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Sample records for increases er-mediated transcription

  1. Transcription-replication collision increases recombination efficiency between plasmids.

    PubMed

    Jialiang, Li; Feng, Chen; Zhen, Xu; Jibing, Chen; Xiang, Lv; Lingling, Zhang; Depei, Liu

    2013-11-01

    It has been proposed that the stalling of the replication forks can induce homologous recombination in several organisms, and that arrested replication forks may offer nuclease targets, thereby providing a substrate for proteins involved in double-strand repair. In this article, we constructed a plasmid with the potential for transcription-replication collision (TRC), in which DNA replication and RNA transcription occur on the same DNA template simultaneously. Theoretically, transcription will impede DNA replication and increase homologous recombination. To validate this hypothesis, another plasmid was constructed that contained a homologous sequence with the exception of some mutated sites. Co-transfection of these two plasmids into 293T cells resulted in increased recombination frequency. The ratio of these two plasmids also affected the recombination frequency. Moreover, we found high expression levels of RAD51, which indicated that the increase in the recombination rate was probably via the homologous recombination pathway. These results indicate that mutant genes in plasmids can be repaired by TRC-induced recombination.

  2. Increased AICD generation does not result in increased nuclear translocation or activation of target gene transcription

    SciTech Connect

    Waldron, Elaine; Isbert, Simone; Kern, Andreas; Jaeger, Sebastian; Martin, Anne M.; Hebert, Sebastien S.; Behl, Christian; Weggen, Sascha; De Strooper, Bart; Pietrzik, Claus U.

    2008-08-01

    A sequence of amyloid precursor protein (APP) cleavages culminates in the sequential release of the APP intracellular domain (AICD) and the amyloid {beta} peptide (A{beta}) and/or p3 fragment. One of the environmental factors favouring the accumulation of AICD appears to be a rise in intracellular pH. Here we further identified the metabolism and subcellular localization of artificially expressed constructs under such conditions. We also co-examined the mechanistic lead up to the AICD accumulation and explored possible significances for its increased expression. We found that most of the AICD generated under pH neutralized conditions is likely cleaved from C83. While the AICD surplus was unable to further activate transcription of a luciferase reporter via a Gal4-DNA-binding domain, it failed entirely via the endogenous promoter regions of proposed target genes, APP and KAI1. The lack of a specific transactivation potential was also demonstrated by the unchanged levels of target gene mRNA. However, rather than translocating to the nucleus, the AICD surplus remains membrane tethered or free in the cytosol where it interacts with Fe65. Therefore we provide strong evidence that an increase in AICD generation does not directly promote gene activation of previously proposed target 0011gen.

  3. Glucagon and Insulin Cooperatively Stimulate Fibroblast Growth Factor 21 Gene Transcription by Increasing the Expression of Activating Transcription Factor 4.

    PubMed

    Alonge, Kimberly M; Meares, Gordon P; Hillgartner, F Bradley

    2017-03-31

    Previous studies have shown that glucagon cooperatively interacts with insulin to stimulate hepatic FGF21 gene expression. Here we investigated the mechanism by which glucagon and insulin increased FGF21 gene transcription in primary hepatocyte cultures. Transfection analyses demonstrated that glucagon plus insulin induction of FGF21 transcription was conferred by two activating transcription factor 4 (ATF4) binding sites in the FGF21 gene. Glucagon plus insulin stimulated a 5-fold increase in ATF4 protein abundance, and knockdown of ATF4 expression suppressed the ability of glucagon plus insulin to increase FGF21 expression. In hepatocytes incubated in the presence of insulin, treatment with a PKA-selective agonist mimicked the ability of glucagon to stimulate ATF4 and FGF21 expression. Inhibition of PKA, PI3K, Akt, and mammalian target of rapamycin complex 1 (mTORC1) suppressed the ability of glucagon plus insulin to stimulate ATF4 and FGF21 expression. Additional analyses demonstrated that chenodeoxycholic acid (CDCA) induced a 6-fold increase in ATF4 expression and that knockdown of ATF4 expression suppressed the ability of CDCA to increase FGF21 gene expression. CDCA increased the phosphorylation of eIF2α, and inhibition of eIF2α signaling activity suppressed CDCA regulation of ATF4 and FGF21 expression. These results demonstrate that glucagon plus insulin increases FGF21 transcription by stimulating ATF4 expression and that activation of cAMP/PKA and PI3K/Akt/mTORC1 mediates the effect of glucagon plus insulin on ATF4 expression. These results also demonstrate that CDCA regulation of FGF21 transcription is mediated at least partially by an eIF2α-dependent increase in ATF4 expression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Dexamethasone Enhances 1α,25-Dihydroxyvitamin D3 Effects by Increasing Vitamin D Receptor Transcription*

    PubMed Central

    Hidalgo, Alejandro A.; Deeb, Kristin K.; Pike, J. Wesley; Johnson, Candace S.; Trump, Donald L.

    2011-01-01

    Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner. PMID:21868377

  5. Increased transcription in hydroxyurea-treated root meristem cells of Vicia faba.

    PubMed

    Winnicki, Konrad; Polit, Justyna Teresa; Maszewski, Janusz

    2013-02-01

    Hydroxyurea (HU), an inhibitor of ribonucleotide reductase, prevents cells from progressing through S phase by depletion of deoxyribonucleoside triphosphates. Concurrently, disruption of DNA replication leads to double-strand DNA breaks. In root meristems of Vicia faba, HU triggers cell cycle arrest (preferentially in G1/S phase) and changes an overall metabolism by global activation of transcription both in the nucleoplasmic and nucleolar regions. High level of transcription is accompanied by an increase in the content of RNA polymerase II large subunit (POLR2A). Changes in transcription activation and POLR2A content correlate with posttranslational modifications of histones that play a role in opening up chromatin for transcription. Increase in the level of H4 Lys5 acetylation indicates that global activation of transcription following HU treatment depends on histone modifications.

  6. Increased expression of the Hutchinson-Gilford progeria syndrome truncated lamin A transcript during cell aging.

    PubMed

    Rodriguez, Sofia; Coppedè, Fabio; Sagelius, Hanna; Eriksson, Maria

    2009-07-01

    Most cases of the segmental progeroid syndrome, Hutchinson-Gilford progeria syndrome (HGPS), are caused by a de novo dominant mutation within a single codon of the LMNA gene. This mutation leads to the increased usage of an internal splice site that generates an alternative lamin A transcript with an internal deletion of 150 nucleotides, called lamin A Delta 150. The LMNA gene encodes two major proteins of the inner nuclear lamina, lamins A and C, but not much is known about their expression levels. Determination of the overall expression levels of the LMNA gene transcripts is an important step to further the understanding of the HGPS. In this study, we have performed absolute quantification of the lamins A, C and A Delta 150 transcripts in primary dermal fibroblasts from HGPS patients and unaffected age-matched and parent controls. We show that the lamin A Delta 150 transcript is present in unaffected controls but its expression is >160-fold lower than that in samples from HGPS patients. Analysis of transcript expression during in vitro aging shows that although the levels of lamin A and lamin C transcripts remain unchanged, the lamin A Delta 150 transcript increases in late passage cells from HGPS patients and parental controls. This study provides a new method for LMNA transcript analysis and insights into the expression of the LMNA gene in HGPS and normal cells.

  7. [NAC transcription factors family and increased tolerance to water deficiency in plants].

    PubMed

    Lip, Sabina; Kurowska, Marzena; Szarejko, Iwona

    2013-01-01

    Transcription factors are proteins that are able to regulate the expression of target genes by specifically binding with DNA sequences and regulating the activity initiation complex of transcription. These proteins are key elements in the adaptation of plants to environmental conditions. Families of transcription factors that are associated with a response to stress are DREB/CBF, AREB/ABF, MYB/MYC and NAC. The NAC gene family is one of the largest families of transcription factors. Members of the NAC family have been identified in many plant species. NAC TFs are involved in the growth, development and response of plants to biotic and abiotic stress. Many transcription factors belonging to the NAC family, including SNAC1, are involved in the response of plants to water deficiency. Drought is the most harmful environmental stress in worldwide agriculture. Obtaining plants with an increased tolerance to water deficiency by using the methods of molecular biology has become a major goal of plant breeding.

  8. Increase of a group of PTC(+) transcripts by curcumin through inhibition of the NMD pathway.

    PubMed

    Feng, Dairong; Su, Ruey-Chyi; Zou, Liping; Triggs-Raine, Barbara; Huang, Shangzhi; Xie, Jiuyong

    2015-08-01

    Nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism, eliminates premature termination codon-containing (PTC⁺) transcripts. For instance, it maintains the homeostasis of splicing factors and degrades aberrant transcripts of human genetic disease genes. Here we examine the inhibitory effect on the NMD pathway and consequent increase of PTC+ transcripts by the dietary compound curcumin. We have found that several PTC⁺ transcripts including that of serine/arginine-rich splicing factor 1 (SRSF1) were specifically increased in cells by curcumin. We also observed a similar curcumin effect on the PTC⁺ mutant transcript from a Tay-Sachs-causing HEXA allele or from a beta-globin reporter gene. The curcumin effect was accompanied by significantly reduced expression of the NMD factors UPF1, 2, 3A and 3B. Consistently, in chromatin immunoprecipitation assays, curcumin specifically reduced the occupancy of acetyl-histone H3 and RNA polymerase II at the promoter region (-376 to -247nt) of human UPF1, in a time- and dosage-dependent way. Importantly, knocking down UPF1 abolished or substantially reduced the difference of PTC(+) transcript levels between control and curcumin-treated cells. The disrupted curcumin effect was efficiently rescued by expression of exogenous Myc-UPF1 in the knockdown cells. Together, our data demonstrate that a group of PTC⁺ transcripts are stabilized by a dietary compound curcumin through the inhibition of UPF factor expression and the NMD pathway.

  9. Exercise induces a transient increase in transcription of the GLUT-4 gene in skeletal muscle.

    PubMed

    Neufer, P D; Dohm, G L

    1993-12-01

    Endurance exercise training elicits an increase in mitochondrial density as well as GLUT-4 glucose transporter protein content in skeletal muscle. Corresponding increases in mRNA for respiratory enzymes and GLUT-4 indicate that pretranslational control mechanisms are involved in this adaptive process. To directly test whether transcription of the GLUT-4 gene is activated in response to exercise training, nuclei were isolated from red hindlimb skeletal muscle of rats after 1 wk of exercise training (8% grade, 32 m/min, 40 min, twice/day). Rats were killed either 30 min, 3 h, or 24 h after the last training session. GLUT-4 transcription, determined by nuclear run-on analysis, was unaltered after 30 min, increased by 1.8-fold after 3 h, but was no longer different from controls 24 h after exercise. A similar transient increase in GLUT-4 transcription was evident, but less pronounced (1.4-fold), in untrained rats after a single bout of exercise, suggesting that the postexercise induction in GLUT-4 gene transcription is enhanced by exercise training. GLUT-4 protein content was increased 1.7-fold after 1 wk of training in the absence of any corresponding change in GLUT-4 mRNA, providing evidence that the initial increase in GLUT-4 expression involves translational and/or posttranslational control mechanisms. These findings demonstrate that muscle GLUT-4 expression in response to exercise training is subject to both transcriptional and posttranscriptional regulation. We propose that the increase in GLUT-4 mRNA evident with extended periods of training may result from a shift to pretranslational control and is the cumulative effect of repeated postexercise transient increases in GLUT-4 gene transcription.

  10. Increased global transcription activity as a mechanism of replication stress in cancer.

    PubMed

    Kotsantis, Panagiotis; Silva, Lara Marques; Irmscher, Sarah; Jones, Rebecca M; Folkes, Lisa; Gromak, Natalia; Petermann, Eva

    2016-10-11

    Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRAS(V12) promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRAS(V12), elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer.

  11. Increased global transcription activity as a mechanism of replication stress in cancer

    PubMed Central

    Kotsantis, Panagiotis; Silva, Lara Marques; Irmscher, Sarah; Jones, Rebecca M.; Folkes, Lisa; Gromak, Natalia; Petermann, Eva

    2016-01-01

    Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRASV12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRASV12, elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer. PMID:27725641

  12. The rice Mybleu transcription factor increases tolerance to oxygen deprivation in Arabidopsis plants.

    PubMed

    Mattana, Monica; Vannini, Candida; Espen, Luca; Bracale, Marcella; Genga, Annamaria; Marsoni, Milena; Iriti, Marcello; Bonazza, Veronica; Romagnoli, Francesco; Baldoni, Elena; Coraggio, Immacolata; Locatelli, Franca

    2007-09-01

    Mybleu is a natural incomplete transcription factor of rice (Oryza sativa), consisting of a partial Myb repeat followed by a short leucine zipper. We previously showed its localization to the apical region of rice roots and coleoptiles. Specifically, in coleoptiles, Mybleu is expressed under both aerobic and anaerobic conditions, whereas in roots, it is expressed only under aerobic conditions. Mybleu is able to dimerize with canonical leucine zippers and to activate transcription selectively. To investigate Mybleu function in vivo, we transformed Arabidopsis thaliana and evaluated several morphological, physiological and biochemical parameters. In agreement with a hypothesized role of Mybleu in cell elongation in the differentiation zone, we found that the constitutive expression of this transcription factor in Arabidopsis induced elongation in the primary roots and in the internodal region of the floral stem; we also observed a modification of the root apex morphology in transformed lines. Based on the high expression of Mybleu in anaerobic rice coleoptiles, we studied the role of this transcription factor in transgenic plants grown under low-oxygen conditions. We found that overexpression of this transcription factor increased tolerance to oxygen deficit. In transgenic plants, this effect may depend both on the maintenance of a higher metabolism during stress and on the higher expression levels of certain genes involved in the anaerobic response.

  13. TATA-Binding Protein Mutants That Increase Transcription from Enhancerless and Repressed Promoters In Vivo

    PubMed Central

    Geisberg, Joseph V.; Struhl, Kevin

    2000-01-01

    Using a genetic screen, we isolated three TATA-binding protein (TBP) mutants that increase transcription from promoters that are repressed by the Cyc8-Tup1 or Sin3-Rpd3 corepressors or that lack an enhancer element, but not from an equivalently weak promoter with a mutated TATA element. Increased transcription is observed when the TBP mutants are expressed at low levels in the presence of wild-type TBP. These TBP mutants are unable to support cell viability, and they are toxic in strains lacking Rpd3 histone deacetylase or when expressed at higher levels. Although these mutants do not detectably bind TATA elements in vitro, genetic and chromatin immunoprecipitation experiments indicate that they act directly at promoters and do not increase transcription by titration of a negative regulatory factor(s). The TBP mutants are mildly defective for associating with promoters responding to moderate or strong activators; in addition, they are severely defective for RNA polymerase (Pol) III but not Pol I transcription. These results suggest that, with respect to Pol II transcription, the TBP mutants specifically increase expression from core promoters. Biochemical analysis indicates that the TBP mutants are unaffected for TFIID complex formation, dimerization, and interactions with either the general negative regulator NC2 or the N-terminal inhibitory domain of TAF130. We speculate that these TBP mutants have an unusual structure that allows them to preferentially access TATA elements in chromatin templates. These TBP mutants define a criterion by which promoters repressed by Cyc8-Tup1 or Sin3-Rpd3 resemble enhancerless, but not TATA-defective, promoters; hence, they support the idea that these corepressors inhibit the function of activator proteins rather than the Pol II machinery. PMID:10669725

  14. During herpes simplex virus type 1 infection of rabbits, the ability to express the latency-associated transcript increases latent-phase transcription of lytic genes.

    PubMed

    Giordani, Nicole V; Neumann, Donna M; Kwiatkowski, Dacia L; Bhattacharjee, Partha S; McAnany, Peterjon K; Hill, James M; Bloom, David C

    2008-06-01

    Trigeminal ganglia (TG) from rabbits latently infected with either wild-type herpes simplex virus type 1 (HSV-1) or the latency-associated transcript (LAT) promoter deletion mutant 17DeltaPst were assessed for their viral chromatin profile and transcript abundance. The wild-type 17syn+ genomes were more enriched in the transcriptionally permissive mark dimethyl H3 K4 than were the 17DeltaPst genomes at the 5' exon and ICP0 and ICP27 promoters. Reverse transcription-PCR analysis revealed significantly more ICP4, tk, and glycoprotein C lytic transcripts in 17syn+ than in 17DeltaPst. These results suggest that, for efficient reactivation from latency in rabbits, the LAT is important for increased transcription of lytic genes during latency.

  15. Increases in Retrograde Injury Signaling Complex-Related Transcripts in Central Axons following Injury

    PubMed Central

    Pathak, Gunja K.; Ornstein, Hannah; Aranda-Espinoza, Helim; Karlsson, Amy J.

    2016-01-01

    Axons in the peripheral nervous system respond to injury by activating retrograde injury signaling (RIS) pathways, which promote local axonal protein synthesis (LPS) and neuronal regeneration. RIS is also initiated following injury of neurons in the central nervous system (CNS). However, regulation of the localization of axonal mRNA required for LPS is not well understood. We used a hippocampal explant system to probe the regulation of axonal levels of RIS-associated transcripts following axonal injury. Axonal levels of importin β1 and RanBP1 were elevated biphasically at 1 and 24 hrs after axotomy. Transcript levels for β-actin, a prototypic axonally synthesized protein, were similarly elevated. Our data suggest differential regulation of axonal transcripts. At 1 hr after injury, deployment of actinomycin revealed that RanBP1, but not importin β1, requires de novo mRNA synthesis. At 24 hrs after injury, use of importazole revealed that the second wave of increased axonal mRNA levels required importin β-mediated nuclear import. We also observed increased importin β1 axonal protein levels at 1 and 6 hrs after injury. RanBP1 levels and vimentin levels fluctuated but were unchanged at 3 and 6 hrs after injury. This study revealed temporally complex regulation of axonal transcript levels, and it has implications for understanding neuronal response to injury in the CNS. PMID:27847648

  16. Over-expression of Dof-type transcription factor increases lipid production in Chlamydomonas reinhardtii.

    PubMed

    Ibáñez-Salazar, Alejandro; Rosales-Mendoza, Sergio; Rocha-Uribe, Alejandro; Ramírez-Alonso, Jocelín Itzel; Lara-Hernández, Ignacio; Hernández-Torres, Araceli; Paz-Maldonado, Luz María Teresita; Silva-Ramírez, Ana Sonia; Bañuelos-Hernández, Bernardo; Martínez-Salgado, José Luis; Soria-Guerra, Ruth Elena

    2014-08-20

    The high demand for less polluting, newer, and cheaper fuel resources has increased the search of the most innovative options for the production of the so-called biofuels. Chlamydomonas reinhardtii is a photosynthetic unicellular algae with multiple biotechnological advantages such as easy handling in the laboratory, a simple scale-up to industrial levels, as well as a feasible genetic modification at nuclear and chloroplast levels. Besides, its fatty acids can be used to produce biofuels. Previous studies in plants have found that the over expression of DOF-type transcription factor genes increases the synthesis and the accumulation of total lipids in seeds. In this context, the over-expression of a DOF-type transcription factor in C. reinhardtii was applied as approach to increase the amount of lipids. The results indicate higher amounts (around 2-fold) of total lipids, which are mainly fatty acids, in the genetically C. reinhardtii modified strains when compared with the non-genetically modified strain. In order to elucidate the possible function of the introduced Dof-type transcription factor, we performed a transcription profile of 8 genes involved in fatty acid biosynthesis and 6 genes involved in glycerolipid biosynthesis, by quantitative real time (qRT-PCR). Differential expression profile was observed, which can explain the increase in lipid accumulation. However, these strains did not show notable changes in the fatty acid profile. This work represents an early effort in generating a strategy to increase fatty acids production in C. reinhardtii and their use in biofuel synthesis.

  17. Mycobacterium tuberculosis increases IP-10 and MIG protein despite inhibition of IP-10 and MIG transcription.

    PubMed

    Bai, Xiyuan; Chmura, Kathryn; Ovrutsky, Alida R; Bowler, Russell P; Scheinman, Robert I; Oberley-Deegan, Rebecca E; Liu, Haiying; Shang, Shaobin; Ordway, Diane; Chan, Edward D

    2011-01-01

    Mycobacterium tuberculosis (MTB) has evolved methods to evade interferon-gamma (IFNγ) mediated protection. We sought to determine the effect of MTB infection on expression of IFNγ-inducible Protein 10 (IP-10) and Monokine Induced by IFNγ (MIG), two chemokines involved in host defense. MTB infection of THP-1 cells inhibited the transcription of IP-10 and MIG. A key mechanism for the inhibition is the disruption of binding of Signal Transduction and Activation of Transcription 1-alpha (STAT1α) to its cis-regulatory element, present in the 5'-flanking region of both IP-10 and MIG promoters. Use of inhibitors specific to the nuclear factor-kappa B (NFκB) and p38 mitogen-activated protein kinase (p38(mapk)) implicate these two signaling pathways in mediating the effect of MTB on the inhibition of IFNγ-induced IP-10 and MIG mRNA expression. Interestingly, despite transcriptional inhibition, there was an unexpected increase in IP-10 and MIG protein production after combined IFNγ and MTB stimulation. MTB also inhibited IFNγ induction of MIG mRNA but augmented MIG protein in primary human monocyte-derived macrophages. The synergy between MTB and IFNγ in the induction of IP-10 and MIG protein appears to involve novel post-transcriptional events that incorporates non-canonical functions of NFκB and p38(mapk). Published by Elsevier Ltd.

  18. Aging increases cell-to-cell transcriptional variability upon immune stimulation

    PubMed Central

    Chen, Hung-Chang; Vallejos, Catalina A.; Kolodziejczyk, Aleksandra A.; Connor, Frances; Stojic, Lovorka; Rayner, Timothy F.; Stubbington, Michael J.T.; Teichmann, Sarah A.; de la Roche, Maike; Marioni, John C.; Odom, Duncan T.

    2017-01-01

    Aging is characterized by progressive loss of physiological and cellular functions, but the molecular basis of this decline remains unclear. We explored how aging impacts transcriptional dynamics using single-cell RNA-sequencing of unstimulated and stimulated naive and effector memory CD4+ T cells from young and old mice from two divergent species. In young animals, immunological activation drives a conserved transcriptomic switch resulting in tightly regulated gene expression, characterized by a strong up-regulation of a core activation program, coupled with a decrease in cell-to-cell variability. Aging perturbed the activation of this core program, and increased expression heterogeneity across populations of cells in both species. These discoveries suggest that increased cell-to-cell transcriptional variability will be a hallmark feature of aging across most, if not all, mammalian tissues. PMID:28360329

  19. Notch Inhibition Enhances Cardiac Reprogramming by Increasing MEF2C Transcriptional Activity.

    PubMed

    Abad, Maria; Hashimoto, Hisayuki; Zhou, Huanyu; Morales, Maria Gabriela; Chen, Beibei; Bassel-Duby, Rhonda; Olson, Eric N

    2017-03-14

    Conversion of fibroblasts into functional cardiomyocytes represents a potential means of restoring cardiac function after myocardial infarction, but so far this process remains inefficient and little is known about its molecular mechanisms. Here we show that DAPT, a classical Notch inhibitor, enhances the conversion of mouse fibroblasts into induced cardiac-like myocytes by the transcription factors GATA4, HAND2, MEF2C, and TBX5. DAPT cooperates with AKT kinase to further augment this process, resulting in up to 70% conversion efficiency. Moreover, DAPT promotes the acquisition of specific cardiomyocyte features, substantially increasing calcium flux, sarcomere structure, and the number of spontaneously beating cells. Transcriptome analysis shows that DAPT induces genetic programs related to muscle development, differentiation, and excitation-contraction coupling. Mechanistically, DAPT increases binding of the transcription factor MEF2C to the promoter regions of cardiac structural genes. These findings provide mechanistic insights into the reprogramming process and may have important implications for cardiac regeneration therapies.

  20. Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC-1.

    PubMed

    Baar, Keith; Wende, Adam R; Jones, Terry E; Marison, Matthew; Nolte, Lorraine A; Chen, May; Kelly, Daniel P; Holloszy, John O

    2002-12-01

    Endurance exercise induces increases in mitochondria and the GLUT4 isoform of the glucose transporter in muscle. Although little is known about the mechanisms underlying these adaptations, new information has accumulated regarding how mitochondrial biogenesis and GLUT4 expression are regulated. This includes the findings that the transcriptional coactivator PGC-1 promotes mitochondrial biogenesis and that NRF-1 and NRF-2 act as transcriptional activators of genes encoding mitochondrial enzymes. We tested the hypothesis that increases in PGC-1, NRF-1, and NRF-2 are involved in the initial adaptive response of muscle to exercise. Five daily bouts of swimming induced increases in mitochondrial enzymes and GLUT4 in skeletal muscle in rats. One exercise bout resulted in approximately twofold increases in full-length muscle PGC-1 mRNA and PGC-1 protein, which were evident 18 h after exercise. A smaller form of PGC-1 increased after exercise. The exercise induced increases in muscle NRF-1 and NRF-2 that were evident 12 to 18 h after one exercise bout. These findings suggest that increases in PGC-1, NRF-1, and NRF-2 represent key regulatory components of the stimulation of mitochondrial biogenesis by exercise and that PGC-1 mediates the coordinated increases in GLUT4 and mitochondria.

  1. Strong negative self regulation of Prokaryotic transcription factors increases the intrinsic noise of protein expression

    PubMed Central

    Stekel, Dov J; Jenkins, Dafyd J

    2008-01-01

    Background Many prokaryotic transcription factors repress their own transcription. It is often asserted that such regulation enables a cell to homeostatically maintain protein abundance. We explore the role of negative self regulation of transcription in regulating the variability of protein abundance using a variety of stochastic modeling techniques. Results We undertake a novel analysis of a classic model for negative self regulation. We demonstrate that, with standard approximations, protein variance relative to its mean should be independent of repressor strength in a physiological range. Consequently, in that range, the coefficient of variation would increase with repressor strength. However, stochastic computer simulations demonstrate that there is a greater increase in noise associated with strong repressors than predicted by theory. The discrepancies between the mathematical analysis and computer simulations arise because with strong repressors the approximation that leads to Michaelis-Menten-like hyperbolic repression terms ceases to be valid. Because we observe that strong negative feedback increases variability and so is unlikely to be a mechanism for noise control, we suggest instead that negative feedback is evolutionarily favoured because it allows the cell to minimize mRNA usage. To test this, we used in silico evolution to demonstrate that while negative feedback can achieve only a modest improvement in protein noise reduction compared with the unregulated system, it can achieve good improvement in protein response times and very substantial improvement in reducing mRNA levels. Conclusion Strong negative self regulation of transcription may not always be a mechanism for homeostatic control of protein abundance, but instead might be evolutionarily favoured as a mechanism to limit the use of mRNA. The use of hyperbolic terms derived from quasi-steady-state approximation should also be avoided in the analysis of stochastic models with strong repressors

  2. Strong negative self regulation of prokaryotic transcription factors increases the intrinsic noise of protein expression.

    PubMed

    Stekel, Dov J; Jenkins, Dafyd J

    2008-01-18

    Many prokaryotic transcription factors repress their own transcription. It is often asserted that such regulation enables a cell to homeostatically maintain protein abundance. We explore the role of negative self regulation of transcription in regulating the variability of protein abundance using a variety of stochastic modeling techniques. We undertake a novel analysis of a classic model for negative self regulation. We demonstrate that, with standard approximations, protein variance relative to its mean should be independent of repressor strength in a physiological range. Consequently, in that range, the coefficient of variation would increase with repressor strength. However, stochastic computer simulations demonstrate that there is a greater increase in noise associated with strong repressors than predicted by theory. The discrepancies between the mathematical analysis and computer simulations arise because with strong repressors the approximation that leads to Michaelis-Menten-like hyperbolic repression terms ceases to be valid. Because we observe that strong negative feedback increases variability and so is unlikely to be a mechanism for noise control, we suggest instead that negative feedback is evolutionarily favoured because it allows the cell to minimize mRNA usage. To test this, we used in silico evolution to demonstrate that while negative feedback can achieve only a modest improvement in protein noise reduction compared with the unregulated system, it can achieve good improvement in protein response times and very substantial improvement in reducing mRNA levels. Strong negative self regulation of transcription may not always be a mechanism for homeostatic control of protein abundance, but instead might be evolutionarily favoured as a mechanism to limit the use of mRNA. The use of hyperbolic terms derived from quasi-steady-state approximation should also be avoided in the analysis of stochastic models with strong repressors.

  3. Statins Increase Plasminogen Activator Inhibitor Type 1 Gene Transcription through a Pregnane X Receptor Regulated Element.

    PubMed

    Stanley, Frederick M; Linder, Kathryn M; Cardozo, Timothy J

    2015-01-01

    Plasminogen activator inhibitor type 1 (PAI-1) is a multifunctional protein that has important roles in inflammation and wound healing. Its aberrant regulation may contribute to many disease processes such as heart disease. The PAI-1 promoter is responsive to multiple inputs including cytokines, growth factors, steroids and oxidative stress. The statin drugs, atorvastatin, mevastatin and rosuvastatin, increased basal and stimulated expression of the PAI-1 promoter 3-fold. A statin-responsive, nuclear hormone response element was previously identified in the PAI-1 promoter, but it was incompletely characterized. We characterized this direct repeat (DR) of AGGTCA with a 3-nucleotide spacer at -269/-255 using deletion and directed mutagenesis. Deletion or mutation of this element increased basal transcription from the promoter suggesting that it repressed PAI-1 transcription in the unliganded state. The half-site spacing and the ligand specificity suggested that this might be a pregnane X receptor (PXR) responsive element. Computational molecular docking showed that atorvastatin, mevastatin and rosuvastatin were structurally compatible with the PXR ligand-binding pocket in its agonist conformation. Experiments with Gal4 DNA binding domain fusion proteins showed that Gal4-PXR was activated by statins while other DR + 3 binding nuclear receptor fusions were not. Overexpression of PXR further enhanced PAI-1 transcription in response to statins. Finally, ChIP experiments using Halo-tagged PXR and RXR demonstrated that both components of the PXR-RXR heterodimer bound to this region of the PAI-1 promoter.

  4. Statins Increase Plasminogen Activator Inhibitor Type 1 Gene Transcription through a Pregnane X Receptor Regulated Element

    PubMed Central

    Stanley, Frederick M.; Linder, Kathryn M.; Cardozo, Timothy J.

    2015-01-01

    Plasminogen activator inhibitor type 1 (PAI-1) is a multifunctional protein that has important roles in inflammation and wound healing. Its aberrant regulation may contribute to many disease processes such as heart disease. The PAI-1 promoter is responsive to multiple inputs including cytokines, growth factors, steroids and oxidative stress. The statin drugs, atorvastatin, mevastatin and rosuvastatin, increased basal and stimulated expression of the PAI-1 promoter 3-fold. A statin-responsive, nuclear hormone response element was previously identified in the PAI-1 promoter, but it was incompletely characterized. We characterized this direct repeat (DR) of AGGTCA with a 3-nucleotide spacer at -269/-255 using deletion and directed mutagenesis. Deletion or mutation of this element increased basal transcription from the promoter suggesting that it repressed PAI-1 transcription in the unliganded state. The half-site spacing and the ligand specificity suggested that this might be a pregnane X receptor (PXR) responsive element. Computational molecular docking showed that atorvastatin, mevastatin and rosuvastatin were structurally compatible with the PXR ligand-binding pocket in its agonist conformation. Experiments with Gal4 DNA binding domain fusion proteins showed that Gal4-PXR was activated by statins while other DR + 3 binding nuclear receptor fusions were not. Overexpression of PXR further enhanced PAI-1 transcription in response to statins. Finally, ChIP experiments using Halo-tagged PXR and RXR demonstrated that both components of the PXR-RXR heterodimer bound to this region of the PAI-1 promoter. PMID:26379245

  5. SNAIL transcription factor increases the motility and invasive capacity of prostate cancer cells

    PubMed Central

    OSORIO, LUIS A.; FARFÁN, NANCY M.; CASTELLÓN, ENRIQUE A.; CONTRERAS, HÉCTOR R.

    2016-01-01

    The incidence and mortality rates of prostate cancer (PCa) are increasing, and PCa is almost the second-leading cause of cancer-associated mortality in men. During tumor progression, epithelial cells decrease the number of adhesion molecules, change their polarity and position, rearrange their cytoskeleton and increase their migratory and invasive capacities. These changes are known under the concept of epithelial-mesenchymal transition (EMT). EMT is characterized by an upregulation of certain transcription factors, including SNAIL1, which represses genes that are characteristic of an epithelial phenotype, including E-cadherin, and indirectly increase the expression levels of genes, which are associated with the mesenchymal phenotype. It has been suggested that the transcription factor, SNAIL1, decreases the proliferation and increases the migratory and invasive capacities of PCa cell lines. The present study was performed using LNCaP and PC3 cell lines, in which the expression levels of SNAIL1 were increased or silenced through the use of lentiviral vectors. The expression levels of EMT markers were quantified using reverse transcription-quantitative polymerase chain reaction and western blot analysis. In addition, cell survival was analyzed using an MTS assay; cell proliferation was examined using an antibody targeting Ki-67; migration on plates with 8 µm pores to allow the passage of cells; and invasiveness was analyzed using a membrane chamber covered in dried basement membrane matrix solution. The levels of apoptosis were determined using a Caspase 3/7 assay containing a substrate modified by caspases 3 and 7. The results demonstrated that the overexpression and silencing of SNAIL1 decreased cell proliferation and survival. However, the overexpression of SNAIL1 decreased apoptosis, compared with cells with the SNAIL1-silenced cells, in which cell apoptosis increased. The migration and invasive capacities increased in the cells overexpressing SNAIL1, and

  6. Using Transcriptional Control To Increase Titers of Secreted Heterologous Proteins by the Type III Secretion System

    PubMed Central

    Metcalf, Kevin J.; Finnerty, Casey; Azam, Anum; Valdivia, Elias

    2014-01-01

    The type III secretion system (T3SS) encoded at the Salmonella pathogenicity island 1 (SPI-1) locus secretes protein directly from the cytosol to the culture media in a concerted, one-step process, bypassing the periplasm. While this approach is attractive for heterologous protein production, product titers are too low for many applications. In addition, the expression of the SPI-1 gene cluster is subject to native regulation, which requires culturing conditions that are not ideal for high-density growth. We used transcriptional control to increase the amount of protein that is secreted into the extracellular space by the T3SS of Salmonella enterica. The controlled expression of the gene encoding SPI-1 transcription factor HilA circumvents the requirement of endogenous induction conditions and allows for synthetic induction of the secretion system. This strategy increases the number of cells that express SPI-1 genes, as measured by promoter activity. In addition, protein secretion titer is sensitive to the time of addition and the concentration of inducer for the protein to be secreted and SPI-1 gene cluster. Overexpression of hilA increases secreted protein titer by >10-fold and enables recovery of up to 28 ± 9 mg/liter of secreted protein from an 8-h culture. We also demonstrate that the protein beta-lactamase is able to adopt an active conformation after secretion, and the increase in secreted titer from hilA overexpression also correlates to increased enzyme activity in the culture supernatant. PMID:25038096

  7. Overexpression of StDREB1 transcription factor increases tolerance to salt in transgenic potato plants.

    PubMed

    Bouaziz, Donia; Pirrello, Julien; Charfeddine, Mariam; Hammami, Asma; Jbir, Rania; Dhieb, Amina; Bouzayen, Mondher; Gargouri-Bouzid, Radhia

    2013-07-01

    It has been established that drought-responsive element binding (DREB) proteins correspond to transcription factors which play important regulatory roles in plant response to abiotic and biotic stresses. In this study, a novel cDNA encoding DREB transcription factor, designated StDREB1, was isolated from potato (Solanum tuberosum L.). This protein was classified in the A-4 group of DREB subfamily based on multiple sequence alignments and phylogenetic characterization. Semi-quantitative RT-PCR showed that StDREB1 is expressed in leaves, stems, and roots under stress conditions and it is greatly induced by NaCl, drought, low temperature, and abscisic acid (ABA) treatments. Overexpression of StDREB1 cDNA in transgenic potato plants exhibited an improved salt and drought stress tolerance in comparison to the non-transformed controls. The enhanced stress tolerance may be associated with the increase in P5CS-RNA expression (δ (1)-pyrroline-5-carboxylate synthetase) and the subsequent accumulation of proline osmoprotectant in addition to a better control of water loss. Overexpression of StDREB1 also activated stress-responsive genes, such as those encoding calcium-dependent protein kinases (CDPKs), in transgenic potatoes under standard and high salt conditions. These data suggest that the StDREB1 transcription factor is involved in the regulation of salt stress tolerance in potato by the activation of different downstream gene expression.

  8. Lead exposure suppressed ALAD transcription by increasing methylation level of the promoter CpG islands.

    PubMed

    Li, Chunping; Xu, Ming; Wang, Sumeng; Yang, Xiaolin; Zhou, Shourong; Zhang, Jingping; Liu, Qizhan; Sun, Yujie

    2011-05-30

    DNA methylation provides a plausible link between the environment and alterations in gene expression that may lead to disease phenotypes. Lead exposure can change DNA methylation status. Here, we hypothesized that the methylation of the ALAD gene promoter may play an important role in lead toxicity. To determine whether the methylation level of the ALAD promoter is associated with the risk of lead poisoning, we conducted a case-control study of 103 workers from a battery plant and 103 healthy volunteers with matching age and gender distribution. We employed real-time PCR and methylation-specific PCR (MSP) in cell models to determine the relationship between ALAD methylation level and transcription level. We found lead exposure to increase the ALAD gene methylation level and down-regulate ALAD transcription. The difference in methylation frequencies between exposures and controls was statistically significant (p=0.002), and individuals with methylated ALAD gene showed an increased risk of lead poisoning (adjusted OR=3.57, 95% CI, 1.55-8.18). This study suggests that the lead-exposure-induced increases in ALAD methylation may be involved in the mechanism of lead toxicity. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  9. Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium.

    PubMed

    Osanai, Takashi; Shirai, Tomokazu; Iijima, Hiroko; Nakaya, Yuka; Okamoto, Mami; Kondo, Akihiko; Hirai, Masami Y

    2015-01-01

    Succinate is a building block compound that the U.S. Department of Energy (DOE) has declared as important in biorefineries, and it is widely used as a commodity chemical. Here, we identified the two genes increasing succinate production of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Succinate was excreted under dark, anaerobic conditions, and its production level increased by knocking out ackA, which encodes an acetate kinase, and by overexpressing sigE, which encodes an RNA polymerase sigma factor. Glycogen catabolism and organic acid biosynthesis were enhanced in the mutant lacking ackA and overexpressing sigE, leading to an increase in succinate production reaching five times of the wild-type levels. Our genetic and metabolomic analyses thus demonstrated the effect of genetic manipulation of a metabolic enzyme and a transcriptional regulator on succinate excretion from this cyanobacterium with the data based on metabolomic technique.

  10. Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium

    PubMed Central

    Osanai, Takashi; Shirai, Tomokazu; Iijima, Hiroko; Nakaya, Yuka; Okamoto, Mami; Kondo, Akihiko; Hirai, Masami Y.

    2015-01-01

    Succinate is a building block compound that the U.S. Department of Energy (DOE) has declared as important in biorefineries, and it is widely used as a commodity chemical. Here, we identified the two genes increasing succinate production of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Succinate was excreted under dark, anaerobic conditions, and its production level increased by knocking out ackA, which encodes an acetate kinase, and by overexpressing sigE, which encodes an RNA polymerase sigma factor. Glycogen catabolism and organic acid biosynthesis were enhanced in the mutant lacking ackA and overexpressing sigE, leading to an increase in succinate production reaching five times of the wild-type levels. Our genetic and metabolomic analyses thus demonstrated the effect of genetic manipulation of a metabolic enzyme and a transcriptional regulator on succinate excretion from this cyanobacterium with the data based on metabolomic technique. PMID:26500619

  11. Increased transcription of Glutathione S-transferases in acaricide exposed scabies mites

    PubMed Central

    2010-01-01

    Background Recent evidence suggests that Sarcoptes scabiei var. hominis mites collected from scabies endemic communities in northern Australia show increasing tolerance to 5% permethrin and oral ivermectin. Previous findings have implicated detoxification pathways in developing resistance to these acaricides. We investigated the contribution of Glutathione S-transferase (GST) enzymes to permethrin and ivermectin tolerance in scabies mites using biochemical and molecular approaches. Results Increased in vitro survival following permethrin exposure was observed in S. scabiei var. hominis compared to acaricide naïve mites (p < 0.0001). The addition of the GST inhibitor diethyl maleate restored in vitro permethrin susceptibility, confirming GST involvement in permethrin detoxification. Assay of GST enzymatic activity in mites demonstrated that S. scabiei var. hominis mites showed a two-fold increase in activity compared to naïve mites (p < 0.0001). Increased transcription of three different GST molecules was observed in permethrin resistant S. scabiei var. canis- mu 1 (p < 0.0001), delta 1 (p < 0.001), and delta 3 (p < 0.0001). mRNA levels of GST mu 1, delta 3 and P-glycoprotein also significantly increased in S. scabiei var. hominis mites collected from a recurrent crusted scabies patient over the course of ivermectin treatment. Conclusions These findings provide further support for the hypothesis that increased drug metabolism and efflux mediate permethrin and ivermectin resistance in scabies mites and highlight the threat of emerging acaricide resistance to the treatment of scabies worldwide. This is one of the first attempts to define specific genes involved in GST mediated acaricide resistance at the transcriptional level, and the first application of such studies to S. scabiei, a historically challenging ectoparasite. PMID:20482766

  12. Testosterone Administration Inhibits Hepcidin Transcription and is Associated with Increased Iron Incorporation into Red Blood Cells

    PubMed Central

    Guo, Wen; Bachman, Eric; Li, Michelle; Roy, Cindy N.; Blusztajn, Jerzy; Wong, Siu; Chan, Stephen Y.; Serra, Carlo; Jasuja, Ravi; Travison, Thomas G.; Muckenthaler, Martina U.; Nemeth, Elizabeta; Bhasin, Shalender

    2013-01-01

    Testosterone administration increases hemoglobin levels and has been used to treat anemia of chronic disease. Erythrocytosis is the most frequent adverse event associated with testosterone therapy of hypogonadal men, especially older men. However, the mechanisms by which testosterone increases hemoglobin remain unknown. Testosterone administration in male and female mice was associated with a greater increase in hemoglobin and hematocrit, reticulocyte count, reticulocyte hemoglobin concentration, and serum iron and transferring saturation than placebo. Testosterone downregulated hepatic hepcidin mRNA expression, upregulated renal erythropoietin mRNA expression, and increased erythropoietin levels. Testosterone-induced suppression of hepcidin expression was independent of its effects on erythropoietin or hypoxia-sensing mechanisms. Transgenic mice with liver-specific constitutive hepcidin over-expression failed to exhibit the expected increase in hemoglobin in response to testosterone administration. Testosterone upregulated splenic ferroportin expression and reduced iron retention in spleen. After intravenous administration of transferrin-bound 58Fe, the amount of 58Fe incorporated into red blood cells was significantly greater in testosterone-treated mice than in placebo-treated mice. Serum from testosterone-treated mice stimulated hemoglobin synthesis in K562 erythroleukemia cells more than that from vehicle-treated mice. Testosterone administration promoted the association of androgen receptor (AR) with Smad1 and Smad4 to reduce their binding to BMP-response elements in hepcidin promoter in the liver. Ectopic expression of AR in hepatocytes suppressed hepcidin transcription; this effect was blocked dose-dependently by AR antagonist flutamide. Testosterone did not affect hepcidin mRNA stability. Conclusion: Testosterone inhibits hepcidin transcription through its interaction with BMP-Smad signaling. Testosterone administration is associated with increased iron

  13. Stress Increases Peripheral Axon Growth and Regeneration through Glucocorticoid Receptor-Dependent Transcriptional Programs

    PubMed Central

    Alexander, Jessica K.; Madalena, Kathryn M.; Motti, Dario; Quach, Tam; Zha, Alicia; Webster Marketon, Jeanette

    2017-01-01

    Abstract Stress and glucocorticoid (GC) release are common behavioral and hormonal responses to injury or disease. In the brain, stress/GCs can alter neuron structure and function leading to cognitive impairment. Stress and GCs also exacerbate pain, but whether a corresponding change occurs in structural plasticity of sensory neurons is unknown. Here, we show that in female mice (Mus musculus) basal GC receptor (Nr3c1, also known as GR) expression in dorsal root ganglion (DRG) sensory neurons is 15-fold higher than in neurons in canonical stress-responsive brain regions (M. musculus). In response to stress or GCs, adult DRG neurite growth increases through mechanisms involving GR-dependent gene transcription. In vivo, prior exposure to an acute systemic stress increases peripheral nerve regeneration. These data have broad clinical implications and highlight the importance of stress and GCs as novel behavioral and circulating modifiers of neuronal plasticity. PMID:28828403

  14. Stress Increases Peripheral Axon Growth and Regeneration through Glucocorticoid Receptor-Dependent Transcriptional Programs.

    PubMed

    Lerch, Jessica K; Alexander, Jessica K; Madalena, Kathryn M; Motti, Dario; Quach, Tam; Dhamija, Akhil; Zha, Alicia; Gensel, John C; Webster Marketon, Jeanette; Lemmon, Vance P; Bixby, John L; Popovich, Phillip G

    2017-01-01

    Stress and glucocorticoid (GC) release are common behavioral and hormonal responses to injury or disease. In the brain, stress/GCs can alter neuron structure and function leading to cognitive impairment. Stress and GCs also exacerbate pain, but whether a corresponding change occurs in structural plasticity of sensory neurons is unknown. Here, we show that in female mice (Mus musculus) basal GC receptor (Nr3c1, also known as GR) expression in dorsal root ganglion (DRG) sensory neurons is 15-fold higher than in neurons in canonical stress-responsive brain regions (M. musculus). In response to stress or GCs, adult DRG neurite growth increases through mechanisms involving GR-dependent gene transcription. In vivo, prior exposure to an acute systemic stress increases peripheral nerve regeneration. These data have broad clinical implications and highlight the importance of stress and GCs as novel behavioral and circulating modifiers of neuronal plasticity.

  15. Transcriptional responses of uropathogenic Escherichia coli to increased environmental osmolality caused by salt or urea.

    PubMed

    Withman, Benjamin; Gunasekera, Thusitha S; Beesetty, Pavani; Agans, Richard; Paliy, Oleg

    2013-01-01

    Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infections in humans. The majority of urinary infections develop via ascending route through the urethra, where bacterial cells come in contact with human urine prior to reaching the bladder or kidneys. Since urine contains significant amounts of inorganic ions and urea, it imposes osmotic and denaturing stresses on bacterial cells. In this study, we determined the transcriptional adaptive responses of UPEC strain CFT073 to the presence of 0.3 M NaCl or 0.6 M urea in the growth medium. The cell responses to these two osmolytes were drastically different. Although most of the genes of the osmotically inducible regulon were overexpressed in medium with salt, urea failed to stimulate osmotic stress response. At the same time, UPEC colonization genes encoding type 1 and F1C fimbriae and capsule biosynthesis were transcriptionally induced in the presence of urea but did not respond to increased salt concentration. We speculate that urea can potentially be sensed by uropathogenic bacteria to initiate infection program. In addition, several molecular chaperone genes were overexpressed in the presence of urea, whereas adding NaCl to the medium led to an upregulation of a number of anaerobic metabolism pathways.

  16. Transcriptional Responses of Uropathogenic Escherichia coli to Increased Environmental Osmolality Caused by Salt or Urea

    PubMed Central

    Withman, Benjamin; Gunasekera, Thusitha S.; Beesetty, Pavani; Agans, Richard

    2013-01-01

    Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infections in humans. The majority of urinary infections develop via ascending route through the urethra, where bacterial cells come in contact with human urine prior to reaching the bladder or kidneys. Since urine contains significant amounts of inorganic ions and urea, it imposes osmotic and denaturing stresses on bacterial cells. In this study, we determined the transcriptional adaptive responses of UPEC strain CFT073 to the presence of 0.3 M NaCl or 0.6 M urea in the growth medium. The cell responses to these two osmolytes were drastically different. Although most of the genes of the osmotically inducible regulon were overexpressed in medium with salt, urea failed to stimulate osmotic stress response. At the same time, UPEC colonization genes encoding type 1 and F1C fimbriae and capsule biosynthesis were transcriptionally induced in the presence of urea but did not respond to increased salt concentration. We speculate that urea can potentially be sensed by uropathogenic bacteria to initiate infection program. In addition, several molecular chaperone genes were overexpressed in the presence of urea, whereas adding NaCl to the medium led to an upregulation of a number of anaerobic metabolism pathways. PMID:23090957

  17. O-GlcNAcylation Increases ChREBP Protein Content and Transcriptional Activity in the Liver

    PubMed Central

    Guinez, Céline; Filhoulaud, Gaëlle; Rayah-Benhamed, Fadila; Marmier, Solenne; Dubuquoy, Céline; Dentin, Renaud; Moldes, Marthe; Burnol, Anne-Françoise; Yang, Xiaoyong; Lefebvre, Tony; Girard, Jean; Postic, Catherine

    2011-01-01

    OBJECTIVE Carbohydrate-responsive element–binding protein (ChREBP) is a key transcription factor that mediates the effects of glucose on glycolytic and lipogenic genes in the liver. We have previously reported that liver-specific inhibition of ChREBP prevents hepatic steatosis in ob/ob mice by specifically decreasing lipogenic rates in vivo. To better understand the regulation of ChREBP activity in the liver, we investigated the implication of O-linked β-N-acetylglucosamine (O-GlcNAc or O-GlcNAcylation), an important glucose-dependent posttranslational modification playing multiple roles in transcription, protein stabilization, nuclear localization, and signal transduction. RESEARCH DESIGN AND METHODS O-GlcNAcylation is highly dynamic through the action of two enzymes: the O-GlcNAc transferase (OGT), which transfers the monosaccharide to serine/threonine residues on a target protein, and the O-GlcNAcase (OGA), which hydrolyses the sugar. To modulate ChREBPOG in vitro and in vivo, the OGT and OGA enzymes were overexpressed or inhibited via adenoviral approaches in mouse hepatocytes and in the liver of C57BL/6J or obese db/db mice. RESULTS Our study shows that ChREBP interacts with OGT and is subjected to O-GlcNAcylation in liver cells. O-GlcNAcylation stabilizes the ChREBP protein and increases its transcriptional activity toward its target glycolytic (L-PK) and lipogenic genes (ACC, FAS, and SCD1) when combined with an active glucose flux in vivo. Indeed, OGT overexpression significantly increased ChREBPOG in liver nuclear extracts from fed C57BL/6J mice, leading in turn to enhanced lipogenic gene expression and to excessive hepatic triglyceride deposition. In the livers of hyperglycemic obese db/db mice, ChREBPOG levels were elevated compared with controls. Interestingly, reducing ChREBPOG levels via OGA overexpression decreased lipogenic protein content (ACC, FAS), prevented hepatic steatosis, and improved the lipidic profile of OGA-treated db/db mice

  18. Global transcriptional changes of Clostridium acetobutylicum cultures with increased butanol:acetone ratios.

    PubMed

    Hönicke, Daniel; Janssen, Holger; Grimmler, Christina; Ehrenreich, Armin; Lütke-Eversloh, Tina

    2012-05-15

    Artificial electron carriers have been widely used to shift the solvent ratio toward butanol in acetone-butanol-ethanol (ABE) fermentation of solventogenic clostridia according to decreased hydrogen production. In this study, first insights on the molecular level were gained to explore the effect of methyl viologen addition to cultures of Clostridium acetobutylicum. Employing batch fermentation in mineral salts medium, the butanol:acetone ratio was successively increased from 2.3 to 12.4 on a 100-ml scale in serum bottles and from 1.4 to 16.5 on a 1300-ml scale in bioreactors, respectively. The latter cultures were used for DNA microarray analyses to provide new information on the transcriptional changes referring to methyl viologen exposure and thus, exhibit gene expression patterns according to the manipulation of the cellular redox balance. Methyl viologen-exposed cultures revealed lower expression levels of the sol operon (CAP0162-0164) and the adjacent adc gene (CAP0165) responsible for solvent formation as well as iron and sulfate transporters and the CAC0105-encoded ferredoxin. On the contrary, genes for riboflavin biosynthesis, for the butyrate/butanol metabolic pathway and genes coding for sugar transport systems were induced. Interestingly, the adhE2-encoded bifunctional NADH-dependent aldhehyde/alcohol-dehydrogenase (CAP0035) was upregulated up to more than 100-fold expression levels as compared to the control culture without methyl viologen addition. The data presented here indicate a transcriptional regulation for decreased acetone biosynthesis and the redox-dependent substitution of adhE1 (CAP0162) by adhE2.

  19. The CCAAT box in the proximal SERCA2 gene promoter regulates basal and stress-induced transcription in cardiomyocytes.

    PubMed

    Fragoso-Medina, Jorge; Rodriguez, Gabriela; Zarain-Herzberg, Angel

    2017-09-07

    The cardiac sarco/endoplasmic reticulum Ca(2+)-ATPase-2a (SERCA2a) is vital for the correct handling of calcium concentration in cardiomyocytes. Recent studies showed that the induction of endoplasmic reticulum (ER) stress (ERS) with the SERCA2 inhibitor Thapsigargin (Tg) increases the mRNA and protein levels of SERCA2a. The SERCA2 gene promoter contains an ERS response element (ERSE) at position -78 bp that is conserved among species and might transcriptionally regulate SERCA2 gene expression. However, its involvement in SERCA2 basal and calcium-mediated transcriptional activation has not been elucidated. In this work, we show that in cellular cultures of neonatal rat ventricular myocytes, the treatment with Tg or the calcium ionophore A23187 increases the SERCA2a mRNA and protein abundance, as well as the transcriptional activity of two chimeric human SERCA2 gene constructs, containing -254 and -2579 bp of 5'-regulatory region cloned in the pGL3-basic vector and transiently transfected in cultured cardiomyocytes. We found that the ERSE present in the SERCA2 proximal promoter contains a CCAAT box that is involved in basal and ERS-mediated hSERCA2 transcriptional activation. The EMSA results showed that the CCAAT box present in the ERSE recruits the NF-Y transcription factor. Additionally, by ChIP assays, we confirmed in vivo binding of NF-Y and C/EBPβ transcription factors to the SERCA2 gene proximal promoter.

  20. Increased abundance of ADAM9 transcripts in the blood is associated with tissue damage

    PubMed Central

    Rinchai, Darawan; Kewcharoenwong, Chidchamai; Kessler, Bianca; Lertmemongkolchai, Ganjana; Chaussabel, Damien

    2016-01-01

    Background: Members of the ADAM (a disintegrin and metalloprotease domain) family have emerged as critical regulators of cell-cell signaling during development and homeostasis. ADAM9 is consistently overexpressed in various human cancers, and has been shown to play an important role in tumorigenesis. However, little is known about the involvement of ADAM9 during immune-mediated processes. Results: Mining of an extensive compendium of transcriptomic datasets identified important gaps in knowledge regarding the possible role of ADAM9 in immunological homeostasis and inflammation: 1) The abundance of ADAM9 transcripts in the blood was increased in patients with acute infection but, 2) changed very little after in vitro exposure to a wide range of pathogen-associated molecular patterns (PAMPs). 3) Furthermore it was found to increase significantly in subjects as a result of tissue injury or tissue remodeling, in absence of infectious processes. Conclusions: Our findings indicate that ADAM9 may constitute a valuable biomarker for the assessment of tissue damage, especially in clinical situations where other inflammatory markers are confounded by infectious processes. PMID:27990250

  1. Methylxanthines Increase Expression of the Splicing Factor SRSF2 by Regulating Multiple Post-transcriptional Mechanisms.

    PubMed

    Shi, Jia; Pabon, Kirk; Scotto, Kathleen W

    2015-06-12

    We have previously reported that the methylxanthine caffeine increases expression of the splicing factor SRSF2, the levels of which are normally controlled by a negative autoregulatory loop. In the present study we have investigated the mechanisms by which methylxanthines induce this aberrant overexpression. RT-PCR analyses suggested little impact of caffeine on SRSF2 total mRNA levels. Instead, caffeine induced changes in the levels of SRSF2 3' UTR splice variants. Although some of these variants were substrates for nonsense-medicated decay (NMD), and could potentially have been stabilized by caffeine-mediated inhibition of NMD, down-regulation of NMD by a genetic approach was not sufficient to reproduce the phenotype. Furthermore, cell-based assays demonstrated that some of the caffeine-induced variants were intrinsically more efficiently translated than others; the addition of caffeine increased the translational efficiency of most SRSF2 transcripts. MicroRNA array analyses revealed a significant caffeine-mediated decrease in the expression of two SRSF2-targeting miRs, both of which were shown to repress translation of specific SRSF2 splice variants. These data support a complex model whereby caffeine down-regulates SRSF2-targeting microRNAs, leading to an increase in SRSF2 translation, which in turn induces SRSF2 splicing. SRSF2 splice variants are then stabilized by caffeine-mediated NMD inhibition, breaking the normal negative feedback loop and allowing the aberrant increase in SRSF2 protein levels. These findings highlight the complexity of SRSF2 gene regulation, and suggest ways in which SRSF2 expression may be dysregulated in disease.

  2. Methylxanthines Increase Expression of the Splicing Factor SRSF2 by Regulating Multiple Post-transcriptional Mechanisms*

    PubMed Central

    Shi, Jia; Pabon, Kirk; Scotto, Kathleen W.

    2015-01-01

    We have previously reported that the methylxanthine caffeine increases expression of the splicing factor SRSF2, the levels of which are normally controlled by a negative autoregulatory loop. In the present study we have investigated the mechanisms by which methylxanthines induce this aberrant overexpression. RT-PCR analyses suggested little impact of caffeine on SRSF2 total mRNA levels. Instead, caffeine induced changes in the levels of SRSF2 3′ UTR splice variants. Although some of these variants were substrates for nonsense-medicated decay (NMD), and could potentially have been stabilized by caffeine-mediated inhibition of NMD, down-regulation of NMD by a genetic approach was not sufficient to reproduce the phenotype. Furthermore, cell-based assays demonstrated that some of the caffeine-induced variants were intrinsically more efficiently translated than others; the addition of caffeine increased the translational efficiency of most SRSF2 transcripts. MicroRNA array analyses revealed a significant caffeine-mediated decrease in the expression of two SRSF2-targeting miRs, both of which were shown to repress translation of specific SRSF2 splice variants. These data support a complex model whereby caffeine down-regulates SRSF2-targeting microRNAs, leading to an increase in SRSF2 translation, which in turn induces SRSF2 splicing. SRSF2 splice variants are then stabilized by caffeine-mediated NMD inhibition, breaking the normal negative feedback loop and allowing the aberrant increase in SRSF2 protein levels. These findings highlight the complexity of SRSF2 gene regulation, and suggest ways in which SRSF2 expression may be dysregulated in disease. PMID:25818199

  3. Post-transcriptional regulation tends to attenuate the mRNA noise and to increase the mRNA gain

    NASA Astrophysics Data System (ADS)

    Shi, Changhong; Wang, Shuqiang; Zhou, Tianshou; Jiang, Yiguo

    2015-10-01

    Post-transcriptional regulation is ubiquitous in prokaryotic and eukaryotic cells, but how it impacts gene expression remains to be fully explored. Here, we analyze a simple gene model in which we assume that mRNAs are produced in a constitutive manner but are regulated post-transcriptionally by a decapping enzyme that switches between the active state and the inactive state. We derive the analytical mRNA distribution governed by a chemical master equation, which can be well used to analyze the mechanism of how post-transcription regulation influences the mRNA expression level including the mRNA noise. We demonstrate that the mean mRNA level in the stochastic case is always higher than that in the deterministic case due to the stochastic effect of the enzyme, but the size of the increased part depends mainly on the switching rates between two enzyme states. More interesting is that we find that in contrast to transcriptional regulation, post-transcriptional regulation tends to attenuate noise in mRNA. Our results provide insight into the role of post-transcriptional regulation in controlling the transcriptional noise.

  4. Post-transcriptional regulation tends to attenuate the mRNA noise and to increase the mRNA gain.

    PubMed

    Shi, Changhong; Wang, Shuqiang; Zhou, Tianshou; Jiang, Yiguo

    2015-08-12

    Post-transcriptional regulation is ubiquitous in prokaryotic and eukaryotic cells, but how it impacts gene expression remains to be fully explored. Here, we analyze a simple gene model in which we assume that mRNAs are produced in a constitutive manner but are regulated post-transcriptionally by a decapping enzyme that switches between the active state and the inactive state. We derive the analytical mRNA distribution governed by a chemical master equation, which can be well used to analyze the mechanism of how post-transcription regulation influences the mRNA expression level including the mRNA noise. We demonstrate that the mean mRNA level in the stochastic case is always higher than that in the deterministic case due to the stochastic effect of the enzyme, but the size of the increased part depends mainly on the switching rates between two enzyme states. More interesting is that we find that in contrast to transcriptional regulation, post-transcriptional regulation tends to attenuate noise in mRNA. Our results provide insight into the role of post-transcriptional regulation in controlling the transcriptional noise.

  5. The transcription factor regulatory factor X1 increases the expression of neuronal glutamate transporter type 3.

    PubMed

    Ma, Kaiwen; Zheng, Shuqiu; Zuo, Zhiyi

    2006-07-28

    Glutamate transporters (excitatory amino acid transporters, EAAT) play an important role in maintaining extracellular glutamate homeostasis and regulating glutamate neurotransmission. However, very few studies have investigated the regulation of EAAT expression. A binding sequence for the regulatory factor X1 (RFX1) exists in the promoter region of the gene encoding for EAAT3, a neuronal EAAT, but not in the promoter regions of the genes encoding for EAAT1 and EAAT2, two glial EAATs. RFX proteins are transcription factors binding to X-boxes of DNA sequences. Although RFX proteins are necessary for the normal function of sensory neurons in Caenorhabditis elegans, their roles in the mammalian brain are not known. We showed that RFX1 increased EAAT3 expression and activity in C6 glioma cells. RFX1 binding complexes were found in the nuclear extracts of C6 cells. The activity of EAAT3 promoter as measured by luciferase reporter activity was increased by RFX1 in C6 cells and the neuron-like SH-SY5Y cells. However, RFX1 did not change the expression of EAAT2 proteins in the NRK52E cells. RFX1 proteins were expressed in the neurons of rat brain. A high expression level of RFX1 proteins was found in the neurons of cerebral cortex and Purkinje cells. Knockdown of the RFX1 expression by RFX1 antisense oligonucleotides decreased EAAT3 expression in rat cortical neurons in culture. These results suggest that RFX1 enhances the activity of EAAT3 promoter to increase the expression of EAAT3 proteins. This study provides initial evidence for the regulation of gene expression in the nervous cells by RFX1.

  6. Transcript Polymorphism Rates in Soybean Seed Tissue Are Increased in a Single Transformant of Glycine max

    PubMed Central

    Whaley, Adam M.; Schlueter, Jessica A.; Piller, Kenneth J.; Bost, Kenneth L.

    2016-01-01

    Transgenic crops have been utilized for decades to enhance agriculture and more recently have been applied as bioreactors for manufacturing pharmaceuticals. Recently, we investigated the gene expression profiles of several in-house transgenic soybean events, finding one transformant group to be consistently different from our controls. In the present study, we examined polymorphisms and sequence variations in the exomes of the same transgenic soybean events. We found that the previously dissimilar soybean line also exhibited markedly increased levels of polymorphisms within mRNA transcripts from seed tissue, many of which are classified as gene expression modifiers. The results from this work will direct future investigations to examine novel SNPs controlling traits of great interest for breeding and improving transgenic soybean crops. Further, this study marks the first work to investigate SNP rates in transgenic soybean seed tissues and demonstrates that while transgenesis may induce abundant unanticipated changes in gene expression and nucleotide variation, phenotypes and overall health of the plants examined remained unaltered. PMID:28025595

  7. Cocaine- and Amphetamine-regulated Transcript (CART) Protects Beta Cells against Glucotoxicity and Increases Cell Proliferation*

    PubMed Central

    Sathanoori, Ramasri; Olde, Björn; Erlinge, David; Göransson, Olga; Wierup, Nils

    2013-01-01

    Cocaine- and amphetamine-regulated transcript (CART) is an islet peptide that promotes glucose-stimulated insulin secretion in beta cells via cAMP/PKA-dependent pathways. In addition, CART is a regulator of neuronal survival. In this study, we examined the effect of exogenous CART 55–102 on beta cell viability and dissected its signaling mechanisms. Evaluation of DNA fragmentation and chromatin condensation revealed that CART 55–102 reduced glucotoxicity-induced apoptosis in both INS-1 (832/13) cells and isolated rat islets. Glucotoxicity in INS-1 (832/13) cells also caused a 50% reduction of endogenous CART protein. We show that CART increased proliferation in INS-1 (832/13) cells, an effect that was blocked by PKA, PKB, and MEK1 inhibitors. In addition, CART induced phosphorylation of CREB, IRS, PKB, FoxO1, p44/42 MAPK, and p90RSK in INS-1 (832/13) cells and isolated rat islets, all key mediators of cell survival and proliferation. Thus, we demonstrate that CART 55-102 protects beta cells against glucotoxicity and promotes proliferation. Taken together our data point to the potential use of CART in therapeutic interventions targeted at enhancing functional beta cell mass and long-term insulin secretion in T2D. PMID:23250745

  8. Increased Transcript Complexity in Genes Associated with Chronic Obstructive Pulmonary Disease

    PubMed Central

    Lackey, Lela; McArthur, Evonne; Laederach, Alain

    2015-01-01

    Genome-wide association studies aim to correlate genotype with phenotype. Many common diseases including Type II diabetes, Alzheimer’s, Parkinson’s and Chronic Obstructive Pulmonary Disease (COPD) are complex genetic traits with hundreds of different loci that are associated with varied disease risk. Identifying common features in the genes associated with each disease remains a challenge. Furthermore, the role of post-transcriptional regulation, and in particular alternative splicing, is still poorly understood in most multigenic diseases. We therefore compiled comprehensive lists of genes associated with Type II diabetes, Alzheimer’s, Parkinson’s and COPD in an attempt to identify common features of their corresponding mRNA transcripts within each gene set. The SERPINA1 gene is a well-recognized genetic risk factor of COPD and it produces 11 transcript variants, which is exceptional for a human gene. This led us to hypothesize that other genes associated with COPD, and complex disorders in general, are highly transcriptionally diverse. We found that COPD-associated genes have a statistically significant enrichment in transcript complexity stemming from a disproportionately high level of alternative splicing, however, Type II Diabetes, Alzheimer’s and Parkinson’s disease genes were not significantly enriched. We also identified a subset of transcriptionally complex COPD-associated genes (~40%) that are differentially expressed between mild, moderate and severe COPD. Although the genes associated with other lung diseases are not extensively documented, we found preliminary data that idiopathic pulmonary disease genes, but not cystic fibrosis modulators, are also more transcriptionally complex. Interestingly, complex COPD transcripts are more often the product of alternative acceptor site usage. To verify the biological importance of these alternative transcripts, we used RNA-sequencing analyses to determine that COPD-associated genes are frequently

  9. Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts.

    PubMed

    Ferizi, Mehrije; Aneja, Manish K; Balmayor, Elizabeth R; Badieyan, Zohreh Sadat; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

    2016-12-15

    Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5'- and 3'-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5'-UTR and/or downstream 3'-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs.

  10. Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

    PubMed Central

    Ferizi, Mehrije; Aneja, Manish K.; Balmayor, Elizabeth R.; Badieyan, Zohreh Sadat; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

    2016-01-01

    Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5′- and 3′-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5′-UTR and/or downstream 3′-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs. PMID:27974853

  11. Pathway-Focused Arrays Reveal Increased Matrix Metalloproteinase-7 (Matrilysin) Transcription in Trachomatous Trichiasis

    PubMed Central

    Jeffries, David; Pattison, Michael; Korr, Gerit; Gall, Alevtina; Joof, Hassan; Manjang, Ahmed; Burton, Matthew J.; Mabey, David C. W.; Bailey, Robin L.

    2010-01-01

    Purpose. Several genes that are associated with protection from or susceptibility to trachomatous trichiasis (TT) have been identified through genetic association studies. Yet there have been few studies in which gene expression profiles were assessed in TT cases and disease-free controls. The purpose was to identify genes that are differentially expressed in the upper tarsal conjunctiva of subjects with TT. Method. Pathway-focused gene arrays were used to screen conjunctival RNA expression of 226 gene transcripts of interest. The screening was followed by validation of differentially expressed genes by qRT-PCR on an independent set of samples. Three different techniques were then used to test for quantitative differences in the recovered conjunctival protein fraction. Results. Focused arrays identified a set of 13 differentially expressed genes. Validation by qRT-PCR confirmed differential expression in four of these genes (COL1A1, COL7A1, MMP7, and TLR6). Increased expression of MMP7 was the only consistent differentially regulated gene in the conjunctival samples of trichiasis subjects. MMP7 was present in isolated conjunctival proteins and in the tissue culture supernatants of peripheral blood lymphocytes after stimulation. Conclusions. There is an imbalance in extracellular matrix turnover with minimal contribution of adaptive immune responses at this stage of trichiasis. There was little evidence of broad differential expression in genes characteristic of polar responses of adaptive T cells or macrophages. The control of the MMP7 response and its activity appears significant in the fibrotic changes observed in TT. PMID:20375326

  12. Use of Transcriptional Control to Increase Secretion of Heterologous Proteins in T3S Systems.

    PubMed

    Metcalf, Kevin J; Tullman-Ercek, Danielle

    2017-01-01

    Heterologous proteins can be produced in a bacterial host and purified from the cellular constituents. Secretion of the protein of interest to the extracellular space simplifies the purification process and is thought to alleviate toxicity problems associated with intracellular accumulation of the protein of interest. In this protocol, we describe a strategy to engineer protein secretion in a bacterial culture using transcriptional control. The transcription factor HilA is inducibly produced to control production of the secretion machine, and in turn signals the production and secretion of a protein of interest. This allows for high titer of secreted protein in optimized culturing conditions and the effect is observed with all proteins tested.

  13. Cyclic AMP-responsive expression of the surfactant protein-A gene is mediated by increased DNA binding and transcriptional activity of thyroid transcription factor-1.

    PubMed

    Li, J; Gao, E; Mendelson, C R

    1998-02-20

    Surfactant protein (SP)-A gene transcription is stimulated by factors that increase cyclic AMP. In the present study, we observed that three thyroid transcription factor-1 (TTF-1) binding elements (TBEs) located within a 255 base pair region flanking the 5'-end of the baboon SP-A2 (bSP-A2) gene are required for maximal cyclic AMP induction of bSP-A2 promoter activity. We found that TTF-1 DNA binding activity was increased in nuclear extracts of pulmonary type II cells cultured in the presence of cyclic AMP. By contrast, the levels of immunoreactive TTF-1 protein were similar in nuclear extracts of control and cyclic AMP-treated type II cells. The incorporation of [32P]orthophosphate into immunoprecipitated TTF-1 protein also was markedly increased by cyclic AMP treatment. Moreover, exposure of nuclear extracts from cyclic AMP-treated type II cells either to potato acid phosphatase or alkaline phosphatase abolished the cyclic AMP-induced increase in TTF-1 DNA-binding activity. Interestingly, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), known to activate protein kinase C, also enhanced incorporation of [32P]orthophosphate into TTF-1 protein; however, the DNA binding activity of TTF-1 was decreased in nuclear extracts of TPA-treated type II cells. Expression vectors encoding TTF-1 and the catalytic subunit of protein kinase A (PKA-cat) were cotransfected into A549 lung adenocarcinoma cells together with an SPA:human growth hormone fusion gene (255 base pairs of 5'-flanking DNA from the baboon SP-A2 gene linked to human growth hormone, as reporter) containing TBEs, or with a reporter gene construct containing three tandem TBEs fused upstream of the bSP-A2 gene TATA box and the transcription initiation site. Coexpression of TTF-1 and PKA-cat increased fusion gene expression 3-4-fold as compared with expression of TTF-1 in the absence of PKA-cat. Moreover, the transcriptional activity of TTF-1 was suppressed by cotransfection of a dominant negative form

  14. CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels.

    PubMed

    Rocha-Perugini, Vera; Suárez, Henar; Álvarez, Susana; López-Martín, Soraya; Lenzi, Gina M; Vences-Catalán, Felipe; Levy, Shoshana; Kim, Baek; Muñoz-Fernández, María A; Sánchez-Madrid, Francisco; Yáñez-Mó, Maria

    2017-09-04

    In this study, we report that the tetraspanin CD81 enhances human immunodeficiency virus (HIV)-1 reverse transcription in HIV-1-infected cells. This is enabled by the direct interaction of CD81 with the deoxynucleoside triphosphate phosphohydrolase SAMHD1. This interaction prevents endosomal accumulation and favours the proteasome-dependent degradation of SAMHD1. Consequently, CD81 depletion results in SAMHD1 increased expression, decreasing the availability of deoxynucleoside triphosphates (dNTP) and thus HIV-1 reverse transcription. Conversely, CD81 overexpression, but not the expression of a CD81 carboxy (C)-terminal deletion mutant, increases cellular dNTP content and HIV-1 reverse transcription. Our results demonstrate that the interaction of CD81 with SAMHD1 controls the metabolic rate of HIV-1 replication by tuning the availability of building blocks for reverse transcription, namely dNTPs. Together with its role in HIV-1 entry and budding into host cells, the data herein indicate that HIV-1 uses CD81 as a rheostat that controls different stages of the infection.CD81 is shown to interact with SAMHD1 and lead to its proteasomal degradation, thereby impacting dNTP availability and enhancing HIV-1 reverse transcription in primary human T cells.

  15. Leukemia patient-derived lymphoblastoid cell lines exhibit increased induction of leukemia-associated transcripts following high-dose irradiation.

    PubMed

    Spencer, A; Granter, N

    1999-09-01

    Improvement in diagnostic cytogenetic techniques has led to the recognition of an increasing number of leukemia-associated chromosomal translocations and inversions. These genetic lesions frequently are associated with the disruption of putative transcription factors and the production of hybrid transcripts that are implicated in leukemogenesis. Epidemiologic evidence suggests that some, but not all, individuals with a history of gamma-irradiation exposure are at increased risk of developing chronic myeloid leukemia (CML). CML is characterized by the Philadelphia chromosome and transcription of the resulting hybrid BCR-ABL gene. Utilizing the leukemia-associated BCR-ABL p210 transcript as a marker, we sought differences in the induction of illegitimate genetic recombination following high-dose gamma-irradiation of karyotypically normal lymphoblastoid cell lines (LCL) derived from individuals with and without a history of myeloid leukemias. Six LCL [4 leukemia patient derived [2 acute myeloid leukemia and 2 CML] and 2 from normal individuals were analyzed with reverse transcriptase polymerase chain reaction for BCR-ABL under stringent conditions following exposure to 0, 50, or 100 Gy of LET gamma-irradiation delivered via a Varian linear accelerator at 4 MV. Transcripts identical to disease-associated b2a2 and b3a2 transcripts were detected both spontaneously (background illegitimate genetic recombination) and following gamma-irradiation. Background BCR-ABL positivity was demonstrable in 4 of the 6 LCL, with no significant difference in detection between leukemic- and nonleukemic-derived LCL. Overall, increasing gamma-irradiation dose resulted in an increased frequency of BCR-ABL transcript detection (0 Gy vs 50 Gy vs 100 Gy,p = 0.0023, Chi-square test). Within the leukemic- but not the nonleukemic-derived LCL there was significantly greater BCR-ABL positivity after gamma-irradiation compared to unirradiated equivalents. Furthermore, the BCR-ABL positivity of both

  16. RNA polymerase II forms transcription networks in rye and Arabidopsis nuclei and its amount increases with endopolyploidy.

    PubMed

    Schubert, Veit

    2014-01-01

    RNA polymerase II (RNAPII) is responsible for the transcription of most eukaryotic genes. In mammalian nuclei, RNAPII is mainly localized in relatively few distinct transcription factories. In this study--applying super-resolution microscopy--it is shown that in plants, inactive (non-phosphorylated) and active (phosphorylated) RNAPII modifications compose distinct 'transcription networks' within the euchromatin. These reticulate structures sometimes attach to each other, but they are absent from heterochromatin and nucleoli. The global RNAPII distribution within nuclei is not influenced by interphase chromatin organization such as Rabl (rye) versus non-Rabl (Arabidopsis thaliana) orientation. Replication of sister chromatids without cell division causes endopolyploidy, a phenomenon widespread in plants and animals. Endopolyploidy raises the number of gene copies per nucleus. Here, it is shown that the amounts of active and inactive RNAPII enzymes in differentiated 2-32C leaf nuclei of A. thaliana proportionally increase with rising endopolyploidy. Thus, increasing the transcriptional activity of cells and tissues seems to be an important function of endopolyploidy.

  17. Stabilised DNA secondary structures with increasing transcription localise hypermutable bases for somatic hypermutation in IGHV3-23.

    PubMed

    Duvvuri, Bhargavi; Duvvuri, Venkata R; Wu, Jianhong; Wu, Gillian E

    2012-07-01

    Somatic hypermutation (SHM) mediated by activation-induced cytidine deaminase (AID) is a transcription-coupled mechanism most responsible for generating high affinity antibodies. An issue remaining enigmatic in SHM is how AID is preferentially targeted during transcription to hypermutable bases in its substrates (WRC motifs) on both DNA strands. AID targets only single stranded DNA. By modelling the dynamical behaviour of IGHV3-23 DNA, a commonly used human variable gene segment, we observed that hypermutable bases on the non-transcribed strand are paired whereas those on transcribed strand are mostly unpaired. Hypermutable bases (both paired and unpaired) are made accessible to AID in stabilised secondary structures formed with increasing transcription levels. This observation provides a rationale for the hypermutable bases on both the strands of DNA being targeted to a similar extent despite having differences in unpairedness. We propose that increasing transcription and RNAP II stalling resulting in the formation and stabilisation of stem-loop structures with AID hotspots in negatively supercoiled region can localise the hypermutable bases of both strands of DNA, to AID-mediated SHM.

  18. Increased epigenetic alterations at the promoters of transcriptional regulators following inadequate maternal gestational weight gain

    PubMed Central

    Kawai, Tomoko; Yamada, Takahiro; Abe, Kosei; Okamura, Kohji; Kamura, Hiromi; Akaishi, Rina; Minakami, Hisanori; Nakabayashi, Kazuhiko; Hata, Kenichiro

    2015-01-01

    Epigenetic modifications are thought to serve as a memory of exposure to in utero environments. However, few human studies have investigated the associations between maternal nutritional conditions during pregnancy and epigenetic alterations in offspring. In this study, we report genome-wide methylation profiles for 33 postpartum placentas from pregnancies of normal and foetal growth restriction with various extents of maternal gestational weight gain. Epigenetic alterations accumulate in the placenta under adverse in utero environments, as shown by application of Smirnov-Grubbs’ outlier test. Moreover, hypermethylation occurs frequently at the promoter regions of transcriptional regulator genes, including polycomb targets and zinc-finger genes, as shown by annotations of the genomic and functional features of loci with altered DNA methylation. Aberrant epigenetic modifications at such developmental regulator loci, if occurring in foetuses as well, will elevate the risk of developing various diseases, including metabolic and mental disorders, later in life. PMID:26415774

  19. Insulin-activated Elk-1 recruits the TIP60/NuA4 complex to increase prolactin gene transcription.

    PubMed

    Mahajan, Muktar A; Stanley, Frederick M

    2014-01-25

    Insulin increases prolactin gene expression in GH4 cells through phosphorylation of Elk-1 (Jacob and Stanley, 2001). We preformed a reverse two-hybrid screen using Elk-1-B42 as bait to identify proteins from GH4 cells that might serve as co-activators or co-repressors for insulin-increased prolactin gene expression. A number of the components of the TIP60/NuA4 complex interacted with Elk-1 suggesting that Elk-1 might activate transcription by recruiting the TIP60 chromatin-remodeling complex to the prolactin promoter. Inhibition of insulin-increased prolactin-luciferase expression by wild type and mutant adenovirus E1A protein provided physiological context for these yeast studies. Inhibition of histone deacetylases dramatically increased both basal and insulin-increased prolactin gene transcription. Co-immune precipitation experiments demonstrated Elk-1 and TIP60 associate in vitro. Transient or stable expression of TIP60 activated insulin-increased prolactin gene expression while a mutated TIP60 blocked insulin-increased prolactin gene expression. Analysis of the prolactin mRNA by quantitative RT-PCR showed that insulin-increased prolactin mRNA accumulation and that this was inhibited in GH4 cells that stably expressed mutant TIP60. Finally, ChIP experiments demonstrate the insulin-dependent occupancy of the prolactin promoter by Elk-1 and TIP60. Our studies suggest that insulin activates prolactin gene transcription by activating Elk-1 that recruits the NuA4 complex to the promoter.

  20. Forkhead transcription factor FOXO3a levels are increased in Huntington disease because of overactivated positive autofeedback loop.

    PubMed

    Kannike, Kaja; Sepp, Mari; Zuccato, Chiara; Cattaneo, Elena; Timmusk, Tõnis

    2014-11-21

    Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an increased number of CAG repeats in the HTT gene coding for huntingtin. Decreased neurotrophic support and increased mitochondrial and excitotoxic stress have been reported in HD striatal and cortical neurons. The members of the class O forkhead (FOXO) transcription factor family, including FOXO3a, act as sensor proteins that are activated upon decreased survival signals and/or increased cellular stress. Using immunocytochemical screening in mouse striatal Hdh(7/7) (wild type), Hdh(7/109) (heterozygous for HD mutation), and Hdh(109/109) (homozygous for HD mutation) cells, we identified FOXO3a as a differentially regulated transcription factor in HD. We report increased nuclear FOXO3a levels in mutant Hdh cells. Additionally, we show that treatment with mitochondrial toxin 3-nitropropionic acid results in enhanced nuclear localization of FOXO3a in wild type Hdh(7/7) cells and in rat primary cortical neurons. Furthermore, mRNA levels of Foxo3a are increased in mutant Hdh cells compared with wild type cells and in 3-nitropropionic acid-treated primary neurons compared with untreated neurons. A similar increase was observed in the cortex of R6/2 mice and HD patient post-mortem caudate tissue compared with controls. Using chromatin immunoprecipitation and reporter assays, we demonstrate that FOXO3a regulates its own transcription by binding to the conserved response element in Foxo3a promoter. Altogether, the findings of this study suggest that FOXO3a levels are increased in HD cells as a result of overactive positive feedback loop.

  1. Forkhead Transcription Factor FOXO3a Levels Are Increased in Huntington Disease Because of Overactivated Positive Autofeedback Loop*

    PubMed Central

    Kannike, Kaja; Sepp, Mari; Zuccato, Chiara; Cattaneo, Elena; Timmusk, Tõnis

    2014-01-01

    Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an increased number of CAG repeats in the HTT gene coding for huntingtin. Decreased neurotrophic support and increased mitochondrial and excitotoxic stress have been reported in HD striatal and cortical neurons. The members of the class O forkhead (FOXO) transcription factor family, including FOXO3a, act as sensor proteins that are activated upon decreased survival signals and/or increased cellular stress. Using immunocytochemical screening in mouse striatal Hdh7/7 (wild type), Hdh7/109 (heterozygous for HD mutation), and Hdh109/109 (homozygous for HD mutation) cells, we identified FOXO3a as a differentially regulated transcription factor in HD. We report increased nuclear FOXO3a levels in mutant Hdh cells. Additionally, we show that treatment with mitochondrial toxin 3-nitropropionic acid results in enhanced nuclear localization of FOXO3a in wild type Hdh7/7 cells and in rat primary cortical neurons. Furthermore, mRNA levels of Foxo3a are increased in mutant Hdh cells compared with wild type cells and in 3-nitropropionic acid-treated primary neurons compared with untreated neurons. A similar increase was observed in the cortex of R6/2 mice and HD patient post-mortem caudate tissue compared with controls. Using chromatin immunoprecipitation and reporter assays, we demonstrate that FOXO3a regulates its own transcription by binding to the conserved response element in Foxo3a promoter. Altogether, the findings of this study suggest that FOXO3a levels are increased in HD cells as a result of overactive positive feedback loop. PMID:25271153

  2. Ribosomal DNA transcription in the dorsal raphe nucleus is increased in residual but not in paranoid schizophrenia.

    PubMed

    Krzyżanowska, Marta; Steiner, Johann; Brisch, Ralf; Mawrin, Christian; Busse, Stefan; Braun, Katharina; Jankowski, Zbigniew; Bernstein, Hans-Gert; Bogerts, Bernhard; Gos, Tomasz

    2015-03-01

    The central serotonergic system is implicated in the pathogenesis of schizophrenia, where the imbalance between dopamine, serotonin and glutamate plays a key pathophysiological role. The dorsal raphe nucleus (DRN) is the main source of serotonergic innervation of forebrain limbic structures disturbed in schizophrenia patients. The study was carried out on paraffin-embedded brains from 17 (8 paranoid and 9 residual) schizophrenia patients and 28 matched controls without mental disorders. The transcriptional activity of ribosomal DNA (rDNA) in DRN neurons was evaluated by the AgNOR silver-staining method. An increased rDNA transcriptional activity was found in schizophrenia patients in the cumulative analysis of all DRN subnuclei (t test, P = 0.02). Further subgroup analysis revealed that it was an effect specific for residual schizophrenia versus paranoid schizophrenia or control groups (ANOVA, P = 0.002). This effect was confounded neither by suicide nor by antipsychotic medication. Our findings suggest that increased activity of rDNA in DRN neurons is a distinct phenomenon in schizophrenia, particularly in residual patients. An activation of the rDNA transcription in DRN neurons may represent a compensatory mechanism to overcome the previously described prefrontal serotonergic hypofunction in this diagnostic subgroup.

  3. [ITF increases the transcriptional activity of ITF promoter via the JAK-STAT3 signal transduction pathway].

    PubMed

    Sun, Yong; LE, Juan; Chen, Baojun; Pan, Xiaofeng; Zhang, Fang

    2014-11-01

    To investigate the eff ect of intestinal trefoil factor (ITF) on the transcriptional activity of ITF promoter and to explore the regulatory mechanism of Janus kinase/signal transducers and activators of transcription (JAK/STAT) on ITF promoter. The 5' flanking sequence of the ITF gene was cloned from human whole blood genomic DNA by PCR. ITF promoter fragment was cloned and inserted into the pGL3-Basic vector to construct recombinant vector. ITF promoter vector was stimulated with ITF at various concentrations and the luciferase activity was measured. The JAK-STAT3 signal transduction pathway was then blocked by a specific inhibitor AG490 to determine the signal pathway involved in ITF promoter activity. Restriction endonuclease analysis and DNA sequencing confirmed that the recombinant plasmid, containing ITF promoter, was constructed successfully. After transient transfection, the activity of ITF promoter was increased significantly in the presence of ITF (P<0.05). Blockage of the JAK-STAT3 signal transduction pathway with AG490 significantly reduced the ITF promoter activity (P<0.05). ITF increases the transcriptional activity of ITF promoter via the JAK-STAT3 signal transduction pathway.

  4. Transcript and metabolite alterations increase ganoderic acid content in Ganoderma lucidum using acetic acid as an inducer.

    PubMed

    Ren, Ang; Li, Xiong-Biao; Miao, Zhi-Gang; Shi, Liang; Jaing, Ai-Liang; Zhao, Ming-Wen

    2014-12-01

    Acetic acid at 5-8 mM increased ganoderic acid (GA) accumulation in Ganoderma lucidum. After optimization by the response surface methodology, the GA content reached 5.5/100 mg dry weight, an increase of 105% compared with the control. The intermediate metabolites of GA biosynthesis, lanosterol and squalene also increased to 47 and 15.8 μg/g dry weight, respectively, in response to acetic acid. Acetic acid significantly induced transcription levels of sqs, lano, hmgs and cyp51 in the GA biosynthesis pathway. An acetic acid-unregulated acetyl coenzyme A synthase (acs) gene was selected from ten candidate homologous acs genes. The results indicate that acetic acid alters the expression of genes related to acetic acid assimilation and increases GA biosynthesis and the metabolic levels of lanosterol, squalene and GA-a, thereby resulting in GA accumulation.

  5. Transcriptional Slippage and RNA Editing Increase the Diversity of Transcripts in Chloroplasts: Insight from Deep Sequencing of Vigna radiata Genome and Transcriptome.

    PubMed

    Lin, Ching-Ping; Ko, Chia-Yun; Kuo, Ching-I; Liu, Mao-Sen; Schafleitner, Roland; Chen, Long-Fang Oliver

    2015-01-01

    We performed deep sequencing of the nuclear and organellar genomes of three mungbean genotypes: Vigna radiata ssp. sublobata TC1966, V. radiata var. radiata NM92 and the recombinant inbred line RIL59 derived from a cross between TC1966 and NM92. Moreover, we performed deep sequencing of the RIL59 transcriptome to investigate transcript variability. The mungbean chloroplast genome has a quadripartite structure including a pair of inverted repeats separated by two single copy regions. A total of 213 simple sequence repeats were identified in the chloroplast genomes of NM92 and RIL59; 78 single nucleotide variants and nine indels were discovered in comparing the chloroplast genomes of TC1966 and NM92. Analysis of the mungbean chloroplast transcriptome revealed mRNAs that were affected by transcriptional slippage and RNA editing. Transcriptional slippage frequency was positively correlated with the length of simple sequence repeats of the mungbean chloroplast genome (R2=0.9911). In total, 41 C-to-U editing sites were found in 23 chloroplast genes and in one intergenic spacer. No editing site that swapped U to C was found. A combination of bioinformatics and experimental methods revealed that the plastid-encoded RNA polymerase-transcribed genes psbF and ndhA are affected by transcriptional slippage in mungbean and in main lineages of land plants, including three dicots (Glycine max, Brassica rapa, and Nicotiana tabacum), two monocots (Oryza sativa and Zea mays), two gymnosperms (Pinus taeda and Ginkgo biloba) and one moss (Physcomitrella patens). Transcript analysis of the rps2 gene showed that transcriptional slippage could affect transcripts at single sequence repeat regions with poly-A runs. It showed that transcriptional slippage together with incomplete RNA editing may cause sequence diversity of transcripts in chloroplasts of land plants.

  6. [VARIOUS ALLELES OF HSF HEAT-SHOCK TRANSCRIPTION FACTOR OF DROSOPHILA MELANOGASTER INCREASE VIABILITY OF ITS CARRIERS IN UNFAVORABLE ENVIRONMENTS].

    PubMed

    Weisman, N Ya; Evgen'ev, M B; Golubovsky, M D

    2015-01-01

    We found increased viability in heterozygous carriers of hsf heat shock transcription factor n comparison with wild type. The effect depends on temperature, sex and direction of crosses. Viability effect is more evident in conditions of soft temperature stress. The males are more sensitive. The maternal effect is observed: if hsf*allele came from mother, the viability effect is stronger. The survival curves of heterozygotes on hsf-1 and hsf-4 alleles are similar in spite of HSF-4 protein is slightly active on normal temperature.

  7. Geminin deletion increases the number of fetal hematopoietic stem cells by affecting the expression of key transcription factors.

    PubMed

    Karamitros, Dimitris; Patmanidi, Alexandra L; Kotantaki, Panoraia; Potocnik, Alexandre J; Bähr-Ivacevic, Tomi; Benes, Vladimir; Lygerou, Zoi; Kioussis, Dimitris; Taraviras, Stavros

    2015-01-01

    Balancing stem cell self-renewal and initiation of lineage specification programs is essential for the development and homeostasis of the hematopoietic system. We have specifically ablated geminin in the developing murine hematopoietic system and observed profound defects in the generation of mature blood cells, leading to embryonic lethality. Hematopoietic stem cells (HSCs) accumulated in the fetal liver following geminin ablation, while committed progenitors were reduced. Genome-wide transcriptome analysis identified key HSC transcription factors as being upregulated upon geminin deletion, revealing a gene network linked with geminin that controls fetal hematopoiesis. In order to obtain mechanistic insight into the ability of geminin to regulate transcription, we examined Hoxa9 as an example of a key gene in definitive hematopoiesis. We demonstrate that in human K562 cells geminin is associated with HOXA9 regulatory elements and its absence increases HOXA9 transcription similarly to that observed in vivo. Moreover, silencing geminin reduced recruitment of the PRC2 component SUZ12 to the HOXA9 locus and resulted in an increase in RNA polymerase II recruitment and H3K4 trimethylation (H3K4me3), whereas the repressive marks H3K9me3 and H3K27me3 were reduced. The chromatin landscape was also modified at the regulatory regions of HOXA10 and GATA1. K562 cells showed a reduced ability to differentiate to erythrocytes and megakaryocytes upon geminin silencing. Our data suggest that geminin is indispensable for fetal hematopoiesis and regulates the generation of a physiological pool of stem and progenitor cells in the fetal hematopoietic system.

  8. Recombinant Mitochondrial Transcription Factor A with N-terminal Mitochondrial Transduction Domain Increases Respiration and Mitochondrial Gene Expression

    PubMed Central

    Iyer, Shilpa; Thomas, Ravindar R.; Portell, Francisco R.; Dunham, Lisa D.; Quigley, Caitlin K.; Bennett, James P.

    2009-01-01

    We developed a scalable procedure to produce human mitochondrial transcription factor A (TFAM) modified with an N-terminal protein transduction domain (PTD) and mitochondrial localization signal (MLS) that allow it to cross membranes and enter mitochondria through its “mitochondrial transduction domain” (MTD=PTD+MLS). Alexa488-labeled MTD-TFAM rapidly entered the mitochondrial compartment of cybrid cells carrying the G11778A LHON mutation. MTD-TFAM reversibly increased respiration and levels of respiratory proteins. In vivo treatment of mice with MTD-TFAM increased motor endurance and complex I-driven respiration in mitochondria from brain and skeletal muscle. MTD-TFAM increases mitochondrial bioenergetics and holds promise for treatment of mitochondrial diseases involving deficiencies of energy production. PMID:19460293

  9. Dioxin Toxicity In Vivo Results from an Increase in the Dioxin-Independent Transcriptional Activity of the Aryl Hydrocarbon Receptor

    PubMed Central

    Céspedes, Miguel Angel; Galindo, Maximo Ibo; Couso, Juan Pablo

    2010-01-01

    The Aryl hydrocarbon receptor (Ahr) is the nuclear receptor mediating the toxicity of dioxins -widespread and persistent pollutants whose toxic effects include tumor promotion, teratogenesis, wasting syndrome and chloracne. Elimination of Ahr in mice eliminates dioxin toxicity but also produces adverse effects, some seemingly unrelated to dioxin. Thus the relationship between the toxic and dioxin-independent functions of Ahr is not clear, which hampers understanding and treatment of dioxin toxicity. Here we develop a Drosophila model to show that dioxin actually increases the in vivo dioxin-independent activity of Ahr. This hyperactivation resembles the effects caused by an increase in the amount of its dimerisation partner Ahr nuclear translocator (Arnt) and entails an increased transcriptional potency of Ahr, in addition to the previously described effect on nuclear translocation. Thus the two apparently different functions of Ahr, dioxin-mediated and dioxin-independent, are in fact two different levels (hyperactivated and basal, respectively) of a single function. PMID:21079739

  10. Coffee modulates transcription factor Nrf2 and highly increases the activity of antioxidant enzymes in rats.

    PubMed

    Vicente, Silvio J V; Ishimoto, Emília Y; Torres, Elizabeth A F S

    2014-01-08

    This study investigated the effect of a 28 day administration of coffee brew on the activity of antioxidant enzymes in rats. After this period of 2.0 mL/day dosages of this beverage, the activities of hepatic superoxide dismutase, catalase, and glutathione peroxidase increased 74.8, 59.4, and 135.2%, respectively, whereas the cytosolic level of Nrf2 increased 131.3%. At the same time, the total antioxidant capacity of the hepatic tissue increased 25.1%, improving the defensive status against oxidative stress. At the end of the experiment, the levels of biomarkers alanine transaminase and aspartate transaminase remained equal to the control group, and no changes were observed in the hepatic histoarchiteture of the animals, suggesting that the liver tissue was not impaired by the exposure to coffee. The changes in enzyme activities and antioxidant capacity were statistically significant (p < 0.05), indicating that coffee could be considered an important alternative against oxidative stress and its correlated degenerative diseases.

  11. Increased expression of BDNF transcript with exon VI in hippocampi of patients with pharmaco-resistant temporal lobe epilepsy.

    PubMed

    Martínez-Levy, G A; Rocha, L; Lubin, F D; Alonso-Vanegas, M A; Nani, A; Buentello-García, R M; Pérez-Molina, R; Briones-Velasco, M; Recillas-Targa, F; Pérez-Molina, A; San-Juan, D; Cienfuegos, J; Cruz-Fuentes, C S

    2016-02-09

    A putative role of the brain-derived neurotrophic factor (BDNF) in epilepsy has emerged from in vitro and animal models, but few studies have analyzed human samples. We assessed the BDNF expression of transcripts with exons I (BDNFI), II (BDNFII), IV (BDNFIV) and VI (BDNFVI) and methylation levels of promoters 4 and 6 in the hippocampi of patients with pharmaco-resistant temporal lobe epilepsy (TLE) (n=24). Hippocampal sclerosis (HS) and pre-surgical pharmacological treatment were considered as clinical independent variables. A statistical significant increase for the BDNFVI (p<0.05) was observed in TLE patients compared to the autopsy control group (n=8). BDNFVI was also increased in anxiety/depression TLE (N=4) when compared to autopsies or to the remaining group of patients (p<0.05). In contrast, the use of the antiepileptic drug Topiramate (TPM) (N=3) was associated to a decrease in BDNFVI expression (p<0.05) when compared to the remaining group of patients. Methylation levels at the BDNF promoters 4 and 6 were similar between TLE and autopsies and in relation to the use of either Sertraline (SRT) or TPM. These results suggest an up-regulated expression of a specific BDNF transcript in patients with TLE, an effect that seems to be dependent on the use of specific drugs. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. Impaired dynamin 2 function leads to increased AP-1 transcriptional activity through the JNK/c-Jun pathway.

    PubMed

    Szymanska, Ewelina; Skowronek, Agnieszka; Miaczynska, Marta

    2016-01-01

    Activation of AP-1 transcription factors, composed of the Jun and Fos proteins, regulates cellular fates, such as proliferation, differentiation or apoptosis. Among other stimuli, the AP-1 pathway can be initiated by extracellular ligands, such as growth factors or cytokines, which undergo internalization in complex with their receptors. Endocytosis has been implicated in the regulation of several signaling pathways; however its possible impact on AP-1 signaling remains unknown. Here we show that inhibition of dynamin 2 (Dyn2), a major regulator of endocytic internalization, strongly stimulates the AP-1 pathway. Specifically, expression of a dominant-negative Dyn2 K44A mutant increases the total levels of c-Jun, its phosphorylation on Ser63/73 and transcription of AP-1 target genes. Interestingly, DNM2 mutations implicated in human neurological disorders exhibit similar effects on AP-1 signaling. Mechanistically, Dyn2 K44A induces AP-1 by increasing phosphorylation of several receptor tyrosine kinases. Their activation is required to initiate a Src- and JNK-dependent signaling cascade converging on c-Jun and stimulating expression of AP-1 target genes. Cumulatively, our data uncover a link between the Dyn2 function and JNK signaling which leads to AP-1 induction.

  13. The Stepwise Increase in the Number of Transcription Factor Families in the Precambrian Predated the Diversification of Plants On Land.

    PubMed

    Catarino, Bruno; Hetherington, Alexander J; Emms, David M; Kelly, Steven; Dolan, Liam

    2016-11-01

    The colonization of the land by streptophytes and their subsequent radiation is a major event in Earth history. We report a stepwise increase in the number of transcription factor (TF) families and subfamilies in Archaeplastida before the colonization of the land. The subsequent increase in TF number on land was through duplication within existing TF families and subfamilies. Almost all subfamilies of the Homeodomain (HD) and basic Helix-Loop-Helix (bHLH) had evolved before the radiation of extant land plant lineages from a common ancestor. We demonstrate that the evolution of these TF families independently followed similar trends in both plants and metazoans; almost all extant HD and bHLH subfamilies were present in the first land plants and in the last common ancestor of bilaterians. These findings reveal that the majority of innovation in plant and metazoan TF families occurred in the Precambrian before the Phanerozoic radiation of land plants and metazoans.

  14. Skipping of an alternative intron in the srsf1 3' untranslated region increases transcript stability.

    PubMed

    Akaike, Yoko; Kurokawa, Ken; Kajita, Keisuke; Kuwano, Yuki; Masuda, Kiyoshi; Nishida, Kensei; Kang, Seung Wan; Tanahashi, Toshihito; Rokutan, Kazuhito

    2011-08-01

    The srsf1 gene encodes serine/arginine-rich splicing factor 1 (SRSF1) that participates in both constitutive and alternative splicing reactions. This gene possesses two ultraconserved elements in the 3' untranslated region (UTR). Skipping of an alternative intron between the two elements has no effect on the protein-coding sequence, but it generates a premature stop codon (PTC)-containing mRNA isoform, whose degradation is considered to depend on nonsense-mediated mRNA decay (NMD). However, several cell lines (HCT116, RKO, HeLa, and WI38 cells) constitutively expressed significant amounts of the srsf1 PTC variant. HCT116 cells expressed the PTC variant nearly equivalent to the major isoform that includes the alternative intron in the 3' UTR. Inhibition of NMD by silencing a key effecter UPF1 or by treatment with cycloheximide failed to increase amounts of the PTC variant in HCT116 cells, and the PTC variant was rather more stable than the major isoform in the presence of actinomycin D. Our results suggest that the original stop codon may escape from the NMD surveillance even in skipping of the alternative intron. The srsf1 gene may produce an alternative splice variant having truncated 3' UTR to relief the microRNA- and/or RNA-binding protein-mediated control of translation or degradation.

  15. Phenolic compounds increase the transcription of mouse intestinal maltase-glucoamylase and sucrase-isomaltase.

    PubMed

    Simsek, Meric; Quezada-Calvillo, Roberto; Nichols, Buford L; Hamaker, Bruce R

    2017-05-24

    Diverse natural phenolic compounds show inhibition activity of intestinal α-glucosidases, which may constitute the molecular basis for their ability to control systemic glycemia. Additionally, phenolics can modify mRNA expression for proteins involved in nutritional, metabolic or immune processes. To explore the possibility that phenolics can regulate the mRNA expression, enzymatic activity, and protein synthesis/processing of intestinal Maltase-Glucoamylase (MGAM) and Sucrase-Isomaltase (SI), small intestinal explants from Balb/c mice were cultured for 24 h in the presence or absence of gallic acid, caffeic acid, and (+)-catechin at 0.1, 0.5, and 1 mM. We measured the levels of MGAM and SI mRNA expression by qRT-PCR, maltase and sucrase activities by a standard colorimetric method and the molecular size distribution of MGAM and SI proteins by western blotting. mRNA expression for MGAM was induced by the three phenolic compounds at 0.1 mM. mRNA expression for SI was induced by caffeic and gallic acids, but not by (+)-catechin. Caffeic acid was the most effective inducer of mRNA expression of these enzymes. Total maltase and sucrase activities were not affected by treatment with phenolics. The proportion of high molecular size forms of MGAM was significantly increased by two of the three phenolic compounds, but little effect was observed on SI proteins. Thus, changes in the protein synthesis/processing, affecting the proportions of the different molecular forms of MGAM, may account for the lack of correlation between mRNA expression and enzymatic activity.

  16. Chromatin Alterations in Response to Forced Swimming Underlie Increased Prodynorphin Transcription

    PubMed Central

    Reed, Brian; Fang, Nancy; Blackwell-Mayer, Brandan; Chen, Shasha; Yuferov, Vadim; Zhou, Yan; Kreek, Mary Jeanne

    2012-01-01

    Antagonism of the kappa opioid receptor (KOR) has been reported to have anti-depressant-like properties. The dynorphin/KOR system is a crucial neurochemical substrate underlying the pathologies of addictive diseases, affective disorders and other disease states. However, the molecular underpinnings and neuroanatomical localization of the dysregulation of this system have not yet been fully elucidated. Utilizing the Porsolt Forced Swim Test (FST), an acute stressor commonly used as in rodent models measuring antidepressant efficacy, male Sprague-Dawley rats were subject to forced swimming for 15 minutes, treated 1 hour with vehicle or nor-BNI (5 or 10 mg/kg), and then 1 day later subject to FST for five minutes. In accordance with previous findings, nor-BNI dose dependently increased climbing time and reduced immobility. In comparison to control animals not exposed to FST, we observed a significant elevation in prodynorphin (pDyn) mRNA levels following FST using real-time optical PCR in the caudate putamen but not in the nucleus accumbens, hypothalamus, amygdala, frontal cortex, or hippocampus. Nor-BNI treatment did not affect pDyn mRNA levels in comparison to animals that received vehicle. The corresponding brain regions from the opposite hemisphere were analyzed for underlying chromatin modifications of the prodynorphin gene promoter region using chromatin immunoprecipitation with antibodies against specifically methylated histones H3K27Me2, H3K27Me3, H3K4Me2, and H3K4Me3, as well as CREB-1 and MeCP2. Significant alterations in proteins bound to DNA in the Cre-3, Cre-4, and Sp1 regions of the prodynorphin promoter were found in the caudate putamen of the FST saline-treated animals compared to control animals, with no changes observed in the hippocampus. Epigenetic changes resulting in elevated dynorphin levels specifically in the caudate putamen may in part underlie the enduring effects of stress. PMID:22698692

  17. Vaginal LPS changed gene transcriptional regulation response to ischemic reperfusion and increased vulnerability of fetal brain hemorrhage.

    PubMed

    Dong, Yupeng; Kimura, Yoshitaka; Ito, Takuya; Velayo, Clarissa; Sato, Takafumi; Sugibayashi, Rika; Funamoto, Kiyoe; Hitomi, Kudo; Iida, Keita; Endo, Miyuki; Sato, Naoaki; Yaegashi, Nobuo

    During pregnancy, both ischemic reperfusion and bacterial agent LPS are known risk factors for fetal brain damage. However, there is a lack of evidence to explain whether vaginal LPS affects the fetus response to ischemic reperfusion. Here we reported that there was more than 2 folds higher vulnerability of fetal brain hemorrhage response to ischemic reperfusion when mother mouse was treated with vaginal LPS. As our previously reported, ischemic reperfusion induces P53-dependent fetal brain damage was based on a molecular mechanism: the transcriptional pattern was changed from HIF-1alpha-dependent to P53-dependent immediately. In the present work, only with vaginal LPS precondition, phosphorylation of activated transcriptional factor (ATF) 2 at Thr71 appeared in response to ischemic reperfusion. Moreover, this phosphorylation was completely blocked by pre-treatment with a P53 inhibitor, pifithrin-α. We concluded that vaginal LPS precondition trigged the p53-dependent phosphorylation of ATF2 in response to ischemic reperfusion, which played an important role of increasing vulnerability to hemorrhage in fetus.

  18. Increase in antioxidant gene transcripts, stress tolerance and biocontrol efficacy of Candida oleophila following sublethal oxidative stress exposure.

    PubMed

    Liu, Jia; Wisniewski, Michael; Droby, Samir; Norelli, John; Hershkovitz, Vera; Tian, Shiping; Farrell, Robert

    2012-06-01

    A pretreatment of the yeast, Candida oleophila, with 5 mM H(2)O(2) for 30 min (sublethal) increased yeast tolerance to subsequent lethal levels of oxidative stress (50 mM H(2)O(2)), high temperature (40 °C), and low pH (pH 4). Compared with non-stress-adapted yeast cells, stress-adapted cells exhibited better control of apple fruit infections by Penicillium expansum and Botrytis cinerea and had initially higher growth rates in apple wounds. Suppression subtractive hybridization analysis was used to identify genes expressed in yeast in response to sublethal oxidative stress. Transcript levels were confirmed using semiquantitative reverse transcription-PCR. Seven antioxidant genes were upregulated. The elevated expression of these genes was associated with less accumulation of reactive oxygen species and a lower level of protein and lipid oxidation under subsequent stresses. These data support the premise that induction of abiotic stress tolerance in biocontrol yeast can improve biocontrol efficacy by upregulation of genes involved in the amelioration of oxidative stress. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  19. A Novel Soybean ERF Transcription Factor, GmERF113, Increases Resistance to Phytophthora sojae Infection in Soybean

    PubMed Central

    Zhao, Yuanling; Chang, Xin; Qi, Dongyue; Dong, Lidong; Wang, Guangjin; Fan, Sujie; Jiang, Liangyu; Cheng, Qun; Chen, Xi; Han, Dan; Xu, Pengfei; Zhang, Shuzhen

    2017-01-01

    Phytophthora root and stem rot of soybean caused by the oomycete Phytophthora sojae, is a destructive disease worldwide. Ethylene response factors (ERFs) play important roles in regulating plant biotic and abiotic stress tolerance. In this study, a new ERF gene, GmERF113, was isolated from the highly resistant soybean ‘Suinong 10.’ Sequence analysis suggested that the protein encoded by GmERF113 contained a conserved AP2/ERF domain of 58 amino acid and belonged to the B-4 subgroup of the ERF subfamily. Expression of GmERF113 was significantly induced by P. sojae, ethylene, and methyl jasmonate. GmERF113 protein localized to the nucleus when transiently expressed in Arabidopsis protoplasts, could bind to the GCC-box, and acted as a transcription activator. In addition, a region of the full-length GmERF113, GmERF113-II, interacted with a basic helix-loop-helix transcription factor (GmbHLH) in yeast cells. Full-length GmERF113 also interacted with GmbHLH in planta. GmERF113-overexpressing transgenic plants in susceptible cultivar ‘Dongnong 50’ soybean exhibited increased resistance to P. sojae and positively regulated the expression of the pathogenesis-related genes, PR1 and PR10-1. These results indicate that GmERF113 may play a crucial role in the defense of soybean against P. sojae infection. PMID:28326092

  20. Transcriptional Activation by NFκB Increases Perlecan/HSPG2 Expression in the Desmoplastic Prostate Tumor Microenvironment

    PubMed Central

    Warren, Curtis R.; Grindel, Brian J.; Francis, Lewis; Carson, Daniel D.; Farach-Carson, Mary C.

    2014-01-01

    Perlecan/HSPG2, a heparan sulfate proteoglycan typically found at tissue borders including those separating epithelia and connective tissue, increases near sites of invasion of primary prostatic tumors as previously shown for other proteins involved in desmoplastic tissue reaction. Studies of prostate cancer cells and stromal cells from both prostate and bone, the major site for prostate cancer metastasis, showed that cancer cells and a subset of stromal cells increased production of perlecan in response to cytokines present in the tumor microenvironment. In silico analysis of the HSPG2 promoter revealed two conserved NFκB binding sites, in addition to the previously reported SMAD3 binding sites. By systematically transfecting cells with a variety of reporter constructs including sequences up to 2.6 kb from the start site of transcription, we identified an active cis element in the distal region of the HSPG2 promoter, and showed that it functions in regulating transcription of HSPG2. Treatment with TNF-α and/or TGFβ1 identified TNF-α as a major cytokine regulator of perlecan production. TNF-α treatment also triggered p65 nuclear translocation and binding to the HSPG2 regulatory region in stromal cells and cancer cells. In addition to stromal induction of perlecan production in the prostate, we identified a matrix-secreting bone marrow stromal cell type that may represent the source for increases in perlecan in the metastatic bone marrow environment. These studies implicate perlecan in cytokine-mediated, innate tissue responses to cancer cell invasion, a process we suggest reflects a modified wound healing tissue response co-opted by prostate cancer cells. PMID:24700612

  1. Overexpression of Transcription Factor Sp2 Inhibits Epidermal Differentiation and Increases Susceptibility to Wound and Carcinogen-Induced Tumorigenesis

    PubMed Central

    Kim, Tae-Hyung; Chiera, Shannon L.; Linder, Keith E.; Trempus, Carol S.; Smart, Robert C.; Horowitz, Jonathan M.

    2010-01-01

    Sp proteins are evolutionarily-conserved transcription factors required for the expression of a wide variety of genes that are critical for development and cell-cycle progression. De-regulated expression of certain Sp proteins is associated with the formation of a variety of human tumors, however direct evidence that any given Sp protein is oncogenic has been lacking. Here we report that Sp2 protein abundance in mice increases in concert with the progression of carcinogen-induced murine squamous cell carcinomas. Transgenic mice specifically overexpressing murine Sp2 in epidermal basal keratinocytes were highly susceptible to wound- and carcinogen-induced papillomagenesis. Transgenic animals that were homozygous rather than hemizygous for the Sp2 transgene exhibited a striking arrest in the epidermal differentiation program, perishing within two weeks of birth. Our results directly support the likelihood that Sp2 overexpression occurring in various human cancers has significant functional impact. PMID:20959487

  2. Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress

    PubMed Central

    Aragon, Anthony D; Quiñones, Gabriel A; Thomas, Edward V; Roy, Sushmita; Werner-Washburne, Margaret

    2006-01-01

    Background As carbon sources are exhausted, Saccharomyces cerevisiae cells exhibit reduced metabolic activity and cultures enter the stationary phase. We asked whether cells in stationary phase cultures respond to additional stress at the level of transcript abundance. Results Microarrays were used to quantify changes in transcript abundance in cells from stationary phase cultures in response to stress. More than 800 mRNAs increased in abundance by one minute after oxidative stress. A significant number of these mRNAs encode proteins involved in stress responses. We tested whether mRNA increases were due to new transcription, rapid poly-adenylation of message (which would not be detected by microarrays), or potential release of mature mRNA present in the cell but resistant to extraction during RNA isolation. Examination of the response to oxidative stress in an RNA polymerase II mutant, rpb1-1, suggested that new transcription was not required. Quantitative RT-PCR analysis of a subset of these transcripts further suggested that the transcripts present in isolated total RNA from stationary phase cultures were polyadenylated. In contrast, over 2,000 transcripts increased after protease treatment of cell-free lysates from stationary phase but not exponentially growing cultures. Different subsets of transcripts were released by oxidative stress and temperature upshift, suggesting that mRNA release is stress-specific. Conclusions Cells in stationary phase cultures contain a large number of extraction-resistant mRNAs in a protease-labile, rapidly releasable form. The transcript release appears to be stress-specific. We hypothesize that these transcripts are associated with P-bodies. PMID:16507144

  3. Mice lacking the transcription factor Mist1 exhibit an altered stress response and increased sensitivity to caerulein-induced pancreatitis.

    PubMed

    Kowalik, Agnes S; Johnson, Charis L; Chadi, Sami A; Weston, Jacqueline Y; Fazio, Elena N; Pin, Christopher L

    2007-04-01

    Several animal models have been developed to investigate the pathobiology of pancreatitis, but few studies have examined the effects that altered pancreatic gene expression have in these models. In this study, the sensitivity to secretagogue-induced pancreatitis was examined in a mouse line that has an altered acinar cell environment due to the targeted deletion of Mist1. Mist1 is an exocrine specific transcription factor important for the complete differentiation and function of pancreatic acinar cells. Mice lacking the Mist1 gene [Mist1 knockout (KO) mice] exhibit cellular disorganization and functional defects in the exocrine pancreas but no gross morphological defects. Following the induction of pancreatitis with caerulein, a CCK analog, we observed elevated serum amylase levels, necrosis, and tissue damage in Mist1 KO mice, indicating increased pancreatic damage. There was also a delay in the regeneration of acinar tissue in Mist1 KO animals. Molecular profiling revealed an altered activation of stress response genes in Mist1 KO pancreatic tissue compared with wild-type (WT) tissue following the induction of pancreatitis. In particular, Western blot analysis for activating transcription factor 3 and phosphorylated eukaryotic initiation factor 2alpha (eIF2alpha), mediators of endoplasmic reticulum (ER) stress, indicated limited activation of this pathway in Mist1 KO animals compared with WT controls. Conversely, Mist1 KO pancreatic tissue exhibits increased expression of growth arrest and DNA damage inducible 34 protein, an inhibitor of eIF2alpha phosphorylation, before and after the induction of pancreatitis. These finding suggest that activation of the ER stress pathway is a protective event in the progression of pancreatitis and highlight the Mist1 KO mouse line as an important new model for studying the molecular events that contribute to the sensitivity to pancreatic injury.

  4. Early Cone Setting in Picea abies acrocona Is Associated with Increased Transcriptional Activity of a MADS Box Transcription Factor1[W][OA

    PubMed Central

    Uddenberg, Daniel; Reimegård, Johan; Clapham, David; Almqvist, Curt; von Arnold, Sara; Emanuelsson, Olof; Sundström, Jens F.

    2013-01-01

    Conifers normally go through a long juvenile period, for Norway spruce (Picea abies) around 20 to 25 years, before developing male and female cones. We have grown plants from inbred crosses of a naturally occurring spruce mutant (acrocona). One-fourth of the segregating acrocona plants initiate cones already in their second growth cycle, suggesting control by a single locus. The early cone-setting properties of the acrocona mutant were utilized to identify candidate genes involved in vegetative-to-reproductive phase change in Norway spruce. Poly(A+) RNA samples from apical and basal shoots of cone-setting and non-cone-setting plants were subjected to high-throughput sequencing (RNA-seq). We assembled and investigated 33,383 expressed putative protein-coding acrocona transcripts. Eight transcripts were differentially expressed between selected sample pairs. One of these (Acr42124_1) was significantly up-regulated in apical shoot samples from cone-setting acrocona plants, and the encoded protein belongs to the MADS box gene family of transcription factors. Using quantitative real-time polymerase chain reaction with independently derived plant material, we confirmed that the MADS box gene is up-regulated in both needles and buds of cone-inducing shoots when reproductive identity is determined. Our results constitute important steps for the development of a rapid cycling model system that can be used to study gene function in conifers. In addition, our data suggest the involvement of a MADS box transcription factor in the vegetative-to-reproductive phase change in Norway spruce. PMID:23221834

  5. Tumor necrosis factor and immune interferon synergistically increase transcription of HLA class I heavy- and light-chain genes in vascular endothelium

    SciTech Connect

    Johnson, D.R.; Pober, J.S. )

    1990-07-01

    Tumor necrosis factor and immune interferon synergistically increase cell-surface expression of class I major histocompatibility complex molecules in cultured human endothelial cells. The authors report that tumor necrosis factor and interferon {gamma} each independently increase mRNA levels and together cause a greater-than-additive (i.e., synergistic) increase in steady-state mRNA levels and transcriptional rates of the class I heavy- and light-chain genes. HLA heavy-chain mRNA is equally stable in cytokine-treated and -untreated endothelial cells. Interferon {gamma} does not increase tumor necrosis factor receptor number or affinity on human endothelial cells. They conclude that the synergistic increase in class I major histocompatibility complex cell-surface expression results principally from the synergistic increase in transcriptional rates. They propose that this increase is caused by the cooperative binding of independently activated transcription factors to the promoter/enhancer sequences of class I genes.

  6. A Wheat CCAAT Box-Binding Transcription Factor Increases the Grain Yield of Wheat with Less Fertilizer Input1

    PubMed Central

    Qu, Baoyuan; He, Xue; Wang, Jing; Zhao, Yanyan; Teng, Wan; Shao, An; Zhao, Xueqiang; Ma, Wenying; Wang, Junyi; Li, Bin; Li, Zhensheng; Tong, Yiping

    2015-01-01

    Increasing fertilizer consumption has led to low fertilizer use efficiency and environmental problems. Identifying nutrient-efficient genes will facilitate the breeding of crops with improved fertilizer use efficiency. This research performed a genome-wide sequence analysis of the A (NFYA), B (NFYB), and C (NFYC) subunits of Nuclear Factor Y (NF-Y) in wheat (Triticum aestivum) and further investigated their responses to nitrogen and phosphorus availability in wheat seedlings. Sequence mining together with gene cloning identified 18 NFYAs, 34 NFYBs, and 28 NFYCs. The expression of most NFYAs positively responded to low nitrogen and phosphorus availability. In contrast, microRNA169 negatively responded to low nitrogen and phosphorus availability and degraded NFYAs. Overexpressing TaNFYA-B1, a low-nitrogen- and low-phosphorus-inducible NFYA transcript factor on chromosome 6B, significantly increased both nitrogen and phosphorus uptake and grain yield under differing nitrogen and phosphorus supply levels in a field experiment. The increased nitrogen and phosphorus uptake may have resulted from the fact that that overexpressing TaNFYA-B1 stimulated root development and up-regulated the expression of both nitrate and phosphate transporters in roots. Our results suggest that TaNFYA-B1 plays essential roles in root development and in nitrogen and phosphorus usage in wheat. Furthermore, our results provide new knowledge and valuable gene resources that should be useful in efforts to breed crops targeting high yield with less fertilizer input. PMID:25489021

  7. A wheat CCAAT box-binding transcription factor increases the grain yield of wheat with less fertilizer input.

    PubMed

    Qu, Baoyuan; He, Xue; Wang, Jing; Zhao, Yanyan; Teng, Wan; Shao, An; Zhao, Xueqiang; Ma, Wenying; Wang, Junyi; Li, Bin; Li, Zhensheng; Tong, Yiping

    2015-02-01

    Increasing fertilizer consumption has led to low fertilizer use efficiency and environmental problems. Identifying nutrient-efficient genes will facilitate the breeding of crops with improved fertilizer use efficiency. This research performed a genome-wide sequence analysis of the A (NFYA), B (NFYB), and C (NFYC) subunits of Nuclear Factor Y (NF-Y) in wheat (Triticum aestivum) and further investigated their responses to nitrogen and phosphorus availability in wheat seedlings. Sequence mining together with gene cloning identified 18 NFYAs, 34 NFYBs, and 28 NFYCs. The expression of most NFYAs positively responded to low nitrogen and phosphorus availability. In contrast, microRNA169 negatively responded to low nitrogen and phosphorus availability and degraded NFYAs. Overexpressing TaNFYA-B1, a low-nitrogen- and low-phosphorus-inducible NFYA transcript factor on chromosome 6B, significantly increased both nitrogen and phosphorus uptake and grain yield under differing nitrogen and phosphorus supply levels in a field experiment. The increased nitrogen and phosphorus uptake may have resulted from the fact that that overexpressing TaNFYA-B1 stimulated root development and up-regulated the expression of both nitrate and phosphate transporters in roots. Our results suggest that TaNFYA-B1 plays essential roles in root development and in nitrogen and phosphorus usage in wheat. Furthermore, our results provide new knowledge and valuable gene resources that should be useful in efforts to breed crops targeting high yield with less fertilizer input.

  8. Increase in cocaine- and amphetamine-regulated transcript (CART) in specific areas of the mouse brain by acute caffeine administration.

    PubMed

    Cho, Jin Hee; Cho, Yun Ha; Kim, Hyo Young; Cha, Seung Ha; Ryu, Hyun; Jang, Wooyoung; Shin, Kyung Ho

    2015-04-01

    Caffeine produces a variety of behavioral effects including increased alertness, reduced food intake, anxiogenic effects, and dependence upon repeated exposure. Although many of the effects of caffeine are mediated by its ability to block adenosine receptors, it is possible that other neural substrates, such as cocaine- and amphetamine-regulated transcript (CART), may be involved in the effects of caffeine. Indeed, a recent study demonstrated that repeated caffeine administration increases CART in the mouse striatum. However, it is not clear whether acute caffeine administration alters CART in other areas of the brain. To explore this possibility, we investigated the dose- and time-dependent changes in CART immunoreactivity (CART-IR) after a single dose of caffeine in mice. We found that a high dose of caffeine (100 mg/kg) significantly increased CART-IR 2 h after administration in the nucleus accumbens shell (AcbSh), dorsal bed nucleus of the stria terminalis (dBNST), central nucleus of the amygdala (CeA), paraventricular hypothalamic nucleus (PVN), arcuate hypothalamic nucleus (Arc), and locus coeruleus (LC), and returned to control levels after 8 h. But this increase was not observed in other brain areas. In addition, caffeine administration at doses of 25 and 50 mg/kg appears to produce dose-dependent increases in CART-IR in these brain areas; however, the magnitude of increase in CART-IR observed at a dose of 50 mg/kg was similar or greater than that observed at a dose of 100 mg/kg. This result suggests that CART-IR in AcbSh, dBNST, CeA, PVN, Arc, and LC is selectively affected by caffeine administration.

  9. Hypertrophy and transcriptional regulation induced in myogenic cell line L6-C5 by an increase of extracellular calcium.

    PubMed

    De Arcangelis, V; Coletti, D; Canato, M; Molinaro, M; Adamo, S; Reggiani, C; Naro, F

    2005-03-01

    Calcium plays a pivotal role in the establishment of the differentiated phenotype in myogenic cells but the involved molecular mechanisms are still matter of debate. Here we studied the effects of exposing L6-C5 myogenic cells to high extracellular Ca2+ concentration ([Ca2+]o), which induces an increase of intracellular calcium ([Ca2+]i) without involving Ca2+ release from the intracellular stores but exclusively due to plasma membrane influx (Naro et al., 2003). Exposure of L6-C5 cells to [Ca2+]o up to 20 mM for 30 min, before shifting them into a differentiative medium, induced the appearance of multinucleated, myosin-positive myotubes, much larger than in control cells with an increased protein/DNA ratio. These large myotubes showed nuclear accumulation of the hypertrophy marker GATA-2. The hypertrophic growth of these cells was blocked by cyclosporin A (CsA), FK506, or overexpression of a calcineurin-dominant negative protein, suggesting the involvement in this process of the Ca2+ responsive phosphatase calcineurin. Furthermore, transient exposure of L6-C5 cells to high [Ca2+]o increased the expression of luciferase reporter driven by myoglobin (Mb) and beta-MHC promoters but not IIB-MHC and MCK promoters. Luciferase transcription driven by CK promoter was, instead, enhanced by mobilizing Ca2+ from the intracellular stores. These data indicate that a transient increase of [Ca2+]i due to plasma-membrane influx is sufficient to induce a hypertrophic phenotype and an increased expression of slow-fiber genes but not fast-fiber genes. 2004 Wiley-Liss, Inc.

  10. M-CSF increases proliferation and phagocytosis while modulating receptor and transcription factor expression in adult human microglia

    PubMed Central

    2013-01-01

    Background Microglia are the primary immune cells of the brain whose phenotype largely depends on their surrounding micro-environment. Microglia respond to a multitude of soluble molecules produced by a variety of brain cells. Macrophage colony-stimulating factor (M-CSF) is a cytokine found in the brain whose receptor is expressed by microglia. Previous studies suggest a critical role for M-CSF in brain development and normal functioning as well as in several disease processes involving neuroinflammation. Methods Using biopsy tissue from patients with intractable temporal epilepsy and autopsy tissue, we cultured primary adult human microglia to investigate their response to M-CSF. Mixed glial cultures were treated with 25 ng/ml M-CSF for 96 hours. Proliferation and phagocytosis assays, and high through-put immunocytochemistry, microscopy and image analysis were performed to investigate microglial phenotype and function. Results We found that the phenotype of primary adult human microglia was markedly changed following exposure to M-CSF. A greater number of microglia were present in the M-CSF- treated cultures as the percentage of proliferating (BrdU and Ki67-positive) microglia was greatly increased. A number of changes in protein expression occurred following M-CSF treatment, including increased transcription factors PU.1 and C/EBPβ, increased DAP12 adaptor protein, increased M-CSF receptor (CSF-1R) and IGF-1 receptor, and reduced HLA-DP, DQ, DR antigen presentation protein. Furthermore, a distinct morphological change was observed with elongation of microglial processes. These changes in phenotype were accompanied by a functional increase in phagocytosis of Aβ1-42 peptide. Conclusions We show here that the cytokine M-CSF dramatically influences the phenotype of adult human microglia. These results pave the way for future investigation of M-CSF-related targets for human therapeutic benefit. PMID:23866312

  11. Improved Insulin Sensitivity despite Increased Visceral Adiposity in Mice Deficient for the Immune Cell Transcription Factor T-bet

    PubMed Central

    Stolarczyk, Emilie; Vong, Chi Teng; Perucha, Esperanza; Jackson, Ian; Cawthorne, Michael A.; Wargent, Edward T.; Powell, Nick; Canavan, James B.; Lord, Graham M.; Howard, Jane K.

    2013-01-01

    Summary Low-grade inflammation in fat is associated with insulin resistance, although the mechanisms are unclear. We report that mice deficient in the immune cell transcription factor T-bet have lower energy expenditure and increased visceral fat compared with wild-type mice, yet paradoxically are more insulin sensitive. This striking phenotype, present in young T-bet−/− mice, persisted with high-fat diet and increasing host age and was associated with altered immune cell numbers and cytokine secretion specifically in visceral adipose tissue. However, the favorable metabolic phenotype observed in T-bet-deficient hosts was lost in T-bet−/− mice also lacking adaptive immunity (T-bet−/−xRag2−/−), demonstrating that T-bet expression in the adaptive rather than the innate immune system impacts host glucose homeostasis. Indeed, adoptive transfer of T-bet-deficient, but not wild-type, CD4+ T cells to Rag2−/− mice improved insulin sensitivity. Our results reveal a role for T-bet in metabolic physiology and obesity-associated insulin resistance. PMID:23562076

  12. Increased expression of acetylcholinesterase T and R transcripts during hematopoietic differentiation is accompanied by parallel elevations in the levels of their respective molecular forms.

    PubMed

    Chan, R Y; Adatia, F A; Krupa, A M; Jasmin, B J

    1998-04-17

    Differentiation of hematopoietic cells is known to be accompanied by profound changes in acetylcholinesterase (AChE) enzyme activity, yet the basic mechanisms underlying this developmental regulation remain unknown. We initiated a series of experiments to examine the molecular mechanisms involved in regulating AChE expression during hematopoiesis. Differentiation of murine erythroleukemia (MEL) cells using dimethyl sulfoxide resulted in a 5- and 10-fold increase in intracellular and secreted AChE enzyme activity, respectively. Interestingly, these increases resulted from a preferential induction of the globular molecular form G1 and a slight increase in G4 instead of an increase in the levels of the G2 membrane-bound form, a molecular form expressed in mature erythrocytes. Concomitantly, expression of the two predominant AChE transcripts (R and T, for read-through and tail, respectively) in MEL cells was induced to a similar extent with differentiation. Nuclear run-on assays performed with nuclei isolated from induced versus uninduced MEL cells revealed that in contrast to the large increases seen in the transcription of the beta-globin gene, the transcriptional activity of the AChE gene remained largely unaffected after differentiation. Determination of the half-lives of the R and T transcripts demonstrated that they both exhibited an increase in stability in induced MEL cells. Taken together, results from these studies indicate that post-transcriptional regulatory mechanisms account for the increased expression of AChE in differentiated hematopoietic cells.

  13. Deficiency of heat shock transcription factor 1 suppresses heat stress-associated increase in slow soleus muscle mass of mice.

    PubMed

    Ohno, Y; Egawa, T; Yokoyama, S; Nakai, A; Sugiura, T; Ohira, Y; Yoshioka, T; Goto, K

    2015-12-01

    Effects of heat shock transcription factor 1 (HSF1) deficiency on heat stress-associated increase in slow soleus muscle mass of mice were investigated. Both HSF1-null and wild-type mice were randomly assigned to control and heat-stressed groups. Mice in heat-stressed group were exposed to heat stress (41 °C for 60 min) in an incubator without anaesthesia. Significant increase in wet and dry weights, and protein content of soleus muscle in wild-type mice was observed seven days after the application of the heat stress. However, heat stress had no impact on soleus muscle mass in HSF1-null mice. Neither type of mice exhibited much effect of heat stress on HSF mRNA expression (HSF1, HSF2 and HSF4). On the other hand, heat stress upregulated heat shock proteins (HSPs) at the mRNA (HSP72) and protein (HSP72 and HSP110) levels in wild-type mice, but not in HSF1-null mice. The population of Pax7-positive nuclei relative to total myonuclei of soleus muscle in wild-type mice was significantly increased by heat stress, but not in HSF1-null mice. Furthermore, the absence of HSF1 gene suppressed heat stress-associated phosphorylation of Akt and p70 S6 kinase (p-p70S6K) in soleus muscle. Heat stress-associated increase in skeletal muscle mass may be induced by HSF1 and/or HSF1-mediated stress response that activates muscle satellite cells and Akt/p70S6K signalling pathway. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  14. Coniferyl Aldehyde Reduces Radiation Damage Through Increased Protein Stability of Heat Shock Transcriptional Factor 1 by Phosphorylation

    SciTech Connect

    Kim, Seo-Young; Lee, Hae-June; Nam, Joo-Won; Seo, Eun-Kyoung; Lee, Yun-Sil

    2015-03-15

    Purpose: We previously screened natural compounds and found that coniferyl aldehyde (CA) was identified as an inducer of HSF1. In this study, we further examined the protective effects of CA against ionizing radiation (IR) in normal cell system. Methods and Materials: Western blotting and reverse transcription-polymerase chain reaction tests were performed to evaluate expression of HSF1, HSP27, and HSP70 in response to CA. Cell death and cleavage of PARP and caspase-3 were analyzed to determine the protective effects of CA in the presence of IR or taxol. The protective effects of CA were also evaluated using animal models. Results: CA increased stability of the HSF1 protein by phosphorylation at Ser326, which was accompanied by increased expression of HSP27 and HSP70. HSF1 phosphorylation at Ser326 by CA was mediated by EKR1/2 activation. Cotreatment of CA with IR or taxol in normal cells induced protective effects with phosphorylation- dependent patterns at Ser326 of HSF1. The decrease in bone marrow (BM) cellularity and increase of terminal deoxynucleotidyl transferase dUTP nick end labeling–positive BM cells by IR were also significantly inhibited by CA in mice (30.6% and 56.0%, respectively). A549 lung orthotopic lung tumor model indicated that CA did not affect the IR-mediated reduction of lung tumor nodules, whereas CA protected normal lung tissues from the therapeutic irradiation. Conclusions: These results suggest that CA may be useful for inducing HSF1 to protect against normal cell damage after IR or chemotherapeutic agents.

  15. Post-fusion treatment with MG132 increases transcription factor expression in somatic cell nuclear transfer embryos in pigs.

    PubMed

    You, Jinyoung; Lee, Joohyeong; Kim, Jinyoung; Park, Junhong; Lee, Eunsong

    2010-02-01

    The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P < 0.05) level in SCNT oocytes that were treated post-fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos. (c) 2009 Wiley-Liss, Inc.

  16. Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell-suppressive capacity.

    PubMed

    van Loosdregt, Jorg; Fleskens, Veerle; Fu, Juan; Brenkman, Arjan B; Bekker, Cornelis P J; Pals, Cornelieke E G M; Meerding, Jenny; Berkers, Celia R; Barbi, Joseph; Gröne, Andrea; Sijts, Alice J A M; Maurice, Madelon M; Kalkhoven, Eric; Prakken, Berent J; Ovaa, Huib; Pan, Fan; Zaiss, Dietmar M W; Coffer, Paul J

    2013-08-22

    Stable Foxp3 expression is required for the development of functional regulatory T (Treg) cells. Here, we demonstrate that the expression of the transcription factor Foxp3 can be regulated through the polyubiquitination of multiple lysine residues, resulting in proteasome-mediated degradation. Expression of the deubiquitinase (DUB) USP7 was found to be upregulated and active in Treg cells, being associated with Foxp3 in the nucleus. Ectopic expression of USP7 decreased Foxp3 polyubiquitination and increased Foxp3 expression. Conversely, either treatment with DUB inhibitor or USP7 knockdown decreased endogenous Foxp3 protein expression and decreased Treg-cell-mediated suppression in vitro. Furthermore, in a murine adoptive-transfer-induced colitis model, either inhibition of DUB activity or USP7 knockdown in Treg cells abrogated their ability to resolve inflammation in vivo. Our data reveal a molecular mechanism in which rapid temporal control of Foxp3 expression in Treg cells can be regulated by USP7, thereby modulating Treg cell numbers and function. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Evidence for karyoplasmic homeostasis during endoreduplication and a ploidy-dependent increase in gene transcription during tomato fruit growth.

    PubMed

    Bourdon, Matthieu; Pirrello, Julien; Cheniclet, Catherine; Coriton, Olivier; Bourge, Mickaël; Brown, Spencer; Moïse, Adeline; Peypelut, Martine; Rouyère, Valérie; Renaudin, Jean-Pierre; Chevalier, Christian; Frangne, Nathalie

    2012-10-01

    Endopolyploidy is a widespread process that corresponds to the amplification of the genome in the absence of mitosis. In tomato, very high ploidy levels (up to 256C) are reached during fruit development, concomitant with very large cell sizes. Using cellular approaches (fluorescence and electron microscopy) we provide a structural analysis of endoreduplicated nuclei at the level of chromatin and nucleolar organisation, nuclear shape and relationship with other cellular organelles such as mitochondria. We demonstrate that endopolyploidy in pericarp leads to the formation of polytene chromosomes and markedly affects nuclear structure. Nuclei manifest a complex shape, with numerous deep grooves that are filled with mitochondria, affording a fairly constant ratio between nuclear surface and nuclear volume. We provide the first direct evidence that endopolyploidy plays a role in increased transcription of rRNA and mRNA on a per-nucleus basis. Overall, our results provide quantitative evidence in favour of the karyoplasmic theory and show that endoreduplication is associated with complex cellular organisation during tomato fruit development.

  18. Phenyl-1-Pyridin-2yl-Ethanone-Based Iron Chelators Increase IκB-α Expression, Modulate CDK2 and CDK9 Activities, and Inhibit HIV-1 Transcription

    PubMed Central

    Kumari, Namita; Iordanskiy, Sergey; Kovalskyy, Dmytro; Breuer, Denitra; Niu, Xiaomei; Lin, Xionghao; Xu, Min; Gavrilenko, Konstantin; Kashanchi, Fatah; Dhawan, Subhash

    2014-01-01

    HIV-1 transcription is activated by the Tat protein, which recruits CDK9/cyclin T1 to the HIV-1 promoter. CDK9 is phosphorylated by CDK2, which facilitates formation of the high-molecular-weight positive transcription elongation factor b (P-TEFb) complex. We previously showed that chelation of intracellular iron inhibits CDK2 and CDK9 activities and suppresses HIV-1 transcription, but the mechanism of the inhibition was not understood. In the present study, we tested a set of novel iron chelators for the ability to inhibit HIV-1 transcription and elucidated their mechanism of action. Novel phenyl-1-pyridin-2yl-ethanone (PPY)-based iron chelators were synthesized and examined for their effects on cellular iron, HIV-1 inhibition, and cytotoxicity. Activities of CDK2 and CDK9, expression of CDK9-dependent and CDK2-inhibitory mRNAs, NF-κB expression, and HIV-1- and NF-κB-dependent transcription were determined. PPY-based iron chelators significantly inhibited HIV-1, with minimal cytotoxicity, in cultured and primary cells chronically or acutely infected with HIV-1 subtype B, but they had less of an effect on HIV-1 subtype C. Iron chelators upregulated the expression of IκB-α, with increased accumulation of cytoplasmic NF-κB. The iron chelators inhibited CDK2 activity and reduced the amount of CDK9/cyclin T1 in the large P-TEFb complex. Iron chelators reduced HIV-1 Gag and Env mRNA synthesis but had no effect on HIV-1 reverse transcription. In addition, iron chelators moderately inhibited basal HIV-1 transcription, equally affecting HIV-1 and Sp1- or NF-κB-driven transcription. By virtue of their involvement in targeting several key steps in HIV-1 transcription, these novel iron chelators have the potential for the development of new therapeutics for the treatment of HIV-1 infection. PMID:25155598

  19. Activating transcription factor 4 is involved in endoplasmic reticulum stress-mediated apoptosis contributing to vascular calcification.

    PubMed

    Duan, Xiao-Hui; Chang, Jin-Rui; Zhang, Jing; Zhang, Bao-Hong; Li, Yu-Lin; Teng, Xu; Zhu, Yi; Du, Jie; Tang, Chao-Shu; Qi, Yong-Fen

    2013-09-01

    Our previous work reported that endoplasmic reticulum stress (ERS)-mediated apoptosis was activated during vascular calcification (VC). Activating transcription factor 4 (ATF4) is a critical transcription factor in osteoblastogenesis and ERS-induced apoptosis. However, whether ATF4 is involved in ERS-mediated apoptosis contributing to VC remains unclear. In the present study, in vivo VC was induced in rats by administering vitamin D3 plus nicotine. Vascular smooth muscle cell (VSMC) calcification in vitro was induced by incubation in calcifying media containing β-glycerophosphate and CaCl2. ERS inhibitors taurine or 4-phenylbutyric acid attenuated ERS and VSMC apoptosis in calcified rat arteries, reduced calcification and retarded the VSMC contractile phenotype transforming into an osteoblast-like phenotype in vivo. Inhibition of ERS retarded the VSMC phenotypic transition into an osteoblast-like cell phenotype and reduced VSMC calcification and apoptosis in vitro. Interestingly, ATF4 was activated in calcified aortas and calcified VSMCs in vitro. ATF4 knockdown attenuated ERS-induced apoptosis in calcified VSMCs. ATF4 deficiency blocked VSMC calcification and negatively regulated the osteoblast phenotypic transition of VSMCs in vitro. Our results demonstrate that ATF4 was involved at least in part in the process of ERS-mediated apoptosis contributing to VC.

  20. Increased Expression of FoxM1 Transcription Factor in Respiratory Epithelium Inhibits Lung Sacculation and Causes Clara Cell Hyperplasia

    PubMed Central

    Wang, I-Ching; Zhang, Yufang; Snyder, Jonathan; Sutherland, Mardi J.; Burhans, Michael S.; Shannon, John M.; Park, Hyun Jung; Whitsett, Jeffrey A.; Kalinichenko, Vladimir V.

    2010-01-01

    Foxm1 is a member of the Forkhead Box (Fox) family of transcription factors. Foxm1 (previously called Foxm1b, HFH-11B, Trident, Win, or MPP2) is expressed in multiple cell types and plays important roles in cellular proliferation, differentiation and tumorigenesis. Genetic deletion of Foxm1 from mouse respiratory epithelium during initial stages of lung development inhibits lung maturation and causes respiratory failure after birth. However, the role of Foxm1 during postnatal lung morphogenesis remains unknown. In the present study, Foxm1 expression was detected in epithelial cells of conducting and peripheral airways and changing dynamically with lung maturation. To discern the biological role of Foxm1 in the prenatal and postnatal lung, a novel transgenic mouse line that expresses a constitutively active form of FoxM1 (FoxM1 N-terminal deletion mutant or FoxM1-ΔN) under the control of lung epithelial-specific SPC promoter was produced. Expression of the FoxM1-ΔN transgene during embryogenesis caused epithelial hyperplasia, inhibited lung sacculation and expression of the type II epithelial marker, pro-SPC. Expression of FoxM1-ΔN mutant during the postnatal period did not influence alveologenesis but caused focal airway hyperplasia and increased proliferation of Clara cells. Likewise, expression of FoxM1-ΔN mutant in conducting airways with Scgb1a1 promoter was sufficient to induce Clara cell hyperplasia. Furthermore, FoxM1-ΔN cooperated with activated K-Ras to induce lung tumor growth in vivo. Increased activity of Foxm1 altered lung sacculation, induced proliferation in the respiratory epithelium and accelerated lung tumor growth, indicating that precise regulation of Foxm1 is critical for normal lung morphogenesis and development of lung cancer. PMID:20816795

  1. Increased expression of X-linked genes in mammals is associated with a higher stability of transcripts and an increased ribosome density.

    PubMed

    Faucillion, Marie-Line; Larsson, Jan

    2015-03-18

    Mammalian sex chromosomes evolved from the degeneration of one homolog of a pair of ancestral autosomes, the proto-Y. This resulted in a gene dose imbalance that is believed to be restored (partially or fully) through upregulation of gene expression from the single active X-chromosome in both sexes by a dosage compensatory mechanism. We analyzed multiple genome-wide RNA stability data sets and found significantly longer average half-lives for X-chromosome transcripts than for autosomal transcripts in various human cell lines, both male and female, and in mice. Analysis of ribosome profiling data shows that ribosome density is higher on X-chromosome transcripts than on autosomal transcripts in both humans and mice, suggesting that the higher stability is causally linked to a higher translation rate. Our results and observations are in accordance with a dosage compensatory upregulation of expressed X-linked genes. We therefore propose that differential mRNA stability and translation rates of the autosomes and sex chromosomes contribute to an evolutionarily conserved dosage compensation mechanism in mammals. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  2. Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

    NASA Astrophysics Data System (ADS)

    House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

    2015-06-01

    Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

  3. Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling.

    PubMed

    House, Carrie D; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T; Annunziata, Christina M; Gutkind, J Silvio; Hales, Tim G; Lee, Norman H

    2015-06-22

    Functional expression of voltage-gated Na(+) channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

  4. Human cytomegalovirus induces the endoplasmic reticulum chaperone BiP through increased transcription and activation of translation by using the BiP internal ribosome entry site.

    PubMed

    Buchkovich, Nicholas J; Yu, Yongjun; Pierciey, Francis J; Alwine, James C

    2010-11-01

    The endoplasmic reticulum (ER) chaperone BiP (immunoglobulin binding protein) plays a major role in the control of the unfolded protein response. We have previously shown that BiP levels are dramatically increased during human cytomegalovirus (HCMV) infection, where BiP performs unique roles in viral assembly and egress. We show that BiP mRNA levels increase during infection due to activation of the BiP promoter by the major immediate-early (MIE) proteins. The BiP promoter, like other ER stress-activated promoters, contains endoplasmic reticulum stress elements (ERSEs), which are activated by unfolded protein response (UPR)-induced transcription factors. However, these elements are not needed for MIE protein-mediated transcriptional activation; thus, a virus-specific transcriptional activation mechanism is used. Transcriptional activation results in only a 3- to 4-fold increase in BiP mRNA, suggesting that additional mechanisms for BiP production are utilized. The BiP mRNA contains an internal ribosome entry site (IRES) which increases the level of BiP mRNA translation. We show that utilization of the BiP IRES is dramatically increased in HCMV-infected cells. Utilization of the BiP IRES can be activated by the La autoantigen, also called Sjögren's syndrome antigen B (SSB). We show that SSB/La levels are significantly increased during HCMV infection, and SSB/La depletion causes the loss of BiP IRES utilization and lowers endogenous BiP levels in infected cells. Our data show that BiP levels increase in HCMV-infected cells through the combination of increased BiP gene transcription mediated by the MIE proteins and increased BiP mRNA translation due to SSB/La-induced utilization of the BiP IRES.

  5. TRAF family member-associated NF-kappa B activator (TANK) expression increases in injured sensory neurons and is transcriptionally regulated by Sox11

    PubMed Central

    Salerno, Kathleen M.; Jing, Xiaotang; Diges, Charlotte M.; Davis, Brian M.; Albers, Kathryn M.

    2013-01-01

    Peripheral nerve injury evokes rapid and complex changes in gene transcription and cellular signaling pathways. Understanding how these changes are functionally related is essential for developing new approaches that accelerate and improve nerve regeneration. Towards this goal we found that nerve injury induces a rapid and significant up-regulation of the transcription factor Sox11 in dorsal root ganglia (DRG) neurons. Gain and loss of function studies have shown this increase is essential for normal axon regeneration. To determine how Sox11 impacts neuronal gene expression, DRG neurons were treated with Sox11 siRNA to identify potential transcriptional targets. One gene significantly reduced by Sox11 knockdown was TRAF (tumor necrosis factor (TNF) receptor-associated factor)-associated NF-κB activator (TANK). Here we show that TANK is expressed in DRG neurons, that TANK expression is increased in response to peripheral nerve injury and that Sox11 overexpression in vitro increases TANK expression. Injury and in vitro overexpression were also found to preferentially increase TANK transcript variant 3 and a larger TANK protein isoform. To determine if Sox11 regulates TANK transcription bioinformatic analysis was used to identify potential Sox binding motifs within 5 kbp of the TANK 5’ untranslated region (UTR) across several mammalian genomes. Two sites in the mouse TANK gene were examined. Luciferase expression assays coupled with site-directed mutagenesis showed each site contributes to enhanced TANK promoter activity. In addition, chromatin immunoprecipitation assays showed direct Sox11 binding in regions containing the two identified Sox motifs in the mouse TANK 5’-UTR. These studies are the first to show that TANK is expressed in DRG neurons, that TANK is increased by peripheral nerve injury and that the regulation of TANK expression is, at least in part, controlled by the injury-associated transcription factor Sox11. PMID:23201825

  6. Engineering of the TetR family transcriptional regulator SAV151 and its target genes increases avermectin production in Streptomyces avermitilis.

    PubMed

    He, Fei; Liu, Wenshuai; Sun, Di; Luo, Shuai; Chen, Zhi; Wen, Ying; Li, Jilun

    2014-01-01

    Avermectins produced by Streptomyces avermitilis are used commercially for broad-spectrum parasite control in medical, veterinary, and agricultural fields. Our previous comparative transcriptome analysis of wild-type strain ATCC31267 vs. avermectin-overproducing strain 76-02-e revealed that the gene SAV151, which encodes a TetR family transcriptional regulator, was downregulated in 76-02-e. In the present study, we investigated the role of SAV151 in avermectin production. Deletion of SAV151 increased avermectin yield ~1-fold in ATCC31267, and this phenotype was complemented by a single copy of SAV151. Overexpression of SAV151 in ATCC31267 reduced avermectin yield by ~70%. RT-PCR analysis showed that the promoting effect of SAV151 deletion on avermectin production was not due to alteration of ave genes at the transcriptional level. SAV151 negatively regulated the transcription of itself and of the adjacent transcriptional unit SAV152-SAV153-SAV154. In chromatin immunoprecipitation and gel shift assays, purified His6-tagged SAV151 protein bound to the bidirectional SAV151-SAV152 promoter region. SAV151 bound to two palindromic sequences in this region and thereby repressed transcription from both directions. Two of the SAV151 target genes, SAV152 (which encodes a putative dehydrogenase) and SAV154 (which encodes a putative hydrolase), had promoting effects on avermectin production. Our findings provide the basis for a strategy to increase avermectin production by controlling SAV151 and its target genes.

  7. Tamoxifen increases nuclear respiratory factor 1 transcription by activating estrogen receptor beta and AP-1 recruitment to adjacent promoter binding sites.

    PubMed

    Ivanova, Margarita M; Luken, Kristen H; Zimmer, Amber S; Lenzo, Felicia L; Smith, Ryan J; Arteel, Maia W; Kollenberg, Tara J; Mattingly, Kathleen A; Klinge, Carolyn M

    2011-04-01

    Little is known about endogenous estrogen receptor β (ERβ) gene targets in human breast cancer. We reported that estradiol (E(2)) induces nuclear respiratory factor-1 (NRF-1) transcription through ERα in MCF-7 breast cancer cells. Here we report that 4-hydroxytamoxifen (4-OHT), with an EC(50) of ~1.7 nM, increases NRF-1 expression by recruiting ERβ, cJun, cFos, CBP, and RNA polymerase II to and dismissing NCoR from the NRF1 promoter. Promoter deletion and transient transfection studies showed that the estrogen response element (ERE) is essential and that an adjacent AP-1 site contributes to maximal 4-OHT-induced NRF-1 transcription. siRNA knockdown of ERβ revealed that ERβ inhibits basal NRF-1 expression and is required for 4-OHT-induced NRF-1 transcription. An AP-1 inhibitor blocked 4-OHT-induced NRF-1 expression. The 4-OHT-induced increase in NRF-1 resulted in increased transcription of NRF-1 target CAPNS1 but not CYC1, CYC2, or TFAM despite increased NRF-1 coactivator PGC-1α protein. The absence of TFAM induction corresponds to a lack of Akt-dependent phosphorylation of NRF-1 with 4-OHT treatment. Overexpression of NRF-1 inhibited 4-OHT-induced apoptosis and siRNA knockdown of NRF-1 increased apoptosis, indicating an antiapoptotic role for NRF-1. Overall, NRF-1 expression and activity is regulated by 4-OHT via endogenous ERβ in MCF-7 cells.

  8. Emergence of hematopoietic stem and progenitor cells involves a Chd1-dependent increase in total nascent transcription

    PubMed Central

    Koh, Fong Ming; Lizama, Carlos O.; Wong, Priscilla; Hawkins, John S.; Ramalho-Santos, Miguel

    2015-01-01

    Lineage specification during development involves reprogramming of transcriptional states, but little is known about how this is regulated in vivo. The chromatin remodeler chomodomain helicase DNA-binding protein 1 (Chd1) promotes an elevated transcriptional output in mouse embryonic stem cells. Here we report that endothelial-specific deletion of Chd1 leads to loss of definitive hematopoietic progenitors, anemia, and lethality by embryonic day (E)15.5. Mutant embryos contain normal numbers of E10.5 intraaortic hematopoietic clusters that express Runx1 and Kit, but these clusters undergo apoptosis and fail to mature into blood lineages in vivo and in vitro. Hematopoietic progenitors emerging from the aorta have an elevated transcriptional output relative to structural endothelium, and this elevation is Chd1-dependent. In contrast, hematopoietic-specific deletion of Chd1 using Vav-Cre has no apparent phenotype. Our results reveal a new paradigm of regulation of a developmental transition by elevation of global transcriptional output that is critical for hemogenesis and may play roles in other contexts. PMID:25831528

  9. Increase of both transcription and translation activities following separate irradiation of the in vitro system components with He-Ne laser.

    PubMed

    Vacca, R A; Marra, E; Quagliariello, E; Greco, M

    1994-09-15

    To gain insight into the mechanism by which cell irradiation with low power continuous wave He-Ne laser (632.8 nm) causes a general stimulation of biosynthetic properties, separate components of transcription and translation systems were irradiated (energy dose 2 Joules/cm2; laser power 12 mW) with measurements made of in vitro RNA and protein synthesis. In addition, all tested components were investigated with respect to influence of laser irradiation on their conformation, as spectroscopically monitored. In all cases an increase in both transcription and translation activities was found with a significant change in absorbance/fluorescence spectra.

  10. Increased Accumulation of Carbohydrates and Decreased Photosynthetic Gene Transcript Levels in Wheat Grown at an Elevated CO2 Concentration in the Field.

    PubMed Central

    Nie, G.; Hendrix, D. L.; Webber, A. N.; Kimball, B. A.; Long, S. P.

    1995-01-01

    Repression of photosynthetic genes by increased soluble carbohydrate concentrations may explain acclimation of photosynthesis to elevated CO2 concentration. This hypothesis was examined in a field crop of spring wheat (Triticum aestivum L.) grown at both ambient (approximately 360 [mu]mol mol-1) and elevated (550 [mu]mol mol-1) atmospheric CO2 concentrations using free-air CO2 enrichment at Maricopa, Arizona. The correspondence of steady-state levels of mRNA transcripts (coding for the 83-kD photosystem I apoprotein, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, phosphoglycerokinase, and the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase) with leaf carbohydrate concentrations (glucose-6-phosphate, glucose, fructose, sucrose, fructans, and starch) was examined at different stages of crop and leaf development and through the diurnal cycle. Overall only a weak correspondence between increased soluble carbohydrate concentrations and decreased levels for nuclear gene transcripts was found. The difference in soluble carbohydrate concentration between leaves grown at elevated and current ambient CO2 concentrations diminished with crop development, whereas the difference in transcript levels increased. In the flag leaf, soluble carbohydrate concentrations declined markedly with the onset of grain filling; yet transcript levels also declined. The results suggest that, whereas the hypothesis may hold well in model laboratory systems, many other factors modified its significance in this field wheat crop. PMID:12228521

  11. Increasing the dynamic control space of mammalian transcription devices by combinatorial assembly of homologous regulatory elements from different bacterial species.

    PubMed

    Bacchus, William; Weber, Wilfried; Fussenegger, Martin

    2013-01-01

    Prokaryotic transcriptional regulatory elements are widely utilized building blocks for constructing regulatory genetic circuits adapted for mammalian cells and have found their way into a broad range of biotechnological applications. Prokaryotic transcriptional repressors, fused to eukaryotic transactivation or repression domains, compose the transcription factor, which binds and adjusts transcription from chimeric promoters containing the repressor-specific operator sequence. Escherichia coli and Chlamydia trachomatis share common features in the regulatory mechanism of the biosynthesis of l-tryptophan. The repressor protein TrpR of C. trachomatis regulates the trpRBA operon and the TrpR of E. coli regulates the trpEDCBA operon, both requiring l-tryptophan as a co-repressor. Fusion of these bacterial repressors to the VP16 transactivation domain of Herpes simplex virus creates synthetic transactivators that could bind and activate chimeric promoters, assembled by placing repressor-specific operator modules adjacent to a minimal promoter, in an l-tryptophan-adjustable manner. Combinations of different transactivator and promoter variants from the same or different bacterial species resulted in a multitude of regulatory systems where l-tryptophan regulation properties, background noise, and maximal gene expression levels were significantly diverse. Different l-tryptophan analogues showed diverse regulatory capacity depending on the promoter/transactivator combination. We believe the systems approach to rationally choose promoters, transactivators and inducer molecules, to obtain desired and predefined genetic expression dynamics and control profiles, will significantly advance the design of new regulatory circuits as well as improving already existing ones. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Inhibition of EHMT2/G9a epigenetically increases the transcription of Beclin-1 via an increase in ROS and activation of NF-κB

    PubMed Central

    Park, Sang Eun; Yi, Hye Jin; Suh, Nayoung; Park, Yun-Yong; Koh, Jae-Young; Jeong, Seong-Yun; Cho, Dong-Hyung; Kim, Choung-Soo; Hwang, Jung Jin

    2016-01-01

    We previously reported that BIX-01294 (BIX), a small molecular inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2/G9a), induces reactive oxygen species (ROS)-dependent autophagy in MCF-7 cells. Herein, we analyzed the epigenetic mechanism that regulates the transcription of Beclin-1, a tumor suppressor and an autophagy-related gene (ATG). Inhibition of EHMT2 reduced dimethylation of lysine 9 on histone H3 (H3K9me2) and dissociated EHMT2 and H3K9me2 from the promoter of Beclin-1. To this promoter, RNA polymerase II and nuclear factor kappa B (NF-κB) were recruited in a ROS-dependent manner, resulting in transcriptional activation. Moreover, treatment with BIX reversed the suppression of Beclin-1 by the cooperative action of EHMT2 and DNA methyltransferase 1 (DNMT1). Accordingly, a combination treatment with BIX and 5-Aza-2′-deoxycytidine (5-Aza-Cd), a DNMT1 inhibitor, exerted a synergistic effect on Beclin-1 expression. Importantly, high levels of EHMT2 expression showed a significant association with low levels of Beclin-1 expression, which was related to a poor prognosis. These findings suggest that EHMT2 can directly repress Beclin-1 and that the inhibition of EHMT2 may be a useful therapeutic approach for cancer prevention by activating autophagy. PMID:27174920

  13. Increased IL17A, IFNG, and FOXP3 Transcripts in Moderate-Severe Psoriasis: A Major Influence Exerted by IL17A in Disease Severity

    PubMed Central

    de Oliveira, Priscilla Stela Santana; Pereira, Michelly Cristiny; Silva de Paula, Simão Kalebe; Lima, Emerson Vasconcelos Andrade; Lima, Mariana Modesto de Andrade; de Arruda, Rodrigo Gomes; Duarte, Ângela Luzia Branco Pinto; Pitta, Ivan da Rocha

    2016-01-01

    Psoriasis is a chronic and recurrent dermatitis, mediated by keratinocytes and T cells. Several proinflammatory cytokines contribute to formation and maintenance of psoriatic plaque. The Th1/Th17 pathways and some of IL-1 family members were involved in psoriasis pathogenesis and could contribute to disease activity. Therefore, we sought to analyse skin transcript levels of IL17A, IL22, RORC, IL8, IFNG, IL33, IL36A, FOXP3, and IL10 and correlate with clinic of patients with plaque-type psoriasis. In order to conduct that, we collected punch biopsies from lesional skin and obtained tissue RNA. After reverse transcription, qRT-PCR quantified the relative mRNA expression. The main results revealed increased transcripts levels of IL17A, IFNG, and FOXP3 in moderate-severe patients. Despite this, only IL17A can increase the chance to worsen disease severity. We also observed many significant positive correlations between each transcript. In conclusion, IL17A is elevated in lesional skin from psoriasis patients and plays crucial role in disease severity. PMID:28042206

  14. Increased IL17A, IFNG, and FOXP3 Transcripts in Moderate-Severe Psoriasis: A Major Influence Exerted by IL17A in Disease Severity.

    PubMed

    de Oliveira, Priscilla Stela Santana; Pereira, Michelly Cristiny; Silva de Paula, Simão Kalebe; Lima, Emerson Vasconcelos Andrade; Lima, Mariana Modesto de Andrade; de Arruda, Rodrigo Gomes; de Oliveira, Wagner Luís Mendes; Duarte, Ângela Luzia Branco Pinto; Pitta, Ivan da Rocha; Rêgo, Moacyr Jesus Melo Barreto; Galdino da Rocha Pitta, Maira

    2016-01-01

    Psoriasis is a chronic and recurrent dermatitis, mediated by keratinocytes and T cells. Several proinflammatory cytokines contribute to formation and maintenance of psoriatic plaque. The Th1/Th17 pathways and some of IL-1 family members were involved in psoriasis pathogenesis and could contribute to disease activity. Therefore, we sought to analyse skin transcript levels of IL17A, IL22, RORC, IL8, IFNG, IL33, IL36A, FOXP3, and IL10 and correlate with clinic of patients with plaque-type psoriasis. In order to conduct that, we collected punch biopsies from lesional skin and obtained tissue RNA. After reverse transcription, qRT-PCR quantified the relative mRNA expression. The main results revealed increased transcripts levels of IL17A, IFNG, and FOXP3 in moderate-severe patients. Despite this, only IL17A can increase the chance to worsen disease severity. We also observed many significant positive correlations between each transcript. In conclusion, IL17A is elevated in lesional skin from psoriasis patients and plays crucial role in disease severity.

  15. A Mutation in a Saccharomyces Cerevisiae Gene (Rad3) Required for Nucleotide Excision Repair and Transcription Increases the Efficiency of Mismatch Correction

    PubMed Central

    Yang, Y.; Johnson, A. L.; Johnston, L. H.; Siede, W.; Friedberg, E. C.; Ramachandran, K.; Kunz, B. A.

    1996-01-01

    RAD3 functions in DNA repair and transcription in Saccharomyces cerevisiae and particular rad3 alleles confer a mutator phenotype, possibly as a consequence of defective mismatch correction. We assessed the potential involvement of the Rad3 protein in mismatch correction by comparing heteroduplex repair in isogenic rad3-1 and wild-type strains. The rad3-1 allele increased the spontaneous mutation rate but did not prevent heteroduplex repair or bias its directionality. Instead, the efficiency of mismatch correction was enhanced in the rad3-1 strain. This surprising result prompted us to examine expression of yeast mismatch repair genes. We determined that MSH2, but not MLH1, is transcriptionally regulated during the cell-cycle like PMS1, and that rad3-1 does not increase the transcript levels for these genes in log phase cells. These observations suggest that the rad3-1 mutation gives rise to an enhanced efficiency of mismatch correction via a process that does not involve transcriptional regulation of mismatch repair. Interestingly, mismatch repair also was more efficient when error-editing by yeast DNA polymerase δ was eliminated. We discuss our results in relation to possible mechanisms that may link the rad3-1 mutation to mismatch correction efficiency. PMID:8889512

  16. Hsp90 inhibition increases SOCS3 transcript and regulates migration and cell death in chronic lymphocytic leukemia

    PubMed Central

    Chen, Timothy L.; Gupta, Nikhil; Lehman, Amy; Ruppert, Amy S.; Yu, Lianbo; Oakes, Christopher C.; Claus, Rainer; Plass, Christoph; Maddocks, Kami J.; Andritsos, Leslie; Jones, Jeffery A.; Lucas, David M.; Johnson, Amy J.; Byrd, John C.; Hertlein, Erin

    2016-01-01

    Epigenetic or transcriptional silencing of important tumor suppressors has been described to contribute to cell survival and tumorigenesis in chronic lymphocytic leukemia (CLL). Using gene expression microarray analysis, we found that thousands of genes are repressed more than 2-fold in CLL compared to normal B cells; however therapeutic approaches to reverse this have been limited in CLL. Following treatment with the Hsp90 inhibitor 17-DMAG, a significant number of these repressed genes were significantly re-expressed. One of the genes significantly repressed in CLL and up-regulated by 17-DMAG was suppressor of cytokine signaling 3, (SOCS3). SOCS3 has been shown to be silenced in solid tumors as well as myeloid leukemia; however little is known about the regulation in CLL. We found that 17-DMAG induces expression of SOCS3 by via the activation of p38 signaling, and subsequently inhibits AKT and STAT3 phosphorylation resulting in downstream effects on cell migration and survival. We therefore suggest that SOCS3 is an important signaling protein in CLL, and Hsp90 inhibitors represent a novel approach to target transcriptional repression in B cell lymphoproliferative disorders which exhibit a substantial degree of gene repression. PMID:27107422

  17. Hsp90 inhibition increases SOCS3 transcript and regulates migration and cell death in chronic lymphocytic leukemia.

    PubMed

    Chen, Timothy L; Gupta, Nikhil; Lehman, Amy; Ruppert, Amy S; Yu, Lianbo; Oakes, Christopher C; Claus, Rainer; Plass, Christoph; Maddocks, Kami J; Andritsos, Leslie; Jones, Jeffery A; Lucas, David M; Johnson, Amy J; Byrd, John C; Hertlein, Erin

    2016-05-10

    Epigenetic or transcriptional silencing of important tumor suppressors has been described to contribute to cell survival and tumorigenesis in chronic lymphocytic leukemia (CLL). Using gene expression microarray analysis, we found that thousands of genes are repressed more than 2-fold in CLL compared to normal B cells; however therapeutic approaches to reverse this have been limited in CLL. Following treatment with the Hsp90 inhibitor 17-DMAG, a significant number of these repressed genes were significantly re-expressed. One of the genes significantly repressed in CLL and up-regulated by 17-DMAG was suppressor of cytokine signaling 3, (SOCS3). SOCS3 has been shown to be silenced in solid tumors as well as myeloid leukemia; however little is known about the regulation in CLL. We found that 17-DMAG induces expression of SOCS3 by via the activation of p38 signaling, and subsequently inhibits AKT and STAT3 phosphorylation resulting in downstream effects on cell migration and survival. We therefore suggest that SOCS3 is an important signaling protein in CLL, and Hsp90 inhibitors represent a novel approach to target transcriptional repression in B cell lymphoproliferative disorders which exhibit a substantial degree of gene repression.

  18. Resveratrol increases anti-aging Klotho gene expression via the activating transcription factor 3/c-Jun complex-mediated signaling pathway.

    PubMed

    Hsu, Shih-Che; Huang, Shih-Ming; Chen, Ann; Sun, Chiao-Yin; Lin, Shih-Hua; Chen, Jin-Shuen; Liu, Shu-Ting; Hsu, Yu-Juei

    2014-08-01

    The Klotho gene functions as an aging suppressor gene. Evidence from animal models suggests that induction of Klotho expression may be a potential treatment for age-associated diseases. However, the molecular mechanism involved in regulating renal Klotho gene expression remains unclear. In this study, we determined that resveratrol, a natural polyphenol, induced renal Klotho expression both in vivo and in vitro. In the mouse kidney, resveratrol administration markedly increased both Klotho mRNA and protein expression. In resveratrol-treated NRK-52E cells, increased Klotho expression was accompanied by the upregulation and nuclear translocation of activating transcription factor 3 (ATF3) and c-Jun. ATF3 or c-Jun overexpression enhanced the transcriptional activation of Klotho. Conversely, resveratrol-induced Klotho expression was attenuated in the presence of dominant-negative ATF3 or c-Jun. Coimmunoprecipitation and a chromatin immunoprecipitation assay revealed that ATF3 physically interacted with c-Jun and that the ATF3/c-Jun complex directly bound to the Klotho promoter through ATF3- and AP-1-binding elements. c-Jun cotransfection augmented the effects of ATF3 on Klotho transcription in vitro. Although Sirtuin 1 mRNA expression was induced by resveratrol and involved in regulating Klotho mRNA expression, it was not the primary cause for the aforementioned ATF3/c-Jun pathway. In summary, resveratrol enhances the renal expression of the anti-aging Klotho gene, and the transcriptional factors ATF3 and c-Jun functionally interact and coordinately regulate the resveratrol-mediated transcriptional activation of Klotho.

  19. Increase of O6-methylguanine-DNA-methyltransferase and N3-methyladenine glycosylase RNA transcripts in rat hepatoma cells treated with DNA-damaging agents

    SciTech Connect

    Laval, F. )

    1991-05-15

    A variety of DNA-damaging agents increase the O6-methylguanine-DNA-methyltransferase (transferase) and the N3-methyladenine (3-meAde)-DNA-glycosylase activities in a rat hepatoma cell line (H4 cells). Using two cDNA expressing either the rat 3-meAde-DNA-glycosylase or the transferase, the level of mRNA transcripts was measured by hybridization in H4 cells treated with three different inducing agents, gamma-rays, cis-dichlorodiammine platinum II or N-methyl-9-hydroxy ellipticinium. The two mRNA increased 24 hours after the cell treatments but this enhanced transcription was a transient phenomenon, as it was no longer observed after 96 hours. No significant DNA amplification was detectable in the treated cells.

  20. Overexpression of the yeast transcription activator Msn2 confers furfural resistance and increases the initial fermentation rate in ethanol production.

    PubMed

    Sasano, Yu; Watanabe, Daisuke; Ukibe, Ken; Inai, Tomomi; Ohtsu, Iwao; Shimoi, Hitoshi; Takagi, Hiroshi

    2012-04-01

    Lignocellulosic biomass is a promising source for bioethanol production, because it is abundant worldwide and has few competing uses. However, the treatment of lignocelllulosic biomass with weak acid to release cellulose and hemicellulose generates many kinds of byproducts including furfural and 5-hydroxymethylfurfural, which inhibit fermentation by yeast, because they generate reactive oxygen species (ROS) in cells. In order to acquire high tolerance to oxidative stress in bioethanol yeast strains, we focused on the transcription activator Msn2 of Saccharomyces cerevisiae, which regulates numerous genes involved in antioxidative stress responses, and constructed bioethanol yeast strains that overexpress Msn2 constitutively. The Msn2-overexpressing bioethanol strains showed tolerance to oxidative stress, probably due to the high-level expression of various antioxidant enzyme genes. Unexpectedly, these strains showed ethanol sensitivity compared with the control strain, probably due to imbalance of the expression level between Msn2 and Msn4. In the presence of furfural, the engineered strains exhibited reduced intracellular ROS levels, and showed rapid growth compared with the control strain. The fermentation test in the presence of furfural revealed that the Msn2-overexpressing strains showed improvement of the initial rate of fermentation. Our results indicate that overexpression of the transcription activator Msn2 in bioethanol yeast strains confers furfural tolerance by reducing the intracellular ROS levels and enhances the initial rate of fermentation in the presence of furfural, suggesting that these strains are capable of adapting rapidly to various compounds that inhibit fermentation by inducing ROS accumulation. Our results not only promise to improve bioethanol production from lignocellulosic biomass, but also provide novel insights for molecular breeding of industrial yeast strains.

  1. Combination of vorinostat and adenovirus-TRAIL exhibits a synergistic antitumor effect by increasing transduction and transcription of TRAIL in lung cancer cells.

    PubMed

    Kim, D R; Park, M-Y; Lee, C-S; Shim, S-H; Yoon, H-I; Lee, J H; Sung, M-W; Kim, Y-S; Lee, C-T

    2011-07-01

    Soluble TRAIL and adenovirus (ad)-TRAIL exhibit a strong antitumor effect by inducing apoptosis. Vorinostat is the histone deacetylase (HDAC) inhibitor that induces cell death in cancer cell lines and regulates the expression of epigenetically silenced genes, such as Coxackie adenoviral receptor (CAR), the receptor for adenoviral entry. We propose a new strategy in which vorinostat will induce high expression of ad-TRAIL and a strong antitumor response, and investigated the mechanism involved. The effect of vorinostat on transcription and expression of TRAIL from ad-TRAIL-transduced lung cancer cells were confirmed by reverse transciption-PCR (RT-PCR), quantitative real time-PCR and western blot assay. Anti-tumor effects were measured after cotreatment of vorinostat and ad-TRAIL, and the drug interactions were analyzed. After combined treatment of vorinostat and ad-TRAIL, apoptosis and western blot assays for Akt, Bcl-2 and caspase were performed. Vorinostat increased the expression of CAR in lung cancer cell lines and increased the expression of luciferase (luc) from ad-luc-transduced cells and TRAIL from ad-TRAIL-transduced cells. RT-PCR and quantitative real time-PCR, after sequential vorinostat treatment, revealed that vorinostat may enhance TRAIL expression from ad-TRAIL by increasing transduction through enhanced CAR expression and increasing adenoviral transgene transcription. Combined vorinostat and ad-TRAIL treatment showed the synergistic anti-tumor effect in lung cancer cell lines. Combined vorinostat and ad-TRAIL induced stronger apoptosis induction, suppression of NF-κB activation and breakdown of the anti-apoptotic molecule Bcl-2. In conclusion, the vorinostat synergistically enhanced the anti-tumor effect of ad-TRAIL by (1) increasing adenoviral transduction through the increased expression of CAR and (2) increasing adenoviral transgene (TRAIL) transcription in lung cancer cell lines.

  2. Requirement of Histone Methyltransferase SMYD3 for Estrogen Receptor-mediated Transcription*

    PubMed Central

    Kim, Hyunjung; Heo, Kyu; Kim, Jeong Hoon; Kim, Kyunghwan; Choi, Jongkyu; An, Woojin

    2009-01-01

    SMYD3 is a SET domain-containing protein with histone methyltransferase activity on histone H3–K4. Recent studies showed that SMYD3 is frequently overexpressed in different types of cancer cells, but how SMYD3 regulates the development and progression of these malignancies remains unknown. Here, we report the previously unrecognized role of SMYD3 in estrogen receptor (ER)-mediated transcription via its histone methyltransferase activity. We demonstrate that SMYD3 functions as a coactivator of ERα and potentiates ERα activity in response to ligand. SMYD3 directly interacts with the ligand binding domain of ER and is recruited to the proximal promoter regions of ER target genes upon gene induction. Importantly, our chromatin immunoprecipitation analyses provide compelling evidence that SMYD3 is responsible for the accumulation of di- and trimethylation of H3–K4 at the induced ER target genes. Furthermore, RNA interference-directed down-regulation of SMYD3 reveals that SMYD3 is required for ER-regulated gene transcription in estrogen signaling pathway. Thus, our results identify SMYD3 as a new coactivator for ER-mediated transcription, providing a possible link between SMYD3 overexpression and breast cancer. PMID:19509295

  3. SND2, a NAC transcription factor gene, regulates genes involved in secondary cell wall development in Arabidopsis fibres and increases fibre cell area in Eucalyptus.

    PubMed

    Hussey, Steven G; Mizrachi, Eshchar; Spokevicius, Antanas V; Bossinger, Gerd; Berger, Dave K; Myburg, Alexander A

    2011-12-01

    NAC domain transcription factors initiate secondary cell wall biosynthesis in Arabidopsis fibres and vessels by activating numerous transcriptional regulators and biosynthetic genes. NAC family member SND2 is an indirect target of a principal regulator of fibre secondary cell wall formation, SND1. A previous study showed that overexpression of SND2 produced a fibre cell-specific increase in secondary cell wall thickness in Arabidopsis stems, and that the protein was able to transactivate the cellulose synthase8 (CesA8) promoter. However, the full repertoire of genes regulated by SND2 is unknown, and the effect of its overexpression on cell wall chemistry remains unexplored. We overexpressed SND2 in Arabidopsis and analyzed homozygous lines with regards to stem chemistry, biomass and fibre secondary cell wall thickness. A line showing upregulation of CesA8 was selected for transcriptome-wide gene expression profiling. We found evidence for upregulation of biosynthetic genes associated with cellulose, xylan, mannan and lignin polymerization in this line, in agreement with significant co-expression of these genes with native SND2 transcripts according to public microarray repositories. Only minor alterations in cell wall chemistry were detected. Transcription factor MYB103, in addition to SND1, was upregulated in SND2-overexpressing plants, and we detected upregulation of genes encoding components of a signal transduction machinery recently proposed to initiate secondary cell wall formation. Several homozygous T4 and hemizygous T1 transgenic lines with pronounced SND2 overexpression levels revealed a negative impact on fibre wall deposition, which may be indirectly attributable to excessive overexpression rather than co-suppression. Conversely, overexpression of SND2 in Eucalyptus stems led to increased fibre cross-sectional cell area. This study supports a function for SND2 in the regulation of cellulose and hemicellulose biosynthetic genes in addition of those

  4. SND2, a NAC transcription factor gene, regulates genes involved in secondary cell wall development in Arabidopsis fibres and increases fibre cell area in Eucalyptus

    PubMed Central

    2011-01-01

    Background NAC domain transcription factors initiate secondary cell wall biosynthesis in Arabidopsis fibres and vessels by activating numerous transcriptional regulators and biosynthetic genes. NAC family member SND2 is an indirect target of a principal regulator of fibre secondary cell wall formation, SND1. A previous study showed that overexpression of SND2 produced a fibre cell-specific increase in secondary cell wall thickness in Arabidopsis stems, and that the protein was able to transactivate the cellulose synthase8 (CesA8) promoter. However, the full repertoire of genes regulated by SND2 is unknown, and the effect of its overexpression on cell wall chemistry remains unexplored. Results We overexpressed SND2 in Arabidopsis and analyzed homozygous lines with regards to stem chemistry, biomass and fibre secondary cell wall thickness. A line showing upregulation of CesA8 was selected for transcriptome-wide gene expression profiling. We found evidence for upregulation of biosynthetic genes associated with cellulose, xylan, mannan and lignin polymerization in this line, in agreement with significant co-expression of these genes with native SND2 transcripts according to public microarray repositories. Only minor alterations in cell wall chemistry were detected. Transcription factor MYB103, in addition to SND1, was upregulated in SND2-overexpressing plants, and we detected upregulation of genes encoding components of a signal transduction machinery recently proposed to initiate secondary cell wall formation. Several homozygous T4 and hemizygous T1 transgenic lines with pronounced SND2 overexpression levels revealed a negative impact on fibre wall deposition, which may be indirectly attributable to excessive overexpression rather than co-suppression. Conversely, overexpression of SND2 in Eucalyptus stems led to increased fibre cross-sectional cell area. Conclusions This study supports a function for SND2 in the regulation of cellulose and hemicellulose biosynthetic

  5. Heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by increasing protein translation of selected transcripts in cancer cells.

    PubMed

    Chang, Elizabeth T; Parekh, Palak R; Yang, Qingyuan; Nguyen, Duc M; Carrier, France

    2016-03-01

    The heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by coordinating the translation of selected transcripts associated with proliferation and survival. hnRNP A18 binds to and stabilizes the transcripts of pro-survival genes harboring its RNA signature motif in their 3'UTRs. hnRNP A18 binds to ATR, RPA, TRX, HIF-1α and several protein translation factor mRNAs on polysomes and increases de novo protein translation under cellular stress. Most importantly, down regulation of hnRNP A18 decreases proliferation, invasion and migration in addition to significantly reducing tumor growth in two mouse xenograft models, melanoma and breast cancer. Moreover, tissue microarrays performed on human melanoma, prostate, breast and colon cancer indicate that hnRNP A18 is over expressed in 40 to 60% of these malignant tissue as compared to normal adjacent tissue. Immunohistochemistry data indicate that hnRNP A18 is over expressed in the stroma and hypoxic areas of human tumors. These data thus indicate that hnRNP A18 can promote tumor growth in in vivo models by coordinating the translation of pro-survival transcripts to support the demands of proliferating cells and increase survival under cellular stress. hnRNP A18 therefore represents a new target to selectively inhibit protein translation in tumor cells.

  6. Heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by increasing protein translation of selected transcripts in cancer cells

    PubMed Central

    Chang, Elizabeth T.; Parekh, Palak R.; Yang, Qingyuan; Nguyen, Duc M.; Carrier, France

    2016-01-01

    The heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by coordinating the translation of selected transcripts associated with proliferation and survival. hnRNP A18 binds to and stabilizes the transcripts of pro-survival genes harboring its RNA signature motif in their 3′UTRs. hnRNP A18 binds to ATR, RPA, TRX, HIF-1α and several protein translation factor mRNAs on polysomes and increases de novo protein translation under cellular stress. Most importantly, down regulation of hnRNP A18 decreases proliferation, invasion and migration in addition to significantly reducing tumor growth in two mouse xenograft models, melanoma and breast cancer. Moreover, tissue microarrays performed on human melanoma, prostate, breast and colon cancer indicate that hnRNP A18 is over expressed in 40 to 60% of these malignant tissue as compared to normal adjacent tissue. Immunohistochemistry data indicate that hnRNP A18 is over expressed in the stroma and hypoxic areas of human tumors. These data thus indicate that hnRNP A18 can promote tumor growth in in vivo models by coordinating the translation of pro-survival transcripts to support the demands of proliferating cells and increase survival under cellular stress. hnRNP A18 therefore represents a new target to selectively inhibit protein translation in tumor cells. PMID:26824423

  7. Effects of Zn fertilization on hordein transcripts at early developmental stage of barley grain and correlation with increased Zn concentration in the mature grain.

    PubMed

    Uddin, Mohammad Nasir; Kaczmarczyk, Agnieszka; Vincze, Eva

    2014-01-01

    Zinc deficiency is causing malnutrition for nearly one third of world populations. It is especially relevant in cereal-based diets in which low amounts of mineral and protein are present. In biological systems, Zn is mainly associated with protein. Cereal grains contain the highest Zn concentration during early developmental stage. Although hordeins are the major storage proteins in the mature barley grain and suggested to be involved in Zn binding, very little information is available regarding the Zn fertilization effects of hordein transcripts at early developmental stage and possible incorporation of Zn with hordein protein of matured grain. Zinc fertilization experiments were conducted in a greenhouse with barley cv. Golden Promise. Zn concentration of the matured grain was measured and the results showed that the increasing Zn fertilization increased grain Zn concentration. Quantitative real time PCR showed increased level of total hordein transcripts upon increasing level of Zn fertilization at 10 days after pollination. Among the hordein transcripts the amount of B-hordeins was highly correlated with the Zn concentration of matured grain. In addition, protein content of the matured grain was analysed and a positive linear relationship was found between the percentage of B-hordein and total grain Zn concentration while C-hordein level decreased. Zn sensing dithizone assay was applied to localize Zn in the matured grain. The Zn distribution was not limited to the embryo and aleurone layer but was also present in the outer part of the endosperm (sub-aleurone layers) which known to be rich in proteins including B-hordeins. Increased Zn fertilization enriched Zn even in the endosperm. Therefore, the increased amount of B-hordein and decreased C-hordein content suggested that B-hordein upregulation or difference between B and C hordein could be one of the key factors for Zn biofortification of cereal grains due to the Zn fertilization.

  8. Effects of Zn Fertilization on Hordein Transcripts at Early Developmental Stage of Barley Grain and Correlation with Increased Zn Concentration in the Mature Grain

    PubMed Central

    Uddin, Mohammad Nasir; Kaczmarczyk, Agnieszka; Vincze, Eva

    2014-01-01

    Zinc deficiency is causing malnutrition for nearly one third of world populations. It is especially relevant in cereal-based diets in which low amounts of mineral and protein are present. In biological systems, Zn is mainly associated with protein. Cereal grains contain the highest Zn concentration during early developmental stage. Although hordeins are the major storage proteins in the mature barley grain and suggested to be involved in Zn binding, very little information is available regarding the Zn fertilization effects of hordein transcripts at early developmental stage and possible incorporation of Zn with hordein protein of matured grain. Zinc fertilization experiments were conducted in a greenhouse with barley cv. Golden Promise. Zn concentration of the matured grain was measured and the results showed that the increasing Zn fertilization increased grain Zn concentration. Quantitative real time PCR showed increased level of total hordein transcripts upon increasing level of Zn fertilization at 10 days after pollination. Among the hordein transcripts the amount of B-hordeins was highly correlated with the Zn concentration of matured grain. In addition, protein content of the matured grain was analysed and a positive linear relationship was found between the percentage of B-hordein and total grain Zn concentration while C-hordein level decreased. Zn sensing dithizone assay was applied to localize Zn in the matured grain. The Zn distribution was not limited to the embryo and aleurone layer but was also present in the outer part of the endosperm (sub-aleurone layers) which known to be rich in proteins including B-hordeins. Increased Zn fertilization enriched Zn even in the endosperm. Therefore, the increased amount of B-hordein and decreased C-hordein content suggested that B-hordein upregulation or difference between B and C hordein could be one of the key factors for Zn biofortification of cereal grains due to the Zn fertilization. PMID:25250985

  9. p53 increase mitochondrial copy number via up-regulation of mitochondrial transcription factor A in colorectal cancer

    PubMed Central

    Zhang, Linhao; Zhou, Hongying; Fang, Dingzhi; Feng, Shi

    2016-01-01

    In colorectal cancer, no study has been carried out discovering the relationship among p53, mitochondrial transcription factor A (TFAM) expression and change of mitochondrial DNA (mtDNA) copy number. In our study, co-expression of p53 and TFAM was observed in colon adenocarcinoma tissues, paracancerous tissues and 9 colorectal cancer cell lines. Then, a significant linear correlation was established between either p53 or TFAM expression and advanced TNM stage, positive lymph nodes and low 5-year survival rate in patients with colon adenocarcinoma. Additionally, advanced TNM stage, large tumor burden, presence of distant metastasis, and high TFAM expression were significantly related to poor overall 5-years survival. Moreover, alteration of p53 expression could change TFAM expression but TFAM could not influence p53 expression, and p53 could enhance TFAM expression via binding to TFAM promoter. While, both of p53 and TFAM expression could incrase mtDNA copy number in vitro. In conclusions, p53 might incrase mtDNA copy number through its regulation on TFAM expression via TFAMpromoter. PMID:27732955

  10. Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes.

    PubMed

    Guerrero, F D; Jones, J T; Mullet, J E

    1990-07-01

    Reduction of turgor in pea shoots caused the accumulation of several poly(A) RNAs. cDNA clones derived from three different poly(A) RNAs which accumulate in wilted pea shoots were isolated, sequenced and expression of the corresponding genes examined. Clone 7a encoded a 289 amino acid protein. The C-terminal 180 amino acids of this protein were homologous to soybean nodulin-26. RNA hybridizing to cDNA 7a was abundant in roots, and induced in shoots by dehydration, heat shock and to a small extent by ABA. Hydropathic plots indicate that the protein encoded by cDNA 7a contains six potential membrane spanning domains similar to proteins which form ion channels. Clone 15a encoded a 363 amino acid protein with high homology to cysteine proteases. RNA hybridizing to cDNA 15a was more abundant in roots than shoots of control plants. Dehydration of pea shoots induced cDNA 15a mRNA levels whereas heat shock or ABA treatment did not. Clone 26g encoded a 508 amino acid protein with 30% residue identity to several aldehyde dehydrogenases. RNA hybridizing to cDNA 26g was induced by dehydration of shoots but not roots and heat shock and ABA did not modulate RNA levels. Levels of the three poly(A) RNAs increased 4-6-fold by 4 h after wilting and this increase was not altered by pretreatment of shoots with cycloheximide. When wilted shoots were rehydrated, RNA hybridizing to cDNA 26g declined to pre-stress levels within 2 h. Run-on transcription experiments using nuclei from pea shoots showed that transcription of the genes which encode the three poly(A) RNAs was induced within 30 min following reduction of shoot turgor. One of the genes showed a further increase in transcription by 4 h after dehydration whereas transcription of the other 2 genes declined. These results indicate that plant cells respond to changes in cell turgor by rapidly increasing transcription of several genes. Furthermore, the expression of the turgor-responsive genes varies with respect to the time course of

  11. Effects of Increasing the Affinity of CarD for RNA Polymerase on Mycobacterium tuberculosis Growth, rRNA Transcription, and Virulence.

    PubMed

    Garner, Ashley L; Rammohan, Jayan; Huynh, Jeremy P; Onder, Lucas M; Chen, James; Bae, Brian; Jensen, Drake; Weiss, Leslie A; Manzano, Ana Ruiz; Darst, Seth A; Campbell, Elizabeth A; Nickels, Bryce E; Galburt, Eric A; Stallings, Christina L

    2017-02-15

    CarD is an essential RNA polymerase (RNAP) interacting protein in Mycobacterium tuberculosis that stimulates formation of RNAP-promoter open complexes. CarD plays a complex role in M. tuberculosis growth and virulence that is not fully understood. Therefore, to gain further insight into the role of CarD in M. tuberculosis growth and virulence, we determined the effect of increasing the affinity of CarD for RNAP. Using site-directed mutagenesis guided by crystal structures of CarD bound to RNAP, we identified amino acid substitutions that increase the affinity of CarD for RNAP. Using these substitutions, we show that increasing the affinity of CarD for RNAP increases the stability of the CarD protein in M. tuberculosis In addition, we show that increasing the affinity of CarD for RNAP increases the growth rate in M. tuberculosis without affecting 16S rRNA levels. We further show that increasing the affinity of CarD for RNAP reduces M. tuberculosis virulence in a mouse model of infection despite the improved growth rate in vitro Our findings suggest that the CarD-RNAP interaction protects CarD from proteolytic degradation in M. tuberculosis, establish that growth rate and rRNA levels can be uncoupled in M. tuberculosis and demonstrate that the strength of the CarD-RNAP interaction has been finely tuned to optimize virulence. Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major global health problem. In order to develop new strategies to battle this pathogen, we must gain a better understanding of the molecular processes involved in its survival and pathogenesis. We have previously identified CarD as an essential transcriptional regulator in mycobacteria. In this study, we detail the effects of increasing the affinity of CarD for RNAP on transcriptional regulation, CarD protein stability, and virulence. These studies expand our understanding of the global transcription regulator CarD, provide insight into how CarD activity is regulated, and

  12. The ENTPD1 promoter polymorphism -860 A > G (rs3814159) is associated with increased gene transcription, protein expression, CD39/NTPDase1 enzymatic activity, and thromboembolism risk.

    PubMed

    Maloney, James P; Branchford, Brian R; Brodsky, Gary L; Cosmic, Maxwell S; Calabrese, David W; Aquilante, Christina L; Maloney, Kelly W; Gonzalez, Joseph R; Zhang, Weiming; Moreau, Kerrie L; Wiggins, Kerri L; Smith, Nicholas L; Broeckel, Ulrich; Di Paola, Jorge

    2017-03-16

    Ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1) degrades the purines ATP and ADP that are key regulators of inflammation and clotting. We hypothesized that NTPDase1 polymorphisms exist and that they regulate this pathway. We sequenced the ENTPD1 gene (encoding NTPDase1) in 216 subjects then assessed genotypes in 2 cohorts comprising 2213 humans to identify ENTPD1 polymorphisms associated with venous thromboembolism (VTE). The G allele of the intron 1 polymorphism rs3176891 was more common in VTE vs. controls (odds ratio 1.26-1.9); it did not affect RNA splicing, but it was in strong linkage disequilibrium with the G allele of the promoter polymorphism rs3814159, which increased transcriptional activity by 8-fold. Oligonucleotides containing the G allele of this promoter region bound nuclear extracts more avidly. Carriers of rs3176891 G had endothelial cells with increased NTPDase1 activity and protein expression, and had platelets with enhanced aggregation. Thus, the G allele of rs3176891 marks a haplotype associated with increased clotting and platelet aggregation attributable to a promoter variant associated with increased transcription, expression, and activity of NTPDase1. We term this gain-of-function phenotype observed with rs3814159 G "CD39 Denver."-Maloney, J. P., Branchford, B. R., Brodsky, G. L., Cosmic, M. S., Calabrese, D. W., Aquilante, C. L., Maloney, K. W., Gonzalez, J. R., Zhang, W., Moreau, K. L., Wiggins, K. L., Smith, N. L., Broeckel, U., Di Paola, J. The ENTPD1 promoter polymorphism -860 A > G (rs3814159) is associated with increased gene transcription, protein expression, CD39/NTPDase1 enzymatic activity, and thromboembolism risk.

  13. Sepsis increases the expression and activity of the transcription factor Forkhead Box O 1 (FOXO1) in skeletal muscle by a glucocorticoid-dependent mechanism.

    PubMed

    Smith, Ira J; Alamdari, Nima; O'Neal, Patrick; Gonnella, Patricia; Aversa, Zaira; Hasselgren, Per-Olof

    2010-05-01

    Sepsis-induced muscle wasting has severe clinical consequences, including muscle weakness, need for prolonged ventilatory support and stay in the intensive care unit, and delayed ambulation with risk for pulmonary and thromboembolic complications. Understanding molecular mechanisms regulating loss of muscle mass in septic patients therefore has significant clinical implications. Forkhead Box O (FOXO) transcription factors have been implicated in muscle wasting, partly reflecting upregulation of the ubiquitin ligases atrogin-1 and MuRF1. The influence of sepsis on FOXO transcription factors in skeletal muscle is poorly understood. We tested the hypothesis that sepsis upregulates expression and activity of FOXO transcription factors in skeletal muscle by a glucocorticoid-dependent mechanism. Sepsis in rats increased muscle FOXO1 and 3a mRNA and protein levels but did not influence FOXO4 expression. Nuclear FOXO1 levels and DNA binding activity were increased in septic muscle whereas FOXO3a nuclear levels were not increased during sepsis. Sepsis-induced expression of FOXO1 was reduced by the glucocorticoid receptor antagonist RU38486 and treatment of rats with dexamethasone increased FOXO1 mRNA levels suggesting that the expression of FOXO1 is regulated by glucocorticoids. Reducing FOXO1, but not FOXO3a, expression by siRNA in cultured L6 myotubes inhibited dexamethasone-induced atrogin-1 and MuRF1 expression, further supporting a role of FOXO1 in glucocorticoid-regulated muscle wasting. Results suggest that sepsis increases FOXO1 expression and activity in skeletal muscle by a glucocorticoid-dependent mechanism and that glucocorticoid-dependent upregulation of atrogin-1 and MuRF1 in skeletal muscle is regulated by FOXO1. The study is significant because it provides novel information about molecular mechanisms involved in sepsis-induced muscle wasting.

  14. The Transcriptional Heat Shock Response of Salmonella Typhimurium Shows Hysteresis and Heated Cells Show Increased Resistance to Heat and Acid Stress

    PubMed Central

    Pin, Carmen; Hansen, Trine; Muñoz-Cuevas, Marina; de Jonge, Rob; Rosenkrantz, Jesper T.; Löfström, Charlotta; Aarts, Henk; Olsen, John E.

    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-replicating stationary phase cells of S. Typhimurium caused by the exposure to 45°C and to pH 5 for 30 min was monitored by microarray hybridizations at the end of the treatment period as well as immediately and 30 minutes after conditions were set back to their initial values, 25°C and pH 7. One hundred and two out of 120 up-regulated genes during the heat shock remained up-regulated 30 minutes after the temperature was set back to 25°C, while only 86 out of 293 down regulated genes remained down regulated 30 minutes after the heat shock ceased. Thus, the majority of the induced genes exhibited hysteresis, i.e., they remained up-regulated after the environmental stress ceased. At 25°C the transcriptional regulation of genes encoding for heat shock proteins was determined by the previous environment. Gene networks constructed with up-regulated genes were significantly more modular than those of down-regulated genes, implying that down-regulation was significantly less synchronized than up-regulation. The hysteretic transcriptional response to heat shock was accompanied by higher resistance to inactivation at 50°C as well as cross-resistance to inactivation at pH 3; however, growth rates and lag times at 43°C and at pH 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 and a deterministic source of variability in gene regulation. PMID:23236453

  15. Gene expression in scrapie. Cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts.

    PubMed

    Dandoy-Dron, F; Guillo, F; Benboudjema, L; Deslys, J P; Lasmézas, C; Dormont, D; Tovey, M G; Dron, M

    1998-03-27

    To define genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies, we analyzed gene expression in scrapie-infected mouse brain using "mRNA differential display." The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. Five of these genes have not previously been reported to exhibit increased expression in this disease: cathepsin S, the C1q B-chain of complement, apolipoprotein D, and two previously unidentified genes denominated scrapie-responsive gene (ScRG)-1 and ScRG-2, which are preferentially expressed in brain tissue. Increased expression of the three remaining genes, beta2 microglobulin, F4/80, and metallothionein II, has previously been reported to occur in experimental scrapie. Kinetic analysis revealed a concomitant increase in the levels of ScRG-1, cathepsin S, the C1q B-chain of complement, and beta2 microglobulin mRNA as well as glial fibrillary acidic protein and F4/80 transcripts, markers of astrocytosis and microglial activation, respectively. In contrast, the level of ScRG-2, apolipoprotein D, and metallothionein II mRNA was only increased at the terminal stage of the disease. ScRG-1 mRNA was found to be preferentially expressed in glial cells and to code for a short protein of 47 amino acids with a strong hydrophobic N-terminal region.

  16. The duplication mutation of Quebec platelet disorder dysregulates PLAU, but not C10orf55, selectively increasing production of normal PLAU transcripts by megakaryocytes but not granulocytes

    PubMed Central

    Soomro, Asim; Waye, John S.; Paterson, Andrew D.; Rivard, Georges E.; Wilson, Michael D.

    2017-01-01

    Quebec Platelet disorder (QPD) is a unique bleeding disorder that markedly increases urokinase plasminogen activator (uPA) in megakaryocytes and platelets but not in plasma or urine. The cause is tandem duplication of a 78 kb region of chromosome 10 containing PLAU (the uPA gene) and C10orf55, a gene of unknown function. QPD increases uPA in platelets and megakaryocytes >100 fold, far more than expected for a gene duplication. To investigate the tissue-specific effect that PLAU duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique PLAU transcripts; 2) increased uPA in leukocytes; 3) altered levels of C10orf55 mRNA and/or protein in megakaryocytes and leukocytes; and 4) global changes in megakaryocyte gene expression. Primary cells and cultured megakaryocytes from donors were prepared for quantitative reverse polymerase chain reaction analyses, RNA-seq and protein expression analyses. Rapidly isolated blood leukocytes from QPD subjects showed only a 3.9 fold increase in PLAU transcript levels, in keeping with the normal to minimally increased uPA in affinity purified, QPD leukocytes. All subjects had more uPA in granulocytes than monocytes and minimal uPA in lymphocytes. QPD leukocytes expressed PLAU alleles in proportions consistent with an extra copy of PLAU on the disease chromosome, unlike QPD megakaryocytes. QPD PLAU transcripts were consistent with reference gene models, with a much higher proportion of reads originating from the disease chromosome in megakaryocytes than granulocytes. QPD and control megakaryocytes contained minimal reads for C10orf55, and C10orf55 protein was not increased in QPD megakaryocytes or platelets. Finally, our QPD megakaryocyte transcriptome analysis revealed a global down regulation of the interferon type 1 pathway. We suggest that the low endogenous levels of uPA in blood are actively regulated, and that the regulatory mechanisms are disrupted in QPD

  17. NF-κB signaling pathway is inhibited by heat shock independently of active transcription factor HSF1 and increased levels of inducible heat shock proteins.

    PubMed

    Janus, Patryk; Pakuła-Cis, Małgorzata; Kalinowska-Herok, Magdalena; Kashchak, Natalia; Szołtysek, Katarzyna; Pigłowski, Wojciech; Widlak, Wieslawa; Kimmel, Marek; Widlak, Piotr

    2011-12-01

    NF-κB transcription factor regulates numerous genes important for inflammation, immune responses and cell survival. HSF1 is the primary transcription factor activated under stress conditions that is responsible for induction of genes encoding heat shock proteins. Previous studies have shown that the NF-κB activation pathway is blocked by heat shock possibly involving heat shock proteins. Here, we investigate whether active HSF1 inhibited this pathway in the absence of stress conditions. Activation of the NF-κB pathway and expression of NF-κB-dependent genes were analyzed in TNFα-stimulated U-2 OS human osteosarcoma cells that were either heat-shocked or engineered to express a constitutively active form of HSF1 in the absence of heat shock. As expected, heat shock resulted in a general blockade in the degradation of the IκBα inhibitor, nuclear translocation of NF-κB and expression of NF-κB-dependent target genes. In marked contrast, the presence of constitutively active HSF1 did not block TNFα-induced activation of the NF-κB pathway or expression of a set of the NF-κB-dependent genes. We conclude that in the absence of heat shock, the NF-κB activation pathway is inhibited by neither active HSF1 transcription factor nor by increased levels of HSF1-induced heat shock proteins.

  18. An ABA-increased interaction of the PYL6 ABA receptor with MYC2 Transcription Factor: A putative link of ABA and JA signaling.

    PubMed

    Aleman, Fernando; Yazaki, Junshi; Lee, Melissa; Takahashi, Yohei; Kim, Alice Y; Li, Zixing; Kinoshita, Toshinori; Ecker, Joseph R; Schroeder, Julian I

    2016-06-30

    Abscisic acid (ABA) is a plant hormone that mediates abiotic stress tolerance and regulates growth and development. ABA binds to members of the PYL/RCAR ABA receptor family that initiate signal transduction inhibiting type 2C protein phosphatases. Although crosstalk between ABA and the hormone Jasmonic Acid (JA) has been shown, the molecular entities that mediate this interaction have yet to be fully elucidated. We report a link between ABA and JA signaling through a direct interaction of the ABA receptor PYL6 (RCAR9) with the basic helix-loop-helix transcription factor MYC2. PYL6 and MYC2 interact in yeast two hybrid assays and the interaction is enhanced in the presence of ABA. PYL6 and MYC2 interact in planta based on bimolecular fluorescence complementation and co-immunoprecipitation of the proteins. Furthermore, PYL6 was able to modify transcription driven by MYC2 using JAZ6 and JAZ8 DNA promoter elements in yeast one hybrid assays. Finally, pyl6 T-DNA mutant plants show an increased sensitivity to the addition of JA along with ABA in cotyledon expansion experiments. Overall, the present study identifies a direct mechanism for transcriptional modulation mediated by an ABA receptor different from the core ABA signaling pathway, and a putative mechanistic link connecting ABA and JA signaling pathways.

  19. Gene expression in the twilight of death: The increase of thousands of transcripts has implications to transplantation, cancer, and forensic research.

    PubMed

    Pozhitkov, Alexander E; Noble, Peter A

    2017-09-01

    After a vertebrate dies, many of its organ systems, tissues, and cells remain functional while its body no longer works as a whole. We define this state as the "twilight of death" - the transition from a living body to a decomposed corpse. We claim that the study of the twilight of death is important to ethical, legal and medical science. We examined gene expression at the twilight of death in the zebrafish and mouse reaching the conclusion that apparently thousands of transcripts significantly increase in abundance from life to several hours/days postmortem relative to live controls. Transcript dynamics of different genes provided "proof-of-principle" that models accurately predict an individual's elapsed-time-of-death (i.e. postmortem interval). While many transcripts were associated with survival and stress compensation, others were associated with epigenetic factors, developmental control, and cancer. Future studies are needed to determine whether the high incidence of cancer in transplant recipients is due to the postmortem processes in donor organs. © 2017 WILEY Periodicals, Inc.

  20. Increase in flavan-3-ols by silencing flavonol synthase mRNA affects the transcript expression and activity levels of antioxidant enzymes in tobacco.

    PubMed

    Mahajan, M; Joshi, R; Gulati, A; Yadav, S K

    2012-09-01

    Flavonoids are plant secondary metabolites widespread throughout the plant kingdom involved in many physiological and biochemical functions. Amongst the flavonoids, flavan-3-ols (catechin and epicatechin) are known for their direct free radical scavenging activity in vitro, but studies on their antioxidant potential and interaction with antioxidant enzymes in vivo are lacking. Here, the flavonoid pathway was engineered by silencing a gene encoding flavonol synthase (FLS) in tobacco to direct the flow of metabolites towards production of flavan-3-ols. FLS silencing reduced flavonol content 17-53%, while it increased catechin and epicatechin content 51-93% and 18-27%, respectively. The silenced lines showed a significant increase in expression of genes for dihydroflavonol reductase and anthocyanidin synthase, a downstream gene towards epicatechin production, with no significant change in expression of other genes of the flavonoid pathway. Effects of accumulation of flavan-3-ols in FLS silenced lines on transcript level and activities of antioxidant enzymes were studied. Transcripts of the antioxidant enzymes glutathione reductase (GR), ascorbate peroxidase (APx), and catalase (CAT) increased, while glutathione-S-transferase (GST), decreased in FLS silenced lines. Enhanced activity of all the antioxidant enzymes was observed in silenced tobacco lines. To validate the affect of flavan-3-ols on the antioxidant system, in vitro experiments were conducted with tobacco seedlings exposed to two concentrations of catechin (10  and 50 μm) for 2 days. In vitro exposed seedlings produced similar levels of transcripts and activity of antioxidant enzymes as FLS silenced seedlings. Results suggest that flavan-3-ols (catechin) might be increasing activity of GR, Apx and CAT by elevating their mRNAs levels. Since these enzymes are involved in scavenging of reactive oxygen species, this strategy would help in tailoring crops for enhanced catechin production as well as making

  1. Disruption of 3D MCF-12A Breast Cell Cultures by Estrogens – An In Vitro Model for ER-Mediated Changes Indicative of Hormonal Carcinogenesis

    PubMed Central

    Marchese, Stephanie; Silva, Elisabete

    2012-01-01

    Introduction Estrogens regulate the proliferation of normal and neoplastic breast epithelium. Although the intracellular mechanisms of estrogens in the breast are largely understood, little is known about how they induce changes in the structure of the mammary epithelium, which are characteristic of breast cancer. In vitro three dimensional (3D) cultures of immortalised breast epithelial cells recapitulate features of the breast epithelium in vivo, including formation of growth arrested acini with hollow lumen and basement membrane. This model can also reproduce features of malignant transformation and breast cancer, such as increased cellular proliferation and filling of the lumen. However, a system where a connection between estrogen receptor (ER) activation and disruption of acini formation can be studied to elucidate the role of estrogens is still missing. Methods/Principal Findings We describe an in vitro 3D model for breast glandular structure development, using breast epithelial MCF-12A cells cultured in a reconstituted basement membrane matrix. These cells are estrogen receptor (ER)α, ERβ and G-protein coupled estrogen receptor 1 (GPER) competent, allowing the investigation of the effects of estrogens on mammary gland formation and disruption. Under normal conditions, MCF-12A cells formed organised acini, with deposition of basement membrane and hollow lumen. However, treatment with 17β-estradiol, and the exogenous estrogens bisphenol A and propylparaben resulted in deformed acini and filling of the acinar lumen. When these chemicals were combined with ER and GPER inhibitors (ICI 182,780 and G-15, respectively), the deformed acini recovered normal features, such as a spheroid shape, proliferative arrest and luminal clearing, suggesting a role for the ER and GPER in the estrogenic disruption of acinar formation. Conclusion This new model offers the opportunity to better understand the role of the ER and GPER in the morphogenesis of breast glandular

  2. High yields and soluble expression of superoxide dismutases in Escherichia coli due to the HIV-1 Tat peptide via increases in mRNA transcription

    PubMed Central

    Sun, Yangdong; Ye, Qiao; Wu, Min; Wu, Yonghong; Zhang, Chenggang; Yan, Weiqun

    2016-01-01

    This study aimed to validate the high yield and soluble expression of proteins carrying the transactivator of transcription (Tat) peptide tag, and further explored the potential mechanism by which the Tat tag increases expression. Escherichia coli superoxide dismutase (SOD) proteins, including SodA, SodB and SodC, were selected for analysis. As expected, the yields and the solubility of Tat-tagged proteins were higher than those of Tat-free proteins, and similar results were observed for the total SOD enzyme activity. Bacterial cells that overexpressed Tat-tagged proteins exhibited increased anti-paraquat activity compared with those expressing Tat-free proteins that manifested as SodA>SodC>SodB. When compared with an MG1655 wild-type strain, the growth of a ΔSodA mutant strain was found to be inhibited after paraquat treatment; the growth of ΔSodB and ΔSodC mutant strains was also slightly inhibited. The mRNA transcript level of genes encoding Tat-tagged proteins was higher than that of genes encoding Tat-free proteins. Furthermore, the α-helix and turn of Tat-tagged proteins were higher than those of Tat-free proteins, but the β-sheet and random coil content was lower. These results indicated that the incorporation of the Tat core peptide as a significant basic membrane transduction peptide in fusion proteins could increase mRNA transcripts and promote the high yield and soluble expression of heterologous proteins in E. coli. PMID:27741225

  3. Cocaine-induced chromatin remodeling increases brain-derived neurotrophic factor transcription in the rat medial prefrontal cortex, which alters the reinforcing efficacy of cocaine.

    PubMed

    Sadri-Vakili, Ghazaleh; Kumaresan, Vidhya; Schmidt, Heath D; Famous, Katie R; Chawla, Prianka; Vassoler, Fair M; Overland, Ryan P; Xia, Eva; Bass, Caroline E; Terwilliger, Ernest F; Pierce, R Christopher; Cha, Jang-Ho J

    2010-09-01

    Cocaine self-administration alters patterns of gene expression in the brain that may underlie cocaine-induced neuronal plasticity. In the present study, male Sprague Dawley rats were allowed to self-administer cocaine (0.25 mg/infusion) 2 h/d for 14 d, followed by 7 d of forced abstinence. Compared with yoked saline control rats, cocaine self-administration resulted in increased brain-derived neurotrophic factor (BDNF) protein levels in the rat medial prefrontal cortex (mPFC). To examine the functional relevance of this finding, cocaine self-administration maintained under a progressive ratio schedule of reinforcement was assessed after short hairpin RNA-induced suppression of BDNF expression in the mPFC. Decreased BDNF expression in the mPFC increased the cocaine self-administration breakpoint. Next, the effect of cocaine self-administration on specific BDNF exons was assessed; results revealed selectively increased BDNF exon IV-containing transcripts in the mPFC. Moreover, there were significant cocaine-induced increases in acetylated histone H3 (AcH3) and phospho-cAMP response element binding protein (pCREB) association with BDNF promoter IV. In contrast, there was decreased methyl-CpG-binding protein 2 (MeCP2) association with BDNF promoter IV in the mPFC of rats that previously self-administered cocaine. Together, these results indicate that cocaine-induced increases in BDNF promoter IV transcript in the mPFC are driven by increased binding of AcH3 and pCREB as well as decreased MeCP2 binding at this BDNF promoter. Collectively, these results indicate that cocaine self-administration remodels chromatin in the mPFC, resulting in increased expression of BDNF, which appears to represent a compensatory neuroadaptation that reduces the reinforcing efficacy of cocaine.

  4. Triptolide increases transcript and protein levels of survival motor neurons in human SMA fibroblasts and improves survival in SMA-like mice.

    PubMed

    Hsu, Ya-Yun; Jong, Yuh-Jyh; Tsai, Hsin-Hung; Tseng, Yu-Ting; An, Li-Mei; Lo, Yi-Ching

    2012-06-01

    Spinal muscular atrophy (SMA) is a progressive neuromuscular disease. Since disease severity is related to the amount of survival motor neuron (SMN) protein, up-regulated functional SMN protein levels from the SMN2 gene are considered a major SMA drug-discovery strategy. In this study, we investigated the possible effects of triptolide, a diterpene triepoxide purified from Tripterygium wilfordii Hook. F., as a new compound for increasing SMN protein. The effects and mechanisms of triptolide on the production of SMA protein were determined by cell-based assays using the motor neuronal cell line NSC34 and skin fibroblasts from SMA patients. Wild-type (Smn(+/+) SMN2(-/-) , C57BL/6) and SMA-like (Smn(-/-) SMN2) mice were injected with triptolide (0.01 or 0.1 mg·kg(-1) ·day(-1) , i.p.) and their survival rate and level of change in SMN protein in neurons and muscle tissue measured. In NSC34 cells and human SMA fibroblasts, pM concentrations of triptolide significantly increased SMN protein expression and the levels of SMN complex component (Gemin2 and Gemin3). In human SMA fibroblasts, triptolide increased SMN-containing nuclear gems and the ratio of full-length transcripts (FL-SMN2) to SMN2 transcripts lacking exon 7 (SMN2Δ7). Furthermore, in SMA-like mice, triptolide significantly increased SMN protein levels in the brain, spinal cord and gastrocnemius muscle. Furthermore, triptolide treatment increased survival and reduced weight loss in SMA-like mice. Triptolide enhanced SMN protein production by promoting SMN2 activation, exon 7 inclusion and increasing nuclear gems, and increased survival in SMA mice, which suggests triptolide might be a potential candidate for SMA therapy. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  5. Increased readthrough transcription across the simian virus 5 M-F gene junction leads to growth defects and a global inhibition of viral mRNA synthesis.

    PubMed

    Parks, G D; Ward, K R; Rassa, J C

    2001-03-01

    Recombinant simian virus 5 (rSV5) mutants containing substitutions in the M-F intergenic region were generated to determine the effect of increased readthrough transcription on the paramyxovirus growth cycle. We have previously shown, using an SV5 dicistronic minigenome, that replacement of the 22-base M-F intergenic region with a foreign sequence results in a template (Rep22) that directs very high levels of M-F readthrough transcription. An rSV5 containing the Rep22 substitution grew slower and to final titers that were 50- to 80-fold lower than those of wild-type (WT) rSV5. Cells infected with the Rep22 virus produced very low levels of monocistronic M and F mRNA, consistent with the M-F readthrough phenotype. Surprisingly, Rep22 virus-infected cells also displayed a global decrease in the accumulation of viral mRNA from genes located upstream and downstream of the M-F junction, and overall viral protein synthesis was reduced. Second-site revertants of the Rep22 virus that had regained WT transcription and growth properties contained a single base substitution that increased the M gene end U tract from four to eight residues, suggesting that the growth defects originated from higher-than-normal M-F readthrough transcription. Thus, the primary growth defect for the Rep22 virus appears to be in viral RNA synthesis and not in morphogenesis. A second rSV5 virus (G14), which contained a different foreign M-F intergenic sequence, grew to similar or slightly higher titers than WT rSV5 in some cell types and produced ~1.5- to 2-fold more mRNA and viral protein. The data support the hypothesis that inhibition of Rep22 virus growth is due to increased access by the polymerase to the 5' end of the genome and to the resulting overexpression of L protein. We propose that the elevated naturally occurring M-F readthrough which is characteristic of many paramyxoviruses serves as a mechanism to fine-tune the level of polymerase that is optimal for virus growth.

  6. Hepatitis B virus X protein induces RNA polymerase III-dependent gene transcription and increases cellular TATA-binding protein by activating the Ras signaling pathway.

    PubMed

    Wang, H D; Trivedi, A; Johnson, D L

    1997-12-01

    Our previous studies have shown that the hepatitis B virus protein, X, activates all three classes of RNA polymerase III (pol III)-dependent promoters by increasing the cellular level of TATA-binding protein (TBP) (H.-D. Wang et al., Mol. Cell. Biol. 15:6720-6728, 1995), a limiting transcription component (A. Trivedi et al., Mol. Cell. Biol. 16:6909-6916, 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.

  7. HIV-1 Infection Leads to Increased Transcription of Human Endogenous Retrovirus HERV-K (HML-2) Proviruses In Vivo but Not to Increased Virion Production

    PubMed Central

    Bhardwaj, Neeru; Maldarelli, Frank; Mellors, John

    2014-01-01

    ABSTRACT Recent studies suggest that human endogenous retrovirus group K (HERV-K) provirus expression plays a role in the pathogenesis of HIV-1 infection. In particular, RNA from the HML-2 subgroup of HERV-K proviruses has been reported to be highly expressed at the cellular level and detectable in the plasma of HIV-1-infected patients, suggestive of virion production and, perhaps, replication. In this study, we developed an HML-2-specific quantitative-PCR assay that detects 51 of the 89 known HML-2 proviruses in the human genome. Plasma and peripheral blood mononuclear cells (PBMCs) from HIV-negative controls and HIV-1-infected patients were collected for analysis of HML-2 RNA expression. Contrary to previous reports, we did not detect high levels of HML-2 RNA in the plasma of HIV-1-infected patients, but we did observe a significant increase of HML-2 RNA in total PBMCs compared to HIV-negative controls. The level of HML-2 expression in PBMCs does not appear to be related to patient use of antiretrovirals or to HIV-1 plasma RNA, cellular RNA, or cellular DNA levels. To investigate the source of HML-2 RNA expression, patient PBMCs were sorted into CD3+ CD4+, CD3+ CD8+, CD3− CD14+, and CD3− CD20+ cell subsets and then analyzed for HML-2 RNA levels. No single cell subset was enriched for HML-2 RNA expression in HIV-1-infected patients, but there appears to be substantial variability in the level of HML-2 expression depending on the cell type. IMPORTANCE Here, we report that human endogenous retrovirus group K (HERV-K) (HML-2) proviruses are expressed at significantly higher levels in peripheral blood mononuclear cells (PBMCs) from patients with HIV-1 infection than in those from uninfected individuals. However, contrary to previous reports, this expression did not lead to detectable virions in the plasma of these patients. In addition, we found that HML-2 proviruses were expressed in multiple blood cell types from HIV-1-infected individuals, and the magnitude of

  8. Rice and Bean Targets for Biofortification Combined with High Carotenoid Content Crops Regulate Transcriptional Mechanisms Increasing Iron Bioavailability.

    PubMed

    Dias, Desirrê Morais; de Castro Moreira, Maria Eliza; Gomes, Mariana Juste Contin; Lopes Toledo, Renata Celi; Nutti, Marilia Regini; Pinheiro Sant'Ana, Helena Maria; Martino, Hércia Stampini Duarte

    2015-11-23

    Iron deficiency affects thousands of people worldwide. Biofortification of staple food crops aims to support the reduction of this deficiency. This study evaluates the effect of combinations of common beans and rice, targets for biofortification, with high carotenoid content crops on the iron bioavailability, protein gene expression, and antioxidant effect. Iron bioavailability was measured by the depletion/repletion method. Seven groups were tested (n = 7): Pontal bean (PB); rice + Pontal bean (R + BP); Pontal bean + sweet potato (PB + SP); Pontal bean + pumpkin (PB + P); Pontal bean + rice + sweet potato (PB + R + P); Pontal bean + rice + sweet potato (PB + R + SP); positive control (Ferrous Sulfate). The evaluations included: hemoglobin gain, hemoglobin regeneration efficiency (HRE), gene expression of divalente metal transporter 1 (DMT-1), duodenal citocromo B (DcytB), ferroportin, hephaestin, transferrin and ferritin and total plasma antioxidant capacity (TAC). The test groups, except the PB, showed higher HRE (p < 0.05) than the control. Gene expression of DMT-1, DcytB and ferroportin increased (p < 0.05) in the groups fed with high content carotenoid crops (sweet potato or pumpkin). The PB group presented lower (p < 0.05) TAC than the other groups. The combination of rice and common beans, and those with high carotenoid content crops increased protein gene expression, increasing the iron bioavailability and antioxidant capacity.

  9. Rice and Bean Targets for Biofortification Combined with High Carotenoid Content Crops Regulate Transcriptional Mechanisms Increasing Iron Bioavailability

    PubMed Central

    Dias, Desirrê Morais; de Castro Moreira, Maria Eliza; Gomes, Mariana Juste Contin; Lopes Toledo, Renata Celi; Nutti, Marilia Regini; Pinheiro Sant’Ana, Helena Maria; Martino, Hércia Stampini Duarte

    2015-01-01

    Iron deficiency affects thousands of people worldwide. Biofortification of staple food crops aims to support the reduction of this deficiency. This study evaluates the effect of combinations of common beans and rice, targets for biofortification, with high carotenoid content crops on the iron bioavailability, protein gene expression, and antioxidant effect. Iron bioavailability was measured by the depletion/repletion method. Seven groups were tested (n = 7): Pontal bean (PB); rice + Pontal bean (R + BP); Pontal bean + sweet potato (PB + SP); Pontal bean + pumpkin (PB + P); Pontal bean + rice + sweet potato (PB + R + P); Pontal bean + rice + sweet potato (PB + R + SP); positive control (Ferrous Sulfate). The evaluations included: hemoglobin gain, hemoglobin regeneration efficiency (HRE), gene expression of divalente metal transporter 1 (DMT-1), duodenal citocromo B (DcytB), ferroportin, hephaestin, transferrin and ferritin and total plasma antioxidant capacity (TAC). The test groups, except the PB, showed higher HRE (p < 0.05) than the control. Gene expression of DMT-1, DcytB and ferroportin increased (p < 0.05) in the groups fed with high content carotenoid crops (sweet potato or pumpkin). The PB group presented lower (p < 0.05) TAC than the other groups. The combination of rice and common beans, and those with high carotenoid content crops increased protein gene expression, increasing the iron bioavailability and antioxidant capacity. PMID:26610564

  10. Progressive recruitment of cortical and striatal regions by inducible postsynaptic density transcripts after increasing doses of antipsychotics with different receptor profiles: insights for psychosis treatment.

    PubMed

    de Bartolomeis, Andrea; Iasevoli, Felice; Marmo, Federica; Buonaguro, Elisabetta F; Eramo, Anna; Rossi, Rodolfo; Avvisati, Livia; Latte, Gianmarco; Tomasetti, Carmine

    2015-04-01

    Antipsychotics may modulate the transcription of multiple gene programs, including those belonging to postsynaptic density (PSD) network, within cortical and subcortical brain regions. Understanding which brain region is activated progressively by increasing doses of antipsychotics and how their different receptor profiles may impact such an activation could be relevant to better correlate the mechanism of action of antipsychotics both with their efficacy and side effects. We analyzed the differential topography of PSD transcripts by incremental doses of two antipsychotics: haloperidol, the prototypical first generation antipsychotic with prevalent dopamine D2 receptors antagonism, and asenapine, a second generation antipsychotic characterized by multiple receptors occupancy. We investigated the expression of PSD genes involved in synaptic plasticity and previously demonstrated to be modulated by antipsychotics: Homer1a, and its related interacting constitutive genes Homer1b/c and PSD95, as well as Arc, C-fos and Zif-268, also known to be induced by antipsychotics administration. We found that increasing acute doses of haloperidol induced immediate-early genes (IEGs) expression in different striatal areas, which were progressively recruited by incremental doses with a dorsal-to-ventral gradient of expression. Conversely, increasing acute asenapine doses progressively de-recruited IEGs expression in cortical areas and increased striatal genes signal intensity. These effects were mirrored by a progressive reduction in locomotor animal activity by haloperidol, and an opposite increase by asenapine. Thus, we demonstrated for the first time that antipsychotics may progressively recruit PSD-related IEGs expression in cortical and subcortical areas when administered at incremental doses and these effects may reflect a fine-tuned dose-dependent modulation of the PSD. Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.

  11. 14-3-3-β and -{varepsilon} contribute to activation of the osmoprotective transcription factor NFAT5 by increasing its protein abundance and its transactivating activity.

    PubMed

    Izumi, Yuichiro; Burg, Maurice B; Ferraris, Joan D

    2014-01-01

    Abstract Having previously found that high NaCl causes rapid exit of 14-3-3 isoforms from the nucleus, we used siRNA-mediated knockdown to test whether 14-3-3s contribute to the high NaCl-induced increase in the activity of the osmoprotective transcription factor NFAT5. We find that, when NaCl is elevated, knockdown of 14-3-3-β and/or 14-3-3-ε decreases NFAT5 transcriptional activity, as assayed both by luciferase reporter and by the mRNA abundance of the NFAT5 target genes aldose reductase and the sodium- and chloride-dependent betaine transporter, BGT1. Knockdown of other 14-3-3 isoforms does not significantly affect NFAT5 activity. 14-3-3-β and/or 14-3-3-ε do not act by affecting the nuclear localization of NFAT5, but by at least two other mechanisms: (1) 14-3-3-β and 14-3-3-ε increase protein abundance of NFAT5 and (2) they increase NFAT5 transactivating activity. When NaCl is elevated, knockdown of 14-3-3-β and/or 14-3-3-ε reduces the protein abundance of NFAT5, as measured by Western blot, without affecting the level of NFAT5 mRNA, and the knockdown also decreases NFAT5 transactivating activity, as measured by luciferase reporter. The 14-3-3s increase NFAT5 protein, not by increasing its translation, but by decreasing the rate at which it is degraded, as measured by cycloheximide chase. It is not clear at this point whether the 14-3-3s affect NFAT5 directly or indirectly through their effects on other proteins that signal activation of NFAT5.

  12. Increased expression of bHLH Transcription Factor E2A (TCF3) in prostate cancer promotes proliferation and confers resistance to doxorubicin induced apoptosis

    PubMed Central

    Patel, Divya; Chaudhary, Jaideep

    2012-01-01

    E2A (TCF3) is a multifunctional basic helix loop helix (bHLH), transcription factor. E2A regulates transcription of target genes by homo- or heterodimerization with cell specific bHLH proteins. In general, E2A promotes cell differentiation, acts as a negative regulator of cell proliferation in normal cells and cancer cell lines and is required for normal B-cell development. Given the diverse biological pathways regulated/ influenced by E2A little is known about its expression in cancer. In this study we investigated the expression of E2A in prostate cancer. Unexpectedly, E2A immuno-histochemistry demonstrated increased E2A expression in prostate cancer as compared to normal prostate. Silencing of E2A in prostate cancer cells DU145 and PC3 led to a significant reduction in proliferation due to G1 arrest that was in part mediated by increased CDKN1A(p21) and decreased Id1, Id3 and c-myc. E2A silencing in prostate cancer cell lines also resulted in increased apoptosis due to increased mitochondrial permeability and caspase 3/7 activation. Moreover, silencing of E2A increased sensitivity to doxorubicin induced apoptosis. Based on our results, we propose that E2A could be an upstream regulator of Id1 and c-Myc which are highly expressed in prostate cancer. These results for the first time demonstrate that E2A could in fact acts as a tumor promoter at least in prostate cancer. PMID:22564737

  13. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase.

    PubMed

    Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj; Oh, Seok Jeong; Lee, Moo Yeol; Kang, Keon Wook

    2010-08-01

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10μg/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  14. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: Essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase

    SciTech Connect

    Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj; Oh, Seok Jeong; Lee, Moo Yeol; Kang, Keon Wook

    2010-08-01

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10 {mu}g/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  15. Increased levels of NOTCH1, NF-kappaB, and other interconnected transcription factors characterize primitive sets of hematopoietic stem cells.

    PubMed

    Panepucci, Rodrigo Alexandre; Oliveira, Lucila Habib B; Zanette, Dalila Luciola; Viu Carrara, Rita de Cassia; Araujo, Amélia Goes; Orellana, Maristela Delgado; Bonini de Palma, Patrícia Vianna; Menezes, Camila C B O; Covas, Dimas Tadeu; Zago, Marco Antonio

    2010-03-01

    As previously shown, higher levels of NOTCH1 and increased NF-kappaB signaling is a distinctive feature of the more primitive umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), as compared to bone marrow (BM). Differences between BM and UCB cell composition also account for this finding. The CD133 marker defines a more primitive cell subset among CD34+ HSC with a proposed hemangioblast potential. To further evaluate the molecular basis related to the more primitive characteristics of UCB and CD133+ HSC, immunomagnetically purified human CD34+ and CD133+ cells from BM and UCB were used on gene expression microarrays studies. UCB CD34+ cells contained a significantly higher proportion of CD133+ cells than BM (70% and 40%, respectively). Cluster analysis showed that BM CD133+ cells grouped with the UCB cells (CD133+ and CD34+) rather than to BM CD34+ cells. Compared with CD34+ cells, CD133+ had a higher expression of many transcription factors (TFs). Promoter analysis on all these TF genes revealed a significantly higher frequency (than expected by chance) of NF-kappaB-binding sites (BS), including potentially novel NF-kappaB targets such as RUNX1, GATA3, and USF1. Selected transcripts of TF related to primitive hematopoiesis and self-renewal, such as RUNX1, GATA3, USF1, TAL1, HOXA9, HOXB4, NOTCH1, RELB, and NFKB2 were evaluated by real-time PCR and were all significantly positively correlated. Taken together, our data indicate the existence of an interconnected transcriptional network characterized by higher levels of NOTCH1, NF-kappaB, and other important TFs on more primitive HSC sets.

  16. Hereditary cutaneous mucinosis in shar pei dogs is associated with increased hyaluronan synthase-2 mRNA transcription by cultured dermal fibroblasts.

    PubMed

    Zanna, Giordana; Docampo, María J; Fondevila, Dolors; Bardagí, Mar; Bassols, Anna; Ferrer, Lluís

    2009-10-01

    Shar pei dogs are known for the distinctive feature of thick, wrinkled skin as a consequence of high dermal mucin content. Excessive dermal deposition of mucinous substance leading to severe skin folding, and/or to the more severe vesicular form characterized by dermal vesicles or bullae, is highly prevalent in this breed and is known as idiopathic mucinosis. Hyaluronic acid (HA) is the main component that accumulates in the dermis, and high levels of HA have also been detected in the serum of shar pei dogs. In this study, the cellular and molecular mechanisms underlying cutaneous mucinosis of shar pei dogs were investigated. Thirteen shar pei dogs and four control dogs of other breeds were included. In primary dermal fibroblast cultures, transcription of the family of hyaluronan synthases (HAS) involved in HA synthesis, and of hyaluronidases (HYAL) involved in HA degradation, were studied by reverse transcriptase polymerase chain reaction. The location of HA in cell cultures was studied by immunofluorescence and confocal laser microscopy. Dermal fibroblasts transcribed HAS2, HAS3, HYAL1 and HYAL2, but no amplification for HAS1 was found. A higher transcription of HAS2 was demonstrated in shar pei dogs compared with control dogs. By confocal microscopy, HA was detected as a more diffuse and intense network-like pattern of green fluorescence in the fibroblast cells of shar pei dogs in comparison with control dogs. Together, these results provide additional evidence that hereditary cutaneous mucinosis in shar pei dogs may be a consequence of over-transcription or increased activity of HAS2.

  17. VrDREB2A, a DREB-binding transcription factor from Vigna radiata, increased drought and high-salt tolerance in transgenic Arabidopsis thaliana.

    PubMed

    Chen, Honglin; Liu, Liping; Wang, Lixia; Wang, Suhua; Cheng, Xuzhen

    2016-03-01

    Mung bean (Vigna radiata L.) is commonly grown in Asia as an important nutritional dry grain legume, as it can survive better in arid conditions than other crops. Abiotic stresses, such as drought and high-salt contents, negatively impact its growth and production. The dehydration-responsive element-binding protein 2 (DREB2) transcription factors play a significant role in the response to these stress stimuli via transcriptional regulation of downstream genes containing the cis-element dehydration-responsive element (DRE). However, the molecular mechanisms involved in the drought tolerance of this species remain elusive, with very few reported candidate genes. No DREB2 ortholog has been reported for mung bean, and the function of mung bean DREB2 is not clear. In this study, a novel VrDREB2A gene with conserved AP2 domains and transactivation ability was isolated from mung bean. A modified VrDREB2A protein lacking the putative negative regulatory domain encoded by nucleotides 394-543 was shown to be localized in the nucleus. Expression of the VrDREB2A gene was induced by drought, high salt concentrations and abscisic acid treatment. Furthermore, comparing with the wild type Arabidopsis, the overexpression of VrDREB2A activated the expression of downstream genes in transgenic Arabidopsis, resulting in enhanced tolerance to drought and high-salt stresses and no growth retardation. The results from this study indicate that VrDREB2A functions as an important transcriptional activator and may help increase the abiotic stress tolerance of the mung bean plant.

  18. Inhibition of p38 MAP kinase increases okadaic acid mediated AP-1 expression and DNA binding but has no effect on TRE dependent transcription.

    PubMed

    Rosenberger, S F; Gupta, A; Bowden, G T

    1999-06-17

    By performing in vitro kinase assays we found in papilloma producing 308 mouse keratinocytes that okadaic acid elevated activities of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinases (MAPKs). This okadaic acid mediated activation of MAP kinases correlated with increased AP-1 binding to a consensus TPA responsive element (TRE) and elevated TRE dependent transcription. To determine the role of p38 MAP kinases in these processes we employed the specific p38 MAP kinase inhibitor SB 203580. Using orthophosphate labeling we showed a decrease in phosphorylation of MAPK activated protein kinase-2 (MAPKAP-K2) indicating reduced activity of p38 MAPKs utilizing this kinase as substrate. In contrast, we found that SB 203580 raised activities of ERK-1/2 and JNKs. Electrophoretic mobility shift assays revealed an increase in TRE binding activity in response to SB 203580 most likely resulting from increased expression of the major TRE binding components JunD and FosB as indicated by Western blot analyses. Increased TRE DNA binding failed to lead to increased transactivation correlating with the inability of SB 203580 to increase phosphorylation of these AP-1 proteins. These data indicate that SB 203580 sensitive p38 MAP kinases are not involved in okadaic acid mediated increases in TRE DNA binding and transactivation.

  19. Increased transforming growth factor β and interleukin 10 transcripts in peripheral blood mononuclear cells of colorectal cancer patients

    PubMed Central

    Stanilov, Noyko S.; Miteva, Lyuba; Cirovski, Geo

    2017-01-01

    Aim of the study The ability of immune cells in peripheral blood to produce certain cytokines affects tumour-elicited inflammation. The aim of this study was to investigate the gene expression of interleukin 12A (IL-12A), IL-12B, IL-23A, IL-10, IL-6, transforming growth factor β (TGF-β), HDAC3, and iNOS in peripheral blood mononuclear cells (PBMC) from colorectal cancer (CRC) patients. Material and methods The venous blood for PBMC isolation was collected preoperatively and 10 days after surgery, from CRC patients. After isolation of total RNA and synthesis of cDNA, quantitative real-time PCR assays were performed. Results Our results demonstrated that among investigated cytokine genes IL-10 and TGF-β were significantly upregulated in patients with CRC compared to the control group, while the expression of IL-23 mRNA was significantly decreased in CRC patients. We observed significantly increased mRNA levels in CRC patients’ PBMC before surgery for IL-10 and TGF-β compared to both postoperative and control groups. We also found a significant upregulation of iNOS in early compared to advanced CRC. Conclusions Based on the results we can assume that PBMC gene expression programming in CRC patients drives local differentiation of Th cells towards Treg instead of the Th1 anti-tumour subpopulation. PMID:28239283

  20. [All signs of metabolic syndrome in the hypertensive ISIAH rats are associated with increased activity of transcription factors PPAR, LXR, PXR, and CAR in the liver].

    PubMed

    Pivovarova, E N; Dushkin, M I; Perepechaeva, M L; Kobzev, V F; Trufakin, V A; Markel', A L

    2011-01-01

    It is known that the metabolic syndrome (MS), which includes hypertension, dislipidemia, glucose intolerance, and obesity leads to cardiovascular diseases. The MS risk is growing catastrophically. Molecular mechanisms allowing to understand the reason of integrated dysfunctions, taking place at MS cases, have remained almost unstudied. The chronical stress plays a crucial role in MS development; therefore in the present work a hypertensive rat strain with Inherited Stress-Induced Arterial Hypertension (ISIAH) was used as a model. It was shown that ISIAH rat strain as compared with the control WAG rat strain is characterized by increased content of triglyceride, VLDL and LDL cholesterols, a decreased content of HDL cholesterol, a high level of apolipoprotein B-100, and decreased level of apolipoprotein A-I. The ISIAH rats body weight was higher as compared with WAG rats; ISIAH rats blood glucose content was higher too. Thus, strain hypertension for ISIAH rat is accompanied by dislipidemia, increased glucose content, and increased body weight, representing a whole set of MS signs. Since at MS cases the systemic abnormalities in lipid and carbohydrate metabolism take place, the functional activity of transcription factors (TFs) participating in integral regulation of lipid and carbohydrate metabolism genes in liver was measured. PPAR, LXR, PXR, CAR DNA-binding activity was increased in ISIAH rats, suggesting involvement of these TFs in MS development. Integrated investigation of PPAR, LXR, PXR, CAR regulatory mechanisms, signal transduction and transcriptional targets will provide insights into the pathogenesis of MS and offer valuable information for designing of drugs for MS treatment.

  1. Niacin increases HDL biogenesis by enhancing DR4-dependent transcription of ABCA1 and lipidation of apolipoprotein A-I in HepG2 cells

    PubMed Central

    Zhang, Lin-Hua; Kamanna, Vaijinath S.; Ganji, Shobha H.; Xiong, Xi-Ming; Kashyap, Moti L.

    2012-01-01

    The lipidation of apoA-I in liver greatly influences HDL biogenesis and plasma HDL levels by stabilizing the secreted apoA-I. Niacin is the most effective lipid-regulating agent clinically available to raise HDL. This study was undertaken to identify regulatory mechanisms of niacin action in hepatic lipidation of apoA-I, a critical event involved in HDL biogenesis. In cultured human hepatocytes (HepG2), niacin increased: association of apoA-I with phospholipids and cholesterol by 46% and 23% respectively, formation of lipid-poor single apoA-I molecule-containing particles up to ∼ 2.4-fold, and pre β 1 and α migrating HDL particles. Niacin dose-dependently stimulated the cell efflux of phospholipid and cholesterol and increased transcription of ABCA1 gene and ABCA1 protein. Mutated DR4, a binding site for nuclear factor liver X receptor alpha (LXR α ) in the ABCA1 promoter, abolished niacin stimulatory effect. Further, knocking down LXR α or ABCA1 by RNA interference eliminated niacin-stimulated apoA-I lipidation. Niacin treatment did not change apoA-I gene expression. The present data indicate that niacin increases apoA-I lipidation by enhancing lipid efflux through a DR4-dependent transcription of ABCA1 gene in HepG2 cells. A stimulatory role of niacin in early hepatic formation of HDL particles suggests a new mechanism that contributes to niacin action to increase the stability of newly synthesized circulating HDL. PMID:22389325

  2. Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata.

    PubMed

    Halitschke, Rayko; Baldwin, Ian T

    2003-12-01

    Inhibition of jasmonic acid (JA) signaling has been shown to decrease herbivore resistance, but the responsible mechanisms are largely unknown because insect resistance is poorly understood in most model plant systems. We characterize three members of the lipoxygenase (LOX) gene family in the native tobacco plant Nicotiana attenuata and manipulate, by antisense expression, a specific, wound- and herbivory-induced isoform (LOX3) involved in JA biosynthesis. In three independent lines, antisense expression reduced wound-induced JA accumulation but not the release of green leaf volatiles (GLVs). The impaired JA signaling reduced two herbivore-induced direct defenses, nicotine and trypsin protease inhibitors (TPI), as well as the potent indirect defense, the release of volatile terpenes that attract generalist predators to feeding herbivores. All these defenses could be fully restored by methyl-JA (MeJA) treatment, with the exception of the increase in TPI activity, which was partially restored, suggesting the involvement of additional signals. The impaired ability to produce chemical defenses resulted in lower resistance to Manduca sexta attack, which could also be restored by MeJA treatment. Expression analysis using a cDNA microarray, specifically designed to analyze M. sexta-induced gene expression in N. attenuata, revealed a pivotal role for LOX3-produced oxylipins in upregulating defense genes (protease inhibitor, PI; xyloglucan endotransglucosylase/hydrolase, XTH; threonine deaminase, TD; hydroperoxide lyase, HPL), suppressing both downregulated growth genes (RUBISCO and photosystem II, PSII) and upregulated oxylipin genes (alpha-dioxygenase, alpha-DOX). By genetically manipulating signaling in a plant with a well-characterized ecology, we demonstrate that the complex phenotypic changes that mediate herbivore resistance are controlled by a specific part of the oxylipin cascade.

  3. Upstream Regulatory Region Alterations Found in Human Papillomavirus Type 16 (HPV-16) Isolates from Cervical Carcinomas Increase Transcription, ori Function, and HPV Immortalization Capacity in Culture▿

    PubMed Central

    Lace, Michael J.; Isacson, Christina; Anson, James R.; Lörincz, Attila T.; Wilczynski, Sharon P.; Haugen, Thomas H.; Turek, Lubomír P.

    2009-01-01

    Human papillomavirus (HPV) DNAs isolated from cervical and head and neck carcinomas frequently contain nucleotide sequence alterations in the viral upstream regulatory region (URR). Our study has addressed the role such sequence changes may play in the efficiency of establishing HPV persistence and altered keratinocyte growth. Genomic mapping of integrated HPV type 16 (HPV-16) genomes from 32 cervical cancers revealed that the viral E6 and E7 oncogenes, as well as the L1 region/URR, were intact in all of them. The URR sequences from integrated and unintegrated viral DNA were found to harbor distinct sets of nucleotide substitutions. A subset of the altered URRs increased the potential of HPV-16 to establish persistent, cell growth-altering viral-genome replication in the cell. This aggressive phenotype in culture was not solely due to increased viral early gene transcription, but also to augmented initial amplification of the viral genome. As revealed in a novel ori-dependent HPV-16 plasmid amplification assay, the altered motifs that led to increased viral transcription from the intact genome also greatly augmented HPV-16 ori function. The nucleotide sequence changes correlate with those previously described in the distinct geographical North American type 1 and Asian-American variants that are associated with more aggressive disease in epidemiologic studies and encompass, but are not limited to, alterations in previously characterized sites for the negative regulatory protein YY1. Our results thus provide evidence that nucleotide alterations in HPV regulatory sequences could serve as potential prognostic markers of HPV-associated carcinogenesis. PMID:19458011

  4. ERα directly activated the MDR1 transcription to increase paclitaxel-resistance of ERα-positive breast cancer cells in vitro and in vivo.

    PubMed

    Shi, Jun-Feng; Yang, Nan; Ding, Hai-Jian; Zhang, Jie-Xin; Hu, Mei-Ling; Leng, Yan; Han, Xiao; Sun, Yu-Jie

    2014-08-01

    Chemotherapy is commonly used to treat early-stage invasive and advanced-stage breast cancer either before or after surgery. Increasing evidence from clinical analysis and in vitro studies has shown that ER-positive breast cancer cells are insensitive to chemotherapy. Complete understanding of how ERα mediates drug resistance is prerequisite to improvement of the chemotherapeutic efficacy. Over-expression of P-glycoprotein (P-gp) encoded by MDR1 gene is one of the major causes of drug resistance. The association between ERα and MDR1 in breast cancer is still unclear and the limited reports are conflict. This study systematically explored intrinsic link between ERα and the P-gp over-expression in paclitaxel-resistant ERα(+) breast cancer cell lines and mouse model in molecular details. Our data showed that ERα activated the MDR1 transcription in MCF-7/PTX breast cancer cells by binding to ERE1/2 and interacting with Sp1 that bridged to the downstream CG-rich element within the MDR1 promoter. Knockdown of MDR1 restrained the effect of ERα in MCF-7 cells and sensitized the cells to paclitaxel. Treatment of ICI 182,780 that selectively suppressed ERα significantly decreased the MDR1 expression and increased the sensitivity of drug resistant breast cancer cells and xenograft tumors to paclitaxel. Our data strongly demonstrated that ERα was able to increase drug resistance of breast cancer cells through activating MDR1 transcription. This novel mechanism provides new insight to how the ERα signaling regulates response of ERα(+) breast tumors to chemotherapy, which may be exploited for developing novel therapeutic strategies for breast cancer in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Toxoplasma gondii Inhibits gamma interferon (IFN-γ)- and IFN-β-induced host cell STAT1 transcriptional activity by increasing the association of STAT1 with DNA.

    PubMed

    Rosowski, Emily E; Nguyen, Quynh P; Camejo, Ana; Spooner, Eric; Saeij, Jeroen P J

    2014-02-01

    The gamma interferon (IFN-γ) response, mediated by the STAT1 transcription factor, is crucial for host defense against the intracellular pathogen Toxoplasma gondii, but prior infection with Toxoplasma can inhibit this response. Recently, it was reported that the Toxoplasma type II NTE strain prevents the recruitment of chromatin remodeling complexes containing Brahma-related gene 1 (BRG-1) to promoters of IFN-γ-induced secondary response genes such as Ciita and major histocompatibility complex class II genes in murine macrophages, thereby inhibiting their expression. We report here that a type I strain of Toxoplasma inhibits the expression of primary IFN-γ response genes such as IRF1 through a distinct mechanism not dependent on the activity of histone deacetylases. Instead, infection with a type I, II, or III strain of Toxoplasma inhibits the dissociation of STAT1 from DNA, preventing its recycling and further rounds of STAT1-mediated transcriptional activation. This leads to increased IFN-γ-induced binding of STAT1 at the IRF1 promoter in host cells and increased global IFN-γ-induced association of STAT1 with chromatin. Toxoplasma type I infection also inhibits IFN-β-induced interferon-stimulated gene factor 3-mediated gene expression, and this inhibition is also linked to increased association of STAT1 with chromatin. The secretion of proteins into the host cell by a type I strain of Toxoplasma without complete parasite invasion is not sufficient to block STAT1-mediated expression, suggesting that the effector protein responsible for this inhibition is not derived from the rhoptries.

  6. Increased Energy Expenditure, Ucp1 Expression, and Resistance to Diet-induced Obesity in Mice Lacking Nuclear Factor-Erythroid-2-related Transcription Factor-2 (Nrf2).

    PubMed

    Schneider, Kevin; Valdez, Joshua; Nguyen, Janice; Vawter, Marquis; Galke, Brandi; Kurtz, Theodore W; Chan, Jefferson Y

    2016-04-01

    The NRF2 (also known as NFE2L2) transcription factor is a critical regulator of genes involved in defense against oxidative stress. Previous studies suggest thatNrf2plays a role in adipogenesisin vitro, and deletion of theNrf2gene protects against diet-induced obesity in mice. Here, we demonstrate that resistance to diet-induced obesity inNrf2(-/-)mice is associated with a 20-30% increase in energy expenditure. Analysis of bioenergetics revealed thatNrf2(-/-)white adipose tissues exhibit greater oxygen consumption. White adipose tissue showed a >2-fold increase inUcp1gene expression. Oxygen consumption is also increased nearly 2.5-fold inNrf2-deficient fibroblasts. Oxidative stress induced by glucose oxidase resulted in increasedUcp1expression. Conversely, antioxidant chemicals (such asN-acetylcysteine and Mn(III)tetrakis(4-benzoic acid)porphyrin chloride) and SB203580 (a known suppressor ofUcp1expression) decreasedUcp1and oxygen consumption inNrf2-deficient fibroblasts. These findings suggest that increasing oxidative stress by limitingNrf2function in white adipocytes may be a novel means to modulate energy balance as a treatment of obesity and related clinical disorders.

  7. Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris.

    PubMed

    Cámara, Elena; Landes, Nils; Albiol, Joan; Gasser, Brigitte; Mattanovich, Diethard; Ferrer, Pau

    2017-03-15

    The methanol-regulated alcohol oxidase promoter (PAOX1) of Pichia pastoris is one of the strongest promoters for heterologous gene expression in this methylotrophic yeast. Although increasing gene dosage is one of the most common strategies to increase recombinant protein productivities, the increase of gene dosage of Rhizopus oryzae lipase (ROL) in P. pastoris has been previously shown to reduce cell growth, lipase production and substrate consumption in high-copy strains. To better assess that physiological response, transcriptomics analysis was performed of a subset of strains with 1 to 15 ROL copies. The macroscopic physiological parameters confirm that growth yield and carbon uptake rate are gene dosage dependent, and were supported by the transcriptomic data, showing the impact of increased dosage of AOX1 promoter-regulated expression cassettes on P. pastoris physiology under steady methanolic growth conditions. Remarkably, increased number of cassettes led to transcription attenuation of the methanol metabolism and peroxisome biogenesis in P. pastoris, concomitant with reduced secretion levels of the heterologous product. Moreover, our data also point to a block in ROL mRNA translation in the higher ROL-copies constructs, while the low productivities of multi-copy strains under steady growth conditions do not appear to be directly related to UPR and ERAD induction.

  8. Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris

    PubMed Central

    Cámara, Elena; Landes, Nils; Albiol, Joan; Gasser, Brigitte; Mattanovich, Diethard; Ferrer, Pau

    2017-01-01

    The methanol-regulated alcohol oxidase promoter (PAOX1) of Pichia pastoris is one of the strongest promoters for heterologous gene expression in this methylotrophic yeast. Although increasing gene dosage is one of the most common strategies to increase recombinant protein productivities, the increase of gene dosage of Rhizopus oryzae lipase (ROL) in P. pastoris has been previously shown to reduce cell growth, lipase production and substrate consumption in high-copy strains. To better assess that physiological response, transcriptomics analysis was performed of a subset of strains with 1 to 15 ROL copies. The macroscopic physiological parameters confirm that growth yield and carbon uptake rate are gene dosage dependent, and were supported by the transcriptomic data, showing the impact of increased dosage of AOX1 promoter-regulated expression cassettes on P. pastoris physiology under steady methanolic growth conditions. Remarkably, increased number of cassettes led to transcription attenuation of the methanol metabolism and peroxisome biogenesis in P. pastoris, concomitant with reduced secretion levels of the heterologous product. Moreover, our data also point to a block in ROL mRNA translation in the higher ROL-copies constructs, while the low productivities of multi-copy strains under steady growth conditions do not appear to be directly related to UPR and ERAD induction. PMID:28295011

  9. Increased Energy Expenditure, Ucp1 Expression, and Resistance to Diet-induced Obesity in Mice Lacking Nuclear Factor-Erythroid-2-related Transcription Factor-2 (Nrf2)*

    PubMed Central

    Schneider, Kevin; Valdez, Joshua; Nguyen, Janice; Vawter, Marquis; Galke, Brandi; Kurtz, Theodore W.; Chan, Jefferson Y.

    2016-01-01

    The NRF2 (also known as NFE2L2) transcription factor is a critical regulator of genes involved in defense against oxidative stress. Previous studies suggest that Nrf2 plays a role in adipogenesis in vitro, and deletion of the Nrf2 gene protects against diet-induced obesity in mice. Here, we demonstrate that resistance to diet-induced obesity in Nrf2−/− mice is associated with a 20–30% increase in energy expenditure. Analysis of bioenergetics revealed that Nrf2−/− white adipose tissues exhibit greater oxygen consumption. White adipose tissue showed a >2-fold increase in Ucp1 gene expression. Oxygen consumption is also increased nearly 2.5-fold in Nrf2-deficient fibroblasts. Oxidative stress induced by glucose oxidase resulted in increased Ucp1 expression. Conversely, antioxidant chemicals (such as N-acetylcysteine and Mn(III)tetrakis(4-benzoic acid)porphyrin chloride) and SB203580 (a known suppressor of Ucp1 expression) decreased Ucp1 and oxygen consumption in Nrf2-deficient fibroblasts. These findings suggest that increasing oxidative stress by limiting Nrf2 function in white adipocytes may be a novel means to modulate energy balance as a treatment of obesity and related clinical disorders. PMID:26841864

  10. Diammonium phosphate stimulates transcription of L-lactate dehydrogenase leading to increased L-lactate production in the thermotolerant Bacillus coagulans strain.

    PubMed

    Sun, Lifan; Li, Yanfeng; Wang, Limin; Wang, Yanping; Yu, Bo

    2016-08-01

    Exploration of cost-effective fermentation substrates for efficient lactate production is an important economic objective. Although some organic nitrogen sources are also cheaper, inorganic nitrogen salts for lactate fermentation have additional advantages in facilitating downstream procedures and significantly improving the commercial competitiveness of lactate production. In this study, we first established an application of diammonium phosphate to replace yeast extract with a reduced 90 % nitrogen cost for a thermotolerant Bacillus coagulans strain. In vivo enzymatic and transcriptional analyses demonstrated that diammonium phosphate stimulates the gene expression of L-lactate dehydrogenase, thus providing higher specific enzyme activity in vivo and increasing L-lactic acid production. This new information provides a foundation for establishing a cost-effective process for polymer-grade L-lactic acid production in an industrial setting.

  11. Increase in gene-transcript levels as indicators of up-regulation of the unfolded protein response in spontaneous canine tumors.

    PubMed

    Elliot, Kirsten; MacDonald-Dickinson, Valerie; Linn, Kathleen; Simko, Elemir; Misra, Vikram

    2014-07-01

    The unfolded protein response (UPR), a conserved cellular response to stressors such as hypoxia and nutrient deprivation, is associated with angiogenesis and metastasis in tumor cells. This article discusses a pilot study conducted to determine whether components of the UPR could be identified in spontaneous canine tumors and whether they were up-regulated within tumor tissue compared with adjacent normal tissue. Tissue samples of various spontaneous canine neoplasms were taken from 13 dogs shortly after surgical excision or euthanasia; control samples were taken from adjacent normal tissue. RNA purification and real-time quantitative reverse-transcription polymerase chain reaction were done to measure the expression of 4 genes associated with the UPR (HERP, CHOP, GRP78, and XBP1s). The results indicated that UPR gene expression can be identified in spontaneous canine tumors and that the UPR is up-regulated, as indicated by significantly increased expression of CHOP and GRP78 within the tumor.

  12. An unusual case of splenomegaly and increased lactate dehydrogenase heralding acute myeloid leukemia with eosinophilia and RUNX1–MECOM fusion transcripts

    PubMed Central

    Forghieri, Fabio; Bigliardi, Sara; Morselli, Monica; Potenza, Leonardo; Fantuzzi, Valeria; Faglioni, Laura; Nasillo, Vincenzo; Messerotti, Andrea; Paolini, Ambra; Luppi, Mario

    2014-01-01

    We report the first case of acute myeloid leukemia (AML) with RUNX1–MECOM fusion transcripts, showing marked eosinophilia. A 63-year old man admitted in August 2013, had previously been observed in April 2013, because of persisting homogeneous splenomegaly and increased LDH, which were initially attributed to both minor β-thalassemia and previous acute myocardial infarction. However, based upon the retrospective analysis of clinical features combined with the documentation of both JAK2 V617F and c-KIT D816V mutations at AML diagnosis, an aggressive leukemic transformation with eosinophilia of a previously unrecognized myeloproliferative neoplasm, rather than the occurrence of de novo AML, may be hypothesized. PMID:25379409

  13. Overexpression of the Transcriptional Regulator WOR1 Increases Susceptibility to Bile Salts and Adhesion to the Mouse Gut Mucosa in Candida albicans

    PubMed Central

    Prieto, Daniel; Román, Elvira; Alonso-Monge, Rebeca; Pla, Jesús

    2017-01-01

    The transcriptional regulator Wor1 has been shown to induce the GUT transition, an environmentally triggered process that increases the fitness of Candida albicans in the mouse gastrointestinal tract. We have developed strains where the expression of this gene is driven from the strong and tightly regulated tetracycline promoter. These cells retain the main characteristics reported for GUT cells albeit they show defects in the initial stages of colonization. They also show a differential colonization along the gastrointestinal tract compared to isogenic strains, which is probably caused by their susceptibility to bile salts. We also show that WOR1 overexpressing cells have an altered metabolic activity, as revealed by a different susceptibility to inhibitors of respiration, and an enhanced adhesion to the mouse mucosa. We propose that this may contribute to their long-term favored ability to colonize the gastrointestinal tract. PMID:28955659

  14. Overexpression of the microRNA hsa-miR-200c leads to reduced expression of transcription factor 8 and increased expression of E-cadherin.

    PubMed

    Hurteau, Gregory J; Carlson, J Andrew; Spivack, Simon D; Brock, Graham J

    2007-09-01

    MicroRNAs are approximately 22-nucleotide sequences thought to interact with multiple mRNAs resulting in either translational repression or degradation. We previously reported that several microRNAs had variable expression in mammalian cell lines, and we examined one, miR-200c, in more detail. A combination of bioinformatics and quantitative reverse transcription-PCR was used to identify potential targets and revealed that the zinc finger transcription factor transcription factor 8 (TCF8; also termed ZEB1, deltaEF1, Nil-2-alpha) had inversely proportional expression levels to miR-200c. Knockout experiments using anti-microRNA oligonucleotides increased TCF8 levels but with nonspecific effects. Therefore, to investigate target predictions, we overexpressed miR-200c in select cells lines. Ordinarily, the expression level of miR-200c in non-small-cell lung cancer A549 cells is low in contrast to normal human bronchial epithelial cells. Stable overexpression of miR-200c in A549 cells results in a loss of TCF8, an increase in expression of its regulatory target, E-cadherin, and altered cell morphology. In MCF7 (estrogen receptor-positive breast cancer) cells, there is endogenous expression of miR-200c and E-cadherin but TCF8 is absent. Conversely, MDA-MB-231 (estrogen receptor-negative) cells lack detectable miR-200c and E-cadherin (the latter reportedly due to promoter region methylation) but express TCF8. The ectopic expression of miR-200c in this cell line also reduced levels of TCF8, restored E-cadherin expression, and altered cell morphology. Because the down-regulation of E-cadherin is a crucial event in epithelial-to-mesenchymal transition, loss of miR-200c expression could play a significant role in the initiation of an invasive phenotype, and, equally, miR-200c overexpression holds potential for its reversal.

  15. Increase of transcription factor EB (TFEB) and lysosomes in rat DRG neurons and their transportation to the central nerve terminal in dorsal horn after nerve injury.

    PubMed

    Jung, J; Uesugi, N; Jeong, N Y; Park, B S; Konishi, H; Kiyama, H

    2016-01-28

    In the spinal dorsal horn (DH), nerve injury activates microglia and induces neuropathic pain. Several studies clarified an involvement of adenosine triphosphate (ATP) in the microglial activation. However, the origin of ATP together with the release mechanism is unclear. Recent in vitro study revealed that an ATP marker, quinacrine, in lysosomes was released from neurite terminal of dorsal root ganglion (DRG) neurons to extracellular space via lysosomal exocytosis. Here, we demonstrate a possibility that the lysosomal ingredient including ATP released from DRG neurons by lysosomal-exocytosis is an additional source of the glial activation in DH after nerve injury. After rat L5 spinal nerve ligation (SNL), mRNA for transcription factor EB (TFEB), a transcription factor controlling lysosomal activation and exocytosis, was induced in the DRG. Simultaneously both lysosomal protein, LAMP1- and vesicular nuclear transporter (VNUT)-positive vesicles were increased in L5 DRG neurons and ipsilateral DH. The quinacrine staining in DH was increased and co-localized with LAMP1 immunoreactivity after nerve injury. In DH, LAMP1-positive vesicles were also co-localized with a peripheral nerve marker, Isolectin B4 (IB4) lectin. Injection of the adenovirus encoding mCherry-LAMP1 into DRG showed that mCherry-positive lysosomes are transported to the central nerve terminal in DH. These findings suggest that activation of lysosome synthesis including ATP packaging in DRG, the central transportation of the lysosome, and subsequent its exocytosis from the central nerve terminal of DRG neurons in response to nerve injury could be a partial mechanism for activation of microglia in DH. This lysosome-mediated microglia activation mechanism may provide another clue to control nociception and pain.

  16. The transcription of MGAT4A glycosyl transferase is increased in white cells of peripheral blood of Type 2 Diabetes patients

    PubMed Central

    López-Orduña, Eduardo; Cruz, Miguel; García-Mena, Jaime

    2007-01-01

    Background Human glycosylase IV is involved in GLUT2 transporter regulation in pancreatic β cells. A KO of this gene along with a high fat diet in a mice model has been associated with the development of type 2 diabetes (T2D). The aims of this study were to measure and compare the MGAT4A mRNA levels in white blood cells (WBC) from T2D subjects and healthy subjects (T2NB), and to measure the half-life of the MGAT4A mRNA. Results We studied a sample of 73 individuals, 40 T2D subjects and 33 T2NB subjects. Anthropometrical and biochemical profiles were registered. The MGAT4A mRNA levels in WBC and the transcript half-life in Jurkat T cells were determined by Real-Time PCR. A blood differential cell counting was made for each individual. Cell counting showed T2D subjects exhibited an increased number of WBC compared to T2NB subjects (P = 0.0001). Biochemical parameters such as fasting glucose (P = 0.0001), and triglycerides (P = 0.002) were statistically significant. T2D subjects had 4.2-fold more MGAT4A transcript compared to T2NB subjects (P = 0.002). The MGAT4A mRNA had a half-life of 2.04 h in Jurkat T cells. Conclusion The results of this work suggest that in T2D subjects, high levels of glucose and triglycerides are accompanied by an increase on MGAT4A mRNA levels and WBC count; condition that suggests a pro-inflammatory state due to a chronic metabolic stress. PMID:17953760

  17. HIV immune activation drives increased Eomes expression in memory CD8 T cells in association with transcriptional downregulation of CD127.

    PubMed

    Hasley, Rebecca B; Hong, Changwan; Li, Wenqing; Friesen, Travis; Nakamura, Yoriko; Kim, Grace Y; Park, Jung-Hyun; Hixon, Julie A; Durum, Scott; Hu, Zonghui; Sneller, Michael C; Oguariri, Raphael; Imamichi, Tomozumi; Lane, H Clifford; Catalfamo, Marta

    2013-07-31

    During HIV infection distinct mechanisms drive immune activation of the CD4 and CD8 T cells leading to CD4 T-cell depletion and expansion of the CD8 T-cell pool. This immune activation is polyclonal and extends beyond HIV-specific T cells. One consequence of this immune activation is a profound decrease in IL-7Rα (CD127) expression on memory CD8 T cells. The mechanisms leading to this are unknown and because of the potential impact of reduced IL-7 signaling in memory T cells specific to HIV and other pathogens, in the present study we examined the molecular mechanisms implicated in this downregulation of CD127. Membrane bound (mIL7RA) and soluble (sIL7RA) mRNA expression was determined by qRT-PCR. CD127, Eomesodermin (Eomes) and T-bet expression in healthy controls and HIV-infected patients were studied by flow cytometry. CD127 downregulation occurs at the transcriptional level for both mIL7RA and sIL7RA alternative spliced forms in the CD127 memory CD8 T cells. CD127 memory CD8 T cells exhibited increased Eomes expression and an 'effector-like' gene profile. These changes were associated with higher HIV-RNA levels. Following combination antiretroviral therapy (cART), there was an increase in CD127 expression over an extended period of time (>5 months) which was associated with decreased Eomes expression. CD127 is downregulated at a transcriptional level in memory CD8 T cells in association with upregulation of Eomes expression.

  18. Increased expression of bHLH transcription factor E2A (TCF3) in prostate cancer promotes proliferation and confers resistance to doxorubicin induced apoptosis

    SciTech Connect

    Patel, Divya; Chaudhary, Jaideep

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer E2A, considered as a tumor suppressor is highly expressed in prostate cancer. Black-Right-Pointing-Pointer Silencing of E2A attenuates cell proliferation and promotes apoptosis. Black-Right-Pointing-Pointer E2A regulates c-myc, Id1, Id3 and CDKN1A expression. Black-Right-Pointing-Pointer Loss of E2A promotes doxorubicin dependent apoptosis in prostate cancer cells. Black-Right-Pointing-Pointer Results suggest that E2A acts as a tumor promoter at least in prostate cancer. -- Abstract: E2A (TCF3) is a multifunctional basic helix loop helix (bHLH), transcription factor. E2A regulates transcription of target genes by homo- or heterodimerization with cell specific bHLH proteins. In general, E2A promotes cell differentiation, acts as a negative regulator of cell proliferation in normal cells and cancer cell lines and is required for normal B-cell development. Given the diverse biological pathways regulated/influenced by E2A little is known about its expression in cancer. In this study we investigated the expression of E2A in prostate cancer. Unexpectedly, E2A immuno-histochemistry demonstrated increased E2A expression in prostate cancer as compared to normal prostate. Silencing of E2A in prostate cancer cells DU145 and PC3 led to a significant reduction in proliferation due to G1 arrest that was in part mediated by increased CDKN1A(p21) and decreased Id1, Id3 and c-myc. E2A silencing in prostate cancer cell lines also resulted in increased apoptosis due to increased mitochondrial permeability and caspase 3/7 activation. Moreover, silencing of E2A increased sensitivity to doxorubicin induced apoptosis. Based on our results, we propose that E2A could be an upstream regulator of Id1 and c-Myc which are highly expressed in prostate cancer. These results for the first time demonstrate that E2A could in fact acts as a tumor promoter at least in prostate cancer.

  19. Ablation of the transcriptional regulator Id1 enhances energy expenditure, increases insulin sensitivity, and protects against age and diet induced insulin resistance, and hepatosteatosis

    PubMed Central

    Satyanarayana, Ande; Klarmann, Kimberly D.; Gavrilova, Oksana; Keller, Jonathan R.

    2012-01-01

    Obesity is a major health concern that contributes to the development of diabetes, hyperlipidemia, coronary artery disease, and cancer. Id proteins are helix-loop-helix transcription factors that regulate the proliferation and differentiation of cells from multiple tissues, including adipocytes. We screened mouse tissues for the expression of Id1 and found that Id1 protein is highly expressed in brown adipose tissue (BAT) and white adipose tissue (WAT), suggesting a role for Id1 in adipogenesis and cell metabolism. Id1−/− mice are viable but show a significant reduction in fat mass (P<0.005) over the life of the animal that was not due to decreased number of adipocytes. Analysis of Id1−/− mice revealed higher energy expenditure, increased lipolysis, and fatty acid oxidation, resulting in reduced triglyceride accumulation in WAT compared to Id1+/+ mice. Serum levels of triglycerides (193.9±32.2 vs. 86.5±33.8, P<0.0005), cholesterol (189.4±33.8 vs. 110.6±8.23, P<0.0005) and leptin (1263±835 vs. 222±260, P<0.005) were significantly lower in aged Id1−/− mice compared to Id1+/+ mice. Id1-deficient mice have higher resting (P<0.005) and total (P<0.05) O2 consumption and lower respiratory exchange ratio (P<0.005), confirming that Id1−/− mice use a higher proportion of lipid as an energy source for the increased energy expenditure. The expression of PGC1α and UCP1 were 2- to 3-fold up-regulated in Id1−/− BAT, suggesting that loss of Id1 increases thermogenesis. As a consequence of higher energy expenditure and reduced fat mass, Id1−/− mice displayed enhanced insulin sensitivity. Id1 deficiency protected mice against age- and high-fat-diet-induced adiposity, insulin resistance, and hepatosteatosis. Our findings suggest that Id1 plays a critical role in the regulation of energy homeostasis and could be a potential target in the treatment of insulin resistance and fatty liver disease.—Satyanarayana, A., Klarmann, K. D., Gavrilova, l O., Keller

  20. Co-evolution between Grapevine rupestris stem pitting-associated virus and Vitis vinifera L. leads to decreased defence responses and increased transcription of genes related to photosynthesis.

    PubMed

    Gambino, Giorgio; Cuozzo, Danila; Fasoli, Marianna; Pagliarani, Chiara; Vitali, Marco; Boccacci, Paolo; Pezzotti, Mario; Mannini, Franco

    2012-10-01

    Grapevine rupestris stem pitting-associated virus (GRSPaV) is a widespread virus infecting Vitis spp. Although it has established a compatible viral interaction in Vitis vinifera without the development of phenotypic alterations, it can occur as distinct variants that show different symptoms in diverse Vitis species. The changes induced by GRSPaV in V. vinifera cv 'Bosco', an Italian white grape variety, were investigated by combining agronomic, physiological, and molecular approaches, in order to provide comprehensive information about the global effects of GRSPaV. In two years, this virus caused a moderate decrease in physiological efficiency, yield performance, and sugar content in berries associated with several transcriptomic alterations. Transcript profiles were analysed by a microarray technique in petiole, leaf, and berry samples collected at véraison and by real-time RT-PCR in a time course carried out at five grapevine developmental stages. Global gene expression analyses showed that transcriptomic changes were highly variable among the different organs and the different phenological phases. GRSPaV triggers some unique responses in the grapevine at véraison, never reported before for other plant-virus interactions. These responses include an increase in transcripts involved in photosynthesis and CO(2) fixation, a moderate reduction in the photosynthesis rate and some defence mechanisms, and an overlap with responses to water and salinity stresses. It is hypothesized that the long co-existence of grapevine and GRSPaV has resulted in the evolution of a form of mutual adaptation between the virus and its host. This study contributes to elucidating alternative mechanisms used by infected plants to contend with viruses.

  1. Variants within MECP2, a key transcriptional regulator, are associated with increased susceptibility to lupus and differential gene expression in lupus patients

    PubMed Central

    Webb, Ryan; Wren, Jonathan D; Jeffries, Matlock; Kelly, Jennifer A; Kaufman, Kenneth M; Tang, Yuhong; Frank, Mark Barton; Merrill, Joan; Alarcón-Riquelme, Marta E; James, Judith A; Vyse, Timothy J; Moser, Kathy L; Gilkeson, Gary S; Harley, John B; Sawalha, Amr H

    2009-01-01

    Objective Both genetic and epigenetic factors play an important role in the pathogenesis of lupus. Herein, we study methyl-CpG-binding protein 2 (MECP2) polymorphism in a large cohort of lupus patients and controls, and determine functional consequences of the lupus-associated MECP2 haplotype. Methods We genotyped 18 SNPs within MECP2, located on chromosome Xq28, in a large cohort of European-derived lupus patients and controls. We studied the functional effects of the lupus-associated MECP2 haplotype by determining gene expression profiles in B cell lines from female lupus patients with and without the lupus-associated MECP2 risk haplotype. Results We confirm, replicate, and extend the genetic association between lupus and genetic markers within MECP2 in a large independent cohort of European-derived lupus patients and controls (OR= 1.35, p= 6.65×10−11). MECP2 is a dichotomous transcriptional regulator that either activates or represses gene expression. We identified 128 genes that are differentially expressed in lupus patients with the disease-associated MECP2 haplotype; most (~81%) are upregulated. Genes that were upregulated have significantly more CpG islands in their promoter regions compared to downregulated genes. Gene ontology analysis using the differentially expressed genes revealed significant association with epigenetic regulatory mechanisms suggesting that these genes are targets for MECP2 regulation in B cells. Further, at least 13 of the 104 upregulated genes are interferon-regulated genes. The disease-risk MECP2 haplotype is associated with increased expression of the MECP2 transcriptional co-activator CREB1, and decreased expression of the co-repressor HDAC1. Conclusion Polymorphism in the MECP2 locus is associated with lupus and, at least in part, contributes to the interferon signature observed in lupus patients. PMID:19333917

  2. Variants within MECP2, a key transcription regulator, are associated with increased susceptibility to lupus and differential gene expression in patients with systemic lupus erythematosus.

    PubMed

    Webb, Ryan; Wren, Jonathan D; Jeffries, Matlock; Kelly, Jennifer A; Kaufman, Kenneth M; Tang, Yuhong; Frank, Mark Barton; Merrill, Joan; Kimberly, Robert P; Edberg, Jeffrey C; Ramsey-Goldman, Rosalind; Petri, Michelle; Reveille, John D; Alarcón, Graciela S; Vilá, Luis M; Alarcón-Riquelme, Marta E; James, Judith A; Vyse, Timothy J; Moser, Kathy L; Gaffney, Patrick M; Gilkeson, Gary S; Harley, John B; Sawalha, Amr H

    2009-04-01

    Both genetic and epigenetic factors play an important role in the pathogenesis of lupus. The aim of this study was to examine methyl-CpG-binding protein 2 gene (MECP2) polymorphisms in a large cohort of patients with lupus and control subjects, and to determine the functional consequences of the lupus-associated MECP2 haplotype. We genotyped 18 single-nucleotide polymorphisms within MECP2, located on chromosome Xq28, in a large cohort of patients with lupus and control subjects of European descent. We studied the functional effects of the lupus-associated MECP2 haplotype by determining gene expression profiles in B cell lines in female lupus patients with and those without the lupus-associated MECP2 risk haplotype. We confirmed, replicated, and extended the genetic association between lupus and genetic markers within MECP2 in a large independent cohort of lupus patients and control subjects of European descent (odds ratio 1.35, P = 6.65 x 10(-11)). MECP2 is a dichotomous transcription regulator that either activates or represses gene expression. We identified 128 genes that are differentially expressed in lupus patients with the disease-associated MECP2 haplotype; most ( approximately 81%) were up-regulated. Genes that were up-regulated had significantly more CpG islands in their promoter regions compared with genes that were down-regulated. Gene ontology analysis using the differentially expressed genes revealed significant association with epigenetic regulatory mechanisms, suggesting that these genes are targets for MECP2 regulation in B cells. Furthermore, at least 13 of the 104 up-regulated genes are regulated by interferon. The disease-risk MECP2 haplotype was associated with increased expression of the MECP2 transcription coactivator CREB1 and decreased expression of the corepressor histone deacetylase 1. Polymorphism in the MECP2 locus is associated with lupus and, at least in part, contributes to the interferon signature observed in lupus patients.

  3. Cdk5 Protein Inhibition and Aβ42 Increase BACE1 Protein Level in Primary Neurons by a Post-transcriptional Mechanism

    PubMed Central

    Sadleir, Katherine R.; Vassar, Robert

    2012-01-01

    The β-secretase enzyme BACE1 initiates production of the amyloid-β (Aβ) peptide that comprises plaques in Alzheimer disease (AD) brain. BACE1 levels are increased in AD, potentially accelerating Aβ generation, but the mechanisms of BACE1 elevation are not fully understood. Cdk5/p25 has been implicated in neurodegeneration and BACE1 regulation, suggesting therapeutic Cdk5 inhibition for AD. In addition, caspase 3 has been implicated in BACE1 elevation. Here, we show that the Cdk5 level and p25:p35 ratio were elevated and correlated with BACE1 level in brains of AD patients and 5XFAD transgenic mice. Mouse primary cortical neurons treated with Aβ42 oligomers had increased BACE1 level and p25:p35 ratio. Surprisingly, the Aβ42-induced BACE1 elevation was not blocked by Cdk5 inhibitors CP68130 and roscovitine, and instead the BACE1 level was increased greater than with Aβ42 treatment alone. Moreover, Cdk5 inhibitors alone elevated BACE1 in a time- and dose-dependent manner that coincided with increased caspase 3 cleavage and decreased Cdk5 level. Caspase 3 inhibitor benzyloxycarbonyl-VAD failed to prevent the Aβ42-induced BACE1 increase. Further experiments suggested that the Aβ42-induced BACE1 elevation was the result of a post-transcriptional mechanism. We conclude that Aβ42 may increase the BACE1 level independently of either Cdk5 or caspase 3 and that Cdk5 inhibition for AD may cause BACE1 elevation, a potentially negative therapeutic outcome. PMID:22223639

  4. MYB and bHLH transcription factor transgenes increase anthocyanin pigmentation in petunia and lisianthus plants, and the petunia phenotypes are strongly enhanced under field conditions

    PubMed Central

    Schwinn, Kathy E.; Boase, Murray R.; Bradley, J. Marie; Lewis, David H.; Deroles, Simon C.; Martin, Cathie R.; Davies, Kevin M.

    2014-01-01

    Petunia line Mitchell [MP, Petunia axillaris × (P. axillaris × P. hybrida)] and Eustoma grandiflorum (lisianthus) plants were produced containing a transgene for over-expression of the R2R3-MYB transcription factor [TF; ROSEA1 (ROS1)] that up-regulates flavonoid biosynthesis in Antirrhinum majus. The petunia lines were also crossed with previously produced MP lines containing a Zea mays flavonoid-related basic helix-loop-helix TF transgene (LEAF COLOR, LC), which induces strong vegetative pigmentation when these 35S:LC plants are exposed to high-light levels. 35S:ROS1 lisianthus transgenics had limited changes in anthocyanin pigmentation, specifically, precocious pigmentation of flower petals and increased pigmentation of sepals. RNA transcript levels for two anthocyanin biosynthetic genes, chalcone synthase and anthocyanidin synthase, were increased in the 35S:ROS1 lisianthus petals compared to those of control lines. With MP, the 35S:ROS1 calli showed novel red pigmentation in culture, but this was generally not seen in tissue culture plantlets regenerated from the calli or young plants transferred to soil in the greenhouse. Anthocyanin pigmentation was enhanced in the stems of mature 35S:ROS1 MP plants, but the MP white-flower phenotype was not complemented. Progeny from a 35S:ROS1 × 35S:LC cross had novel pigmentation phenotypes that were not present in either parental line or MP. In particular, there was increased pigment in the petal throat region, and the anthers changed from yellow to purple pigmentation. An outdoor field trial was conducted with the 35S:ROS1, 35S:LC, 35S:ROS1 × 35S:LC and control MP lines. Field conditions rapidly induced intense foliage pigmentation in 35S:LC plants, a phenotype not observed in control MP or equivalent 35S:LC plants maintained in a greenhouse. No difference in plant stature, seed germination, or plant survival was observed between transgenic and control plants. PMID:25414715

  5. MYB and bHLH transcription factor transgenes increase anthocyanin pigmentation in petunia and lisianthus plants, and the petunia phenotypes are strongly enhanced under field conditions.

    PubMed

    Schwinn, Kathy E; Boase, Murray R; Bradley, J Marie; Lewis, David H; Deroles, Simon C; Martin, Cathie R; Davies, Kevin M

    2014-01-01

    Petunia line Mitchell [MP, Petunia axillaris × (P. axillaris × P. hybrida)] and Eustoma grandiflorum (lisianthus) plants were produced containing a transgene for over-expression of the R2R3-MYB transcription factor [TF; ROSEA1 (ROS1)] that up-regulates flavonoid biosynthesis in Antirrhinum majus. The petunia lines were also crossed with previously produced MP lines containing a Zea mays flavonoid-related basic helix-loop-helix TF transgene (LEAF COLOR, LC), which induces strong vegetative pigmentation when these 35S:LC plants are exposed to high-light levels. 35S:ROS1 lisianthus transgenics had limited changes in anthocyanin pigmentation, specifically, precocious pigmentation of flower petals and increased pigmentation of sepals. RNA transcript levels for two anthocyanin biosynthetic genes, chalcone synthase and anthocyanidin synthase, were increased in the 35S:ROS1 lisianthus petals compared to those of control lines. With MP, the 35S:ROS1 calli showed novel red pigmentation in culture, but this was generally not seen in tissue culture plantlets regenerated from the calli or young plants transferred to soil in the greenhouse. Anthocyanin pigmentation was enhanced in the stems of mature 35S:ROS1 MP plants, but the MP white-flower phenotype was not complemented. Progeny from a 35S:ROS1 × 35S:LC cross had novel pigmentation phenotypes that were not present in either parental line or MP. In particular, there was increased pigment in the petal throat region, and the anthers changed from yellow to purple pigmentation. An outdoor field trial was conducted with the 35S:ROS1, 35S:LC, 35S:ROS1 × 35S:LC and control MP lines. Field conditions rapidly induced intense foliage pigmentation in 35S:LC plants, a phenotype not observed in control MP or equivalent 35S:LC plants maintained in a greenhouse. No difference in plant stature, seed germination, or plant survival was observed between transgenic and control plants.

  6. The overexpression of the pine transcription factor PpDof5 in Arabidopsis leads to increased lignin content and affects carbon and nitrogen metabolism.

    PubMed

    Rueda-López, Marina; Cañas, Rafael A; Canales, Javier; Cánovas, Francisco M; Ávila, Concepción

    2015-12-01

    PpDof 5 is a regulator of the expression of glutamine synthetase (GS; EC 6.3.1.2) genes in photosynthetic and non-photosynthetic tissues of maritime pine. We have used Arabidopsis thaliana as a model system to study PpDof 5 function in planta, generating transgenic lines overexpressing the pine transcription factor. The overexpression of PpDof 5 resulted in a substantial increase of lignin content with a simultaneous regulation of carbon and nitrogen key genes. In addition, partitioning in carbon and nitrogen compounds was spread via various secondary metabolic pathways. These results suggest pleiotropic effects of PpDof 5 expression on various metabolic pathways of carbon and nitrogen metabolism. Plants overexpressing PpDof 5 exhibited upregulation of genes encoding enzymes for sucrose and starch biosynthesis, with a parallel increase in the content of soluble sugars. When the plants were grown under nitrate as the sole nitrogen source, they exhibited a significant regulation of the expression of genes involved mainly in signaling, but similar growth rates to wild-type plants. However, plants grown under ammonium exhibited major induction of the expression of photosynthetic genes and differential expression of ammonium and nitrate transporters. All these data suggest that in addition to controlling ammonium assimilation, PpDof 5 could be also involved in the regulation of other pathways in carbon and nitrogen metabolism in pine trees.

  7. The neurotoxicant PCB-95 by increasing the neuronal transcriptional repressor REST down-regulates caspase-8 and increases Ripk1, Ripk3 and MLKL expression determining necroptotic neuronal death.

    PubMed

    Guida, Natascia; Laudati, Giusy; Serani, Angelo; Mascolo, Luigi; Molinaro, Pasquale; Montuori, Paolo; Di Renzo, Gianfranco; Canzoniero, Lorella M T; Formisano, Luigi

    2017-10-15

    Our previous study showed that the environmental neurotoxicant non-dioxin-like polychlorinated biphenyl (PCB)-95 increases RE1-silencing transcription factor (REST) expression, which is related to necrosis, but not apoptosis, of neurons. Meanwhile, necroptosis is a type of a programmed necrosis that is positively regulated by receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL) and negatively regulated by caspase-8. Here we evaluated whether necroptosis contributes to PCB-95-induced neuronal death through REST up-regulation. Our results demonstrated that in cortical neurons PCB-95 increased RIPK1, RIPK3, and MLKL expression and decreased caspase-8 at the gene and protein level. Furthermore, the RIPK1 inhibitor necrostatin-1 or siRNA-mediated RIPK1, RIPK3 and MLKL expression knockdown significantly reduced PCB-95-induced neuronal death. Intriguingly, PCB-95-induced increases in RIPK1, RIPK3, MLKL expression and decreases in caspase-8 expression were reversed by knockdown of REST expression with a REST-specific siRNA (siREST). Notably, in silico analysis of the rat genome identified a REST consensus sequence in the caspase-8 gene promoter (Casp8-RE1), but not the RIPK1, RIPK3 and MLKL promoters. Interestingly, in PCB-95-treated neurons, REST binding to the Casp8-RE1 sequence increased in parallel with a reduction in its promoter activity, whereas under the same experimental conditions, transfection of siREST or mutation of the Casp8-RE1 sequence blocked PCB-95-induced caspase-8 reduction. Since RIPK1, RIPK3 and MLKL rat genes showed no putative REST binding site, we assessed whether the transcription factor cAMP Responsive Element Binding Protein (CREB), which has a consensus sequence in all three genes, affected neuronal death. In neurons treated with PCB-95, CREB protein expression decreased in parallel with a reduction in binding to the RIPK1, RIPK3 and MLKL gene promoter sequence. Furthermore, CREB overexpression was

  8. c-MYC Generates Repair Errors via Increased Transcription of Alternative-NHEJ Factors, LIG3 and PARP1, in Tyrosine Kinase-Activated Leukemias.

    PubMed

    Muvarak, Nidal; Kelley, Shannon; Robert, Carine; Baer, Maria R; Perrotti, Danilo; Gambacorti-Passerini, Carlo; Civin, Curt; Scheibner, Kara; Rassool, Feyruz V

    2015-04-01

    Leukemias expressing the constitutively activated tyrosine kinases (TK) BCR-ABL1 and FLT3/ITD activate signaling pathways that increase genomic instability through generation of reactive oxygen species (ROS), DNA double-strand breaks (DSB), and error-prone repair. The nonhomologous end-joining (NHEJ) pathway is a major pathway for DSB repair and is highly aberrant in TK-activated leukemias; an alternative form of NHEJ (ALT-NHEJ) predominates, evidenced by increased expression of DNA ligase IIIα (LIG3) and PARP1, increased frequency of large genomic deletions, and repair using DNA sequence microhomologies. This study, for the first time, demonstrates that the TK target c-MYC plays a role in transcriptional activation and subsequent expression of LIG3 and PARP1 and contributes to the increased error-prone repair observed in TK-activated leukemias. c-MYC negatively regulates microRNAs miR-150 and miR-22, which demonstrate an inverse correlation with LIG3 and PARP1 expression in primary and cultured leukemia cells and chronic myelogenous leukemia human patient samples. Notably, inhibition of c-MYC and overexpression of miR-150 and -22 decreases ALT-NHEJ activity. Thus, BCR-ABL1 or FLT3/ITD induces c-MYC expression, leading to genomic instability via augmented expression of ALT-NHEJ repair factors that generate repair errors. In the context of TK-activated leukemias, c-MYC contributes to aberrant DNA repair through downstream targets LIG3 and PARP1, which represent viable and attractive therapeutic targets. ©2015 American Association for Cancer Research.

  9. Downhill running and exercise in hot environments increase leukocyte Hsp72 (HSPA1A) and Hsp90α (HSPC1) gene transcripts.

    PubMed

    Tuttle, James A; Castle, Paul C; Metcalfe, Alan J; Midgley, Adrian W; Taylor, Lee; Lewis, Mark P

    2015-04-15

    Stressors within humans and other species activate Hsp72 and Hsp90α mRNA transcription, although it is unclear which environmental temperature or treadmill gradient induces the largest increase. To determine the optimal stressor for priming the Hsp system, physically active but not heat-acclimated participants (19.8 ± 1.9 and 20.9 ± 3.6 yr) exercised at lactate threshold in either temperate (20°C, 50% relative humidity; RH) or hot (30°C, 50% RH) environmental conditions. Within each condition, participants completed a flat running (temperate flat or hot flat) and a downhill running (temperate downhill or hot downhill) experimental trial in a randomized counterbalanced order separated by at least 7 days. Venous blood samples were taken immediately before (basal), immediately after exercise, and 3 and 24 h postexercise. RNA was extracted from leukocytes and RT-quantitative PCR conducted to determine Hsp72 and Hsp90α mRNA relative expression. Leukocyte Hsp72 mRNA was increased immediately after exercise following downhill running (1.9 ± 0.9-fold) compared with flat running (1.3 ± 0.4-fold; P = 0.001) and in hot (1.9 ± 0.6-fold) compared with temperate conditions (1.1 ± 0.5-fold; P = 0.003). Leukocyte Hsp90α mRNA increased immediately after exercise following downhill running (1.4 ± 0.8-fold) compared with flat running (0.9 ± 0.6-fold; P = 0.002) and in hot (1.6 ± 1.0-fold) compared with temperate conditions (0.9 ± 0.6-fold; P = 0.003). Downhill running and exercise in hot conditions induced the largest stimuli for leukocyte Hsp72 and Hsp90α mRNA increases.

  10. Loss of ULK1 increases RPS6KB1-NCOR1 repression of NR1H/LXR-mediated Scd1 transcription and augments lipotoxicity in hepatic cells

    PubMed Central

    Sinha, Rohit Anthony; Singh, Brijesh K.; Zhou, Jin; Xie, Sherwin; Farah, Benjamin L.; Lesmana, Ronny; Ohba, Kenji; Tripathi, Madhulika; Ghosh, Sujoy; Hollenberg, Anthony N.; Yen, Paul M.

    2017-01-01

    ABSTRACT Lipotoxicity caused by saturated fatty acids (SFAs) induces tissue damage and inflammation in metabolic disorders. SCD1 (stearoyl-coenzyme A desaturase 1) converts SFAs to mono-unsaturated fatty acids (MUFAs) that are incorporated into triglycerides and stored in lipid droplets. SCD1 thus helps protect hepatocytes from lipotoxicity and its reduced expression is associated with increased lipotoxic injury in cultured hepatic cells and mouse models. To further understand the role of SCD1 in lipotoxicity, we examined the regulation of Scd1 in hepatic cells treated with palmitate, and found that NR1H/LXR (nuclear receptor subfamily 1 group H) ligand, GW3965, induced Scd1 expression and lipid droplet formation to improve cell survival. Surprisingly, ULK1/ATG1 (unc-51 like kinase) played a critical role in protecting hepatic cells from SFA-induced lipotoxicity via a novel mechanism that did not involve macroautophagy/autophagy. Specific loss of Ulk1 blocked the induction of Scd1 gene transcription by GW3965, decreased lipid droplet formation, and increased apoptosis in hepatic cells exposed to palmitate. Knockdown of ULK1 increased RPS6KB1 (ribosomal protein S6 kinase, polypeptide 1) signaling that, in turn, induced NCOR1 (nuclear receptor co-repressor 1) nuclear uptake, interaction with NR1H/LXR, and recruitment to the Scd1 promoter. These events abrogated the stimulation of Scd1 gene expression by GW3965, and increased lipotoxicity in hepatic cells. In summary, we have identified a novel autophagy-independent role of ULK1 that regulates NR1H/LXR signaling, Scd1 expression, and intracellular lipid homeostasis in hepatic cells exposed to a lipotoxic environment. PMID:27846372

  11. The Nitrate-Inducible NAC Transcription Factor TaNAC2-5A Controls Nitrate Response and Increases Wheat Yield1[OPEN

    PubMed Central

    He, Xue; Qu, Baoyuan; Li, Wenjing; Zhao, Xueqiang; Teng, Wan; Ma, Wenying; Ren, Yongzhe; Li, Bin; Li, Zhensheng; Tong, Yiping

    2015-01-01

    Nitrate is a major nitrogen resource for cereal crops; thus, understanding nitrate signaling in cereal crops is valuable for engineering crops with improved nitrogen use efficiency. Although several regulators have been identified in nitrate sensing and signaling in Arabidopsis (Arabidopsis thaliana), the equivalent information in cereals is missing. Here, we isolated a nitrate-inducible and cereal-specific NAM, ATAF, and CUC (NAC) transcription factor, TaNAC2-5A, from wheat (Triticum aestivum). A chromatin immunoprecipitation assay showed that TaNAC2-5A could directly bind to the promoter regions of the genes encoding nitrate transporter and glutamine synthetase. Overexpression of TaNAC2-5A in wheat enhanced root growth and nitrate influx rate and, hence, increased the root’s ability to acquire nitrogen. Furthermore, we found that TaNAC2-5A-overexpressing transgenic wheat lines had higher grain yield and higher nitrogen accumulation in aerial parts and allocated more nitrogen in grains in a field experiment. These results suggest that TaNAC2-5A is involved in nitrate signaling and show that it is an exciting gene resource for breeding crops with more efficient use of fertilizer. PMID:26371233

  12. Integrated stress response of Escherichia coli to methylglyoxal: transcriptional readthrough from the nemRA operon enhances protection through increased expression of glyoxalase I.

    PubMed

    Ozyamak, Ertan; de Almeida, Camila; de Moura, Alessandro P S; Miller, Samantha; Booth, Ian R

    2013-06-01

    Methylglyoxal (MG) elicits activation of K(+) efflux systems to protect cells against the toxicity of the electrophile. ChIP-chip targeting RNA polymerase, supported by a range of other biochemical measurements and mutant creation, was used to identify genes transcribed in response to MG and which complement this rapid response. The SOS DNA repair regulon is induced at cytotoxic levels of MG, even when exposure to MG is transient. Glyoxalase I alone among the core MG protective systems is induced in response to MG exposure. Increased expression is an indirect consequence of induction of the upstream nemRA operon, encoding an enzyme system that itself does not contribute to MG detoxification. Moreover, this induction, via nemRA only occurs when cells are exposed to growth inhibitory concentrations of MG. We show that the kdpFABCDE genes are induced and that this expression occurs as a result of depletion of cytoplasmic K(+) consequent upon activation of the KefGB K(+) efflux system. Finally, our analysis suggests that the transcriptional changes in response to MG are a culmination of the damage to DNA and proteins, but that some integrate specific functions, such as DNA repair, to augment the allosteric activation of the main protective system, KefGB.

  13. A missense mutation in the transcription factor ETV5 leads to sterility, increased embryonic and perinatal death, postnatal growth restriction, renal asymmetry and polydactyly in the mouse.

    PubMed

    Jamsai, Duangporn; Clark, Brett J; Smith, Stephanie J; Whittle, Belinda; Goodnow, Christopher C; Ormandy, Christopher J; O'Bryan, Moira K

    2013-01-01

    ETV5 (Ets variant gene 5) is a transcription factor that is required for fertility. In this study, we demonstrate that ETV5 plays additional roles in embryonic and postnatal developmental processes in the mouse. Through a genome-wide mouse mutagenesis approach, we generated a sterile mouse line that carried a nonsense mutation in exon 12 of the Etv5 gene. The mutation led to the conversion of lysine at position 412 into a premature termination codon (PTC) within the ETS DNA binding domain of the protein. We showed that the PTC-containing allele produced a highly unstable mRNA, which in turn resulted in an undetectable level of ETV5 protein. The Etv5 mutation resulted in male and female sterility as determined by breeding experiments. Mutant males were sterile due to a progressive loss of spermatogonia, which ultimately resulted in a Sertoli cell only phenotype by 8 week-of-age. Further, the ETV5 target genes Cxcr4 and Ccl9 were significantly down-regulated in mutant neonate testes. CXCR4 and CCL9 have been implicated in the maintenance and migration of spermatogonia, respectively. Moreover, the Etv5 mutation resulted in several developmental abnormalities including an increased incidence of embryonic and perinatal lethality, postnatal growth restriction, polydactyly and renal asymmetry. Thus, our data define a physiological role for ETV5 in many aspects of development including embryonic and perinatal survival, postnatal growth, limb patterning, kidney development and fertility.

  14. Integrated stress response of Escherichia coli to methylglyoxal: transcriptional readthrough from the nemRA operon enhances protection through increased expression of glyoxalase I

    PubMed Central

    Ozyamak, Ertan; Almeida, Camila; de Moura, Alessandro P S; Miller, Samantha; Booth, Ian R

    2013-01-01

    Methylglyoxal (MG) elicits activation of K+ efflux systems to protect cells against the toxicity of the electrophile. ChIP-chip targeting RNA polymerase, supported by a range of other biochemical measurements and mutant creation, was used to identify genes transcribed in response to MG and which complement this rapid response. The SOS DNA repair regulon is induced at cytotoxic levels of MG, even when exposure to MG is transient. Glyoxalase I alone among the core MG protective systems is induced in response to MG exposure. Increased expression is an indirect consequence of induction of the upstream nemRA operon, encoding an enzyme system that itself does not contribute to MG detoxification. Moreover, this induction, via nemRA only occurs when cells are exposed to growth inhibitory concentrations of MG. We show that the kdpFABCDE genes are induced and that this expression occurs as a result of depletion of cytoplasmic K+ consequent upon activation of the KefGB K+ efflux system. Finally, our analysis suggests that the transcriptional changes in response to MG are a culmination of the damage to DNA and proteins, but that some integrate specific functions, such as DNA repair, to augment the allosteric activation of the main protective system, KefGB. PMID:23646895

  15. Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility.

    PubMed

    Preston, R S; Philp, A; Claessens, T; Gijezen, L; Dydensborg, A B; Dunlop, E A; Harper, K T; Brinkhuizen, T; Menko, F H; Davies, D M; Land, S C; Pause, A; Baar, K; van Steensel, M A M; Tee, A R

    2011-03-10

    Under conditions of reduced tissue oxygenation, hypoxia-inducible factor (HIF) controls many processes, including angiogenesis and cellular metabolism, and also influences cell proliferation and survival decisions. HIF is centrally involved in tumour growth in inherited diseases that give rise to renal cell carcinoma (RCC), such as Von Hippel-Lindau syndrome and tuberous sclerosis complex. In this study, we examined whether HIF is involved in tumour formation of RCC in Birt-Hogg-Dubé syndrome. For this, we analysed a Birt-Hogg-Dubé patient-derived renal tumour cell line (UOK257) that is devoid of the Birt-Hogg-Dubé protein (BHD) and observed high levels of HIF activity. Knockdown of BHD expression also caused a threefold activation of HIF, which was not as a consequence of more HIF1α or HIF2α protein. Transcription of HIF target genes VEGF, BNIP3 and CCND1 was also increased. We found nuclear localization of HIF1α and increased expression of VEGF, BNIP3 and GLUT1 in a chromophobe carcinoma from a Birt-Hogg-Dubé patient. Our data also reveal that UOK257 cells have high lactate dehydrogenase, pyruvate kinase and 3-hydroxyacyl-CoA dehydrogenase activity. We observed increased expression of pyruvate dehydrogenase kinase 1 (a HIF gene target), which in turn leads to increased phosphorylation and inhibition of pyruvate dehydrogenase. Together with increased protein levels of GLUT1, our data reveal that UOK257 cells favour glycolytic rather than lipid metabolism (a cancer phenomenon termed the 'Warburg effect'). UOK257 cells also possessed a higher expression level of the L-lactate influx monocarboxylate transporter 1 and consequently utilized L-lactate as a metabolic fuel. As a result of their higher dependency on glycolysis, we were able to selectively inhibit the growth of these UOK257 cells by treatment with 2-deoxyglucose. This work suggests that targeting glycolytic metabolism may be used therapeutically to treat Birt-Hogg-Dubé-associated renal lesions.

  16. Ectopic expression of a novel peach (Prunus persica) CBF transcription factor in apple (Malus × domestica) results in short-day induced dormancy and increased cold hardiness.

    PubMed

    Wisniewski, Michael; Norelli, John; Bassett, Carole; Artlip, Timothy; Macarisin, Dumitru

    2011-05-01

    Low, non-freezing temperatures and/or short daylength (SD) regulates cold acclimation and dormancy in fruit trees. Regarding cold acclimation, C-repeat binding factor (CBF/DREB) transcriptional activator genes have the well-documented ability to induce the expression of a suite of genes associated with increased cold tolerance. We isolated a full-length cDNA of a peach CBF gene, designated PpCBF1 (GenBank Accession HM992943), and constitutively expressed it using an enhanced 35S promoter in apple. Unexpectedly, constitutive overexpression of the PpCBF1 in apple resulted in strong sensitivity to short daylength. Growth cessation and leaf senescence were induced in transgenic lines exposed to SD and optimal growth temperatures of 25°C over a 4-week period. Following 1-4 weeks of SD and 25°C trees were returned to LD and 25°C in the greenhouse. Control (untransformed) plants continued to grow while transgenic lines receiving two or more weeks of SD remained dormant and began to drop leaves. Constitutive overexpression of the PpCBF1 in apple resulted in a 4-6°C increase in freezing tolerance in both the non-acclimated and acclimated states, respectively, compared with untransformed M.26 trees. This is the first instance that constitutive overexpression of a CBF gene has resulted in SD-induction of dormancy and to our knowledge the first time apple has been shown to strongly respond to short daylength as a result of the insertion of a transgene.

  17. Increased utility in the CNS of a powerful neuron-specific tetracycline-regulatable adenoviral system developed using a post-transcriptional enhancer.

    PubMed

    Lee, Youn-Bok; Cosgrave, A Siobhan; Glover, Colin P J; Bienemann, Alison; Heywood, Darren; Hobson, Russell J; Uney, James B

    2005-05-01

    In previous studies we have found that the tetracycline (Tet)-regulatable system functions best in recombinant adenoviral (Ad) vectors when the Tet transactivators and the Tet-regulatable element (TRE) are incorporated into separate viral vectors. However, such a dual vector system is disadvantaged by the need to use relatively high titres that may elicit an immune response. Therefore, to develop a system that could be used at low titres while mediating strong, tightly regulatable gene expression in the central nervous system (CNS), we incorporated the woodchuck hepatitis virus post-transcriptional enhancer (WPRE) into a neuron-specific Tet-regulatable Ad system. The WPRE was incorporated into Ad vectors encoding the Tet-Off (tTA) transactivator driven by the synapsin-1 and CMV promoters and encoding the TRE driving EGFP expression (TRE)-EGFP. The addition of the WPRE to the neuron-specific Tet-regulatable system mediated a greater than three-fold increase in transgene expression in primary hippocampal neurons with no loss of gene regulation. The results also showed that the addition of the WPRE enhanced transgene expression in the CNS without the loss of neuron specificity and without affecting the ability to regulate transgene expression. We have further developed a tetracycline-regulatable neuron-specific expression system such that it can now be used at low titres with no loss of transgene expression or ability to regulate transgene expression. It should therefore be of significant value to studies investigating neuronal gene function and to those seeking to develop effective neuronal gene therapy strategies. Copyright (c) 2004 John Wiley & Sons, Ltd.

  18. Ectopic Overexpression of SlHsfA3, a Heat Stress Transcription Factor from Tomato, Confers Increased Thermotolerance and Salt Hypersensitivity in Germination in Transgenic Arabidopsis

    PubMed Central

    Li, Zhenjun; Zhang, Lili; Wang, Aoxue; Xu, Xiangyang; Li, Jingfu

    2013-01-01

    Plant heat stress transcription factors (Hsfs) are the critical components involved in mediating responses to various environmental stressors. However, the detailed roles of many plant Hsfs are far from fully understood. In this study, an Hsf (SlHsfA3) was isolated from the cultivated tomato (Solanum lycopersicum, Sl) and functionally characterized at the genetic and developmental levels. The nucleus-localized SlHsfA3 was basally and ubiquitously expressed in different plant organs. The expression of SlHsfA3 was induced dramatically by heat stress, moderately by high salinity, and slightly by drought, but was not induced by abscisic acid (ABA). The ectopic overexpression of SlHsfA3 conferred increased thermotolerance and late flowering phenotype to transgenic Arabidopsis plants. Moreover, SlHsfA3 played a negative role in controlling seed germination under salt stress. RNA-sequencing data demonstrated that a number of heat shock proteins (Hsps) and stress-associated genes were induced in Arabidopsis plants overexpressing SlHsfA3. A gel shift experiment and transient expression assays in Nicotiana benthamiana leaves demonstrated that SlHsfA3 directly activates the expression of SlHsp26.1-P and SlHsp21.5-ER. Taken together, our results suggest that SlHsfA3 behaves as a typical Hsf to contribute to plant thermotolerance. The late flowering and seed germination phenotypes and the RNA-seq data derived from SlHsfA3 overexpression lines lend more credence to the hypothesis that plant Hsfs participate in diverse physiological and biochemical processes related to adverse conditions. PMID:23349984

  19. Bisphenol A increases aP2 expression in 3T3L1 by enhancing the transcriptional activity of nuclear receptors at the promoter

    PubMed Central

    Atlas, Ella; Pope, Louise; Wade, Mike G; Kawata, Alice; Boudreau, Adele; Boucher, Jonathan G

    2014-01-01

    Environmental pollutants, such as bisphenol A (BPA), have the potential to affect the differentiation processes and the biology of the adipose tissue. The 3T3-L1 model is one of the murine cell models used extensively for the investigation of the molecular events that govern the differentiation of adipocytes from a committed preadipocyte to a mature, lipid laden adipocyte. Most of the studies investigating the effects of BPA on preadipocyte differentiation have investigated the effects of this chemical in the presence of an optimal differentiation cocktail containing high concentrations of the synthetic glucocorticoid dexamethasone, conditions that result in 90% to 100% of differentiated adipocytes. Our studies employed the 3T3-L1 cell model in the absence of exogenous glucocorticoids. We show that BPA is able to increase the differentiation of the 3T3-L1 cells under these conditions. Furthermore, the effect of BPA was observed in the absence of the synthetic glucocorticoid (dexamethasone), a hormone known to be required for the differentiation of the 3T3-L1 cells. In addition, BPA upregulated the mRNA expression and protein levels of the terminal marker of adipogenesis the fatty acid binding protein (aP2) in these cells. Interestingly, the known modulators of adipogenesis such as the peroxisome proliferator-activated receptor (PPAR) γ or CCAAT enhancer binding protein (C/EBP) α were not elevated at the mRNA or protein level in response to BPA. Furthermore, BPA upregulated the expression levels of the marker of adipogenesis aP2, through an effect on the transcriptional activity of C/EBPδ and the glucocorticoid receptor (GR) at its promoter. PMID:25068083

  20. Chronic high fat feeding increases anxiety-like behaviour and reduces transcript abundance of glucocorticoid signalling genes in the hippocampus of female rats.

    PubMed

    Sivanathan, Shathveekan; Thavartnam, Kabriya; Arif, Shahneen; Elegino, Trisha; McGowan, Patrick O

    2015-06-01

    The consumption of diets high in saturated fats and obesity have been associated with impaired physical and mental health. Previous studies indicate that chronic high fat diet consumption leads to systemic inflammation in humans and non-human animal models. Studies in non-human animals suggest that altered physiological responses to stress are also a consequence of high fat diet consumption. Glucocorticoid signalling mechanisms may link immune and stress-related pathways in the brain, and were shown to be significantly altered in the brains of female rat offspring of mothers exposed to chronic high fat diet during pregnancy and lactation. For adult females, the consequence of chronic high fat diet consumption on these signalling pathways and their relationship to stress-related behaviour is not known. In this study, we examined the effects of chronic consumption of a high fat diet compared to a low fat control diet among adult female Long Evans rats. We found significant differences in weight gain, caloric intake, anxiety-related behaviours, and glucocorticoid-related gene expression over a 10-week exposure period. As expected, rats in the high fat diet group gained the most weight and consumed the greatest number of calories. Rats in the high fat diet group showed significantly greater levels of anxiety-related behaviour in the Light Dark and Open Field tasks compared to rats in the low fat diet group. Rats consuming high fat diet also exhibited reduced transcript abundance in the hippocampus of stress-related mineralocorticoid receptor and glucocorticoid receptor genes, as well as nuclear factor kappa beta gene expression, implicated in inflammatory processes. Together, these data indicate that chronic high fat diet consumption may increase anxiety-like behaviour at least in part via alterations in glucocorticoid signalling mechanisms in limbic brain regions.

  1. Heme and nitric oxide binding by the transcriptional regulator DnrF from the marine bacterium Dinoroseobacter shibae increases napD promoter affinity.

    PubMed

    Ebert, Matthias; Schweyen, Peter; Bröring, Martin; Laass, Sebastian; Härtig, Elisabeth; Jahn, Dieter

    2017-09-15

    Under oxygen-limiting conditions, the marine bacterium Dinoroseobacter shibae DFL12(T) generates energy via denitrification, a respiratory process in which nitric oxide (NO) is an intermediate. Accumulation of NO may cause cytotoxic effects. The response to this nitrosative (NO-triggered) stress is controlled by the Crp/Fnr-type transcriptional regulator DnrF. We analyzed the response to NO and the mechanism of NO sensing by the DnrF regulator. Using reporter gene fusions and transcriptomics, here we report that DnrF selectively repressed nitrate reductase (nap) genes, preventing further NO formation. In addition, DnrF induced the expression of the NO reductase genes (norCB), which promote NO consumption. We used UV-visible and EPR spectroscopy to characterize heme binding to DnrF and subsequent NO coordination. DnrF detects NO via its bound heme cofactor. We found that the dimeric DnrF bound one molecule of heme per subunit. Purified recombinant apo-DnrF bound its target promoter sequences (napD, nosR2, norC, hemA, and dnrE) in electromobility shift assays, and we identified a specific palindromic DNA-binding site 5'-TTGATN4ATCAA-3' in these target sequences via mutagenesis studies. Most importantly, successive addition of heme as well as heme and NO to purified recombinant apo-DnrF protein increased affinity of the holo-DnrF for its specific binding motif in the napD promoter. On the basis of these results, we propose a model for the DnrF-mediated NO stress response of this marine bacterium. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. The PPARgamma ligand rosiglitazone influences triacylglycerol metabolism in non-obese males, without increasing the transcriptional activity of PPARgamma in the subcutaneous adipose tissue.

    PubMed

    Joosen, Annemiek M C P; Bakker, Arjen H F; Kersten, Sander; Westerterp, Klaas R

    2008-03-01

    PPARgamma is obligatory for fat mass generation and is thought to determine the amount of TAG stored per fat cell. We investigated whether ligand availability for PPARgamma is rate limiting in fat mass generation and substrate metabolism. Twenty healthy men (20-29 years) were randomly assigned to receive the PPARgamma ligand rosiglitazone (RSG) (8 mg/d) (n 10) or a placebo (n 10) during a stay of 7 d in a respiration chamber. Food intake was ad libitum, resulting in positive energy balances of 32.2 MJ (placebo) and 44.7 MJ (RSG). Fat cell size and expression of PPARgamma, adipocyte fatty acid-binding protein (aP2), adipsin, adiponectin and fasting-induced adipose factor (FIAF) were determined in subcutaneous abdominal fat biopsies. The total amount of fat stored and the amount of TAG per fat cell were not different between groups. For the entire group, fat cell size was decreased after overeating (P = 0.02). FIAF mRNA levels were decreased after overeating in the RSG group (P = 0.01), with a trend towards a decrease in the placebo group. Unexpectedly, RSG treatment did not influence the expression levels of PPARgamma and of the PPARgamma responsive genes aP2, adiponectin and adipsin. In addition, RSG resulted in a larger increase in plasma TAG during overeating than placebo treatment. These results suggest that in healthy, non-obese males the PPARgamma ligand RSG influences TAG metabolism, independent of its PPARgamma transcriptional activity in the subcutaneous adipose tissue.

  3. Ectopic overexpression of SlHsfA3, a heat stress transcription factor from tomato, confers increased thermotolerance and salt hypersensitivity in germination in transgenic Arabidopsis.

    PubMed

    Li, Zhenjun; Zhang, Lili; Wang, Aoxue; Xu, Xiangyang; Li, Jingfu

    2013-01-01

    Plant heat stress transcription factors (Hsfs) are the critical components involved in mediating responses to various environmental stressors. However, the detailed roles of many plant Hsfs are far from fully understood. In this study, an Hsf (SlHsfA3) was isolated from the cultivated tomato (Solanum lycopersicum, Sl) and functionally characterized at the genetic and developmental levels. The nucleus-localized SlHsfA3 was basally and ubiquitously expressed in different plant organs. The expression of SlHsfA3 was induced dramatically by heat stress, moderately by high salinity, and slightly by drought, but was not induced by abscisic acid (ABA). The ectopic overexpression of SlHsfA3 conferred increased thermotolerance and late flowering phenotype to transgenic Arabidopsis plants. Moreover, SlHsfA3 played a negative role in controlling seed germination under salt stress. RNA-sequencing data demonstrated that a number of heat shock proteins (Hsps) and stress-associated genes were induced in Arabidopsis plants overexpressing SlHsfA3. A gel shift experiment and transient expression assays in Nicotiana benthamiana leaves demonstrated that SlHsfA3 directly activates the expression of SlHsp26.1-P and SlHsp21.5-ER. Taken together, our results suggest that SlHsfA3 behaves as a typical Hsf to contribute to plant thermotolerance. The late flowering and seed germination phenotypes and the RNA-seq data derived from SlHsfA3 overexpression lines lend more credence to the hypothesis that plant Hsfs participate in diverse physiological and biochemical processes related to adverse conditions.

  4. P2Y2 Receptor Transcription Is Increased by NF-κB and Stimulates Cyclooxygenase-2 Expression and PGE2 Released by Intestinal Epithelial Cells1

    PubMed Central

    Degagné, Emilie; Grbic, Djordje M.; Dupuis, Andrée-Anne; Lavoie, Elise G.; Langlois, Christine; Jain, Nishant; Weisman, Gary A.; Sévigny, Jean; Gendron, Fernand-Pierre

    2013-01-01

    Inflammatory stresses associated with inflammatory bowel diseases up-regulate P2Y2 mRNA receptor expression in the human colon adenocarcinoma cell line Caco-2, the noncancerous IEC-6 cells and in colonic tissues of patient suffering from Crohn’s disease and ulcerative colitis. However, the transcriptional events regulating P2Y2 receptor (P2Y2R) expression are not known. We have identified a putative transcription start site in the P2Y2R gene and demonstrated acetylation of Lys14 on histone H3 and Lys8 on histone H4, thus suggesting that the chromatin associated with the P2Y2 promoter is accessible to transcription factors. We also showed that the transcription factor NF-κB p65 regulates P2Y2R transcription under both proinflammatory and basal conditions. A NF-κB-responsive element was identified at −181 to −172 bp in the promoter region of P2Y2. Hence, activation of P2Y2R by ATP and UTP stimulated cyclooxygenase-2 expression and PGE2 secretion by intestinal epithelial cells. These findings demonstrate that P2Y2R expression is regulated during intestinal inflammation through an NF-κB p65-dependent mechanism and could contribute not only to inflammatory bowel disease but also to other inflammatory diseases by regulating PG release. PMID:19734210

  5. P2Y2 receptor transcription is increased by NF-kappa B and stimulates cyclooxygenase-2 expression and PGE2 released by intestinal epithelial cells.

    PubMed

    Degagné, Emilie; Grbic, Djordje M; Dupuis, Andrée-Anne; Lavoie, Elise G; Langlois, Christine; Jain, Nishant; Weisman, Gary A; Sévigny, Jean; Gendron, Fernand-Pierre

    2009-10-01

    Inflammatory stresses associated with inflammatory bowel diseases up-regulate P2Y(2) mRNA receptor expression in the human colon adenocarcinoma cell line Caco-2, the noncancerous IEC-6 cells and in colonic tissues of patient suffering from Crohn's disease and ulcerative colitis. However, the transcriptional events regulating P2Y(2) receptor (P2Y(2)R) expression are not known. We have identified a putative transcription start site in the P2Y(2)R gene and demonstrated acetylation of Lys(14) on histone H3 and Lys(8) on histone H4, thus suggesting that the chromatin associated with the P2Y(2) promoter is accessible to transcription factors. We also showed that the transcription factor NF-kappaB p65 regulates P2Y(2)R transcription under both proinflammatory and basal conditions. A NF-kappaB-responsive element was identified at -181 to -172 bp in the promoter region of P2Y(2). Hence, activation of P2Y(2)R by ATP and UTP stimulated cyclooxygenase-2 expression and PGE(2) secretion by intestinal epithelial cells. These findings demonstrate that P2Y(2)R expression is regulated during intestinal inflammation through an NF-kappaB p65-dependent mechanism and could contribute not only to inflammatory bowel disease but also to other inflammatory diseases by regulating PG release.

  6. MCG101-induced cancer anorexia-cachexia features altered expression of hypothalamic Nucb2 and Cartpt and increased plasma levels of cocaine- and amphetamine-regulated transcript peptides.

    PubMed

    Burgos, Jonathan R; Iresjö, Britt-Marie; Smedh, Ulrika

    2016-04-01

    The aim of the present study was to explore central and peripheral host responses to an anorexia-cachexia producing tumor. We focused on neuroendocrine anorexigenic signals in the hypothalamus, brainstem, pituitary and from the tumor per se. Expression of mRNA for corticotropin-releasing hormone (CRH), cocaine- and amphetamine-regulated transcript (CART), nesfatin-1, thyrotropin (TSH) and the TSH receptor were explored. In addition, we examined changes in plasma TSH, CART peptides (CARTp) and serum amyloid P component (SAP). C57BL/6 mice were implanted with MCG101 tumors or sham-treated. A sham-implanted, pair‑fed (PF) group was included to delineate between primary tumor and secondary effects from reduced feeding. Food intake and body weight were measured daily. mRNA levels from microdissected mouse brain samples were assayed using qPCR, and plasma levels were determined using ELISA. MCG101 tumors expectedly induced anorexia and loss of body weight. Tumor-bearing (TB) mice exhibited an increase in nesfatin-1 mRNA as well as a decrease in CART mRNA in the paraventricular area (PVN). The CART mRNA response was secondary to reduced caloric intake whereas nesfatin-1 mRNA appeared to be tumor-specifically induced. In the pituitary, CART and TSH mRNA were upregulated in the TB and PF animals compared to the freely fed controls. Plasma levels for CARTp were significantly elevated in TB but not PF mice whereas levels of TSH were unaffected. The plasma CARTp response was correlated to the degree of inflammation represented by SAP. The increase in nesfatin-1 mRNA in the PVN highlights nesfatin-1 as a plausible candidate for causing tumor-induced anorexia. CART mRNA expression in the PVN is likely an adaptation to reduced caloric intake secondary to a cancer anorexia-cachexia syndrome (CACS)‑inducing tumor. The MCG101 tumor did not express CART mRNA, thus the elevation of plasma CARTp is host derived and likely driven by inflammation.

  7. Transcription Regulation in Archaea

    PubMed Central

    Gehring, Alexandra M.; Walker, Julie E.

    2016-01-01

    The known diversity of metabolic strategies and physiological adaptations of archaeal species to extreme environments is extraordinary. Accurate and responsive mechanisms to ensure that gene expression patterns match the needs of the cell necessitate regulatory strategies that control the activities and output of the archaeal transcription apparatus. Archaea are reliant on a single RNA polymerase for all transcription, and many of the known regulatory mechanisms employed for archaeal transcription mimic strategies also employed for eukaryotic and bacterial species. Novel mechanisms of transcription regulation have become apparent by increasingly sophisticated in vivo and in vitro investigations of archaeal species. This review emphasizes recent progress in understanding archaeal transcription regulatory mechanisms and highlights insights gained from studies of the influence of archaeal chromatin on transcription. PMID:27137495

  8. A Forum To Expand Advanced Placement Opportunities: Increasing Access and Improving Preparation in High Schools. Transcript of Proceedings (Washington, D.C., February 11, 2000).

    ERIC Educational Resources Information Center

    Department of Education, Washington, DC.

    This transcript reproduces a Department of Education/College Board-sponsored discussion on ways to expand advanced-placement (AP) opportunities in high schools. The deliberations opened with a presentation by Terry Peterson, Senior Advisor to Education Secretary Richard Riley, in which he focused on the importance of AP courses for minority and…

  9. Opposing Control by Transcription Factors MYB61 and MYB3 Increases Freezing Tolerance by Relieving C-Repeat Binding Factor Suppression1[OPEN

    PubMed Central

    Zhang, Yunqin; Miao, Zhenyan; Xie, Can; Meng, Xiangzhao; Deng, Jie; Mysore, Kirankumar S.; Frugier, Florian; Wang, Tao

    2016-01-01

    Cold acclimation is an important process by which plants respond to low temperature and enhance their winter hardiness. C-REPEAT BINDING FACTOR1 (CBF1), CBF2, and CBF3 genes were shown previously to participate in cold acclimation in Medicago truncatula. In addition, MtCBF4 is transcriptionally induced by salt, drought, and cold stresses. We show here that MtCBF4, shown previously to enhance drought and salt tolerance, also positively regulates cold acclimation and freezing tolerance. To identify molecular factors acting upstream and downstream of the MtCBF4 transcription factor (TF) in cold responses, we first identified genes that are differentially regulated upon MtCBF4 overexpression using RNAseq Digital Gene Expression Profiling. Among these, we showed that MtCBF4 directly activates the transcription of the COLD ACCLIMATION SPECIFIC15 (MtCAS15) gene. To gain insights into how MtCBF4 is transcriptionally regulated in response to cold, an R2R3-MYB TF, MtMYB3, was identified based on a yeast one-hybrid screen as binding directly to MYB cis-elements in the MtCBF4 promoter, leading to the inhibition of MtCBF4 expression. In addition, another MYB TF, MtMYB61, identified as an interactor of MtMYB3, can relieve the inhibitory effect of MtMYB3 on MtCBF4 transcription. This study, therefore, supports a model describing how MtCBF4 is regulated by antagonistic MtMYB3/MtMYB61 TFs, leading to the up-regulation of downstream targets such as MtCAS15 acting in cold acclimation in M. truncatula. PMID:27578551

  10. Cloning of a long HIV-1 readthrough transcript and detection of an increased level of early growth response protein-1 (Egr-1) mRNA in chronically infected U937 cells.

    PubMed

    Dron, M; Hameau, L; Benboudjema, L; Guymarho, J; Cajean-Feroldi, C; Rizza, P; Godard, C; Jasmin, C; Tovey, M G; Lang, M C

    1999-01-01

    To identify the pathways involved in HIV-1 modification of cellular gene expression, chronically infected U937 cells were screened by mRNA differential display. A chimeric transcript consisting of the 3' end of the LTR of a HIV-1 provirus, followed by 3.7 kb of cellular RNA was identified suggesting that long readthrough transcription might be one of the mechanisms by which gene expression could be modified in individual infected cells. Such a phenomenon may also be the first step towards the potential transduction of cellular sequences. Furthermore, the mRNA encoding for the transcription factor Egr-1 was detected as an over-represented transcript in infected cells. Northern blot analysis confirmed the increase of Egr-1 mRNA content in both HIV-1 infected promonocytic U937 cells and T cell lines such as Jurkat and CEM. Interestingly a similar increase of Egr-1 mRNA has previously been reported to occur in HTLV-1 and HTLV-2 infected T cell lines. Despite the consistent increase in the level of Egr-1 mRNA, the amount of the encoded protein did not appear to be modified in HIV-1 infected cells, suggesting an increased turn over of the protein in chronically infected cells.

  11. Changes in transcription of cytokinin metabolism and signalling genes in grape (Vitis vinifera L.) berries are associated with the ripening-related increase in isopentenyladenine.

    PubMed

    Böttcher, Christine; Burbidge, Crista A; Boss, Paul K; Davies, Christopher

    2015-09-16

    Cytokinins are known to play an important role in fruit set and early fruit growth, but their involvement in later stages of fruit development is less well understood. Recent reports of greatly increased cytokinin concentrations in the flesh of ripening kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang & A.R. Ferguson) and grapes (Vitis vinifera L.) have suggested that these hormones are implicated in the control of ripening-related processes. A similar pattern of isopentenyladenine (iP) accumulation was observed in the ripening fruit of several grapevine cultivars, strawberry (Fragaria ananassa Duch.) and tomato (Solanum lycopersicum Mill.), suggesting a common, ripening-related role for this cytokinin. Significant differences in maximal iP concentrations between grapevine cultivars and between fruit species might reflect varying degrees of relevance or functional adaptations of this hormone in the ripening process. Grapevine orthologues of five Arabidopsis (Arabidopsis thaliana L.) gene families involved in cytokinin metabolism and signalling were identified and analysed for their expression in developing grape berries and a range of other grapevine tissues. Members of each gene family were characterised by distinct expression profiles during berry development and in different grapevine organs, suggesting a complex regulation of cellular cytokinin activities throughout the plant. The post-veraison-specific expression of a set of biosynthesis, activation, perception and signalling genes together with a lack of expression of degradation-related genes during the ripening phase were indicative of a local control of berry iP concentrations leading to the observed accumulation of iP in ripening grapes. The transcriptional analysis of grapevine genes involved in cytokinin production, degradation and response has provided a possible explanation for the ripening-associated accumulation of iP in grapes and other fruit. The pre- and post-veraison-specific expression of

  12. The Transcriptional Response of Listeria monocytogenes during Adaptation to Growth on Lactate and Diacetate Includes Synergistic Changes That Increase Fermentative Acetoin Production▿†

    PubMed Central

    Stasiewicz, Matthew J.; Wiedmann, Martin; Bergholz, Teresa M.

    2011-01-01

    The organic acids lactate and diacetate are commonly used in combination in ready-to-eat foods because they show synergistic ability to inhibit the growth of Listeria monocytogenes. Full-genome microarrays were used to investigate the synergistic transcriptomic responses of two L. monocytogenes strains, H7858 (serotype 4b) and F6854 (serotype 1/2a), to these two organic acids under conditions representing osmotic and cold stress encountered in foods. Strains were exposed to brain heart infusion (BHI) broth at 7°C with 4.65% water-phase (w.p.) NaCl at pH 6.1 with (i) 2% w.p. potassium lactate, (ii) 0.14% w.p. sodium diacetate, (iii) the combination of both at the same levels, or (iv) no organic acids as a control. RNA was extracted 8 h after exposure, during lag phase, to capture gene transcription changes during adaptation to the organic acid stress. Significant differential transcription of 1,041 genes in H7858 and 640 genes in F6854 was observed in at least one pair of the 4 different treatments. The effects of combined treatment with lactate and diacetate included (i) synergistic transcription differences for 474 and 209 genes in H7858 and F6854, respectively, (ii) differential transcription of genes encoding cation transporters and ABC transporters of metals, and (iii) altered metabolism, including induction of a nutrient-limiting stress response, reduction of menaquinone biosynthesis, and a shift from fermentative production of acetate and lactate to energetically less favorable, neutral acetoin. These data suggest that additional treatments that interfere with cellular energy generation processes could more efficiently inhibit the growth of L. monocytogenes. PMID:21666015

  13. The transcriptional response of Listeria monocytogenes during adaptation to growth on lactate and diacetate includes synergistic changes that increase fermentative acetoin production.

    PubMed

    Stasiewicz, Matthew J; Wiedmann, Martin; Bergholz, Teresa M

    2011-08-01

    The organic acids lactate and diacetate are commonly used in combination in ready-to-eat foods because they show synergistic ability to inhibit the growth of Listeria monocytogenes. Full-genome microarrays were used to investigate the synergistic transcriptomic responses of two L. monocytogenes strains, H7858 (serotype 4b) and F6854 (serotype 1/2a), to these two organic acids under conditions representing osmotic and cold stress encountered in foods. Strains were exposed to brain heart infusion (BHI) broth at 7°C with 4.65% water-phase (w.p.) NaCl at pH 6.1 with (i) 2% w.p. potassium lactate, (ii) 0.14% w.p. sodium diacetate, (iii) the combination of both at the same levels, or (iv) no organic acids as a control. RNA was extracted 8 h after exposure, during lag phase, to capture gene transcription changes during adaptation to the organic acid stress. Significant differential transcription of 1,041 genes in H7858 and 640 genes in F6854 was observed in at least one pair of the 4 different treatments. The effects of combined treatment with lactate and diacetate included (i) synergistic transcription differences for 474 and 209 genes in H7858 and F6854, respectively, (ii) differential transcription of genes encoding cation transporters and ABC transporters of metals, and (iii) altered metabolism, including induction of a nutrient-limiting stress response, reduction of menaquinone biosynthesis, and a shift from fermentative production of acetate and lactate to energetically less favorable, neutral acetoin. These data suggest that additional treatments that interfere with cellular energy generation processes could more efficiently inhibit the growth of L. monocytogenes.

  14. Nuclear F-actin enhances the transcriptional activity of β-catenin by increasing its nuclear localization and binding to chromatin.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; de Lanerolle, Primal; Harata, Masahiko

    2016-04-01

    Actin plays multiple roles both in the cytoplasm and in the nucleus. Cytoplasmic actin, in addition to its structural role in the cytoskeleton, also contributes to the subcellular localization of transcription factors by interacting with them or their partners. The transcriptional cofactor β-catenin, which acts as an intracellular transducer of canonical Wnt signaling, indirectly associates with the cytoplasmic filamentous actin (F-actin). Recently, it has been observed that F-actin is transiently formed within the nucleus in response to serum stimulation and integrin signaling, and also during gene reprogramming. Despite these earlier observations, information about the function of nuclear F-actin is poorly defined. Here, by facilitating the accumulation of nuclear actin artificially, we demonstrate that polymerizing nuclear actin enhanced the nuclear accumulation and transcriptional function of β-catenin. Our results also show that the nuclear F-actin colocalizes with β-catenin and enhances the binding of β-catenin to the downstream target genes of the Wnt/β-catenin signaling pathway, including the genes for the cell cycle regulators c-myc and cyclin D, and the OCT4 gene. Nuclear F-actin itself also associated with these genes. Since Wnt/β-catenin signaling has important roles in cell differentiation and pluripotency, our observations suggest that nuclear F-actin formed during these biological processes is involved in regulating Wnt/β-catenin signaling.

  15. Flexible Transcription Testing

    ERIC Educational Resources Information Center

    Carr-Smith, Norma

    1976-01-01

    Flexible structure in a San Francisco State University shorthand course is described as a way to provide motivation for students. Topics discussed are transcription testing plan, method of evaluation, practice tests, increasing difficulty of tests, and classroom results. (TA)

  16. Overexpression of a bHLH1 Transcription Factor of Pyrus ussuriensis Confers Enhanced Cold Tolerance and Increases Expression of Stress-Responsive Genes

    PubMed Central

    Jin, Cong; Huang, Xiao-San; Li, Kong-Qing; Yin, Hao; Li, Lei-Ting; Yao, Zheng-Hong; Zhang, Shao-Ling

    2016-01-01

    The basic helix-loop-helix (bHLH) transcription factors are involved in arrays of physiological and biochemical processes. However, knowledge concerning the functions of bHLHs in cold tolerance remains poorly understood. In this study, a PubHLH1 gene isolated from Pyrus ussuriensis was characterized for its function in cold tolerance. PubHLH1 was upregulated by cold, salt, and dehydration, with the greatest induction under cold conditions. PubHLH1 had the transactivational activity and localized in the nucleus. Ectopic expression of PubHLH1 in transgenic tobacco conferred enhanced tolerance to cold stress. The transgenic lines had higher survival rates, higher chlorophyll, higher proline contents, lower electrolyte leakages and MDA when compared with wild type (WT). In addition, transcript levels of eight genes associated with ROS scavenging, regulation, and stress defense were higher in the transgenic plants relative to the WT under the chilling stress. Taken together, these results demonstrated that PubHLH1 played a key role in cold tolerance and, at least in part, contributed to activation of stress-responsive genes. PMID:27092159

  17. Stochastic and nonstochastic post-transcriptional silencing of chitinase and beta-1,3-glucanase genes involves increased RNA turnover-possible role for ribosome-independent RNA degradation.

    PubMed Central

    Holtorf, H; Schöb, H; Kunz, C; Waldvogel, R; Meins, F

    1999-01-01

    Stochastic and nonstochastic post-transcriptional gene silencing (PTGS) in Nicotiana sylvestris plants carrying tobacco class I chitinase (CHN) and beta-1,3-glucanase transgenes differs in incidence, stability, and pattern of expression. Measurements with inhibitors of RNA synthesis (cordycepin, actinomycin D, and alpha-amanitin) showed that both forms of PTGS are associated with increased sequence-specific degradation of transcripts, suggesting that increased RNA turnover may be a general feature of PTGS. The protein synthesis inhibitors cycloheximide and verrucarin A did not inhibit degradation of CHN RNA targeted for PTGS, confirming that PTGS-related RNA degradation does not depend on ongoing protein synthesis. Because verrucarin A, unlike cycloheximide, dissociates mRNA from ribosomes, our results also suggest that ribosome-associated RNA degradation pathways may not be involved in CHN PTGS. PMID:10072405

  18. Increasing diversity of human thyroperoxidase generated by alternative splicing. Characterized by molecular cloning of new transcripts with single- and multispliced mRNAs.

    PubMed

    Ferrand, Mireille; Le Fourn, Valérie; Franc, Jean-Louis

    2003-02-07

    The human thyroperoxidase (hTPO) gene is composed of 17 exons. The longest complete cDNA sequence determined so far contains a full-length hTPO (TPO1) encoding a 933-amino acid polypeptide. Several mRNA species encoding for hTPO isoforms are present in normal thyroid tissues, including TPO2 with exon 10 deleted and TPOzanelli with exon 16 deleted. In the present study, we established the existence of two new single-spliced transcripts, TPO4 and TPO5, lacking exons 14 and 8, respectively. Upon transfecting the TPO4 cDNA into Chinese hamster ovary cells, it was observed that TPO4 is able to reach the cell surface, is enzymatically active, and is able to be recognized by a panel of 12 monoclonal antibodies directed against hTPO, whereas TPO5 does not fold correctly and is unable to reach the cell surface. In normal tissues, the expression of TPO4 mRNA was examined by performing quantitative reverse transcription PCR. This deleted TPO mRNA amounted to 32 +/- 11% of the total TPO mRNAs. In the same tissues, the TPO2, TPOzanelli, and TPO5 amounted to 35 +/- 12%, 36 +/- 14%, and approximately 10%, respectively. The sum of these four species (not including TPO1) was more than 100%, possibly due to the presence of multispliced mRNAs. This possibility was tested, and three new variants were identified: TPO2/3, lacking exons 10 and 16, TPO2/4, lacking exons 10 and 14, and an unexpected variant, TPO6, corresponding to the deletion of exons 10, 12, 13, 14, and 16. In conclusion, these results indicate the existence of five new transcripts. One of them, TPO4, codes for an enzymatically active protein, whereas TPO5 is unable to fold correctly. The functional significance of the other newly spliced mRNA variants still remains to be elucidated, but these results might help to explain the heterogeneity of the hTPO purified from the thyroid gland.

  19. Overexpression of a Novel NAC Domain-Containing Transcription Factor Gene (AaNAC1) Enhances the Content of Artemisinin and Increases Tolerance to Drought and Botrytis cinerea in Artemisia annua.

    PubMed

    Lv, Zongyou; Wang, Shu; Zhang, Fangyuan; Chen, Lingxian; Hao, Xiaolong; Pan, Qifang; Fu, Xueqing; Li, Ling; Sun, Xiaofen; Tang, Kexuan

    2016-09-01

    The NAC (NAM, ATAF and CUC) superfamily is one of the largest plant-specific transcription factor families. NAC transcription factors always play important roles in response to various abiotic stresses. A NAC transcription factor gene AaNAC1 containing a complete open reading frame (ORF) of 864 bp was cloned from Artemisia annua. The expression of AaNAC1 could be induced by dehydration, cold, salicylic acid (SA) and methyl jasmonate (MJ), suggesting that it might be a key regulator of stress signaling pathways in A. annua. AaNAC1 was shown to be localized to the nuclei by transforming tobacco leaf epidermal cells. When AaNAC1 was overexpressed in A. annua, the content of artemisinin and dihydroartemisinic acid was increased by 79% and 150%, respectively. The expression levels of artemisinin biosynthetic pathway genes, i.e. amorpha-4,11-diene synthase (ADS), artemisinic aldehyde Δ11(13) reductase (DBR2) and aldehyde dehydrogenase 1 (ALDH1), were increased. Dual luciferase (dual-LUC) assays showed that AaNAC1 could activate the transcription of ADS in vivo. The transgenic A. annua exhibited increased tolerance to drought and resistance to Botrytis cinerea. When AaNAC1 was overexpressed in Arabidopsis, the transgenic Arabidopsis were markedly more tolerant to drought. The transgenic Arabidopsis showed increased resistance to B. cinerea. These results indicate that AaNAC1 can potentially be used in transgenic breeding for improving the content of artemisinin and drought tolerance in A. annua. © 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.

  20. Transcription factories

    PubMed Central

    Rieder, Dietmar; Trajanoski, Zlatko; McNally, James G.

    2012-01-01

    There is considerable evidence that transcription does not occur homogeneously or diffusely throughout the nucleus, but rather at a number of specialized, discrete sites termed transcription factories. The factories are composed of ~4–30 RNA polymerase molecules, and are associated with many other molecules involved in transcriptional activation and mRNA processing. Some data suggest that the polymerase molecules within a factory remain stationary relative to the transcribed DNA, which is thought to be reeled through the factory site. There is also some evidence that transcription factories could help organize chromatin and nuclear structure, contributing to both the formation of chromatin loops and the clustering of active and co-regulated genes. PMID:23109938

  1. Increased transcript level of poly(ADP-ribose) polymerase (PARP-1) in human tricuspid compared with bicuspid aortic valves correlates with the stenosis severity

    SciTech Connect

    Nagy, Edit; Caidahl, Kenneth; Franco-Cereceda, Anders; Baeck, Magnus

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Oxidative stress has been implicated in the pathomechanism of calcific aortic valve stenosis. Black-Right-Pointing-Pointer We assessed the transcript levels for PARP-1 (poly(ADP-ribose) polymerase), acts as a DNA damage nick sensor in stenotic valves. Black-Right-Pointing-Pointer Early stage of diseased tricuspid valves exhibited higher mRNA levels for PARP-1 compared to bicuspid valves. Black-Right-Pointing-Pointer The mRNA levels for PARP-1 inversely correlated with the clinical stenosis severity in tricuspid valves. Black-Right-Pointing-Pointer Our data demonstrated that DNA damage pathways might be associated with stenosis severity only in tricuspid valves. -- Abstract: Oxidative stress may contribute to the hemodynamic progression of aortic valve stenosis, and is associated with activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) 1. The aim of the present study was to assess the transcriptional profile and the topological distribution of PARP-1 in human aortic valves, and its relation to the stenosis severity. Human stenotic aortic valves were obtained from 46 patients undergoing aortic valve replacement surgery and used for mRNA extraction followed by quantitative real-time PCR to correlate the PARP-1 expression levels with the non invasive hemodynamic parameters quantifying the stenosis severity. Primary isolated valvular interstitial cells (VICs) were used to explore the effects of cytokines and leukotriene C{sub 4} (LTC{sub 4}) on valvular PARP-1 expression. The thickened areas of stenotic valves with tricuspid morphology expressed significantly higher levels of PARP-1 mRNA compared with the corresponding part of bicuspid valves (0.501 vs 0.243, P = 0.01). Furthermore, the quantitative gene expression levels of PARP-1 were inversely correlated with the aortic valve area (AVA) (r = -0.46, P = 0.0469) and AVA indexed for body surface area (BSA) (r = -0.498; P = 0.0298) only in tricuspid aortic valves

  2. High NaCl–induced activation of CDK5 increases phosphorylation of the osmoprotective transcription factor TonEBP/OREBP at threonine 135, which contributes to its rapid nuclear localization

    PubMed Central

    Gallazzini, Morgan; Heussler, Gary E.; Kunin, Margarita; Izumi, Yuichiro; Burg, Maurice B.; Ferraris, Joan D.

    2011-01-01

    When activated by high NaCl, tonicity-responsive enhancer–binding protein/osmotic response element–binding protein (TonEBP/OREBP) increases transcription of osmoprotective genes. High NaCl activates TonEBP/OREBP by increasing its phosphorylation, nuclear localization, and transactivating activity. In HEK293 cells, mass spectrometry shows phosphorylation of TonEBP/OREBP-S120, -S134, -T135, and -S155. When those residues are individually mutated to alanine, nuclear localization is greater for S155A, less for S134A and T135A, and unchanged for S120A. High osmolality increases phosphorylation at T135 in HEK293 cells and in rat renal inner medullas in vivo. In HEK293 cells, high NaCl activates cyclin-dependent kinase 5 (CDK5), which directly phosphorylates TonEBP/OREBP-T135. Inhibition of CDK5 activity reduces the rapid high NaCl–induced nuclear localization of TonEBP/OREBP but does not affect its transactivating activity. High NaCl induces nuclear localization of TonEBP/OREBP faster (≤2 h) than it increases its overall protein abundance (≥6 h). Inhibition of CDK5 reduces the increase in TonEBP/OREBP transcriptional activity that has occurred by 4 h after NaCl is raised, associated with less nuclear TonEBP/OREBP at that time, but does not reduce either activity or nuclear TonEBP/OREBP after 16 h. Thus high NaCl–induced increase of the overall abundance of TonEBP/OREBP, by itself, eventually raises its effective level in the nucleus, but its rapid CDK5-dependent nuclear localization accelerates the process, speeding transcription of osmoprotective target genes. PMID:21209322

  3. The pesticide deltamethrin increases free radical production and promotes nuclear translocation of the stress response transcription factor Nrf2 in rat brain

    PubMed Central

    Li, HY; Wu, SY; Ma, Q; Shi, N

    2015-01-01

    The transcription factor NF-E2-related factor 2 (Nrf2) plays a critical role in the mammalian response to chemical and oxidative stress through induction of phase II detoxification enzymes and oxidative stress response proteins. We reported that Nrf2 expression was activated by deltamethrin (DM), a prototype of the widely used pyrithroid pesticides, in PC12 cells. However, no study has examined Nrf2 nuclear translocation and free radical production, two hallmarks of oxidative stress, in the mammalian brain in vivo. To this end, we examined translocation of Nrf2 and production of free radicals in rat brain exposed to DM. Indeed, DM initiated nuclear translocation of Nrf2 in a dose-dependent manner. Furthermore, Nrf2 translocation was accompanied by the expression of heme oxygenase-1 gene, an Nrf2-regulated gene linked to free radical production. Deltamethrin exposure promoted free radical formation in rat brain and reactive oxygen species generation in PC12 cells. Translocation of Nrf2 may be a response to DM-dependent induction of free radicals and DM may act as a mammalian neurotoxin by initiating oxidative stress. PMID:21398409

  4. High Glucose and Interferon Gamma Synergistically Stimulate MMP-1 Expression in U937 Macrophages by Increasing Transcription Factor STAT1 Activity

    PubMed Central

    Nareika, Alena; Sundararaj, Kamala P; Im, Yeong-Bin; Game, Bryan A.; Lopes-Virella, Maria F.; Huang, Yan

    2009-01-01

    Recent Diabetes Control and Complications Trial and Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) and other clinical studies have reported that glucose control in patients with diabetes leads to a significant reduction of cardiovascular events and atherosclerosis, indicating that hyperglycemia plays an essential role in cardiovascular disease in diabetic patients. Although several mechanisms by which hyperglycemia promotes atherosclerosis have been proposed, it remains unclear how hyperglycemia promotes atherosclerosis by interaction with inflammatory cytokines. To test our hypothesis that hyperglycemia interplays with interferon gamma (IFNγ), a key factor involved in atherosclerosis, to up-regulate the expression of genes such as matrix metalloproteinases (MMPs) and cytokines that are involved in plaque destabilization, U937 macrophages cultured in medium containing either normal or high glucose were challenged with IFNγ and the expression of MMPs and cytokines were then quantified by real-time PCR and ELISA. Results showed that high glucose and IFNγ had a synergistic effect on the expression of MMP-1, MMP-9 and IL-1β. High glucose also enhanced IFNγ-induced priming effect on LPS-stimulated MMP-1 secretion. Furthermore, high glucose and IFNγ exert the synergistic effect on MMP-1 expression by enhancing STAT1 phosphorylation and STAT1 transcriptional activity. In summary, this study revealed a novel mechanism potentially involved in diabetes-promoted cardiovascular disease. PMID:18586252

  5. Increase in transcript accumulation of Psy1 and e-Lcy genes in grain development is associated with differences in seed carotenoid content between durum wheat and tritordeum.

    PubMed

    Rodríguez-Suárez, Cristina; Mellado-Ortega, Elena; Hornero-Méndez, Dámaso; Atienza, Sergio G

    2014-04-01

    Carotenoid rich diets have been associated with lower risk of certain diseases. The great importance of cereals in human diet has directed breeding programs towards carotenoid enhancement to alleviate these deficiencies in developing countries and to offer new functional foods in the developed ones. The new cereal tritordeum (×Tritordeum Ascherson et Graebener) derived from durum wheat (Triticum turgidum ssp. durum) and the wild barley Hordeum chilense, naturally presents carotenoid levels 5-8 times higher than those of durum wheat. The improvement of tritordeum properties as a new functional food requires the elucidation of biosynthetic steps for carotenoid accumulation in seeds that differ from durum wheat. In this work expression patterns of nine genes from the isoprenoid and carotenoid biosynthetic pathways were monitored during grain development in durum wheat and tritordeum. Additionally, a fine identification and quantification of pigments (chlorophylls and carotenoids) during grain development and in mature seeds has been addressed. Transcript levels of Psy1, Psy2, Zds, e-Lcy and b-Lcy were found to correlate to carotenoid content in mature grains. The specific activation of the homeologous genes Psy1, e-Lcy from H. chilense and the high lutein esterification found in tritordeum may serve to explain the differences with durum wheat in carotenoid accumulation.

  6. Berry Phenolic Compounds Increase Expression of Hepatocyte Nuclear Factor-1α (HNF-1α) in Caco-2 and Normal Colon Cells Due to High Affinities with Transcription and Dimerization Domains of HNF-1α.

    PubMed

    Real Hernandez, Luis M; Fan, Junfeng; Johnson, Michelle H; Gonzalez de Mejia, Elvira

    2015-01-01

    Hepatocyte nuclear factor-1α (HNF-1α) is found in the kidneys, spleen, thymus, testis, skin, and throughout the digestive organs. It has been found to promote the transcription of various proteins involved in the management of type II diabetes, including dipeptidyl peptidase-IV (DPP-IV). Phenolic compounds from berries and citrus fruits are known to inhibit DPP-IV, but have not been tested for their interactions with wild-type HNF-1α. By studying the interactions of compounds from berries and citrus fruits have with HNF-1α, pre-transcriptional mechanisms that inhibit the expression of proteins such as DPP-IV may be elucidated. In this study, the interactions of berry phenolic compounds and citrus flavonoids with the dimerization and transcriptional domains of HNF-1α were characterized using the molecular docking program AutoDock Vina. The anthocyanin delphinidin-3-O-arabinoside had the highest binding affinity for the dimerization domain as a homodimer (-7.2 kcal/mol) and transcription domain (-8.3 kcal/mol) of HNF-1α. Anthocyanins and anthocyanidins had relatively higher affinities than resveratrol and citrus flavonoids for both, the transcription domain and the dimerization domain as a homodimer. The flavonoid flavone had the highest affinity for a single unit of the dimerization domain (-6.5 kcal/mol). Nuclear expression of HNF-1α was measured in Caco-2 and human normal colon cells treated with blueberry and blackberry anthocyanin extracts. All extracts tested increased significantly (P < 0.05) the nuclear expression of HNF-1α in Caco-2 cells by 85.2 to 260% compared to a control. The extracts tested increased significantly (P < 0.02) the nuclear expression of HNF-1α in normal colon cells by 48.6 to 243%. It was confirmed that delphinidin-3-O-glucoside increased by 3-fold nuclear HNF-1α expression in Caco-2 cells (P < 0.05). Anthocyanins significantly increased nuclear HNF-1α expression, suggesting that these compounds might regulate the genes HNF-1

  7. E2F transcription factor-1 deficiency reduces pathophysiology in the mouse model of Duchenne muscular dystrophy through increased muscle oxidative metabolism.

    PubMed

    Blanchet, Emilie; Annicotte, Jean-Sébastien; Pradelli, Ludivine A; Hugon, Gérald; Matecki, Stéfan; Mornet, Dominique; Rivier, François; Fajas, Lluis

    2012-09-01

    E2F1 deletion leads to increased mitochondrial number and function, increased body temperature in response to cold and increased resistance to fatigue with exercise. Since E2f1-/- mice show increased muscle performance, we examined the effect of E2f1 genetic inactivation in the mdx background, a mouse model of Duchenne muscular dystrophy (DMD). E2f1-/-;mdx mice demonstrated a strong reduction of physiopathological signs of DMD, including preservation of muscle structure, decreased inflammatory profile, increased utrophin expression, resulting in better endurance and muscle contractile parameters, comparable to normal mdx mice. E2f1 deficiency in the mdx genetic background increased the oxidative metabolic gene program, mitochondrial activity and improved muscle functions. Interestingly, we observed increased E2F1 protein levels in DMD patients, suggesting that E2F1 might represent a promising target for the treatment of DMD.

  8. RNA editing of mat-r transcripts in maize and soybean increases similarity of the encoded protein to fungal and bryophyte group II intron maturases: evidence that mat-r encodes a functional protein.

    PubMed Central

    Thomson, M C; Macfarlane, J L; Beagley, C T; Wolstenholme, D R

    1994-01-01

    We present evidence that transcripts of the mat-r (maturase-related) genes of maize and soybean contain 15 and 14 uridines (U), respectively, at positions occupied by cytosines (C) in the mat-r gene sequences. Eleven and twelve of these C-->U edits result in an amino acid replacement. Ten C-->U edits are at corresponding nucleotides in the maize and soybean transcripts and, except for a single silent edit, the remainder are at positions in one species that are Us in the other species. This results in an increase in amino acid sequence similarity of the maize and soybean MAT-R proteins. Further, of those amino acids in maize and soybean MAT-R proteins specified by edited codons, ten are conserved in the reverse transcriptase-associated and RNA splicing-associated sequences of the cox1-I2 and/or the cox1-I1 maturases of the fungus Saccharomyces cerevisiae and the bryophyte, Marchantia polymorpha, respectively. The implied strong selection for amino acid sequence conservation indicates that the MAT-R protein is functional. The possibility is discussed that initiation of translation of the mat-r transcripts is at a four nucleotide codon, ATAA or ATGA. PMID:7838731

  9. Maintenance of CCL5 mRNA stores by post-effector and memory CD8 T cells is dependent on transcription and is coupled to increased mRNA stability.

    PubMed

    Marçais, Antoine; Tomkowiak, Martine; Walzer, Thierry; Coupet, Charles-Antoine; Ravel-Chapuis, Aymeric; Marvel, Jacqueline

    2006-10-01

    Immunological memory is associated with the display of improved effector functions by cells of the adaptive immune system. The storage of untranslated mRNA coding for the CCL5 chemokine by CD8 memory cells is a new process supporting the immediate display of an effector function. Here, we show that, after induction during the primary response, high CCL5 mRNA levels are specifically preserved in CD8 T cells. We have investigated the mechanisms involved in the long-term maintenance of CCL5 mRNA levels by memory CD8 T cells. We demonstrate that the CCL5 mRNA half-life is increased in memory CD8 T cells and that these cells constitutively transcribe ccl5 gene. By inhibiting ccl5 transcription using IL-4, we demonstrate the essential role of transcription in the maintenance of CCL5 mRNA stores. Finally, we show that these stores are spontaneously reconstituted when the inhibitory signal is removed, indicating that the transcription of ccl5 is a default feature of memory CD8 T cells imprinted in their genetic program.

  10. RNA editing of mat-r transcripts in maize and soybean increases similarity of the encoded protein to fungal and bryophyte group II intron maturases: evidence that mat-r encodes a functional protein.

    PubMed

    Thomson, M C; Macfarlane, J L; Beagley, C T; Wolstenholme, D R

    1994-12-25

    We present evidence that transcripts of the mat-r (maturase-related) genes of maize and soybean contain 15 and 14 uridines (U), respectively, at positions occupied by cytosines (C) in the mat-r gene sequences. Eleven and twelve of these C-->U edits result in an amino acid replacement. Ten C-->U edits are at corresponding nucleotides in the maize and soybean transcripts and, except for a single silent edit, the remainder are at positions in one species that are Us in the other species. This results in an increase in amino acid sequence similarity of the maize and soybean MAT-R proteins. Further, of those amino acids in maize and soybean MAT-R proteins specified by edited codons, ten are conserved in the reverse transcriptase-associated and RNA splicing-associated sequences of the cox1-I2 and/or the cox1-I1 maturases of the fungus Saccharomyces cerevisiae and the bryophyte, Marchantia polymorpha, respectively. The implied strong selection for amino acid sequence conservation indicates that the MAT-R protein is functional. The possibility is discussed that initiation of translation of the mat-r transcripts is at a four nucleotide codon, ATAA or ATGA.

  11. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-{alpha}-dependent pathway in human dermal fibroblasts

    SciTech Connect

    Yamane, Takumi; Kobayashi-Hattori, Kazuo; Oishi, Yuichi

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. Black-Right-Pointing-Pointer Adiponectin also increases the phosphorylation of AMPK. Black-Right-Pointing-Pointer A pharmacological activator of AMPK increases mRNA levels of PPAR{alpha} and HAS2. Black-Right-Pointing-Pointer Adiponectin-induced HAS2 mRNA expression is blocked by a PPAR{alpha} antagonist. Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis via an AMPK/PPAR{alpha}-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1{beta}-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPAR{alpha} antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPAR{alpha}-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  12. Transcription elongation

    PubMed Central

    Imashimizu, Masahiko; Shimamoto, Nobuo; Oshima, Taku; Kashlev, Mikhail

    2014-01-01

    Regulation of transcription elongation via pausing of RNA polymerase has multiple physiological roles. The pausing mechanism depends on the sequence heterogeneity of the DNA being transcribed, as well as on certain interactions of polymerase with specific DNA sequences. In order to describe the mechanism of regulation, we introduce the concept of heterogeneity into the previously proposed alternative models of elongation, power stroke and Brownian ratchet. We also discuss molecular origins and physiological significances of the heterogeneity. PMID:25764114

  13. Increased neurovirulence and reactivation of the herpes simplex virus type 1 latency associated transcript (LAT) negative mutant dLAT2903 with a disrupted LAT miR-H2

    PubMed Central

    Jiang, Xianzhi; Brown, Don; Osorio, Nelson; Hsiang, Chinhui; BenMohamed, Lbachir; Wechsler, Steven L.

    2015-01-01

    At least six microRNAs (miRNAs) appear to be encoded by the latency associated transcript (LAT) of herpes simplex virus type 1 (HSV-1). The gene for ICP0, an important immediate early (IE) viral protein, is antisense to, and overlaps with, the region of LAT from which miRNA H2 (miR-H2) is derived. We recently reported that a mutant (McK-ΔH2) disrupted for miR-H2 on the wild type HSV-1 strain McKrae genomic background has increased ICP0 expression, increased neurovirulence, and slightly more rapid reactivation. We report here that HSV-1 mutants deleted for the LAT promoter nonetheless make significant amounts of miR-H2 during lytic tissue culture infection, presumably via readthrough transcription from an upstream promoter. To determine if miR-H2 might also play a role in the HSV-1 latency-reactivation cycle of a LAT negative mutant, we constructed dLAT-ΔH2, in which miR-H2 is disrupted in dLAT2903 without altering the predicted amino acid sequence of the overlapping ICP0 open reading frame. Similar to McK-ΔH2, dLAT-ΔH2 expressed more ICP0, was more neurovirulent, and had increased reactivation in the mouse TG explant induced reactivation model of HSV-1 compared to its parental virus. Interestingly, although the increased reactivation of McK-ΔH2 compared to its parental wt virus was subtle and only detected at very early times after explant TG induced reactivation, the increased reactivation of dLAT-ΔH2 compared to its dLAT2903 parental virus appeared more robust and was significantly increased even at late times after induction. These results confirm that miR-H2 plays a role in modulating the HSV-1 reactivation phenotype. PMID:26069184

  14. Vitex Agnus Castus Extract Improves Learning and Memory and Increases the Transcription of Estrogen Receptor α in Hippocampus of Ovariectomized Rats.

    PubMed

    Allahtavakoli, Mohammad; Honari, Najmeh; Pourabolli, Iran; Kazemi Arababadi, Mohammad; Ghafarian, Hossein; Roohbakhsh, Ali; Esmaeili Nadimi, Ali; Shamsizadeh, Ali

    2015-07-01

    Lower level of estrogen hormone is considered as an important factor for loss of learning and memory in postmenopausal women. Although estrogen replacement therapy is used for compensation, but long-term usage of estrogen is associated with a higher risk of hormone-dependent cancers. Phytoestrogens, due to fewer side effects, have been proposed to prevent menopause-related cognitive decline. 24 female Wistar rats weighing 180-220 g were used in this study. The animals were ovariectomized and randomly divided into four groups including, control and two groups which received 8 and 80 mg/kg Vitex agnus castus (VAC) ethanolic extract orally. The last groups were treated with 40 μg/kg of estradiol valerat. Step-through passive avoidance (STPA) test was used for the evaluation of learning and memory. The hippocampal estrogen receptor α (ERα) expression was measured using Real-Time PCR. The results demonstrated that VAC extract or estradiol had better performance on step-through passive avoidance test than control group (all P<0.05). Moreover, administration of either estradiol or VAC extract increased the hippocampal mRNA level of ERα and prevented the decrease in uterine weight of ovariectomized rats. Based on our data, VAC extract improves learning and memory in ovariectomized rats. The positive effect of VAC extract on learning and memory is possibly associated with an increase in ERα gene expression in the hippocampal formation.

  15. Vitex Agnus Castus Extract Improves Learning and Memory and Increases the Transcription of Estrogen Receptor α in Hippocampus of Ovariectomized Rats

    PubMed Central

    Allahtavakoli, Mohammad; Honari, Najmeh; Pourabolli, Iran; Kazemi Arababadi, Mohammad; Ghafarian, Hossein; Roohbakhsh, Ali; Esmaeili Nadimi, Ali; Shamsizadeh, Ali

    2015-01-01

    Introduction: Lower level of estrogen hormone is considered as an important factor for loss of learning and memory in postmenopausal women. Although estrogen replacement therapy is used for compensation, but long-term usage of estrogen is associated with a higher risk of hormone-dependent cancers. Phytoestrogens, due to fewer side effects, have been proposed to prevent menopause-related cognitive decline. Methods: 24 female Wistar rats weighing 180–220 g were used in this study. The animals were ovariectomized and randomly divided into four groups including, control and two groups which received 8 and 80 mg/kg Vitex agnus castus (VAC) ethanolic extract orally. The last groups were treated with 40 μg/kg of estradiol valerat. Step-through passive avoidance (STPA) test was used for the evaluation of learning and memory. The hippocampal estrogen receptor α (ERα) expression was measured using Real-Time PCR. Results: The results demonstrated that VAC extract or estradiol had better performance on step-through passive avoidance test than control group (all P<0.05). Moreover, administration of either estradiol or VAC extract increased the hippocampal mRNA level of ERα and prevented the decrease in uterine weight of ovariectomized rats. Discussion: Based on our data, VAC extract improves learning and memory in ovariectomized rats. The positive effect of VAC extract on learning and memory is possibly associated with an increase in ERα gene expression in the hippocampal formation. PMID:26904176

  16. Pervasive transcription: detecting functional RNAs in bacteria.

    PubMed

    Lybecker, Meghan; Bilusic, Ivana; Raghavan, Rahul

    2014-01-01

    Pervasive, or genome-wide, transcription has been reported in all domains of life. In bacteria, most pervasive transcription occurs antisense to protein-coding transcripts, although recently a new class of pervasive RNAs was identified that originates from within annotated genes. Initially considered to be non-functional transcriptional noise, pervasive transcription is increasingly being recognized as important in regulating gene expression. The function of pervasive transcription is an extensively debated question in the field of transcriptomics and regulatory RNA biology. Here, we highlight the most recent contributions addressing the purpose of pervasive transcription in bacteria and discuss their implications.

  17. A gene variant in the transcription factor 7-like 2 (TCF7L2) is associated with an increased risk of gestational diabetes mellitus.

    PubMed

    Pagán, Ana; Sabater-Molina, María; Olza, Josune; Prieto-Sánchez, María T; Blanco-Carnero, Jose E; Parrilla, Juan J; Gil, Ángel; Larqué, Elvira

    2014-09-01

    Adipokines play an important role in the pathogenesis of insulin resistance during pregnancy. We studied the association of genetic variants linked with type 2 diabetes in gestational diabetes mellitus (GDM) subjects and its influence on maternal adipokines. We recruited 25 healthy pregnant women (Controls) and 45 women with GDM at 24-28 weeks of gestation. Maternal blood samples were collected at recruitment and delivery. Adipokines were determined at both sampling times. Genomic DNA was extracted from recruitment samples and FTO rs9939609, TCF7L2 rs4506565, rs7901695, rs12243326, rs12255372 and rs7903146, INSIG2 rs7566605, SREBF1 rs114001633, rs45535737 and rs12941356 and FATP4 rs2003560 genotyped. Serum adiponectin was significantly lower in GDM than Controls at recruitment and showed a similar trend at delivery (p=0.060). In contrast, resistin tended to higher levels in GDM only at recruitment. TCF7L2 rs4506565 (OR=2.31, 95% CI: 1.97-5.01; p=0.031) and FTO rs9939609 (OR=2.17, 95% CI: 1.07-4.41; p=0.039) were associated with GDM risk. Women carrying the T allele of TCF7L2 rs4506565 had increases in plasma resistin of 9.38 μg/L (95% CI 1.39-17.37; p=0.022) per allele; this association remained significant after adjusting for pre-gestational body weight. TCF7L2 rs4506565 variant (T/T) is associated with increased risk of GDM and plasma resistin concentrations in women with GDM. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  18. Increased Stability and DNA Site Discrimination of Single Chain Variants of the Dimeric beta-Barrel DNA Binding Domain of the Human Papillomavirus E2 Transcriptional Regulator

    SciTech Connect

    Dellarole,M.; Sanchez, I.; Freire, E.; de Prat-Gay, G.

    2007-01-01

    Human papillomavirus infects millions of people worldwide and is a causal agent of cervical cancer in women. The HPV E2 protein controls the expression of all viral genes through binding of its dimeric C-terminal domain (E2C) to its target DNA site. We engineered monomeric versions of the HPV16 E2C, in order to probe the link of the dimeric {beta}-barrel fold to stability, dimerization, and DNA binding. Two single-chain variants, with 6 and 12 residue linkers (scE2C-6 and scE2C-12), were purified and characterized. Spectroscopy and crystallography show that the native structure is unperturbed in scE2C-12. The single chain variants are stabilized with respect to E2C, with effective concentrations of 0.6 to 6 mM. The early folding events of the E2C dimer and scE2C-12 are very similar and include formation of a compact species in the submillisecond time scale and a non-native monomeric intermediate with a half-life of 25 ms. However, monomerization changes the unfolding mechanism of the linked species from two-state to three-state, with a high-energy intermediate. Binding to the specific target site is up to 5-fold tighter in the single chain variants. Nonspecific DNA binding is up to 7-fold weaker in the single chain variants, leading to an overall 10-fold increased site discrimination capacity, the largest described so far for linked DNA binding domains. Titration calorimetric binding analysis, however, shows almost identical behavior for dimer and single-chain species, suggesting very subtle changes behind the increased specificity. Global analysis of the mechanisms probed suggests that the dynamics of the E2C domain, rather than the structure, are responsible for the differential properties. Thus, the plastic and dimeric nature of the domain did not evolve for a maximum affinity, specificity, and stability of the quaternary structure, likely because of regulatory reasons and for roles other than DNA binding played by partly folded dimeric or monomeric conformers.

  19. The Translation Initiation Factor 1A (TheIF1A) from Tamarix hispida Is Regulated by a Dof Transcription Factor and Increased Abiotic Stress Tolerance

    PubMed Central

    Yang, Guiyan; Yu, Lili; Wang, Yucheng; Wang, Chao; Gao, Caiqiu

    2017-01-01

    Eukaryotic translation initiation factor 1A (eIF1A) functions as an mRNA scanner and AUG initiation codon locator. However, few studies have clarified the role of eIF1A in abiotic stress. In this study, we cloned eIF1A (TheIF1A) from Tamarix hispida and found its expression to be induced by NaCl and polyethylene glycol (PEG) in roots, stems, and leaves. Compared to control, TheIF1A root expression was increased 187.63-fold when exposed to NaCl for 6 h, suggesting a potential abiotic stress response for this gene. Furthermore, transgenic tobacco plants overexpressing TheIF1A exhibited enhanced seed germination and a higher total chlorophyll content under salt and mannitol stresses. Increased superoxide dismutase, peroxidase, glutathione transferase and glutathione peroxidase activities, as well as decreased electrolyte leakage rates and malondialdehyde contents, were observed in TheIF1A-transgenic tobacco and T. hispida seedlings under salt and mannitol stresses. Histochemical staining suggested that TheIF1A improves reactive oxygen species (ROS) scavenging in plants. Moreover, TheIF1A may regulate expression of stress-related genes, including TOBLTP, GST, MnSOD, NtMPK9, poxN1, and CDPK15. Moreover, a 1352-bp promoter fragment of TheIF1A was isolated, and cis-elements were identified. Yeast one-hybrid assays showed that ThDof can specifically bind to the Dof motif present in the promoter. In addition, ThDof showed expression patterns similar to those of TheIF1A under NaCl and PEG stresses. These findings suggest the potential mechanism and physiological roles of TheIF1A. ThDof may be an upstream regulator of TheIF1A, and TheIF1A may function as a stress response regulator to improve plant salt and osmotic stress tolerance via regulation of associated enzymes and ROS scavenging, thereby reducing cell damage under stress conditions. PMID:28439284

  20. High tumor necrosis factor alpha (TNF-alpha) production in Trypanosoma cruzi-infected pregnant mice and increased TNF-alpha gene transcription in their offspring.

    PubMed Central

    Rivera, M T; Marques de Araujo, S; Lucas, R; Deman, J; Truyens, C; Defresne, M P; de Baetselier, P; Carlier, Y

    1995-01-01

    Since tumor necrosis factor alpha (TNF-alpha) is known to be involved in the feto-maternal relationship, this cytokine was studied in Trypanosoma cruzi-infected pregnant BALB/c mice and their fetuses and offspring. Pregnant chronically infected mice displayed significantly higher levels of circulating TNF-alpha than animals either only infected or only pregnant. TNF-alpha was undetectable in sera of uninfected and nonpregnant mice as well as in breast milk obtained from infected and uninfected animals. Fetuses from infected mice exhibited significantly more cells containing TNF-alpha mRNA in their thymus than fetuses from uninfected mothers. When infected 2 months after birth, offspring born to infected and uninfected mothers displayed similar amounts of circulating TNF-alpha during chronic infection, whereas this cytokine was only weakly detectable during the acute phase of the disease. An intravenous injection of lipopolysaccharide during acute infection strongly increased the production of TNF-alpha in offspring born to infected mothers to levels higher than those in progeny from uninfected mice. These results suggest that TNF-alpha is an important cytokine in the feto-maternal relationship during T. cruzi infection and that fetuses and offspring of infected mothers are primed to produce elevated levels of TNF-alpha. PMID:7822027

  1. SNP in human ARHGEF3 promoter is associated with DNase hypersensitivity, transcript level and platelet function, and Arhgef3 KO mice have increased mean platelet volume.

    PubMed

    Zou, Siying; Teixeira, Alexandra M; Kostadima, Myrto; Astle, William J; Radhakrishnan, Aparna; Simon, Lukas Mikolaj; Truman, Lucy; Fang, Jennifer S; Hwa, John; Zhang, Ping-Xia; van der Harst, Pim; Bray, Paul F; Ouwehand, Willem H; Frontini, Mattia; Krause, Diane S

    2017-01-01

    Genome-wide association studies have identified a genetic variant at 3p14.3 (SNP rs1354034) that strongly associates with platelet number and mean platelet volume in humans. While originally proposed to be intronic, analysis of mRNA expression in primary human hematopoietic subpopulations reveals that this SNP is located directly upstream of the predominantly expressed ARHGEF3 isoform in megakaryocytes (MK). We found that ARHGEF3, which encodes a Rho guanine exchange factor, is dramatically upregulated during both human and murine MK maturation. We show that the SNP (rs1354034) is located in a DNase I hypersensitive region in human MKs and is an expression quantitative locus (eQTL) associated with ARHGEF3 expression level in human platelets, suggesting that it may be the causal SNP that accounts for the variations observed in human platelet traits and ARHGEF3 expression. In vitro human platelet activation assays revealed that rs1354034 is highly correlated with human platelet activation by ADP. In order to test whether ARHGEF3 plays a role in MK development and/or platelet function, we developed an Arhgef3 KO/LacZ reporter mouse model. Reflecting changes in gene expression, LacZ expression increases during MK maturation in these mice. Although Arhgef3 KO mice have significantly larger platelets, loss of Arhgef3 does not affect baseline MK or platelets nor does it affect platelet function or platelet recovery in response to antibody-mediated platelet depletion compared to littermate controls. In summary, our data suggest that modulation of ARHGEF3 gene expression in humans with a promoter-localized SNP plays a role in human MKs and human platelet function-a finding resulting from the biological follow-up of human genetic studies. Arhgef3 KO mice partially recapitulate the human phenotype.

  2. Severe psychosocial deprivation in early childhood is associated with increased DNA methylation across a region spanning the transcription start site of CYP2E1

    PubMed Central

    Kumsta, R; Marzi, S J; Viana, J; Dempster, E L; Crawford, B; Rutter, M; Mill, J; Sonuga-Barke, E J S

    2016-01-01

    Exposure to adverse rearing environments including institutional deprivation and severe childhood abuse is associated with an increased risk for mental and physical health problems across the lifespan. Although the mechanisms mediating these effects are not known, recent work in rodent models suggests that epigenetic processes may be involved. We studied the impact of severe early-life adversity on epigenetic variation in a sample of adolescents adopted from the severely depriving orphanages of the Romanian communist era in the 1980s. We quantified buccal cell DNA methylation at ~400 000 sites across the genome in Romanian adoptees exposed to either extended (6–43 months; n=16) or limited duration (<6 months; n=17) of severe early-life deprivation, in addition to a matched sample of UK adoptees (n=16) not exposed to severe deprivation. Although no probe-wise differences remained significant after controlling for the number of probes tested, we identified an exposure-associated differentially methylated region (DMR) spanning nine sequential CpG sites in the promoter-regulatory region of the cytochrome P450 2E1 gene (CYP2E1) on chromosome 10 (corrected P=2.98 × 10−5). Elevated DNA methylation across this region was also associated with deprivation-related clinical markers of impaired social cognition. Our data suggest that environmental insults of sufficient biological impact during early development are associated with long-lasting epigenetic changes, potentially reflecting a biological mechanism linking the effects of early-life adversity to cognitive and neurobiological phenotypes. PMID:27271856

  3. Activating transcription factor 4, a mediator of the integrated stress response, is increased in the dorsal root ganglia following painful facet joint distraction

    PubMed Central

    Dong, Ling; Guarino, Benjamin B.; Jordan-Sciutto, Kelly L.; Winkelstein, Beth A.

    2011-01-01

    Chronic neck pain is one of the most common musculoskeletal disorders in the US. Although biomechanical and clinical studies have implicated the facet joint as a primary source of neck pain, specific cellular mechanisms still remain speculative. The purpose of this study was to investigate whether a mediator (ATF4) of the integrated stress response (ISR) is involved in facet-mediated pain. Holtzman rats underwent C6/C7 facet joint loading that produces either painful (n=16) or nonpainful (n=8) responses. A sham group (n=9) was also included as surgical controls. Behavioral sensitivity was measured and the C6 DRGs were harvested on day 7 to evaluate the total and neuronal ATF4 expression. In separate groups, an intra-articular ketorolac injection was administered either immediately (D0 ketorolac) or 1 day (D1 ketorolac) after painful facet joint loading. Allodynia was measured at days 1 and 7 after injury to assess the effects on behavioral responses. ATF4 and BiP (an indicator of ISR activation) were separately quantified at day 7. Facet joint loading sufficient to elicit behavioral hypersensitivity produced a 3-fold increase in total and neuronal ATF4 expression in the DRG. After ketorolac treatment at the time of injury, ATF4 expression was significantly (p<0.01) reduced despite not producing any attenuation of behavioral responses. Interestingly, ketorolac treatment at day 1 significantly (p<0.001) alleviated behavioral sensitivity at day 7, but did not modify ATF4 expression. BiP expression was unchanged after either intervention time. Results suggest that ATF4-dependent activation of the ISR does not directly contribute to persistent pain, but may sensitize neurons responsible for pain initiation. These behavioral and immunohistochemical findings imply that facet-mediated pain may be sustained through other pathways of the ISR. PMID:21821103

  4. A peripherally administered, centrally acting angiotensin II AT2 antagonist selectively increases brain AT1 receptors and decreases brain tyrosine hydroxylase transcription, pituitary vasopressin and ACTH

    PubMed Central

    Macova, Miroslava; Pavel, Jaroslav; Saavedra, Juan M.

    2009-01-01

    The physiological actions of brain Angiotensin II AT2 receptors and their relationship to Angiotensin II AT1 receptors remain controversial. To further clarify their role, we determined to what extent systemic administration of an AT2 receptor antagonist affected AT2 receptor binding within the brain and the expression of AT1 receptors. For this purpose, we subcutaneously administered the AT2 receptor antagonist PD123319 (1 mg/kg/day) to adult male rats for two weeks via osmotic minipumps. We also studied the content of pituitary adrenocorticotropic hormone and vasopressin, representative of hypothalamic–pituitary–adrenal axis activation, and the tyrosine hydroxylase gene expression in the locus coeruleus as a measure of central norepinephrine function. We found significant decreases in AT2 receptor binding in brain areas inside the blood brain barrier, the inferior olive and the locus coeruleus. AT2 receptor blockade increased AT1 receptor binding and mRNA expression not only in the subfornical organ and the median eminence, situated outside the blood brain barrier, but also in the hypothalamic paraventricular nucleus, located inside the blood brain barrier. These changes paralleled decreased expression of tyrosine hydroxylase mRNA in the locus coeruleus and decreased pituitary adrenocorticotropic and vasopressin content. Our results demonstrate that sustained peripheral administration of an AT2 antagonist decreases binding to brain AT2 receptors, indicating that this drug is a useful tool for the study of their central role. AT2 receptor activity inhibition up-regulates AT1 receptor expression in specific brain areas. Blockade of brain AT2 receptors is compatible with enhanced hypothalamic–pituitary–adrenal axis and decreased central sympathetic system activity. PMID:19038235

  5. Cadmium, cobalt and lead cause stress response, cell cycle deregulation and increased steroid as well as xenobiotic metabolism in primary normal human bronchial epithelial cells which is coordinated by at least nine transcription factors.

    PubMed

    Glahn, Felix; Schmidt-Heck, Wolfgang; Zellmer, Sebastian; Guthke, Reinhard; Wiese, Jan; Golka, Klaus; Hergenröder, Roland; Degen, Gisela H; Lehmann, Thomas; Hermes, Matthias; Schormann, Wiebke; Brulport, Marc; Bauer, Alexander; Bedawy, Essam; Gebhardt, Rolf; Hengstler, Jan G; Foth, Heidi

    2008-08-01

    Workers occupationally exposed to cadmium, cobalt and lead have been reported to have increased levels of DNA damage. To analyze whether in vivo relevant concentrations of heavy metals cause systematic alterations in RNA expression patterns, we performed a gene array study using primary normal human bronchial epithelial cells. Cells were incubated with 15 microg/l Cd(II), 25 microg/l Co(II) and 550 microg/l Pb(II) either with individual substances or in combination. Differentially expressed genes were filtered out and used to identify enriched GO categories as well as KEGG pathways and to identify transcription factors whose binding sites are enriched in a given set of promoters. Interestingly, combined exposure to Cd(II), Co(II) and Pb(II) caused a coordinated response of at least seven stress response-related transcription factors, namely Oct-1, HIC1, TGIF, CREB, ATF4, SRF and YY1. A stress response was further corroborated by up regulation of genes involved in glutathione metabolism. A second major response to heavy metal exposure was deregulation of the cell cycle as evidenced by down regulation of the transcription factors ELK-1 and the Ets transcription factor GABP, as well as deregulation of genes involved in purine and pyrimidine metabolism. A third and surprising response was up regulation of genes involved in steroid metabolism, whereby promoter analysis identified up regulation of SRY that is known to play a role in sex determination. A forth response was up regulation of xenobiotic metabolising enzymes, particularly of dihydrodiol dehydrogenases 1 and 2 (AKR1C1, AKR1C2). Incubations with individual heavy metals showed that the response of AKR1C1 and AKR1C2 was predominantly caused by lead. In conclusion, we have shown that in vivo relevant concentrations of Cd(II), Co(II) and Pb(II) cause a complex and coordinated response in normal human bronchial epithelial cells. This study gives an overview of the most responsive genes.

  6. Eukaryotic transcriptional control.

    PubMed

    Kornberg, R D

    1999-12-01

    Some 30 years ago, following the elucidation of transcriptional control in prokaryotes, attention turned to the corresponding problem in eukaryotes: how are so many genes transcribed in a cell-type-specific, developmentally regulated manner? The answer has been found in two modes of regulation, one involving chromatin and the other the chief transcribing enzyme, RNA polymerase II. Although basic features of the prokaryotic mechanism have been preserved, the demands of eukaryotic transcription control are met by a huge increase in complexity and by the addition of new layers to the transcription apparatus. Discovering the components of this apparatus has been a major theme of research over the past three decades; unravelling the mechanisms is a challenge for the future.

  7. Estrogen Signaling Multiple Pathways to Impact Gene Transcription

    PubMed Central

    Marino, Maria; Galluzzo, Paola; Ascenzi, Paolo

    2006-01-01

    Steroid hormones exert profound effects on cell growth, development, differentiation, and homeostasis. Their effects are mediated through specific intracellular steroid receptors that act via multiple mechanisms. Among others, the action mechanism starting upon 17β-estradiol (E2) binds to its receptors (ER) is considered a paradigmatic example of how steroid hormones function. Ligand-activated ER dimerizes and translocates in the nucleus where it recognizes specific hormone response elements located in or near promoter DNA regions of target genes. Behind the classical genomic mechanism shared with other steroid hormones, E2 also modulates gene expression by a second indirect mechanism that involves the interaction of ER with other transcription factors which, in turn, bind their cognate DNA elements. In this case, ER modulates the activities of transcription factors such as the activator protein (AP)-1, nuclear factor-κB (NF-κB) and stimulating protein-1 (Sp-1), by stabilizing DNA-protein complexes and/or recruiting co-activators. In addition, E2 binding to ER may also exert rapid actions that start with the activation of a variety of signal transduction pathways (e.g. ERK/MAPK, p38/MAPK, PI3K/AKT, PLC/PKC). The debate about the contribution of different ER-mediated signaling pathways to coordinate the expression of specific sets of genes is still open. This review will focus on the recent knowledge about the mechanism by which ERs regulate the expression of target genes and the emerging field of integration of membrane and nuclear receptor signaling, giving examples of the ways by which the genomic and non-genomic actions of ERs on target genes converge. PMID:18369406

  8. Exposure to static magnetic fields increases insulin secretion in rat INS-1 cells by activating the transcription of the insulin gene and up-regulating the expression of vesicle-secreted proteins.

    PubMed

    Mao, Libin; Wang, Huiqin; Ma, Fenghui; Guo, Zhixia; He, Hongpeng; Zhou, Hao; Wang, Nan

    2017-08-01

    To evaluate the effect of static magnetic fields (SMFs) on insulin secretion and explore the mechanisms underlying exposure to SMF-induced insulin secretion in rat insulinoma INS-1 cells. INS-1 cells were exposed to a 400 mT SMF for 72 h, and the proliferation of INS-1 cells was detected by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The secretion of insulin was measured with an enzyme linked immunosorbent assays (ELISA), the expression of genes was detected by real-time PCR, and the expression of proteins was measured by Western blotting. Exposure to an SMF increased the expression and secretion of insulin by INS-1 cells but did not affect cell proliferation. Moreover, SMF exposure up-regulated the expression of several pancreas-specific transcriptional factors. Specifically, the activity of the rat insulin promoter was enhanced in INS-1 cells exposed to an SMF, and the expression levels of synaptosomal-associated protein 25 (SNAP-25) and syntaxin-1A were up-regulated after exposure to an SMF. SMF exposure can promote insulin secretion in rat INS-1 cells by activating the transcription of the insulin gene and up-regulating the expression of vesicle-secreted proteins.

  9. A Novel R2R3-MYB Transcription Factor BpMYB106 of Birch (Betula platyphylla) Confers Increased Photosynthesis and Growth Rate through Up-regulating Photosynthetic Gene Expression.

    PubMed

    Zhou, Chenguang; Li, Chenghao

    2016-01-01

    We isolated a R2R3-MYB transcription factor BpMYB106, which regulates photosynthesis in birch (Betula platyphylla Suk.). BpMYB106 mainly expresses in the leaf and shoot tip of birch, and its protein is localized in the nucleus. We further fused isolated a 1588 bp promoter of BpMYB106 and analyzed it by PLACE, which showed some cis-acting elements related to photosynthesis. BpMYB106 promoter β-glucuronidase (GUS) reporter fusion studies gene, the result, showed the GUS reporter gene in transgenic birch with BpMYB106 promoter showed strong activities in shoot tip, cotyledon margins, and mature leaf trichomes. The overexpression of BpMYB106 in birch resulted in significantly increased trichome density, net photosynthetic rate, and growth rate as compared with the wild-type birch. RNA-Seq profiling revealed the upregulation of several photosynthesis-related genes in the photosynthesis and oxidative phosphorylation pathways in the leaves of transgenic plants. Yeast one-hybrid analysis, coupled with transient assay in tobacco, revealed that BpMYB106 binds a MYB binding site MYB2 in differentially expressed gene promoters. Thus, BpMYB106 may directly activate the expression of a range of photosynthesis related genes through interacting with the MYB2 element in their promoters. Our study demonstrating the overexpression of BpMYB106-a R2R3-MYB transcription factor-upregulates the genes of the photosynthesis and oxidative phosphorylation pathways to improve photosynthesis.

  10. Improved Methods for Teaching Machine Transcription.

    ERIC Educational Resources Information Center

    Smith, Clara J.

    1980-01-01

    The increased use of machine transcription in business and industry demands that business educators attract and train more highly skilled machine transcriptionists. Realistic production measurement and appropriate vocabulary should be taught to link machine transcription to word processing. (Author)

  11. Increased neurovirulence and reactivation of the herpes simplex virus type 1 latency-associated transcript (LAT)-negative mutant dLAT2903 with a disrupted LAT miR-H2.

    PubMed

    Jiang, Xianzhi; Brown, Don; Osorio, Nelson; Hsiang, Chinhui; BenMohamed, Lbachir; Wechsler, Steven L

    2016-02-01

    At least six microRNAs (miRNAs) appear to be encoded by the latency-associated transcript (LAT) of herpes simplex virus type 1 (HSV-1). The gene for ICP0, an important immediate early (IE) viral protein, is anti-sense to, and overlaps with, the region of LAT from which miRNA H2 (miR-H2) is derived. We recently reported that a mutant (McK-ΔH2) disrupted for miR-H2 on the wild-type HSV-1 strain McKrae genomic background has increased ICP0 expression, increased neurovirulence, and slightly more rapid reactivation. We report here that HSV-1 mutants deleted for the LAT promoter nonetheless make significant amounts of miR-H2 during lytic tissue culture infection, presumably via readthrough transcription from an upstream promoter. To determine if miR-H2 might also play a role in the HSV-1 latency/reactivation cycle of a LAT-negative mutant, we constructed dLAT-ΔH2, in which miR-H2 is disrupted in dLAT2903 without altering the predicted amino acid sequence of the overlapping ICP0 open reading frame. Similar to McK-ΔH2, dLAT-ΔH2 expressed more ICP0, was more neurovirulent, and had increased reactivation in the mouse TG explant-induced reactivation model of HSV-1 compared with its parental virus. Interestingly, although the increased reactivation of McK-ΔH2 compared with its parental wild-type (wt) virus was subtle and only detected at very early times after explant TG induced reactivation, the increased reactivation of dLAT-ΔH2 compared with its dLAT2903 parental virus appeared more robust and was significantly increased even at late times after induction. These results confirm that miR-H2 plays a role in modulating the HSV-1 reactivation phenotype.

  12. Glyceollin I Enantiomers Distinctly Regulate ER-Mediated Gene Expression

    USDA-ARS?s Scientific Manuscript database

    Glyceollins (GLYs) are pterocarpan, phytoalexins elicited in high concentrations when soybeans are stressed. We have previously reported that the three most common glyceollin isomers (GLY I, II, and III) exhibit antiestrogenic properties, which may have significant biological effects following human...

  13. 14‐3‐3‐β and ‐ε contribute to activation of the osmoprotective transcription factor NFAT5 by increasing its protein abundance and its transactivating activity

    PubMed Central

    Izumi, Yuichiro; Burg, Maurice B.; Ferraris, Joan D.

    2014-01-01

    Abstract Having previously found that high NaCl causes rapid exit of 14‐3‐3 isoforms from the nucleus, we used siRNA‐mediated knockdown to test whether 14‐3‐3s contribute to the high NaCl‐induced increase in the activity of the osmoprotective transcription factor NFAT5. We find that, when NaCl is elevated, knockdown of 14‐3‐3‐β and/or 14‐3‐3‐ε decreases NFAT5 transcriptional activity, as assayed both by luciferase reporter and by the mRNA abundance of the NFAT5 target genes aldose reductase and the sodium‐ and chloride‐dependent betaine transporter, BGT1. Knockdown of other 14‐3‐3 isoforms does not significantly affect NFAT5 activity. 14‐3‐3‐β and/or 14‐3‐3‐ε do not act by affecting the nuclear localization of NFAT5, but by at least two other mechanisms: (1) 14‐3‐3‐β and 14‐3‐3‐ε increase protein abundance of NFAT5 and (2) they increase NFAT5 transactivating activity. When NaCl is elevated, knockdown of 14‐3‐3‐β and/or 14‐3‐3‐ε reduces the protein abundance of NFAT5, as measured by Western blot, without affecting the level of NFAT5 mRNA, and the knockdown also decreases NFAT5 transactivating activity, as measured by luciferase reporter. The 14‐3‐3s increase NFAT5 protein, not by increasing its translation, but by decreasing the rate at which it is degraded, as measured by cycloheximide chase. It is not clear at this point whether the 14‐3‐3s affect NFAT5 directly or indirectly through their effects on other proteins that signal activation of NFAT5. PMID:24771694

  14. Methyl protodioscin increases ABCA1 expression and cholesterol efflux while inhibiting gene expressions for synthesis of cholesterol and triglycerides by suppressing SREBP transcription and microRNA 33a/b levels.

    PubMed

    Ma, Weilie; Ding, Hang; Gong, Xiaohua; Liu, Zhen; Lin, Yalin; Zhang, Zhizhen; Lin, Guorong

    2015-04-01

    Sterol regulatory element-binding proteins (SREBPs) regulate homeostasis of LDL, HDL and triglycerides. This study was aimed to determine if inhibition of SREBPs by methyl protodioscin (MPD) regulates downstream gene and protein expressions of lipid metabolisms. In THP-1 macrophages, MPD increases levels of ABCA1 mRNA and protein in dose- and time-dependent manners, and apoA-1-mediated cholesterol efflux. The underlying mechanisms for the effects is that MPD inhibits the transcription of SREBP1c and SREBP2, and decreases levels of microRNA 33a/b hosted in the introns of SREBPs, which leads to reciprocally increase ABCA1 levels. In HepG2 cells, MPD shows the same effects as these observed in THP-1 macrophages. MPD also decreases the gene expressions of HMGCR, FAS and ACC for cholesterol and fatty acid synthesis. MPD further promotes LDL receptor through reducing the PCSK9 level. Collectively, the study demonstrates that MPD potentially increase HDL cholesterol while reducing LDL cholesterol and triglycerides. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. A spontaneous deletion of α-synuclein is associated with an increase in CB1 mRNA transcript and receptor expression in the hippocampus and amygdala: effects on alcohol consumption

    PubMed Central

    López-Jiménez, Alejandro; Walter, Nicole A. R.; Giné, Elena; Santos, Ángel; Echeverry-Alzate, Victor; Bühler, Kora-Mareen; Olmos, Pedro; Giezendanner, Stéphanie; Moratalla, Rosario; Montoliu, Lluis; Buck, Kari J.; López-Moreno, Jose Antonio

    2014-01-01

    α-Synuclein (α-syn) protein and endocannabinoid CB1 receptors are primarily located in presynaptic terminals. An association between α-syn and CB1 receptors has recently been established in Parkinson’s disease, but it is completely unknown whether there is an association between these two proteins in alcohol addiction. Therefore, we aimed to examine the α-syn mRNA transcript and protein expression levels in the prefrontal cortex, striatum, amygdala and hippocampus. These brain regions are the most frequently implicated in alcohol and other drug addiction. In these studies, we used C57BL/6 mice carrying a spontaneous deletion of the α-syn gene (C57BL/6Snca−/−) and their respective controls (C57BL/6Snca+/+). These animals were monitored for spontaneous alcohol consumption (3–10%) and their response to a hypnotic-sedative dose of alcohol (3 g/kg) was also assessed. Compared with the C57BL/6Snca+/+ mice, we found that the C57BL/6Snca−/− mice exhibited a higher expression level of the CB1 mRNA transcript and CB1 receptor in the hippocampus and amygdala. Furthermore, C57BL/6Snca−/− mice showed an increase in alcohol consumption when offered a 10% alcohol solution. There was no significant difference in sleep time after the injection of 3 g/kg alcohol. These results are the first to reveal an association between α-syn and the CB1 receptor in the brain regions that are most frequently implicated in alcohol and other drug addictions. PMID:23345080

  16. A Novel R2R3-MYB Transcription Factor BpMYB106 of Birch (Betula platyphylla) Confers Increased Photosynthesis and Growth Rate through Up-regulating Photosynthetic Gene Expression

    PubMed Central

    Zhou, Chenguang; Li, Chenghao

    2016-01-01

    We isolated a R2R3-MYB transcription factor BpMYB106, which regulates photosynthesis in birch (Betula platyphylla Suk.). BpMYB106 mainly expresses in the leaf and shoot tip of birch, and its protein is localized in the nucleus. We further fused isolated a 1588 bp promoter of BpMYB106 and analyzed it by PLACE, which showed some cis-acting elements related to photosynthesis. BpMYB106 promoter β-glucuronidase (GUS) reporter fusion studies gene, the result, showed the GUS reporter gene in transgenic birch with BpMYB106 promoter showed strong activities in shoot tip, cotyledon margins, and mature leaf trichomes. The overexpression of BpMYB106 in birch resulted in significantly increased trichome density, net photosynthetic rate, and growth rate as compared with the wild-type birch. RNA-Seq profiling revealed the upregulation of several photosynthesis-related genes in the photosynthesis and oxidative phosphorylation pathways in the leaves of transgenic plants. Yeast one-hybrid analysis, coupled with transient assay in tobacco, revealed that BpMYB106 binds a MYB binding site MYB2 in differentially expressed gene promoters. Thus, BpMYB106 may directly activate the expression of a range of photosynthesis related genes through interacting with the MYB2 element in their promoters. Our study demonstrating the overexpression of BpMYB106—a R2R3-MYB transcription factor—upregulates the genes of the photosynthesis and oxidative phosphorylation pathways to improve photosynthesis. PMID:27047502

  17. Cdk5 protein inhibition and Aβ42 increase BACE1 protein level in primary neurons by a post-transcriptional mechanism: implications of CDK5 as a therapeutic target for Alzheimer disease.

    PubMed

    Sadleir, Katherine R; Vassar, Robert

    2012-03-02

    The β-secretase enzyme BACE1 initiates production of the amyloid-β (Aβ) peptide that comprises plaques in Alzheimer disease (AD) brain. BACE1 levels are increased in AD, potentially accelerating Aβ generation, but the mechanisms of BACE1 elevation are not fully understood. Cdk5/p25 has been implicated in neurodegeneration and BACE1 regulation, suggesting therapeutic Cdk5 inhibition for AD. In addition, caspase 3 has been implicated in BACE1 elevation. Here, we show that the Cdk5 level and p25:p35 ratio were elevated and correlated with BACE1 level in brains of AD patients and 5XFAD transgenic mice. Mouse primary cortical neurons treated with Aβ42 oligomers had increased BACE1 level and p25:p35 ratio. Surprisingly, the Aβ42-induced BACE1 elevation was not blocked by Cdk5 inhibitors CP68130 and roscovitine, and instead the BACE1 level was increased greater than with Aβ42 treatment alone. Moreover, Cdk5 inhibitors alone elevated BACE1 in a time- and dose-dependent manner that coincided with increased caspase 3 cleavage and decreased Cdk5 level. Caspase 3 inhibitor benzyloxycarbonyl-VAD failed to prevent the Aβ42-induced BACE1 increase. Further experiments suggested that the Aβ42-induced BACE1 elevation was the result of a post-transcriptional mechanism. We conclude that Aβ42 may increase the BACE1 level independently of either Cdk5 or caspase 3 and that Cdk5 inhibition for AD may cause BACE1 elevation, a potentially negative therapeutic outcome.

  18. Silencing the Menkes copper-transporting ATPase (Atp7a) gene in rat intestinal epithelial (IEC-6) cells increases iron flux via transcriptional induction of ferroportin 1 (Fpn1).

    PubMed

    Gulec, Sukru; Collins, James F

    2014-01-01

    The Menkes copper-transporting ATPase (Atp7a) gene is induced in rat duodenum during iron deficiency, consistent with copper accumulation in the intestinal mucosa and liver. To test the hypothesis that ATP7A influences intestinal iron metabolism, the Atp7a gene was silenced in rat intestinal epithelial (IEC-6) cells using short hairpin RNA (shRNA) technology. Perturbations in intracellular copper homeostasis were noted in knockdown cells, consistent with the dual roles of ATP7A in pumping copper into the trans-Golgi (for cuproenzyme synthesis) and exporting copper from cells. Intracellular iron concentrations were unaffected by Atp7a knockdown. Unexpectedly, however, vectorial iron ((59)Fe) transport increased (∼33%) in knockdown cells grown in bicameral inserts and increased further (∼70%) by iron deprivation (compared with negative control shRNA-transfected cells). Additional experiments were designed to elucidate the molecular mechanism of increased transepithelial iron flux. Enhanced iron uptake by knockdown cells was associated with increased expression of a ferrireductase (duodenal cytochrome b) and activity of a cell-surface ferrireductase. Increased iron efflux from knockdown cells was likely mediated via transcriptional activation of the ferroportin 1 gene (by an unknown mechanism). Moreover, Atp7a knockdown significantly attenuated expression of an iron oxidase [hephaestin (HEPH); by ∼80%] and membrane ferroxidase activity (by ∼50%). Cytosolic ferroxidase activity, however, was retained in knockdown cells (75% of control cells), perhaps compensating for diminished HEPH activity. This investigation has thus documented alterations in iron homeostasis associated with Atp7a knockdown in enterocyte-like cells. Alterations in copper transport, trafficking, or distribution may underlie the increase in transepithelial iron flux noted when ATP7A activity is diminished.

  19. Silencing the Menkes Copper-Transporting ATPase (Atp7a) Gene in Rat Intestinal Epithelial (IEC-6) Cells Increases Iron Flux via Transcriptional Induction of Ferroportin 1 (Fpn1)123

    PubMed Central

    Gulec, Sukru; Collins, James F.

    2014-01-01

    The Menkes copper-transporting ATPase (Atp7a) gene is induced in rat duodenum during iron deficiency, consistent with copper accumulation in the intestinal mucosa and liver. To test the hypothesis that ATP7A influences intestinal iron metabolism, the Atp7a gene was silenced in rat intestinal epithelial (IEC-6) cells using short hairpin RNA (shRNA) technology. Perturbations in intracellular copper homeostasis were noted in knockdown cells, consistent with the dual roles of ATP7A in pumping copper into the trans-Golgi (for cuproenzyme synthesis) and exporting copper from cells. Intracellular iron concentrations were unaffected by Atp7a knockdown. Unexpectedly, however, vectorial iron (59Fe) transport increased (∼33%) in knockdown cells grown in bicameral inserts and increased further (∼70%) by iron deprivation (compared with negative control shRNA-transfected cells). Additional experiments were designed to elucidate the molecular mechanism of increased transepithelial iron flux. Enhanced iron uptake by knockdown cells was associated with increased expression of a ferrireductase (duodenal cytochrome b) and activity of a cell-surface ferrireductase. Increased iron efflux from knockdown cells was likely mediated via transcriptional activation of the ferroportin 1 gene (by an unknown mechanism). Moreover, Atp7a knockdown significantly attenuated expression of an iron oxidase [hephaestin (HEPH); by ∼80%] and membrane ferroxidase activity (by ∼50%). Cytosolic ferroxidase activity, however, was retained in knockdown cells (75% of control cells), perhaps compensating for diminished HEPH activity. This investigation has thus documented alterations in iron homeostasis associated with Atp7a knockdown in enterocyte-like cells. Alterations in copper transport, trafficking, or distribution may underlie the increase in transepithelial iron flux noted when ATP7A activity is diminished. PMID:24174620

  20. Transcriptional regulatory factor X6 (Rfx6) increases gastric inhibitory polypeptide (GIP) expression in enteroendocrine K-cells and is involved in GIP hypersecretion in high fat diet-induced obesity.

    PubMed

    Suzuki, Kazuyo; Harada, Norio; Yamane, Shunsuke; Nakamura, Yasuhiko; Sasaki, Kazuki; Nasteska, Daniela; Joo, Erina; Shibue, Kimitaka; Harada, Takanari; Hamasaki, Akihiro; Toyoda, Kentaro; Nagashima, Kazuaki; Inagaki, Nobuya

    2013-01-18

    Gastric inhibitory polypeptide (GIP) is an incretin released from enteroendocrine K-cells in response to nutrient ingestion. GIP potentiates glucose-stimulated insulin secretion and induces energy accumulation into adipose tissue, resulting in obesity. Plasma GIP levels are reported to be increased in the obese state. However, the molecular mechanisms of GIP secretion and high fat diet (HFD)-induced GIP hypersecretion remain unclear, primarily due to difficulties in separating K-cells from other intestinal epithelial cells in vivo. In this study, GIP-GFP knock-in mice that enable us to visualize K-cells by enhanced GFP were established. Microarray analysis of isolated K-cells from these mice revealed that transcriptional regulatory factor X6 (Rfx6) is expressed exclusively in K-cells. In vitro experiments using the mouse intestinal cell line STC-1 showed that knockdown of Rfx6 decreased mRNA expression, cellular content, and secretion of GIP. Rfx6 bound to the region in the gip promoter that regulates gip promoter activity, and overexpression of Rfx6 increased GIP mRNA expression. HFD induced obesity and GIP hypersecretion in GIP-GFP heterozygous mice in vivo. Immunohistochemical and flow cytometry analysis showed no significant difference in K-cell number between control fat diet-fed (CFD) and HFD-fed mice. However, GIP content in the upper small intestine and GIP mRNA expression in K-cells were significantly increased in HFD-fed mice compared with those in CFD-fed mice. Furthermore, expression levels of Rfx6 mRNA were increased in K-cells of HFD-fed mice. These results suggest that Rfx6 increases GIP expression and content in K-cells and is involved in GIP hypersecretion in HFD-induced obesity.

  1. The nuclear retention of transcription factor FOXO3a correlates with a DNA damage response and increased glutamine synthetase expression by astrocytes suggesting a neuroprotective role in the ageing brain.

    PubMed

    Fluteau, Adeline; Ince, Paul G; Minett, Thais; Matthews, Fiona E; Brayne, Carol; Garwood, Claire J; Ratcliffe, Laura E; Morgan, Sarah; Heath, Paul R; Shaw, Pamela J; Wharton, Stephen B; Simpson, Julie E

    2015-11-16

    The accumulation of reactive oxygen species leading to oxidative damage and cell death plays an important role in a number of neurodegenerative disorders. FOXO3a, the main isoform of FOXO transcription factors, mediates the cellular response to oxidative stress by regulating the expression of genes involved in DNA repair and glutamine metabolism, including glutamine synthetase (GS). Immunohistochemical investigation of the population-based neuropathology cohort of the Medical Research Council's Cognitive Function and Ageing Study (MRC CFAS) demonstrates that nuclear retention of FOXO3a significantly correlates with a DNA damage response and with GS expression by astrocytes. Furthermore, we show that GS expression correlates with increasing Alzheimer-type pathology in this ageing cohort. Our findings suggest that in response to oxidative stress, the nuclear retention of FOXO3a in astrocytes upregulates expression of GS as a neuroprotective mechanism. However, the activity of GS may be compromised by increasing levels of oxidative stress in the ageing brain resulting in dysfunctional enzyme activity, neuronal excitotoxic damage and cognitive impairment.

  2. The nuclear retention of transcription factor FOXO3a correlates with a DNA damage response and increased glutamine synthetase expression by astrocytes suggesting a neuroprotective role in the ageing brain

    PubMed Central

    Fluteau, Adeline; Ince, Paul G.; Minett, Thais; Matthews, Fiona E.; Brayne, Carol; Garwood, Claire J.; Ratcliffe, Laura E.; Morgan, Sarah; Heath, Paul R.; Shaw, Pamela J.; Wharton, Stephen B.; Simpson, Julie E.

    2015-01-01

    The accumulation of reactive oxygen species leading to oxidative damage and cell death plays an important role in a number of neurodegenerative disorders. FOXO3a, the main isoform of FOXO transcription factors, mediates the cellular response to oxidative stress by regulating the expression of genes involved in DNA repair and glutamine metabolism, including glutamine synthetase (GS). Immunohistochemical investigation of the population-based neuropathology cohort of the Medical Research Council’s Cognitive Function and Ageing Study (MRC CFAS) demonstrates that nuclear retention of FOXO3a significantly correlates with a DNA damage response and with GS expression by astrocytes. Furthermore, we show that GS expression correlates with increasing Alzheimer-type pathology in this ageing cohort. Our findings suggest that in response to oxidative stress, the nuclear retention of FOXO3a in astrocytes upregulates expression of GS as a neuroprotective mechanism. However, the activity of GS may be compromised by increasing levels of oxidative stress in the ageing brain resulting in dysfunctional enzyme activity, neuronal excitotoxic damage and cognitive impairment. PMID:26455863

  3. Lithium-induced neuroprotection in stroke involves increased miR-124 expression, reduced RE1-silencing transcription factor abundance and decreased protein deubiquitination by GSK3β inhibition-independent pathways.

    PubMed

    Doeppner, Thorsten R; Kaltwasser, Britta; Sanchez-Mendoza, Eduardo H; Caglayan, Ahmet B; Bähr, Mathias; Hermann, Dirk M

    2017-03-01

    Lithium promotes acute poststroke neuronal survival, which includes mechanisms that are not limited to GSK3β inhibition. However, whether lithium induces long-term neuroprotection and enhanced brain remodeling is unclear. Therefore, mice were exposed to transient middle cerebral artery occlusion and lithium (1 mg/kg bolus followed by 2 mg/kg/day over up to 7 days) was intraperitoneally administered starting 0-9 h after reperfusion onset. Delivery of lithium no later than 6 h reduced infarct volume on day 2 and decreased brain edema, leukocyte infiltration, and microglial activation, as shown by histochemistry and flow cytometry. Lithium-induced neuroprotection persisted throughout the observation period of 56 days and was associated with enhanced neurological recovery. Poststroke angioneurogenesis and axonal plasticity were also enhanced by lithium. On the molecular level, lithium increased miR-124 expression, reduced RE1-silencing transcription factor abundance, and decreased protein deubiquitination in cultivated cortical neurons exposed to oxygen-glucose deprivation and in brains of mice exposed to cerebral ischemia. Notably, this effect was not mimicked by pharmacological GSK3β inhibition. This study for the first time provides efficacy data for lithium in the postacute ischemic phase, reporting a novel mechanism of action, i.e. increased miR-124 expression facilitating REST degradation by which lithium promotes postischemic neuroplasticity and angiogenesis.

  4. Expression pattern of transcription factors and intracellular cytokines reveals that clinically cured tuberculosis is accompanied by an increase in Mycobacterium-specific Th1, Th2, and Th17 cells.

    PubMed

    da Silva, Marcos V; Massaro Junior, Vladimir J; Machado, Juliana R; Silva, Djalma A A; Castellano, Lúcio R; Alexandre, Patricia B D; Rodrigues, Denise B R; Rodrigues, Virmondes

    2015-01-01

    Tuberculosis (TB) remains a major global health problem and is the second biggest cause of death by infectious disease worldwide. Here, we investigate in vitro the Th1, Th2, Th17, and Treg cytokines and transcriptional factors produced after Mycobacterium-specific antigen stimulation in patients with active pulmonary tuberculosis, clinically cured pulmonary tuberculosis, and healthy donors with a positive tuberculin skin test (TST+). Together, our data indicate that clinical cure after treatment increases the percentages of Mycobacterium-specific Th1, Th2, and Th17 cells compared with those found in active-TB and TST+ healthy donors. These results show that the host-parasite equilibrium in latent TB breaks in favor of the microorganism and that the subsequent clinical recovery posttreatment does not return the percentage levels of such cells to those observed in latent tuberculosis. Additionally, our results indicate that rather than showing an increase in the percentage of Mycobacterium-specific Tregs, active-TB patients display lower Th1 : Treg and Th17 : Treg ratios. These data, together with lower Th1 : Th2 and Th17 : Th2 ratios, may indicate a mechanism by which the breakdown of the host-parasite equilibrium leads to active-TB and changes in the repertoire of Mycobacterium-specific Th cells that are associated with clinical cure after treatment of pulmonary tuberculosis.

  5. TBX21-1993T/C (rs4794067) polymorphism is associated with increased risk of chronic periodontitis and increased T-bet expression in periodontal lesions, but does not significantly impact the IFN-g transcriptional level or the pattern of periodontophatic bacterial infection

    PubMed Central

    Cavalla, Franco; Biguetti, Claudia Cristina; Colavite, Priscila Maria; Silveira, Elcia Varise; Martins, Walter; Letra, Ariadne; Trombone, Ana Paula Favaro; Silva, Renato Menezes; Garlet, Gustavo Pompermaier

    2015-01-01

    Th1-polarized host response, mediated by IFN-γ, has been associated with increased severity of periodontal disease as well as control of periodontal infection. The functional polymorphism TBX21-1993T/C (rs4794067) increases the transcriptional activity of the TBX21 gene (essential for Th1 polarization) resulting in a predisposition to a Th-1 biased immune response. Thus, we conducted a case-control study, including a population of healthy controls (H, n = 218), chronic periodontitis (CP, n = 197), and chronic gingivitis patients (CG, n = 193), to investigate if genetic variations in TBX21 could impact the development of Th1 responses, and consequently influence the pattern of bacterial infection and periodontitis outcome. We observed that the polymorphic allele T was significantly enriched in the CP patients compared to CG subjects, while the H controls demonstrated and intermediate genotype. Also, investigating the putative functionality TBX21-1993T/C in the modulation of local response, we observed that the transcripts levels of T-bet, but not of IFN-γ, were upregulated in homozygote and heterozygote polymorphic subjects. In addition, TBX21-1993T/C did not influence the pattern of bacterial infection or the clinical parameters of disease severity, being the presence/absence of red complex bacteria the main factor associated with the disease status and the subrogate variable probing depth (PD) in the logistic regression analysis. PMID:25832120

  6. Herpes Simplex Virus Type 1 Serum Neutralizing Antibody Titers Increase during Latency in Rabbits Latently Infected with Latency-Associated Transcript (LAT)-Positive but Not LAT-Negative Viruses

    PubMed Central

    Perng, Guey-Chuen; Slanina, Susan M.; Yukht, Ada; Ghiasi, Homayon; Nesburn, Anthony B.; Wechsler, Steven L.

    1999-01-01

    The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation in the rabbit ocular model of HSV-1 latency and reactivation. LAT is also the only viral gene abundantly expressed during latency. Rabbits were ocularly infected with the wild-type HSV-1 strain McKrae or the McKrae-derived LAT null mutant dLAT2903. Serum neutralizing antibody titers were determined at various times during acute and latent infection. The neutralizing antibody titers induced by both viruses increased and were similar throughout the first 45 days after infection (P > 0.05). However, by day 59 postinfection (approximately 31 to 45 days after latency had been established), the neutralizing antibody titers induced by wild-type virus and dLAT2903 diverged significantly (P = 0.0005). The dLAT2903-induced neutralizing antibody titers decreased, while the wild-type virus-induced neutralizing antibody titers continued to increase. A rescuant of dLAT2903, in which spontaneous reactivation was fully restored, induced wild-type neutralizing antibody levels on day 59 postinfection. A second LAT mutant with impaired spontaneous reactivation had neutralizing antibody levels comparable to those of dLAT2903. In contrast to the results obtained in rabbits, in mice, neutralizing antibody titers did not increase over time during latency with any of the viruses. Since LAT is expressed in both rabbits and mice during latency, the difference in neutralizing antibody titers between these animals is unlikely to be due to expression of a LAT protein during latency. In contrast, LAT-positive (LAT+), but not LAT-negative (LAT−), viruses undergo efficient spontaneous reactivation in rabbits, while neither LAT+ nor LAT− viruses undergo efficient spontaneous reactivation in mice. Thus, the increase in neutralizing antibody titers in rabbits latently infected with LAT+ viruses may have been due to continued restimulation of the immune system by

  7. Herpes simplex virus type 1 serum neutralizing antibody titers increase during latency in rabbits latently infected with latency-associated transcript (LAT)-positive but not LAT-negative viruses.

    PubMed

    Perng, G C; Slanina, S M; Yukht, A; Ghiasi, H; Nesburn, A B; Wechsler, S L

    1999-11-01

    The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation in the rabbit ocular model of HSV-1 latency and reactivation. LAT is also the only viral gene abundantly expressed during latency. Rabbits were ocularly infected with the wild-type HSV-1 strain McKrae or the McKrae-derived LAT null mutant dLAT2903. Serum neutralizing antibody titers were determined at various times during acute and latent infection. The neutralizing antibody titers induced by both viruses increased and were similar throughout the first 45 days after infection (P > 0.05). However, by day 59 postinfection (approximately 31 to 45 days after latency had been established), the neutralizing antibody titers induced by wild-type virus and dLAT2903 diverged significantly (P = 0.0005). The dLAT2903-induced neutralizing antibody titers decreased, while the wild-type virus-induced neutralizing antibody titers continued to increase. A rescuant of dLAT2903, in which spontaneous reactivation was fully restored, induced wild-type neutralizing antibody levels on day 59 postinfection. A second LAT mutant with impaired spontaneous reactivation had neutralizing antibody levels comparable to those of dLAT2903. In contrast to the results obtained in rabbits, in mice, neutralizing antibody titers did not increase over time during latency with any of the viruses. Since LAT is expressed in both rabbits and mice during latency, the difference in neutralizing antibody titers between these animals is unlikely to be due to expression of a LAT protein during latency. In contrast, LAT-positive (LAT(+)), but not LAT-negative (LAT(-)), viruses undergo efficient spontaneous reactivation in rabbits, while neither LAT(+) nor LAT(-) viruses undergo efficient spontaneous reactivation in mice. Thus, the increase in neutralizing antibody titers in rabbits latently infected with LAT(+) viruses may have been due to continued restimulation of the immune system

  8. Antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing

    PubMed Central

    Dang, Yunkun; Cheng, Jiasen; Sun, Xianyun; Zhou, Zhipeng; Liu, Yi

    2016-01-01

    In eukaryotes, antisense transcription can regulate sense transcription by induction of epigenetic modifications. We showed previously that antisense transcription triggers Dicer-independent siRNA (disiRNA) production and disiRNA locus DNA methylation (DLDM) in Neurospora crassa. Here we show that the conserved exonuclease ERI-1 (enhanced RNAi-1) is a critical component in this process. Antisense transcription and ERI-1 binding to target RNAs are necessary and sufficient to trigger DLDM. Convergent transcription causes stalling of RNA polymerase II during transcription, which permits ERI-1 to bind nascent RNAs in the nucleus and recruit a histone methyltransferase complex that catalyzes chromatin modifications. Furthermore, we show that, in the cytoplasm, ERI-1 targets hundreds of transcripts from loci without antisense transcription to regulate RNA stability. Together, our results demonstrate a critical role for transcription kinetics in long noncoding RNA-mediated epigenetic modifications and identify ERI-1 as an important regulator of cotranscriptional gene silencing and post-transcriptional RNA metabolism. PMID:27856616

  9. Benzimidazoles diminish ERE transcriptional activity and cell growth in breast cancer cells

    SciTech Connect

    Payton-Stewart, Florastina; Tilghman, Syreeta L.; Williams, LaKeisha G.; Winfield, Leyte L.

    2014-08-08

    Highlights: • The methyl-substituted benzimidazole was more effective at inhibiting growth in MDA-MB 231 cells. • The naphthyl-substituted benzimidazole was more effective at inhibiting growth in MCF-7 cells than ICI. • The benzimidazole molecules demonstrated a dose-dependent reduction in ERE transcriptional activity. • The benzimidazole molecules had binding mode in ERα and ERβ comparable to that of the co-crystallized ligand. - Abstract: Estrogen receptors (ERα and ERβ) are members of the nuclear receptor superfamily. They regulate the transcription of estrogen-responsive genes and mediate numerous estrogen related diseases (i.e., fertility, osteoporosis, cancer, etc.). As such, ERs are potentially useful targets for developing therapies and diagnostic tools for hormonally responsive human breast cancers. In this work, two benzimidazole-based sulfonamides originally designed to reduce proliferation in prostate cancer, have been evaluated for their ability to modulate growth in estrogen dependent and independent cell lines (MCF-7 and MDA-MB 231) using cell viability assays. The molecules reduced growth in MCF-7 cells, but differed in their impact on the growth of MDA-MB 231 cells. Although both molecules reduced estrogen response element (ERE) transcriptional activity in a dose dependent manner, the contrasting activity in the MDA-MB-231 cells seems to suggest that the molecules may act through alternate ER-mediated pathways. Further, the methyl analog showed modest selectivity for the ERβ receptor in an ER gene expression array panel, while the naphthyl analog did not significantly alter gene expression. The molecules were docked in the ligand binding domains of the ERα-antagonist and ERβ-agonist crystal structures to evaluate the potential of the molecules to interact with the receptors. The computational analysis complimented the results obtained in the assay of transcriptional activity and gene expression suggesting that the molecules

  10. Increased levels of the T-helper 22-associated cytokine (interleukin-22) and transcription factor (aryl hydrocarbon receptor) in patients with periodontitis are associated with osteoclast resorptive activity and severity of the disease.

    PubMed

    Díaz-Zúñiga, J; Melgar-Rodríguez, S; Rojas, L; Alvarez, C; Monasterio, G; Carvajal, P; Vernal, R

    2017-10-01

    Two new T-helper (Th) phenotypes have been recently described and named Th9 and Th22 lymphocytes; however, their role in the pathogenesis of periodontitis remains unclear. This study was aimed to assess whether Th9 and Th22 lymphocytes, through interleukin (IL)-9 and IL-22 production, respectively, are associated with the severity of periodontitis and bone resorption. Gingival crevicular fluid samples and biopsies were obtained from patients with moderate-to-advanced chronic periodontitis and gingivitis, and healthy controls. The levels for the Th9 and Th22-associated cytokines and master-switch transcription factors Spi-B and aryl hydrocarbon receptor (AhR) were quantified by enzyme-linked immunosorbent assay, real-time reverse-transcription quantitative polymerase chain reaction and flow cytometry. In addition, the osteoclast activity in response to tissue homogenates from periodontitis and healthy samples was analyzed quantifying the number of TRAP-positive cells and areas of bone resorption pits produced, in the presence or absence of recombinant human IL-22 and anti-IL-22 neutralization antibody. Higher levels of IL-22 and AhR were detected in patients with periodontitis compared with gingivitis and healthy individuals. In addition, higher levels of IL-9 and Spi-B were detected in gingivitis patients compared with periodontitis and healthy individuals. In patients with periodontitis, a significant positive correlation was detected between secreted levels of IL-22 and clinical attachment level of the sampled periodontal pockets. When osteoclasts were exposed to tissue homogenates obtained from patients with periodontitis, higher levels of resorptive activity were observed as compared with the same cells exposed to tissue homogenates obtained from healthy individuals, and this increment was dependent on the presence and neutralization of IL-22. Increased levels of IL-22 produced by Th22 lymphocytes are associated with the pathogenesis of periodontitis, in

  11. Overexpression of Grain Amaranth (Amaranthus hypochondriacus) AhERF or AhDOF Transcription Factors in Arabidopsis thaliana Increases Water Deficit- and Salt-Stress Tolerance, Respectively, via Contrasting Stress-Amelioration Mechanisms

    PubMed Central

    Massange-Sánchez, Julio A.; Palmeros-Suárez, Paola A.; Espitia-Rangel, Eduardo; Rodríguez-Arévalo, Isaac; Sánchez-Segura, Lino; Martínez-Gallardo, Norma A.; Alatorre-Cobos, Fulgencio; Tiessen, Axel; Délano-Frier, John P.

    2016-01-01

    Two grain amaranth transcription factor (TF) genes were overexpressed in Arabidopsis plants. The first, coding for a group VII ethylene response factor TF (i.e., AhERF-VII) conferred tolerance to water-deficit stress (WS) in transgenic Arabidopsis without affecting vegetative or reproductive growth. A significantly lower water-loss rate in detached leaves coupled to a reduced stomatal opening in leaves of plants subjected to WS was associated with this trait. WS tolerance was also associated with an increased antioxidant enzyme activity and the accumulation of putative stress-related secondary metabolites. However, microarray and GO data did not indicate an obvious correlation between WS tolerance, stomatal closure, and abscisic acid (ABA)-related signaling. This scenario suggested that stomatal closure during WS in these plants involved ABA-independent mechanisms, possibly involving reactive oxygen species (ROS). WS tolerance may have also involved other protective processes, such as those employed for methyl glyoxal detoxification. The second, coding for a class A and cluster I DNA binding with one finger TF (i.e., AhDof-AI) provided salt-stress (SS) tolerance with no evident fitness penalties. The lack of an obvious development-related phenotype contrasted with microarray and GO data showing an enrichment of categories and genes related to developmental processes, particularly flowering. SS tolerance also correlated with increased superoxide dismutase activity but not with augmented stomatal closure. Additionally, microarray and GO data indicated that, contrary to AhERF-VII, SS tolerance conferred by AhDof-AI in Arabidopsis involved ABA-dependent and ABA-independent stress amelioration mechanisms. PMID:27749893

  12. Association of MMP7 -181A→G Promoter Polymorphism with Gastric Cancer Risk: INFLUENCE OF NICOTINE IN DIFFERENTIAL ALLELE-SPECIFIC TRANSCRIPTION VIA INCREASED PHOSPHORYLATION OF cAMP-RESPONSE ELEMENT-BINDING PROTEIN (CREB).

    PubMed

    Kesh, Kousik; Subramanian, Lakshmi; Ghosh, Nillu; Gupta, Vinayak; Gupta, Arnab; Bhattacharya, Samir; Mahapatra, Nitish R; Swarnakar, Snehasikta

    2015-06-05

    Elevated expression of matrix metalloproteinase7 (MMP7) has been demonstrated to play a pivotal role in cancer invasion. The -181A→G (rs11568818) polymorphism in the MMP7 promoter modulates gene expression and possibly affects cancer progression. Here, we evaluated the impact of -181A→G polymorphism on MMP7 promoter activity and its association with gastric cancer risk in eastern Indian case-control cohorts (n = 520). The GG genotype as compared with the AA genotype was predisposed (p = 0.02; odds ratio = 1.9, 95% confidence interval = 1.1-3.3) to gastric cancer risk. Stratification analysis showed that tobacco addiction enhanced gastric cancer risk in GG subjects when compared with AA subjects (p = 0.03, odds ratio = 2.46, and 95% confidence interval = 1.07-5.68). Meta-analysis revealed that tobacco enhanced the risk for cancer more markedly in AG and GG carriers. Activity and expression of MMP7 were significantly higher in GG than in AA carriers. In support, MMP7 promoter-reporter assays showed greater transcriptional activity toward A to G transition under basal/nicotine-induced/cAMP-response element-binding protein (CREB) overexpressed conditions in gastric adenocarcinoma cells. Moreover, nicotine (a major component of tobacco) treatment significantly up-regulated MMP7 expression due to enhanced CREB phosphorylation followed by its nuclear translocation in gastric adenocarcinoma cells. Furthermore, chromatin immunoprecipitation experiments revealed higher binding of phosphorylated CREB with the -181G than the -181A allele. Altogether, specific binding of phosphorylated CREB to the G allele-carrying promoter enhances MMP7 gene expression that is further augmented by nicotine due to increased CREB phosphorylation and thereby increases the risk for gastric cancer.

  13. AthaMap, integrating transcriptional and post-transcriptional data

    PubMed Central

    Bülow, Lorenz; Engelmann, Stefan; Schindler, Martin; Hehl, Reinhard

    2009-01-01

    The AthaMap database generates a map of predicted transcription factor binding sites (TFBS) for the whole Arabidopsis thaliana genome. AthaMap has now been extended to include data on post-transcriptional regulation. A total of 403 173 genomic positions of small RNAs have been mapped in the A. thaliana genome. These identify 5772 putative post-transcriptionally regulated target genes. AthaMap tools have been modified to improve the identification of common TFBS in co-regulated genes by subtracting post-transcriptionally regulated genes from such analyses. Furthermore, AthaMap was updated to the TAIR7 genome annotation, a graphic display of gene analysis results was implemented, and the TFBS data content was increased. AthaMap is freely available at http://www.athamap.de/. PMID:18842622

  14. Control of Transcriptional Elongation

    PubMed Central

    Kwak, Hojoong; Lis, John T.

    2014-01-01

    Elongation is becoming increasingly recognized as a critically controlled step in transcriptional regulation. While traditional genetic and biochemical studies have identified major players of transcriptional elongation, our understanding of the importance and roles of these factors is evolving rapidly through the recent advances in genome-wide and single-molecule technologies. Here we focus on how elongation can modulate the transcriptional outcome through the rate-liming step of RNA polymerase II pausing near promoters, and how the participating factors were identified. Among the factors we describe are NELF and DSIF, the pausing factors, and P-TEFb, the key player in pause release. We also describe non-exclusive models for how pausing is achieved by making use of high resolution genome-wide mapping of paused Pol II relative to promoter elements and the first nucleosome. We also discuss Pol II elongation through the bodies of genes and the roles of FACT and Spt6, the factors that allow Pol II to move through nucleosomes. PMID:24050178

  15. Antagonistic regulation of growth and immunity by the Arabidopsis basic helix-loop-helix transcription factor homolog of brassinosteroid enhanced expression2 interacting with increased leaf inclination1 binding bHLH1.

    PubMed

    Malinovsky, Frederikke Gro; Batoux, Martine; Schwessinger, Benjamin; Youn, Ji Hyun; Stransfeld, Lena; Win, Joe; Kim, Seong-Ki; Zipfel, Cyril

    2014-03-01

    Plants need to finely balance resources allocated to growth and immunity to achieve optimal fitness. A tradeoff between pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and brassinosteroid (BR)-mediated growth was recently reported, but more information about the underlying mechanisms is needed. Here, we identify the basic helix-loop-helix (bHLH) transcription factor homolog of brassinosteroid enhanced expression2 interacting with IBH1 (HBI1) as a negative regulator of PTI signaling in Arabidopsis (Arabidopsis thaliana). HBI1 expression is down-regulated in response to different PAMPs. HBI1 overexpression leads to reduced PAMP-triggered responses. This inhibition correlates with reduced steady-state expression of immune marker genes, leading to increased susceptibility to the bacterium Pseudomonas syringae. Overexpression of the HBI1-related bHLHs brassinosteroid enhanced expression2 (BEE2) and cryptochrome-interacting bHLH (CIB1) partially inhibits immunity, indicating that BEE2 and CIB1 may act redundantly with HBI1. In contrast to its expression pattern upon PAMP treatment, HBI1 expression is enhanced by BR treatment. Also, HBI1-overexpressing plants are hyperresponsive to BR and more resistant to the BR biosynthetic inhibitor brassinazole. HBI1 is nucleus localized, and a mutation in a conserved leucine residue within the first helix of the protein interaction domain impairs its function in BR signaling. Interestingly, HBI1 interacts with several inhibitory atypical bHLHs, which likely keep HBI1 under negative control. Hence, HBI1 is a positive regulator of BR-triggered responses, and the negative effect of PTI is likely due to the antagonism between BR and PTI signaling. This study identifies a novel component involved in the complex tradeoff between innate immunity and BR-regulated growth.

  16. Activation of Epithelial Signal Transducer and Activator of Transcription 1 by Interleukin 28 Controls Mucosal Healing in Mice With Colitis and Is Increased in Mucosa of Patients With Inflammatory Bowel Disease.

    PubMed

    Chiriac, Mircea T; Buchen, Barbara; Wandersee, Alexandra; Hundorfean, Gheorghe; Günther, Claudia; Bourjau, Yvonne; Doyle, Sean E; Frey, Benjamin; Ekici, Arif B; Büttner, Christian; Weigmann, Benno; Atreya, Raja; Wirtz, Stefan; Becker, Christoph; Siebler, Jürgen; Neurath, Markus F

    2017-07-01

    We investigated the roles of interleukin 28A (also called IL28A or interferon λ2) in intestinal epithelial cell (IEC) activation, studying its effects in mouse models of inflammatory bowel diseases (IBD) and intestinal mucosal healing. Colitis was induced in C57BL/6JCrl mice (controls), mice with IEC-specific disruption of Stat1 (Stat1IEC-KO), mice with disruption of the interferon λ receptor 1 gene (Il28ra(-/-)), and mice with disruption of the interferon regulatory factor 3 gene (Irf3(-/-)), with or without disruption of Irf7 (Irf7(-/-)). We used high-resolution mini-endoscopy and in vivo imaging methods to assess colitis progression. We used 3-dimensional small intestine and colon organoids, along with RNA-Seq and gene ontology methods, to characterize the effects of IL28 on primary IECs. We studied the effects of IL28 on the human intestinal cancer cell line Caco-2 in a wound-healing assay, and in mice colon wounds. Colonic biopsies and resected tissue from patients with IBD (n = 62) and patients without colon inflammation (controls, n = 23) were analyzed by quantitative polymerase chain rection to measure expression of IL28A, IL28RA, and other related cytokines; biopsy samples were also analyzed by immunofluorescence to identify sources of IL28 production. IECs were isolated from patient tissues and incubated with IL28; signal transducer and activator of transcription 1 (STAT1) phosphorylation was measured by immunoblots and confocal imaging. Lamina propria cells in colon tissues of patients with IBD, and mice with colitis, had increased expression of IL28 compared with controls; levels of IL28R were increased in the colonic epithelium of patients with IBD and mice with colitis. Administration of IL28 induced phosphorylation of STAT1 in primary human and mouse IECs, increasing with dose. Il28ra(-/-), Irf3(-/-), Irf3(-/-)Irf7(-/-), as well as Stat1IEC-KO mice, developed more severe colitis after administration of dextran sulfate sodium than control mice

  17. Chromatin potentiates transcription

    PubMed Central

    Nagai, Shigeki; Davis, Ralph E.; Mattei, Pierre Jean; Eagen, Kyle Patrick; Kornberg, Roger D.

    2017-01-01

    Chromatin isolated from the chromosomal locus of the PHO5 gene of yeast in a transcriptionally repressed state was transcribed with 12 pure proteins (80 polypeptides): RNA polymerase II, six general transcription factors, TFIIS, the Pho4 gene activator protein, and the SAGA, SWI/SNF, and Mediator complexes. Contrary to expectation, a nucleosome occluding the TATA box and transcription start sites did not impede transcription but rather, enhanced it: the level of chromatin transcription was at least sevenfold greater than that of naked DNA, and chromatin gave patterns of transcription start sites closely similar to those occurring in vivo, whereas naked DNA gave many aberrant transcripts. Both histone acetylation and trimethylation of H3K4 (H3K4me3) were important for chromatin transcription. The nucleosome, long known to serve as a general gene repressor, thus also performs an important positive role in transcription. PMID:28137832

  18. The Absence of the Transcription Factor Yrr1p, Identified from Comparative Genome Profiling, Increased Vanillin Tolerance Due to Enhancements of ABC Transporters Expressing, rRNA Processing and Ribosome Biogenesis in Saccharomyces cerevisiae

    PubMed Central

    Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Shen, Yu; Bao, Xiaoming

    2017-01-01

    Enhancing the tolerance of Saccharomyces cerevisiae to inhibitors derived from lignocellulose is conducive to producing biofuel and chemicals using abundant lignocellulosic materials. Vanillin is a major type of phenolic inhibitor in lignocellulose hydrolysates for S. cerevisiae. In the present work, the factors beneficial to vanillin resistance in yeast were identified from the vanillin-resistant strain EMV-8, which was derived from strain NAN-27 by adaptive evolution. We found 450 SNPs and 44 genes with InDels in the vanillin-tolerant strain EMV-8 by comparing the genome sequences of EMV-8 and NAN-27. To investigate the effects of InDels, InDels were deleted in BY4741, respectively. We demonstrated that the deletion of YRR1 improved vanillin tolerance of strain. In the presence of 6 mM vanillin, deleting YRR1 increase the maximum specific growth rate and the vanillin consumption rate by 142 and 51%, respectively. The subsequent transcriptome analysis revealed that deleting YRR1 resulted in changed expression of over 200 genes in the presence of 5 mM vanillin. The most marked changes were the significant up-regulation of the dehydrogenase ADH7, several ATP-binding cassette (ABC) transporters, and dozens of genes involved in ribosome biogenesis and rRNA processing. Coincidently, the crude enzyme solution of BY4741(yrr1Δ) exhibited higher NADPH-dependent vanillin reduction activity than control. In addition, overexpressing the ABC transporter genes PDR5, YOR1, and SNQ2, as well as the RNA helicase gene DBP2, increased the vanillin tolerance of strain. Interestingly, unlike the marked changes we mentioned above, under vanillin-free conditions, there are only limited transcriptional differences between wildtype and yrr1Δ. This indicated that vanillin might act as an effector in Yrr1p-related regulatory processes. The new findings of the relationship between YRR1 and vanillin tolerance, as well as the contribution of rRNA processing and ribosome biogenesis to

  19. Isolation and characterization of transcription fidelity mutants.

    PubMed

    Strathern, Jeffrey N; Jin, Ding Jun; Court, Donald L; Kashlev, Mikhail

    2012-07-01

    Accurate transcription is an essential step in maintaining genetic information. Error-prone transcription has been proposed to contribute to cancer, aging, adaptive mutagenesis, and mutagenic evolution of retroviruses and retrotransposons. The mechanisms controlling transcription fidelity and the biological consequences of transcription errors are poorly understood. Because of the transient nature of mRNAs and the lack of reliable experimental systems, the identification and characterization of defects that increase transcription errors have been particularly challenging. In this review we describe novel genetic screens for the isolation of fidelity mutants in both Saccharomyces cerevisiae and Escherichia coli RNA polymerases. We obtained and characterized two distinct classes of mutants altering NTP misincorporation and transcription slippage both in vivo and in vitro. Our study not only validates the genetic schemes for the isolation of RNA polymerase mutants that alter fidelity, but also sheds light on the mechanism of transcription accuracy. This article is part of a Special Issue entitled: Chromatin in time and space.

  20. Effects of elongation delay in transcription dynamics.

    PubMed

    Zhang, Xuan; Jin, Huiqin; Yang, Zhuoqin; Lei, Jinzhi

    2014-12-01

    In the transcription process, elongation delay is induced by the movement of RNA polymerases (RNAP) along the DNA sequence, and can result in changes in the transcription dynamics. This paper studies the transcription dynamics that involved the elongation delay and effects of cell division and DNA replication. The stochastic process of gene expression is modeled with delay chemical master equation with periodic coefficients, and is studied numerically through the stochastic simulation algorithm with delay. We show that the average transcription level approaches to a periodic dynamics over cell cycles at homeostasis, and the elongation delay can reduce the transcription level and increase the transcription noise. Moreover, the transcription elongation can induce bimodal distribution of mRNA levels that can be measured by the techniques of flow cytometry.

  1. Transcriptional enhancers: Transcription, function and flexibility.

    PubMed

    Melamed, Philippa; Yosefzon, Yahav; Rudnizky, Sergei; Pnueli, Lilach

    2016-01-01

    Active transcriptional enhancers are often transcribed to eRNAs, whose changing levels mirror those of the target gene mRNA. We discuss some of the reported functions of these eRNAs and their likely diversity to allow utilization of distinct cis regulatory regions to enhance transcription in diverse developmental and cellular contexts.

  2. A Forum To Expand Advanced Placement Opportunities: Increasing Access and Improving Preparation in High Schools. Strategies To Overcome Challenges in Rural and Small Schools. Transcript of Proceedings (Washington, D.C., February 11, 2000).

    ERIC Educational Resources Information Center

    Department of Education, Washington, DC.

    This transcript reproduces a Department of Education/College Board sponsored discussion on ways to expand advanced placement (AP) opportunities in high schools. The deliberations opened with a presentation by Phil Chavez, an assistant principal in San Antonio, Texas, who outlined the genesis and development of the AP program in his predominantly…

  3. Transcription in archaea

    NASA Technical Reports Server (NTRS)

    Kyrpides, N. C.; Ouzounis, C. A.; Woese, C. R. (Principal Investigator)

    1999-01-01

    Using the sequences of all the known transcription-associated proteins from Bacteria and Eucarya (a total of 4,147), we have identified their homologous counterparts in the four complete archaeal genomes. Through extensive sequence comparisons, we establish the presence of 280 predicted transcription factors or transcription-associated proteins in the four archaeal genomes, of which 168 have homologs only in Bacteria, 51 have homologs only in Eucarya, and the remaining 61 have homologs in both phylogenetic domains. Although bacterial and eukaryotic transcription have very few factors in common, each exclusively shares a significantly greater number with the Archaea, especially the Bacteria. This last fact contrasts with the obvious close relationship between the archaeal and eukaryotic transcription mechanisms per se, and in particular, basic transcription initiation. We interpret these results to mean that the archaeal transcription system has retained more ancestral characteristics than have the transcription mechanisms in either of the other two domains.

  4. Transcription in archaea

    NASA Technical Reports Server (NTRS)

    Kyrpides, N. C.; Ouzounis, C. A.; Woese, C. R. (Principal Investigator)

    1999-01-01

    Using the sequences of all the known transcription-associated proteins from Bacteria and Eucarya (a total of 4,147), we have identified their homologous counterparts in the four complete archaeal genomes. Through extensive sequence comparisons, we establish the presence of 280 predicted transcription factors or transcription-associated proteins in the four archaeal genomes, of which 168 have homologs only in Bacteria, 51 have homologs only in Eucarya, and the remaining 61 have homologs in both phylogenetic domains. Although bacterial and eukaryotic transcription have very few factors in common, each exclusively shares a significantly greater number with the Archaea, especially the Bacteria. This last fact contrasts with the obvious close relationship between the archaeal and eukaryotic transcription mechanisms per se, and in particular, basic transcription initiation. We interpret these results to mean that the archaeal transcription system has retained more ancestral characteristics than have the transcription mechanisms in either of the other two domains.

  5. Widespread Inducible Transcription Downstream of Human Genes

    PubMed Central

    Vilborg, Anna; Passarelli, Maria C.; Yario, Therese A.; Tycowski, Kazimierz T.; Steitz, Joan A.

    2015-01-01

    Summary Pervasive transcription of the human genome generates RNAs whose mode of formation and functions are largely uncharacterized. Here, we combine RNA-Seq with detailed mechanistic studies to describe a transcript type derived from protein-coding genes. The resulting RNAs, which we call DoGs for downstream of gene containing transcripts, possess long non-coding regions (often >45 kb) and remain chromatin bound. DoGs are inducible by osmotic stress through an IP3 receptor signaling-dependent pathway, indicating active regulation. DoG levels are increased by decreased termination of the upstream transcript, a previously undescribed mechanism for rapid transcript induction. Relative depletion of polyA signals in DoG regions correlates with increased levels of DoGs after osmotic stress. We detect DoG transcription in several human cell lines and provide evidence for thousands of DoGs genome-wide. PMID:26190259

  6. Nuclear transport and transcription.

    PubMed

    Komeili, A; O'Shea, E K

    2000-06-01

    The compartmentalization of DNA in the nucleus of eukaryotic cells establishes a connection between the nuclear transport machinery and the transcriptional apparatus. General transcription factors, as well as specific transcriptional activators and repressors, such as p53 and NF-AT, need to be imported into the nucleus following their translation. In addition, nuclear transport plays a crucial role in regulating the activity of many transcription factors.

  7. WRKY transcription factors.

    PubMed

    Rushton, Paul J; Somssich, Imre E; Ringler, Patricia; Shen, Qingxi J

    2010-05-01

    WRKY transcription factors are one of the largest families of transcriptional regulators in plants and form integral parts of signalling webs that modulate many plant processes. Here, we review recent significant progress in WRKY transcription factor research. New findings illustrate that WRKY proteins often act as repressors as well as activators, and that members of the family play roles in both the repression and de-repression of important plant processes. Furthermore, it is becoming clear that a single WRKY transcription factor might be involved in regulating several seemingly disparate processes. Mechanisms of signalling and transcriptional regulation are being dissected, uncovering WRKY protein functions via interactions with a diverse array of protein partners, including MAP kinases, MAP kinase kinases, 14-3-3 proteins, calmodulin, histone deacetylases, resistance proteins and other WRKY transcription factors. WRKY genes exhibit extensive autoregulation and cross-regulation that facilitates transcriptional reprogramming in a dynamic web with built-in redundancy. 2010 Elsevier Ltd. All rights reserved.

  8. Transcription and recombination: when RNA meets DNA.

    PubMed

    Aguilera, Andrés; Gaillard, Hélène

    2014-08-01

    A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription-replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  9. Theoretical analysis of transcription process with polymerase stalling

    NASA Astrophysics Data System (ADS)

    Li, Jingwei; Zhang, Yunxin

    2015-05-01

    Experimental evidence shows that in gene transcription RNA polymerase has the possibility to be stalled at a certain position of the transcription template. This may be due to the template damage or protein barriers. Once stalled, polymerase may backtrack along the template to the previous nucleotide to wait for the repair of the damaged site, simply bypass the barrier or damaged site and consequently synthesize an incorrect messenger RNA, or degrade and detach from the template. Thus, the effective transcription rate (the rate to synthesize correct product mRNA) and the transcription effectiveness (the ratio of the effective transcription rate to the effective transcription initiation rate) are both influenced by polymerase stalling events. So far, no theoretical model has been given to discuss the gene transcription process including polymerase stalling. In this study, based on the totally asymmetric simple exclusion process, the transcription process including polymerase stalling is analyzed theoretically. The dependence of the effective transcription rate, effective transcription initiation rate, and transcription effectiveness on the transcription initiation rate, termination rate, as well as the backtracking rate, bypass rate, and detachment (degradation) rate when stalling, are discussed in detail. The results showed that backtracking restart after polymerase stalling is an ideal mechanism to increase both the effective transcription rate and the transcription effectiveness. Without backtracking, detachment of stalled polymerase can also help to increase the effective transcription rate and transcription effectiveness. Generally, the increase of the bypass rate of the stalled polymerase will lead to the decrease of the effective transcription rate and transcription effectiveness. However, when both detachment rate and backtracking rate of the stalled polymerase vanish, the effective transcription rate may also be increased by the bypass mechanism.

  10. Theoretical analysis of transcription process with polymerase stalling.

    PubMed

    Li, Jingwei; Zhang, Yunxin

    2015-05-01

    Experimental evidence shows that in gene transcription RNA polymerase has the possibility to be stalled at a certain position of the transcription template. This may be due to the template damage or protein barriers. Once stalled, polymerase may backtrack along the template to the previous nucleotide to wait for the repair of the damaged site, simply bypass the barrier or damaged site and consequently synthesize an incorrect messenger RNA, or degrade and detach from the template. Thus, the effective transcription rate (the rate to synthesize correct product mRNA) and the transcription effectiveness (the ratio of the effective transcription rate to the effective transcription initiation rate) are both influenced by polymerase stalling events. So far, no theoretical model has been given to discuss the gene transcription process including polymerase stalling. In this study, based on the totally asymmetric simple exclusion process, the transcription process including polymerase stalling is analyzed theoretically. The dependence of the effective transcription rate, effective transcription initiation rate, and transcription effectiveness on the transcription initiation rate, termination rate, as well as the backtracking rate, bypass rate, and detachment (degradation) rate when stalling, are discussed in detail. The results showed that backtracking restart after polymerase stalling is an ideal mechanism to increase both the effective transcription rate and the transcription effectiveness. Without backtracking, detachment of stalled polymerase can also help to increase the effective transcription rate and transcription effectiveness. Generally, the increase of the bypass rate of the stalled polymerase will lead to the decrease of the effective transcription rate and transcription effectiveness. However, when both detachment rate and backtracking rate of the stalled polymerase vanish, the effective transcription rate may also be increased by the bypass mechanism.

  11. LaAP2L1, a heterosis-associated AP2/EREBP transcription factor of Larix, increases organ size and final biomass by affecting cell proliferation in Arabidopsis.

    PubMed

    Li, Ai; Zhou, Yanan; Jin, Chuan; Song, Wenqin; Chen, Chengbin; Wang, Chunguo

    2013-11-01

    In Larix and in some crops, heterosis is prevalent and has been widely used in breeding to produce excellent varieties. However, the molecular basis of heterosis in Larix remains ambiguous. LaAP2L1, a member of the AP2/EREBP transcription factor family, has been suggested to be involved in heterosis in Larix hybrids. Here, the function and regulation of LaAP2L1 were further explored. Overexpression of LaAP2L1 led to markedly enlarged organs and heterosis-like traits in Arabidopsis. Fresh weight of leaves was almost twice as great as in vector controls. Likewise, seed yield of 35S::LaAP2L1 individual plants was >200% greater than that of control plants. The enlarged organs and heterosis-like traits displayed by 35S::LaAP2L1 plants were mainly due to enhanced cell proliferation and prolonged growth duration. At the molecular level, LaAP2L1 upregulated the expression of ANT, EBP1, and CycD3;1 and inhibited the expression of ARGOS in 35S::LaAP2L1 plants, suggesting an important molecular role of LaAP2L1 in regulating plant organ development. These findings provide new insights into the formation of heterosis in woody plants and suggest that LaAP2L1 has potential applications in breeding high-yielding crops and energy plants. In addition, 50 AP2/EREBP transcription factors, including LaAP2L1, in Larix were identified by transcriptome sequencing, and phylogenetic analysis was conducted. This provided information that will be important in further revealing the functions of these transcription factors.

  12. Transcriptional Regulation by Competing Transcription Factor Modules

    PubMed Central

    Hermsen, Rutger; Tans, Sander; ten Wolde, Pieter Rein

    2006-01-01

    Gene regulatory networks lie at the heart of cellular computation. In these networks, intracellular and extracellular signals are integrated by transcription factors, which control the expression of transcription units by binding to cis-regulatory regions on the DNA. The designs of both eukaryotic and prokaryotic cis-regulatory regions are usually highly complex. They frequently consist of both repetitive and overlapping transcription factor binding sites. To unravel the design principles of these promoter architectures, we have designed in silico prokaryotic transcriptional logic gates with predefined input–output relations using an evolutionary algorithm. The resulting cis-regulatory designs are often composed of modules that consist of tandem arrays of binding sites to which the transcription factors bind cooperatively. Moreover, these modules often overlap with each other, leading to competition between them. Our analysis thus identifies a new signal integration motif that is based upon the interplay between intramodular cooperativity and intermodular competition. We show that this signal integration mechanism drastically enhances the capacity of cis-regulatory domains to integrate signals. Our results provide a possible explanation for the complexity of promoter architectures and could be used for the rational design of synthetic gene circuits. PMID:17140283

  13. Organization of Transcription

    PubMed Central

    Chakalova, Lyubomira; Fraser, Peter

    2010-01-01

    Investigations into the organization of transcription have their origins in cell biology. Early studies characterized nascent transcription in relation to discernable nuclear structures and components. Advances in light microscopy, immunofluorescence, and in situ hybridization helped to begin the difficult task of naming the countless individual players and components of transcription and placing them in context. With the completion of mammalian genome sequences, the seemingly boundless task of understanding transcription of the genome became finite and began a new period of rapid advance. Here we focus on the organization of transcription in mammals drawing upon information from lower organisms where necessary. The emerging picture is one of a highly organized nucleus with specific conformations of the genome adapted for tissue-specific programs of transcription and gene expression. PMID:20668006

  14. Increased glutamine in leaves of poplar transgenic with pine GS1a caused greater anthranilate synthetase α-subunit (ASA1) transcript and protein abundances: an auxin-related mechanism for enhanced growth in GS transgenics?

    PubMed

    Man, Huimin; Pollmann, Stephan; Weiler, Elmar W; Kirby, Edward G

    2011-08-01

    The initial reaction in the pathway leading to the production of indole-3-acetic acid (IAA) in plants is the reaction between chorismate and glutamine to produce anthranilate, catalysed by the enzyme anthranilate synthase (ASA; EC 4.1.3.27). Compared with non-transgenic controls, leaves of transgenic poplar with ectopic expression of the pine cytosolic glutamine synthetase (GS1a; EC 6.3.1.2) produced significantly greater glutamine and significantly enhanced ASA α-subunit (ASA1) transcript and protein (approximately 130% and 120% higher than in the untransformed controls, respectively). Similarly, tobacco leaves fed with 30 mM glutamine and 2 mM chorismate showed enhanced ASA1 transcript and protein (175% and 90% higher than controls, respectively). Furthermore, free IAA was significantly elevated both in leaves of GS1a transgenic poplar and in tobacco leaves fed with 30 mM glutamine and 2 mM chorismate. These results indicated that enhanced cellular glutamine may account for the enhanced growth in GS transgenic poplars through the regulation of auxin biosynthesis. © 2011 The Author(s).

  15. WRKY transcription factors

    PubMed Central

    Bakshi, Madhunita; Oelmüller, Ralf

    2014-01-01

    WRKY transcription factors are one of the largest families of transcriptional regulators found exclusively in plants. They have diverse biological functions in plant disease resistance, abiotic stress responses, nutrient deprivation, senescence, seed and trichome development, embryogenesis, as well as additional developmental and hormone-controlled processes. WRKYs can act as transcriptional activators or repressors, in various homo- and heterodimer combinations. Here we review recent progress on the function of WRKY transcription factors in Arabidopsis and other plant species such as rice, potato, and parsley, with a special focus on abiotic, developmental, and hormone-regulated processes. PMID:24492469

  16. A Nonnatural Transcriptional Coactivator

    NASA Astrophysics Data System (ADS)

    Nyanguile, Origene; Uesugi, Motonari; Austin, David J.; Verdine, Gregory L.

    1997-12-01

    In eukaryotes, sequence-specific DNA-binding proteins activate gene expression by recruiting the transcriptional apparatus and chromatin remodeling proteins to the promoter through protein-protein contacts. In many instances, the connection between DNA-binding proteins and the transcriptional apparatus is established through the intermediacy of adapter proteins known as coactivators. Here we describe synthetic molecules with low molecular weight that act as transcriptional coactivators. We demonstrate that a completely nonnatural activation domain in one such molecule is capable of stimulating transcription in vitro and in vivo. The present strategy provides a means of gaining external control over gene activation through intervention using small molecules.

  17. Significantly increased expression of beta-glucuronidase in the central nervous system of mucopolysaccharidosis type VII mice from the latency-associated transcript promoter in a nonpathogenic herpes simplex virus type 1 vector.

    PubMed

    Zhu, J; Kang, W; Wolfe, J H; Fraser, N W

    2000-07-01

    Herpes simplex virus (HSV) has the ability to establish life-long latent infections in postmitotic neurons and to remain transcriptionally active, continuously expressing latency-associated transcripts (LAT) while producing minimal disease. These properties have made HSV an excellent candidate for neuronal gene transfer. Previously, we have shown that in mucopolysaccharidosis type VII mice (MPS VII, beta-glucuronidase deficiency) the LAT promoter is capable of expressing beta-glucuronidase (GUSB) in the trigeminal ganglion and the brainstem after latency is established. However, the number of neurons expressing GUSB is much lower than the number expressing 2-kb LAT following a wild-type virus infection. In this study, we have evaluated the effect of the position of the coding sequence relative to the LAT promoter on beta-glucuronidase gene expression in the central nervous system (CNS). Non-neurovirulent (ICP-34.5-deleted HSV-1) vectors were used, allowing direct intracranial injection. Significantly more GUSB activity was detected in brains of MPS VII mice inoculated with a recombinant virus (HSV-LAT-GUSB-JS) in which the GUSB cDNA was inserted near the LAT promoter, compared to viruses where it was inserted farther downstream in either the LAT exon 1 or overlapping exon 1 and the 2-kb LAT intron. This vector produced more than 100 times the number of positive cells than the other constructs. During acute infection, the distribution of viral replication differed from the distribution of GUSB enzyme expression. Viral antigen was predominately present in cells around the site of injection in the caudate putamen and in ependymal cells lining the ventricles. In contrast, GUSB expression was present mainly in cells of the thalamus and hypothalamus, which did not exhibit viral antigen, suggesting that GUSB enzyme activity was expressed from latently but not acutely infected neuronal cells. This vector design should be useful for high-level expression of various genes in

  18. Mechanical Properties of Transcription

    NASA Astrophysics Data System (ADS)

    Sevier, Stuart A.; Levine, Herbert

    2017-06-01

    The mechanical properties of transcription have recently been shown to play a central role in gene expression. However, a full physical characterization of this central biological process is lacking. In this Letter, we introduce a simple description of the basic physical elements of transcription where RNA elongation, RNA polymerase rotation, and DNA supercoiling are coupled. The resulting framework describes the relative amount of RNA polymerase rotation and DNA supercoiling that occurs during RNA elongation. Asymptotic behavior is derived and can be used to experimentally extract unknown mechanical parameters of transcription. Mechanical limits to transcription are incorporated through the addition of a DNA supercoiling-dependent RNA polymerase velocity. This addition can lead to transcriptional stalling and resulting implications for gene expression, chromatin structure and genome organization are discussed.

  19. HIV-1 Reverse Transcription

    PubMed Central

    Hu, Wei-Shau; Hughes, Stephen H.

    2012-01-01

    Reverse transcription and integration are the defining features of the Retroviridae; the common name “retrovirus” derives from the fact that these viruses use a virally encoded enzyme, reverse transcriptase (RT), to convert their RNA genomes into DNA. Reverse transcription is an essential step in retroviral replication. This article presents an overview of reverse transcription, briefly describes the structure and function of RT, provides an introduction to some of the cellular and viral factors that can affect reverse transcription, and discusses fidelity and recombination, two processes in which reverse transcription plays an important role. In keeping with the theme of the collection, the emphasis is on HIV-1 and HIV-1 RT. PMID:23028129

  20. Transcription and Recombination: When RNA Meets DNA

    PubMed Central

    Aguilera, Andrés; Gaillard, Hélène

    2014-01-01

    A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription–replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. PMID:25085910

  1. Stochastic Model of Supercoiling-Dependent Transcription

    NASA Astrophysics Data System (ADS)

    Brackley, C. A.; Johnson, J.; Bentivoglio, A.; Corless, S.; Gilbert, N.; Gonnella, G.; Marenduzzo, D.

    2016-07-01

    We propose a stochastic model for gene transcription coupled to DNA supercoiling, where we incorporate the experimental observation that polymerases create supercoiling as they unwind the DNA helix and that these enzymes bind more favorably to regions where the genome is unwound. Within this model, we show that when the transcriptionally induced flux of supercoiling increases, there is a sharp crossover from a regime where torsional stresses relax quickly and gene transcription is random, to one where gene expression is highly correlated and tightly regulated by supercoiling. In the latter regime, the model displays transcriptional bursts, waves of supercoiling, and up regulation of divergent or bidirectional genes. It also predicts that topological enzymes which relax twist and writhe should provide a pathway to down regulate transcription.

  2. On schemes of combinatorial transcription logic.

    PubMed

    Buchler, Nicolas E; Gerland, Ulrich; Hwa, Terence

    2003-04-29

    Cells receive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate specific genetic responses. Here we explore theoretically the potentials and limitations of combinatorial signal integration at the level of cis-regulatory transcription control. Our analysis suggests that many complex transcription-control functions of the type encountered in higher eukaryotes are already implementable within the much simpler bacterial transcription system. Using a quantitative model of bacterial transcription and invoking only specific protein-DNA interaction and weak glue-like interaction between regulatory proteins, we show explicit schemes to implement regulatory logic functions of increasing complexity by appropriately selecting the strengths and arranging the relative positions of the relevant protein-binding DNA sequences in the cis-regulatory region. The architectures that emerge are naturally modular and evolvable. Our results suggest that the transcription regulatory apparatus is a "programmable" computing machine, belonging formally to the class of Boltzmann machines. Crucial to our results is the ability to regulate gene expression at a distance. In bacteria, this can be achieved for isolated genes via DNA looping controlled by the dimerization of DNA-bound proteins. However, if adopted extensively in the genome, long-distance interaction can cause unintentional intergenic cross talk, a detrimental side effect difficult to overcome by the known bacterial transcription-regulation systems. This may be a key factor limiting the genome-wide adoption of complex transcription control in bacteria. Implications of our findings for combinatorial transcription control in eukaryotes are discussed.

  3. Overexpression of OrbHLH001, a putative helix-loop-helix transcription factor, causes increased expression of AKT1 and maintains ionic balance under salt stress in rice.

    PubMed

    Chen, Yuan; Li, Fei; Ma, Yan; Chong, Kang; Xu, Yunyuan

    2013-01-01

    The basic helix-loop-helix family of proteins, which function as transcription factors, have been intensively studied in plants and animals. However, the molecular mechanism of these factors contributing to stress tolerance is unknown. Here, we report on the overexpression of OrbHLH001 from Dongxiang wild rice (Oryza rufipogon) conferring salt tolerance in transgenic rice plants. The expression of OrbHLH001 was tissue specific, mainly in phloem tissues throughout the plant. Ion assay with the scanning ion-selective electrode technique showed that NaCl stress has a greater influence on Na(+) efflux and K(+) influx in OrbHLH001-overexpressed plants than the wild type. OrbHLH001 protein can induce the expression of OsAKT1 to regulate the Na(+)/K(+) ratio in OrbHLH001-overexpressed plants by specifically binding to an E-box motif in the promoter region of OsAKT1. The mechanism may have potential use in rice molecular breeding. Copyright © 2012 Elsevier GmbH. All rights reserved.

  4. Transcriptional Tools: Small Molecules for Modulating CBP KIX-dependent Transcriptional Activators

    PubMed Central

    Bates, Caleb A.; Pomerantz, William C.; Mapp, Anna K.

    2010-01-01

    Previously it was demonstrated that amphipathic isoxazolidines are able to functionally replace the transcriptional activation domains of endogenous transcriptional activators. In addition, in vitro binding studies suggested that a key binding partner of these molecules is the Creb Binding Protein (CBP), more specifically the KIX domain with this protein. Here we show that CBP and the KIX domain play an essential role in the ability of isoxazolidine transcriptional activation domains to activate transcription in cells. Consistent with this model, isoxazolidines are able to function as competitive inhibitors of the activators MLL and Jun, both of which utilize a binding interaction with KIX to up-regulate transcription. Further, modification of the N2 side chain produced two analogs with enhanced potency against Jun-mediated transcription, although increased cytotoxicity was also observed. Collectively these small KIX-binding molecules will be useful tools for dissecting the role of the KIX domain in a variety of pathological processes. PMID:20882601

  5. Transcription factor abundance controlled by an auto-regulatory mechanism involving a transcription start site switch.

    PubMed

    Ngondo, Richard Patryk; Carbon, Philippe

    2014-02-01

    A transcriptional feedback loop is the simplest and most direct means for a transcription factor to provide an increased stability of gene expression. In this work performed in human cells, we reveal a new negative auto-regulatory mechanism involving an alternative transcription start site (TSS) usage. Using the activating transcription factor ZNF143 as a model, we show that the ZNF143 low-affinity binding sites, located downstream of its canonical TSS, play the role of protein sensors to induce the up- or down-regulation of ZNF143 gene expression. We uncovered that the TSS switch that mediates this regulation implies the differential expression of two transcripts with an opposite protein production ability due to their different 5' untranslated regions. Moreover, our analysis of the ENCODE data suggests that this mechanism could be used by other transcription factors to rapidly respond to their own aberrant expression level.

  6. ASTP Onboard Voice Transcription

    NASA Technical Reports Server (NTRS)

    1975-01-01

    The transcription is presented of the Apollo-Soyuz Test Project voice communications as recorded on the command module data storage equipment. Data from this recorder are telemetered (dumped) to Space Tracking and Data Network sites for retransmission to the Johnson Space Center. The transcript is divided into three columns -- time, speaker, and text. The Greenwich mean time column consists of three two-digit numbers representing hours, minutes, and seconds (e.g., 22 34 14) for the Julian dates shown at the top of the page on which a new day begins. The speaker column indicates the source of a transmission; the text column contains the verbatim transcript of the communications.

  7. Antisense-mediated FLC transcriptional repression requires the P-TEFb transcription elongation factor

    PubMed Central

    Wang, Zhi-Wei; Wu, Zhe; Raitskin, Oleg; Sun, Qianwen; Dean, Caroline

    2014-01-01

    The functional significance of noncoding transcripts is currently a major question in biology. We have been studying the function of a set of antisense transcripts called COOLAIR that encompass the whole transcription unit of the Arabidopsis floral repressor FLOWERING LOCUS C (FLC). Alternative polyadenylation of COOLAIR transcripts correlates with different FLC sense expression states. Suppressor mutagenesis aimed at understanding the importance of this sense–antisense transcriptional circuitry has identified a role for Arabidopsis cyclin-dependent kinase C (CDKC;2) in FLC repression. CDKC;2 functions in an Arabidopsis positive transcription elongation factor b (P-TEFb) complex and influences global RNA polymerase II (Pol II) Ser2 phosphorylation levels. CDKC;2 activity directly promotes COOLAIR transcription but does not affect an FLC transgene missing the COOLAIR promoter. In the endogenous gene context, however, the reduction of COOLAIR transcription by cdkc;2 disrupts a COOLAIR-mediated repression mechanism that increases FLC expression. This disruption then feeds back to indirectly increase COOLAIR expression. This tight interconnection between sense and antisense transcription, together with differential promoter sensitivity to P-TEFb, is central to quantitative regulation of this important floral repressor gene. PMID:24799695

  8. DNA supercoiling during transcription.

    PubMed

    Ma, Jie; Wang, Michelle D

    2016-11-01

    The twin-supercoiled-domain model describes how transcription can drive DNA supercoiling, and how DNA supercoiling, in turn plays an important role in regulating gene transcription. In vivo and in vitro experiments have disclosed many details of the complex interactions in this relationship, and recently new insights have been gained with the help of genome-wide DNA supercoiling mapping techniques and single molecule methods. This review summarizes the general mechanisms of the interplay between DNA supercoiling and transcription, considers the biological implications, and focuses on recent important discoveries and technical advances in this field. We highlight the significant impact of DNA supercoiling in transcription, but also more broadly in all processes operating on DNA.

  9. DNA supercoiling during transcription

    PubMed Central

    Ma, Jie; Wang, Michelle D.

    2017-01-01

    The twin-supercoiled-domain model describes how transcription can drive DNA supercoiling, and how DNA supercoiling, in turn plays an important role in regulating gene transcription. In vivo and in vitro experiments have disclosed many details of the complex interactions in this relationship, and recently new insights have been gained with the help of genome-wide DNA supercoiling mapping techniques and single molecule methods. This review summarizes the general mechanisms of the interplay between DNA supercoiling and transcription, considers the biological implications, and focuses on recent important discoveries and technical advances in this field. We highlight the significant impact of DNA supercoiling in transcription, but also more broadly in all processes operating on DNA.

  10. Thyrotropin controls transcription of the thyroglobulin gene.

    PubMed

    Van Heuverswyn, B; Streydio, C; Brocas, H; Refetoff, S; Dumont, J; Vassart, G

    1984-10-01

    The availability of rat thyroglobulin cDNA clones was exploited to study the regulation of thyroglobulin gene transcription by thyrotropin (TSH). Groups of rats were subjected to treatments leading to reduction or increase in the rat serum TSH (rTSH) levels. Thyroid gland nuclei were isolated, incubated in vitro in the presence of 32P-labeled uridine triphosphate, and thyroglobulin transcripts were quantitated by hybridization to immobilized rat thyroglobulin cDNA clones. Transcription of the thyroglobulin gene was found to be very active in thyroid nuclei from control animals. It represented about 10% of total RNA polymerase II activity. Chronic hyperstimulation of the thyroid glands with endogenous rTSH was achieved in rats treated with the goitrogen propylthiouracil. No significant increase of thyroglobulin gene transcription could be measured in thyroid nuclei from these animals. On the contrary, a dramatic decrease in thyroglobulin gene transcription was observed in those animals in which endogenous rTSH levels had been suppressed by hypophysectomy or by the administration of triiodothyronine. Injection of exogenous bovine TSH in such animals readily restored transcriptional activity of the gene. Our results identify transcription as an important regulatory step involved in TSH action. They suggest that normal TSH levels induce close to maximal expression of the thyroglobulin gene but that continuous presence of TSH is required in order to maintain the gene in an activated state.

  11. Transcription termination maintains chromosome integrity.

    PubMed

    Washburn, Robert S; Gottesman, Max E

    2011-01-11

    DNA replication fork movement is impeded by collisions with transcription elongation complexes (TEC). We propose that a critical function of transcription termination factors is to prevent TEC from blocking DNA replication and inducing replication fork arrest, one consequence of which is DNA double-strand breaks. We show that inhibition of Rho-dependent transcription termination by bicyclomycin in Escherichia coli induced double-strand breaks. Cells deleted for Rho-cofactors nusA and nusG were hypersensitive to bicyclomycin, and had extensive chromosome fragmentation even in the absence of the drug. An RNA polymerase mutation that destabilizes TEC (rpoB*35) increased bicyclomycin resistance >40-fold. Double-strand break formation depended on DNA replication, and can be explained by replication fork collapse. Deleting recombination genes required for replication fork repair (recB and ruvC) increased sensitivity to bicyclomycin, as did loss of the replication fork reloading helicases rep and priA. We propose that Rho responds to a translocating replisome by releasing obstructing TEC.

  12. Transcription and cancer.

    PubMed Central

    Cox, P. M.; Goding, C. R.

    1991-01-01

    The normal growth, development and function of an organism requires precise and co-ordinated control of gene expression. A major part of this control is exerted by regulating messenger RNA (mRNA) production and involves complex interactions between an array of transcriptionally active proteins and specific regulatory DNA sequences. The combination of such proteins and DNA sequences is specific for given gene or group of genes in a particular cell type and the proteins regulating the same gene may vary between cell types. In addition the expression or activity of these regulatory proteins may be modified depending on the state of differentiation of a cell or in response to an external stimulus. Thus, the differentiation of embryonic cells into diverse tissues is achieved and the mature structure and function of the organism is maintained. This review focusses on the role of perturbations of these transcriptional controls in neoplasia. Deregulation of transcription may result in the failure to express genes responsible for cellular differentiation, or alternatively, in the transcription of genes involved in cell division, through the inappropriate expression or activation of positively acting transcription factors and nuclear oncogenes. Whether the biochemical abnormalities that lead to the disordered growth and differentiation of a malignant tumour affect cell surface receptors, membrane or cytoplasmic signalling proteins or nuclear transcription factors, the end result is the inappropriate expression of some genes and failure to express others. Current research is starting to elucidate which of the elements of this complicated system are important in neoplasia. PMID:1645561

  13. Colon cancer associated transcripts in human cancers.

    PubMed

    Chen, Yincong; Xie, Haibiao; Gao, Qunjun; Zhan, Hengji; Xiao, Huizhong; Zou, Yifan; Zhang, Fuyou; Liu, Yuchen; Li, Jianfa

    2017-08-02

    Long non-coding RNAs serve as important regulators in complicated cellular activities, including cell differentiation, proliferation and death. Dysregulation of long non-coding RNAs occurs in the formation and progression of cancers. The family of colon cancer associated transcripts, long non-coding RNAs colon cancer associated transcript-1 and colon cancer associated transcript-2 are known as oncogenes involved in various cancers. Colon cancer associated transcript-1 is a novel lncRNA located in 8q24.2, and colon cancer associated transcript-2 maps to the 8q24.21 region encompassing rs6983267. Colon cancer associated transcripts have close associations with clinical characteristics, such as lymph node metastasis, high TNM stage and short overall survival. Knockdown of them can reverse the malignant phenotypes of cancer cells, including proliferation, migration, invasion and apoptosis. Moreover, they can increase the expression level of c-MYC and oncogenic microRNAs via activating a series of complex mechanisms. In brief, the family of colon cancer associated transcripts may serve as potential biomarkers or therapeutic targets for human cancers. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  14. Mitochondrial biology. Replication-transcription switch in human mitochondria.

    PubMed

    Agaronyan, Karen; Morozov, Yaroslav I; Anikin, Michael; Temiakov, Dmitry

    2015-01-30

    Coordinated replication and expression of the mitochondrial genome is critical for metabolically active cells during various stages of development. However, it is not known whether replication and transcription can occur simultaneously without interfering with each other and whether mitochondrial DNA copy number can be regulated by the transcription machinery. We found that interaction of human transcription elongation factor TEFM with mitochondrial RNA polymerase and nascent transcript prevents the generation of replication primers and increases transcription processivity and thereby serves as a molecular switch between replication and transcription, which appear to be mutually exclusive processes in mitochondria. TEFM may allow mitochondria to increase transcription rates and, as a consequence, respiration and adenosine triphosphate production without the need to replicate mitochondrial DNA, as has been observed during spermatogenesis and the early stages of embryogenesis.

  15. Transcriptional activation of ribosomal RNA genes during compensatory renal hypertrophy

    SciTech Connect

    Ouellette, A.J.; Moonka, R.; Zelenetz, A.; Malt, R.A.

    1986-05-01

    The overall rate of rDNA transcription increases by 50% during the first 24 hours of compensatory renal hypertrophy in the mouse. To study mechanisms of ribosome accumulation after uninephrectomy, transcription rates were measured in isolated kidneys by transcriptional runoff. /sup 32/P-labeled nascent transcripts were hybridized to blots containing linearized, denatured cloned rDNA, and hybridization was quantitated autoradiographically and by direct counting. Overall transcriptional activity of rDNA was increased by 30% above control levels at 6 hrs after nephrectomy and by 50% at 12, 18, and 24 hrs after operation. Hybridizing RNA was insensitive to inhibiby alpha-amanitin, and no hybridization was detected to vector DNA. Thus, accelerated rDNA transcription is one regulatory element in the accretion of ribosomes in renal growth, and the regulatory event is an early event. Mechanisms of activation may include enhanced transcription of active genes or induction of inactive DNA.

  16. Increases in CYP3A Expression and Glucocorticoid-Inducibility in Liver of Rats Fed Soy Protein Isolate (SPI) Involves Post-Transcriptional Effects on mRNA Processing

    USDA-ARS?s Scientific Manuscript database

    We analyzed a time course of dexamethasone (DEX)-induction at PND25 and PND60 in male and female rats fed soy protein isolate (SPI) or casein (CAS) based AIN93G diets throughout development to examine molecular mechanisms underlying increased CYP3A expression and inducibility after SPI-feeding. At ...

  17. Regulation of Transcript Elongation

    PubMed Central

    Belogurov, Georgiy A.; Artsimovitch, Irina

    2015-01-01

    Bacteria lack subcellular compartments and harbor a single RNA polymerase that synthesizes both structural and protein-coding RNAs, which are cotranscriptionally processed by distinct pathways. Nascent rRNAs fold into elaborate secondary structures and associate with ribosomal proteins, whereas nascent mRNAs are translated by ribosomes. During elongation, nucleic acid signals and regulatory proteins modulate concurrent RNA-processing events, instruct RNA polymerase where to pause and terminate transcription, or act as roadblocks to the moving enzyme. Communications among complexes that carry out transcription, translation, repair, and other cellular processes ensure timely execution of the gene expression program and survival under conditions of stress. This network is maintained by auxiliary proteins that act as bridges between RNA polymerase, ribosome, and repair enzymes, blurring boundaries between separate information-processing steps and making assignments of unique regulatory functions meaningless. Understanding the regulation of transcript elongation thus requires genome-wide approaches, which confirm known and reveal new regulatory connections. PMID:26132790

  18. Smad transcription factors.

    PubMed

    Massagué, Joan; Seoane, Joan; Wotton, David

    2005-12-01

    Smad transcription factors lie at the core of one of the most versatile cytokine signaling pathways in metazoan biology-the transforming growth factor-beta (TGFbeta) pathway. Recent progress has shed light into the processes of Smad activation and deactivation, nucleocytoplasmic dynamics, and assembly of transcriptional complexes. A rich repertoire of regulatory devices exerts control over each step of the Smad pathway. This knowledge is enabling work on more complex questions about the organization, integration, and modulation of Smad-dependent transcriptional programs. We are beginning to uncover self-enabled gene response cascades, graded Smad response mechanisms, and Smad-dependent synexpression groups. Our growing understanding of TGFbeta signaling through the Smad pathway provides general principles for how animal cells translate complex inputs into concrete behavior.

  19. Transcriptional Regulation by CHIP/LDB Complexes

    PubMed Central

    Bronstein, Revital; Levkovitz, Liron; Yosef, Nir; Yanku, Michaela; Ruppin, Eytan; Sharan, Roded; Westphal, Heiner; Oliver, Brian; Segal, Daniel

    2010-01-01

    It is increasingly clear that transcription factors play versatile roles in turning genes “on” or “off” depending on cellular context via the various transcription complexes they form. This poses a major challenge in unraveling combinatorial transcription complex codes. Here we use the powerful genetics of Drosophila combined with microarray and bioinformatics analyses to tackle this challenge. The nuclear adaptor CHIP/LDB is a major developmental regulator capable of forming tissue-specific transcription complexes with various types of transcription factors and cofactors, making it a valuable model to study the intricacies of gene regulation. To date only few CHIP/LDB complexes target genes have been identified, and possible tissue-dependent crosstalk between these complexes has not been rigorously explored. SSDP proteins protect CHIP/LDB complexes from proteasome dependent degradation and are rate-limiting cofactors for these complexes. By using mutations in SSDP, we identified 189 down-stream targets of CHIP/LDB and show that these genes are enriched for the binding sites of APTEROUS (AP) and PANNIER (PNR), two well studied transcription factors associated with CHIP/LDB complexes. We performed extensive genetic screens and identified target genes that genetically interact with components of CHIP/LDB complexes in directing the development of the wings (28 genes) and thoracic bristles (23 genes). Moreover, by in vivo RNAi silencing we uncovered novel roles for two of the target genes, xbp1 and Gs-alpha, in early development of these structures. Taken together, our results suggest that loss of SSDP disrupts the normal balance between the CHIP-AP and the CHIP-PNR transcription complexes, resulting in down-regulation of CHIP-AP target genes and the concomitant up-regulation of CHIP-PNR target genes. Understanding the combinatorial nature of transcription complexes as presented here is crucial to the study of transcription regulation of gene batteries required

  20. The Transcription Factor Encyclopedia

    PubMed Central

    2012-01-01

    Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe. PMID:22458515

  1. The transcription factor encyclopedia.

    PubMed

    Yusuf, Dimas; Butland, Stefanie L; Swanson, Magdalena I; Bolotin, Eugene; Ticoll, Amy; Cheung, Warren A; Zhang, Xiao Yu Cindy; Dickman, Christopher T D; Fulton, Debra L; Lim, Jonathan S; Schnabl, Jake M; Ramos, Oscar H P; Vasseur-Cognet, Mireille; de Leeuw, Charles N; Simpson, Elizabeth M; Ryffel, Gerhart U; Lam, Eric W-F; Kist, Ralf; Wilson, Miranda S C; Marco-Ferreres, Raquel; Brosens, Jan J; Beccari, Leonardo L; Bovolenta, Paola; Benayoun, Bérénice A; Monteiro, Lara J; Schwenen, Helma D C; Grontved, Lars; Wederell, Elizabeth; Mandrup, Susanne; Veitia, Reiner A; Chakravarthy, Harini; Hoodless, Pamela A; Mancarelli, M Michela; Torbett, Bruce E; Banham, Alison H; Reddy, Sekhar P; Cullum, Rebecca L; Liedtke, Michaela; Tschan, Mario P; Vaz, Michelle; Rizzino, Angie; Zannini, Mariastella; Frietze, Seth; Farnham, Peggy J; Eijkelenboom, Astrid; Brown, Philip J; Laperrière, David; Leprince, Dominique; de Cristofaro, Tiziana; Prince, Kelly L; Putker, Marrit; del Peso, Luis; Camenisch, Gieri; Wenger, Roland H; Mikula, Michal; Rozendaal, Marieke; Mader, Sylvie; Ostrowski, Jerzy; Rhodes, Simon J; Van Rechem, Capucine; Boulay, Gaylor; Olechnowicz, Sam W Z; Breslin, Mary B; Lan, Michael S; Nanan, Kyster K; Wegner, Michael; Hou, Juan; Mullen, Rachel D; Colvin, Stephanie C; Noy, Peter John; Webb, Carol F; Witek, Matthew E; Ferrell, Scott; Daniel, Juliet M; Park, Jason; Waldman, Scott A; Peet, Daniel J; Taggart, Michael; Jayaraman, Padma-Sheela; Karrich, Julien J; Blom, Bianca; Vesuna, Farhad; O'Geen, Henriette; Sun, Yunfu; Gronostajski, Richard M; Woodcroft, Mark W; Hough, Margaret R; Chen, Edwin; Europe-Finner, G Nicholas; Karolczak-Bayatti, Magdalena; Bailey, Jarrod; Hankinson, Oliver; Raman, Venu; LeBrun, David P; Biswal, Shyam; Harvey, Christopher J; DeBruyne, Jason P; Hogenesch, John B; Hevner, Robert F; Héligon, Christophe; Luo, Xin M; Blank, Marissa Cathleen; Millen, Kathleen Joyce; Sharlin, David S; Forrest, Douglas; Dahlman-Wright, Karin; Zhao, Chunyan; Mishima, Yuriko; Sinha, Satrajit; Chakrabarti, Rumela; Portales-Casamar, Elodie; Sladek, Frances M; Bradley, Philip H; Wasserman, Wyeth W

    2012-01-01

    Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.

  2. Transcriptional response of Enterococcus faecalis to sunlight.

    PubMed

    Sassoubre, Lauren M; Ramsey, Matthew M; Gilmore, Michael S; Boehm, Alexandria B

    2014-01-05

    Microarrays were used to investigate the transcriptional response of Enterococcus faecalis to photostress. E. faecalis are Gram-positive bacteria used as indicators of water quality and have been shown to vary diurnally in response to sunlight. E. faecalis in filtered seawater microcosms were exposed to artificial sunlight for 12h and then placed in the dark for 12h. Transcript abundance was measured at 0, 2, 6, 12, and 24h in the sunlit microcosm and a dark control using microarrays. Culturable E. faecalis concentrations decreased 6-7 orders of magnitude within the first 6h of light exposure. After 12h in the dark, no evidence of dark-repair was observed. Expression data collected after 12h of sunlight exposure revealed a difference in transcript abundance in the light relative to dark microcosms for 35 unique ORFs, 33 ORFs showed increased transcript abundance and 2 ORFs showed reduced transcript abundance. A majority (51%) of the ORFs with increased transcript abundance in the sunlit relative to dark microcosms encoded hypothetical proteins; others were associated with protein synthesis, oxidative stress and DNA repair. Results suggest that E. faecalis exposed to sunlight actively transcribe RNA in response to photostress.

  3. Canine Distemper Virus Infection Leads to an Inhibitory Phenotype of Monocyte-Derived Dendritic Cells In Vitro with Reduced Expression of Co-Stimulatory Molecules and Increased Interleukin-10 Transcription

    PubMed Central

    Herder, Vanessa; Stein, Veronika M.; Tipold, Andrea; Urhausen, Carola; Günzel-Apel, Anne-Rose; Rohn, Karl; Baumgärtner, Wolfgang; Beineke, Andreas

    2014-01-01

    Canine distemper virus (CDV) exhibits a profound lymphotropism that causes immunosuppression and increased susceptibility of affected dogs to opportunistic infections. Similar to human measles virus, CDV is supposed to inhibit terminal differentiation of dendritic cells (DCs), responsible for disturbed repopulation of lymphoid tissues and diminished antigen presenting function in dogs. In order to testify the hypothesis that CDV-infection leads to an impairment of professional antigen presenting cells, canine DCs have been generated from peripheral blood monocytes in vitro and infected with CDV. Virus infection was confirmed and quantified by transmission electron microscopy, CDV-specific immunofluorescence, and virus titration. Flow cytometric analyses revealed a significant down-regulation of the major histocompatibility complex class II and co-stimulatory molecules CD80 and CD86 in CDV-infected DCs, indicative of disturbed antigen presenting capacity. Molecular analyses revealed an increased expression of the immune inhibitory cytokine interleukin-10 in DCs following infection. Results of the present study demonstrate that CDV causes phenotypical changes and altered cytokine expression of DCs, which represent potential mechanisms to evade host immune responses and might contribute to immune dysfunction and virus persistence in canine distemper. PMID:24769532

  4. Canine distemper virus infection leads to an inhibitory phenotype of monocyte-derived dendritic cells in vitro with reduced expression of co-stimulatory molecules and increased interleukin-10 transcription.

    PubMed

    Qeska, Visar; Barthel, Yvonne; Herder, Vanessa; Stein, Veronika M; Tipold, Andrea; Urhausen, Carola; Günzel-Apel, Anne-Rose; Rohn, Karl; Baumgärtner, Wolfgang; Beineke, Andreas

    2014-01-01

    Canine distemper virus (CDV) exhibits a profound lymphotropism that causes immunosuppression and increased susceptibility of affected dogs to opportunistic infections. Similar to human measles virus, CDV is supposed to inhibit terminal differentiation of dendritic cells (DCs), responsible for disturbed repopulation of lymphoid tissues and diminished antigen presenting function in dogs. In order to testify the hypothesis that CDV-infection leads to an impairment of professional antigen presenting cells, canine DCs have been generated from peripheral blood monocytes in vitro and infected with CDV. Virus infection was confirmed and quantified by transmission electron microscopy, CDV-specific immunofluorescence, and virus titration. Flow cytometric analyses revealed a significant down-regulation of the major histocompatibility complex class II and co-stimulatory molecules CD80 and CD86 in CDV-infected DCs, indicative of disturbed antigen presenting capacity. Molecular analyses revealed an increased expression of the immune inhibitory cytokine interleukin-10 in DCs following infection. Results of the present study demonstrate that CDV causes phenotypical changes and altered cytokine expression of DCs, which represent potential mechanisms to evade host immune responses and might contribute to immune dysfunction and virus persistence in canine distemper.

  5. Mapping Yeast Transcriptional Networks

    PubMed Central

    Hughes, Timothy R.; de Boer, Carl G.

    2013-01-01

    The term “transcriptional network” refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face. For most yeast TFs, data have been collected on their sequence preferences, in vivo promoter occupancy, and gene expression profiles in deletion mutants. These systematic studies have led to the identification of new regulators of numerous cellular functions and shed light on the overall organization of yeast gene regulation. However, many yeast TFs appear to be inactive under standard laboratory growth conditions, and many of the available data were collected using techniques that have since been improved. Perhaps as a consequence, comprehensive and accurate mapping among TF sequence preferences, promoter binding, and gene expression remains an open challenge. We propose that the time is ripe for renewed systematic efforts toward a complete mapping of yeast transcriptional regulatory mechanisms. PMID:24018767

  6. Fungal CSL transcription factors

    PubMed Central

    Převorovský, Martin; Půta, František; Folk, Petr

    2007-01-01

    Background The CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factor family members are well-known components of the transmembrane receptor Notch signaling pathway, which plays a critical role in metazoan development. They function as context-dependent activators or repressors of transcription of their responsive genes, the promoters of which harbor the GTG(G/A)GAA consensus elements. Recently, several studies described Notch-independent activities of the CSL proteins. Results We have identified putative CSL genes in several fungal species, showing that this family is not confined to metazoans. We have analyzed their sequence conservation and identified the presence of well-defined domains typical of genuine CSL proteins. Furthermore, we have shown that the candidate fungal protein sequences contain highly conserved regions known to be required for sequence-specific DNA binding in their metazoan counterparts. The phylogenetic analysis of the newly identified fungal CSL proteins revealed the existence of two distinct classes, both of which are present in all the species studied. Conclusion Our findings support the evolutionary origin of the CSL transcription factor family in the last common ancestor of fungi and metazoans. We hypothesize that the ancestral CSL function involved DNA binding and Notch-independent regulation of transcription and that this function may still be shared, to a certain degree, by the present CSL family members from both fungi and metazoans. PMID:17629904

  7. Mapping yeast transcriptional networks.

    PubMed

    Hughes, Timothy R; de Boer, Carl G

    2013-09-01

    The term "transcriptional network" refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face. For most yeast TFs, data have been collected on their sequence preferences, in vivo promoter occupancy, and gene expression profiles in deletion mutants. These systematic studies have led to the identification of new regulators of numerous cellular functions and shed light on the overall organization of yeast gene regulation. However, many yeast TFs appear to be inactive under standard laboratory growth conditions, and many of the available data were collected using techniques that have since been improved. Perhaps as a consequence, comprehensive and accurate mapping among TF sequence preferences, promoter binding, and gene expression remains an open challenge. We propose that the time is ripe for renewed systematic efforts toward a complete mapping of yeast transcriptional regulatory mechanisms.

  8. Focus on Refugees. Transcript.

    ERIC Educational Resources Information Center

    Brandel, Sarah; And Others

    This is the transcript of the "Focus on Refugees," proqram conducted by the Overseas Development Council. Remarks from the following participants are included: (1) Sarah Brandel, Associate Fellow at the Overseas Development Council; (2) Gary Perkins, Chief of Mission of the Washington Office of the United Nations High Commissioner for Refugees…

  9. Transcription of mitochondrial DNA.

    PubMed

    Tabak, H F; Grivell, L A; Borst, P

    1983-01-01

    While mitochondrial DNA (mtDNA) is the simplest DNA in nature, coding for rRNAs and tRNAs, results of DNA sequence, and transcript analysis have demonstrated that both the synthesis and processing of mitochondrial RNAs involve remarkably intricate events. At one extreme, genes in animal mtDNAs are tightly packed, both DNA strands are completely transcribed (symmetric transcription), and the appearance of specific mRNAs is entirely dependent on processing at sites signalled by the sequences of the tRNAs, which abut virtually every gene. At the other extreme, gene organization in yeast (Saccharomyces) is anything but compact, with long stretches of AT-rich DNA interspaced between coding sequences and no obvious logic to the order of genes. Transcription is asymmetric and several RNAs are initiated de novo. Nevertheless, extensive RNA processing occurs due largely to the presence of split genes. RNA splicing is complex, is controlled by both mitochondrial and nuclear genes, and in some cases is accompanied by the formation of RNAs that behave as covalently closed circles. The present article reviews current knowledge of mitochondrial transcription and RNA processing in relation to possible mechanisms for the regulation of mitochondrial gene expression.

  10. DNA damage response and transcription.

    PubMed

    Lagerwerf, Saskia; Vrouwe, Mischa G; Overmeer, René M; Fousteri, Maria I; Mullenders, Leon H F

    2011-07-15

    A network of DNA damage surveillance systems is triggered by sensing of DNA lesions and the initiation of a signal transduction cascade that activates genome-protection pathways including nucleotide excision repair (NER). NER operates through coordinated assembly of repair factors into pre- and post-incision complexes. Recent work identifies RPA as a key regulator of the transition from dual incision to repair-synthesis in UV-irradiated non-cycling cells, thereby averting the generation of unprocessed repair intermediates. These intermediates could lead to recombinogenic events and trigger a persistent ATR-dependent checkpoint signaling. It is now evident that DNA damage signaling is not limited to NER proficient cells. ATR-dependent checkpoint activation also occurs in UV-exposed non-cycling repair deficient cells coinciding with the formation of endonuclease APE1-mediated DNA strand breaks. In addition, the encounter of elongating RNA polymerase II (RNAPIIo) with DNA damage lesions and its persistent stalling provides a strong DNA damage signaling leading to cell cycle arrest, apoptosis and increased mutagenesis. The mechanism underlying the strong and strand specific induction of UV-induced mutations in NER deficient cells has been recently resolved by the finding that gene transcription itself increases UV-induced mutagenesis in a strand specific manner via increased deamination of cytosines. The cell removes the RNAPIIo-blocking DNA lesions by transcription-coupled repair (TC-NER) without displacement of the DNA damage stalled RNAPIIo. Deficiency in TC-NER associates with mutations in the CSA and CSB genes giving rise to the rare human disorder Cockayne syndrome (CS). CSB functions as a repair coupling factor to attract NER proteins, chromatin remodelers and the CSA-E3-ubiquitin ligase complex to the stalled RNAPIIo; CSA is dispensable for attraction of NER proteins, yet in cooperation with CSB is required to recruit XAB2, the nucleosomal binding protein HMGN1

  11. Transcription errors induce proteotoxic stress and shorten cellular lifespan.

    PubMed

    Vermulst, Marc; Denney, Ashley S; Lang, Michael J; Hung, Chao-Wei; Moore, Stephanie; Moseley, M Arthur; Mosely, Arthur M; Thompson, J Will; Thompson, William J; Madden, Victoria; Gauer, Jacob; Wolfe, Katie J; Summers, Daniel W; Schleit, Jennifer; Sutphin, George L; Haroon, Suraiya; Holczbauer, Agnes; Caine, Joanne; Jorgenson, James; Cyr, Douglas; Kaeberlein, Matt; Strathern, Jeffrey N; Duncan, Mara C; Erie, Dorothy A

    2015-08-25

    Transcription errors occur in all living cells; however, it is unknown how these errors affect cellular health. To answer this question, we monitor yeast cells that are genetically engineered to display error-prone transcription. We discover that these cells suffer from a profound loss in proteostasis, which sensitizes them to the expression of genes that are associated with protein-folding diseases in humans; thus, transcription errors represent a new molecular mechanism by which cells can acquire disease phenotypes. We further find that the error rate of transcription increases as cells age, suggesting that transcription errors affect proteostasis particularly in aging cells. Accordingly, transcription errors accelerate the aggregation of a peptide that is implicated in Alzheimer's disease, and shorten the lifespan of cells. These experiments reveal a previously unappreciated role for transcriptional fidelity in cellular health and aging.

  12. Modulation of RNA polymerase assembly dynamics in transcriptional regulation

    PubMed Central

    Gorski, Stanislaw A.; Snyder, Sara K.; John, Sam; Grummt, Ingrid; Misteli, Tom

    2008-01-01

    The interaction of transcription factors with target genes is highly dynamic. Whether the dynamic nature of these interactions is merely an intrinsic property of transcriptions factors or serves a regulatory role is unknown. Here, we have used single cell fluorescence imaging combined with computational modeling and chromatin immunoprecipitation to analyze transcription complex dynamics in gene regulation during the cell cycle in living cells. We demonstrate a link between the dynamics of RNA polymerase I (RNA pol I) assembly and transcriptional output. We show that transcriptional upregulation is accompanied by prolonged retention of RNA pol I components at the promoter, resulting in longer promoter dwell time, and an increase in the steady state population of assembling polymerase. As a consequence, polymerase assembly efficiency, and ultimately, an rate of entry into processive elongation are elevated. Our results show that regulation of rDNA transcription in vivo occurs via modulation of the efficiency of transcription complex subunit capture and assembly. PMID:18498750

  13. Using both strands: The fundamental nature of antisense transcription.

    PubMed

    Murray, Struan C; Mellor, Jane

    2016-01-01

    Non-coding transcription across the antisense strands of genes is an abundant, pervasive process in eukaryotes from yeast to humans, however its biological function remains elusive. Here, we provide commentary on a recent study of ours, which demonstrates a genome-wide role for antisense transcription: establishing a unique, dynamic chromatin architecture over genes. Antisense transcription increases the level of nucleosome occupancy and histone acetylation at the promoter and body of genes, without necessarily modulating the level of protein-coding sense transcription. It is also associated with high levels of histone turnover. By allowing genes to sample a wider range of chromatin configurations, antisense transcription could serve to make genes more sensitive to changing signals, priming them for responses to developmental programs or stressful cellular environments. Given the abundance of antisense transcription and the breadth of these chromatin changes, we propose that antisense transcription represents a fundamental, canonical feature of eukaryotic genes.

  14. Endoplasmic reticulum stress induced by tunicamycin increases resistin messenger ribonucleic acid through the pancreatic endoplasmic reticulum eukaryotic initiation factor 2α kinase-activating transcription factor 4-CAAT/enhancer binding protein-α homologous protein pathway in THP-1 human monocytes.

    PubMed

    Hamada, Junpei; Onuma, Hiroshi; Ochi, Fumihiro; Hirai, Hiroki; Takemoto, Koji; Miyoshi, Akiko; Matsushita, Manami; Kadota, Yuko; Ohashi, Jun; Kawamura, Ryoichi; Takata, Yasunori; Nishida, Wataru; Hashida, Seiichi; Ishii, Eiichi; Osawa, Haruhiko

    2016-05-01

    Resistin, secreted from adipocytes, causes insulin resistance in mice. In humans, the resistin gene is mainly expressed in monocytes and macrophages. Tunicamycin is known to induce endoplasmic reticulum (ER) stress, and reduce resistin gene expression in 3T3-L1 mouse adipocytes. The aim of the present study was to examine whether ER stress affects resistin gene expression in human monocytes. The relationship between resistin messenger ribonucleic acid (mRNA) and ER stress markers mRNA was analyzed by reverse transcription polymerase chain reaction in isolated monocytes of 30 healthy volunteers. The effect of endotoxin/lipopolysaccharides or tunicamycin on resistin gene expression was analyzed in THP-1 human monocytes. Signaling pathways leading to resistin mRNA were assessed by the knockdown using small interfering RNA or overexpression of key molecules involved in unfolded protein response. Resistin mRNA was positively associated with immunoglobulin heavy chain-binding protein (BiP) or CAAT/enhancer binding protein-α homologous protein (CHOP) mRNA in human isolated monocytes. In THP-1 cells, lipopolysaccharides increased mRNA of BiP, pancreatic endoplasmic reticulum eukaryotic initiation factor 2α kinase (PERK) and CHOP, as well as resistin. Tunicamycin also increased resistin mRNA. This induction appeared to be dose- and time-dependent. Tunicamycin-induced resistin mRNA was inhibited by chemical chaperone, 4-phenylbutyric acid. The knockdown of either PERK, activating transcription factor 4 (ATF4) or CHOP reduced tunicamycin-induced resistin mRNA. Conversely, overexpression of ATF4 or CHOP increased resistin mRNA. Endoplasmic reticulum stress induced by tunicamycin increased resistin mRNA through the PERK-ATF4-CHOP pathway in THP-1 human monocytes. ER stress could lead to insulin resistance through enhanced resistin gene expression in human monocytes.

  15. Strategies to identify natural antisense transcripts.

    PubMed

    Sun, Yulong; Li, Dijie; Zhang, Ru; Peng, Shang; Zhang, Ge; Yang, Tuanmin; Qian, Airong

    2017-01-01

    Natural antisense transcripts, originally considered as transcriptional noises arising from so-called "junk DNA″, are recently recognized as important modulators for gene regulation. They are prevalent in nearly all realms of life and have been found to modulate gene expression positively or negatively. By affecting almost all stages of gene expression range from pre-transcriptional, transcriptional and post-transcriptional to translation, NATs are fundamentally involved in various biological processes. However, compared to increasing huge data from transcriptional analysis especially high-throughput sequencing technologies (such as RNA-seq), limited functional NATs (around 70) are so far reported, which hinder our advanced comprehensive understanding for this field. Hence, efficient strategies for identifying NATs are urgently desired. In this review, we discussed the current strategies for identifying NATs, with a focus on the advantages, disadvantages, and applications of methods isolating functional NATs. Moreover, publicly available databases for NATs were also discussed. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  16. Proofreading of misincorporated nucleotides in DNA transcription

    NASA Astrophysics Data System (ADS)

    Voliotis, Margaritis; Cohen, Netta; Molina-París, Carmen; Liverpool, Tanniemola B.

    2012-06-01

    The accuracy of DNA transcription is crucial for the proper functioning of the cell. Although RNA polymerases demonstrate selectivity for correct nucleotides, additional active mechanisms of transcriptional error correction are required to achieve observed levels of fidelity. Recent experimental findings have shed light on a particular mechanism of transcriptional error correction involving: (i) diffusive translocation of the RNA polymerase along the DNA (backtracking) and (ii) irreversible RNA cleavage. This mechanism achieves preferential cleavage of misincorporated nucleotides by biasing the local rates of translocation. Here, we study how misincorporated nucleotides affect backtracking dynamics and how this effect determines the level of transcriptional fidelity. We consider backtracking as a diffusive process in a periodic, one-dimensional energy landscape, which at a coarse-grained level gives rise to a hopping process between neighboring local minima. We propose a model for how misincorporated nucleotides deform this energy landscape and hence affect the hopping rates. In particular, we show that this model can be used to derive both the theoretical limit on the fidelity (i.e. the minimum fraction of misincorporated nucleotides) and the actual fidelity relative to this optimum, achieved for specific combinations of the cleavage and polymerization rates. Finally, we study how external factors influencing backtracking dynamics affect transcriptional fidelity. We show that biologically relevant loads, similar to those exerted by nucleosomes or other transcriptional barriers, increase error correction.

  17. Proofreading of misincorporated nucleotides in DNA transcription

    NASA Astrophysics Data System (ADS)

    Voliotis, Margaritis; Cohen, Netta; Molina-París, Carmen; Liverpool, Tanniemola B.

    2012-06-01

    The accuracy of DNA transcription is crucial for the proper functioning of the cell. Although RNA polymerases demonstrate selectivity for correct nucleotides, additional active mechanisms of transcriptional error correction are required to achieve observed levels of fidelity. Recent experimental findings have shed light on a particular mechanism of transcriptional error correction involving: (i) diffusive translocation of the RNA polymerase along the DNA (backtracking) and (ii) irreversible RNA cleavage. This mechanism achieves preferential cleavage of misincorporated nucleotides by biasing the local rates of translocation. Here, we study how misincorporated nucleotides affect backtracking dynamics and how this effect determines the level of transcriptional fidelity. We consider backtracking as a diffusive process in a periodic, one-dimensional energy landscape, which at a coarse-grained level gives rise to a hopping process between neighbouring local minima. We propose a model for how misincorporated nucleotides deform this energy landscape and hence affect the hopping rates. In particular, we show that this model can be used to derive both the theoretical limit on the fidelity (i.e. the minimum fraction of misincorporated nucleotides) and the actual fidelity relative to this optimum, achieved for specific combinations of the cleavage and polymerization rates. Finally, we study how external factors influencing backtracking dynamics affect transcriptional fidelity. We show that biologically relevant loads, similar to those exerted by nucleosomes or other transcriptional barriers, increase error correction.

  18. Transcriptional proofreading in dense RNA polymerase traffic

    NASA Astrophysics Data System (ADS)

    Sahoo, Mamata; Klumpp, Stefan

    2011-12-01

    The correction of errors during transcription involves the diffusive backward translocation (backtracking) of RNA polymerases (RNAPs) on the DNA. A trailing RNAP on the same template can interfere with backtracking as it progressively restricts the space that is available for backward translocation and thereby ratchets the backtracked RNAP forward. We analyze the resulting negative impact on proofreading theoretically using a driven lattice gas model of transcription under conditions of dense RNAP traffic. The fraction of errors that are corrected is calculated exactly for the case of a single RNAP; for multi-RNAP transcription, we use simulations and an analytical approximation and find a decrease with increasing traffic density. Moreover, we ask how the parameters of the system have to be set to keep down the impact of the interference of a trailing RNAP. Our analysis uncovers a surprisingly simple picture of the design of the error correction system: its efficiency is essentially determined by the rate for the initial backtracking step, while the value of the cleavage rate ensures that the correction mechanism remains efficient at high transcription rates. Finally, we argue that our analysis can also be applied to cases with transcription-translation coupling where the leading ribosome on the transcript assumes the role of the trailing RNAP.

  19. Chromatin looping and eRNA transcription precede the transcriptional activation of gene in the β-globin locus

    PubMed Central

    Kim, Yea Woon; Lee, Sungkung; Yun, Jangmi; Kim, AeRi

    2015-01-01

    Enhancers are closely positioned with actively transcribed target genes by chromatin looping. Non-coding RNAs are often transcribed on active enhancers, referred to as eRNAs (enhancer RNAs). To explore the kinetics of enhancer–promoter looping and eRNA transcription during transcriptional activation, we induced the β-globin locus by chemical treatment and analysed cross-linking frequency between the β-globin gene and locus control region (LCR) and the amount of eRNAs transcribed on the LCR in a time course manner. The cross-linking frequency was increased after chemical induction but before the transcriptional activation of gene in the β-globin locus. Transcription of eRNAs was increased in concomitant with the increase in cross-linking frequency. These results show that chromatin looping and eRNA transcription precedes the transcriptional activation of gene. Concomitant occurrence of the two events suggests functional relationship between them. PMID:25588787

  20. Machine Transcription--Practically Speaking.

    ERIC Educational Resources Information Center

    Clippinger, Dorinda A.

    1984-01-01

    Draws transcription teaching principles from Gagne's theories about learning. Recommends 12-16 weeks of instruction, pre-transcription development of related skills, frequent feedback, and use of teaching materials that are arranged to take advantage of learning cycles. (SK)

  1. Transcription Dynamics in Plant Immunity

    PubMed Central

    Moore, John W.; Loake, Gary J.; Spoel, Steven H.

    2011-01-01

    Plant cells maintain sophisticated gene transcription programs to regulate their development, communication, and response to the environment. Environmental stress cues, such as pathogen encounter, lead to dramatic reprogramming of transcription to favor stress responses over normal cellular functions. Transcription reprogramming is conferred by the concerted action of myriad transcription (co)factors that function directly or indirectly to recruit or release RNA Polymerase II. To establish an effective defense response, cells require transcription (co)factors to deploy their activity rapidly, transiently, spatially, and hierarchically. Recent findings suggest that in plant immunity these requirements are met by posttranslational modifications that accurately regulate transcription (co)factor activity as well as by sequential pulse activation of specific gene transcription programs that provide feedback and feedforward properties to the defense gene network. Here, we integrate these recent findings from plant defense studies into the emerging field of transcription dynamics in eukaryotes. PMID:21841124

  2. Divergent transcription is associated with promoters of transcriptional regulators

    PubMed Central

    2013-01-01

    Background Divergent transcription is a wide-spread phenomenon in mammals. For instance, short bidirectional transcripts are a hallmark of active promoters, while longer transcripts can be detected antisense from active genes in conditions where the RNA degradation machinery is inhibited. Moreover, many described long non-coding RNAs (lncRNAs) are transcribed antisense from coding gene promoters. However, the general significance of divergent lncRNA/mRNA gene pair transcription is still poorly understood. Here, we used strand-specific RNA-seq with high sequencing depth to thoroughly identify antisense transcripts from coding gene promoters in primary mouse tissues. Results We found that a substantial fraction of coding-gene promoters sustain divergent transcription of long non-coding RNA (lncRNA)/mRNA gene pairs. Strikingly, upstream antisense transcription is significantly associated with genes related to transcriptional regulation and development. Their promoters share several characteristics with those of transcriptional developmental genes, including very large CpG islands, high degree of conservation and epigenetic regulation in ES cells. In-depth analysis revealed a unique GC skew profile at these promoter regions, while the associated coding genes were found to have large first exons, two genomic features that might enforce bidirectional transcription. Finally, genes associated with antisense transcription harbor specific H3K79me2 epigenetic marking and RNA polymerase II enrichment profiles linked to an intensified rate of early transcriptional elongation. Conclusions We concluded that promoters of a class of transcription regulators are characterized by a specialized transcriptional control mechanism, which is directly coupled to relaxed bidirectional transcription. PMID:24365181

  3. Non-transcriptional regulatory processes shape transcriptional network dynamics.

    PubMed

    Ray, J Christian J; Tabor, Jeffrey J; Igoshin, Oleg A

    2011-10-11

    Information about the extra- or intracellular environment is often captured as biochemical signals that propagate through regulatory networks. These signals eventually drive phenotypic changes, typically by altering gene expression programmes in the cell. Reconstruction of transcriptional regulatory networks has given a compelling picture of bacterial physiology, but transcriptional network maps alone often fail to describe phenotypes. Cellular response dynamics are ultimately determined by interactions between transcriptional and non-transcriptional networks, with dramatic implications for physiology and evolution. Here, we provide an overview of non-transcriptional interactions that can affect the performance of natural and synthetic bacterial regulatory networks.

  4. Antibiotics trapping transcription initiation intermediates

    PubMed Central

    2011-01-01

    Promoter DNA melting, culminating in the loading of the single-stranded DNA template into the RNA polymerase active site, is a key step in transcription initiation. Recently, the first transcription inhibitors found to block distinct steps of promoter melting were characterized. Here, the impact of these studies is discussed with respect to the current models of transcription initiation. PMID:21468230

  5. Gene transcription and electromagnetic fields

    SciTech Connect

    Henderson, A.S.

    1992-01-01

    Our overall aim is to obtain sufficient information to allow us to ultimately determine whether ELF EM field exposure is an initiating factor in neoplastic transformation and/or if exposure can mimic characteristics of the second-step counterpart in neoplastic disease. This aim is based on our previous findings that levels of some transcripts are increased in cells exposed to EM fields. While the research is basic in nature, the ramifications have bearing on the general safety of exposure to EM fields in industrial and everyday life. A large array of diverse biological effects are reported to occur as the result of exposure to elf EM fields, suggesting that the cell response to EM fields is at a basic level, presumably initiated by molecular and/or biophysical events at the cell membrane. The hypothesized route is a signal transduction pathway involving membrane calcium fluxes. Information flow resulting from signal transduction can mediate the induction of regulatory factors in the cell, and directly affect how transcription is regulated.

  6. Cooperative activation of Xenopus rhodopsin transcription by paired-like transcription factors

    PubMed Central

    2014-01-01

    Background In vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx5. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. Cooperative action by these transcription factors activates rod-specific genes such as rhodopsin. However, a quantitative mechanistic explanation of transcriptional rate determinants is lacking. Results We investigated the contributions of various paired-like transcription factors and their cognate cis-elements to rhodopsin gene activation using cultured cells to quantify activity. The Xenopus rhodopsin promoter (XOP) has a bipartite structure, with ~200 bp proximal to the start site (RPP) coordinating cooperative activation by Nrl/LNrl-Crx/Otx5 and the adjacent 5300 bp upstream sequence increasing the overall expression level. The synergistic activation by Nrl/LNrl-Crx/Otx5 also occurred when XOP was stably integrated into the genome. We determined that Crx/Otx5 synergistically activated transcription independently and additively through the two Pax-like cis-elements, BAT1 and Ret4, but not through Ret1. Other Pax-like family members, Rax1 and Rax2, do not synergistically activate XOP transcription with Nrl/LNrl and/or Crx/Otx5; rather they act as co-activators via the Ret1 cis-element. Conclusions We have provided a quantitative model of cooperative transcriptional activation of the rhodopsin promoter through interaction of Crx/Otx5 with Nrl/LNrl at two paired-like cis-elements proximal to the NRE and TATA binding site. Further, we have shown that Rax genes act in cooperation with Crx/Otx5 with Nrl/LNrl as co-activators of rhodopsin transcription. PMID:24499263

  7. reSETting chromatin during transcription elongation

    PubMed Central

    Smolle, Michaela; Workman, Jerry L.; Venkatesh, Swaminathan

    2013-01-01

    Maintenance of ordered chromatin structure over the body of genes is vital for the regulation of transcription. Increased access to the underlying DNA sequence results in the recruitment of RNA polymerase II to inappropriate, promoter-like sites within genes, resulting in unfettered transcription. Two new papers show how the Set2-mediated methylation of histone H3 on Lys36 (H3K36me) maintains chromatin structure by limiting histone dynamics over gene bodies, either by recruiting chromatin remodelers that preserve ordered nucleosomal distribution or by lowering the binding affinity of histone chaperones for histones, preventing their removal. PMID:23257840

  8. Transcriptional and Epigenetic Mechanisms of Addiction

    PubMed Central

    Robison, Alfred J.; Nestler, Eric J.

    2012-01-01

    Preface Investigations of long-term changes in brain structure and function that accompany chronic exposure to drugs of abuse suggest that alterations in gene regulation contribute importantly to the addictive phenotype. We review multiple mechanisms by which drugs alter the transcriptional potential of genes, from the mobilization or repression of the transcriptional machinery to epigenetics — including alterations in the accessibility of genes within their native chromatin structure and the regulation of gene expression by non-coding RNAs. Increasing evidence implicates these various mechanisms of gene regulation in the lasting changes that drugs of abuse induce in brain, and offer novel inroads for addiction therapy. PMID:21989194

  9. Depleting Mycobacterium tuberculosis of the transcription termination factor Rho causes pervasive transcription and rapid death

    PubMed Central

    Botella, Laure; Vaubourgeix, Julien; Livny, Jonathan; Schnappinger, Dirk

    2017-01-01

    Rifampicin, which inhibits bacterial RNA polymerase, provides one of the most effective treatments for tuberculosis. Inhibition of the transcription termination factor Rho is used to treat some bacterial infections, but its importance varies across bacteria. Here we show that Rho of Mycobacterium tuberculosis functions to both define the 3′ ends of mRNAs and silence substantial fragments of the genome. Brief inactivation of Rho affects over 500 transcripts enriched for genes of foreign DNA elements and bacterial virulence factors. Prolonged inactivation of Rho causes extensive pervasive transcription, a genome-wide increase in antisense transcripts, and a rapid loss of viability of replicating and non-replicating M. tuberculosis in vitro and during acute and chronic infection in mice. Collectively, these data suggest that inhibition of Rho may provide an alternative strategy to treat tuberculosis with an efficacy similar to inhibition of RNA polymerase. PMID:28348398

  10. Depleting Mycobacterium tuberculosis of the transcription termination factor Rho causes pervasive transcription and rapid death.

    PubMed

    Botella, Laure; Vaubourgeix, Julien; Livny, Jonathan; Schnappinger, Dirk

    2017-03-28

    Rifampicin, which inhibits bacterial RNA polymerase, provides one of the most effective treatments for tuberculosis. Inhibition of the transcription termination factor Rho is used to treat some bacterial infections, but its importance varies across bacteria. Here we show that Rho of Mycobacterium tuberculosis functions to both define the 3' ends of mRNAs and silence substantial fragments of the genome. Brief inactivation of Rho affects over 500 transcripts enriched for genes of foreign DNA elements and bacterial virulence factors. Prolonged inactivation of Rho causes extensive pervasive transcription, a genome-wide increase in antisense transcripts, and a rapid loss of viability of replicating and non-replicating M. tuberculosis in vitro and during acute and chronic infection in mice. Collectively, these data suggest that inhibition of Rho may provide an alternative strategy to treat tuberculosis with an efficacy similar to inhibition of RNA polymerase.

  11. The Smad3 linker region contains a transcriptional activation domain.

    PubMed

    Wang, Guannan; Long, Jianyin; Matsuura, Isao; He, Dongming; Liu, Fang

    2005-02-15

    Transforming growth factor-beta (TGF-beta)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-beta/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-beta transcriptional activation responses, although it can be phosphorylated by the TGF-beta receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-beta. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control.

  12. HIFs Enhance the Transcriptional Activation and Splicing of Adrenomedullin

    PubMed Central

    Sena, Johnny A.; Wang, Liyi; Pawlus, Matthew R.; Hu, Cheng-Jun

    2014-01-01

    Adrenomedullin (ADM) is important for tumor angiogenesis, tumor cell growth and survival. Under normoxic conditions, the ADM gene was found to produce two alternative transcripts, a fully-spliced transcript that produces AM and PAMP peptides and a intron-3-retaining transcript that produces a less functionally significant PAMP peptide only. ADM is a well-established hypoxia inducible gene; however, it is not clear which ADM isoform is induced by hypoxia. In this study, it was determined that various cancer and normal cells express two predominant types of ADM transcripts, a AM/PAMP peptide producing FL transcript in which all introns are removed, and a non-protein producing I1-3 transcript in which all introns are retained. Interestingly, hypoxia preferentially induced the FL isoform. Moreover, HIFs, but not hypoxia per se are necessary and sufficient to increase splicing of ADM pre-mRNA. ADM splicing reporters confirmed that transcriptional activation by HIF or other transcription factors is sufficient to enhance splicing. However, HIFs are more potent in enhancing ADM pre-mRNA splicing than other transcriptional activators. Thus, ADM intron retention is not a consequence of abnormal splicing, but is an important mechanism to regulate ADM expression. These results demonstrate a novel function of HIFs in regulating ADM expression by enhancing its pre-mRNA splicing. Importantly, using endogenous and cloned ADM gene, further evidence is provided for the coupling of transcription and RNA splicing. PMID:24523299

  13. Natural antisense and noncoding RNA transcripts as potential drug targets.

    PubMed

    Wahlestedt, Claes

    2006-06-01

    Information on the complexity of mammalian RNA transcription has increased greatly in the past few years. Notably, thousands of sense transcripts (conventional protein-coding genes) have antisense transcript partners, most of which are noncoding. Interestingly, a number of antisense transcripts regulate the expression of their sense partners, either in a discordant (antisense knockdown results in sense-transcript elevation) or concordant (antisense knockdown results in concomitant sense-transcript reduction) manner. Two new pharmacological strategies based on the knockdown of antisense RNA transcripts by siRNA (or another RNA targeting principle) are proposed in this review. In the case of discordant regulation, knockdown of antisense transcript elevates the expression of the conventional (sense) gene, thereby conceivably mimicking agonist-activator action. In the case of concordant regulation, knockdown of antisense transcript, or concomitant knockdown of antisense and sense transcripts, results in an additive or even synergistic reduction of the conventional gene expression. Although both strategies have been demonstrated to be valid in cell culture, it remains to be seen whether they provide advantages in other contexts.

  14. The Transcription Unit Architecture of the Escherichia Coli Genome

    SciTech Connect

    Cho, Byung-Kwan; Zengler, Karsten; Qiu, Yu; Park, Young S.; Knight, Eric M.; Barrett, Christian; Gao, Yuan; Palsson, Bernhard O.

    2009-11-01

    Under EMSL User Proposal 25660, the authors reported that bacterial genomes are organized by structural and functional elements, including promoters, transcription start and termination sites, open reading frames, regulatory noncoding regions, untranslated regions and transcription units. Here, we iteratively integrate high-throughput, genome-wide measurements of RNA polymerase binding locations and mRNA transcript abundance, 5' sequences and translation into proteins to determine the organizational structure of the Escherichia coli K-12 MG1655 genome. Integration of the organizational elements provides an experimentally annotated transcription unit architecture, including alternative transcription start sites, 5' untranslated region, boundaries and open reading frames of each transcription unit. A total of 4,661 transcription units were identified, representing an increase of >530% over current knowledge. This comprehensive transcription unit architecture allows for the elucidation of condition-specific uses of alternative sigma factors at the genome scale. Furthermore, the transcription unit architecture provides a foundation on which to construct genome-scale transcriptional and translational regulatory networks.

  15. Transcription bypass of DNA lesions enhances cell survival but attenuates transcription coupled DNA repair.

    PubMed

    Li, Wentao; Selvam, Kathiresan; Ko, Tengyu; Li, Shisheng

    2014-12-01

    Transcription-coupled DNA repair (TCR) is a subpathway of nucleotide excision repair (NER) dedicated to rapid removal of DNA lesions in the transcribed strand of actively transcribed genes. The precise nature of the TCR signal and how the repair machinery gains access to lesions imbedded in stalled RNA polymerase II (RNAP II) complexes in eukaryotic cells are still enigmatic. RNAP II has an intrinsic capacity for transcription bypass of DNA lesions by incorporation or misincorporation of nucleotides across the lesions. It has been suggested that transcription bypass of lesions, which exposes the lesions, may be required for TCR. Here, we show that E1103G mutation of Rpb1, the largest subunit of RNAP II, which promotes transcription bypass of UV-induced cyclobutane pyrimidine dimers (CPDs), increases survival of UV irradiated yeast cells but attenuates TCR. The increased cell survival is independent of any NER subpathways. In contrast, G730D mutation of Rpb1, which impairs transcription bypass of CPDs, enhances TCR. Our results suggest that transcription bypass of lesions attenuates TCR but enhances cell tolerance to DNA lesions. Efficient stalling of RNAP II is essential for efficient TCR. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Transcriptional Regulatory Elements in Fungal Secondary Metabolism

    PubMed Central

    Yin, Wenbing; Keller, Nancy P.

    2013-01-01

    Filamentous fungi produce a variety of secondary metabolites of diverse beneficial and detrimental activities to humankind. The genes encoding the enzymatic machinery required to make these metabolites are typically clustered in fungal genomes. There is considerable evidence that secondary metabolite gene regulation is, in part, by transcriptional control through hierarchical levels of transcriptional regulatory elements involved in secondary metabolite cluster regulation. Identification of secondary metabolism regulatory elements could potentially provide a means of increasing production of beneficial metabolites, decreasing production of detrimental metabolites, aid in the identification of ‘silent’ natural products and also contribute to a broader understanding of molecular mechanisms by which secondary metabolites are produced. This review summarizes regulation of secondary metabolism associated on transcriptional regulatory elements from a broad view as well as tremendous advances in discovery of cryptic or novel secondary metabolites by genomic mining in the basis of this knowledge. PMID:21717315

  17. Epigenetic regulation of transcription in intermediate heterochromatin.

    PubMed

    Habu, Yoshiki; Mathieu, Olivier; Tariq, Muhammad; Probst, Aline V; Smathajitt, Chotika; Zhu, Tong; Paszkowski, Jerzy

    2006-12-01

    Constitutive heterochromatin is a compact, transcriptionally inert structure formed in gene-poor and repeat- and transposon-rich regions. In Arabidopsis, constitutive heterochromatin is characterized by hypermethylated DNA and histone H3 dimethylated at lysine (K) 9 (H3K9me2) together with depletion of histone H3 dimethylated at lysine 4 (H3K4me2). Here, we describe loci with intermediate properties of heterochromatin in which transcription downregulation is inherited in a manner similar to constitutive heterochromatin, although the loci are associated with opposing histone marks--H3K4me2 and H3K9me2. In the ddm1 (decrease in DNA methylation 1) mutants, their transcriptional activation is accompanied by the expected shift in the H3 modifications--depletion of H3K9me2 and enrichment in H3K4me2. In mom1 (Morpheus' molecule 1) mutants, however, a marked increase in transcription is not accompanied by detectable changes in the levels of H3K4me2 and H3K9me2. Therefore, transcriptional regulation in the intermediate heterochromatin involves two distinct epigenetic mechanisms. Interestingly, silent transgenic inserts seem to acquire properties characteristic of the intermediate heterochromatin.

  18. Coupling pre-mRNA processing to transcription on the RNA factory assembly line

    PubMed Central

    Lee, Kuo-Ming; Tarn, Woan-Yuh

    2013-01-01

    It has been well-documented that nuclear processing of primary transcripts of RNA polymerase II occurs co-transcriptionally and is functionally coupled to transcription. Moreover, increasing evidence indicates that transcription influences pre-mRNA splicing and even several post-splicing RNA processing events. In this review, we discuss the issues of how RNA polymerase II modulates co-transcriptional RNA processing events via its carboxyl terminal domain, and the protein domains involved in coupling of transcription and RNA processing events. In addition, we describe how transcription influences the expression or stability of mRNAs through the formation of distinct mRNP complexes. Finally, we delineate emerging findings that chromatin modifications function in the regulation of RNA processing steps, especially splicing, in addition to transcription. Overall, we provide a comprehensive view that transcription could integrate different control systems, from epigenetic to post-transcriptional control, for efficient gene expression. PMID:23392244

  19. Regulated assembly of transcription factors and control of transcription initiation.

    PubMed

    Beckett, D

    2001-11-30

    Proteins that function in regulation of transcription initiation are typically homo or hetero-oligomeric. Results of recent biophysical studies of transcription regulators indicate that the assembly of these proteins is often subject to regulation. This regulation of assembly dictates the frequency of transcription initiation via its influence on the affinity of a transcription regulator for DNA and its affect on target site selection. Factors that modulate transcription factor assembly include binding of small molecules, post-translational modification, DNA binding and interactions with other proteins. Here, the results of recent structural and/or thermodynamic studies of a number of transcription regulators that are subject to regulated assembly are reviewed. The accumulated data indicate that this phenomenon is ubiquitous and that mechanisms utilized in eukaryotes and prokaryotes share common features. Copyright 2001 Academic Press.

  20. [Genetic transcription in eukaryotes: from transcriptional factors to disease].

    PubMed

    Moreno Rocha, J C; Revol de Mendoza, A; Barrera Saldaña, H A

    1999-01-01

    The organisms' genetic information is stored as DNA sequences: the genes. The most important level of gene expression regulation is exerted at the transfer process of this information from the genes into messenger RNA molecules; this process is called transcription and is carried out by a molecular machinery conformed by hundreds of different proteins which are assembled in an ordered step way. These proteins or transcriptional factors are classified according to their mode of action in 4 groups: general transcriptional factors, activators, coactivators and repressors. There are diseases like. Aniridia, the Rubinstein-Taybi syndrome and Hodgkin's disease, in which some transcriptional factor have been involved and in some, the molecular cause i.e. the mutations responsible for the molecular dysfunction in a transcriptional factor has been elucidated. Understanding at the molecular level the transcription process will help to comprehend the relationship of it with the development and health of the organism.

  1. Home Study Academic Transcripts. NHSC Occasional Paper Number 10.

    ERIC Educational Resources Information Center

    Welch, Sally R.

    Academic transcripts are becoming increasingly important in home study environments because they are gaining wider use as "sales tools" by home study program graduates seeking acceptance of their home study work by employers and registrars. Well-designed transcripts have become an excellent alumni service and subtle marketing device that all…

  2. Transcriptional control of secondary growth and wood formation.

    Treesearch

    Juan Du; Andrew Groover

    2010-01-01

    Secondary growth and wood formation are products of the vascular cambium, a lateral meristem. Although the mechanisms have only recently begun to be uncovered, transcriptional regulation appears increasingly central to the regulation of secondary growth. The importance of transcriptional regulation is illustrated by the correlation of expression of specific classes of...

  3. Transcriptional profiling of lymphoblast lines from subjects with panic disorder.

    PubMed

    Philibert, Robert A; Crowe, Raymond; Ryu, Gi-Yung; Yoon, Jae-Geun; Secrest, Dianna; Sandhu, Harinder; Madan, Anup

    2007-07-05

    In attempts to isolate genetic vulnerability factors for panic disorder (PD), a number of investigators have used genome-wide linkage or association analyses. But these attempts have been only modestly successful which suggests that alternative approaches may be needed to define the biology of PD. Therefore, using recently developed genome-wide gene expression profiling, we explored whether transcriptional signatures associated with PD are present in lymphoblast cell line. The expression of 2,469 transcripts in lymphoblast cell lines from 16 subjects was arithmetically increased in every line and significantly increased overall and 354 transcripts was arithmetically decreased in every cell line and significantly decreased overall as compared to those lymphoblast lines from 17 subjects without a history of behavioral illness. Further sex specific analyses showed that in those 10 lines derived from female probands, the expression of a further 67 transcripts was arithmetically increased in every line and significantly increased overall and a further 332 transcripts was arithmetically decreased in every cell line and significantly decreased. Conversely, in cell lines from the six male probands, the expression of an additional 212 was arithmetically increased in every line and significantly increased overall and a further 332 transcripts was arithmetically decreased in every cell line. We conclude that lymphoblast cell lines derived from subjects with PD have significant, partially sex dependent changes in gene transcription. Further studies are necessary to correlate these changes in these hemopoetically derived cells with those changes postulated to occur in the CNS in association with PD.

  4. Ubiquitin and Proteasomes in Transcription

    PubMed Central

    Geng, Fuqiang; Wenzel, Sabine; Tansey, William P.

    2013-01-01

    Regulation of gene transcription is vitally important for the maintenance of normal cellular homeostasis. Failure to correctly regulate gene expression, or to deal with problems that arise during the transcription process, can lead to cellular catastrophe and disease. One of the ways cells cope with the challenges of transcription is by making extensive use of the proteolytic and nonproteolytic activities of the ubiquitin-proteasome system (UPS). Here, we review recent evidence showing deep mechanistic connections between the transcription and ubiquitin-proteasome systems. Our goal is to leave the reader with a sense that just about every step in transcription—from transcription initiation through to export of mRNA from the nucleus—is influenced by the UPS and that all major arms of the system—from the first step in ubiquitin (Ub) conjugation through to the proteasome—are recruited into transcriptional processes to provide regulation, directionality, and deconstructive power. PMID:22404630

  5. Transients in chloroplast gene transcription

    SciTech Connect

    Puthiyaveetil, Sujith; Allen, John F.

    2008-04-18

    Transcriptional regulation of chloroplast genes is demonstrated by Quantitative Polymerase Chain Reaction (qPCR). These genes encode apoproteins of the reaction centres of photosystem I and photosystem II. Their transcription is regulated by changes in wavelength of light selectively absorbed by photosystem I and photosystem II, and therefore by the redox state of an electron carrier located between the two photosystems. Chloroplast transcriptional redox regulation is shown to have greater amplitude, and the kinetics of transcriptional changes are more complex, than suggested by previous experiments using only DNA probes in Northern blot experiments. Redox effects on chloroplast transcription appear to be superimposed on an endogenous rhythm of mRNA abundance. The functional significance of these transients in chloroplast gene transcription is discussed.

  6. DNA topology and transcription

    PubMed Central

    Kouzine, Fedor; Levens, David; Baranello, Laura

    2014-01-01

    Chromatin is a complex assembly that compacts DNA inside the nucleus while providing the necessary level of accessibility to regulatory factors conscripted by cellular signaling systems. In this superstructure, DNA is the subject of mechanical forces applied by variety of molecular motors. Rather than being a rigid stick, DNA possesses dynamic structural variability that could be harnessed during critical steps of genome functioning. The strong relationship between DNA structure and key genomic processes necessitates the study of physical constrains acting on the double helix. Here we provide insight into the source, dynamics, and biology of DNA topological domains in the eukaryotic cells and summarize their possible involvement in gene transcription. We emphasize recent studies that might inspire and impact future experiments on the involvement of DNA topology in cellular functions. PMID:24755522

  7. Transcriptional elongation factor ENL phosphorylated by ATM recruits polycomb and switches off transcription for DSB repair.

    PubMed

    Ui, Ayako; Nagaura, Yuko; Yasui, Akira

    2015-05-07

    Transcription is repressed if a DNA double-strand break (DSB) is introduced in close proximity to a transcriptional activation site at least in part by H2A-ubiquitination. While ATM signaling is involved, how it controls H2A-ubiquitination remains unclear. Here, we identify that, in response to DSBs, a transcriptional elongation factor, ENL (MLLT1), is phosphorylated by ATM at conserved SQ sites. This phosphorylation increases the interaction between ENL and the E3-ubiquitin-ligase complex of Polycomb Repressive Complex 1 (PRC1) via BMI1. This interaction promotes enrichment of PRC1 at transcription elongation sites near DSBs to ubiquitinate H2A leading to transcriptional repression. ENL SQ sites and BMI1 are necessary for KU70 accumulation at DSBs near active transcription sites and cellular resistance to DSBs. Our data suggest that ATM-dependent phosphorylation of ENL functions as switch from elongation to Polycomb-mediated repression to preserve genome integrity. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Imaging Transcription in Living Cells

    PubMed Central

    Darzacq, Xavier; Yao, Jie; Larson, Daniel R.; Causse, Sebastien Z.; Bosanac, Lana; de Turris, Valeria; Ruda, Vera M.; Lionnet, Timothee; Zenklusen, Daniel; Guglielmi, Benjamin; Tjian, Robert; Singer, Robert H.

    2011-01-01

    The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding. PMID:19416065

  9. Promoter-mediated transcriptional dynamics.

    PubMed

    Zhang, Jiajun; Zhou, Tianshou

    2014-01-21

    Genes in eukaryotic cells are typically regulated by complex promoters containing multiple binding sites for a variety of transcription factors, but how promoter dynamics affect transcriptional dynamics has remained poorly understood. In this study, we analyze gene models at the transcriptional regulation level, which incorporate the complexity of promoter structure (PS) defined as transcriptional exits (i.e., ON states of the promoter) and the transition pattern (described by a matrix consisting of transition rates among promoter activity states). We show that multiple exits of transcription are the essential origin of generating multimodal distributions of mRNA, but promoters with the same transition pattern can lead to multimodality of different modes, depending on the regulation of transcriptional factors. In turn, for similar mRNA distributions in the models, the mean ON or OFF time distributions may exhibit different characteristics, thus providing the supplemental information on PS. In addition, we demonstrate that the transcriptional noise can be characterized by a nonlinear function of mean ON and OFF times. These results not only reveal essential characteristics of promoter-mediated transcriptional dynamics but also provide signatures useful for inferring PS based on characteristics of transcriptional outputs. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Agouti regulates adipocyte transcription factors.

    PubMed

    Mynatt, R L; Stephens, J M

    2001-04-01

    Agouti is a secreted paracrine factor that regulates pigmentation in hair follicle melanocytes. Several dominant mutations cause ectopic expression of agouti, resulting in a phenotype characterized by yellow fur, adult-onset obesity and diabetes, increased linear growth and skeletal mass, and increased susceptibility to tumors. Humans also produce agouti protein, but the highest levels of agouti in humans are found in adipose tissue. To mimic the human agouti expression pattern in mice, transgenic mice (aP2-agouti) that express agouti in adipose tissue were generated. The transgenic mice develop a mild form of obesity, and they are sensitized to the action of insulin. We correlated the levels of specific regulators of insulin signaling and adipocyte differentiation with these phenotypic changes in adipose tissue. Signal transducers and activators of transcription (STAT)1, STAT3, and peroxisome proliferator-activated receptor (PPAR)-gamma protein levels were elevated in the transgenic mice. Treatment of mature 3T3-L1 adipocytes recapitulated these effects. These data demonstrate that agouti has potent effects on adipose tissue. We hypothesize that agouti increases adiposity and promotes insulin sensitivity by acting directly on adipocytes via PPAR-gamma.

  11. Transcriptional landscape of the human cell cycle.

    PubMed

    Liu, Yin; Chen, Sujun; Wang, Su; Soares, Fraser; Fischer, Martin; Meng, Feilong; Du, Zhou; Lin, Charles; Meyer, Clifford; DeCaprio, James A; Brown, Myles; Liu, X Shirley; He, Housheng Hansen

    2017-03-28

    Steady-state gene expression across the cell cycle has been studied extensively. However, transcriptional gene regulation and the dynamics of histone modification at different cell-cycle stages are largely unknown. By applying a combination of global nuclear run-on sequencing (GRO-seq), RNA sequencing (RNA-seq), and histone-modification Chip sequencing (ChIP-seq), we depicted a comprehensive transcriptional landscape at the G0/G1, G1/S, and M phases of breast cancer MCF-7 cells. Importantly, GRO-seq and RNA-seq analysis identified different cell-cycle-regulated genes, suggesting a lag between transcription and steady-state expression during the cell cycle. Interestingly, we identified genes actively transcribed at early M phase that are longer in length and have low expression and are accompanied by a global increase in active histone 3 lysine 4 methylation (H3K4me2) and histone 3 lysine 27 acetylation (H3K27ac) modifications. In addition, we identified 2,440 cell-cycle-regulated enhancer RNAs (eRNAs) that are strongly associated with differential active transcription but not with stable expression levels across the cell cycle. Motif analysis of dynamic eRNAs predicted Kruppel-like factor 4 (KLF4) as a key regulator of G1/S transition, and this identification was validated experimentally. Taken together, our combined analysis characterized the transcriptional and histone-modification profile of the human cell cycle and identified dynamic transcriptional signatures across the cell cycle.

  12. The LIM Homeodomain Transcription Factor LHX6

    PubMed Central

    Zhang, Zichao; Gutierrez, Diana; Li, Xiao; Bidlack, Felicitas; Cao, Huojun; Wang, Jianbo; Andrade, Kelsey; Margolis, Henry C.; Amendt, Brad A.

    2013-01-01

    LHX6 is a LIM-homeobox transcription factor expressed during embryogenesis; however, the molecular mechanisms regulating LHX6 transcriptional activities are unknown. LHX6 and the PITX2 homeodomain transcription factor have overlapping expression patterns during tooth and craniofacial development, and in this report, we demonstrate new transcriptional mechanisms for these factors. PITX2 and LHX6 are co-expressed in the oral and dental epithelium and epithelial cell lines. Lhx6 expression is increased in Pitx2c transgenic mice and decreased in Pitx2 null mice. PITX2 activates endogenous Lhx6 expression and the Lhx6 promoter, whereas LHX6 represses its promoter activity. Chromatin immunoprecipitation experiments reveal endogenous PITX2 binding to the Lhx6 promoter. LHX6 directly interacts with PITX2 to inhibit PITX2 transcriptional activities and activation of multiple promoters. Bimolecular fluorescence complementation assays reveal an LHX6·PITX2 nuclear interaction in living cells. LHX6 has a dominant repressive effect on the PITX2 synergistic activation with LEF-1 and β-catenin co-factors. Thus, LHX6 acts as a transcriptional repressor and represses the expression of several genes involved in odontogenesis. We have identified specific defects in incisor, molar, mandible, bone, and root development and late stage enamel formation in Lhx6 null mice. Amelogenin and ameloblastin expression is reduced and/or delayed in the Lhx6 null mice, potentially resulting from defects in dentin deposition and ameloblast differentiation. Our results demonstrate that LHX6 regulates cell proliferation in the cervical loop and promotes cell differentiation in the anterior region of the incisor. We demonstrate new molecular mechanisms for LHX6 and an interaction with PITX2 for normal craniofacial and tooth development. PMID:23229549

  13. Stochastic models of transcription: from single molecules to single cells.

    PubMed

    Sanchez, Alvaro; Choubey, Sandeep; Kondev, Jane

    2013-07-15

    Genes in prokaryotic and eukaryotic cells are typically regulated by complex promoters containing multiple binding sites for a variety of transcription factors leading to a specific functional dependence between regulatory inputs and transcriptional outputs. With increasing regularity, the transcriptional outputs from different promoters are being measured in quantitative detail in single-cell experiments thus providing the impetus for the development of quantitative models of transcription. We describe recent progress in developing models of transcriptional regulation that incorporate, to different degrees, the complexity of multi-state promoter dynamics, and its effect on the transcriptional outputs of single cells. The goal of these models is to predict the statistical properties of transcriptional outputs and characterize their variability in time and across a population of cells, as a function of the input concentrations of transcription factors. The interplay between mathematical models of different regulatory mechanisms and quantitative biophysical experiments holds the promise of elucidating the molecular-scale mechanisms of transcriptional regulation in cells, from bacteria to higher eukaryotes. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Transcription factor IIS impacts UV-inhibited transcription.

    PubMed

    Jensen, Anne; Mullenders, Leon H F

    2010-11-10

    Inhibition of transcription elongation can cause severe developmental and neurological abnormalities notably manifested by the rare recessive progeroid disorder Cockayne syndrome (CS). DNA alterations can cause permanent blocks to an elongating RNA polymerase II (RNAPII) leading to transcriptional arrest. Abrogation of transcription arrest requires removal of transcription blocking lesions through transcription-coupled nucleotide excision repair (TC-NER) a process defective in CS. Transcription elongation factor IIS (TFIIS) has been found to localize with the TC-NER complex after cellular exposure to UV-C light and in vitro addition of TFIIS to a damage arrested RNAPII causes transcript shortening. Hence default TFIIS activity might mimic or contribute to the severe phenotype of Cockayne syndrome. Here we show that down regulation of TFIIS by siRNA treatment of human cells lead to impaired RNA synthesis recovery and elevated levels of hyper-phosphorylated RNAPII after UV-irradiation. TFIIS knock down does not affect TC-NER, the reappearance of hypo-phosphorylated RNAPII post-UV-irradiation, UV sensitivity or the p53 damage response. These findings reveal a role for TFIIS in transcription recovery and re-establishment of the balance between hypo- and hyper-phosphorylated RNAPII after DNA damage repair. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. Mechanosensitive mechanisms in transcriptional regulation.

    PubMed

    Mammoto, Akiko; Mammoto, Tadanori; Ingber, Donald E

    2012-07-01

    Transcriptional regulation contributes to the maintenance of pluripotency, self-renewal and differentiation in embryonic cells and in stem cells. Therefore, control of gene expression at the level of transcription is crucial for embryonic development, as well as for organogenesis, functional adaptation, and regeneration in adult tissues and organs. In the past, most work has focused on how transcriptional regulation results from the complex interplay between chemical cues, adhesion signals, transcription factors and their co-regulators during development. However, chemical signaling alone is not sufficient to explain how three-dimensional (3D) tissues and organs are constructed and maintained through the spatiotemporal control of transcriptional activities. Accumulated evidence indicates that mechanical cues, which include physical forces (e.g. tension, compression or shear stress), alterations in extracellular matrix (ECM) mechanics and changes in cell shape, are transmitted to the nucleus directly or indirectly to orchestrate transcriptional activities that are crucial for embryogenesis and organogenesis. In this Commentary, we review how the mechanical control of gene transcription contributes to the maintenance of pluripotency, determination of cell fate, pattern formation and organogenesis, as well as how it is involved in the control of cell and tissue function throughout embryogenesis and adult life. A deeper understanding of these mechanosensitive transcriptional control mechanisms should lead to new approaches to tissue engineering and regenerative medicine.

  16. Mechanosensitive mechanisms in transcriptional regulation

    PubMed Central

    Mammoto, Akiko; Mammoto, Tadanori; Ingber, Donald E.

    2012-01-01

    Summary Transcriptional regulation contributes to the maintenance of pluripotency, self-renewal and differentiation in embryonic cells and in stem cells. Therefore, control of gene expression at the level of transcription is crucial for embryonic development, as well as for organogenesis, functional adaptation, and regeneration in adult tissues and organs. In the past, most work has focused on how transcriptional regulation results from the complex interplay between chemical cues, adhesion signals, transcription factors and their co-regulators during development. However, chemical signaling alone is not sufficient to explain how three-dimensional (3D) tissues and organs are constructed and maintained through the spatiotemporal control of transcriptional activities. Accumulated evidence indicates that mechanical cues, which include physical forces (e.g. tension, compression or shear stress), alterations in extracellular matrix (ECM) mechanics and changes in cell shape, are transmitted to the nucleus directly or indirectly to orchestrate transcriptional activities that are crucial for embryogenesis and organogenesis. In this Commentary, we review how the mechanical control of gene transcription contributes to the maintenance of pluripotency, determination of cell fate, pattern formation and organogenesis, as well as how it is involved in the control of cell and tissue function throughout embryogenesis and adult life. A deeper understanding of these mechanosensitive transcriptional control mechanisms should lead to new approaches to tissue engineering and regenerative medicine. PMID:22797927

  17. Adaptation with transcriptional regulation

    NASA Astrophysics Data System (ADS)

    Shi, Wenjia; Ma, Wenzhe; Xiong, Liyang; Zhang, Mingyue; Tang, Chao

    2017-02-01

    Biochemical adaptation is one of the basic functions that are widely implemented in biological systems for a variety of purposes such as signal sensing, stress response and homeostasis. The adaptation time scales span from milliseconds to days, involving different regulatory machineries in different processes. The adaptive networks with enzymatic regulation (ERNs) have been investigated in detail. But it remains unclear if and how other forms of regulation will impact the network topology and other features of the function. Here, we systematically studied three-node transcriptional regulatory networks (TRNs), with three different types of gene regulation logics. We found that the topologies of adaptive gene regulatory networks can still be grouped into two general classes: negative feedback loop (NFBL) and incoherent feed-forward loop (IFFL), but with some distinct topological features comparing to the enzymatic networks. Specifically, an auto-activation loop on the buffer node is necessary for the NFBL class. For IFFL class, the control node can be either a proportional node or an inversely-proportional node. Furthermore, the tunability of adaptive behavior differs between TRNs and ERNs. Our findings highlight the role of regulation forms in network topology, implementation and dynamics.

  18. Adaptation with transcriptional regulation

    PubMed Central

    Shi, Wenjia; Ma, Wenzhe; Xiong, Liyang; Zhang, Mingyue; Tang, Chao

    2017-01-01

    Biochemical adaptation is one of the basic functions that are widely implemented in biological systems for a variety of purposes such as signal sensing, stress response and homeostasis. The adaptation time scales span from milliseconds to days, involving different regulatory machineries in different processes. The adaptive networks with enzymatic regulation (ERNs) have been investigated in detail. But it remains unclear if and how other forms of regulation will impact the network topology and other features of the function. Here, we systematically studied three-node transcriptional regulatory networks (TRNs), with three different types of gene regulation logics. We found that the topologies of adaptive gene regulatory networks can still be grouped into two general classes: negative feedback loop (NFBL) and incoherent feed-forward loop (IFFL), but with some distinct topological features comparing to the enzymatic networks. Specifically, an auto-activation loop on the buffer node is necessary for the NFBL class. For IFFL class, the control node can be either a proportional node or an inversely-proportional node. Furthermore, the tunability of adaptive behavior differs between TRNs and ERNs. Our findings highlight the role of regulation forms in network topology, implementation and dynamics. PMID:28233824

  19. Adaptation with transcriptional regulation.

    PubMed

    Shi, Wenjia; Ma, Wenzhe; Xiong, Liyang; Zhang, Mingyue; Tang, Chao

    2017-02-24

    Biochemical adaptation is one of the basic functions that are widely implemented in biological systems for a variety of purposes such as signal sensing, stress response and homeostasis. The adaptation time scales span from milliseconds to days, involving different regulatory machineries in different processes. The adaptive networks with enzymatic regulation (ERNs) have been investigated in detail. But it remains unclear if and how other forms of regulation will impact the network topology and other features of the function. Here, we systematically studied three-node transcriptional regulatory networks (TRNs), with three different types of gene regulation logics. We found that the topologies of adaptive gene regulatory networks can still be grouped into two general classes: negative feedback loop (NFBL) and incoherent feed-forward loop (IFFL), but with some distinct topological features comparing to the enzymatic networks. Specifically, an auto-activation loop on the buffer node is necessary for the NFBL class. For IFFL class, the control node can be either a proportional node or an inversely-proportional node. Furthermore, the tunability of adaptive behavior differs between TRNs and ERNs. Our findings highlight the role of regulation forms in network topology, implementation and dynamics.

  20. Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.

    PubMed

    Cecere, Germano; Hoersch, Sebastian; O'Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

    2014-04-01

    Argonaute proteins and their small RNA cofactors short interfering RNAs are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) that are antisense to germline transcripts. However, its role in gene expression regulation remains controversial. Here we used genome-wide profiling of nascent RNA transcripts and found that the CSR-1 RNA interference pathway promoted sense-oriented RNA polymerase II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. On the basis of these findings, we propose that the CSR-1 pathway helps maintain the directionality of active transcription, thereby propagating the distinction between transcriptionally active and silent genomic regions.

  1. Mutually exclusive sense–antisense transcription at FLC facilitates environmentally induced gene repression

    PubMed Central

    Rosa, Stefanie; Duncan, Susan; Dean, Caroline

    2016-01-01

    Antisense transcription through genic regions is pervasive in most genomes; however, its functional significance is still unclear. We are studying the role of antisense transcripts (COOLAIR) in the cold-induced, epigenetic silencing of Arabidopsis FLOWERING LOCUS C (FLC), a regulator of the transition to reproduction. Here we use single-molecule RNA FISH to address the mechanistic relationship of FLC and COOLAIR transcription at the cellular level. We demonstrate that while sense and antisense transcripts can co-occur in the same cell they are mutually exclusive at individual loci. Cold strongly upregulates COOLAIR transcription in an increased number of cells and through the mutually exclusive relationship facilitates shutdown of sense FLC transcription in cis. COOLAIR transcripts form dense clouds at each locus, acting to influence FLC transcription through changed H3K36me3 dynamics. These results may have general implications for other loci showing both sense and antisense transcription. PMID:27713408

  2. Transcriptional regulation during Drosophila spermatogenesis

    PubMed Central

    Lim, Cindy; Tarayrah, Lama; Chen, Xin

    2012-01-01

    Drosophila spermatogenesis has become a paradigmatic system for the study of mechanisms that regulate adult stem cell maintenance, proliferation and differentiation. The dramatic cellular differentiation process from germline stem cell (GSC) to mature sperm is accompanied by dynamic changes in gene expression, which are regulated at transcriptional, post-transcriptional (including translational) and post-translational levels. Post-transcriptional regulation has been proposed as a unique feature of germ cells. However, recent studies have provided new insights into transcriptional regulation during Drosophila spermatogenesis. Both signaling pathways and epigenetic mechanisms act to orchestrate the transcriptional regulation of distinct genes at different germ cell differentiation stages. Many of the regulatory pathways that control male gamete differentiation in Drosophila are conserved in mammals. Therefore, studies using Drosophila spermatogenesis will provide insight into the molecular mechanisms that regulate mammalian germ cell differentiation pathways. PMID:23087835

  3. Structural basis of transcription elongation.

    PubMed

    Martinez-Rucobo, Fuensanta W; Cramer, Patrick

    2013-01-01

    For transcription elongation, all cellular RNA polymerases form a stable elongation complex (EC) with the DNA template and the RNA transcript. Since the millennium, a wealth of structural information and complementary functional studies provided a detailed three-dimensional picture of the EC and many of its functional states. Here we summarize these studies that elucidated EC structure and maintenance, nucleotide selection and addition, translocation, elongation inhibition, pausing and proofreading, backtracking, arrest and reactivation, processivity, DNA lesion-induced stalling, lesion bypass, and transcriptional mutagenesis. In the future, additional structural and functional studies of elongation factors that control the EC and their possible allosteric modes of action should result in a more complete understanding of the dynamic molecular mechanisms underlying transcription elongation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Structural basis of transcription activation.

    PubMed

    Feng, Yu; Zhang, Yu; Ebright, Richard H

    2016-06-10

    Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)-promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σ(A) holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme. Copyright © 2016, American Association for the Advancement of Science.

  5. Thermodynamic Model of Transcription Elongation

    NASA Astrophysics Data System (ADS)

    Tadigotla, Vasisht; O'Maoileidigh, Daibhid; Sengupta, Anirvan; Epshtein, Vitaly; Ebright, Richard; Nudler, Evgeny; Ruckenstein, Andrei

    2006-03-01

    We present a statistical mechanics approach to the prediction of backtracked pauses in prokaryotic transcription elongation derived from structural models of the transcription elongation complex (TEC). Our algorithm is based on the thermodynamic stability of TEC along the DNA template calculated from the sequence dependent free-energy of DNA-DNA, DNA-RNA and RNA-RNA base pairing associated with (a) the translocation and size fluctuations of the transcription bubble; (b) the changes in the DNA-RNA hybrid; and (c) the changes in the RNA folding free-energy. The calculations involve no adjustable parameters apart from a cutoff used to discriminate paused from non-paused complexes. When applied to 100 experimental pauses in transcription elongation by E. coli RNA polymerase on ten DNA templates the approach produces highly statistically significant results. Transcription elongation is an inherently kinetic process and a simplified kinetic model with the same predictive power is presented separately.

  6. Ultraviolet B Regulation of Transcription Factor Families

    PubMed Central

    Cooper, S.J.; Bowden, G.T.

    2008-01-01

    Prolonged and repeated exposure of the skin to ultraviolet light (UV) leads not only to aging of the skin but also increases the incidence of non-melanoma skin cancer (NMSC). Damage of cells induced by ultraviolet B (UVB) light both at the DNA level and molecular level initiates the activation of transcription factor pathways, which in turn regulate the expression of a number of genes termed the “UV response genes”. Two such transcription factor families that are activated in this way are those of the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) families. These two transcription factor families have been identified to be involved in the processes of cell proliferation, cell differentiation and cell survival and therefore play important roles in tumorigenesis. The study of these two transcription factor pathways and the cross-talk between them in response to UVB exposure may help with the development of new chemopreventive strategies for the prevention of UVB-induced skin carcinogenesis. PMID:17979627

  7. Effect of selenium deficiency on gene transcription

    SciTech Connect

    Christensen, M.J.; Burgener, K.W. )

    1991-03-11

    To investigate the general effects of dietary selenium (Se) deficiency on gene transcription, weanling male Sprague-Dawley rats were fed a basal Se-deficient Torula yeast-based diet or the same diet supplemented with 0.5 ppm Se as sodium selenite for 40 days. At that time three rats in each dietary group were sacrificed. Livers were excised and divided into two portions for isolation of nuclei and for assay of cytosolic Se-glutathione peroxidase (Se-GPX) activity. Se-GPX activity was 279 {plus minus} 4 (mean {plus minus} SEM) mUnits/mg protein in Se-adequate livers, and 10 {plus minus} 2 mUnits/mg protein in Se-deficient livers. One aliquot of nuclei from each dietary group was used in a run-on transcription assay, employing {alpha}-{sup 32}P-UTP to label nascent transcripts. Equal quantities of radioactivity from these nuclei were hybridized with cDNA probes bound to nitrocellulose. Message bound to each probe was quantitated by laser densitometry of autoradiographs, and by scintillation counting of dot blotted nitrocellulose. Transcription of most genes tested, including Se-GPX, was not significantly affected by dietary Se intake. However, the amount of hybridization to a murine oncogene probe (v-fos) was increased in Se deficiency.

  8. ER-mediated control for abundance, quality, and signaling of transmembrane immune receptors in plants

    PubMed Central

    Tintor, Nico; Saijo, Yusuke

    2014-01-01

    Plants recognize a wide range of microbes with cell-surface and intracellular immune receptors. Transmembrane pattern recognition receptors (PRRs) initiate immune responses upon recognition of cognate ligands characteristic of microbes or aberrant cellular states, designated microbe-associated molecular patterns or danger-associated molecular patterns (DAMPs), respectively.Pattern-triggered immunity provides a first line of defense that restricts the invasion and propagation of both adapted and non-adapted pathogens. Receptor kinases (RKs) and receptor-like proteins (RLPs) with an extracellular leucine-rich repeat or lysine-motif (LysM) domain are extensively used as PRRs. The correct folding of the extracellular domain of these receptors is under quality control (QC) in the endoplasmic reticulum (ER), which thus provides a critical step in plant immunity. Genetic and structural insight suggests that ERQC regulates not only the abundance and quality of transmembrane receptors but also affects signal sorting between multi-branched pathways downstream of the receptor. However, ERQC dysfunction can also positively stimulate plant immunity, possibly through cell death and DAMP signaling pathways. PMID:24616730

  9. Evolution of transcriptional control from prokaryotic beginnings to eukaryotic complexities.

    PubMed

    Huang, L; Guan, R J; Pardee, A B

    1999-01-01

    Mechanisms for regulating gene transcription became increasingly complex as organisms evolved. In prokaryotes the relatively simple mechanism of repression is based on a few proteins that bind to specific DNA sequences in a ligand-dependent fashion. In eukaryotes large complexes that include ligand binding proteins regulate transcription. Lower eukaryotes developed an additional level of control based on protein complexes that include modifying enzymes. The DNA/histone complex, in combination with gene-specific transcriptional factors, is the basis of gene regulation in eukaryotes. Higher eukaryotes took regulation a level further by methylating CpGs in promoter sequences of DNA, thereby allowing binding of histone deacetylases and inhibiting transcription. Finally, long-lasting "superrepression" provides another mechanism for coordinate transcriptional regulation of large blocks of genes.

  10. Transcriptional regulation of secondary growth and wood formation.

    PubMed

    Du, Juan; Groover, Andrew

    2010-01-01

    Secondary growth and wood formation are products of the vascular cambium, a lateral meristem. Although the mechanisms have only recently begun to be uncovered, transcriptional regulation appears increasingly central to the regulation of secondary growth. The importance of transcriptional regulation is illustrated by the correlation of expression of specific classes of genes with related biological processes occurring at specific stages of secondary growth, including cell division, cell expansion, and cell differentiation. At the same time, transcription factors have been characterized that affect specific aspects of secondary growth, including regulation of the cambium and differentiation of cambial daughter cells. In the present review, we summarize evidence pointing to transcription as a major mechanism for regulation of secondary growth, and outline future approaches for comprehensively describing transcriptional networks underlying secondary growth.

  11. Transcriptional Amplification in Tumor Cells with Elevated c-Myc

    PubMed Central

    Lin, Charles Y.; Lovén, Jakob; Rahl, Peter B.; Paranal, Ronald M.; Burge, Christopher B.; Bradner, James E.; Lee, Tong Ihn; Young, Richard A.

    2012-01-01

    Summary Elevated expression of the c-Myc transcription factor occurs frequently in human cancers and is associated with tumor aggression and poor clinical outcome. The effect of high levels of c-Myc on global gene regulation is poorly understood, but is widely thought to involve newly activated or repressed “Myc target genes”. We report here that in tumor cells expressing high levels of c-Myc, the transcription factor accumulates in the promoter regions of active genes and causes transcriptional amplification, producing increased levels of transcripts within the cell's gene expression program. Thus, rather than binding and regulating a new set of genes, c-Myc amplifies the output of the existing gene expression program. These results provide an explanation for the diverse effects of oncogenic c-Myc on gene expression in different tumor cells and suggest that transcriptional amplification reduces rate-limiting constraints for tumor cell growth and proliferation. PMID:23021215

  12. Prolyl isomerases in gene transcription

    PubMed Central

    Hanes, Steven D.

    2014-01-01

    Background Peptidyl-prolyl isomerases (PPIases) are enzymes that assist in the folding of newly-synthesized proteins and regulate the stability, localization, and activity of mature proteins. They do so by catalyzing reversible (cis-trans) rotation about the peptide bond that precedes proline, inducing conformational changes in target proteins. Scope of Review This review will discuss how PPIases regulate gene transcription by controlling the activity of (1) DNA-binding transcription regulatory proteins, (2) RNA polymerase II, and (3) chromatin and histone modifying enzymes. Major Conclusions Members of each family of PPIase (cyclophilins, FKBPs, and parvulins) regulate gene transcription at multiple levels. In all but a few cases, the exact mechanisms remain elusive. Structure studies, development of specific inhibitors, and new methodologies for studying cis/trans isomerization in vivo represent some of the challenges in this new frontier that merges two important fields. General Significance Prolyl isomerases have been found to play key regulatory roles in all phases of the transcription process. Moreover, PPIases control upstream signaling pathways that regulate gene-specific transcription during development, hormone response and environmental stress. More broadly, although transcription is often rate-limiting in the production of enzymes and structural proteins, post-transcriptional modifications are also critical, and PPIases play key roles here as well (see other reviews in this issue). PMID:25450176

  13. Transcription of Trypanosoma brucei maxicircles

    SciTech Connect

    Michelotti, E.F.; Hajduk, S.L.

    1987-05-01

    Trypanosoma brucei is a protozoan parasite which developmentally regulates mitochondrial activity. In the mammal T. brucei produces ATP entirely by glycolysis while cytochrome mediated respiration resumes in the life-stage in the midgut of the insect vector. Using quantitative S1 nuclease protection assays two types of regulation of the steady state levels of the mitochondrial transcripts were found. Transcription of cytochrome b, cytochrome oxidase, and the rRNA genes is repressed in early bloodstream developmental stages, undergoes dramatic activation in later bloodstream stages, and finally a lesser activation in the insect developmental stage. Transcription of NADH dehydrogenase genes, however, is unregulated. Mitochondrial transcripts with a 5' triphosphate terminus, representing the site of transcription initiation, were capped using guanylyl transferase. The in vitro capped RNA hybridized to only one of eight mitochondrial restriction fragments on a Southern blot, however, hybridization of Southern blots with RNA from ..cap alpha..-/sup 32/P-UTP pulsed mitochondria labelled all restriction fragments equally. These results suggest that each DNA strand has a single promoter which directs the transcription of a full-length RNA which is subsequently processed. Different mitochondrial genes, despite being expressed on the same precursor RNA molecule, are independently regulated by both transcription initiation and RNA processing.

  14. Transcriptional regulation of plant phosphate transporters

    PubMed Central

    Muchhal, Umesh S.; Raghothama, K. G.

    1999-01-01

    Phosphorus is acquired by plant roots primarily via the high-affinity inorganic phosphate (Pi) transporters. The transcripts for Pi transporters are highly inducible upon Pi starvation, which also results in enhanced Pi uptake when Pi is resupplied. Using antibodies specific to one of the tomato Pi transporters (encoded by LePT1), we show that an increase in the LePT1 transcript under Pi starvation leads to a concurrent increase in the transporter protein, suggesting a transcriptional regulation for Pi acquisition. LePT1 protein accumulates rapidly in tomato roots in response to Pi starvation. The level of transporter protein accumulation depends on the Pi concentration in the medium, and it is reversible upon resupply of Pi. LePT1 protein accumulates all along the roots under Pi starvation and is localized primarily in the plasma membranes. These results clearly demonstrate that plants increase their capacity for Pi uptake during Pi starvation by synthesis of additional transporter molecules. PMID:10318976

  15. Swinger RNA self-hybridization and mitochondrial non-canonical swinger transcription, transcription systematically exchanging nucleotides.

    PubMed

    Seligmann, Hervé

    2016-06-21

    Stem-loop hairpins punctuate mitochondrial post-transcriptional processing. Regulation of mitochondrial swinger transcription, transcription producing RNAs matching the mitogenome only assuming systematic exchanges between nucleotides (23 bijective transformations along 9 symmetric exchanges X<>Y, e.g. A<>G, and 14 asymmetric exchanges X>Y>Z>X, e.g. A>G>C>A) remains unknown. Does swinger RNA self-hybridization regulate swinger, as regular, transcription? Groups of 8 swinger transformations share canonical self-hybridization properties within each group, group 0 includes identity (regular) transcription. The human mitogenome has more stem-loop hairpins than randomized sequences for all groups. Group 2 transformations reveal complementarity of the light strand replication origin (OL) loop and a neighboring tRNA gene, detecting the longtime presumed OL/tRNA homology. Non-canonical G=U pairings in hairpins increases with swinger RNA detection. These results confirm biological relevancy of swinger-transformed DNA/RNA, independently of, and in combination with, previously detected swinger DNA/RNA and swinger peptides. Swinger-transformed mitogenomes include unsuspected multilayered information.

  16. The Transcription Factor THO Promotes Transcription Initiation and Elongation by RNA Polymerase I.

    PubMed

    Zhang, Yinfeng; French, Sarah L; Beyer, Ann L; Schneider, David A

    2016-02-05

    Although ribosomal RNA represents the majority of cellular RNA, and ribosome synthesis is closely connected to cell growth and proliferation rates, a complete understanding of the factors that influence transcription of ribosomal DNA is lacking. Here, we show that the THO complex positively affects transcription by RNA polymerase I (Pol I). We found that THO physically associates with the rDNA repeat and interacts genetically with Pol I transcription initiation factors. Pol I transcription in hpr1 or tho2 null mutants is dramatically reduced to less than 20% of the WT level. Pol I occupancy of the coding region of the rDNA in THO mutants is decreased to ~50% of WT level. Furthermore, although the percentage of active rDNA repeats remains unaffected in the mutant cells, the overall rDNA copy number increases ~2-fold compared with WT. Together, these data show that perturbation of THO function impairs transcription initiation and elongation by Pol I, identifying a new cellular target for the conserved THO complex. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Nascent transcription affected by RNA polymerase IV in Zea mays.

    PubMed

    Erhard, Karl F; Talbot, Joy-El R B; Deans, Natalie C; McClish, Allison E; Hollick, Jay B

    2015-04-01

    All eukaryotes use three DNA-dependent RNA polymerases (RNAPs) to create cellular RNAs from DNA templates. Plants have additional RNAPs related to Pol II, but their evolutionary role(s) remain largely unknown. Zea mays (maize) RNA polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs), and transcriptional regulation of specific alleles. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based regulation. Comparisons of WT and rpd1 mutant GRO-seq profiles indicate that Pol IV globally affects transcription at both transcriptional start sites and immediately downstream of polyadenylation addition sites. We found no evidence of divergent transcription from gene promoters as seen in mammalian GRO-seq profiles. Statistical comparisons identify genes and TEs whose transcription is affected by RPD1. Most examples of significant increases in genic antisense transcription appear to be initiated by 3'-proximal long terminal repeat retrotransposons. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for specific regions of the maize genome including genes having developmental significance.

  18. Nascent Transcription Affected by RNA Polymerase IV in Zea mays

    PubMed Central

    Erhard, Karl F.; Talbot, Joy-El R. B.; Deans, Natalie C.; McClish, Allison E.; Hollick, Jay B.

    2015-01-01

    All eukaryotes use three DNA-dependent RNA polymerases (RNAPs) to create cellular RNAs from DNA templates. Plants have additional RNAPs related to Pol II, but their evolutionary role(s) remain largely unknown. Zea mays (maize) RNA polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs), and transcriptional regulation of specific alleles. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based regulation. Comparisons of WT and rpd1 mutant GRO-seq profiles indicate that Pol IV globally affects transcription at both transcriptional start sites and immediately downstream of polyadenylation addition sites. We found no evidence of divergent transcription from gene promoters as seen in mammalian GRO-seq profiles. Statistical comparisons identify genes and TEs whose transcription is affected by RPD1. Most examples of significant increases in genic antisense transcription appear to be initiated by 3ʹ-proximal long terminal repeat retrotransposons. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for specific regions of the maize genome including genes having developmental significance. PMID:25653306

  19. Transcription regulation mechanisms of bacteriophages

    PubMed Central

    Yang, Haiquan; Ma, Yingfang; Wang, Yitian; Yang, Haixia; Shen, Wei; Chen, Xianzhong

    2014-01-01

    Phage diversity significantly contributes to ecology and evolution of new bacterial species through horizontal gene transfer. Therefore, it is essential to understand the mechanisms underlying phage-host interactions. After initial infection, the phage utilizes the transcriptional machinery of the host to direct the expression of its own genes. This review presents a view on the transcriptional regulation mechanisms of bacteriophages, and its contribution to phage diversity and classification. Through this review, we aim to broaden the understanding of phage-host interactions while providing a reference source for researchers studying the regulation of phage transcription. PMID:25482231

  20. Effects of hemorrhage on cytokine gene transcription.

    PubMed

    Shenkar, R; Abraham, E

    1993-08-01

    Injury and blood loss are often followed by infection and the rapid development of organ system dysfunction, frequently involving mucosal sites, such as the lung and intestine. To examine possible mechanisms contributing to these conditions, we used semiquantitative polymerase chain reactions to determine cytokine mRNA expression among cellular populations isolated from mucosal and systemic anatomic sites of mice at predetermined time points following 30% blood volume hemorrhage with resuscitation 1 hr later. Within 1 hr after hemorrhage, significant increases were observed in mRNA levels for IL-1 alpha, IL-1 beta, IL-5, and TGF-beta in intraparenchymal pulmonary mononuclear cells. The levels of TGF-beta transcripts among alveolar macrophages were increased 1 hr following blood loss, and increase in IL-1 alpha transcripts was found starting 2 hr posthemorrhage. Cells from Peyer's patches showed significant increases in mRNA levels for IL-1 beta, IL-2, IL-5, IL-6, IFN-gamma, and TGF-beta during the 4 hr following hemorrhage. Significant increases in mRNA levels for IL-1 beta, TNF-alpha, and TGF-beta were present within 4 hr of blood loss among cells isolated from mesenteric lymph nodes. The expression of mRNA for most cytokines was not significantly altered in splenocytes or peripheral blood mononuclear cells at any time point following hemorrhage. These experiments demonstrate that blood loss, even if resuscitated, produces significant increases in proinflammatory and immunoregulatory cytokine gene transcription as early as 1 hr following hemorrhage. These posthemorrhage alterations in cytokine mRNA expression were particularly prominent at mucosal sites, suggesting a mechanism for the increased incidence of pulmonary and intestinal involvement in organ system failure following severe blood loss and injury.

  1. Zinc triggers a complex transcriptional and post-transcriptional regulation of the metal homeostasis gene FRD3 in Arabidopsis relatives

    PubMed Central

    Charlier, Jean-Benoit; Polese, Catherine; Nouet, Cécile; Carnol, Monique; Bosman, Bernard; Krämer, Ute; Motte, Patrick; Hanikenne, Marc

    2015-01-01

    In Arabidopsis thaliana, FRD3 (FERRIC CHELATE REDUCTASE DEFECTIVE 3) plays a central role in metal homeostasis. FRD3 is among a set of metal homeostasis genes that are constitutively highly expressed in roots and shoots of Arabidopsis halleri, a zinc hyperaccumulating and hypertolerant species. Here, we examined the regulation of FRD3 by zinc in both species to shed light on the evolutionary processes underlying the evolution of hyperaccumulation in A. halleri. We combined gene expression studies with the use of β-glucuronidase and green fluorescent protein reporter constructs to compare the expression profile and transcriptional and post-transcriptional regulation of FRD3 in both species. The AtFRD3 and AhFRD3 genes displayed a conserved expression profile. In A. thaliana, alternative transcription initiation sites from two promoters determined transcript variants that were differentially regulated by zinc supply in roots and shoots to favour the most highly translated variant under zinc-excess conditions. In A. halleri, a single transcript variant with higher transcript stability and enhanced translation has been maintained. The FRD3 gene thus undergoes complex transcriptional and post-transcriptional regulation in Arabidopsis relatives. Our study reveals that a diverse set of mechanisms underlie increased gene dosage in the A. halleri lineage and illustrates how an environmental challenge can alter gene regulation. PMID:25900619

  2. Zinc triggers a complex transcriptional and post-transcriptional regulation of the metal homeostasis gene FRD3 in Arabidopsis relatives.

    PubMed

    Charlier, Jean-Benoit; Polese, Catherine; Nouet, Cécile; Carnol, Monique; Bosman, Bernard; Krämer, Ute; Motte, Patrick; Hanikenne, Marc

    2015-07-01

    In Arabidopsis thaliana, FRD3 (FERRIC CHELATE REDUCTASE DEFECTIVE 3) plays a central role in metal homeostasis. FRD3 is among a set of metal homeostasis genes that are constitutively highly expressed in roots and shoots of Arabidopsis halleri, a zinc hyperaccumulating and hypertolerant species. Here, we examined the regulation of FRD3 by zinc in both species to shed light on the evolutionary processes underlying the evolution of hyperaccumulation in A. halleri. We combined gene expression studies with the use of β-glucuronidase and green fluorescent protein reporter constructs to compare the expression profile and transcriptional and post-transcriptional regulation of FRD3 in both species. The AtFRD3 and AhFRD3 genes displayed a conserved expression profile. In A. thaliana, alternative transcription initiation sites from two promoters determined transcript variants that were differentially regulated by zinc supply in roots and shoots to favour the most highly translated variant under zinc-excess conditions. In A. halleri, a single transcript variant with higher transcript stability and enhanced translation has been maintained. The FRD3 gene thus undergoes complex transcriptional and post-transcriptional regulation in Arabidopsis relatives. Our study reveals that a diverse set of mechanisms underlie increased gene dosage in the A. halleri lineage and illustrates how an environmental challenge can alter gene regulation. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  3. Reinitiation enhances reliable transcriptional responses in eukaryotes

    PubMed Central

    Liu, Bo; Yuan, Zhanjiang; Aihara, Kazuyuki; Chen, Luonan

    2014-01-01

    Gene transcription is a noisy process carried out by the transcription machinery recruited to the promoter. Noise reduction is a fundamental requirement for reliable transcriptional responses which in turn are crucial for signal transduction. Compared with the relatively simple transcription initiation in prokaryotes, eukaryotic transcription is more complex partially owing to its additional reinitiation mechanism. By theoretical analysis, we showed that reinitiation reduces noise in eukaryotic transcription independent of the transcription level. Besides, a higher reinitiation rate enables a stable scaffold complex an advantage in noise reduction. Finally, we showed that the coupling between scaffold formation and transcription can further reduce transcription noise independent of the transcription level. Furthermore, compared with the reinitiation mechanism, the noise reduction effect of the coupling can be of more significance in the case that the transcription level is low and the intrinsic noise dominates. Our results uncover a mechanistic route which eukaryotes may use to facilitate a more reliable response in the noisy transcription process. PMID:24850905

  4. Comparison of voice-automated transcription and human transcription in generating pathology reports.

    PubMed

    Al-Aynati, Maamoun M; Chorneyko, Katherine A

    2003-06-01

    Software that can convert spoken words into written text has been available since the early 1980s. Early continuous speech systems were developed in 1994, with the latest commercially available editions having a claimed accuracy of up to 98% of speech recognition at natural speech rates. To evaluate the efficacy of one commercially available voice-recognition software system with pathology vocabulary in generating pathology reports and to compare this with human transcription. To draw cost analysis conclusions regarding human versus computer-based transcription. Two hundred six routine pathology reports from the surgical pathology material handled at St Joseph's Healthcare, Hamilton, Ontario, were generated simultaneously using computer-based transcription and human transcription. The following hardware and software were used: a desktop 450-MHz Intel Pentium III processor with 192 MB of RAM, a speech-quality sound card (Sound Blaster), noise-canceling headset microphone, and IBM ViaVoice Pro version 8 with pathology vocabulary support (Voice Automated, Huntington Beach, Calif). The cost of the hardware and software used was approximately Can 2250 dollars. A total of 23 458 words were transcribed using both methods with a mean of 114 words per report. The mean accuracy rate was 93.6% (range, 87.4%-96%) using the computer software, compared to a mean accuracy of 99.6% (range, 99.4%-99.8%) for human transcription (P <.001). Time needed to edit documents by the primary evaluator (M.A.) using the computer was on average twice that needed for editing the documents produced by human transcriptionists (range, 1.4-3.5 times). The extra time needed to edit documents was 67 minutes per week (13 minutes per day). Computer-based continuous speech-recognition systems in pathology can be successfully used in pathology practice even during the handling of gross pathology specimens. The relatively low accuracy rate of this voice-recognition software with resultant increased editing

  5. The grammar of transcriptional regulation

    PubMed Central

    Weingarten-Gabbay, Shira; Segal, Eran

    2014-01-01

    Eukaryotes employ combinatorial strategies to generate a variety of expression patterns from a relatively small set of regulatory DNA elements. As in any other language, deciphering the mapping between DNA and expression requires an understanding of the set of rules that govern basic principles in transcriptional regulation, the functional elements involved, and the ways in which they combine to orchestrate a transcriptional output. Here, we review current understanding of various grammatical rules, including the effect on expression of the number of transcription factor binding-sites, their location, orientation, affinity and activity; co-association with different factors; and intrinsic nucleosome organization. We review different methods that are used to study the grammar of transcription regulation, highlight gaps in current understanding, and discuss how recent technological advances may be utilized to bridge them. PMID:24390306

  6. Coupling transcription and alternative splicing.

    PubMed

    Kornblihtt, Alberto R

    2007-01-01

    Alternative splicing regulation not only depends on the interaction of splicing factors with splicing enhancers and silencers in the pre-mRNA, but also on the coupling between transcription and splicing. This coupling is possible because splicing is often cotranscriptional and promoter identity and occupation may affect alternative splicing. We discuss here the different mechanisms by which transcription regulates alternative splicing. These include the recruitment of splicing factors to the transcribing polymerase and "kinetic coupling", which involves changes in the rate of transcriptional elongation that in turn affect the timing in which splice sites are presented to the splicing machinery. The recruitment mechanism may depend on the particular features of the carboxyl terminal domain of RNA polymerase II, whereas kinetic coupling seems to be linked to how changes in chromatin structure and other factors affect transcription elongation.

  7. Nucleolar localization of myc transcripts.

    PubMed Central

    Bond, V C; Wold, B

    1993-01-01

    In situ hybridization has revealed a striking subnuclear distribution of c-myc RNA transcripts. A major fraction of the sense-strand nuclear c-myc transcripts was localized to the nucleoli. myc intron 1-containing RNAs were noticeably absent from nucleoli, accumulating instead in the nucleoplasm. The localization of myc RNA to nucleoli was shown to be common to a number of diverse cell types, including primary Sertoli cells and several cell lines. Furthermore, nucleolar localization was not restricted to c-myc and N-myc and myoD transcripts also displayed this phenomenon. In contrast, gamma-actin or lactate dehydrogenase transcripts did not display nucleolar localization. These observations suggest a new role for the nucleolus in transport and/or turnover of potential mRNAs. Images PMID:7684491

  8. RNA-guided transcriptional regulation

    DOEpatents

    Church, George M.; Mali, Prashant G.; Esvelt, Kevin M.

    2016-02-23

    Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

  9. Transcript maturation in apicomplexan parasites

    PubMed Central

    Suvorova, Elena S.; White, Michael W.

    2014-01-01

    Summary The complex life cycles of apicomplexan parasites are associated with dynamic changes of protein repertoire. In Toxoplasma gondii, global analysis of gene expression demonstrates that dynamic changes in mRNA levels unfold in a serial cascade during asexual replication and up to 50% of encoded genes are unequally expressed in development. Recent studies indicate transcription as well as mRNA processing have important roles in fulfilling the “just-in-time” delivery of proteins to parasite growth and development. The prominence of post-transcriptional mechanisms in the Apicomplexa was demonstrated by mechanistic studies of the critical RNA-binding proteins and regulatory kinases. However, it is still early in our understanding of how transcription and post-transcriptional mechanisms are balanced to produce adequate numbers of specialized forms that is required to complete the parasite life cycle. PMID:24934558

  10. Genetic analysis of transcription-associated mutation in Saccharomyces cerevisiae.

    PubMed

    Morey, N J; Greene, C N; Jinks-Robertson, S

    2000-01-01

    High levels of transcription are associated with elevated mutation rates in yeast, a phenomenon referred to as transcription-associated mutation (TAM). The transcription-associated increase in mutation rates was previously shown to be partially dependent on the Rev3p translesion bypass pathway, thus implicating DNA damage in TAM. In this study, we use reversion of a pGAL-driven lys2DeltaBgl allele to further examine the genetic requirements of TAM. We find that TAM is increased by disruption of the nucleotide excision repair or recombination pathways. In contrast, elimination of base excision repair components has only modest effects on TAM. In addition to the genetic studies, the lys2DeltaBgl reversion spectra of repair-proficient low and high transcription strains were obtained. In the low transcription spectrum, most of the frameshift events correspond to deletions of AT base pairs whereas in the high transcription strain, deletions of GC base pairs predominate. These results are discussed in terms of transcription and its role in DNA damage and repair.

  11. The eukaryotic gene transcription machinery.

    PubMed

    Kornberg, R D

    2001-08-01

    Seven purified proteins may be combined to reconstitute regulated, promoter-dependent RNA polymerase II transcription: five general transcription factors, Mediator, and RNA polymerase II. The entire system has been conserved across species from yeast to humans. The structure of RNA polymerase II, consisting of 10 polypeptides with a mass of about 500 kDa, has been determined at atomic resolution. On the basis of this structure, that of an actively transcribing RNA polymerase II complex has been determined as well.

  12. [Regulation of bacterial transcription elongation].

    PubMed

    Proshkin, S A; Mironov, A S

    2011-01-01

    The elongation complex, which involves RNA polymerase, DNA template and nascent RNA, is a central intermediate in transcription cycle. It is elongation complex that represents the main target for the action of different regulatory factors. Over the past several years, many structural and biochemical data have been obtained that shed light upon the molecular details of RNA polymerase function. Cooperation between RNA polymerase elongation complex and translating ribosome was established recently. Here we discuss the mechanisms of the regulation of bacterial transcription elongation.

  13. Transcriptional regulation of lignin biosynthesis.

    PubMed

    Zhong, Ruiqin; Ye, Zheng-Hua

    2009-11-01

    Lignin is the second most abundant plant biopolymer mainly present in the secondary walls of tracheary elements and fibers in wood. Understanding how lignin is biosynthesized has long been an interest to plant biologists and will have a significant impact on tree biotechnology. Lignin is polymerized from monolignols that are synthesized through the lignin biosynthetic pathway. To make lignin, all the genes in the lignin biosynthetic pathway need to be coordinately turned on. It has been shown that a common cis-element, namely the AC element, is present in the majority of the lignin biosynthetic genes and required for their expression in lignifying cells. Important progress has been made in the identification of transcription factors that bind to the AC elements and are potentially involved in the coordinated regulation of lignin biosynthesis. The Arabidopsis MYB58 and MYB63 as well as their poplar ortholog PtrMYB28 are transcriptional activators of the lignin biosynthetic pathway, whereas the eucalyptus EgMYB2 and pine PtMYB4 transcription factors are likely Arabidopsis MYB46 orthologs involved in the regulation of the entire secondary wall biosynthetic program. It was found that the transcriptional regulation of lignin biosynthesis is under the control of the same transcriptional network regulating the biosynthesis of other secondary wall components, including cellulose and xylan. The identification of transcription factors directly activating lignin biosynthetic genes provides unprecedented tools to potentially manipulate the amount of lignin in wood and other plant products based on our needs.

  14. GOLDEN 2-LIKE Transcription Factors of Plants

    PubMed Central

    Chen, Min; Ji, Meiling; Wen, Binbin; Liu, Li; Li, Shaoxuan; Chen, Xiude; Gao, Dongsheng; Li, Ling

    2016-01-01

    Golden2-like (GLK) transcription factors are members of the GARP family of Myb transcription factors with an established relationship to chloroplast development in the plant kingdom. In the last century, Golden2 was proposed as a second golden producing factor and identified as controlling cellular differentiation in maize leaves. Then, GLKs were also found to play roles in disease defense and their function is conserved in regulating chloroplast development. Recently, research on GLKs has rapidly increased and shown that GLKs control chloroplast development in green and non-green tissues. Moreover, links between phytohormones and GLKs were verified. In this mini-review, we summarize the history, conservation, function, potential targets and degradation of GLKs. PMID:27757121

  15. Transcriptional regulation of epithelial-mesenchymal transition.

    PubMed

    Teng, Yingqi; Zeisberg, Michael; Kalluri, Raghu

    2007-02-01

    It has become increasingly obvious that the notion of a terminally differentiated cell is likely a simplified concept. Epithelial-mesenchymal transition (EMT), during which epithelial cells assume a mesenchymal phenotype, is a key event occurring during normal development and pathological processes. Multiple extracellular stimuli and transcriptional regulators can trigger EMT, but how such distinct signaling pathways orchestrate the complex cellular events that facilitate EMT is not well understood. In this issue of the JCI, Venkov et al. report on their examination of fibroblasts resulting from EMT and describe a novel protein-DNA complex that is essential for transcription of fibroblast-specific protein 1 (FSP1) and sufficient to induce early EMT events (see the related article beginning on page 482). Collectively, their results suggest that this complex is an important regulator of the EMT transcriptome.

  16. Transcriptional regulation of epithelial-mesenchymal transition

    PubMed Central

    Teng, Yingqi; Zeisberg, Michael; Kalluri, Raghu

    2007-01-01

    It has become increasingly obvious that the notion of a terminally differentiated cell is likely a simplified concept. Epithelial-mesenchymal transition (EMT), during which epithelial cells assume a mesenchymal phenotype, is a key event occurring during normal development and pathological processes. Multiple extracellular stimuli and transcriptional regulators can trigger EMT, but how such distinct signaling pathways orchestrate the complex cellular events that facilitate EMT is not well understood. In this issue of the JCI, Venkov et al. report on their examination of fibroblasts resulting from EMT and describe a novel protein-DNA complex that is essential for transcription of fibroblast-specific protein 1 (FSP1) and sufficient to induce early EMT events (see the related article beginning on page 482). Collectively, their results suggest that this complex is an important regulator of the EMT transcriptome. PMID:17273552

  17. Transcriptional repressor DREAM regulates trigeminal noxious perception.

    PubMed

    Benedet, Tomaso; Gonzalez, Paz; Oliveros, Juan C; Dopazo, Jose M; Ghimire, Kedar; Palczewska, Malgorzata; Mellstrom, Britt; Naranjo, Jose R

    2017-05-01

    Expression of the downstream regulatory element antagonist modulator (DREAM) protein in dorsal root ganglia and spinal cord is related to endogenous control mechanisms of acute and chronic pain. In primary sensory trigeminal neurons, high levels of endogenous DREAM protein are preferentially localized in the nucleus, suggesting a major transcriptional role. Here, we show that transgenic mice expressing a dominant active mutant of DREAM in trigeminal neurons show increased responses following orofacial sensory stimulation, which correlates with a decreased expression of prodynorphin and brain-derived neurotrophic factor in trigeminal ganglia. Genome-wide analysis of trigeminal neurons in daDREAM transgenic mice identified cathepsin L and the monoglyceride lipase as two new DREAM transcriptional targets related to pain. Our results suggest a role for DREAM in the regulation of trigeminal nociception. This article is part of the special article series "Pain". © 2016 International Society for Neurochemistry.

  18. The world according to GARP transcription factors.

    PubMed

    Safi, Alaeddine; Medici, Anna; Szponarski, Wojciech; Ruffel, Sandrine; Lacombe, Benoît; Krouk, Gabriel

    2017-10-01

    Plant specific GARP transcription factor family (made of ARR-B and G2-like) contains genes with very diverse in planta functions: nutrient sensing, root and shoot development, floral transition, chloroplast development, circadian clock oscillation maintenance, hormonal transport and signaling. In this work we review: first, their structural but distant relationships with MYB transcription factors, second, their role in planta, third, the diversity of their Cis-regulatory elements, fourth, their potential protein partners. We conclude that the GARP family may hold keys to understand the interactions between nutritional signaling pathways (nitrogen and phosphate at least) and development. Understanding how plant nutrition and development are coordinated is central to understand how to adapt plants to an ever-changing environment. Consequently GARPs are likely to attract increasing research attentions, as they are likely at the crossroads of these fundamental processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. SUMOylation of ROR{alpha} potentiates transcriptional activation function

    SciTech Connect

    Hwang, Eun Ju; Lee, Ji Min; Jeong, Jiyeong; Park, Joo Hyeon; Yang, Young; Lim, Jong-Seok; Kim, Jung Hwa; Baek, Sung Hee; Kim, Keun Il

    2009-01-16

    SUMOylation regulates a variety of cellular processes, including control of transcriptional activities of nuclear receptors. Here, we present SUMOylation of orphan nuclear receptor, ROR{alpha} by both SUMO-1 and SUMO-2. SUMOylation of ROR{alpha} occurred on the 240th lysine residue at the hinge region of human protein. PIAS family members, PIASx{alpha}, PIAS3, and PIASy, increased SUMOylation of ROR{alpha}, whereas SENP2 specifically removed SUMO from ROR{alpha}. SUMOylation-defective mutant form of ROR{alpha} exhibited decreased transcriptional activity on ROR{alpha}-responsive promoters indicating that SUMOylation may positively regulate transcriptional function of ROR{alpha}.

  20. The transcriptional landscape of the mammalian genome.

    PubMed

    Carninci, P; Kasukawa, T; Katayama, S; Gough, J; Frith, M C; Maeda, N; Oyama, R; Ravasi, T; Lenhard, B; Wells, C; Kodzius, R; Shimokawa, K; Bajic, V B; Brenner, S E; Batalov, S; Forrest, A R R; Zavolan, M; Davis, M J; Wilming, L G; Aidinis, V; Allen, J E; Ambesi-Impiombato, A; Apweiler, R; Aturaliya, R N; Bailey, T L; Bansal, M; Baxter, L; Beisel, K W; Bersano, T; Bono, H; Chalk, A M; Chiu, K P; Choudhary, V; Christoffels, A; Clutterbuck, D R; Crowe, M L; Dalla, E; Dalrymple, B P; de Bono, B; Della Gatta, G; di Bernardo, D; Down, T; Engstrom, P; Fagiolini, M; Faulkner, G; Fletcher, C F; Fukushima, T; Furuno, M; Futaki, S; Gariboldi, M; Georgii-Hemming, P; Gingeras, T R; Gojobori, T; Green, R E; Gustincich, S; Harbers, M; Hayashi, Y; Hensch, T K; Hirokawa, N; Hill, D; Huminiecki, L; Iacono, M; Ikeo, K; Iwama, A; Ishikawa, T; Jakt, M; Kanapin, A; Katoh, M; Kawasawa, Y; Kelso, J; Kitamura, H; Kitano, H; Kollias, G; Krishnan, S P T; Kruger, A; Kummerfeld, S K; Kurochkin, I V; Lareau, L F; Lazarevic, D; Lipovich, L; Liu, J; Liuni, S; McWilliam, S; Madan Babu, M; Madera, M; Marchionni, L; Matsuda, H; Matsuzawa, S; Miki, H; Mignone, F; Miyake, S; Morris, K; Mottagui-Tabar, S; Mulder, N; Nakano, N; Nakauchi, H; Ng, P; Nilsson, R; Nishiguchi, S; Nishikawa, S; Nori, F; Ohara, O; Okazaki, Y; Orlando, V; Pang, K C; Pavan, W J; Pavesi, G; Pesole, G; Petrovsky, N; Piazza, S; Reed, J; Reid, J F; Ring, B Z; Ringwald, M; Rost, B; Ruan, Y; Salzberg, S L; Sandelin, A; Schneider, C; Schönbach, C; Sekiguchi, K; Semple, C A M; Seno, S; Sessa, L; Sheng, Y; Shibata, Y; Shimada, H; Shimada, K; Silva, D; Sinclair, B; Sperling, S; Stupka, E; Sugiura, K; Sultana, R; Takenaka, Y; Taki, K; Tammoja, K; Tan, S L; Tang, S; Taylor, M S; Tegner, J; Teichmann, S A; Ueda, H R; van Nimwegen, E; Verardo, R; Wei, C L; Yagi, K; Yamanishi, H; Zabarovsky, E; Zhu, S; Zimmer, A; Hide, W; Bult, C; Grimmond, S M; Teasdale, R D; Liu, E T; Brusic, V; Quackenbush, J; Wahlestedt, C; Mattick, J S; Hume, D A; Kai, C; Sasaki, D; Tomaru, Y; Fukuda, S; Kanamori-Katayama, M; Suzuki, M; Aoki, J; Arakawa, T; Iida, J; Imamura, K; Itoh, M; Kato, T; Kawaji, H; Kawagashira, N; Kawashima, T; Kojima, M; Kondo, S; Konno, H; Nakano, K; Ninomiya, N; Nishio, T; Okada, M; Plessy, C; Shibata, K; Shiraki, T; Suzuki, S; Tagami, M; Waki, K; Watahiki, A; Okamura-Oho, Y; Suzuki, H; Kawai, J; Hayashizaki, Y

    2005-09-02

    This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

  1. Downregulation of rRNA transcription triggers cell differentiation.

    PubMed

    Hayashi, Yuki; Kuroda, Takao; Kishimoto, Hiroyuki; Wang, Changshan; Iwama, Atsushi; Kimura, Keiji

    2014-01-01

    Responding to various stimuli is indispensable for the maintenance of homeostasis. The downregulation of ribosomal RNA (rRNA) transcription is one of the mechanisms involved in the response to stimuli by various cellular processes, such as cell cycle arrest and apoptosis. Cell differentiation is caused by intra- and extracellular stimuli and is associated with the downregulation of rRNA transcription as well as reduced cell growth. The downregulation of rRNA transcription during differentiation is considered to contribute to reduced cell growth. However, the downregulation of rRNA transcription can induce various cellular processes; therefore, it may positively regulate cell differentiation. To test this possibility, we specifically downregulated rRNA transcription using actinomycin D or a siRNA for Pol I-specific transcription factor IA (TIF-IA) in HL-60 and THP-1 cells, both of which have differentiation potential. The inhibition of rRNA transcription induced cell differentiation in both cell lines, which was demonstrated by the expression of the common differentiation marker CD11b. Furthermore, TIF-IA knockdown in an ex vivo culture of mouse hematopoietic stem cells increased the percentage of myeloid cells and reduced the percentage of immature cells. We also evaluated whether differentiation was induced via the inhibition of cell cycle progression because rRNA transcription is tightly coupled to cell growth. We found that cell cycle arrest without affecting rRNA transcription did not induce differentiation. To the best of our knowledge, our results demonstrate the first time that the downregulation of rRNA levels could be a trigger for the induction of differentiation in mammalian cells. Furthermore, this phenomenon was not simply a reflection of cell cycle arrest. Our results provide a novel insight into the relationship between rRNA transcription and cell differentiation.

  2. CDK9-dependent RNA polymerase II pausing controls transcription initiation.

    PubMed

    Gressel, Saskia; Schwalb, Björn; Decker, Tim Michael; Qin, Weihua; Leonhardt, Heinrich; Eick, Dirk; Cramer, Patrick

    2017-10-10

    Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a 'multi-omics' approach to demonstrate that the duration of polymerase pausing generally limits the productive frequency of transcription initiation in human cells ('pause-initiation limit'). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. CDK9 activity decreases the pause duration but also increases the productive initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.

  3. Transcriptional regulation by post-transcriptional modification--role of phosphorylation in Sp1 transcriptional activity.

    PubMed

    Chu, Shijian

    2012-10-15

    Sp1 is a ubiquitously expressed transcription factor involved in the regulation of a large number of genes including housekeeping genes as well as actively regulated genes. Although Sp1 was discovered nearly three decades ago, its functional diversity is still not completely understood. One of the ways that make Sp1 versatile in transcriptional regulation is its post-transcriptional modification, which alters Sp1 structure in different cells and at different times. Compared to other types of modifications of the Sp1 protein, phosphorylation has been studied far more extensively. This review focuses on the inducers, pathways, enzymes, and biological effects of Sp1 phosphorylation. Recent data are beginning to reveal the biological significance and universal presence of Sp1 phosphorylation-related cell/molecular responses. Studies in this field provide a quick glance at how a simple chemical modification of a transcription factor could produce significant functional diversity of the protein.

  4. Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation

    PubMed Central

    Chauvier, Adrien; Picard-Jean, Frédéric; Berger-Dancause, Jean-Christophe; Bastet, Laurène; Naghdi, Mohammad Reza; Dubé, Audrey; Turcotte, Pierre; Perreault, Jonathan; Lafontaine, Daniel A.

    2017-01-01

    On the basis of nascent transcript sequencing, it has been postulated but never demonstrated that transcriptional pausing at translation start sites is important for gene regulation. Here we show that the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch contains a regulatory pause site in the translation initiation region that acts as a checkpoint for thiC expression. By biochemically probing nascent transcription complexes halted at defined positions, we find a narrow transcriptional window for metabolite binding, in which the downstream boundary is delimited by the checkpoint. We show that transcription complexes at the regulatory pause site favour the formation of a riboswitch intramolecular lock that strongly prevents TPP binding. In contrast, cotranscriptional metabolite binding increases RNA polymerase pausing and induces Rho-dependent transcription termination at the checkpoint. Early transcriptional pausing may provide a general mechanism, whereby transient transcriptional windows directly coordinate the sensing of environmental cues and bacterial mRNA regulation. PMID:28071751

  5. Repairing RNA Transcripts that Mediate Breast Cancer Susceptibility

    DTIC Science & Technology

    2005-08-01

    is actually the yield of TES product plus the yield of cryptic is in contrast to hammerhead and hairpin ribozymes , which products. This increases the...therapeutics. To this end, we have developed a novel biomolecule (a ribozyme ) that can specifically excise regions from RNA transcripts. In this work, we...designed a ribozyme that excises an insertion mutation that is linked to breast cancer predisposition from a short mimic of the p53 transcript in a

  6. Transcriptional effects on double-strand break-induced gene conversion tracts.

    PubMed

    Weng, Y S; Xing, D; Clikeman, J A; Nickoloff, J A

    2000-10-16

    Transcription stimulates spontaneous homologous recombination, but prior studies have not investigated the effects of transcription on double-strand break (DSB)-induced recombination in yeast. We examined products of five ura3 direct repeat substrates in yeast using alleles that were transcribed at low or high levels. In each strain, recombination was stimulated by DSBs created in vivo at an HO site in one copy of ura3. Increasing transcription levels in donor or recipient alleles did not further stimulate DSB-induced recombination, nor did it alter the relative frequencies of conversion and deletion (pop-out) events. This result is consistent with the idea that transcription enhances spontaneous recombination by increasing initiation. Gene conversion tracts were measured using silent restriction fragment length polymorphisms (RFLPs) at approximately 100bp intervals. Transcription did not alter average tract lengths, but increased transcription in donor alleles increased both the frequency of promoter-proximal (5') unidirectional tracts and conversion of 5' markers. Increased transcription in recipient alleles increased the frequency of bidirectional tracts. We demonstrate that these effects are due to transcription per se, and not just transcription factor binding. These results suggest that transcription influences aspects of gene conversion after initiation, such as strand invasion and/or mismatch repair (MMR).

  7. Opposing Transcriptional Mechanisms Regulate Toxoplasma Development

    PubMed Central

    Hong, Dong-Pyo; Radke, Joshua B.

    2017-01-01

    ABSTRACT The Toxoplasma biology that underlies human chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite stage. We investigated the roles of two alkaline-stress-induced ApiAP2 transcription factors, AP2IV-3 and AP2IX-9, in bradyzoite development. These factors were expressed in two overlapping waves during bradyzoite development, with AP2IX-9 increasing expression earlier than AP2IV-3, which peaked as AP2IX-9 expression was declining. Disruption of the AP2IX-9 gene enhanced, while deletion of AP2IV-3 gene decreased, tissue cyst formation, demonstrating that these factors have opposite functions in bradyzoite development. Conversely, conditional overexpression of FKBP-modified AP2IX-9 or AP2IV-3 with the small molecule Shield 1 had a reciprocal effect on tissue cyst formation, confirming the conclusions of the knockout experiments. The AP2IX-9 repressor and AP2IV-3 activator tissue cyst phenotypes were borne out in gene expression studies that determined that many of the same bradyzoite genes were regulated in an opposite manner by these transcription factors. A common gene target was the canonical bradyzoite marker BAG1, and mechanistic experiments determined that, like AP2IX-9, AP2IV-3 regulates a BAG1 promoter-luciferase reporter and specifically binds the BAG1 promoter in parasite chromatin. Altogether, these results suggest that the AP2IX-9 transcriptional repressor and the AP2IV-3 transcriptional activator likely compete to control bradyzoite gene expression, which may permit Toxoplasma to better adapt to different tissue environments and select a suitable host cell for long-term survival of the dormant tissue cyst. IMPORTANCE Toxoplasma infections are lifelong because of the development of the bradyzoite tissue cyst, which is effectively invisible to the immune system. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control tissue

  8. Appetite - increased

    MedlinePlus

    ... Have you noticed any other symptoms such as anxiety, palpitations , increased thirst , vomiting , frequent urination , or unintentional weight gain? Tests that may be done include: Blood tests, ...

  9. Vespucci: a system for building annotated databases of nascent transcripts

    PubMed Central

    Allison, Karmel A.; Kaikkonen, Minna U.; Gaasterland, Terry; Glass, Christopher K.

    2014-01-01

    Global run-on sequencing (GRO-seq) is a recent addition to the series of high-throughput sequencing methods that enables new insights into transcriptional dynamics within a cell. However, GRO-sequencing presents new algorithmic challenges, as existing analysis platforms for ChIP-seq and RNA-seq do not address the unique problem of identifying transcriptional units de novo from short reads located all across the genome. Here, we present a novel algorithm for de novo transcript identification from GRO-sequencing data, along with a system that determines transcript regions, stores them in a relational database and associates them with known reference annotations. We use this method to analyze GRO-sequencing data from primary mouse macrophages and derive novel quantitative insights into the extent and characteristics of non-coding transcription in mammalian cells. In doing so, we demonstrate that Vespucci expands existing annotations for mRNAs and lincRNAs by defining the primary transcript beyond the polyadenylation site. In addition, Vespucci generates assemblies for un-annotated non-coding RNAs such as those transcribed from enhancer-like elements. Vespucci thereby provides a robust system for defining, storing and analyzing diverse classes of primary RNA transcripts that are of increasing biological interest. PMID:24304890

  10. Genetic Regulation of Transcriptional Variation in Natural Arabidopsis thaliana Accessions

    PubMed Central

    Zan, Yanjun; Shen, Xia; Forsberg, Simon K. G.; Carlborg, Örjan

    2016-01-01

    An increased knowledge of the genetic regulation of expression in Arabidopsis thaliana is likely to provide important insights about the basis of the plant’s extensive phenotypic variation. Here, we reanalyzed two publicly available datasets with genome-wide data on genetic and transcript variation in large collections of natural A. thaliana accessions. Transcripts from more than half of all genes were detected in the leaves of all accessions, and from nearly all annotated genes in at least one accession. Thousands of genes had high transcript levels in some accessions, but no transcripts at all in others, and this pattern was correlated with the genome-wide genotype. In total, 2669 eQTL were mapped in the largest population, and 717 of them were replicated in the other population. A total of 646 cis-eQTL-regulated genes that lacked detectable transcripts in some accessions was found, and for 159 of these we identified one, or several, common structural variants in the populations that were shown to be likely contributors to the lack of detectable RNA transcripts for these genes. This study thus provides new insights into the overall genetic regulation of global gene expression diversity in the leaf of natural A. thaliana accessions. Further, it also shows that strong cis-acting polymorphisms, many of which are likely to be structural variations, make important contributions to the transcriptional variation in the worldwide A. thaliana population. PMID:27226169

  11. Abscisic acid represses the transcription of chloroplast genes*

    PubMed Central

    Yamburenko, Maria V.; Zubo, Yan O.; Börner, Thomas

    2013-01-01

    Numerous studies have shown effects of abscisic acid (ABA) on nuclear genes encoding chloroplast-localized proteins. ABA effects on the transcription of chloroplast genes, however, have not been investigated yet thoroughly. This work, therefore, studied the effects of ABA (75 μM) on transcription and steady-state levels of transcripts in chloroplasts of basal and apical segments of primary leaves of barley (Hordeum vulgare L.). Basal segments consist of young cells with developing chloroplasts, while apical segments contain the oldest cells with mature chloroplasts. Exogenous ABA reduced the chlorophyll content and caused changes of the endogenous concentrations not only of ABA but also of cytokinins to different extents in the basal and apical segments. It repressed transcription by the chloroplast phage-type and bacteria-type RNA polymerases and lowered transcript levels of most investigated chloroplast genes drastically. ABA did not repress the transcription of psbD and a few other genes and even increased psbD mRNA levels under certain conditions. The ABA effects on chloroplast transcription were more pronounced in basal vs. apical leaf segments and enhanced by light. Simultaneous application of cytokinin (22 μM 6-benzyladenine) minimized the ABA effects on chloroplast gene expression. These data demonstrate that ABA affects the expression of chloroplast genes differentially and points to a role of ABA in the regulation and coordination of the activities of nuclear and chloroplast genes coding for proteins with functions in photosynthesis. PMID:24078671

  12. Retroactivity effects dependency on the transcription factors binding mechanisms.

    PubMed

    Pantoja-Hernández, Libertad; Álvarez-Buylla, Elena; Aguilar-Ibáñez, Carlos F; Garay-Arroyo, Adriana; Soria-López, Alberto; Martínez-García, Juan Carlos

    2016-12-07

    Downstream connection effects on transcription are caused by retroactivity. When biomolecular dynamical systems interconnect retroactivity is a property that becomes important. The biological functional meaning of these effects is increasingly becoming an area of interest. Downstream targets, which are operator binding sites in transcriptional networks, may induce behaviors such as ultrasensitive responses or even represent an undesired issue in regulation. To the best of our knowledge, the role of the binding mechanisms of transcription factors in relation to minimizing - or enhancing - retroactivity effects has not been previously addressed. Our aim is to evaluate retroactivity effects considering how the binding mechanism impacts the number of free functional transcription factor (FFTF) molecules using a simple model via deterministic and stochastic simulations. We study four transcription factor binding mechanisms (BM): simple monomer binding (SMB), dimer binding (DB), cooperative sequential binding (CSB) and cooperative sequential binding with dimerization (CSB_D). We consider weak and strong binding regimes for each mechanism, where we contrast the cases when the FFTF is bound or unbound to the downstream loads. Upon interconnection, the number of FFTF molecules changed less for the SMB mechanism while for DB they changed the most. Our results show that for the chosen mechanisms (in terms of the corresponding described dynamics), retroactivity effects depend on transcription binding mechanisms. This contributes to the understanding of how the transcription factor regulatory function-such as decision making-and its dynamic needs for the response, may determine the nature of the selected binding mechanism.

  13. Abscisic acid represses the transcription of chloroplast genes.

    PubMed

    Yamburenko, Maria V; Zubo, Yan O; Vanková, Radomíra; Kusnetsov, Victor V; Kulaeva, Olga N; Börner, Thomas

    2013-11-01

    Numerous studies have shown effects of abscisic acid (ABA) on nuclear genes encoding chloroplast-localized proteins. ABA effects on the transcription of chloroplast genes, however, have not been investigated yet thoroughly. This work, therefore, studied the effects of ABA (75 μM) on transcription and steady-state levels of transcripts in chloroplasts of basal and apical segments of primary leaves of barley (Hordeum vulgare L.). Basal segments consist of young cells with developing chloroplasts, while apical segments contain the oldest cells with mature chloroplasts. Exogenous ABA reduced the chlorophyll content and caused changes of the endogenous concentrations not only of ABA but also of cytokinins to different extents in the basal and apical segments. It repressed transcription by the chloroplast phage-type and bacteria-type RNA polymerases and lowered transcript levels of most investigated chloroplast genes drastically. ABA did not repress the transcription of psbD and a few other genes and even increased psbD mRNA levels under certain conditions. The ABA effects on chloroplast transcription were more pronounced in basal vs. apical leaf segments and enhanced by light. Simultaneous application of cytokinin (22 μM 6-benzyladenine) minimized the ABA effects on chloroplast gene expression. These data demonstrate that ABA affects the expression of chloroplast genes differentially and points to a role of ABA in the regulation and coordination of the activities of nuclear and chloroplast genes coding for proteins with functions in photosynthesis.

  14. Transcriptional control of Sost in bone [Transcriptional control of Sclerostin

    DOE PAGES

    Sebastian, Aimy; Loots, Gabriela G.

    2016-10-19

    Sclerostin is an osteocyte derived negative regulator of bone formation. A highly specific expression pattern and the exclusive bone phenotype have made Sclerostin an attractive target for therapeutic intervention in treating metabolic bone diseases such as osteoporosis and in facilitating fracture repair. Understanding the molecular mechanisms that regulate Sclerostin transcription is of great interest as it may unveil new avenues for therapeutic approaches. Such studies may also elucidate how various signaling pathways intersect to modulate bone metabolism. Furthermore we review the current understanding of the upstream molecular mechanisms that regulate Sost/SOST transcription, in bone.

  15. Transcriptional Signatures in Huntington's Disease

    PubMed Central

    2007-01-01

    While selective neuronal death has been an influential theme in Huntington's disease (HD), there is now a preponderance of evidence that significant neuronal dysfunction precedes frank neuronal death. The best evidence for neuronal dysfunction is the observation that gene expression is altered in HD brain, suggesting that transcriptional dysregulation is a central mechanism. Studies of altered gene expression began with careful observations of post-mortem human HD brain and subsequently were accelerated by the development of transgenic mouse models. The application of DNA microarray technology has spurred tremendous progress with respect to the altered transcriptional processes that occur in HD, through gene expression studies of both transgenic mouse models as well as cellular models of HD. Gene expression profiles are remarkably comparable across these models, bolstering the idea that transcriptional signatures reflect an essential feature of disease pathogenesis. Finally, gene expression studies have been applied to human HD, thus not only validating the approach of using model systems, but also solidifying the idea that altered transcription is a key mechanism in HD pathogenesis. In the future, gene expression profiling will be used as a readout in clinical trials aimed at correcting transcriptional dysregulation in Huntington's disease. PMID:17467140

  16. Kinetic Modelling of Transcription Elongation

    NASA Astrophysics Data System (ADS)

    O'Maoileidigh, Daibhid; Tadigotla, Vasisht; Sengupta, Anirvan; Epshtein, Vitaly; Ebright, Richard; Nudler, Evgeny; Ruckenstein, Andrei

    2006-03-01

    Transcription is the first step in gene expression and it is at this stage that most of genetic regulation occurs. The enzyme RNA polymerase (RNAP) walks along DNA creating an RNA transcript at a highly non-uniform rate. We discuss how many non-intuitive features of the system may be experimentally and physically motivated and present first a model, which agrees qualitatively with a host of experimental evidence. We also examine intrinsic pauses where it is thought that the RNAP will move backwards along the DNA template without changing the length of the RNA transcript. We describe a simplified kinetic scheme for the recovery of intrinsic pauses with the same degree of predictive power as our thermodynamic model (presented separately). The separation of timescales between the movement of the RNAP and global changes in the RNA secondary structure is seen to be crucial for the function of RNAP. This is essentially a model of a Brownian ratchet where RNAP executes a 1D random walk in a sequence dependent potential over a range determined by the co-transcriptional RNA fold for each transcript length

  17. Transcriptional gene silencing in humans

    PubMed Central

    Weinberg, Marc S.; Morris, Kevin V.

    2016-01-01

    It has been over a decade since the first observation that small non-coding RNAs can functionally modulate epigenetic states in human cells to achieve functional transcriptional gene silencing (TGS). TGS is mechanistically distinct from the RNA interference (RNAi) gene-silencing pathway. TGS can result in long-term stable epigenetic modifications to gene expression that can be passed on to daughter cells during cell division, whereas RNAi does not. Early studies of TGS have been largely overlooked, overshadowed by subsequent discoveries of small RNA-directed post-TGS and RNAi. A reappraisal of early work has been brought about by recent findings in human cells where endogenous long non-coding RNAs function to regulate the epigenome. There are distinct and common overlaps between the proteins involved in small and long non-coding RNA transcriptional regulatory mechanisms, suggesting that the early studies using small non-coding RNAs to modulate transcription were making use of a previously unrecognized endogenous mechanism of RNA-directed gene regulation. Here we review how non-coding RNA plays a role in regulation of transcription and epigenetic gene silencing in human cells by revisiting these earlier studies and the mechanistic insights gained to date. We also provide a list of mammalian genes that have been shown to be transcriptionally regulated by non-coding RNAs. Lastly, we explore how TGS may serve as the basis for development of future therapeutic agents. PMID:27060137

  18. The vascular endothelial growth factor receptor inhibitor PTK787/ZK222584 inhibits aromatase.

    PubMed

    Banerjee, Susana; Zvelebil, Marketa; Furet, Pascal; Mueller-Vieira, Ursula; Evans, Dean B; Dowsett, Mitch; Martin, Lesley-Ann

    2009-06-01

    Endocrine therapy is well established for the treatment of breast cancer, and antiangiogenic agents are showing considerable promise. Targeting the vascular endothelial growth factor (VEGF) and estrogen receptor (ER) signaling pathways concomitantly may provide enhanced therapeutic benefit in ER-positive breast cancer. Therefore, the effects of the VEGF receptor (VEGFR) tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) were investigated using human breast cancer cell lines engineered to express aromatase. As expected in this system, estrogen (E2) or androstenedione induced a proliferative response and increased ER-mediated transcription in ER-positive cell lines expressing aromatase. However, surprisingly, in the presence of androstenedione, PTK/ZK suppressed both the androstenedione-stimulated proliferation and ER-mediated transcription. PTK/ZK alone and in the presence of E2 had no observable effect on proliferation or ER-mediated transcription. These effects result from PTK/ZK having previously unrecognized antiaromatase activity and PTK/ZK being a competitive aromatase inhibitor. Computer-assisted molecular modeling showed that PTK/ZK could potentially bind directly to aromatase. The demonstration that PTK/ZK inhibits aromatase and VEGFR indicates that agents cross-inhibiting two important classes of targets in breast cancer could be developed.

  19. Cockayne syndrome: defective repair of transcription?

    PubMed Central

    van Gool, A J; van der Horst, G T; Citterio, E; Hoeijmakers, J H

    1997-01-01

    In the past years, it has become increasingly evident that basal metabolic processes within the cell are intimately linked and influenced by one another. One such link that recently has attracted much attention is the close interplay between nucleotide excision DNA repair and transcription. This is illustrated both by the preferential repair of the transcribed strand of active genes (a phenomenon known as transcription-coupled repair, TCR) as well as by the distinct dual involvement of proteins in both processes. The mechanism of TCR in eukaryotes is still largely unknown. It was first discovered in mammals by the pioneering studies of Hanawalt and colleagues, and subsequently identified in yeast and Escherichia coli. In the latter case, one protein, the transcription repair-coupling factor, was found to accomplish this function in vitro, and a plausible model for its activity was proposed. While the E. coli model still functions as a paradigm for TCR in eukaryotes, recent observations prompt us to believe that the situation in eukaryotes is much more complex, involving dual functionality of multiple proteins. PMID:9250659

  20. Innate immunity and inflammation: a transcriptional paradigm.

    PubMed

    Hawiger, J

    2001-01-01

    The innate immune response and the process of inflammation are interwoven. Excessive and continuing cytokine production in response to bacterial lipopolysacharides (LPS) or superantigens is a hallmark of the systemic inflammatory response (IR), which can be life-threatening. Dissemination of these bacterial products induces waves of proinflammatory cytokines that cause vascular injury and multiple organ dysfunction. Both LPS and superantigens induce signaling to the nucleus in mononuclear phagocytes and T cells, respectively. These signaling pathways are mediated by NF-kappaB and other stress-responsive transcription factors (SRTFs), which play a critical role in reprogramming gene expression. The nuclear import of NF-kappaB allows transcriptional activation of over 100 genes that encode mediators of inflammatory and immune responses. We have developed a novel method to block nuclear import of NF-kappaB through cell-permeable peptide transduction in monocytes, macrophages, T lymphocytes, and endothelial cells. Strikingly, a cell-permeable peptide that antagonizes nuclear import of NF-kappaB and other SRTFs, suppressed the systemic production of proinflammatory cytokines (TNFalpha and interferon gamma) in mice challenged with a lethal dose of LPS, and increased their survival by at least 90%. Thus, systemic inflammatory responses are critically dependent on the transcriptional activation ofcytokine genes that are controlled by NF-kappaB and other SRTFs.

  1. RNA Polymerase II Collision Interrupts Convergent Transcription

    PubMed Central

    Hobson, David J.; Wei, Wu; Steinmetz, Lars M.; Svejstrup, Jesper Q.

    2012-01-01

    Summary Antisense noncoding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical and genetic approaches in yeast to show that polymerases transcribing opposite DNA strands cannot bypass each other. RNAPII stops but does not dissociate upon head-to-head collision in vitro, suggesting that opposing polymerases represent insurmountable obstacles for each other. Head-to-head collision in vivo also results in RNAPII stopping, and removal of collided RNAPII from the DNA template can be achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII polyubiquitylation, the half-life of collided polymerases increases, so that they can be detected between convergent genes. These results provide insight into fundamental mechanisms of gene traffic control and point to an unexplored effect of antisense transcription on gene regulation via polymerase collision. PMID:23041286

  2. Transcription of tandemly repetitive DNA: functional roles.

    PubMed

    Biscotti, Maria Assunta; Canapa, Adriana; Forconi, Mariko; Olmo, Ettore; Barucca, Marco

    2015-09-01

    A considerable fraction of the eukaryotic genome is made up of satellite DNA constituted of tandemly repeated sequences. These elements are mainly located at centromeres, pericentromeres, and telomeres and are major components of constitutive heterochromatin. Although originally satellite DNA was thought silent and inert, an increasing number of studies are providing evidence on its transcriptional activity supporting, on the contrary, an unexpected dynamicity. This review summarizes the multiple structural roles of satellite noncoding RNAs at chromosome level. Indeed, satellite noncoding RNAs play a role in the establishment of a heterochromatic state at centromere and telomere. These highly condensed structures are indispensable to preserve chromosome integrity and genome stability, preventing recombination events, and ensuring the correct chromosome pairing and segregation. Moreover, these RNA molecules seem to be involved also in maintaining centromere identity and in elongation, capping, and replication of telomere. Finally, the abnormal variation of centromeric and pericentromeric DNA transcription across major eukaryotic lineages in stress condition and disease has evidenced the critical role that these transcripts may play and the potentially dire consequences for the organism.

  3. Transgenerational analysis of transcriptional silencing in zebrafish

    PubMed Central

    Akitake, Courtney M.; Macurak, Michelle; Halpern, Marnie E.; Goll, Mary G.

    2011-01-01

    The yeast Gal4/UAS transcriptional activation system is a powerful tool for regulating gene expression in Drosophila and has been increasing in popularity for developmental studies in zebrafish. It is also useful for studying the basis of de novo transcriptional silencing. Fluorescent reporter genes under the control of multiple tandem copies of the upstream activator sequence (UAS) often show evidence of variegated expression and DNA methylation in transgenic zebrafish embryos. To characterize this systematically, we monitored the progression of transcriptional silencing of UAS-regulated transgenes that differ in their integration sites and in the repetitive nature of the UAS. Transgenic larvae were examined in three generations for tissue-specific expression of a green fluorescent protein (GFP) reporter and DNA methylation at the UAS. Single insertions containing four distinct upstream activator sequences were far less susceptible to methylation than insertions containing fourteen copies of the same UAS. In addition, transgenes that integrated in or adjacent to transposon sequence exhibited silencing regardless of the number of UAS sites included in the transgene. Placement of promoter-driven Gal4 upstream of UAS-regulated responder genes in a single bicistronic construct also appeared to accelerate silencing and methylation. The results demonstrate the utility of the zebrafish for efficient tracking of gene silencing mechanisms across several generations, as well as provide useful guidelines for optimal Gal4-regulated gene expression in organisms subject to DNA methylation. PMID:21223961

  4. Modulation of transcription factors by curcumin.

    PubMed

    Shishodia, Shishir; Singh, Tulika; Chaturvedi, Madan M

    2007-01-01

    Curcumin is the active ingredient of turmeric that has been consumed as a dietary spice for ages. Turmeric i