediated susceptibility to RBSDV infection. A mixture of MeJA and BL treatment resulted in a significant reduction in RBSDV infection compared with a single BL treatment. MeJA application efficiently suppressed the expression of BR pathway genes, and this inhibition depended on the JA coreceptor OsCOI1. Collectively, our results reveal that JA-mediated defense can suppress the BR-mediated susceptibility to RBSDV infection. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  20. Key mediators of intracellular amino acids signaling to mTORC1 activation.

    Science.gov (United States)

    Duan, Yehui; Li, Fengna; Tan, Kunrong; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Tang, Yulong; Wu, Guoyao; Yin, Yulong

    2015-05-01

    Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

  1. The obesity-induced transcriptional regulator TRIP-Br2 mediates visceral fat endoplasmic reticulum stress-induced inflammation.

    Science.gov (United States)

    Qiang, Guifen; Kong, Hyerim Whang; Fang, Difeng; McCann, Maximilian; Yang, Xiuying; Du, Guanhua; Blüher, Matthias; Zhu, Jinfang; Liew, Chong Wee

    2016-04-25

    The intimate link between location of fat accumulation and metabolic disease risk and depot-specific differences is well established, but how these differences between depots are regulated at the molecular level remains largely unclear. Here we show that TRIP-Br2 mediates endoplasmic reticulum (ER) stress-induced inflammatory responses in visceral fat. Using in vitro, ex vivo and in vivo approaches, we demonstrate that obesity-induced circulating factors upregulate TRIP-Br2 specifically in visceral fat via the ER stress pathway. We find that ablation of TRIP-Br2 ameliorates both chemical and physiological ER stress-induced inflammatory and acute phase response in adipocytes, leading to lower circulating levels of inflammatory cytokines. Using promoter assays, as well as molecular and pharmacological experiments, we show that the transcription factor GATA3 is responsible for the ER stress-induced TRIP-Br2 expression in visceral fat. Taken together, our study identifies molecular regulators of inflammatory response in visceral fat that-given that these pathways are conserved in humans-might serve as potential therapeutic targets in obesity.

  2. Members of the Dof transcription factor family in Triticum aestivum are associated with light-mediated gene regulation.

    Science.gov (United States)

    Shaw, Lindsay M; McIntyre, C Lynne; Gresshoff, Peter M; Xue, Gang-Ping

    2009-11-01

    DNA binding with One Finger (Dof) protein is a plant-specific transcription factor implicated in the regulation of many important plant-specific processes, including photosynthesis and carbohydrate metabolism. This study has identified 31 Dof genes (TaDof) in bread wheat through extensive analysis of current nucleotide databases. Phylogenetic analysis suggests that the TaDof family can be divided into four clades. Expression analysis of the TaDof family across all major organs using quantitative RT-PCR and searches of the wheat genome array database revealed that the majority of TaDof members were predominately expressed in vegetative organs. A large number of TaDof members were down-regulated by drought and/or were responsive to the light and dark cycle. Further expression analysis revealed that light up-regulated TaDof members were highly correlated in expression with a number of genes that are involved in photosynthesis or sucrose transport. These data suggest that the TaDof family may have an important role in light-mediated gene regulation, including involvement in the photosynthetic process.

  3. New insights into the acid mediated disproportionation of pentavalent uranyl

    Energy Technology Data Exchange (ETDEWEB)

    Mougel, Victor; Biswas, Biplab; Pecaut, Jacques; Mazzanti, Marinella [Laboratoire de Reconnaissance Ionique et Chimie de Coordination, SCIB, UMR-E 3 CEA-UJF FRE 3200 CNRS, INAC, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 09 (France)

    2010-07-01

    The reaction of benzoic acid with the uranyl(V) complex [(UO{sub 2}Py{sub 5})(KI{sub 2}Py{sub 2})] in pyridine leads to immediate disproportionation with formation of a hexa-nuclear U(IV) benzoate cluster, a bis-benzoate complex of uranyl(VI) and water. (authors)

  4. Fulvic Acid Mediated Photolysis of Ibuprofen in Water.

    Science.gov (United States)

    Photolysis of the nonsteroidal anti-inflammatory drug ibuprofen was studied in solutions of fulvic acid (FA) isolated from Pony Lake, Antarctica; Suwannee River, GA, USA; and Old Woman Creek, OH, USA. At an initial concentration of 10 µM ibuprofen degrades by direct photolysis...

  5. Gene Transcription and Virulence Potential of Listeria monocytogenes Strains After Exposure to Acidic and NaCl Stress

    DEFF Research Database (Denmark)

    Olesen, Inger; Vogensen, Finn Kvist; Jespersen, Lene

    2009-01-01

    transcription were observed both after exposure to shock (six genes) and after long-term adaptation to stress (18 genes). In the shock experiments, a transient induction of clpC and clpE was seen for both strains, while transient induction of sigB, inlA, and inlB was observed for strain 4140 only; actA was only...... induced in EGD-e after NaCl shock. The longterm stress experiments were included to imitate the stress conditions encountered by L. monocytogenes when present in food products. Long-term adaptation of EGD-e to acidic stress induced transcription of iap and repressed flaA, while genes related to stress......Gene transcription and virulence potential of two strains of Listeria monocytogenes, EGD-e and 4140, were compared by quantitative real-time polymerase chain reaction and in a Caco-2 in vitro model after exposure to acidic (pH 5.5) and NaCl (4.5% w=v) stress. Strain-dependent differences in gene...

  6. A Canonical DREB2-Type Transcription Factor in Lily Is Post-translationally Regulated and Mediates Heat Stress Response

    Directory of Open Access Journals (Sweden)

    Ze Wu

    2018-03-01

    Full Text Available Based on studies of monocot crops and eudicot model plants, the DREB2 class of AP2-type transcription factor has been shown to play crucial roles in various abiotic stresses, especially in the upstream of the heat stress response; however, research on DREB2s has not been reported in non-gramineous monocot plants. Here, we identified a novel DREB2 (LlDREB2B from lily (Lilium longiflorum, which was homologous to AtDREB2A of Arabidopsis, OsDREB2B of rice, and ZmDREB2A of maize. LlDREB2B was induced by heat, cold, salt, and mannitol stress, and its protein had transcriptional activity, was located in the nucleus, was able to bind to the dehydration-responsive element (DRE, and participated in the heat-responsive pathway of HsfA3. Overexpression of LlDREB2B in Arabidopsis activated expression of downstream genes and improved thermotolerance. LlDREB2B was not regulated by alternative splicing; functional transcripts accumulated under either normal or heat-stress conditions. A potential PEST sequence was predicted in LlDREB2B, but the stability of the LlDREB2B protein was not positively affected when the predicated PEST sequence was deleted. Further analysis revealed that the predicated PEST sequence lacked a SBC or SBC-like motif allowing interaction with BPMs and required for negative regulation. Nevertheless, LlDREB2B was still regulated at the post-translational level by interaction with AtDRIP1 and AtDRIP2 of Arabidopsis. In addition, LlDREB2B also interacted with AtRCD1 and LlRCD1 via a potential RIM motif located at amino acids 215–245. Taken together, our results show that LlDREB2B participated in the establishment of thermotolerance, and its regulation was different from that of the orthologs of gramineous and eudicot plants.

  7. A Canonical DREB2-Type Transcription Factor in Lily Is Post-translationally Regulated and Mediates Heat Stress Response.

    Science.gov (United States)

    Wu, Ze; Liang, Jiahui; Zhang, Shuai; Zhang, Bing; Zhao, Qingcui; Li, Guoqing; Yang, Xi; Wang, Chengpeng; He, Junna; Yi, Mingfang

    2018-01-01

    Based on studies of monocot crops and eudicot model plants, the DREB2 class of AP2-type transcription factor has been shown to play crucial roles in various abiotic stresses, especially in the upstream of the heat stress response; however, research on DREB2s has not been reported in non-gramineous monocot plants. Here, we identified a novel DREB2 (LlDREB2B) from lily ( Lilium longiflorum ), which was homologous to AtDREB2A of Arabidopsis, OsDREB2B of rice, and ZmDREB2A of maize. LlDREB2B was induced by heat, cold, salt, and mannitol stress, and its protein had transcriptional activity, was located in the nucleus, was able to bind to the dehydration-responsive element (DRE), and participated in the heat-responsive pathway of HsfA3. Overexpression of LlDREB2B in Arabidopsis activated expression of downstream genes and improved thermotolerance. LlDREB2B was not regulated by alternative splicing; functional transcripts accumulated under either normal or heat-stress conditions. A potential PEST sequence was predicted in LlDREB2B, but the stability of the LlDREB2B protein was not positively affected when the predicated PEST sequence was deleted. Further analysis revealed that the predicated PEST sequence lacked a SBC or SBC-like motif allowing interaction with BPMs and required for negative regulation. Nevertheless, LlDREB2B was still regulated at the post-translational level by interaction with AtDRIP1 and AtDRIP2 of Arabidopsis. In addition, LlDREB2B also interacted with AtRCD1 and LlRCD1 via a potential RIM motif located at amino acids 215-245. Taken together, our results show that LlDREB2B participated in the establishment of thermotolerance, and its regulation was different from that of the orthologs of gramineous and eudicot plants.

  8. The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Christine E Cucinotta

    2015-08-01

    Full Text Available Eukaryotes regulate gene expression and other nuclear processes through the posttranslational modification of histones. In S. cerevisiae, the mono-ubiquitylation of histone H2B on lysine 123 (H2B K123ub affects nucleosome stability, broadly influences gene expression and other DNA-templated processes, and is a prerequisite for additional conserved histone modifications that are associated with active transcription, namely the methylation of lysine residues in H3. While the enzymes that promote these chromatin marks are known, regions of the nucleosome required for the recruitment of these enzymes are undefined. To identify histone residues required for H2B K123ub, we exploited a functional interaction between the ubiquitin-protein ligase, Rkr1/Ltn1, and H2B K123ub in S. cerevisiae. Specifically, we performed a synthetic lethal screen with cells lacking RKR1 and a comprehensive library of H2A and H2B residue substitutions, and identified H2A residues that are required for H2B K123ub. Many of these residues map to the nucleosome acidic patch. The substitutions in the acidic patch confer varying histone modification defects downstream of H2B K123ub, indicating that this region contributes differentially to multiple histone modifications. Interestingly, substitutions in the acidic patch result in decreased recruitment of H2B K123ub machinery to active genes and defects in transcription elongation and termination. Together, our findings reveal a role for the nucleosome acidic patch in recruitment of histone modification machinery and maintenance of transcriptional integrity.

  9. The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages

    OpenAIRE

    Czimmerer, Zsolt; Daniel, Bence; Horvath, Attila; Rückerl, Dominik; Nagy, Gergely; Kiss, Mate; Peloquin, Matthew; Budai, Marietta M.; Cuaranta-Monroy, Ixchelt; Simandi, Zoltan; Steiner, Laszlo; Nagy, Bela; Poliska, Szilard; Banko, Csaba; Bacso, Zsolt

    2018-01-01

    Summary The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription fac...

  10. PtrWRKY73, a salicylic acid-inducible poplar WRKY transcription factor, is involved in disease resistance in Arabidopsis thaliana.

    Science.gov (United States)

    Duan, Yanjiao; Jiang, Yuanzhong; Ye, Shenglong; Karim, Abdul; Ling, Zhengyi; He, Yunqiu; Yang, Siqi; Luo, Keming

    2015-05-01

    A salicylic acid-inducible WRKY gene, PtrWRKY73, from Populus trichocarpa , was isolated and characterized. Overexpression of PtrWRKY73 in Arabidopsis thaliana increased resistance to biotrophic pathogens but reduced resistance against necrotrophic pathogens. WRKY transcription factors are commonly involved in plant defense responses. However, limited information is available about the roles of the WRKY genes in poplar defense. In this study, we isolated a salicylic acid (SA)-inducible WRKY gene, PtrWRKY73, from Populus trichocarpa, belonging to group I family and containing two WRKY domains, a D domain and an SP cluster. PtrWRKY73 was expressed predominantly in roots, old leaves, sprouts and stems, especially in phloem and its expression was induced in response to treatment with exogenous SA. PtrWRKY73 was localized to the nucleus of plant cells and exhibited transcriptional activation. Overexpression of PtrWRKY73 in Arabidopsis thaliana resulted in increased resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae (PstDC3000), but more sensitivity to the necrotrophic fungal pathogen Botrytis cinerea. The SA-mediated defense-associated genes, such as PR1, PR2 and PAD4, were markedly up-regulated in transgenic plants overexpressing PtrWRKY73. Arabidopsis non-expressor of PR1 (NPR1) was not affected, whereas a defense-related gene PAL4 had reduced in PtrWRKY73 overexpressor plants. Together, these results indicated that PtrWRKY73 plays a positive role in plant resistance to biotrophic pathogens but a negative effect on resistance against necrotrophic pathogens.

  11. Role of the N-terminal activation domain of coactivator CoCoA in mediating transcriptional activation by β-catenin*

    OpenAIRE

    Yang, Catherine K.; Kim, Jeong Hoon; Stallcup, Michael R.

    2006-01-01

    The coiled-coil coactivator (CoCoA) is involved in transcriptional activation of target genes by nuclear receptors and the xenobiotic aryl hydrocarbon receptor, as well as target genes of the Wnt signaling pathway, which is mediated by the lymphocyte enhancer factor (LEF)/T cell factor transcription factors and the coactivator β-catenin. The recruitment of CoCoA by nuclear receptors is accomplished by the interaction of the central coiled-coiled domain of CoCoA with p160 coactivators; the C-t...

  12. Silicon Dioxide Thin Film Mediated Single Cell Nucleic Acid Isolation

    Science.gov (United States)

    Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

    2013-01-01

    A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube. PMID:23874571

  13. Strong transcription blockage mediated by R-loop formation within a G-rich homopurine-homopyrimidine sequence localized in the vicinity of the promoter.

    Science.gov (United States)

    Belotserkovskii, Boris P; Soo Shin, Jane Hae; Hanawalt, Philip C

    2017-06-20

    Guanine-rich (G-rich) homopurine-homopyrimidine nucleotide sequences can block transcription with an efficiency that depends upon their orientation, composition and length, as well as the presence of negative supercoiling or breaks in the non-template DNA strand. We report that a G-rich sequence in the non-template strand reduces the yield of T7 RNA polymerase transcription by more than an order of magnitude when positioned close (9 bp) to the promoter, in comparison to that for a distal (∼250 bp) location of the same sequence. This transcription blockage is much less pronounced for a C-rich sequence, and is not significant for an A-rich sequence. Remarkably, the blockage is not pronounced if transcription is performed in the presence of RNase H, which specifically digests the RNA strands within RNA-DNA hybrids. The blockage also becomes less pronounced upon reduced RNA polymerase concentration. Based upon these observations and those from control experiments, we conclude that the blockage is primarily due to the formation of stable RNA-DNA hybrids (R-loops), which inhibit successive rounds of transcription. Our results could be relevant to transcription dynamics in vivo (e.g. transcription 'bursting') and may also have practical implications for the design of expression vectors. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. The transcriptional regulatory network mediated by banana (Musa acuminata) dehydration-responsive element binding (MaDREB) transcription factors in fruit ripening.

    Science.gov (United States)

    Kuang, Jian-Fei; Chen, Jian-Ye; Liu, Xun-Cheng; Han, Yan-Chao; Xiao, Yun-Yi; Shan, Wei; Tang, Yang; Wu, Ke-Qiang; He, Jun-Xian; Lu, Wang-Jin

    2017-04-01

    Fruit ripening is a complex, genetically programmed process involving the action of critical transcription factors (TFs). Despite the established significance of dehydration-responsive element binding (DREB) TFs in plant abiotic stress responses, the involvement of DREBs in fruit ripening is yet to be determined. Here, we identified four genes encoding ripening-regulated DREB TFs in banana (Musa acuminata), MaDREB1, MaDREB2, MaDREB3, and MaDREB4, and demonstrated that they play regulatory roles in fruit ripening. We showed that MaDREB1-MaDREB4 are nucleus-localized, induced by ethylene and encompass transcriptional activation activities. We performed a genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) experiment for MaDREB2 and identified 697 genomic regions as potential targets of MaDREB2. MaDREB2 binds to hundreds of loci with diverse functions and its binding sites are distributed in the promoter regions proximal to the transcriptional start site (TSS). Most of the MaDREB2-binding targets contain the conserved (A/G)CC(G/C)AC motif and MaDREB2 appears to directly regulate the expression of a number of genes involved in fruit ripening. In combination with transcriptome profiling (RNA sequencing) data, our results indicate that MaDREB2 may serve as both transcriptional activator and repressor during banana fruit ripening. In conclusion, our study suggests a hierarchical regulatory model of fruit ripening in banana and that the MaDREB TFs may act as transcriptional regulators in the regulatory network. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  15. NF-κB Directly Regulates Fas Transcription to Modulate Fas-mediated Apoptosis and Tumor Suppression*

    Science.gov (United States)

    Liu, Feiyan; Bardhan, Kankana; Yang, Dafeng; Thangaraju, Muthusamy; Ganapathy, Vadivel; Waller, Jennifer L.; Liles, Georgia B.; Lee, Jeffrey R.; Liu, Kebin

    2012-01-01

    Fas is a member of the death receptor family. Stimulation of Fas leads to induction of apoptotic signals, such as caspase 8 activation, as well as “non-apoptotic” cellular responses, notably NF-κB activation. Convincing experimental data have identified NF-κB as a critical promoter of cancer development, creating a solid rationale for the development of antitumor therapy that suppresses NF-κB activity. On the other hand, compelling data have also shown that NF-κB activity enhances tumor cell sensitivity to apoptosis and senescence. Furthermore, although stimulation of Fas activates NF-κB, the function of NF-κB in the Fas-mediated apoptosis pathway remains largely undefined. In this study, we observed that deficiency of either Fas or FasL resulted in significantly increased incidence of 3-methylcholanthrene-induced spontaneous sarcoma development in mice. Furthermore, Fas-deficient mice also exhibited significantly greater incidence of azoxymethane and dextran sodium sulfate-induced colon carcinoma. In addition, human colorectal cancer patients with high Fas protein in their tumor cells had a longer time before recurrence occurred. Engagement of Fas with FasL triggered NF-κB activation. Interestingly, canonical NF-κB was found to directly bind to the FAS promoter. Blocking canonical NF-κB activation diminished Fas expression, whereas blocking alternate NF-κB increased Fas expression in human carcinoma cells. Moreover, although canonical NF-κB protected mouse embryo fibroblast (MEF) cells from TNFα-induced apoptosis, knocking out p65 diminished Fas expression in MEF cells, resulting in inhibition of FasL-induced caspase 8 activation and apoptosis. In contrast, knocking out p52 increased Fas expression in MEF cells. Our observations suggest that canonical NF-κB is a Fas transcription activator and alternate NF-κB is a Fas transcription repressor, and Fas functions as a suppressor of spontaneous sarcoma and colon carcinoma. PMID:22669972

  16. Antisense-mediated suppression of C-hordein biosynthesis in the barley grain results in correlated changes in the transcriptome, protein profile, and amino acid composition

    DEFF Research Database (Denmark)

    Hansen, Mette; Lange, Marianne; Friis, Carsten

    2007-01-01

    Antisense- or RNAi-mediated suppression of the biosynthesis of nutritionally inferior storage proteins is a promising strategy for improving the amino acid profile of seeds. However, the potential pleiotropic effects of this on interconnected pathways and the agronomic quality traits need...... to be addressed. In the current study, a transcriptomic analysis of an antisense C-hordein line of barley was performed, using a grain-specific cDNA array. The C-hordein antisense line is characterized by marked changes in storage protein and amino acid profiles, while the seed weight is within the normal range...... and no external morphological irregularities were observed. The results of the transcriptome analysis showed excellent correlation with data on changes in the relative proportions of storage proteins and amino acid composition. The antisense line had a lower C-hordein level and down-regulated transcript encoding...

  17. Dose-dependent transitions in Nrf2-mediated adaptive response and related stress responses to hypochlorous acid in mouse macrophages

    International Nuclear Information System (INIS)

    Woods, Courtney G.; Fu Jingqi; Xue Peng; Hou Yongyong; Pluta, Linda J.; Yang Longlong; Zhang Qiang; Thomas, Russell S.; Andersen, Melvin E.; Pi Jingbo

    2009-01-01

    Hypochlorous acid (HOCl) is potentially an important source of cellular oxidative stress. Human HOCl exposure can occur from chlorine gas inhalation or from endogenous sources of HOCl, such as respiratory burst by phagocytes. Transcription factor Nrf2 is a key regulator of cellular redox status and serves as a primary source of defense against oxidative stress. We recently demonstrated that HOCl activates Nrf2-mediated antioxidant response in cultured mouse macrophages in a biphasic manner. In an effort to determine whether Nrf2 pathways overlap with other stress pathways, gene expression profiling was performed in RAW 264.7 macrophages exposed to HOCl using whole genome mouse microarrays. Benchmark dose (BMD) analysis on gene expression data revealed that Nrf2-mediated antioxidant response and protein ubiquitination were the most sensitive biological pathways that were activated in response to low concentrations of HOCl (< 0.35 mM). Genes involved in chromatin architecture maintenance and DNA-dependent transcription were also sensitive to very low doses. Moderate concentrations of HOCl (0.35 to 1.4 mM) caused maximal activation of the Nrf2 pathway and innate immune response genes, such as IL-1β, IL-6, IL-10 and chemokines. At even higher concentrations of HOCl (2.8 to 3.5 mM) there was a loss of Nrf2-target gene expression with increased expression of numerous heat shock and histone cluster genes, AP-1-family genes, cFos and Fra1 and DNA damage-inducible Gadd45 genes. These findings confirm an Nrf2-centric mechanism of action of HOCl in mouse macrophages and provide evidence of interactions between Nrf2, inflammatory, and other stress pathways.

  18. Free-radical-mediated conjugate additions. Enantioselective synthesis of butyrolactone natural products: (-)-enterolactone, (-)-arctigenin, (-)-isoarctigenin, (-)-nephrosteranic acid, and (-)-roccellaric acid.

    Science.gov (United States)

    Sibi, Mukund P; Liu, Pingrong; Ji, Jianguo; Hajra, Saumen; Chen, Jian-xie

    2002-03-22

    Lewis acid-mediated conjugate addition of alkyl radicals to a differentially protected fumarate 10 produced the monoalkylated succinates with high chemical efficiency and excellent stereoselectivity. A subsequent alkylation or an aldol reaction furnished the disubstituted succinates with syn configuration. The chiral auxiliary, 4-diphenylmethyl-2-oxazolidinone, controlled the stereoselectivity in both steps. Manipulation of the disubstituted succinates obtained by alkylation furnished the natural products (-)-enterolactone, (-)-arctigenin, and (-)-isoarctigenin. The overall yields for the target natural products were 20-26% over six steps. Selective functionalization of the disubstituted succinates obtained by aldol condensation gave the paraconic acid natural products (-)-nephrosteranic acid (8) and (-)-roccellaric acid (9). The overall yield of the natural products 8 and 9 over four steps was 53% and 42%, respectively.

  19. Generation and transcriptional programming of intestinal dendritic cells: essential role of retinoic acid

    DEFF Research Database (Denmark)

    Zeng, R.; Bscheider, M; Lahl, Katharina

    2016-01-01

    programs, and suppressing proinflammatory nuclear factor-κB-dependent gene expression. Thus, RA is required for transcriptional programming and maturation of intestinal cDC, and with GM-CSF and Flt3L provides a minimal environment for in vitro generation of intestinal cDC1- and cDC2-like cDC from...

  20. Critical role of RanBP2-mediated SUMOylation of Small Heterodimer Partner in maintaining bile acid homeostasis

    Science.gov (United States)

    Kim, Dong-Hyun; Kwon, Sanghoon; Byun, Sangwon; Xiao, Zhen; Park, Sean; Wu, Shwu-Yuan; Chiang, Cheng-Ming; Kemper, Byron; Kemper, Jongsook Kim

    2016-01-01

    Bile acids (BAs) are recently recognized signalling molecules that profoundly affect metabolism. Because of detergent-like toxicity, BA levels must be tightly regulated. An orphan nuclear receptor, Small Heterodimer Partner (SHP), plays a key role in this regulation, but how SHP senses the BA signal for feedback transcriptional responses is not clearly understood. We show an unexpected function of a nucleoporin, RanBP2, in maintaining BA homoeostasis through SUMOylation of SHP. Upon BA signalling, RanBP2 co-localizes with SHP at the nuclear envelope region and mediates SUMO2 modification at K68, which facilitates nuclear transport of SHP and its interaction with repressive histone modifiers to inhibit BA synthetic genes. Mice expressing a SUMO-defective K68R SHP mutant have increased liver BA levels, and upon BA- or drug-induced biliary insults, these mice exhibit exacerbated cholestatic pathologies. These results demonstrate a function of RanBP2-mediated SUMOylation of SHP in maintaining BA homoeostasis and protecting from the BA hepatotoxicity. PMID:27412403

  1. Phytochrome B Mediates the Regulation of Chlorophyll Biosynthesis through Transcriptional Regulation of ChlH and GUN4 in Rice Seedlings

    Science.gov (United States)

    Kagawa, Takatoshi; Tanaka, Ayumi; Ueno, Osamu; Shimada, Hiroaki; Takano, Makoto

    2015-01-01

    Accurate regulation of chlorophyll synthesis is crucial for chloroplast formation during the greening process in angiosperms. In this study, we examined the role of phytochrome B (phyB) in the regulation of chlorophyll synthesis in rice seedlings (Oryza sativa L.) through the characterization of a pale-green phenotype observed in the phyB mutant grown under continuous red light (Rc) irradiation. Our results show that the Rc-induced chlorophyll accumulation can be divided into two components—a phyB-dependent and a phyB-independent component, and that the pale-green phenotype is caused by the absence of the phyB-dependent component. To elucidate the role of the missing component we established an Rc-induced greening experiment, the results of which revealed that several genes encoding proteins on the chlorophyll branch were repressed in the phyB mutant. Notable among them were ChlH and GUN4 genes, which encode subunit H and an activating factor of magnesium chelatase (Mg-chelatase), respectively, that were largely repressed in the mutant. Moreover, the kinetic profiles of chlorophyll precursors suggested that Mg-chelatase activity simultaneously decreased with the reduction in the transcript levels of ChlH and GUN4. These results suggest that phyB mediates the regulation of chlorophyll synthesis through transcriptional regulation of these two genes, whose products exert their action at the branching point of the chlorophyll biosynthesis pathway. Reduction of 5-aminolevulinic acid (5-ALA) synthesis could be detected in the mutant, but the kinetic profiles of chlorophyll precursors indicated that it was an event posterior to the reduction of the Mg-chelatase activity. It means that the repression of 5-ALA synthesis should not be a triggering event for the appearance of the pale-green phenotype. Instead, the repression of 5-ALA synthesis might be important for the subsequent stabilization of the pale-green phenotype for preventing excessive accumulation of hazardous

  2. Transcriptional dysregulation of γ-aminobutyric acid transporter in parvalbumin-containing inhibitory neurons in the prefrontal cortex in schizophrenia.

    Science.gov (United States)

    Bitanihirwe, Byron K Y; Woo, Tsung-Ung W

    2014-12-30

    Parvalbumin (PV)-containing neurons are functionally compromised in schizophrenia. Using double in situ hybridization in postmortem human prefrontal cortex, we found that the messenger RNA (mRNA) for the γ-aminobutyric acid (GABA) transporter GAT-1 was undetectable in 22-41% of PV neurons in layers 3-4 in schizophrenia. In the remaining PV neurons with detectable GAT-1 mRNA, transcript expression was decreased by 26% in layer 3. Hence, the dysfunction of PV neurons involves the molecular dysregulation of presynaptic GABA reuptake. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Pentavalent Bismuth-Mediated Glycosylation Methods to Activate Sialic and Uronic Acids and the Incorporation of Sialic Acids Into Insulin

    Science.gov (United States)

    Kabotso, Daniel Elorm Kwame

    The negative charge at physiological pH of carboxylic acid-containing monosaccharides modulate the properties of many natural biomolecules such as oligosaccharides and glycoconjugates. Unfortunately, these altered electronic properties also make the incorporation of such acidic sugars more challenging as compared to the more commonly studied neutral sugars. Herein are reported the first demonstration of glycosylation reactions mediated by triphenylbis(1,1,1-trifluoromethanesulfonato)-bismuth with thioglycosides containing carboxylic acid substituents protected as esters. Unlike with many neutral sugar substrates, the addition of 1-propanethiol to the reactions proved critical to obtaining good yields of the desired glycosylation products using sialic acid, galacturonic acid, and glucuronic acid. The protocol was demonstrated to be amenable to automation using a liquid-handling platform. The consequences of artificially incorporating carboxylic-acid-containing sugars into proteins were tested by the design of a linker containing 1 to 4 sialic acids--a sugar found in many human proteins and brain tissues--that was attached via reductive amination of trityl thiopropylaldehyde at the phenyl alanine terminal end of the protein insulin produced through solid-phase peptide synthesis. Removal of the trityl group with neat trifluoroacetic acid furnished the thiol-free modified insulin that was ligated via a disulfide bond to the peptide scaffold bearing acetyl protected sialic acids. A 14-15% ammonium hydroxide solution was found to be effective in deprotecting the acetyl groups without degradation of the disulfide bond. In addition to maintaining the potency and bioactivity of insulin, the sialic acid-containing linker rendered insulin more resistant to aggregation due to heat and mechanical agitation compared to the unmodified protein.

  4. Visual detection of the human metapneumovirus using reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye

    Directory of Open Access Journals (Sweden)

    Wang Xiang

    2012-07-01

    Full Text Available Abstract Background Human metapneumovirus (hMPV is a major cause of acute respiratory infections ranging from wheezing to bronchiolitis and pneumonia in children worldwide. The objective of this study is to develop a visual reverse transcription loop-mediated isothermal amplification (RT-LAMP assay for the detection of hMPV and applied to the clinical samples. Results In this study, visual RT-LAMP assay for hMPV was performed in one step with the addition of hydroxynaphthol blue (HNB, and were used to detect respiratory samples. Six primers, including two outer primers (F3 and B3, two inner primers (FIP, BIP and two loop primers (LF and LB, were designed for hMPV N gene by the online software. Moreover, the RT-LAMP assay showed good specificity and no cross-reactivity was observed with human rhinovirus (HRV, human respiratory syncytial Virus (RSV, or influenza virus A/PR/8/34 (H1N1. The detection limit of the RT-LAMP assay was approximately ten viral RNA copies, lower than that of traditional reverse transcriptase polymerase chain reaction (RT-PCR 100 RNA copies. In the 176 nasopharyngeal samples, 23 (13.1% were conformed as hMPV positive by RT-LAMP, but 18 (10.2% positive by RT-PCR. Conclusion Compared with conventional RT-PCR, the visual hMPV RT-LAMP assay performed well in the aspect of detect time, sensitivity, specificity and visibility. It is anticipated that the RT-LAMP will be used for clinical tests in hospital or field testing during outbreaks and in emergency.

  5. Differential transcriptional modulation of duplicated fatty acid-binding protein genes by dietary fatty acids in zebrafish (Danio rerio: evidence for subfunctionalization or neofunctionalization of duplicated genes

    Directory of Open Access Journals (Sweden)

    Denovan-Wright Eileen M

    2009-09-01

    Full Text Available Abstract Background In the Duplication-Degeneration-Complementation (DDC model, subfunctionalization and neofunctionalization have been proposed as important processes driving the retention of duplicated genes in the genome. These processes are thought to occur by gain or loss of regulatory elements in the promoters of duplicated genes. We tested the DDC model by determining the transcriptional induction of fatty acid-binding proteins (Fabps genes by dietary fatty acids (FAs in zebrafish. We chose zebrafish for this study for two reasons: extensive bioinformatics resources are available for zebrafish at zfin.org and zebrafish contains many duplicated genes owing to a whole genome duplication event that occurred early in the ray-finned fish lineage approximately 230-400 million years ago. Adult zebrafish were fed diets containing either fish oil (12% lipid, rich in highly unsaturated fatty acid, sunflower oil (12% lipid, rich in linoleic acid, linseed oil (12% lipid, rich in linolenic acid, or low fat (4% lipid, low fat diet for 10 weeks. FA profiles and the steady-state levels of fabp mRNA and heterogeneous nuclear RNA in intestine, liver, muscle and brain of zebrafish were determined. Result FA profiles assayed by gas chromatography differed in the intestine, brain, muscle and liver depending on diet. The steady-state level of mRNA for three sets of duplicated genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, and fabp11a/fabp11b, was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR. In brain, the steady-state level of fabp7b mRNAs was induced in fish fed the linoleic acid-rich diet; in intestine, the transcript level of fabp1b.1 and fabp7b were elevated in fish fed the linolenic acid-rich diet; in liver, the level of fabp7a mRNAs was elevated in fish fed the low fat diet; and in muscle, the level of fabp7a and fabp11a mRNAs were elevated in fish fed the linolenic acid-rich or the low fat diets. In all cases

  6. The marine sponge-derived polyketide endoperoxide plakortide F acid mediates its antifungal activity by interfering with calcium homeostasis

    Science.gov (United States)

    Plakortide F acid (PFA) is a marine-derived polyketide endoperoxide exhibiting strong inhibitory activity against several clinically important fungal pathogens. In the present study, transcriptional profiling coupled with mutant and biochemical analyses were conducted using the model organism Sacch...

  7. The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity

    International Nuclear Information System (INIS)

    Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji; Miura, Ryota; Hirayama, Jun; Nishina, Hiroshi

    2014-01-01

    Highlights: •Loss of the PDZ-binding motif inhibits constitutively active YAP (5SA)-induced oncogenic cell transformation. •The PDZ-binding motif of YAP promotes its nuclear localization in cultured cells and mouse liver. •Loss of the PDZ-binding motif inhibits YAP (5SA)-induced CTGF transcription in cultured cells and mouse liver. -- Abstract: YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains a highly conserved PDZ-binding motif that regulates YAP’s functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP’s co-activation of TEAD-mediated CTGF transcription

  8. The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity

    Energy Technology Data Exchange (ETDEWEB)

    Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji; Miura, Ryota; Hirayama, Jun, E-mail: hirayama.dbio@mri.tmd.ac.jp; Nishina, Hiroshi, E-mail: nishina.dbio@mri.tmd.ac.jp

    2014-01-17

    Highlights: •Loss of the PDZ-binding motif inhibits constitutively active YAP (5SA)-induced oncogenic cell transformation. •The PDZ-binding motif of YAP promotes its nuclear localization in cultured cells and mouse liver. •Loss of the PDZ-binding motif inhibits YAP (5SA)-induced CTGF transcription in cultured cells and mouse liver. -- Abstract: YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains a highly conserved PDZ-binding motif that regulates YAP’s functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP’s co-activation of TEAD-mediated CTGF transcription.

  9. Mediator and p300/CBP-Steroid Receptor Coactivator Complexes Have Distinct Roles, but Function Synergistically, during Estrogen Receptor α-Dependent Transcription with Chromatin Templates

    OpenAIRE

    Acevedo, Mari Luz; Kraus, W. Lee

    2003-01-01

    Ligand-dependent transcriptional activation by nuclear receptors involves the recruitment of various coactivators to the promoters of hormone-regulated genes assembled into chromatin. Nuclear receptor coactivators include histone acetyltransferase complexes, such as p300/CBP-steroid receptor coactivator (SRC), as well as the multisubunit mediator complexes (“Mediator”), which may help recruit RNA polymerase II to the promoter. We have used a biochemical approach, including an in vitro chromat...

  10. TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thaliana viral infection.

    Science.gov (United States)

    Rodriguez, Maria Cecilia; Conti, Gabriela; Zavallo, Diego; Manacorda, Carlos Augusto; Asurmendi, Sebastian

    2014-08-03

    Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.

  11. The murine cytomegalovirus M35 protein antagonizes type I IFN induction downstream of pattern recognition receptors by targeting NF-κB mediated transcription.

    Directory of Open Access Journals (Sweden)

    Baca Chan

    2017-05-01

    Full Text Available The type I interferon (IFN response is imperative for the establishment of the early antiviral immune response. Here we report the identification of the first type I IFN antagonist encoded by murine cytomegalovirus (MCMV that shuts down signaling following pattern recognition receptor (PRR sensing. Screening of an MCMV open reading frame (ORF library identified M35 as a novel and strong negative modulator of IFNβ promoter induction following activation of both RNA and DNA cytoplasmic PRR. Additionally, M35 inhibits the proinflammatory cytokine response downstream of Toll-like receptors (TLR. Using a series of luciferase-based reporters with specific transcription factor binding sites, we determined that M35 targets NF-κB-, but not IRF-mediated, transcription. Expression of M35 upon retroviral transduction of immortalized bone marrow-derived macrophages (iBMDM led to reduced IFNβ transcription and secretion upon activation of stimulator of IFN genes (STING-dependent signaling. On the other hand, M35 does not antagonize interferon-stimulated gene (ISG 56 promoter induction or ISG transcription upon exogenous stimulation of the type I IFN receptor (IFNAR. M35 is present in the viral particle and, upon MCMV infection of fibroblasts, is immediately shuttled to the nucleus where it exerts its immunomodulatory effects. Deletion of M35 from the MCMV genome and hence from the viral particle resulted in elevated type I IFN transcription and secretion in vitro and in vivo. In the absence of M35, lower viral titers are observed during acute infection of the host, and productive infection in the salivary glands was not detected. In conclusion, the M35 protein is released by MCMV immediately upon infection in order to deftly inhibit the antiviral type I IFN response by targeting NF-κB-mediated transcription. The identification of this novel viral protein reinforces the importance of timely countermeasures in the complex relationship between virus and host.

  12. Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells

    International Nuclear Information System (INIS)

    Wallden, Brett; Emond, Mary; Swift, Mari E; Disis, Mary L; Swisshelm, Karen

    2005-01-01

    The retinoic acid receptor beta 2 (RARβ2) gene modulates proliferation and survival of cultured human breast cancer cells. Previously we showed that ectopic expression of RARβ2 in a mouse xenograft model prevented metastasis, even in the absence of the ligand, all-trans retinoic acid. We investigated both cultured cells and xenograft tumors in order to delineate the gene expression profiles responsible for an antimetastatic phenotype. RNA from MDA-MB-435 human breast cancer cells transduced with RARβ2 or empty retroviral vector (LXSN) was analyzed using Agilent Human 1A Oligo microarrays. The one hundred probes with the greatest differential intensity (p < 0.004, jointly) were determined by selecting the top median log ratios from eight-paired microarrays. Validation of differences in expression was done using Northern blot analysis and quantitative RT-PCR (qRT-PCR). We determined expression of selected genes in xenograft tumors. RARβ2 cells exhibit gene profiles with overrepresentation of genes from Xq28 (p = 2 × 10 -8 ), a cytogenetic region that contains a large portion of the cancer/testis antigen gene family. Other functions or factors impacted by the presence of exogenous RARβ2 include mediators of the immune response and transcriptional regulatory mechanisms. Thirteen of fifteen (87%) of the genes evaluated in xenograft tumors were consistent with differences we found in the cell cultures (p = 0.007). Antimetastatic RARβ2 signalling, direct or indirect, results in an elevation of expression for genes such as tumor-cell antigens (CTAG1 and CTAG2), those involved in innate immune response (e.g., RIG-I/DDX58), and tumor suppressor functions (e.g., TYRP1). Genes whose expression is diminished by RARβ2 signalling include cell adhesion functions (e.g, CD164) nutritional or metabolic processes (e.g., FABP6), and the transcription factor, JUN

  13. Physiological and transcriptional response of Lactobacillus casei ATCC 334 to acid stress.

    Science.gov (United States)

    Broadbent, Jeff R; Larsen, Rebecca L; Deibel, Virginia; Steele, James L

    2010-05-01

    This study investigated features of the acid tolerance response (ATR) in Lactobacillus casei ATCC 334. To optimize ATR induction, cells were acid adapted for 10 or 20 min at different pH values (range, 3.0 to 5.0) and then acid challenged at pH 2.0. Adaptation over a broad range of pHs improved acid tolerance, but the highest survival was noted in cells acid adapted for 10 or 20 min at pH 4.5. Analysis of cytoplasmic membrane fatty acids (CMFAs) in acid-adapted cells showed that they had significantly (P L. casei survival at pH 2.5 was improved at least 100-fold by chemical induction of the stringent response or by the addition of 30 mM malate or 30 mM histidine to the acid challenge medium. To our knowledge, this is the first report that intracellular histidine accumulation may be involved in bacterial acid resistance.

  14. Bile acid effects are mediated by ATP release and purinergic signalling in exocrine pancreatic cells

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena; Haanes, Kristian Agmund; Christensen, Nynne

    2015-01-01

    BACKGROUND: In many cells, bile acids (BAs) have a multitude of effects, some of which may be mediated by specific receptors such the TGR5 or FXR receptors. In pancreas systemic BAs, as well as intra-ductal BAs from bile reflux, can affect pancreatic secretion. Extracellular ATP and purinergic...

  15. Hypoxia induces cancer-associated cAMP/PKA signalling through HIF-mediated transcriptional control of adenylyl cyclases VI and VII.

    Science.gov (United States)

    Simko, Veronika; Iuliano, Filippo; Sevcikova, Andrea; Labudova, Martina; Barathova, Monika; Radvak, Peter; Pastorekova, Silvia; Pastorek, Jaromir; Csaderova, Lucia

    2017-08-31

    Hypoxia is a phenomenon often arising in solid tumours, linked to aggressive malignancy, bad prognosis and resistance to therapy. Hypoxia-inducible factor-1 has been identified as a key mediator of cell and tissue adaptation to hypoxic conditions through transcriptional activation of many genes involved in glucose metabolism and other cancer-related processes, such as angiogenesis, cell survival and cell invasion. Cyclic adenosine 3'5'-monophosphate is one of the most ancient and evolutionarily conserved signalling molecules and the cAMP/PKA signalling pathway plays an important role in cellular adaptation to hypoxia. We have investigated possible new mechanisms behind hypoxic activation of the cAMP/PKA pathway. For the first time, we have shown that hypoxia induces transcriptional up-regulation of the system of adenylyl cyclases, enzymes responsible for cAMP production, in a panel of carcinoma cell lines of various origin. Our data prove functional relevance of the hypoxic increase of adenylyl cyclases VI and VII at least partially mediated by HIF-1 transcription factor. We have identified adenylyl cyclase VI and VII isoforms as mediators of cellular response to hypoxia, which led to the elevation of cAMP levels and enhanced PKA activity, with an impact on cell migration and pH regulation.

  16. PRDM16 enhances nuclear receptor-dependent transcription of the brown fat-specific Ucp1 gene through interactions with Mediator subunit MED1.

    Science.gov (United States)

    Iida, Satoshi; Chen, Wei; Nakadai, Tomoyoshi; Ohkuma, Yoshiaki; Roeder, Robert G

    2015-02-01

    PR domain-containing 16 (PRDM16) induces expression of brown fat-specific genes in brown and beige adipocytes, although the underlying transcription-related mechanisms remain largely unknown. Here, in vitro studies show that PRDM16, through its zinc finger domains, directly interacts with the MED1 subunit of the Mediator complex, is recruited to the enhancer of the brown fat-specific uncoupling protein 1 (Ucp1) gene through this interaction, and enhances thyroid hormone receptor (TR)-driven transcription in a biochemically defined system in a Mediator-dependent manner, thus providing a direct link to the general transcription machinery. Complementary cell-based studies show that upon forskolin treatment, PRDM16 induces Ucp1 expression in undifferentiated murine embryonic fibroblasts, that this induction depends on MED1 and TR, and, consistent with a direct effect, that PRDM16 is recruited to the Ucp1 enhancer. Related studies have defined MED1 and PRDM16 interaction domains important for Ucp1 versus Ppargc1a induction by PRDM16. These results reveal novel mechanisms for PRDM16 function through the Mediator complex. © 2015 Iida et al.; Published by Cold Spring Harbor Laboratory Press.

  17. The use of transcription inhibitors in the study of the mechanism of abscisic acid action in germinating triticale caryopses

    Directory of Open Access Journals (Sweden)

    Stanisław Weidner

    2014-01-01

    Full Text Available The study was conducted on germinating triticale (var. Grado caryopses. The purpose of the experiments was to compare the effect of selected inhibitors of transcription with the action of abscisic acid during germination of caryopses. The following inhibitors were used: α-amanitin, cordycepin, cycloheximide and 5-fluorouracil. Studied were the synthesis of total and polyribosomal RNA, the process of polyribosome formation and the synthesis of ribosomal proteins. The effect of exogenous ABA, especially in the early stages of germination, was not similar to any of the four above inhibitors of transcription. After 12 h of imbibition at a lowered temperature and 3 h of germination, ABA caused a relatively low level of inhibition of RNA synthesis, whereas all of the inhibitors used halted RNA synthesis in embryos by about 50-60%. After 6 h of germination, the same proportion of polyribosomes in the total ribosome fraction (46% was found in both the embryos from the control sample and treated with ABA. The use of inhibitors brought this figure down to below 40%. The conclusion is drawn that in the early stages of germination, regulation of protein synthesis by ABA in triticale caryopses must occur on a level other than transcription.

  18. Rational Design of a Green-Light-Mediated Unimolecular Platform for Fast Switchable Acidic Sensing.

    Science.gov (United States)

    Zhou, Yunyun; Zou, Qi; Qiu, Jing; Wang, Linjun; Zhu, Liangliang

    2018-02-01

    A controllable sensing ability strongly connects to complex and precise events in diagnosis and treatment. However, imposing visible light into the molecular-scale mediation of sensing processes is restricted by the lack of structural relevance. To address this critical challenge, we present the rational design, synthesis, and in vitro studies of a novel cyanostyryl-modified azulene system for green-light-mediated fast switchable acidic sensing. The advantageous features of the design include a highly efficient green-light-driven Z/E-isomerization (a quantum yield up to 61.3%) for fast erasing chromatic and luminescent expressions and a superior compatibility with control of ratiometric protonation. Significantly, these merits of the design enable the development of a microfluidic system to perform a green-light-mediated reusable sensing function toward a gastric acid analyte in a miniaturized platform. The results may provide new insights for building future integrated green materials.

  19. Current concepts on the physiology and genetics of neurotransmitters-mediating enzyme-aromatic L-amino acid decarboxylase

    International Nuclear Information System (INIS)

    Rahman, M.K.

    1993-03-01

    Two most important neurotransmitters, dopamine and serotonin are mediated by the enzyme aromatic L-amino acid decarboxylase (AADC). Because of their importance in the regulation of neuronal functions, behaviour and emotion of higher animals, many researchers are working on this enzyme to elucidate its physiological properties, structure and genetic aspects. We have discovered this enzyme in the mammalian blood, we established sensitive assay methods for the assay of the activities of this enzyme. We have made systematic studies on this enzyme in the tissues and brains of rats, and human subjects. We have found an endogenous inhibitor of this enzyme in the monkey's blood. The amino acid sequences of human AADC has been compared to rat or bovine. A full-length cDNA clone encoding human AADC has been isolated. Very recently the structure of human AADC gene including 5'-flaking region has been characterized and the transcriptional starting point has been determined. The human AADC gene assigned to chromosome 7. Up-to-date research data have shown that AADC is encoded by a single gene. Recently two patients with AADC deficiency were reported. This paper describes the systematic up-to-date review studies on AADC. (author). 62 refs, 5 figs, 8 tabs

  20. Functional characterization of tobacco transcription factor TGA2.1

    DEFF Research Database (Denmark)

    Kegler, C.; Lenk, I.; Krawczyk, S.

    2004-01-01

    Activation sequence-1 (as-1)-like regulatory cis elements mediate transcriptional activation in response to increased levels of plant signalling molecules auxin and salicylic acid (SA). Our earlier work has shown that tobacco cellular as-1-binding complex SARP (salicylic acid responsive protein...

  1. Amino acid limitation induces down-regulation of WNT5a at transcriptional level

    International Nuclear Information System (INIS)

    Wang Zuguang; Chen Hong

    2009-01-01

    An aberrant WNT signaling contributes to the development and progression of multiple cancers. WNT5a is one of the WNT signaling molecules. This study was designed to test the hypothesis that amino acid deprivation induces changes in the WNT signaling pathway in colon cancer cells. Results showed that targets of the amino acid response pathway, ATF3 and p21, were induced in the human colon cancer cell line SW480 during amino acid limitation. There was a significant decrease in the WNT5a mRNA level following amino acid deprivation. The down-regulation of WNT5a mRNA by amino acid deprivation is not due to mRNA destabilization. There is a reduction of nuclear β-catenin protein level by amino acid limitation. Under amino acid limitation, phosphorylation of ERK1/2 was increased and the blockage of ERK1/2 by the inhibitor U0126 partially restored WNT5a mRNA level. In conclusion, amino acid limitation in colon cancer cells induces phosphorylation of ERK1/2, which then down-regulates WNT5a expression.

  2. Docosahexaenoic acid inhibits IL-6 expression via PPARγ-mediated expression of catalase in cerulein-stimulated pancreatic acinar cells.

    Science.gov (United States)

    Song, Eun Ah; Lim, Joo Weon; Kim, Hyeyoung

    2017-07-01

    Cerulein pancreatitis mirrors human acute pancreatitis. In pancreatic acinar cells exposed to cerulein, reactive oxygen species (ROS) mediate inflammatory signaling by Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3, and cytokine induction. Docosahexaenoic acid (DHA) acts as an agonist of peroxisome proliferator activated receptor γ (PPARγ), which mediates the expression of some antioxidant enzymes. We hypothesized that DHA may induce PPARγ-target catalase expression and reduce ROS levels, leading to the inhibition of JAK2/STAT3 activation and IL-6 expression in cerulein-stimulated acinar cells. Pancreatic acinar AR42J cells were treated with DHA in the presence or absence of the PPARγ antagonist GW9662, or treated with the PPARγ agonist troglitazone, and then stimulated with cerulein. Expression of IL-6 and catalase, ROS levels, JAK2/STAT3 activation, and nuclear translocation of PPARγ were assessed. DHA suppressed the increase in ROS, JAK2/STAT3 activation, and IL-6 expression induced nuclear translocation of PPARγ and catalase expression in cerulein-stimulated AR42J cells. Troglitazone inhibited the cerulein-induced increase in ROS and IL-6 expression, but induced catalase expression similar to DHA in AR42J cells. GW9662 abolished the inhibitory effect of DHA on cerulein-induced increase in ROS and IL-6 expression in AR42J cells. DHA-induced expression of catalase was suppressed by GW9662 in cerulein-stimulated AR42J cells. Thus, DHA induces PPARγ activation and catalase expression, which inhibits ROS-mediated activation of JAK2/STAT3 and IL-6 expression in cerulein-stimulated pancreatic acinar cells. Copyright © 2017. Published by Elsevier Ltd.

  3. Biochemical and redox characterization of the mediator complex and its associated transcription factor GeBPL, a GLABROUS1 enhancer binding protein.

    Science.gov (United States)

    Shaikhali, Jehad; Davoine, Céline; Brännström, Kristoffer; Rouhier, Nicolas; Bygdell, Joakim; Björklund, Stefan; Wingsle, Gunnar

    2015-06-15

    The eukaryotic mediator integrates regulatory signals from promoter-bound transcription factors (TFs) and transmits them to RNA polymerase II (Pol II) machinery. Although redox signalling is important in adjusting plant metabolism and development, nothing is known about a possible redox regulation of mediator. In the present study, using pull-down and yeast two-hybrid assays, we demonstrate the association of mediator (MED) subunits MED10a, MED28 and MED32 with the GLABROUS1 (GL1) enhancer-binding protein-like (GeBPL), a plant-specific TF that binds a promoter containing cryptochrome 1 response element 2 (CryR2) element. All the corresponding recombinant proteins form various types of covalent oligomers linked by intermolecular disulfide bonds that are reduced in vitro by the thioredoxin (TRX) and/or glutathione/glutaredoxin (GRX) systems. The presence of recombinant MED10a, MED28 and MED32 subunits or changes of its redox state affect the DNA-binding capacity of GeBPL suggesting that redox-driven conformational changes might modulate its activity. Overall, these results advance our understanding of how redox signalling affects transcription and identify mediator as a novel actor in redox signalling pathways, relaying or integrating redox changes in combination with specific TFs as GeBPL. © The Authors Journal compilation © 2015 Biochemical Society.

  4. CD36 Mediated Fatty Acid-Induced Podocyte Apoptosis via Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Wei Hua

    Full Text Available Hyperlipidemia-induced apoptosis mediated by fatty acid translocase CD36 is associated with increased uptake of ox-LDL or fatty acid in macrophages, hepatocytes and proximal tubular epithelial cells, leading to atherosclerosis, liver damage and fibrosis in obese patients, and diabetic nephropathy (DN, respectively. However, the specific role of CD36 in podocyte apoptosis in DN with hyperlipidemia remains poorly investigated.The expression of CD36 was measured in paraffin-embedded kidney tissue samples (Ctr = 18, DN = 20 by immunohistochemistry and immunofluorescence staining. We cultured conditionally immortalized mouse podocytes (MPC5 and treated cells with palmitic acid, and measured CD36 expression by real-time PCR, Western blot analysis and immunofluorescence; lipid uptake by Oil red O staining and BODIPY staining; apoptosis by flow cytometry assay, TUNEL assay and Western blot analysis; and ROS production by DCFH-DA fluorescence staining. All statistical analyses were performed using SPSS 21.0 statistical software.CD36 expression was increased in kidney tissue from DN patients with hyperlipidemia. Palmitic acid upregulated CD36 expression and promoted its translocation from cytoplasm to plasma membrane in podocytes. Furthermore, palmitic acid increased lipid uptake, ROS production and apoptosis in podocytes, Sulfo-N-succinimidyloleate (SSO, the specific inhibitor of the fatty acid binding site on CD36, decreased palmitic acid-induced fatty acid accumulation, ROS production, and apoptosis in podocytes. Antioxidant 4-hydroxy-2,2,6,6- tetramethylpiperidine -1-oxyl (tempol inhibited the overproduction of ROS and apoptosis in podocytes induced by palmitic acid.CD36 mediated fatty acid-induced podocyte apoptosis via oxidative stress might participate in the process of DN.

  5. A novel branched chain amino acids responsive transcriptional regulator, BCARR, negatively acts on the proteolytic system in Lactobacillus helveticus.

    Directory of Open Access Journals (Sweden)

    Taketo Wakai

    Full Text Available Transcriptional negative regulation of the proteolytic system of Lactobacillus helveticus CM4 in response to amino acids seems to be very important for the control of antihypertensive peptide production; however, it remains poorly understood. A 26-kDa protein with N-terminal cystathionine β-synthase domains (CBS domain protein, which seems to be involved in the regulatory system, was purified by using a DNA-sepharose bound 300-bp DNA fragment corresponding to the upstream regions of the six proteolytic genes that are down-regulated by amino acids. The CBS domain protein bound to a DNA fragment corresponding to the region upstream of the pepV gene in response to branched chain amino acids (BCAAs. The expression of the pepV gene in Escherichia coli grown in BCAA-enriched medium was repressed when the CBS domain protein was co-expressed. These results reveal that the CBS domain protein acts as a novel type of BCAA-responsive transcriptional regulator (BCARR in L. helveticus. From comparative analysis of the promoter regions of the six proteolysis genes, a palindromic AT-rich motif, 5'-AAAAANNCTWTTATT-3', was predicted as the consensus DNA motif for the BCARR protein binding. Footprint analysis using the pepV promotor region and gel shift analyses with the corresponding short DNA fragments strongly suggested that the BCARR protein binds adjacent to the pepV promoter region and affects the transcription level of the pepV gene in the presence of BCAAs. Homology search analysis of the C-terminal region of the BCARR protein suggested the existence of a unique βαββαβ fold structure that has been reported in a variety of ACT (aspartate kinase-chorismate mutase-tyrA domain proteins for sensing amino acids. These results also suggest that the sensing of BCAAs by the ACT domain might promote the binding of the BCARR to DNA sequences upstream of proteolysis genes, which affects the gene expression of the proteolytic system in L. helveticus.

  6. Induction of time-dependent oxidative stress and related transcriptional effects of perfluorododecanoic acid in zebrafish liver

    Energy Technology Data Exchange (ETDEWEB)

    Liu Yang [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Datun Road, Beijing 100101 (China); Graduate School of the Chinese Academy of Sciences, Beijing 100080 (China); Wang Jianshe; Wei Yanhong; Zhang Hongxia; Xu Muqi [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Datun Road, Beijing 100101 (China); Dai Jiayin [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Datun Road, Beijing 100101 (China)], E-mail: daijy@ioz.ac.cn

    2008-09-29

    The effects of acute perfluorododecanoic acid (PFDoA) exposure on the induction of oxidative stress and alteration of mitochondrial gene expression were studied in the livers of female zebrafish (Danio rerio). Female zebrafish were exposed to PFDoA via a single intraperitoneal injection (0, 20, 40, or 80 {mu}g PFDoA/g body weight) and were then sacrificed 48 h, 96 h, or seven days post-PFDoA administration. PFDoA-treated fish exhibited histopathological liver damage, including swollen hepatocytes, vacuolar degeneration, and nuclei pycnosis. Glutathione (GSH) content and catalase (CAT) activity decreased significantly at 48 h post-injection while superoxide dismutase (SOD) activity was initially decreased at 48 h post-injection but was then elevated by seven days post-injection. The activity of glutathione peroxidase (GPx) increased at 48 h and seven days compared to control fish, although the increased level at seven days post-injection was decreased compared to the level at 48 h post-injection. Lipid peroxidation levels were increased at seven days post-injection, while no apparent induction was observed at 48 h or 96 h post-injection. The mRNA expression of medium-chain fatty acid dehydrogenase (MCAD) was induced, while the transcriptional expression of liver fatty acid binding protein (L-FABP), peroxisome proliferating activating receptor {alpha} (PPAR{alpha}), carnitine palmitoyl-transferase I (CPT-I), uncoupling protein 2 (UCP-2), and Bcl-2 were significantly inhibited. Furthermore, the transcriptional expression of peroxisomal fatty acyl-CoA oxidase (ACOX), very long-chain acyl-CoA dehydrogenase (VLCAD), long-chain acyl-CoA dehydrogenase (LCAD) did not exhibit significant changes following PFDoA treatment. No significant changes were noted in the transcriptional expression of genes involved in mitochondrial respiratory chain and ATP synthesis, including cytochrome c oxidase subunit I (COXI), NADH dehydrogenase subunit I (NDI), and ATP synthase F0 subunit 6

  7. Induction of time-dependent oxidative stress and related transcriptional effects of perfluorododecanoic acid in zebrafish liver

    International Nuclear Information System (INIS)

    Liu Yang; Wang Jianshe; Wei Yanhong; Zhang Hongxia; Xu Muqi; Dai Jiayin

    2008-01-01

    The effects of acute perfluorododecanoic acid (PFDoA) exposure on the induction of oxidative stress and alteration of mitochondrial gene expression were studied in the livers of female zebrafish (Danio rerio). Female zebrafish were exposed to PFDoA via a single intraperitoneal injection (0, 20, 40, or 80 μg PFDoA/g body weight) and were then sacrificed 48 h, 96 h, or seven days post-PFDoA administration. PFDoA-treated fish exhibited histopathological liver damage, including swollen hepatocytes, vacuolar degeneration, and nuclei pycnosis. Glutathione (GSH) content and catalase (CAT) activity decreased significantly at 48 h post-injection while superoxide dismutase (SOD) activity was initially decreased at 48 h post-injection but was then elevated by seven days post-injection. The activity of glutathione peroxidase (GPx) increased at 48 h and seven days compared to control fish, although the increased level at seven days post-injection was decreased compared to the level at 48 h post-injection. Lipid peroxidation levels were increased at seven days post-injection, while no apparent induction was observed at 48 h or 96 h post-injection. The mRNA expression of medium-chain fatty acid dehydrogenase (MCAD) was induced, while the transcriptional expression of liver fatty acid binding protein (L-FABP), peroxisome proliferating activating receptor α (PPARα), carnitine palmitoyl-transferase I (CPT-I), uncoupling protein 2 (UCP-2), and Bcl-2 were significantly inhibited. Furthermore, the transcriptional expression of peroxisomal fatty acyl-CoA oxidase (ACOX), very long-chain acyl-CoA dehydrogenase (VLCAD), long-chain acyl-CoA dehydrogenase (LCAD) did not exhibit significant changes following PFDoA treatment. No significant changes were noted in the transcriptional expression of genes involved in mitochondrial respiratory chain and ATP synthesis, including cytochrome c oxidase subunit I (COXI), NADH dehydrogenase subunit I (NDI), and ATP synthase F0 subunit 6 (ATPo6). These

  8. Grr1p is required for transcriptional induction of amino acid permease genes and proper transcriptional regulation of genes in carbon metabolism of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine; Regenberg, Birgitte; Nielsen, Jens

    2005-01-01

    and a grr1 Delta strain and adding citrulline in the exponential phase. Whole-genome transcription analyses were performed on samples from each cultivation, both immediately before and 30 min after citrulline addition. Transcriptional induction of the AAP genes AGP1, BAP2, BAP3, DIP5, GNP1 and TAT1 is fully...

  9. Apoptosis-inducing factor (Aif1) mediates anacardic acid-induced apoptosis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Muzaffar, Suhail; Chattoo, Bharat B

    2017-03-01

    Anacardic acid is a medicinal phytochemical that inhibits proliferation of fungal as well as several types of cancer cells. It induces apoptotic cell death in various cell types, but very little is known about the mechanism involved in the process. Here, we used budding yeast Saccharomyces cerevisiae as a model to study the involvement of some key elements of apoptosis in the anacardic acid-induced cell death. Plasma membrane constriction, chromatin condensation, DNA degradation, and externalization of phosphatidylserine (PS) indicated that anacardic acid induces apoptotic cell death in S. cerevisiae. However, the exogenous addition of broad-spectrum caspase inhibitor Z-VAD-FMK or deletion of the yeast caspase Yca1 showed that the anacardic acid-induced cell death is caspase independent. Apoptosis-inducing factor (AIF1) deletion mutant was resistant to the anacardic acid-induced cell death, suggesting a key role of Aif1. Overexpression of Aif1 made cells highly susceptible to anacardic acid, further confirming that Aif1 mediates anacardic acid-induced apoptosis. Interestingly, instead of the increase in the intracellular reactive oxygen species (ROS) normally observed during apoptosis, anacardic acid caused a decrease in the intracellular ROS levels. Quantitative real-time PCR analysis showed downregulation of the BIR1 survivin mRNA expression during the anacardic acid-induced apoptosis.

  10. Tannic Acid Induces Endoplasmic Reticulum Stress-Mediated Apoptosis in Prostate Cancer.

    Science.gov (United States)

    Nagesh, Prashanth K B; Hatami, Elham; Chowdhury, Pallabita; Kashyap, Vivek K; Khan, Sheema; Hafeez, Bilal B; Chauhan, Subhash C; Jaggi, Meena; Yallapu, Murali M

    2018-03-07

    Endoplasmic reticulum (ER) stress is an intriguing target with significant clinical importance in chemotherapy. Interference with ER functions can lead to the accumulation of unfolded proteins, as detected by transmembrane sensors that instigate the unfolded protein response (UPR). Therefore, controlling induced UPR via ER stress with natural compounds could be a novel therapeutic strategy for the management of prostate cancer. Tannic acid (a naturally occurring polyphenol) was used to examine the ER stress mediated UPR pathway in prostate cancer cells. Tannic acid treatment inhibited the growth, clonogenic, invasive, and migratory potential of prostate cancer cells. Tannic acid demonstrated activation of ER stress response (Protein kinase R-like endoplasmic reticulum kinase (PERK) and inositol requiring enzyme 1 (IRE1)) and altered its regulatory proteins (ATF4, Bip, and PDI) expression. Tannic acid treatment affirmed upregulation of apoptosis-associated markers (Bak, Bim, cleaved caspase 3, and cleaved PARP), while downregulation of pro-survival proteins (Bcl-2 and Bcl-xL). Tannic acid exhibited elevated G₁ population, due to increase in p18 INK4C and p21 WAF1/CIP1 expression, while cyclin D1 expression was inhibited. Reduction of MMP2 and MMP9, and reinstated E-cadherin signifies the anti-metastatic potential of this compound. Altogether, these results demonstrate that tannic acid can promote apoptosis via the ER stress mediated UPR pathway, indicating a potential candidate for cancer treatment.

  11. Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents

    Science.gov (United States)

    Benoit, Stephen C.; Kemp, Christopher J.; Elias, Carol F.; Abplanalp, William; Herman, James P.; Migrenne, Stephanie; Lefevre, Anne-Laure; Cruciani-Guglielmacci, Céline; Magnan, Christophe; Yu, Fang; Niswender, Kevin; Irani, Boman G.; Holland, William L.; Clegg, Deborah J.

    2009-01-01

    Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-θ, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-θ was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-θ to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-θ nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-θ attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKC-θ activation, resulting in reduced insulin activity. PMID:19726875

  12. Palmitic acid mediates hypothalamic insulin resistance by altering PKC-theta subcellular localization in rodents.

    Science.gov (United States)

    Benoit, Stephen C; Kemp, Christopher J; Elias, Carol F; Abplanalp, William; Herman, James P; Migrenne, Stephanie; Lefevre, Anne-Laure; Cruciani-Guglielmacci, Céline; Magnan, Christophe; Yu, Fang; Niswender, Kevin; Irani, Boman G; Holland, William L; Clegg, Deborah J

    2009-09-01

    Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-theta, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-theta was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-theta to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-theta nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-theta attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKC-theta activation, resulting in reduced insulin activity.

  13. The N-Terminus of the Floral Arabidopsis TGA Transcription Factor PERIANTHIA Mediates Redox-Sensitive DNA-Binding.

    Directory of Open Access Journals (Sweden)

    Nora Gutsche

    Full Text Available The Arabidopsis TGA transcription factor (TF PERIANTHIA (PAN regulates the formation of the floral organ primordia as revealed by the pan mutant forming an abnormal pentamerous arrangement of the outer three floral whorls. The Arabidopsis TGA bZIP TF family comprises 10 members, of which PAN and TGA9/10 control flower developmental processes and TGA1/2/5/6 participate in stress-responses. For the TGA1 protein it was shown that several cysteines can be redox-dependently modified. TGA proteins interact in the nucleus with land plant-specific glutaredoxins, which may alter their activities posttranslationally. Here, we investigated the DNA-binding of PAN to the AAGAAT motif under different redox-conditions. The AAGAAT motif is localized in the second intron of the floral homeotic regulator AGAMOUS (AG, which controls stamen and carpel development as well as floral determinacy. Whereas PAN protein binds to this regulatory cis-element under reducing conditions, the interaction is strongly reduced under oxidizing conditions in EMSA studies. The redox-sensitive DNA-binding is mediated via a special PAN N-terminus, which is not present in other Arabidopsis TGA TFs and comprises five cysteines. Two N-terminal PAN cysteines, Cys68 and Cys87, were shown to form a disulfide bridge and Cys340, localized in a C-terminal putative transactivation domain, can be S-glutathionylated. Comparative land plant analyses revealed that the AAGAAT motif exists in asterid and rosid plant species. TGA TFs with N-terminal extensions of variable length were identified in all analyzed seed plants. However, a PAN-like N-terminus exists only in the rosids and exclusively Brassicaceae homologs comprise four to five of the PAN N-terminal cysteines. Redox-dependent modifications of TGA cysteines are known to regulate the activity of stress-related TGA TFs. Here, we show that the N-terminal PAN cysteines participate in a redox-dependent control of the PAN interaction with a highly

  14. A systems approach for discovering linoleic acid derivatives that potentially mediate pain and itch

    Science.gov (United States)

    Ramsden, Christopher E.; Domenichiello, Anthony F.; Yuan, Zhi-Xin; Sapio, Matthew R.; Keyes, Gregory S.; Mishra, Santosh K.; Gross, Jacklyn R.; Majchrzak-Hong, Sharon; Zamora, Daisy; Horowitz, Mark S.; Davis, John M.; Sorokin, Alexander V.; Dey, Amit; LaPaglia, Danielle M.; Wheeler, Joshua J.; Vasko, Michael R.; Mehta, Nehal N.; Mannes, Andrew J.; Iadarola, Michael J.

    2018-01-01

    Chronic pain and itch are common hypersensitivity syndromes that are affected by endogenous mediators. We applied a systems-based, translational approach to predict, discover, and characterize mediators of pain and itch that are regulated by diet and inflammation. Profiling of tissue-specific precursor abundance and biosynthetic gene expression predicted that inflamed skin would be abundant in four previously unknown 11-hydroxy-epoxy-or 11-keto-epoxy-octadecenoate linoleic acid derivatives and four previously identified 9- or 13-hydroxy-epoxy- or 9- or 13-keto-epoxy-octadecenoate linoleic acid derivatives. All of these mediators were confirmed to be abundant in rat and human skin by mass spectrometry. However, only the two 11-hydroxy-epoxy-octadecenoates sensitized rat dorsal root ganglion neurons to release more calcitonin gene–related peptide (CGRP), which is involved in pain transmission, in response to low pH (which mimics an inflammatory state) or capsaicin (which activates ion channels involved in nociception). The two 11-hydroxy-epoxy-octadecenoates share a 3-hydroxy-Z-pentenyl-E-epoxide moiety, thus suggesting that this substructure could mediate nociceptor sensitization. In rats, intradermal hind paw injection of 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate elicited C-fiber–mediated sensitivity to thermal pain. In a randomized trial testing adjunctive strategies to manage refractory chronic headaches, reducing the dietary intake of linoleic acid was associated with decreases in plasma 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate, which correlated with clinical pain reduction. Human psoriatic skin had 30-fold higher 9-keto-12,13-trans-epoxy-(10E)-octadecenoate compared to control skin, and intradermal injection of this compound induced itch-related scratching behavior in mice. Collectively, these findings define a family of endogenous mediators with potential roles in pain and itch. PMID:28831021

  15. Fatty Acid and Transcript Profiling in Developing Seeds of Three Brassica napus Cultivars

    Directory of Open Access Journals (Sweden)

    Petkova Mariana

    2015-12-01

    Full Text Available Fatty acid levels and gene expression profiles for selected genes associated with the synthesis of fatty acids (FA, triacylglycerol, and oil body proteins were examined in three oilseed rape (Brassica napus cultivars that have utility for cultivar development in our spring canola breeding program. The seed oil content of Bronowski, Q2, and Westar was 39.0, 40.1, and 40.6%, respectively at 40 days after flowering (DAF. During the 20 to 40 day period of seed development, cultivars had varying levels of palmitic, stearic, oleic, linoleic, α-linolenic, eicosenoic, and erucic acid. In general, the percentage of each FA was similar among the cultivars during seed development. However, the level of oleic acid was lower and the levels of eicosenoic acid and erucic acid were higher in Bronowski than in Q2 and Westar seeds; linoleic acid also tended to be lower in Bronowski. Gene expression among the cultivars was similar from 10 to 40 DAF. The few exceptions were that expression of KAS1 and SAD were higher in Westar and Q2 than in Bronowski at 25 DAF, SAD was highest in Q2, intermediate in Westar, and lowest in Bronowski at 35 DAF, FAD2 was higher in Q2 than in Bronowski at 35 DAF, FAD3 was higher in Q2 than in Bronowski at 15 DAF and Q2 and Westar at 25 and 30 DAF, and FAE1 was higher in Westar and Q2 than in Bronowski at 30 DAF. Correlation analysis for gene expression against DAF for each genotype supported a common trend in gene expression among the three cultivars with gene expression tending to decrease over time; except for LPAAT, which tended to increase. The correlation between the level of FAs and expression of genes by genotype indicated no general trend; rather correlations seem to depend on the genotype.

  16. Developing Novel Anticancer DNA-binding Drugs to Disrupt ETS-Mediated Transcription Associated with Breast Cancer: Use of the c-fos Serum Response Element as a Model System

    National Research Council Canada - National Science Library

    White, Christine

    2002-01-01

    Disregulated transcription factor (TF)-mediated activation of gene expression can play a key role in oncogenesis, especially in breast cancer, preventing TF/DNA interactions using small molecule DNA-reactive agents may decrease oncogenic...

  17. Transcriptional responses of earthworm (Eisenia fetida) exposed to naphthenic acids in soil

    International Nuclear Information System (INIS)

    Wang, Jie; Cao, Xiaofeng; Sun, Jinhua; Chai, Liwei; Huang, Yi; Tang, Xiaoyan

    2015-01-01

    In this study, earthworms (Eisenia fetida) were exposed to commercial NAs contaminated soil, and changes in the levels of reactive oxygen species (ROS) and gene expressions of their defense system were monitored. The effects on the gene expression involved in reproduction and carcinogenesis were also evaluated. Significant increases in ROS levels was observed in NAs exposure groups, and the superoxide dismutase (SOD) and catalase (CAT) genes were both up-regulated at low and medium exposure doses, which implied NAs might exert toxicity by oxidative stress. The transcription of CRT and HSP70 coincided with oxidative stress, which implied both chaperones perform important functions in the protection against oxidative toxicity. The upregulation of TCTP gene indicated a potential adverse effect of NAs to terrestrial organisms through induction of carcinogenesis, and the downregulation of ANN gene indicated that NAs might potentially result in deleterious reproduction effects. - Highlights: • The first attempt to study gene ecotoxicity of NAs in terrestrial environment. • NAs exert toxicity by oxidative stress on earthworm. • NAs might cause carcinogenesis and reproductive disruption to earthworm. - NAs induced oxidative stress and altered transcriptions of genes involved in defense, reproduction, and carcinogenesis

  18. Ezh2 regulates transcriptional and post-translational expression of T-bet and promotes Th1 cell responses mediating aplastic anemia in mice1

    Science.gov (United States)

    Tong, Qing; He, Shan; Xie, Fang; Mochizuki, Kazuhiro; Liu, Yongnian; Mochizuki, Izumi; Meng, Lijun; Sun, Hongxing; Zhang, Yanyun; Guo, Yajun; Hexner, Elizabeth; Zhang, Yi

    2014-01-01

    Acquired aplastic anemia (AA) is a potentially fatal bone marrow (BM) failure syndrome. IFN-γ-producing T helper (Th)1 CD4+ T cells mediate the immune destruction of hematopoietic cells, and are central to the pathogenesis. However, the molecular events that control the development of BM-destructive Th1 cells remain largely unknown. Ezh2 is a chromatin-modifying enzyme that regulates multiple cellular processes primarily by silencing gene expression. We recently reported that Ezh2 is crucial for inflammatory T cell responses after allogeneic BM transplantation. To elucidate whether Ezh2 mediates pathogenic Th1 responses in AA and the mechanism of Ezh2 action in regulating Th1 cells, we studied the effects of Ezh2 inhibition in CD4+ T cells using a mouse model of human AA. Conditionally deleting Ezh2 in mature T cells dramatically reduced the production of BM-destructive Th1 cells in vivo, decreased BM-infiltrating Th1 cells, and rescued mice from BM failure. Ezh2 inhibition resulted in significant decrease in the expression of Tbx21 and Stat4 (which encode transcription factors T-bet and STAT4, respectively). Introduction of T-bet but not STAT4 into Ezh2-deficient T cells fully rescued their differentiation into Th1 cells mediating AA. Ezh2 bound to the Tbx21 promoter in Th1 cells, and directly activated Tbx21 transcription. Unexpectedly, Ezh2 was also required to prevent proteasome-mediated degradation of T-bet protein in Th1 cells. Our results identify T-bet as the transcriptional and post-translational Ezh2 target that acts together to generate BM-destructive Th1 cells, and highlight the therapeutic potential of Ezh2 inhibition in reducing AA and other autoimmune diseases. PMID:24760151

  19. Penta-acetyl geniposide-induced apoptosis involving transcription of NGF/p75 via MAPK-mediated AP-1 activation in C6 glioma cells

    International Nuclear Information System (INIS)

    Peng, C.-H.; Huang, C.-N.; Hsu, S.-P.; Wang, C.-J.

    2007-01-01

    We have demonstrated the herbal derivative penta-acetyl geniposide ((Ac) 5 GP) induces C6 glioma cell apoptosis through the critical sphingomyelinase (SMase)/nerve growth factor (NGF)/p75 and its downstream signals. It has been reported mitogen-activated protein kinase (MAPK) mediates NGF synthesis induced by SMase activation. In this study, ERK, p38 and JNK are shown to mediate (Ac) 5 GP-induced glioma cell apoptosis and elevation of NGF and p75. Treatment of PD98059 (ERK-specific inhibitor), SB203580 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) decreases the elevation of NGF and p75 mRNA induced by (Ac) 5 GP, indicating possible transcription regulation via MAPKs. The results of nuclear extract blotting and EMSA further confirm (Ac) 5 GP maximally increases AP-1 and NF-κB DNA binding at 6 h. Inhibition of ERK, p38 and JNK block the activation of AP-1 and NF-κB, suggesting these MAPKs are involved in (Ac) 5 GP-induced transcription regulation. We thereby used RT-PCR to analyze cells treated with (Ac) 5 GP, with or without AP-1 or NF-κB inhibitors. AP-1 inhibitor NDGA decreases NGF/p75 and expression of FasL and caspase 3 induced by (Ac) 5 GP, suggesting the importance of AP-1 in mediating NGF/p75 and their downstream apoptotic signals. However, FasL and caspase 3 do not change with the NF-κB inhibitor PDTC; NF-κB might be linked to other cellular events. Overall, we demonstrate that MAPK mediates (Ac) 5 GP-induced activation of AP-1, promoting the transcription of NGF/p75 and downstream apoptotic signals. These results further highlight the potential therapeutic effects of (Ac) 5 GP in chemoprevention or as an anti-tumor agent

  20. An ortholog of farA of Aspergillus nidulans is implicated in the transcriptional activation of genes involved in fatty acid utilization in the yeast Yarrowia lipolytica

    International Nuclear Information System (INIS)

    Poopanitpan, Napapol; Kobayashi, Satoshi; Fukuda, Ryouichi; Horiuchi, Hiroyuki; Ohta, Akinori

    2010-01-01

    Research highlights: → POR1 is a Yarrowia lipolytica ortholog of farA involved in fatty acid response in A. nidulans. → Deletion of POR1 caused growth defects on fatty acids. → Δpor1 strain exhibited defects in the induction of genes involved in fatty acid utilization. -- Abstract: The yeast Yarrowia lipolytica effectively utilizes hydrophobic substrates such as fatty acids and n-alkanes. To identify a gene(s) regulating fatty acid utilization in Y. lipolytica, we first studied homologous genes to OAF1 and PIP2 of Saccharomyces cerevisiae, but their disruption did not change growth on oleic acid at all. We next characterized a Y. lipolytica gene, POR1 (primary oleate regulator 1), an ortholog of farA encoding a transcriptional activator that regulates fatty acid utilization in Aspergillus nidulans. The deletion mutant of POR1 was defective in the growth on various fatty acids, but not on glucose, glycerol, or n-hexadecane. It exhibited slight defect on n-decane. The transcriptional induction of genes involved in β-oxidation and peroxisome proliferation by oleate was distinctly diminished in the Δpor1 strains. These data suggest that POR1 encodes a transcriptional activator widely regulating fatty acid metabolism in Y. lipolytica.

  1. PNA binding to the non-template DNA strand interferes with transcription, suggesting a blockage mechanism mediated by R-loop formation.

    Science.gov (United States)

    Belotserkovskii, Boris P; Hanawalt, Philip C

    2015-11-01

    Peptide Nucleic Acids (PNAs) are artificial DNA mimics with superior nucleic acid binding capabilities. T7 RNA polymerase (T7 RNAP) transcription upon encountering PNA bound to the non-template DNA strand was studied in vitro. A characteristic pattern of blockage signals was observed, extending downstream from the PNA binding site, similar to that produced by G-rich homopurine-homopyrimidine (hPu-hPy) sequences and likely caused by R-loop formation. Since blocked transcription complexes in association with stable R-loops may interfere with replication and in some cases trigger apoptosis, targeted R-loop formation might be employed to inactivate selected cells, such as those in tumors, based upon their unique complement of expressed genes. © 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

  2. Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000.

    Science.gov (United States)

    Lewis, Laura A; Polanski, Krzysztof; de Torres-Zabala, Marta; Jayaraman, Siddharth; Bowden, Laura; Moore, Jonathan; Penfold, Christopher A; Jenkins, Dafyd J; Hill, Claire; Baxter, Laura; Kulasekaran, Satish; Truman, William; Littlejohn, George; Prusinska, Justyna; Mead, Andrew; Steinbrenner, Jens; Hickman, Richard; Rand, David; Wild, David L; Ott, Sascha; Buchanan-Wollaston, Vicky; Smirnoff, Nick; Beynon, Jim; Denby, Katherine; Grant, Murray

    2015-11-01

    Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae. © 2015 American Society of Plant Biologists. All rights reserved.

  3. The Transcriptional Heat Shock Response of Salmonella Typhimurium Shows Hysteresis and Heated Cells Show Increased Resistance to Heat and Acid Stress

    DEFF Research Database (Denmark)

    Pin, C.; Hansen, Trine; Munoz-Cuevas, M.

    2012-01-01

    We investigated if the transcriptional response of Salmonella Typhimurium to temperature and acid variations was hysteretic, i.e. whether the transcriptional regulation caused by environmental stimuli showed memory and remained after the stimuli ceased. The transcriptional activity of non......, implying that down-regulation was significantly less synchronized than upregulation. The hysteretic transcriptional response to heat shock was accompanied by higher resistance to inactivation at 50uC as well as cross-resistance to inactivation at pH 3; however, growth rates and lag times at 43uC and at p......H 4.5 were not affected. The exposure to pH 5 only caused up-regulation of 12 genes and this response was neither hysteretic nor accompanied of increased resistance to inactivation conditions. Cellular memory at the transcriptional level may represent a mechanism of adaptation to the environment...

  4. Zinc-fingers and homeoboxes 1 (ZHX1) binds DNA methyltransferase (DNMT) 3B to enhance DNMT3B-mediated transcriptional repression

    International Nuclear Information System (INIS)

    Kim, Sung-Hak; Park, Jinah; Choi, Moon-Chang; Kim, Hwang-Phill; Park, Jung-Hyun; Jung, Yeonjoo; Lee, Ju-Hee; Oh, Do-Youn; Im, Seock-Ah; Bang, Yung-Jue; Kim, Tae-You

    2007-01-01

    DNA methyltransferases (DNMT) 3B is a de novo DNMT that represses transcription independent of DNMT activity. In order to gain a better insight into DNMT3B-mediated transcriptional repression, we performed a yeast two-hybrid analysis using DNMT3B as a bait. Of the various binding candidates, ZHX1, a member of zinc-finger and homeobox protein, was found to interact with DNMT3B in vivo and in vitro. N-terminal PWWP domain of DNMT3B was required for its interaction with homeobox motifs of ZHX1. ZHX1 contains nuclear localization signal at C-terminal homeobox motif, and both ZHX1 and DNMT3B were co-localized in nucleus. Furthermore, we found that ZHX1 enhanced the transcriptional repression mediated by DNMT3B when DNMT3B is directly targeted to DNA. These results showed for First the direct linkage between DNMT and zinc-fingers homeoboxes protein, leading to enhanced gene silencing by DNMT3B

  5. Mediator 1 contributes to enamel mineralization as a coactivator for Notch1 signaling and stimulates transcription of the alkaline phosphatase gene.

    Science.gov (United States)

    Yoshizaki, Keigo; Hu, Lizhi; Nguyen, Thai; Sakai, Kiyoshi; Ishikawa, Masaki; Takahashi, Ichiro; Fukumoto, Satoshi; DenBesten, Pamela K; Bikle, Daniel D; Oda, Yuko; Yamada, Yoshihiko

    2017-08-18

    Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1 -deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1-RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene.

  6. Roles of arabidopsis WRKY18, WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress

    OpenAIRE

    Chen Zhixiang; Xiao Yong; Shi Junwei; Lai Zhibing; Chen Han; Xu Xinping

    2010-01-01

    Abstract Background WRKY transcription factors are involved in plant responses to both biotic and abiotic stresses. Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors interact both physically and functionally in plant defense responses. However, their role in plant abiotic stress response has not been directly analyzed. Results We report that the three WRKYs are involved in plant responses to abscisic acid (ABA) and abiotic stress. Through analysis of single, double, and triple muta...

  7. The antagonistic regulation of abscisic acid-inhibited root growth by brassinosteroids is partially mediated via direct suppression of ABSCISIC ACID INSENSITIVE 5 expression by BRASSINAZOLE RESISTANT 1.

    Science.gov (United States)

    Yang, Xiaorui; Bai, Yang; Shang, Jianxiu; Xin, Ruijiao; Tang, Wenqiang

    2016-09-01

    Brassinosteroids (BRs) and abscisic acid (ABA) are plant hormones that antagonistically regulate many aspects of plant growth and development; however, the mechanisms that regulate the crosstalk of these two hormones are still not well understood. BRs regulate plant growth and development by activating BRASSINAZOLE RESISTANT 1 (BZR1) family transcription factors. Here we show that the crosstalk between BRs and ABA signalling is partially mediated by BZR1 regulated gene expression. bzr1-1D is a dominant mutant with enhanced BR signalling; our results showed that bzr1-1D mutant is less sensitive to ABA-inhibited primary root growth. By RNA sequencing, a subset of BZR1 regulated ABA-responsive root genes were identified. Of these genes, the expression of a major ABA signalling component ABA INSENSITIVE 5 (ABI5) was found to be suppressed by BR and by BZR1. Additional evidences showed that BZR1 could bind strongly with several G-box cis-elements in the promoter of ABI5, suppress the expression of ABI5 and make plants less sensitive to ABA. Our study demonstrated that ABI5 is a direct target gene of BZR1, and modulating the expression of ABI5 by BZR1 plays important roles in regulating the crosstalk between the BR and ABA signalling pathways. © 2016 John Wiley & Sons Ltd.

  8. TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter

    OpenAIRE

    Wright, Kevin J.; Marr, Michael T.; Tjian, Robert

    2006-01-01

    Activator-dependent recruitment of TFIID initiates formation of the transcriptional preinitiation complex. TFIID binds core promoter DNA elements and directs the assembly of other general transcription factors, leading to binding of RNA polymerase II and activation of RNA synthesis. How TATA box-binding protein (TBP) and the TBP-associated factors (TAFs) are assembled into a functional TFIID complex with promoter recognition and coactivator activities in vivo remains unknown. Here, we use RNA...

  9. Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome-dependent and -independent downregulation of specificity proteins (Sp transcription factors

    Directory of Open Access Journals (Sweden)

    Pathi Satya

    2011-08-01

    Full Text Available Abstract Background Betulinic acid (BA inhibits growth of several cancer cell lines and tumors and the effects of BA have been attributed to its mitochondriotoxicity and inhibition of multiple pro-oncogenic factors. Previous studies show that BA induces proteasome-dependent degradation of specificity protein (Sp transcription factors Sp1, Sp3 and Sp4 in prostate cancer cells and this study focused on the mechanism of action of BA in colon cancer cells. Methods The effects of BA on colon cancer cell proliferation and apoptosis and tumor growth in vivo were determined using standardized assays. The effects of BA on Sp proteins and Sp-regulated gene products were analyzed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a and ZBTB10 mRNA expression. Results BA inhibited growth and induced apoptosis in RKO and SW480 colon cancer cells and inhibited tumor growth in athymic nude mice bearing RKO cells as xenograft. BA also decreased expression of Sp1, Sp3 and Sp4 transcription factors which are overexpressed in colon cancer cells and decreased levels of several Sp-regulated genes including survivin, vascular endothelial growth factor, p65 sub-unit of NFκB, epidermal growth factor receptor, cyclin D1, and pituitary tumor transforming gene-1. The mechanism of action of BA was dependent on cell context, since BA induced proteasome-dependent and proteasome-independent downregulation of Sp1, Sp3 and Sp4 in SW480 and RKO cells, respectively. In RKO cells, the mechanism of BA-induced repression of Sp1, Sp3 and Sp4 was due to induction of reactive oxygen species (ROS, ROS-mediated repression of microRNA-27a, and induction of the Sp repressor gene ZBTB10. Conclusions These results suggest that the anticancer activity of BA in colon cancer cells is due, in part, to downregulation of Sp1, Sp3 and Sp4 transcription factors; however, the mechanism of this response is cell context-dependent.

  10. Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome-dependent and -independent downregulation of specificity proteins (Sp) transcription factors

    International Nuclear Information System (INIS)

    Chintharlapalli, Sudhakar; Papineni, Sabitha; Lei, Ping; Pathi, Satya; Safe, Stephen

    2011-01-01

    Betulinic acid (BA) inhibits growth of several cancer cell lines and tumors and the effects of BA have been attributed to its mitochondriotoxicity and inhibition of multiple pro-oncogenic factors. Previous studies show that BA induces proteasome-dependent degradation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 in prostate cancer cells and this study focused on the mechanism of action of BA in colon cancer cells. The effects of BA on colon cancer cell proliferation and apoptosis and tumor growth in vivo were determined using standardized assays. The effects of BA on Sp proteins and Sp-regulated gene products were analyzed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a) and ZBTB10 mRNA expression. BA inhibited growth and induced apoptosis in RKO and SW480 colon cancer cells and inhibited tumor growth in athymic nude mice bearing RKO cells as xenograft. BA also decreased expression of Sp1, Sp3 and Sp4 transcription factors which are overexpressed in colon cancer cells and decreased levels of several Sp-regulated genes including survivin, vascular endothelial growth factor, p65 sub-unit of NFκB, epidermal growth factor receptor, cyclin D1, and pituitary tumor transforming gene-1. The mechanism of action of BA was dependent on cell context, since BA induced proteasome-dependent and proteasome-independent downregulation of Sp1, Sp3 and Sp4 in SW480 and RKO cells, respectively. In RKO cells, the mechanism of BA-induced repression of Sp1, Sp3 and Sp4 was due to induction of reactive oxygen species (ROS), ROS-mediated repression of microRNA-27a, and induction of the Sp repressor gene ZBTB10. These results suggest that the anticancer activity of BA in colon cancer cells is due, in part, to downregulation of Sp1, Sp3 and Sp4 transcription factors; however, the mechanism of this response is cell context-dependent

  11. Identification and functional analysis of two alternatively spliced transcripts of ABSCISIC ACID INSENSITIVE3 (ABI3) in linseed flax (Linum usitatissimum L.).

    Science.gov (United States)

    Wang, Yanyan; Zhang, Tianbao; Song, Xiaxia; Zhang, Jianping; Dang, Zhanhai; Pei, Xinwu; Long, Yan

    2018-01-01

    Alternative splicing is a popular phenomenon in different types of plants. It can produce alternative spliced transcripts that encode proteins with altered functions. Previous studies have shown that one transcription factor, ABSCISIC ACID INSENSITIVE3 (ABI3), which encodes an important component in abscisic acid (ABA) signaling, is subjected to alternative splicing in both mono- and dicotyledons. In the current study, we identified two homologs of ABI3 in the genome of linseed flax. We screened two alternatively spliced flax LuABI3 transcripts, LuABI3-2 and LuABI3-3, and one normal flax LuABI3 transcript, LuABI3-1. Sequence analysis revealed that one of the alternatively spliced transcripts, LuABI3-3, retained a 6 bp intron. RNA accumulation analysis showed that all three transcripts were expressed during seed development, while subcellular localization and transgene experiments showed that LuABI3-3 had no biological function. The two normal transcripts, LuABI3-1 and LuABI3-2, are the important functional isoforms in flax and play significant roles in the ABA regulatory pathway during seed development, germination, and maturation.

  12. Identification and functional analysis of two alternatively spliced transcripts of ABSCISIC ACID INSENSITIVE3 (ABI3 in linseed flax (Linum usitatissimum L..

    Directory of Open Access Journals (Sweden)

    Yanyan Wang

    Full Text Available Alternative splicing is a popular phenomenon in different types of plants. It can produce alternative spliced transcripts that encode proteins with altered functions. Previous studies have shown that one transcription factor, ABSCISIC ACID INSENSITIVE3 (ABI3, which encodes an important component in abscisic acid (ABA signaling, is subjected to alternative splicing in both mono- and dicotyledons. In the current study, we identified two homologs of ABI3 in the genome of linseed flax. We screened two alternatively spliced flax LuABI3 transcripts, LuABI3-2 and LuABI3-3, and one normal flax LuABI3 transcript, LuABI3-1. Sequence analysis revealed that one of the alternatively spliced transcripts, LuABI3-3, retained a 6 bp intron. RNA accumulation analysis showed that all three transcripts were expressed during seed development, while subcellular localization and transgene experiments showed that LuABI3-3 had no biological function. The two normal transcripts, LuABI3-1 and LuABI3-2, are the important functional isoforms in flax and play significant roles in the ABA regulatory pathway during seed development, germination, and maturation.

  13. Amino acid-dependent signaling via S6K1 and MYC is essential for regulation of rDNA transcription

    Science.gov (United States)

    Kang, Jian; Kusnadi, Eric P.; Ogden, Allison J.; Hicks, Rodney J.; Bammert, Lukas; Kutay, Ulrike; Hung, Sandy; Sanij, Elaine; Hannan, Ross D.; Hannan, Katherine M.; Pearson, Richard B.

    2016-01-01

    Dysregulation of RNA polymerase I (Pol I)-dependent ribosomal DNA (rDNA) transcription is a consistent feature of malignant transformation that can be targeted to treat cancer. Understanding how rDNA transcription is coupled to the availability of growth factors and nutrients will provide insight into how ribosome biogenesis is maintained in a tumour environment characterised by limiting nutrients. We demonstrate that modulation of rDNA transcription initiation, elongation and rRNA processing is an immediate, co-regulated response to altered amino acid abundance, dependent on both mTORC1 activation of S6K1 and MYC activity. Growth factors regulate rDNA transcription initiation while amino acids modulate growth factor-dependent rDNA transcription by primarily regulating S6K1-dependent rDNA transcription elongation and processing. Thus, we show for the first time amino acids regulate rRNA synthesis by a distinct, post-initiation mechanism, providing a novel model for integrated control of ribosome biogenesis that has implications for understanding how this process is dysregulated in cancer. PMID:27385002

  14. WRKY transcription factors involved in salicylic acid-induced defense gene expression

    NARCIS (Netherlands)

    Verk, Marcel Cristiaan van

    2010-01-01

    The salicylic acid (SA) signaling pathway triggered by attack of biotrophic pathogens leads to broad spectrum resistance against a plethora of pathogenic fungi, bacteria and viruses and is known as systemic acquired resistance (SAR). One of the hallmarks of SAR is the accumulation of PR proteins and

  15. Identification of two CiGADs from Caragana intermedia and their transcriptional responses to abiotic stresses and exogenous abscisic acid.

    Science.gov (United States)

    Ji, Jing; Zheng, Lingyu; Yue, Jianyun; Yao, Xiamei; Chang, Ermei; Xie, Tiantian; Deng, Nan; Chen, Lanzhen; Huang, Yuwen; Jiang, Zeping; Shi, Shengqing

    2017-01-01

    Glutamate decarboxylase (GAD), as a key enzyme in the γ -aminobutyric acid (GABA) shunt, catalyzes the decarboxylation of L-glutamate to form GABA. This pathway has attracted much interest because of its roles in carbon and nitrogen metabolism, stress responses, and signaling in higher plants. The aim of this study was to isolate and characterize genes encoding GADs from Caragana intermedia , an important nitrogen-fixing leguminous shrub. Two full-length cDNAs encoding GADs (designated as CiGAD1 and CiGAD2 ) were isolated and characterized. Multiple alignment and phylogenetic analyses were conducted to evaluate their structures and identities to each other and to homologs in other plants. Tissue expression analyses were conducted to evaluate their transcriptional responses to stress (NaCl, ZnSO 4 , CdCl 2 , high/low temperature, and dehydration) and exogenous abscisic acid. The CiGAD s contained the conserved PLP domain and calmodulin (CaM)-binding domain in the C-terminal region. The phylogenetic analysis showed that they were more closely related to the GADs of soybean, another legume, than to GADs of other model plants. According to Southern blotting analysis, CiGAD1 had one copy and CiGAD2 -related genes were present as two copies in C. intermedia . In the tissue expression analyses, there were much higher transcript levels of CiGAD2 than CiGAD1 in bark, suggesting that CiGAD2 might play a role in secondary growth of woody plants. Several stress treatments (NaCl, ZnSO 4 , CdCl 2 , high/low temperature, and dehydration) significantly increased the transcript levels of both CiGAD s, except for CiGAD2 under Cd stress. The CiGAD1 transcript levels strongly increased in response to Zn stress (74.3-fold increase in roots) and heat stress (218.1-fold increase in leaves). The transcript levels of both CiGAD s significantly increased as GABA accumulated during a 24-h salt treatment. Abscisic acid was involved in regulating the expression of these two CiGAD s under salt

  16. Trans-splicing of plastid rps12 transcripts, mediated by AtPPR4, is essential for embryo patterning in Arabidopsis thaliana.

    Science.gov (United States)

    Tadini, Luca; Ferrari, Roberto; Lehniger, Marie-Kristin; Mizzotti, Chiara; Moratti, Fabio; Resentini, Francesca; Colombo, Monica; Costa, Alex; Masiero, Simona; Pesaresi, Paolo

    2018-04-23

    AtPPR4-mediated trans-splicing of plastid rps12 transcripts is essential for key embryo morphogenetic events such as development of cotyledons, determination of provascular tissue, and organization of the shoot apical meristem (SAM), but not for the formation of the protodermal layer. Members of the pentatricopeptide repeat (PPR) containing protein family have emerged as key regulators of the organelle post-transcriptional processing and to be essential for proper plant embryo development. In this study, we report the functional characterization of the AtPPR4 (At5g04810) gene encoding a plastid nucleoid PPR protein. In-situ hybridization analysis reveals the presence of AtPPR4 transcripts already at the transition stage of embryo development. As a consequence, embryos lacking the AtPPR4 protein arrest their development at the transition/early-heart stages and show defects in the determination of the provascular tissue and organization of SAM. This complex phenotype is due to the specific role of AtPPR4 in the trans-splicing of the plastid rps12 transcripts, as shown by northern and slot-blot hybridizations, and the consequent defect in 70S ribosome accumulation and plastid protein synthesis, in agreement with the role proposed for the maize orthologue, ZmPPR4.

  17. Computational studies on non-succinimide-mediated stereoinversion mechanism of aspartic acid residues assisted by phosphate

    Science.gov (United States)

    Nakayoshi, Tomoki; Fukuyoshi, Shuichi; Takahashi, Ohgi; Oda, Akifumi

    2018-03-01

    Although nearly all of the amino acids that constitute proteins are l-amino acids, d-amino acid residues in human proteins have been recently reported. d-amino acid residues cause a change in the three-dimensional structure of proteins, and d-aspartic acid (Asp) residues are considered to be one of the causes of age-related diseases. The stereoinversion of Asp residues in peptides and proteins is thought to proceed via a succinimide intermediate; however, it has been reported that stereoinversion can occur even under conditions where a succinimide intermediate cannot be formed. In order to elucidate the non-succinimide-mediated stereoinversion pathway, we investigated the stereoinversion of l-Asp to d-Asp catalysed by phosphate and estimated the activation barrier using B3LYP/6-31+G(d,p) density functional theory (DFT) calculations. For the DFT calculations, a model compound in which the Asp residue is capped with acetyl and methyl-amino groups on the N- and C-termini, respectively, was used. The calculated activation barrier was not excessively high for the stereoinversion to occur in vivo. Therefore, this stereoinversion mechanism may compete with the succinimide-mediated mechanism.

  18. Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression.

    Science.gov (United States)

    DeSmet, Marsha L; Fleet, James C

    2017-10-01

    High vitamin D status is associated with reduced colon cancer risk but these studies ignore the diversity in the molecular etiology of colon cancer. RAS activating mutations are common in colon cancer and they activate pro-proliferative signaling pathways. We examined the impact of RAS activating mutations on 1,25 dihydroxyvitamin D (1,25(OH) 2 D)-mediated gene expression in cultured colon and intestinal cell lines. Transient transfection of Caco-2 cells with a constitutively active mutant K-RAS (G12 V) significantly reduced 1,25(OH) 2 D-induced activity of both a human 25-hydroxyvitamin D, 24 hydroxyase (CYP24A1) promoter-luciferase and an artificial 3X vitamin D response element (VDRE) promoter-luciferase reporter gene. Young Adult Mouse Colon (YAMC) and Rat Intestinal Epithelial (RIE) cell lines with stable expression of mutant H-RAS had suppressed 1,25(OH) 2 D-mediated induction of CYP24A1 mRNA. The RAS effects were associated with lower Vitamin D receptor (VDR) mRNA and protein levels in YAMC and RIE cells and they could be partially reversed by VDR overexpression. RAS-mediated suppression of VDR levels was not due to either reduced VDR mRNA stability or increased VDR gene methylation. However, chromatin accessibility to the VDR gene at the proximal promoter (-300bp), an enhancer region at -6kb, and an enhancer region located in exon 3 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS). These data show that constitutively active RAS signaling suppresses 1,25(OH) 2 D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription but the mechanism for this suppression is not yet known. These data suggest that cancers with RAS-activating mutations may be less responsive to vitamin D mediated treatment or chemoprevention. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents

    OpenAIRE

    Benoit, Stephen C.; Kemp, Christopher J.; Elias, Carol F.; Abplanalp, William; Herman, James P.; Migrenne, Stephanie; Lefevre, Anne-Laure; Cruciani-Guglielmacci, Céline; Magnan, Christophe; Yu, Fang; Niswender, Kevin; Irani, Boman G.; Holland, William L.; Clegg, Deborah J.

    2009-01-01

    Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-θ, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-θ was expressed in discrete neuronal populations of ...

  20. Antagonism between abscisic acid and gibberellins is partially mediated by ascorbic acid during seed germination in rice.

    Science.gov (United States)

    Ye, Nenghui; Zhang, Jianhua

    2012-05-01

    The antagonism between abscisic acid (ABA) and gibberellin (GA) plays a key role in controlling seed germination, but the mechanism of antagonism during this process is not known. In the associated study, we investigated the relationship among ABA, reactive oxygen species (ROS), ascorbic acid (ASC) and GA during rice seed germination. ROS production is reduced by ABA, which hence results in decreasing ASC accumulation during imbibition. GA accumulation was also suppressed by a reduced ROS and ASC level, whereas application of exogenous ASC can partially rescue seed germination from ABA treatment. Further results show that production of ASC, which acts as a substrate in GA biosynthesis, was significantly inhibited by lycorine which thus suppressed the accumulation of GA. Consequently, expression of GA biosynthesis genes was suppressed by the low levels of ROS and ASC in ABA-treated seeds. These studies reveal a new role for ASC in mediating the antagonism between ABA and GA during seed germination in rice.

  1. Therapeutic Applicability of Anti-Inflammatory and Proresolving Polyunsaturated Fatty Acid-Derived Lipid Mediators

    Directory of Open Access Journals (Sweden)

    Gerard L. Bannenberg

    2010-01-01

    Full Text Available The enzymatic oxygenation of polyunsaturated fatty acids by lipoxygenases and cyclo-oxygenases is a resourceful mode of formation of specific autacoids that regulate the extent and pace of the inflammatory response. Arachidonate-derived eicosanoids, such as lipoxin A4, prostaglandin (PGD2, PGF2α, PGE2, and PGD2-derived cyclopentenones exert specific roles in counter-regulating inflammation and turning on resolution. Recently recognized classes of autacoids derived from long-chain ω-3 polyunsaturated fatty acids, the E- and D-series resolvins, protectin D1, and maresin 1, act as specialized mediators to dampen inflammation actively, afford tissue protection, stimulate host defense, and activate resolution. It is held that counter-regulatory lipid mediators and the specific molecular pathways activated by such endogenous agonists may be suitable for pharmacological use in the treatment of inflammatory disease. The anti-inflammatory drug aspirin is a striking example of a drug that is able to act in such a manner, namely through triggering the formation of 15-epi-lipoxin A4 and aspirin-triggered resolvins. Different aspects of the therapeutic applicability of lipid mediators have been addressed here, and indicate that the development of innovative pharmacotherapy based on anti-inflammatory and proresolution lipid mediators presents novel prospects for the treatment of inflammatory disease.

  2. Two Theobroma cacao genotypes with contrasting pathogen tolerance show aberrant transcriptional and ROS responses after salicylic acid treatment.

    Science.gov (United States)

    Fister, Andrew S; O'Neil, Shawn T; Shi, Zi; Zhang, Yufan; Tyler, Brett M; Guiltinan, Mark J; Maximova, Siela N

    2015-10-01

    Understanding the genetic basis of pathogen susceptibility in various crop plants is crucial to increasing the stability of food, feed, and fuel production. Varietal differences in defence responses provide insights into the mechanisms of resistance and are a key resource for plant breeders. To explore the role of salicylic acid in the regulation of defence in cacao, we demonstrated that SA treatment decreased susceptibility to a pod rot pathogen, Phytophthora tropicalis in two genotypes, Scavina 6 and Imperial College Selection 1, which differ in their resistance to several agriculturally important pathogens. Transient overexpression of TcNPR1, a major transcriptional regulator of the SA-dependent plant immune system, also increased pathogen tolerance in cacao leaves. To explore further the genetic basis of resistance in cacao, we used microarrays to measure gene expression profiles after salicylic acid (SA) treatment in these two cacao genotypes. The two genotypes displayed distinct transcriptional responses to SA. Unexpectedly, the expression profile of the susceptible genotype ICS1 included a larger number of pathogenesis-related genes that were induced by SA at 24h after treatment, whereas genes encoding many chloroplast and mitochondrial proteins implicated in reactive oxygen species production were up-regulated in the resistant genotype, Sca6. Sca6 accumulated significantly more superoxide at 24h after treatment of leaves with SA. These experiments revealed critical insights regarding the molecular differences between cacao varieties, which will allow a better understanding of defence mechanisms to help guide breeding programmes. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  3. Jasmonic acid-isoleucine formation in grapevine (Vitis vinifera L.) by two enzymes with distinct transcription profiles.

    Science.gov (United States)

    Böttcher, Christine; Burbidge, Crista A; di Rienzo, Valentina; Boss, Paul K; Davies, Christopher

    2015-07-01

    The plant hormone jasmonic acid (JA) is essential for stress responses and the formation of reproductive organs, but its role in fruit development and ripening is unclear. Conjugation of JA to isoleucine is a crucial step in the JA signaling pathway since only JA-Ile is recognized by the jasmonate receptor. The conjugation reaction is catalyzed by JA-amido synthetases, belonging to the family of Gretchen Hagen3 (GH3) proteins. Here, in vitro studies of two grapevine (Vitis vinifera L. cv Shiraz) GH3 enzymes, VvGH3-7 and VvGH3-9, demonstrated JA-conjugating activities with an overlapping range of amino acid substrates, including isoleucine. Expression studies of the corresponding genes in grape berries combined with JA and JA-Ile measurements suggested a primary role for JA signaling in fruit set and cell division and did not support an involvement of JA in the ripening process. In response to methyl JA (MeJA) treatment, and in wounded and unwounded (distal) leaves, VvGH3-9 transcripts accumulated, indicating a participation in the JA response. In contrast, VvGH3-7 was unresponsive to MeJA and local wounding, demonstrating a differential transcriptional regulation of VvGH3-7 and VvGH3-9. The transient induction of VvGH3-7 in unwounded, distal leaves was suggestive of the involvement of an unknown mobile wound signal. © 2014 Institute of Botany, Chinese Academy of Sciences.

  4. C/EBPβ (CCAAT/enhancer-binding protein β) mediates progesterone production through transcriptional regulation in co-operation with SF-1 (steroidogenic factor-1).

    Science.gov (United States)

    Mizutani, Tetsuya; Ju, Yunfeng; Imamichi, Yoshitaka; Osaki, Tsukasa; Yazawa, Takashi; Kawabe, Shinya; Ishikane, Shin; Matsumura, Takehiro; Kanno, Masafumi; Kamiki, Yasue; Kimura, Kohei; Minamino, Naoto; Miyamoto, Kaoru

    2014-06-15

    The transcription factor SF-1 (steroidogenic factor-1) is a master regulator of steroidogenesis. Previously, we have found that SF-1 induces the differentiation of mesenchymal stem cells into steroidogenic cells. To elucidate the molecular mechanisms of SF-1-mediated functions, we attempted to identify protein components of the SF-1 nuclear protein complex in differentiated cells. SF-1 immunoaffinity chromatography followed by MS/MS analysis was performed, and 24 proteins were identified. Among these proteins, we focused on C/EBPβ (CCAAT/enhancer-binding protein β), which is an essential transcription factor for ovulation and luteinization, as the transcriptional mechanisms of C/EBPβ working together with SF-1 are poorly understood. C/EBPβ knockdown attenuated cAMP-induced progesterone production in granulosa tumour-derived KGN cells by altering STAR (steroidogenic acute regulatory protein), CYP11A1 (cytochrome P450, family 11, subfamily A, polypeptide 1) and HSD3B2 (hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2) expression. EMSA and ChIP assays revealed novel C/EBPβ-binding sites in the upstream regions of the HSD3B2 and CYP11A1 genes. These interactions were enhanced by cAMP stimulation. Luciferase assays showed that C/EBPβ-responsive regions were found in each promoter and C/EBPβ is involved in the cAMP-induced transcriptional activity of these genes together with SF-1. These results indicate that C/EBPβ is an important mediator of progesterone production by working together with SF-1, especially under tropic hormone-stimulated conditions.

  5. Phospholipid-binding protein EhC2A mediates calcium-dependent translocation of transcription factor URE3-BP to the plasma membrane of Entamoeba histolytica.

    Science.gov (United States)

    Moreno, Heriberto; Linford, Alicia S; Gilchrist, Carol A; Petri, William A

    2010-05-01

    The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) is a transcription factor that binds DNA in a Ca(2+)-inhibitable manner. The protein is located in both the nucleus and the cytoplasm but has also been found to be enriched in the plasma membrane of amebic trophozoites. We investigated the reason for the unusual localization of URE3-BP at the amebic plasma membrane. Here we identify and characterize a 22-kDa Ca(2+)-dependent binding partner of URE3-BP, EhC2A, a novel member of the C2-domain superfamily. Immunoprecipitations of URE3-BP and EhC2A showed that the proteins interact and that such interaction was enhanced in the presence of Ca(2+). Recombinant and native EhC2A bound phospholipid liposomes in a Ca(2+)-dependent manner, with half-maximal binding occurring at 3.4 muM free Ca(2+). A direct interaction between EhC2A and URE3-BP was demonstrated by the ability of recombinant EhC2A to recruit recombinant URE3-BP to phospholipid liposomes in a Ca(2+)-dependent manner. URE3-BP and EhC2A were observed to translocate to the amebic plasma membrane upon an increase in the intracellular Ca(2+) concentration of trophozoites, as revealed by subcellular fractionation and immunofluorescent staining. Short hairpin RNA-mediated knockdown of EhC2A protein expression significantly modulated the mRNA levels of URE3-BP-regulated transcripts. Based on these results, we propose a model for EhC2A-mediated regulation of the transcriptional activities of URE3-BP via Ca(2+)-dependent anchoring of the transcription factor to the amebic plasma membrane.

  6. Digital gene expression analysis of male and female bud transition in Metasequoia reveals high activity of MADS-box transcription factors and hormone-mediated sugar pathways.

    Science.gov (United States)

    Zhao, Ying; Liang, Haiying; Li, Lan; Tang, Sha; Han, Xiao; Wang, Congpeng; Xia, Xinli; Yin, Weilun

    2015-01-01

    Metasequoia glyptostroboides is a famous redwood tree of ecological and economic importance, and requires more than 20 years of juvenile-to-adult transition before producing female and male cones. Previously, we induced reproductive buds using a hormone solution in juvenile Metasequoia trees as young as 5-to-7 years old. In the current study, hormone-treated shoots found in female and male buds were used to identify candidate genes involved in reproductive bud transition in Metasequoia. Samples from hormone-treated cone reproductive shoots and naturally occurring non-cone setting shoots were analyzed using 24 digital gene expression (DGE) tag profiles using Illumina, generating a total of 69,520 putative transcripts. Next, 32 differentially and specifically expressed transcripts were determined using quantitative real-time polymerase chain reaction, including the upregulation of MADS-box transcription factors involved in male bud transition and flowering time control proteins involved in female bud transition. These differentially expressed transcripts were associated with 243 KEGG pathways. Among the significantly changed pathways, sugar pathways were mediated by hormone signals during the vegetative-to-reproductive phase transition, including glycolysis/gluconeogenesis and sucrose and starch metabolism pathways. Key enzymes were identified in these pathways, including alcohol dehydrogenase (NAD) and glutathione dehydrogenase for the glycolysis/gluconeogenesis pathway, and glucanphosphorylase for sucrose and starch metabolism pathways. Our results increase our understanding of the reproductive bud transition in gymnosperms. In addition, these studies on hormone-mediated sugar pathways increase our understanding of the relationship between sugar and hormone signaling during female and male bud initiation in Metasequoia.

  7. Digital gene expression analysis of male and female bud transition in Metasequoia reveals high activity of MADS-box transcription factors and hormone-mediated sugar pathways

    Directory of Open Access Journals (Sweden)

    Ying eZhao

    2015-06-01

    Full Text Available Metasequoiaglyptostroboidies is a famous redwood tree of ecological and economic importance, and requires more than 20 years of juvenile-to-adult transition before producing female and male cones. Previously, we induced reproductive buds using a hormone solution in juvenile Metasequoia trees as young as5-to-7years old. In the current study, hormone-treated shoots found in female and male buds were used to identify candidate genes involved in reproductive bud transition in Metasequoia. Samples from hormone-treated cone reproductive shoots and naturally occurring non-cone setting shoots were analyzed using 24 digital gene expression (DGE tag profiles using Illumina, generating a total of 69,520 putative transcripts. Next, 32 differentially and specifically expressed transcripts were determined using quantitative real-time polymerase chain reaction, including the upregulation of MADS-box transcription factors involved in male bud transition and flowering time control proteins involved in female bud transition. These differentially expressed transcripts were associated with 243 KEGG pathways. Among the significantly changed pathways, sugar pathways were mediated by hormone signals during the vegetative-to-reproductive phase transition, including glycolysis/gluconeogenesis and sucrose and starch metabolism pathways. Key enzymes were identified in these pathways, including alcohol dehydrogenase (NAD and glutathione dehydrogenase for the glycolysis/gluconeogenesis pathway, and glucanphosphorylase for sucrose and starch metabolism pathways. Our results increase our understanding of the reproductive bud transition in gymnosperms. In addition, these studies on hormone-mediated sugar pathways increase our understanding of the relationship between sugar and hormone signaling during female and male bud initiation in Metasequoia.

  8. Okadaic acid and trifluoperazine enhance Agrobacterium-mediated transformation in eastern white pine.

    Science.gov (United States)

    Tang, Wei; Lin, Jinxing; Newton, Ronald J

    2007-05-01

    Mature zygotic embryos of recalcitrant Christmas tree species eastern white pine (Pinus strobus L.) were used as explants for Agrobacterium tumefaciens strain GV3101-mediated transformation using the uidA (beta-Glucuronidase) gene as a reporter. Influence of the time of sonication and the concentrations of protein phosphatase inhibitor (okadaic acid) and kinase inhibitor (trifluoperazine) on Agrobacterium-mediated transformation have been evaluated. A high transformation frequency was obtained after embryos were sonicated for 45-50 s, or treated with 1.5-2.0 microM okadaic acid or treated with 100-200 microM trifluoperazine, respectively. Protein phosphatase and kinase inhibitors enhance Agrobacterium-mediated transformation in eastern white pine. A 2-3.5-fold higher rate of hygromycin-resistant callus was obtained with an addition of 2 microM okadaic acid or 150 microM trifluoperazine or sonicated embryos for 45 s. Stable integration of the uidA gene in the plant genome of eastern white pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in eastern white pine and this system would provide an opportunity to transfer economically important genes into this Christmas tree species.

  9. v-src induction of the TIS10/PGS2 prostaglandin synthase gene is mediated by an ATF/CRE transcription response element.

    Science.gov (United States)

    Xie, W; Fletcher, B S; Andersen, R D; Herschman, H R

    1994-10-01

    We recently reported the cloning of a mitogen-inducible prostaglandin synthase gene, TIS10/PGS2. In addition to growth factors and tumor promoters, the v-src oncogene induces TIS10/PGS2 expression in 3T3 cells. Deletion analysis, using luciferase reporters, identifies a region between -80 and -40 nucleotides 5' of the TIS10/PGS2 transcription start site that mediates pp60v-src induction in 3T3 cells. This region contains the sequence CGTCACGTG, which includes overlapping ATF/CRE (CGTCA) and E-box (CACGTG) sequences. Gel shift-oligonucleotide competition experiments with nuclear extracts from cells stably transfected with a temperature-sensitive v-src gene demonstrate that the CGTCACGTG sequence can bind proteins at both the ATF/CRE and E-box sequences. Dominant-negative CREB and Myc proteins that bind DNA, but do not transactivate, block v-src induction of a luciferase reporter driven by the first 80 nucleotides of the TIS10/PGS2 promoter. Mutational analysis distinguishes which TIS10/PGS2 cis-acting element mediates pp60v-src induction. E-box mutation has no effect on the fold induction in response to pp60v-src. In contrast, ATF/CRE mutation attenuates the pp60v-src response. Antibody supershift and methylation interference experiments demonstrate that CREB and at least one other ATF transcription factor in these extracts bind to the TIS10/PGS2 ATF/CRE element. Expression of a dominant-negative ras gene also blocks TIS10/PGS2 induction by v-src. Our data suggest that Ras mediates pp60v-src activation of an ATF transcription factor, leading to induced TIS10/PGS2 expression via the ATF/CRE element of the TIS10/PGS2 promoter. This is the first description of v-src activation of gene expression via an ATF/CRE element.

  10. The crystal structure of the AhRR-ARNT heterodimer reveals the structural basis of the repression of AhR-mediated transcription.

    Science.gov (United States)

    Sakurai, Shunya; Shimizu, Toshiyuki; Ohto, Umeharu

    2017-10-27

    2,3,7,8-Tetrachlorodibenzo- p -dioxin and related compounds are extraordinarily potent environmental toxic pollutants. Most of the 2,3,7,8-tetrachlorodibenzo- p -dioxin toxicities are mediated by aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor belonging to the basic helix-loop-helix (bHLH) Per-ARNT-Sim (PAS) family. Upon ligand binding, AhR forms a heterodimer with AhR nuclear translocator (ARNT) and induces the expression of genes involved in various biological responses. One of the genes induced by AhR encodes AhR repressor (AhRR), which also forms a heterodimer with ARNT and represses the activation of AhR-dependent transcription. The control of AhR activation is critical for managing AhR-mediated diseases, but the mechanisms by which AhRR represses AhR activation remain poorly understood, because of the lack of structural information. Here, we determined the structure of the AhRR-ARNT heterodimer by X-ray crystallography, which revealed an asymmetric intertwined domain organization presenting structural features that are both conserved and distinct among bHLH-PAS family members. The structures of AhRR-ARNT and AhR-ARNT were similar in the bHLH-PAS-A region, whereas the PAS-B of ARNT in the AhRR-ARNT complex exhibited a different domain arrangement in this family reported so far. The structure clearly disclosed that AhRR competitively represses AhR binding to ARNT and target DNA and further suggested the existence of an AhRR-ARNT-specific repression mechanism. This study provides a structural basis for understanding the mechanism by which AhRR represses AhR-mediated gene transcription. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Detection of influenza viruses by coupling multiplex reverse-transcription loop-mediated isothermal amplification with cascade invasive reaction using nanoparticles as a sensor

    Directory of Open Access Journals (Sweden)

    Ge Y

    2017-04-01

    Full Text Available Yiyue Ge,1 Qiang Zhou,2 Kangchen Zhao,1 Ying Chi,1 Bin Liu,3 Xiaoyan Min,4 Zhiyang Shi,1 Bingjie Zou,2 Lunbiao Cui1 1Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health, Jiangsu Provincial Center for Disease Control and Prevention, 2Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, 3Department of Biomedical Engineering, Nanjing Medical University, 4Department of Geriatrics, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of China Abstract: Influenza virus infections represent a worldwide public health and economic problem due to the significant morbidity and mortality caused by seasonal epidemics and pandemics. Sensitive and convenient methodologies for detection of influenza viruses are essential for further disease control. Loop-mediated isothermal amplification (LAMP is the most commonly used method of nucleic acid isothermal amplification. However, with regard to multiplex LAMP, differentiating the ladder-like LAMP products derived from multiple targets is still challenging today. The requirement of specialized instruments has further hindered the on-site application of multiplex LAMP. We have developed an integrated assay coupling multiplex reverse transcription LAMP with cascade invasive reaction using nanoparticles (mRT-LAMP-CIRN as a sensor for the detection of three subtypes of influenza viruses: A/H1N1pdm09, A/H3 and influenza B. The analytic sensitivities of the mRT-LAMP-CIRN assay were 101 copies of RNA for both A/H1N1pdm09 and A/H3, and 102 copies of RNA for influenza B. This assay demonstrated highly specific detection of target viruses and could differentiate them from other genetically or clinically related viruses. Clinical specimen analysis showed the mRT-LAMP-CIRN assay had an overall sensitivity and specificity of 98.3% and 100%, respectively. In summary, the mRT-LAMP-CIRN assay is

  12. Responsiveness to acidity via metal ion regulators mediates virulence in the gastric pathogen Helicobacter pylori.

    Science.gov (United States)

    Bury-Moné, Stéphanie; Thiberge, Jean-Michel; Contreras, Monica; Maitournam, Aboubakar; Labigne, Agnès; De Reuse, Hilde

    2004-07-01

    The virulence of pathogenic bacteria is dependent on their adaptation to and survival in the stressful conditions encountered in their hosts. Helicobacter pylori exclusively colonizes the acid stomach of primates, making it an ideal study model. Little is known about how H. pylori responds to the moderately acidic conditions encountered at its colonization site, the gastric mucus layer. Thus, we compared gene expression profiles of H. pylori 26695 grown at neutral and acidic pH, and validated the data for a selection of genes by real-time polymerase chain reaction, dot-blots or enzymatic assays. During growth in acidic conditions, 56 genes were upregulated and 45 genes downregulated. We found that acidity is a signal modulating the expression of several virulence factors. Regulation of genes related to metal ion homeostasis suggests protective mechanisms involving diminished transport and enhanced storage. Genes encoding subunits of the F0F1 ATPase and of a newly identified Na+/H+ antiporter (NhaC-HP0946) were downregulated, revealing that this bacterium uses original mechanisms to control proton entry. Five of the upregulated genes encoded proteins controlling intracellular ammonia synthesis, including urease, amidase and formamidase, underlining the major role of this buffering compound in the protection against acidity in H. pylori. Regulatory networks and transcriptome analysis as well as enzymatic assays implicated two metal-responsive transcriptional regulators (NikR and Fur) and an essential two-component response regulator (HP0166, OmpR-like) as effectors of the H. pylori acid response. Finally, a nikR-fur mutant is attenuated in the mouse model, emphasizing the link between response to acidity, metal metabolism and virulence in this gastric pathogen.

  13. Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

    International Nuclear Information System (INIS)

    Junker, L.H.; Davis, R.A.

    1989-01-01

    The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of [14C]cholesterol from [2-14C]acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of [14C]cholesterol from [2-14C]acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis

  14. v-src induction of the TIS10/PGS2 prostaglandin synthase gene is mediated by an ATF/CRE transcription response element.

    OpenAIRE

    Xie, W; Fletcher, B S; Andersen, R D; Herschman, H R

    1994-01-01

    We recently reported the cloning of a mitogen-inducible prostaglandin synthase gene, TIS10/PGS2. In addition to growth factors and tumor promoters, the v-src oncogene induces TIS10/PGS2 expression in 3T3 cells. Deletion analysis, using luciferase reporters, identifies a region between -80 and -40 nucleotides 5' of the TIS10/PGS2 transcription start site that mediates pp60v-src induction in 3T3 cells. This region contains the sequence CGTCACGTG, which includes overlapping ATF/CRE (CGTCA) and E...

  15. MED14 tethers mediator to the N-terminal domain of peroxisome proliferator-activated receptor gamma and is required for full transcriptional activity and adipogenesis

    DEFF Research Database (Denmark)

    Grøntved, Lars; Madsen, Maria S; Boergesen, Michael

    2010-01-01

    and proximal promoter of the PPARgamma target gene Fabp4 is also independent of MED1. Using a small interfering RNA (siRNA)-based approach, we identify MED14 as a novel critical Mediator component for PPARgamma-dependent transactivation, and we demonstrate that MED14 interacts directly with the N terminus...... of PPARgamma in a ligand-independent manner. Interestingly, MED14 knockdown does not affect the recruitment of PPARgamma, MED6, and MED8 to the Fabp4 enhancer but does reduce their occupancy of the Fabp4 proximal promoter. In agreement with the necessity of MED14 for PPARgamma transcriptional activity, we show...

  16. The Zygosaccharomyces bailii transcription factor Haa1 is required for acetic acid and copper stress responses suggesting subfunctionalization of the ancestral bifunctional protein Haa1/Cup2.

    Science.gov (United States)

    Palma, Margarida; Dias, Paulo Jorge; Roque, Filipa de Canaveira; Luzia, Laura; Guerreiro, Joana Fernandes; Sá-Correia, Isabel

    2017-01-13

    The food spoilage yeast species Zygosaccharomyces bailii exhibits an extraordinary capacity to tolerate weak acids, in particular acetic acid. In Saccharomyces cerevisiae, the transcription factor Haa1 (ScHaa1) is considered the main player in genomic expression reprogramming in response to acetic acid stress, but the role of its homologue in Z. bailii (ZbHaa1) is unknown. In this study it is demonstrated that ZbHaa1 is a ScHaa1 functional homologue by rescuing the acetic acid susceptibility phenotype of S. cerevisiae haa1Δ. The disruption of ZbHAA1 in Z. bailii IST302 and the expression of an extra ZbHAA1 copy confirmed ZbHAA1 as a determinant of acetic acid tolerance. ZbHaa1 was found to be required for acetic acid stress-induced transcriptional activation of Z. bailii genes homologous to ScHaa1-target genes. An evolutionary analysis of the Haa1 homologues identified in 28 Saccharomycetaceae species genome sequences, including Z bailii, was carried out using phylogenetic and gene neighbourhood approaches. Consistent with previous studies, this analysis revealed a group containing pre-whole genome duplication species Haa1/Cup2 single orthologues, including ZbHaa1, and two groups containing either Haa1 or Cup2 orthologues from post-whole genome duplication species. S. cerevisiae Cup2 (alias Ace1) is a transcription factor involved in response and tolerance to copper stress. Taken together, these observations led us to hypothesize and demonstrate that ZbHaa1 is also involved in copper-induced transcriptional regulation and copper tolerance. The transcription factor ZbHaa1 is required for adaptive response and tolerance to both acetic acid and copper stresses. The subfunctionalization of the single ancestral Haa1/Cup2 orthologue that originated Haa1 and Cup2 paralogues after whole genome duplication is proposed.

  17. ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) regulates jasmonic acid and abscisic acid biosynthesis and signaling through binding to a novel cis-element.

    Science.gov (United States)

    Chen, Hsing-Yu; Hsieh, En-Jung; Cheng, Mei-Chun; Chen, Chien-Yu; Hwang, Shih-Ying; Lin, Tsan-Piao

    2016-07-01

    ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) of Arabidopsis thaliana is an AP2/ERF domain transcription factor that regulates jasmonate (JA) biosynthesis and is induced by methyl JA treatment. The regulatory mechanism of ORA47 remains unclear. ORA47 is shown to bind to the cis-element (NC/GT)CGNCCA, which is referred to as the O-box, in the promoter of ABI2. We proposed that ORA47 acts as a connection between ABA INSENSITIVE1 (ABI1) and ABI2 and mediates an ABI1-ORA47-ABI2 positive feedback loop. PORA47:ORA47-GFP transgenic plants were used in a chromatin immunoprecipitation (ChIP) assay to show that ORA47 participates in the biosynthesis and/or signaling pathways of nine phytohormones. Specifically, many abscisic acid (ABA) and JA biosynthesis and signaling genes were direct targets of ORA47 under stress conditions. The JA content of the P35S:ORA47-GR lines was highly induced under wounding and moderately induced under water stress relative to that of the wild-type plants. The wounding treatment moderately increased ABA accumulation in the transgenic lines, whereas the water stress treatment repressed the ABA content. ORA47 is proposed to play a role in the biosynthesis of JA and ABA and in regulating the biosynthesis and/or signaling of a suite of phytohormone genes when plants are subjected to wounding and water stress. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  18. Anti-inflammatory effects of benfotiamine are mediated through the regulation of the arachidonic acid pathway in macrophages.

    Science.gov (United States)

    Shoeb, Mohammad; Ramana, Kota V

    2012-01-01

    Benfotiamine, a lipid-soluble analogue of vitamin B1, is a potent antioxidant that is used as a food supplement for the treatment of diabetic complications. Our recent study (U.C. Yadav et al., Free Radic. Biol. Med. 48:1423-1434, 2010) indicates a novel role for benfotiamine in the prevention of bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in murine macrophages. Nevertheless, it remains unclear how benfotiamine mediates anti-inflammatory effects. In this study, we investigated the anti-inflammatory role of benfotiamine in regulating arachidonic acid (AA) pathway-generated inflammatory lipid mediators in RAW264.7 macrophages. Benfotiamine prevented the LPS-induced activation of cPLA2 and release of AA metabolites such as leukotrienes, prostaglandin E2, thromboxane 2 (TXB2), and prostacyclin (PGI2) in macrophages. Further, LPS-induced expression of AA-metabolizing enzymes such as COX-2, LOX-5, TXB synthase, and PGI2 synthase was significantly blocked by benfotiamine. Furthermore, benfotiamine prevented the LPS-induced phosphorylation of ERK1/2 and expression of transcription factors NF-κB and Egr-1. Benfotiamine also prevented the LPS-induced oxidative stress and protein-HNE adduct formation. Most importantly, compared to specific COX-2 and LOX-5 inhibitors, benfotiamine significantly prevented LPS-induced macrophage death and monocyte adhesion to endothelial cells. Thus, our studies indicate that the dual regulation of the COX and LOX pathways in AA metabolism could be a novel mechanism by which benfotiamine exhibits its potential anti-inflammatory response. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Anti-Inflammatory Effects of Benfotiamine are Mediated Through the Regulation of Arachidonic Acid Pathway in Macrophages

    Science.gov (United States)

    Shoeb, Mohammad; Ramana, Kota V

    2011-01-01

    Benfotiamine, a lipid-soluble analogue of vitamin B1, is a potent anti-oxidant that is used as a food supplement for the treatment of diabetic complications. Our recent study indicates a novel role of benfotiamine in the prevention of bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in murine macrophages. Nevertheless, it remains unclear how benfotiamine mediates anti-inflammatory effects. In this study, we investigated the anti-inflammatory role of benfotiamine in regulating the arachidonic acid (AA) pathway generated inflammatory lipid mediators in RAW 264.7 macrophages. Benfotiamine prevented the LPS-induced activation of cPLA2 and release of AA metabolites such as leukotrienes (LTB4), prostaglandin E2 (PGE2), thromboxanes 2 (TXB2) and prostacyclin (PGI2) in macrophages. Further, LPS-induced expressions of AA metabolizing enzymes such as COX-2, LOX-5, TXB synthase and PGI2 synthase were significantly blocked by benfotiamine. Furthermore, benfotiamine prevented the LPS-induced phosphorylation of ERK1/2 and expression of transcription factors NF-kB, and Egr-1. Benfotiamine also prevented the LPS-induced oxidative stress and protein-HNE adducts formation. Most importantly, as compared to specific COX-2 and LOX-5 inhibitors, benfotiamine significantly prevented the LPS-induced macrophage death and monocytes adhesion to endothelial cells. Thus, our studies indicate that the dual regulation of COX and LOX pathways in AA metabolism could be a novel mechanism by which benfotiamine exhibits its potential anti-inflammatory response. PMID:22067901

  20. GlnR-Mediated Regulation of ectABCD Transcription Expands the Role of the GlnR Regulon to Osmotic Stress Management.

    Science.gov (United States)

    Shao, ZhiHui; Deng, WanXin; Li, ShiYuan; He, JuanMei; Ren, ShuangXi; Huang, WeiRen; Lu, YinHua; Zhao, GuoPing; Cai, ZhiMing; Wang, Jin

    2015-10-01

    Ectoine and hydroxyectoine are excellent compatible solutes for bacteria to deal with environmental osmotic stress and temperature damages. The biosynthesis cluster of ectoine and hydroxyectoine is widespread among microorganisms, and its expression is activated by high salinity and temperature changes. So far, little is known about the mechanism of the regulation of the transcription of ect genes and only two MarR family regulators (EctR1 in methylobacteria and the EctR1-related regulator CosR in Vibrio cholerae) have been found to negatively regulate the expression of ect genes. Here, we characterize GlnR, the global regulator for nitrogen metabolism in actinomycetes, as a negative regulator for the transcription of ectoine/hydroxyectoine biosynthetic genes (ect operon) in Streptomyces coelicolor. The physiological role of this transcriptional repression by GlnR is proposed to protect the intracellular glutamate pool, which acts as a key nitrogen donor for both the nitrogen metabolism and the ectoine/hydroxyectoine biosynthesis. High salinity is deleterious, and cells must evolve sophisticated mechanisms to cope with this osmotic stress. Although production of ectoine and hydroxyectoine is one of the most frequently adopted strategies, the in-depth mechanism of regulation of their biosynthesis is less understood. So far, only two MarR family negative regulators, EctR1 and CosR, have been identified in methylobacteria and Vibrio, respectively. Here, our work demonstrates that GlnR, the global regulator for nitrogen metabolism, is a negative transcriptional regulator for ect genes in Streptomyces coelicolor. Moreover, a close relationship is found between nitrogen metabolism and osmotic resistance, and GlnR-mediated regulation of ect transcription is proposed to protect the intracellular glutamate pool. Meanwhile, the work reveals the multiple roles of GlnR in bacterial physiology. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. NF-κB mediates the transcription of mouse calsarcin-1 gene, but not calsarcin-2, in C2C12 cells

    Directory of Open Access Journals (Sweden)

    Mu Yulian

    2007-03-01

    Full Text Available Abstract Background The calsarcins comprise a novel family of muscle-specific calcineurin-interaction proteins that play an important role in modulating both the function and substrate specificity of calcineurin in muscle cells. The expression of calsarcin-1 (CS-1 is restricted to slow-twitch skeletal muscle fibres, whereas that of both calsarcin-2 (CS-2 and calsarcin-3 (CS-3 is enriched in fast-twitch fibres. However, the transcriptional control of this selective expression has not been previously elucidated. Results Our real-time RT-PCR analyses suggest that the expression of CS-1 and CS-2 is increased during the myogenic differentiation of mouse C2C12 cells. Promoter deletion analysis further suggests that an NF-κB binding site within the CS-1 promoter is responsible for the up-regulation of CS-1 transcription, but no similar mechanism was evident for CS-2. These findings are further supported by the results of EMSA analysis, as well as by overexpression and inhibition experiments in which NF-κB function was blocked by treatment with its inhibitor, PDTC. In addition, the overexpression of NFATc4 induces both the CS-1 and CS-2 promoters, whereas MEF2C only activates CS-1. Conclusion Our present data suggest that NF-κB is required for the transcription of mouse CS-1 but not CS-2, and that the regulation of the calsarcins is mediated also by the NFAT and MEF2 transcription factors. These results provide new insights into the molecular mechanisms governing transcription in specific muscle fibre cells. The calsarcins may also serve as a valuable mechanistic tool to better understand the regulation of calcineurin signalling during muscle differentiation.

  2. A critical role for very long-chain fatty acid elongases in oleic acid-mediated Saccharomyces cerevisiae cytotoxicity.

    Science.gov (United States)

    Wang, Qiao; Du, Xiuxiu; Ma, Ke; Shi, Ping; Liu, Wenbin; Sun, Jing; Peng, Min; Huang, Zhiwei

    2018-03-01

    Elongases FEN1/ELO2 and SUR4/ELO3 are important enzymes involved in the elongation of long-chain fatty acids (LCFAs) to very long-chain fatty acids (VLCFAs) in Saccharomyces cerevisiae. The molecular mechanism of the involvement of these elongases in lipotoxicity is unclear. In the present study, we investigated the role of VLCFA elongases in oleic acid-mediated yeast cytotoxicity. The spot test showed that yeast strains with the deletion of ELO2 or ELO3 were strikingly sensitive to oleic acid, while there was no change on the growth of strain with deleted ELO1 which was involved in the elongation of C 14 fatty acid (FA) to C 16 FA. By using GC-MS, the unsaturation index was increased in elo2△ and elo3△ mutants after treatment with oleic acid (OLA). However, the proportion of VLCFAs was increased in response to OLA in the wild-type strain. The growth inhibition of elo2△ and elo3△ could be partially rescued by two commonly used antioxidant agents N-acetyl cysteine (NAC) and Ascorbic acid (VC). The further study showed that exposure to excess OLA led to an increase in the levels of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS), and a decline in the quantity of reduced glutathione (GSH) in both the wild type and mutant strains. However, the antioxidant enzyme activities of superoxide dismutase (SOD) and catalase (CAT) were increased in the wild type and elo1△ strains, while they were significantly decreased in the mutants of elo2△ and elo3△ after treated with excess OLA. Thus, oxidative damage mainly contributed to the cell death induced by OLA in ole2△ and ole3△. Taken together, although disruption of ELO2 or ELO3 did not affect the cellular lipid unsaturation, they altered the distribution and propotion of cellular VLCFAs, leading to the cell membrane impairment, which augmented the ability of OLA to permeabilize the plasma membrane. The data suggest that the very long-chain fatty acids elongases ELO2 and ELO3

  3. Method for determining transcriptional linkage by means of inhibition of deoxyribonucleic acid transcription by ultraviolet irradiation: evaluation in application to the investigation of in vivo transcription in bacteriophage T7

    International Nuclear Information System (INIS)

    Brautigam, A.R.

    1975-01-01

    A technique is presented for mapping promotor sites that utilizes the introduction of transcription-terminating lesions in DNA through uv irradiation which prevents transcription of genes in proportion to their distance from the promotor. This technique was applied to and evaluated in investigations of the transcriptional linkage of bacteriophage T7. All results substantiate the hypothesis that transcription in vivo does not proceed beyond the first uv lesion encountered in the template DNA and that such premature termination of transcription is the principal effect of the uv irradiation on the transcriptional template function of DNA. UV-induced inhibition of the initiation of transcription is insignificant by comparison. Uv inactivation of expression of individual T7 genes was found to follow pseudo first-order kinetics, allowing a gene-specific uv inactivation cross section to be evaluated for each gene. Promotor locations were inferred from the discontinuity in the numerical values of inactivation cross sections arising at the start of each new unit. By such analysis the bacteriophage T7 genome was found to consist of seven transcription units. In vivo E. coli RNA polymerase transcribes the T7 early region as a single unit from a pomotor region located at the left end of the genome. The T7 late region was found to consist of six transcription units, with promotors located just ahead of genes 1.7, 7, 9, 11, 13 and 17

  4. The role of basic leucine zipper transcription factor E4BP4 in the immune system and immune-mediated diseases.

    Science.gov (United States)

    Yin, Jinghua; Zhang, Jian; Lu, Qianjin

    2017-07-01

    Basic leucine zipper transcription factor E4BP4 (also known as NFIL3) has been implicated in the molecular and cellular mechanisms of functions and activities in mammals. The interactions between E4BP4 and major regulators of cellular processes have triggered significant interest in the roles of E4BP4 in the pathogenesis of certain chronic diseases. Indeed, novel discoveries have been emerging to illustrate the involvement of E4BP4 in multiple disorders. It is recognized that E4BP4 is extensively involved in some immune-mediated diseases, but the mechanisms of E4BP4 involvement in these complex diseases remain poorly defined. Here we review the regulatory mechanisms of E4BP4 engaging in not only the biological function but also the development of immune-mediated diseases, paving the way for future therapies. Copyright © 2017. Published by Elsevier Inc.

  5. Proteomic characterization of the acid tolerance response in Lactobacillus delbrueckii subsp. bulgaricus CAUH1 and functional identification of a novel acid stress-related transcriptional regulator Ldb0677.

    Science.gov (United States)

    Zhai, Zhengyuan; Douillard, François P; An, Haoran; Wang, Guohong; Guo, Xinghua; Luo, Yunbo; Hao, Yanling

    2014-06-01

    To overcome the deleterious effects of acid stress, Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) elicits an adaptive response to acid stress. In this study, proteomics approach complemented by transcriptional analysis revealed some cellular changes in L. bulgaricus CAUH1 during acid adaptation. We observed an increase of glycolysis-associated proteins, promoting an optimal utilization of carbohydrates. Also, rerouting of the pyruvate metabolism to fatty acid biosynthesis was observed, indicating a possible modification of the cell membrane rigidity and impermeability. In addition, expression of ribosomal protein S1 (RpsA) was repressed; however, the expression of EF-Tu, EF-G and TypA was up-regulated at both protein and transcript levels. This suggests a reduction of protein synthesis in response to acid stress along with possible enhancement of the translational accuracy and protein folding. It is noteworthy that the putative transcriptional regulator Ldb0677 was 1.84-fold up-regulated. Heterologous expression of Ldb0677 was shown to significantly enhance acid resistance in host strain Lactococcus lactis. To clarify its role in transcriptional regulation network, the DNA-binding specificity of Ldb0677 was determined using bacterial one-hybrid and electrophoretic mobility shift assay. The identification of a binding motif (SSTAGACR) present in the promoter regions of 22 genes indicates that it might function as a major regulator in acid stress response in L. bulgaricus. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  6. Tumour–stromal interactions in acid-mediated invasion: A mathematical model

    KAUST Repository

    Martin, Natasha K.

    2010-12-01

    It is well established that the tumour microenvironment can both promote and suppress tumour growth and invasion, however, most mathematical models of invasion view the normal tissue as inhibiting tumour progression via immune modulation or spatial constraint. In particular, the production of acid by tumour cells and the subsequent creation of a low extracellular pH environment has been explored in several \\'acid-mediated tumour invasion\\' models where the acidic environment facilitates normal cell death and permits tumour invasion. In this paper, we extend the acid-invasion model developed by Gatenby and Gawlinski (1996) to include both the competitive and cooperative interactions between tumour and normal cells, by incorporating the influence of extracellular matrix and protease production at the tumour-stroma interface. Our model predicts an optimal level of tumour acidity which produces both cell death and matrix degradation. Additionally, very aggressive tumours prevent protease production and matrix degradation by excessive normal cell destruction, leading to an acellular (but matrix filled) gap between the tumour and normal tissue, a feature seen in encapsulated tumours. These results suggest, counterintuitively, that increasing tumour acidity may, in some cases, prevent tumour invasion.

  7. Transcriptional and electrophysiological consequences of KChIP2-mediated regulation of CaV1.2

    DEFF Research Database (Denmark)

    Thomsen, Morten B; Foster, Erika; Nguyen, Katherine H

    2009-01-01

    previous finding. Using gene-chip and real-time PCR techniques, we find that KChIP2(-/-) mice have an increased transcriptional activity of the calcium channel beta(2) subunit, CACNB2, whereas the expression of Ca(V)1.2 is preserved. Although I(to,f) is absent and I(Ca,L) is decreased in myocytes from KCh...

  8. Bicarbonate-mediated transcriptional activation of divergent operons by the virulence regulatory protein, RegA, from Citrobacter rodentium.

    Science.gov (United States)

    Yang, Ji; Hart, Emily; Tauschek, Marija; Price, G Dean; Hartland, Elizabeth L; Strugnell, Richard A; Robins-Browne, Roy M

    2008-04-01

    Regulation of virulence gene expression plays a central role in the pathogenesis of enteric bacteria as they encounter diverse environmental conditions in the gastrointestinal tract of their hosts. In this study, we investigated environmental regulation of two putative virulence determinants adcA and kfc by RegA, an AraC/XylS-like regulator, from Citrobacter rodentium, and identified bicarbonate as the environmental signal which induced transcription of adcA and kfc through RegA. Primer extension experiments showed that adcA and kfc were divergently transcribed from sigma(70) promoters. In vivo and in vitro experiments demonstrated that bicarbonate facilitated and stabilized the binding of RegA to an operator located between the two promoters. The interaction of RegA with its DNA target resulted in the formation of a nucleosome-like structure, which evidently displaced the histone-like proteins, H-NS and StpA, from the adcA and kfc promoter regions, leading to transcriptional derepression. In addition, our results indicated that RegA also behaved as a Class I activator by directly stimulating transcription initiation by RNA polymerase. This is the first report to describe the molecular mechanism by which an environmental chemical stimulates transcription of virulence-associated genes of an enteric pathogen through an AraC/XlyS-like activator.

  9. Transcription factor ZBED6 mediates IGF2 gene expression by regulating promoter activity and DNA methylation in myoblasts

    Science.gov (United States)

    Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were up regulated during C2C12 differentiation. The IGF2 expression levels wer...

  10. Repression of transcription mediated at a thyroid hormone response element by the v-erb-A oncogene product

    DEFF Research Database (Denmark)

    Sap, J; Muñoz, A; Schmitt, J

    1989-01-01

    Several recent observations, such as the identification of the cellular homologue of the v-erb-A oncogene as a thyroid-hormone receptor, have strongly implicated nuclear oncogenes in transcriptional control mechanisms. The v-erb-A oncogene blocks the differentiation of erythroid cells, and changes...

  11. Inhibition of Different Histone Acetyltransferases (HATs) Uncovers Transcription-Dependent and -Independent Acetylation-Mediated Mechanisms in Memory Formation

    Science.gov (United States)

    Merschbaecher, Katja; Hatko, Lucyna; Folz, Jennifer; Mueller, Uli

    2016-01-01

    Acetylation of histones changes the efficiency of the transcription processes and thus contributes to the formation of long-term memory (LTM). In our comparative study, we used two inhibitors to characterize the contribution of different histone acetyl transferases (HATs) to appetitive associative learning in the honeybee. For one we applied…

  12. An Interspecies Signaling System Mediated by Fusaric Acid Has Parallel Effects on Antifungal Metabolite Production by Pseudomonas protegens Strain Pf-5 and Antibiosis of Fusarium spp.

    Science.gov (United States)

    Quecine, Maria Carolina; Kidarsa, Teresa A.; Goebel, Neal C.; Shaffer, Brenda T.; Henkels, Marcella D.; Zabriskie, T. Mark

    2015-01-01

    Pseudomonas protegens strain Pf-5 is a rhizosphere bacterium that suppresses soilborne plant diseases and produces at least seven different secondary metabolites with antifungal properties. We derived mutants of Pf-5 with single and multiple mutations in biosynthesis genes for seven antifungal metabolites: 2,4-diacetylphoroglucinol (DAPG), pyrrolnitrin, pyoluteorin, hydrogen cyanide, rhizoxin, orfamide A, and toxoflavin. These mutants were tested for inhibition of the pathogens Fusarium verticillioides and Fusarium oxysporum f. sp. pisi. Rhizoxin, pyrrolnitrin, and DAPG were found to be primarily responsible for fungal antagonism by Pf-5. Previously, other workers showed that the mycotoxin fusaric acid, which is produced by many Fusarium species, including F. verticillioides, inhibited the production of DAPG by Pseudomonas spp. In this study, amendment of culture media with fusaric acid decreased DAPG production, increased pyoluteorin production, and had no consistent influence on pyrrolnitrin or orfamide A production by Pf-5. Fusaric acid also altered the transcription of biosynthetic genes, indicating that the mycotoxin influenced antibiotic production by Pf-5 at the transcriptional level. Addition of fusaric acid to the culture medium reduced antibiosis of F. verticillioides by Pf-5 and derivative strains that produce DAPG but had no effect on antibiosis by Pf-5 derivatives that suppressed F. verticillioides due to pyrrolnitrin or rhizoxin production. Our results demonstrated the importance of three compounds, rhizoxin, pyrrolnitrin, and DAPG, in suppression of Fusarium spp. by Pf-5 and confirmed that an interspecies signaling system mediated by fusaric acid had parallel effects on antifungal metabolite production and antibiosis by the bacterial biological control organism. PMID:26655755

  13. An Interspecies Signaling System Mediated by Fusaric Acid Has Parallel Effects on Antifungal Metabolite Production by Pseudomonas protegens Strain Pf-5 and Antibiosis of Fusarium spp.

    Science.gov (United States)

    Quecine, Maria Carolina; Kidarsa, Teresa A; Goebel, Neal C; Shaffer, Brenda T; Henkels, Marcella D; Zabriskie, T Mark; Loper, Joyce E

    2015-12-11

    Pseudomonas protegens strain Pf-5 is a rhizosphere bacterium that suppresses soilborne plant diseases and produces at least seven different secondary metabolites with antifungal properties. We derived mutants of Pf-5 with single and multiple mutations in biosynthesis genes for seven antifungal metabolites: 2,4-diacetylphoroglucinol (DAPG), pyrrolnitrin, pyoluteorin, hydrogen cyanide, rhizoxin, orfamide A, and toxoflavin. These mutants were tested for inhibition of the pathogens Fusarium verticillioides and Fusarium oxysporum f. sp. pisi. Rhizoxin, pyrrolnitrin, and DAPG were found to be primarily responsible for fungal antagonism by Pf-5. Previously, other workers showed that the mycotoxin fusaric acid, which is produced by many Fusarium species, including F. verticillioides, inhibited the production of DAPG by Pseudomonas spp. In this study, amendment of culture media with fusaric acid decreased DAPG production, increased pyoluteorin production, and had no consistent influence on pyrrolnitrin or orfamide A production by Pf-5. Fusaric acid also altered the transcription of biosynthetic genes, indicating that the mycotoxin influenced antibiotic production by Pf-5 at the transcriptional level. Addition of fusaric acid to the culture medium reduced antibiosis of F. verticillioides by Pf-5 and derivative strains that produce DAPG but had no effect on antibiosis by Pf-5 derivatives that suppressed F. verticillioides due to pyrrolnitrin or rhizoxin production. Our results demonstrated the importance of three compounds, rhizoxin, pyrrolnitrin, and DAPG, in suppression of Fusarium spp. by Pf-5 and confirmed that an interspecies signaling system mediated by fusaric acid had parallel effects on antifungal metabolite production and antibiosis by the bacterial biological control organism. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  14. PGE2 mediates EGFR internalization and nuclear translocation via caveolin endocytosis promoting its transcriptional activity and proliferation in human NSCLC cells.

    Science.gov (United States)

    Bazzani, Lorenzo; Donnini, Sandra; Giachetti, Antonio; Christofori, Gerhard; Ziche, Marina

    2018-03-13

    Prostaglandin E 2 (PGE 2 ) contributes to tumor progression by promoting cancer cell growth, invasion and by creating a favorable pro-tumor microenvironment. PGE 2 has been reported to transactivate and internalize into the nucleus receptor tyrosine kinases such as Epidermal growth factor receptor (EGFR), thereby supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, PGE 2 induces EGFR nuclear translocation via different dynamin-dependent endocytic pathways, promotes the formation of an EGFR-STAT3 complex, affects nuclear EGFR target gene expression and mediates tumor cell proliferation. Indeed, we find that PGE 2 induces EGFR internalization and consequent nuclear import through Clathrin- and Caveolin-mediated endocytosis and through the interaction of EGFR with Importin β1. Within the nucleus, EGFR forms a complex with STAT3, an event blocked by ablation of Clathrin Heavy Chain or Caveolin-1. The combination of EGF and PGE 2 prolongs nuclear EGFR transcriptional activity manifested by the upregulation of CCND1 , PTGS2 , MYC and NOS2 mRNA levels and potentiates nuclear EGFR-induced NSCLC cell proliferation. Additionally, NSCLC patients with high expression of a nuclear EGFR gene signature display shorter survival times than those with low expression, thus showing a putative correlation between nuclear EGFR and poor prognosis in NSCLC. Together, our findings indicate a complex mechanism underlying PGE 2 -induced EGF/EGFR signaling and transcriptional control, which plays a key role in cancer progression.

  15. A BMP-mediated transcriptional cascade involving Cash1 and Tlx-3 specifies first-order relay sensory neurons in the developing hindbrain.

    Science.gov (United States)

    Hornbruch, Amata; Ma, Grace; Ballermann, Mark A; Tumova, Katerina; Liu, Dan; Cairine Logan, C

    2005-07-01

    The divergent homeobox-containing transcription factor, Tlx-3 (also known as Hox11L2/Rnx), is required for proper formation of first-order relay sensory neurons in the developing vertebrate brainstem. To date, however, the inductive signals and transcriptional regulatory cascade underlying their development are poorly understood. We previously isolated the chick Tlx-3 homologue and showed it is expressed early (i.e. beginning at HH15) in distinct subcomponents of both the trigeminal/solitary and vestibular nuclei. Here we show via in vivo rhombomere inversions that expression of Tlx-3 is under control of local environmental signals. Our RNA in situ analysis shows expression of the BMP-specific receptor, Bmpr-1b, correlates well with Tlx-3. Furthermore, manipulation of the BMP signaling pathway in vivo via electroporation of expression vectors encoding either BMP or NOGGIN coupled with MASH1 gain-of-function experiments demonstrate that a BMP-mediated transcriptional cascade involving Cash1 and Tlx-3 specifies first-order relay sensory neurons in the developing brainstem. Notably, high-level Noggin misexpression results in an increase in newly differentiated Tlx-3+ neurons that correlates with a corresponding increase in the number of Calretinin+ neurons in vestibular nuclei at later developmental stages strongly suggesting that Tlx-3, in addition to being required for proper formation of somatic as well as visceral sensory neurons in the trigeminal and solitary nuclei, respectively, is sufficient for proper formation of special somatic sensory neurons in vestibular nuclei.

  16. In vivo identification of promoter elements and transcription factors mediating activation of hepatic HMG-CoA reductase by T{sub 3}

    Energy Technology Data Exchange (ETDEWEB)

    Boone, Lindsey R.; Niesen, Melissa I. [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL (United States); Jaroszeski, Mark [Department of Chemical and Biomedical Engineering, College of Engineering, University of South Florida, Tampa, FL (United States); Ness, Gene C., E-mail: gness@hsc.usf.edu [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL (United States)

    2009-07-31

    The promoter elements and transcription factors necessary for triiodothyronine (T{sub 3}) induction of hepatic HMG-CoA reductase (HMGR) were investigated by transfecting rat livers with wild type and mutant HMGR promoter-luciferase constructs using in vivo electroporation. Mutations in the sterol response element (SRE), nuclear factor-y (NF-Y) site, and the newly identified upstream transcription factor-2 (USF-2) site essentially abolished the T{sub 3} response. Chromatin immunoprecipitation (ChIP) analysis demonstrated that T{sub 3} treatment caused a 4-fold increase in in vivo binding of USF-2 to the HMGR promoter. Co-transfection of the wild type HMGR promoter with siRNAs to USF-2, SREBP-2, or NF-Y nearly abolished the T{sub 3} induction, as measured by promoter activity. These data provide in vivo evidence for functional roles for USF-2, SREBP-2, and NF-Y in mediating the T{sub 3}-induction of hepatic HMGR transcription.

  17. Cell Cycle-Dependent Recruitment of Polycomb Proteins to the ASNS Promoter Counteracts C/ebp-Mediated Transcriptional Activation in Bombyx mori

    Science.gov (United States)

    Li, Zhiqing; Cheng, Daojun; Mon, Hiroaki; Zhu, Li; Xu, Jian; Tatsuke, Tsuneyuki; Lee, Jae Man; Xia, Qingyou; Kusakabe, Takahiro

    2013-01-01

    Epigenetic modifiers and transcription factors contribute to developmentally programmed gene expression. Here, we establish a functional link between epigenetic regulation by Polycomb group (PcG) proteins and transcriptional regulation by C/ebp that orchestrates the correct expression of Bombyx mori asparagine synthetase (BmASNS), a gene involved in the biosynthesis of asparagine. We show that the cis-regulatory elements of YY1-binding motifs and the CpG island present on the BmASNS promoter are required for the recruitment of PcG proteins and the subsequent deposition of the epigenetic repression mark H3K27me3. RNAi-mediated knockdown of PcG genes leads to derepression of the BmASNS gene via the recruitment of activators, including BmC/ebp, to the promoter. Intriguingly, we find that PcG proteins and BmC/ebp can dynamically modulate the transcriptional output of the BmASNS target in a cell cycle-dependent manner. It will be essential to suppress BmASNS expression by PcG proteins at the G2/M phase of the cell cycle in the presence of BmC/ebp activator. Thus, our results provide a novel insight into the molecular mechanism underlying the recruitment and regulation of the PcG system at a discrete gene locus in Bombyx mori. PMID:23382816

  18. Activation of transcription factor AP-2 mediates UVA radiation- and singlet oxygen-induced expression of the human intercellular adhesion molecule 1 gene

    International Nuclear Information System (INIS)

    Grether-Beck, S.; Olaizola-Horn, S.; Schmitt, H.; Grewe, M.

    1996-01-01

    UVA radiation is the major component of the UV solar spectrum that reaches the earth, and the therapeutic application of UVA radiation is increasing in medicine. Analysis of the cellular effects of UVA radiation has revealed that exposure of human cells to UVA radiation at physiological doses leads to increased gene expression and that this UVA response is primarily mediated through the generation of singlet oxygen. In this study, the mechanisms by which UVA radiation induces transcriptional activation of the human intercellular adhesion molecule 1 (ICAM-1) were examined. UVA radiation was capable of inducing activation of the human ICAM-1 promoter and increasing OCAM-1 mRNA and protein expression. These UVA radiation effects were inhibited by singlet oxygen quenchers, augmented by enhancement of singlet oxygen life-time, and mimicked in unirradiated cells by a singlet oxygen-generating system. UVA radiation as well as singlet oxygen-induced ICAM-1 promoter activation required activation of the transcription factor AP-2. Accordingly, both stimuli activated AP-2, and deletion of the putative AP-2-binding site abrogated ICAM-1 promoter activation in this system. This study identified the AP-2 site as the UVA radiation- and singlet oxygen-responsive element of the human ICAM-1 gene. The capacity of UVA radiation and/or singlet oxygen to induce human gene expression through activation of AP-2 indicates a previously unrecognized role of this transcription factor in the mammalian stress response. 38 refs., 3 figs., 3 tabs

  19. Decoding genome-wide GadEWX-transcriptional regulatory networks reveals multifaceted cellular responses to acid stress in Escherichia coli

    DEFF Research Database (Denmark)

    Seo, Sang Woo; Kim, Donghyuk; O'Brien, Edward J.

    2015-01-01

    The regulators GadE, GadW and GadX (which we refer to as GadEWX) play a critical role in the transcriptional regulation of the glutamate-dependent acid resistance (GDAR) system in Escherichia coli K-12 MG1655. However, the genome-wide regulatory role of GadEWX is still unknown. Here we comprehens...

  20. A Novel WRKY transcription factor is required for induction of PR-1a gene expression by salicylic acid and bacterial elicitors

    NARCIS (Netherlands)

    van Verk, Marcel C|info:eu-repo/dai/nl/327618671; Pappaioannou, Dimitri; Neeleman, Lyda; Bol, John F; Linthorst, Huub J M

    PR-1a is a salicylic acid-inducible defense gene of tobacco (Nicotiana tabacum). One-hybrid screens identified a novel tobacco WRKY transcription factor (NtWRKY12) with specific binding sites in the PR-1a promoter at positions -564 (box WK(1)) and -859 (box WK(2)). NtWRKY12 belongs to the class of

  1. Carnitine palmitoyltransferase 1A (CPT1A): a transcriptional target of PAX3-FKHR and mediates PAX3-FKHR–dependent motility in alveolar rhabdomyosarcoma cells

    International Nuclear Information System (INIS)

    Liu, Lingling; Wang, Yong-Dong; Wu, Jing; Cui, Jimmy; Chen, Taosheng

    2012-01-01

    Alveolar rhabdomyosarcoma (ARMS) has a high propensity to metastasize, leading to its aggressiveness and a poor survival rate among those with the disease. More than 80% of aggressive ARMSs harbor a PAX3-FKHR fusion transcription factor, which regulates cell migration and promotes metastasis, most likely by regulating the fusion protein’s transcriptional targets. Therefore, identifying druggable transcription targets of PAX3-FKHR that are also downstream effectors of PAX3-FKHR–mediated cell migration and metastasis may lead to novel therapeutic approaches for treating ARMS. To identify genes whose expression is directly affected by the level of PAX3-FKHR in an ARMS cellular-context, we first developed an ARMS cell line in which PAX3-FKHR is stably down-regulated, and showed that stably downregulating PAX3-FKHR in ARMS cells significantly decreased the cells’ motility. We used microarray analysis to identify genes whose expression level decreased when PAX3-FKHR was downregulated. We used mutational analysis, promoter reporter assays, and electrophoretic mobility shift assays to determine whether PAX3-FKHR binds to the promoter region of the target gene. We used siRNA and pharmacologic inhibitor to downregulate the target gene of PAX3-FKHR and investigated the effect of such downregulation on cell motility. We found that when PAX3-FKHR was downregulated, the expression of carnitine palmitoyltransferase 1A (CPT1A) decreased. We showed that PAX3-FKHR binds to a paired-domain binding-site in the CPT1A promoter region, indicating that CPT1A is a novel transcriptional target of PAX3-FKHR. Furthermore, downregulating CPT1A decreased cell motility in ARMS cells, indicating that CPT1A is a downstream effector of PAX3-FKHR–mediated cell migration and metastasis. Taken together, we have identified CPT1A as a novel transcriptional target of PAX3-FKHR and revealed the novel function of CPT1A in promoting cell motility. CPT1A may represent a novel therapeutic target for

  2. Microarray Analysis of Transcriptional Responses to Abscisic Acid and Salt Stress in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Yucheng Wang

    2013-05-01

    Full Text Available Abscisic acid (ABA plays a crucial role in plant responses to abiotic stress. To investigate differences in plant responses to salt and ABA stimulus, differences in gene expression in Arabidopsis in response to salt and ABA were compared using an Agilent oligo microarray. A total of 144 and 139 genes were significantly up- and downregulated, respectively, under NaCl stress, while 406 and 381 genes were significantly up- and downregulated, respectively, under ABA stress conditions. In addition, 31 genes were upregulated by both NaCl and ABA stresses, and 23 genes were downregulated by these stressors, suggesting that these genes may play similar roles in plant responses to salt and ABA stress. Gene ontology (GO analysis revealed four subgroups of genes, including genes in the GO categories “Molecular transducer activity”, “Growth”, “Biological adhesion” and “Pigmentation”, which were expressed in response to ABA stress but not NaCl stress. In addition, genes that play specific roles during salt or ABA stress were identified. Our results may help elucidate differences in the response of plants to salt and ABA stress.

  3. Activation of the Arabidopsis membrane-bound transcription factor bZIP28 is mediated by site-2 protease, but not site-1 protease.

    Science.gov (United States)

    Iwata, Yuji; Ashida, Makoto; Hasegawa, Chisa; Tabara, Kazuki; Mishiba, Kei-Ichiro; Koizumi, Nozomu

    2017-08-01

    The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  4. Activation of the omega-3 fatty acid receptor GPR120 mediates anti-inflammatory actions in immortalized hypothalamic neurons.

    Science.gov (United States)

    Wellhauser, Leigh; Belsham, Denise D

    2014-03-27

    Overnutrition and the ensuing hypothalamic inflammation is a major perpetuating factor in the development of metabolic diseases, such as obesity and diabetes. Inflamed neurons of the CNS fail to properly regulate energy homeostasis leading to pathogenic changes in glucose handling, feeding, and body weight. Hypothalamic neurons are particularly sensitive to pro-inflammatory signals derived locally and peripherally, and it is these neurons that become inflamed first upon high fat feeding. Given the prevalence of metabolic disease, efforts are underway to identify therapeutic targets for this inflammatory state. At least in the periphery, omega-3 fatty acids and their receptor, G-protein coupled receptor 120 (GPR120), have emerged as putative targets. The role for GPR120 in the hypothalamus or CNS in general is poorly understood. Here we introduce a novel, immortalized cell model derived from the rat hypothalamus, rHypoE-7, to study GPR120 activation at the level of the individual neuron. Gene expression levels of pro-inflammatory cytokines were studied by quantitative reverse transcriptase-PCR (qRT-PCR) upon exposure to tumor necrosis factor α (TNFα) treatment in the presence or absence of the polyunsaturated omega-3 fatty acid docosahexaenoic acid (DHA). Signal transduction pathway involvement was also studied using phospho-specific antibodies to key proteins by western blot analysis. Importantly, rHypoE-7 cells exhibit a transcriptional and translational inflammatory response upon exposure to TNFα and express abundant levels of GPR120, which is functionally responsive to DHA. DHA pretreatment prevents the inflammatory state and this effect was inhibited by the reduction of endogenous GPR120 levels. GPR120 activates both AKT (protein kinase b) and ERK (extracellular signal-regulated kinase); however, the anti-inflammatory action of this omega-3 fatty acid (FA) receptor is AKT- and ERK-independent and likely involves the GPR120-transforming growth factor

  5. PGC-1α-mediated branched-chain amino acid metabolism in the skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Yukino Hatazawa

    Full Text Available Peroxisome proliferator-activated receptor (PPAR γ coactivator 1α (PGC-1α is a coactivator of various nuclear receptors and other transcription factors, which is involved in the regulation of energy metabolism, thermogenesis, and other biological processes that control phenotypic characteristics of various organ systems including skeletal muscle. PGC-1α in skeletal muscle is considered to be involved in contractile protein function, mitochondrial function, metabolic regulation, intracellular signaling, and transcriptional responses. Branched-chain amino acid (BCAA metabolism mainly occurs in skeletal muscle mitochondria, and enzymes related to BCAA metabolism are increased by exercise. Using murine skeletal muscle overexpressing PGC-1α and cultured cells, we investigated whether PGC-1α stimulates BCAA metabolism by increasing the expression of enzymes involved in BCAA metabolism. Transgenic mice overexpressing PGC-1α specifically in the skeletal muscle had increased the expression of branched-chain aminotransferase (BCAT 2, branched-chain α-keto acid dehydrogenase (BCKDH, which catabolize BCAA. The expression of BCKDH kinase (BCKDK, which phosphorylates BCKDH and suppresses its enzymatic activity, was unchanged. The amount of BCAA in the skeletal muscle was significantly decreased in the transgenic mice compared with that in the wild-type mice. The amount of glutamic acid, a metabolite of BCAA catabolism, was increased in the transgenic mice, suggesting the activation of muscle BCAA metabolism by PGC-1α. In C2C12 cells, the overexpression of PGC-1α significantly increased the expression of BCAT2 and BCKDH but not BCKDK. Thus, PGC-1α in the skeletal muscle is considered to significantly contribute to BCAA metabolism.

  6. PGC-1α-mediated branched-chain amino acid metabolism in the skeletal muscle.

    Science.gov (United States)

    Hatazawa, Yukino; Tadaishi, Miki; Nagaike, Yuta; Morita, Akihito; Ogawa, Yoshihiro; Ezaki, Osamu; Takai-Igarashi, Takako; Kitaura, Yasuyuki; Shimomura, Yoshiharu; Kamei, Yasutomi; Miura, Shinji

    2014-01-01

    Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α) is a coactivator of various nuclear receptors and other transcription factors, which is involved in the regulation of energy metabolism, thermogenesis, and other biological processes that control phenotypic characteristics of various organ systems including skeletal muscle. PGC-1α in skeletal muscle is considered to be involved in contractile protein function, mitochondrial function, metabolic regulation, intracellular signaling, and transcriptional responses. Branched-chain amino acid (BCAA) metabolism mainly occurs in skeletal muscle mitochondria, and enzymes related to BCAA metabolism are increased by exercise. Using murine skeletal muscle overexpressing PGC-1α and cultured cells, we investigated whether PGC-1α stimulates BCAA metabolism by increasing the expression of enzymes involved in BCAA metabolism. Transgenic mice overexpressing PGC-1α specifically in the skeletal muscle had increased the expression of branched-chain aminotransferase (BCAT) 2, branched-chain α-keto acid dehydrogenase (BCKDH), which catabolize BCAA. The expression of BCKDH kinase (BCKDK), which phosphorylates BCKDH and suppresses its enzymatic activity, was unchanged. The amount of BCAA in the skeletal muscle was significantly decreased in the transgenic mice compared with that in the wild-type mice. The amount of glutamic acid, a metabolite of BCAA catabolism, was increased in the transgenic mice, suggesting the activation of muscle BCAA metabolism by PGC-1α. In C2C12 cells, the overexpression of PGC-1α significantly increased the expression of BCAT2 and BCKDH but not BCKDK. Thus, PGC-1α in the skeletal muscle is considered to significantly contribute to BCAA metabolism.

  7. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... to these general questions by distinguishing between two concepts: mediation and mediatization. The media effects tradition generally considers the effects of the media to be a result of individuals being exposed to media content, i.e. effects are seen as an outcome of mediated communication. Mediatization...... research is concerned with long-term structural changes involving media, culture, and society, i.e. the influences of the media are understood in relation to how media are implicated in social and cultural changes and how these processes come to create new conditions for human communication and interaction...

  8. KIT(D816V) Induces SRC-Mediated Tyrosine Phosphorylation of MITF and Altered Transcription Program in Melanoma

    DEFF Research Database (Denmark)

    Phung, Bengt; Kazi, Julhash U; Lundby, Alicia

    2017-01-01

    The oncogenic D816V mutation of the KIT receptor is well characterized in systemic mastocytosis and acute myeloid leukemia. Although KIT(D816V) has been found in melanoma, its function and involvement in this malignancy is not understood. Here we show that KIT(D816V) induces tyrosine phosphorylat......The oncogenic D816V mutation of the KIT receptor is well characterized in systemic mastocytosis and acute myeloid leukemia. Although KIT(D816V) has been found in melanoma, its function and involvement in this malignancy is not understood. Here we show that KIT(D816V) induces tyrosine.......Implications: This study demonstrates that an oncogenic tyrosine kinase mutant, KIT(D816V), can alter the transcriptional program of the transcription factor MITF in melanoma Mol Cancer Res; 15(9); 1265-74. ©2017 AACR....

  9. Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activati