Science.gov

Sample records for active transcription factors

  1. In vitro squelching of activated transcription by serum response factor: evidence for a common coactivator used by multiple transcriptional activators.

    PubMed Central

    Prywes, R; Zhu, H

    1992-01-01

    Low amounts of serum response factor (SRF) activate transcription in vitro from a fos promoter construct containing an SRF binding site. Using this human HeLa cell-derived in vitro transcription system, we have found that high amounts of SRF inhibited, or 'squelched', transcription from this construct. Transcription from several other promoters activated by different gene-specific factors, including CREB and the acidic activator VP16, was also inhibited by high amounts of SRF. Basal transcription, from TATA-only promoters, however, was not inhibited. These results suggest that SRF binds to a common factor(s) (termed coactivator) required for activated transcription by a diverse group of transcriptional activators. Inhibition of transcription by SRF could be blocked by a double stranded oligonucleotide containing an SRF binding site. Mutations in SRF which abolished its DNA binding activity also reduced its ability to inhibit transcription. In addition, a C-terminal truncation of SRF which reduced its ability to activate transcription also reduced SRF's ability to inhibit transcription. These results suggest that activation and inhibition of transcription may be mediated by SRF binding to the same factor and that SRF can only bind to this factor when SRF is bound to plasmid DNA. Images PMID:1531519

  2. 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

  3. Sumoylation delays the ATF7 transcription factor subcellular localization and inhibits its transcriptional activity.

    PubMed

    Hamard, Pierre-Jacques; Boyer-Guittaut, Michaël; Camuzeaux, Barbara; Dujardin, Denis; Hauss, Charlotte; Oelgeschläger, Thomas; Vigneron, Marc; Kedinger, Claude; Chatton, Bruno

    2007-01-01

    Over the past few years, small ubiquitin-like modifier (SUMO) modification has emerged as an important regulator of diverse pathways and activities including protein localization and transcriptional regulation. We identified a consensus sumoylation motif (IKEE), located within the N-terminal activation domain of the ATF7 transcription factor and thus investigated the role of this modification. ATF7 is a ubiquitously expressed transcription factor, homologous to ATF2, that binds to CRE elements within specific promoters. This protein is able to heterodimerize with Jun or Fos proteins and its transcriptional activity is mediated by interaction with TAF12, a subunit of the general transcription factor TFIID. In the present article, we demonstrate that ATF7 is sumoylated in vitro (using RanBP2 as a E3-specific ligase) and in vivo. Moreover, we show that ATF7 sumoylation affects its intranuclear localization by delaying its entry into the nucleus. Furthermore, SUMO conjugation inhibits ATF7 transactivation activity by (i) impairing its association with TAF12 and (ii) blocking its binding-to-specific sequences within target promoters.

  4. The EDLL motif: a potent plant transcriptional activation domain from AP2/ERF transcription factors.

    PubMed

    Tiwari, Shiv B; Belachew, Alemu; Ma, Siu Fong; Young, Melinda; Ade, Jules; Shen, Yu; Marion, Colleen M; Holtan, Hans E; Bailey, Adina; Stone, Jeffrey K; Edwards, Leslie; Wallace, Andreah D; Canales, Roger D; Adam, Luc; Ratcliffe, Oliver J; Repetti, Peter P

    2012-06-01

    In plants, the ERF/EREBP family of transcriptional regulators plays a key role in adaptation to various biotic and abiotic stresses. These proteins contain a conserved AP2 DNA-binding domain and several uncharacterized motifs. Here, we describe a short motif, termed 'EDLL', that is present in AtERF98/TDR1 and other clade members from the same AP2 sub-family. We show that the EDLL motif, which has a unique arrangement of acidic amino acids and hydrophobic leucines, functions as a strong activation domain. The motif is transferable to other proteins, and is active at both proximal and distal positions of target promoters. As such, the EDLL motif is able to partly overcome the repression conferred by the AtHB2 transcription factor, which contains an ERF-associated amphiphilic repression (EAR) motif. We further examined the activation potential of EDLL by analysis of the regulation of flowering time by NF-Y (nuclear factor Y) proteins. Genetic evidence indicates that NF-Y protein complexes potentiate the action of CONSTANS in regulation of flowering in Arabidopsis; we show that the transcriptional activation function of CONSTANS can be substituted by direct fusion of the EDLL activation motif to NF-YB subunits. The EDLL motif represents a potent plant activation domain that can be used as a tool to confer transcriptional activation potential to heterologous DNA-binding proteins.

  5. Resveratrol regulates gene transcription via activation of stimulus-responsive transcription factors.

    PubMed

    Thiel, Gerald; Rössler, Oliver G

    2017-03-01

    Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin of grapes and other fruits and plants, is a common constituent of our diet and of dietary supplements. Many health-promoting benefits have been connected with resveratrol in the treatment of cardiovascular diseases, cancer, diabetes, inflammation, neurodegeneration, and diseases connected with aging. To explain the pleiotropic effects of resveratrol, the molecular targets of this compound have to be identified on the cellular level. Resveratrol induces intracellular signal transduction pathways which ultimately lead to changes in the gene expression pattern of the cells. Here, we review the effect of resveratrol on the activation of the stimulus-responsive transcription factors CREB, AP-1, Egr-1, Elk-1, and Nrf2. Following activation, these transcription factors induce transcription of delayed response genes. The gene products of these delayed response genes are ultimately responsible for the changes in the biochemistry and physiology of resveratrol-treated cells. The activation of stimulus-responsive transcription factors may explain many of the intracellular activities of resveratrol. However, results obtained in vitro may not easily be transferred to in vivo systems.

  6. Theory on the dynamic memory in the transcription-factor-mediated transcription activation

    NASA Astrophysics Data System (ADS)

    Murugan, R.

    2011-04-01

    We develop a theory to explain the origin of the static and dynamical memory effects in transcription-factor-mediated transcription activation. Our results suggest that the following inequality conditions should be satisfied to observe such memory effects: (a) τL≫max(τR,τE), (b) τLT≫τT, and (c) τI⩾(τEL+τTR) where τL is the average time required for the looping-mediated spatial interactions of enhancer—transcription-factor complex with the corresponding promoter—RNA-polymerase or eukaryotic RNA polymerase type II (PolII in eukaryotes) complex that is located L base pairs away from the cis-acting element, (τR,τE) are respectively the search times required for the site-specific binding of the RNA polymerase and the transcription factor with the respective promoter and the cis-regulatory module, τLT is the time associated with the relaxation of the looped-out segment of DNA that connects the cis-acting site and promoter, τT is the time required to generate a complete transcript, τI is the transcription initiation time, τEL is the elongation time, and τTR is the termination time. We have theoretically derived the expressions for the various searching, looping, and loop-relaxation time components. Using the experimentally determined values of various time components we further show that the dynamical memory effects cannot be experimentally observed whenever the segment of DNA that connects the cis-regulatory element with the promoter is not loaded with bulky histone bodies. Our analysis suggests that the presence of histone-mediated compaction of the connecting segment of DNA can result in higher values of looping and loop-relaxation times, which is the origin of the static memory in the transcription activation that is mediated by the memory gene loops in eukaryotes.

  7. Theory on the dynamic memory in the transcription-factor-mediated transcription activation.

    PubMed

    Murugan, R

    2011-04-01

    We develop a theory to explain the origin of the static and dynamical memory effects in transcription-factor-mediated transcription activation. Our results suggest that the following inequality conditions should be satisfied to observe such memory effects: (a) τ(L)≫max(τ(R),τ(E)), (b) τ(LT)≫τ(T), and (c) τ(I)≥(τ(EL)+τ(TR)) where τ(L) is the average time required for the looping-mediated spatial interactions of enhancer-transcription-factor complex with the corresponding promoter--RNA-polymerase or eukaryotic RNA polymerase type II (PolII in eukaryotes) complex that is located L base pairs away from the cis-acting element, (τ(R),τ(E)) are respectively the search times required for the site-specific binding of the RNA polymerase and the transcription factor with the respective promoter and the cis-regulatory module, τ(LT) is the time associated with the relaxation of the looped-out segment of DNA that connects the cis-acting site and promoter, τ(T) is the time required to generate a complete transcript, τ(I) is the transcription initiation time, τ(EL) is the elongation time, and τ(TR) is the termination time. We have theoretically derived the expressions for the various searching, looping, and loop-relaxation time components. Using the experimentally determined values of various time components we further show that the dynamical memory effects cannot be experimentally observed whenever the segment of DNA that connects the cis-regulatory element with the promoter is not loaded with bulky histone bodies. Our analysis suggests that the presence of histone-mediated compaction of the connecting segment of DNA can result in higher values of looping and loop-relaxation times, which is the origin of the static memory in the transcription activation that is mediated by the memory gene loops in eukaryotes.

  8. Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease.

    PubMed

    Naranjo, José R; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C; Arrabal, María D; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt

    2016-02-01

    Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD.

  9. Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease

    PubMed Central

    Naranjo, José R.; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M.; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C.; Arrabal, María D.; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt

    2016-01-01

    Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD. PMID:26752648

  10. Drosophila factor 2, an RNA polymerase II transcript release factor, has DNA-dependent ATPase activity.

    PubMed

    Xie, Z; Price, D

    1997-12-12

    Drosophila factor 2 has been identified as a component of negative transcription elongation factor (N-TEF) that causes the release of RNA polymerase II transcripts in an ATP-dependent manner (Xie, Z. and Price D. H. (1996) J. Biol. Chem. 271, 11043-11046). We show here that the transcript release activity of factor 2 requires ATP or dATP and that adenosine 5'-O-(thiotriphosphate) (ATPgammaS), adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), or other NTPs do not support the activity. Factor 2 demonstrated a strong DNA-dependent ATPase activity that correlated with its transcript release activity. At 20 microg/ml DNA, the ATPase activity of factor 2 had an apparent Km(ATP) of 28 microM and an estimated Kcat of 140 min-1. Factor 2 caused the release of nascent transcripts associated with elongation complexes generated by RNA polymerase II on a dC-tailed template. Therefore, no other protein cofactors are required for the transcript release activity of factor 2. Using the dC-tailed template assay, it was found that renaturation of the template was required for factor 2 function.

  11. The Transcription Factor p53 Influences Microglial Activation Phenotype

    PubMed Central

    Jayadev, Suman; Nesser, Nicole K.; Hopkins, Stephanie; Myers, Scott J.; Case, Amanda; Lee, Rona J.; Seaburg, Luke A.; Uo, Takuma; Murphy, Sean P.; Morrison, Richard S.; Garden, Gwenn A.

    2011-01-01

    Several neurodegenerative diseases are influenced by the innate immune response in the central nervous system (CNS). Microglia, have pro-inflammatory and subsequently neurotoxic actions as well as anti-inflammatory functions that promote recovery and repair. Very little is known about the transcriptional control of these specific microglial behaviors. We have previously shown that in HIV associated neurocognitive disorders (HAND), the transcription factor p53 accumulates in microglia and that microglial p53 expression is required for the in vitro neurotoxicity of the HIV coat glycoprotein gp120. These findings suggested a novel function for p53 in regulating microglial activation. Here we report that in the absence of p53, microglia demonstrate a blunted response to interferon-γ, failing to increase expression of genes associated with classical macrophage activation or secrete pro-inflammatory cytokines. Microarray analysis of global gene expression profiles revealed increased expression of genes associated with anti-inflammatory functions, phagocytosis and tissue repair in p53 knockout (p53−/−) microglia compared with those cultured from strain matched p53 expressing (p53+/+) mice. We further observed that p53−/− microglia demonstrate increased phagocytic activity in vitro and expression of markers for alternative macrophage activation both in vitro and in vivo. In HAND brain tissue, the alternative activation marker CD163 was expressed in a separate subset of microglia than those demonstrating p53 accumulation. These data suggest that p53 influences microglial behavior, supporting the adoption of a pro-inflammatory phenotype, while p53 deficiency promotes phagocytosis and gene expression associated with alternative activation and anti-inflammatory functions. PMID:21598312

  12. Activating transcription factor 3 regulates immune and metabolic homeostasis.

    PubMed

    Rynes, Jan; Donohoe, Colin D; Frommolt, Peter; Brodesser, Susanne; Jindra, Marek; Uhlirova, Mirka

    2012-10-01

    Integration of metabolic and immune responses during animal development ensures energy balance, permitting both growth and defense. Disturbed homeostasis causes organ failure, growth retardation, and metabolic disorders. Here, we show that the Drosophila melanogaster activating transcription factor 3 (Atf3) safeguards metabolic and immune system homeostasis. Loss of Atf3 results in chronic inflammation and starvation responses mounted primarily by the larval gut epithelium, while the fat body suffers lipid overload, causing energy imbalance and death. Hyperactive proinflammatory and stress signaling through NF-κB/Relish, Jun N-terminal kinase, and FOXO in atf3 mutants deregulates genes important for immune defense, digestion, and lipid metabolism. Reducing the dose of either FOXO or Relish normalizes both lipid metabolism and gene expression in atf3 mutants. The function of Atf3 is conserved, as human ATF3 averts some of the Drosophila mutant phenotypes, improving their survival. The single Drosophila Atf3 may incorporate the diversified roles of two related mammalian proteins.

  13. Transcriptional Regulation in Saccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

    PubMed Central

    Hahn, Steven; Young, Elton T.

    2011-01-01

    Here we review recent advances in understanding the regulation of mRNA synthesis in Saccharomyces cerevisiae. Many fundamental gene regulatory mechanisms have been conserved in all eukaryotes, and budding yeast has been at the forefront in the discovery and dissection of these conserved mechanisms. Topics covered include upstream activation sequence and promoter structure, transcription factor classification, and examples of regulated transcription factor activity. We also examine advances in understanding the RNA polymerase II transcription machinery, conserved coactivator complexes, transcription activation domains, and the cooperation of these factors in gene regulatory mechanisms. PMID:22084422

  14. Activating Transcription Factor 3 Regulates Immune and Metabolic Homeostasis

    PubMed Central

    Rynes, Jan; Donohoe, Colin D.; Frommolt, Peter; Brodesser, Susanne; Jindra, Marek

    2012-01-01

    Integration of metabolic and immune responses during animal development ensures energy balance, permitting both growth and defense. Disturbed homeostasis causes organ failure, growth retardation, and metabolic disorders. Here, we show that the Drosophila melanogaster activating transcription factor 3 (Atf3) safeguards metabolic and immune system homeostasis. Loss of Atf3 results in chronic inflammation and starvation responses mounted primarily by the larval gut epithelium, while the fat body suffers lipid overload, causing energy imbalance and death. Hyperactive proinflammatory and stress signaling through NF-κB/Relish, Jun N-terminal kinase, and FOXO in atf3 mutants deregulates genes important for immune defense, digestion, and lipid metabolism. Reducing the dose of either FOXO or Relish normalizes both lipid metabolism and gene expression in atf3 mutants. The function of Atf3 is conserved, as human ATF3 averts some of the Drosophila mutant phenotypes, improving their survival. The single Drosophila Atf3 may incorporate the diversified roles of two related mammalian proteins. PMID:22851689

  15. Identification of Post-Transcriptional Modulators of Breast Cancer Transcription Factor Activity Using MINDy

    PubMed Central

    Campbell, Thomas M.; Castro, Mauro A. A.; Ponder, Bruce A. J.

    2016-01-01

    We have recently identified transcription factors (TFs) that are key drivers of breast cancer risk. To better understand the pathways or sub-networks in which these TFs mediate their function we sought to identify upstream modulators of their activity. We applied the MINDy (Modulator Inference by Network Dynamics) algorithm to four TFs (ESR1, FOXA1, GATA3 and SPDEF) that are key drivers of estrogen receptor-positive (ER+) breast cancer risk, as well as cancer progression. Our computational analysis identified over 500 potential modulators. We assayed 189 of these and identified 55 genes with functional characteristics that were consistent with a role as TF modulators. In the future, the identified modulators may be tested as potential therapeutic targets, able to alter the activity of TFs that are critical in the development of breast cancer. PMID:27997592

  16. Building gene expression signatures indicative of transcription factor activation to predict AOP modulation

    EPA Science Inventory

    Building gene expression signatures indicative of transcription factor activation to predict AOP modulation Adverse outcome pathways (AOPs) are a framework for predicting quantitative relationships between molecular initiatin...

  17. Transcription Factor Arabidopsis Activating Factor1 Integrates Carbon Starvation Responses with Trehalose Metabolism.

    PubMed

    Garapati, Prashanth; Feil, Regina; Lunn, John Edward; Van Dijck, Patrick; Balazadeh, Salma; Mueller-Roeber, Bernd

    2015-09-01

    Plants respond to low carbon supply by massive reprogramming of the transcriptome and metabolome. We show here that the carbon starvation-induced NAC (for NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON) transcription factor Arabidopsis (Arabidopsis thaliana) Transcription Activation Factor1 (ATAF1) plays an important role in this physiological process. We identified TREHALASE1, the only trehalase-encoding gene in Arabidopsis, as a direct downstream target of ATAF1. Overexpression of ATAF1 activates TREHALASE1 expression and leads to reduced trehalose-6-phosphate levels and a sugar starvation metabolome. In accordance with changes in expression of starch biosynthesis- and breakdown-related genes, starch levels are generally reduced in ATAF1 overexpressors but elevated in ataf1 knockout plants. At the global transcriptome level, genes affected by ATAF1 are broadly associated with energy and carbon starvation responses. Furthermore, transcriptional responses triggered by ATAF1 largely overlap with expression patterns observed in plants starved for carbon or energy supply. Collectively, our data highlight the existence of a positively acting feedforward loop between ATAF1 expression, which is induced by carbon starvation, and the depletion of cellular carbon/energy pools that is triggered by the transcriptional regulation of downstream gene regulatory networks by ATAF1.

  18. Prediction of Pathway Activation by Xenobiotic-Responsive Transcription Factors in the Mouse Liver

    EPA Science Inventory

    Many drugs and environmentally-relevant chemicals activate xenobioticresponsive transcription factors (TF). Identification of target genes of these factors would be useful in predicting pathway activation in in vitro chemical screening. Starting with a large compendium of Affymet...

  19. Functional Interplay between CBP and PCAF in Acetylation and Regulation of Transcription Factor KLF13 Activity

    PubMed Central

    Song, Chao-Zhong; Keller, Kimberly; Chen, Yangchao; Stamatoyannopoulos, George

    2010-01-01

    The transcriptional co-activators CBP/p300 and PCAF participate in transcriptional activation by many factors. We have shown that both CBP/p300 and PCAF stimulate the transcriptional activation by KLF13, a member of the KLF/Sp1 family, either individually or cooperatively. Here we further investigated how CBP and PCAF acetylation regulate KLF13 activity, and how these two co-activators functionally interplay in the regulation of KLF13 activity. We found that CBP and PCAF acetylated KLF13 at specific lysine residues in the zinc finger domain of KLF13. The acetylation by CBP, however, resulted in disruption of KLF13 DNA binding. Although the acetyltransferase activity of CBP is not required for stimulating the DNA binding activity of all of the transcription factors that we have examined, the disruption of factor DNA binding by CBP acetylation is factor-specific. We further showed that PCAF and CBP act synergistically and antagonistically to regulate KLF13 DNA binding depending on the status of acetylation. PCAF blocked CBP acetylation and disruption of KLF13 DNA binding. Conversely, acetylation of KLF13 by CBP prevented PCAF stimulation of KLF13 DNA binding. PCAF blocked CBP disruption of KLF13 DNA binding by preventing CBP acetylation of KLF13. These results demonstrate that acetylation by CBP has distinct effects on transcription factor DNA binding, and that CBP and PCAF regulate each other functionally in their regulation of transcription factor DNA binding. PMID:12758070

  20. Nuclear Factor of Activated T Cells Transcription Factor Nfatp Controls Superantigen-Induced Lethal Shock

    PubMed Central

    Tsytsykova, Alla V.; Goldfeld, Anne E.

    2000-01-01

    Tumor necrosis factor α (TNF-α) is the key mediator of superantigen-induced T cell lethal shock. Here, we show that nuclear factor of activated T cells transcription factor, NFATp, controls susceptibility to superantigen-induced lethal shock in mice through its activation of TNF-α gene transcription. In NFATp-deficient mice, T cell stimulation leads to delayed induction and attenuation of TNF-α mRNA levels, decreased TNF-α serum levels, and resistance to superantigen-induced lethal shock. By contrast, after lipopolysaccharide (LPS) challenge, serum levels of TNF-α and susceptibility to shock are unaffected. These results demonstrate that NFATp is an essential activator of immediate early TNF-α gene expression in T cells and they present in vivo evidence of the inducer- and cell type–specific regulation of TNF-α gene expression. Furthermore, they suggest NFATp as a potential selective target in the treatment of superantigen-induced lethal shock. PMID:10952728

  1. Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression.

    EPA Science Inventory

    Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression Exposure to many drugs and environmentally-relevant chemicals can cause adverse outcomes. These adverse outcomes, such as cancer, have been linked to mol...

  2. Tcra enhancer activation by inducible transcription factors downstream of pre-TCR signaling.

    PubMed

    del Blanco, Beatriz; García-Mariscal, Alberto; Wiest, David L; Hernández-Munain, Cristina

    2012-04-01

    The Tcra enhancer (Eα) is essential for pre-TCR-mediated activation of germline transcription and V(D)J recombination. Eα is considered an archetypical enhanceosome that acts through the functional synergy and cooperative binding of multiple transcription factors. Based on dimethylsulfate genomic footprinting experiments, there has been a long-standing paradox regarding Eα activation in the absence of differences in enhancer occupancy. Our data provide the molecular mechanism of Eα activation and an explanation of this paradox. We found that germline transcriptional activation of Tcra is dependent on constant phospholipase Cγ, as well as calcineurin- and MAPK/ERK-mediated signaling, indicating that inducible transcription factors are crucially involved. NFAT, AP-1, and early growth response factor 1, together with CREB-binding protein/p300 coactivators, bind to Eα as part of an active enhanceosome assembled during pre-TCR signaling. We favor a scenario in which the binding of lymphoid-restricted and constitutive transcription factors to Eα prior to its activation forms a regulatory scaffold to recruit factors induced by pre-TCR signaling. Thus, the combinatorial assembly of tissue- and signal-specific transcription factors dictates the Eα function. This mechanism for enhancer activation may represent a general paradigm in tissue-restricted and stimulus-responsive gene regulation.

  3. Altered activities of transcription factors and their related gene expression in cardiac tissues of diabetic rats.

    PubMed

    Nishio, Y; Kashiwagi, A; Taki, H; Shinozaki, K; Maeno, Y; Kojima, H; Maegawa, H; Haneda, M; Hidaka, H; Yasuda, H; Horiike, K; Kikkawa, R

    1998-08-01

    Gene regulation in the cardiovascular tissues of diabetic subjects has been reported to be altered. To examine abnormal activities in transcription factors as a possible cause of this altered gene regulation, we studied the activity of two redox-sensitive transcription factors--nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1)--and the change in the mRNA content of heme oxygenase-1, which is regulated by these transcription factors in the cardiac tissues of rats with streptozotocin-induced diabetes. Increased activity of NF-kappaB and AP-1 but not nuclear transcription-activating factor, as determined by an electrophoretic mobility shift assay, was found in the hearts of 4-week diabetic rats. Glycemic control by a subcutaneous injection of insulin prevented these diabetes-induced changes in transcription factor activity. In accordance with these changes, the mRNA content of heme oxygenase-1 was increased fourfold in 4-week diabetic rats and threefold in 24-week diabetic rats as compared with control rats (P < 0.01 and P < 0.05, respectively). Insulin treatment also consistently prevented changes in the mRNA content of heme oxygenase-1. The oral administration of an antioxidant, probucol, to these diabetic rats partially prevented the elevation of the activity of both NF-kappaB and AP-1, and normalized the mRNA content of heme oxygenase-1 without producing any change in the plasma glucose concentration. These results suggest that elevated oxidative stress is involved in the activation of the transcription factors NF-kappaB and AP-1 in the cardiac tissues of diabetic rats, and that these abnormal activities of transcription factors could be associated with the altered gene regulation observed in the cardiovascular tissues of diabetic rats.

  4. Feedback regulation of PRL secretion is mediated by the transcription factor, signal transducer, and activator of transcription 5b.

    PubMed

    Grattan, D R; Xu, J; McLachlan, M J; Kokay, I C; Bunn, S J; Hovey, R C; Davey, H W

    2001-09-01

    PRL secretion from the anterior pituitary gland is inhibited by dopamine produced in the tuberoinfundibular dopamine neurons of the hypothalamus. The activity of tuberoinfundibular dopamine neurons is stimulated by PRL; thus, PRL regulates its own secretion by a negative feedback mechanism. PRL receptors are expressed on tuberoinfundibular dopamine neurons, but the intracellular signaling pathway is not known. We have observed that mice with a disrupted signal transducer and activator of transcription 5b gene have grossly elevated serum PRL concentrations. Despite this hyperprolactinemia, mRNA levels and immunoreactivity of tyrosine hydroxylase, the key enzyme in dopamine synthesis, were significantly lower in the tuberoinfundibular dopamine neurons of these signal transducer and activator of transcription 5b-deficient mice. Concentrations of the dopamine metabolite dihydroxyphenylacetic acid in the median eminence were also significantly lower in signal transducer and activator of transcription 5b-deficient mice than in wild-type mice. No changes were observed in nonhypothalamic dopaminergic neuronal populations, indicating that the effects were selective to tuberoinfundibular dopamine neurons. These data indicate that in the absence of signal transducer and activator of transcription 5b, PRL signal transduction in tuberoinfundibular dopamine neurons is impaired, and they demonstrate that this transcription factor plays an obligatory and nonredundant role in mediating the negative feedback action of PRL on tuberoinfundibular dopamine neurons.

  5. A transcription factor active on the epidermal growth factor receptor gene.

    PubMed Central

    Kageyama, R; Merlino, G T; Pastan, I

    1988-01-01

    We have developed an in vitro transcription system for the epidermal growth factor receptor (EGFR) oncogene by using nuclear extracts of A431 human epidermoid carcinoma cells, which overproduce EGFR. We found that a nuclear factor, termed EGFR-specific transcription factor (ETF), specifically stimulated EGFR transcription by 5- to 10-fold. In this report, ETF, purified by using sequence-specific oligonucleotide affinity chromatography, is shown by renaturing material eluted from a NaDodSO4/polyacrylamide gel to be a protein with a molecular mass of 120 kDa. ETF binds to the promoter region, as measured by DNase I "footprinting" and gel-mobility-shift assays, and specifically stimulates the transcription of the EGFR gene in a reconstituted in vitro transcription system. These results suggest that ETF could play a role in the overexpression of the cellular oncogene EGFR. Images PMID:3393529

  6. How glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering.

    PubMed

    Ratman, Dariusz; Vanden Berghe, Wim; Dejager, Lien; Libert, Claude; Tavernier, Jan; Beck, Ilse M; De Bosscher, Karolien

    2013-11-05

    The activity of the glucocorticoid receptor (GR), a nuclear receptor transcription factor belonging to subclass 3C of the steroid/thyroid hormone receptor superfamily, is typically triggered by glucocorticoid hormones. Apart from driving gene transcription via binding onto glucocorticoid response elements in regulatory regions of particular target genes, GR can also inhibit gene expression via transrepression, a mechanism largely based on protein:protein interactions. Hereby GR can influence the activity of other transcription factors, without contacting DNA itself. GR is known to inhibit the activity of a growing list of immune-regulating transcription factors. Hence, GCs still rule the clinic for treatments of inflammatory disorders, notwithstanding concomitant deleterious side effects. Although patience is a virtue when it comes to deciphering the many mechanisms GR uses to influence various signaling pathways, the current review is testimony of the fact that groundbreaking mechanistic work has been accumulating over the past years and steadily continues to grow.

  7. Angiotensin II activates the proinflammatory transcription factor nuclear factor-kappaB in human monocytes.

    PubMed

    Kranzhöfer, R; Browatzki, M; Schmidt, J; Kübler, W

    1999-04-21

    The renin-angiotensin system may contribute to the pathogenesis of atherosclerosis. A common feature of all stages of atherosclerosis is inflammation of the vessel wall. The transcription factor nuclear factor-kappaB (NF-kappaB) participates in most signaling pathways involved in inflammation. This study therefore examined the effect of angiotensin (ANG) II on NF-kappaB activation in monocytic cells, a major cellular component of human atheroma, by electrophoretic mobility shift assay. ANG II, like TNFalpha, caused rapid activation of NF-kappaB in human mononuclear cells isolated from peripheral blood by Ficoll density gradient. This ANG II effect was blocked by the angiotensin AT1 receptor antagonist losartan. Specificity of ANG II-induced NF-kappaB activation was ascertained by supershift and competition experiments. Moreover, ANG II stimulated NF-kappaB activation in human monocytes, but not in lymphocytes from the same preparation. Together, the data demonstrate the ability of the vasoactive peptide ANG II to activate inflammatory pathways in human monocytes.

  8. Nuclear actin activates human transcription factor genes including the OCT4 gene.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; Tokunaga, Makio; Sakata-Sogawa, Kumiko; Harata, Masahiko

    2015-01-01

    RNA microarray analyses revealed that nuclear actin activated many human transcription factor genes including OCT4, which is required for gene reprogramming. Oct4 is known to be activated by nuclear actin in Xenopus oocytes. Our findings imply that this process of OCT4 activation is conserved in vertebrates and among cell types and could be used for gene reprogramming of human cells.

  9. C/EBP Transcription Factors Mediate Epicardial Activation During Heart Development and Injury

    PubMed Central

    Huang, Guo N.; Thatcher, Jeffrey E.; McAnally, John; Kong, Yongli; Qi, Xiaoxia; Tan, Wei; DiMaio, J. Michael; Amatruda, James F.; Gerard, Robert D.; Hill, Joseph A.; Bassel-Duby, Rhonda; Olson, Eric N.

    2013-01-01

    The epicardium encapsulates the heart and functions as a source of multipotent progenitor cells and paracrine factors essential for cardiac development and repair. Injury of the adult heart results in reactivation of a developmental gene program in the epicardium, but the transcriptional basis of epicardial gene expression has not been delineated. We established a mouse embryonic heart organ culture and gene expression system that facilitated the identification of epicardial enhancers activated during heart development and injury. Epicardial activation of these enhancers depends on a combinatorial transcriptional code centered on CCAAT/enhancer binding protein (C/EBP) transcription factors. Disruption of C/EBP signaling in the adult epicardium reduced injury-induced neutrophil infiltration and improved cardiac function. These findings reveal a transcriptional basis for epicardial activation and heart injury, providing a platform for enhancing cardiac regeneration. PMID:23160954

  10. Direct transcriptional activation of BT genes by NLP transcription factors is a key component of the nitrate response in Arabidopsis.

    PubMed

    Sato, Takeo; Maekawa, Shugo; Konishi, Mineko; Yoshioka, Nozomi; Sasaki, Yuki; Maeda, Haruna; Ishida, Tetsuya; Kato, Yuki; Yamaguchi, Junji; Yanagisawa, Shuichi

    2017-01-29

    Nitrate modulates growth and development, functioning as a nutrient signal in plants. Although many changes in physiological processes in response to nitrate have been well characterized as nitrate responses, the molecular mechanisms underlying the nitrate response are not yet fully understood. Here, we show that NLP transcription factors, which are key regulators of the nitrate response, directly activate the nitrate-inducible expression of BT1 and BT2 encoding putative scaffold proteins with a plant-specific domain structure in Arabidopsis. Interestingly, the 35S promoter-driven expression of BT2 partially rescued growth inhibition caused by reductions in NLP activity in Arabidopsis. Furthermore, simultaneous disruption of BT1 and BT2 affected nitrate-dependent lateral root development. These results suggest that direct activation of BT1 and BT2 by NLP transcriptional activators is a key component of the molecular mechanism underlying the nitrate response in Arabidopsis.

  11. Transcription factor Runx3 regulates interleukin-15-dependent natural killer cell activation.

    PubMed

    Levanon, Ditsa; Negreanu, Varda; Lotem, Joseph; Bone, Karen Rae; Brenner, Ori; Leshkowitz, Dena; Groner, Yoram

    2014-03-01

    Natural killer cells belong to the family of innate lymphoid cells comprising the frontline defense against infected and transformed cells. Development and activation of natural killer cells is highly dependent on interleukin-15 signaling. However, very little is known about the transcription program driving this process. The transcription factor Runx3 is highly expressed in natural killer cells, but its function in these cells is largely unknown. We show that loss of Runx3 impaired interleukin-15-dependent accumulation of mature natural killer cells in vivo and under culture conditions and pregnant Runx3(-/-) mice completely lack the unique population of interleukin-15-dependent uterine natural killer cells. Combined chromatin immunoprecipitation sequencing and differential gene expression analysis of wild-type versus Runx3-deficient in vivo activated splenic natural killer cells revealed that Runx3 cooperates with ETS and T-box transcription factors to drive the interleukin-15-mediated transcription program during activation of these cells. Runx3 functions as a nuclear regulator during interleukin-15-dependent activation of natural killer cells by regulating the expression of genes involved in proliferation, maturation, and migration. Similar studies with additional transcription factors will allow the construction of a more detailed transcriptional network that controls natural killer cell development and function.

  12. Expression and Activation of STAT Transcription Factors in Breast Cancer

    DTIC Science & Technology

    1998-05-08

    several other studies suggest that estrogen given in low doses to relieve menopausal symptoms probably does not increase the incidence of breast cancer...breast cancer. ’It was recently demonstrated that, while .overall STAT DNA-bindinq activity is low in normal breast and benign lesions, it is...adjuvant anticancer treatment, particularly in chronic myelogenous leukemia, maliqnant melanoma, low · grade lymphoma, multiple myeloma, and midgut

  13. Repression of the heat shock factor 1 transcriptional activation domain is modulated by constitutive phosphorylation.

    PubMed Central

    Kline, M P; Morimoto, R I

    1997-01-01

    Heat shock transcription factor 1 (HSF1) is constitutively expressed in mammalian cells and negatively regulated for DNA binding and transcriptional activity. Upon exposure to heat shock and other forms of chemical and physiological stress, these activities of HSF1 are rapidly induced. In this report, we demonstrate that constitutive phosphorylation of HSF1 at serine residues distal to the transcriptional activation domain functions to repress transactivation. Tryptic phosphopeptide analysis of a collection of chimeric GAL4-HSF1 deletion and point mutants identified a region of constitutive phosphorylation encompassing serine residues 303 and 307. The significance of phosphorylation at serines 303 and 307 in the regulation of HSF1 transcriptional activity was demonstrated by transient transfection and assay of a chloramphenicol acetyltransferase reporter construct. Whereas the transfected wild-type GAL4-HSF1 chimera is repressed for transcriptional activity and derepressed by heat shock, mutation of serines 303 and 307 to alanine results in derepression to a high level of constitutive activity. Similar results were obtained with mutation of these serine residues in the context of full-length HSF1. These data reveal that constitutive phosphorylation of serines 303 and 307 has an important role in the negative regulation of HSF1 transcriptional activity at control temperatures. PMID:9121459

  14. Cytokinin Response Factor 5 has transcriptional activity governed by its C-terminal domain.

    PubMed

    Striberny, Bernd; Melton, Anthony E; Schwacke, Rainer; Krause, Kirsten; Fischer, Karsten; Goertzen, Leslie R; Rashotte, Aaron M

    2017-02-01

    Cytokinin Response Factors (CRFs) are AP2/ERF transcription factors involved in cytokinin signal transduction. CRF proteins consist of a N-terminal dimerization domain (CRF domain), an AP2 DNA-binding domain, and a clade-specific C-terminal region of unknown function. Using a series of sequential deletions in yeast-2-hybrid assays, we provide evidence that the C-terminal region of Arabidopsis CRF5 can confer transactivation activity. Although comparative analyses identified evolutionarily conserved protein sequence within the C-terminal region, deletion experiments suggest that this transactivation domain has a partially redundant modular structure required for activation of target gene transcription.

  15. Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators.

    PubMed

    Bogerd, Hal P; Kornepati, Anand V R; Marshall, Joy B; Kennedy, Edward M; Cullen, Bryan R

    2015-12-29

    Whereas several mammalian proteins can restrict the replication of HIV-1 and other viruses, these are often not expressed in relevant target cells. A potential method to inhibit viral replication might therefore be to use synthetic transcription factors to induce restriction factor expression. In particular, mutants of the RNA-guided DNA binding protein Cas9 that have lost their DNA cleavage activity could be used to recruit transcription activation domains to specific promoters. However, initial experiments revealed only weak activation unless multiple promoter-specific single guide RNAs (sgRNAs) were used. Recently, the recruitment of multiple transcription activation domains by a single sgRNA, modified to contain MS2-derived stem loops that recruit fusion proteins consisting of the MS2 coat protein linked to transcription activation domains, was reported to induce otherwise silent cellular genes. Here, we demonstrate that such "synergistic activation mediators" can induce the expression of two restriction factors, APOBEC3G (A3G) and APOBEC3B (A3B), in human cells that normally lack these proteins. We observed modest activation of endogenous A3G or A3B expression using single sgRNAs but high expression when two sgRNAs were used. Whereas the induced A3G and A3B proteins both blocked infection by an HIV-1 variant lacking a functional vif gene by inducing extensive dC-to-dU editing, only the induced A3B protein inhibited wild-type HIV-1. These data demonstrate that Cas9-derived transcriptional activators have the potential to be used for screens for endogenous genes that affect virus replication and raise the possibility that synthetic transcription factors might prove clinically useful if efficient delivery mechanisms could be developed.

  16. PDGF inactivates forkhead family transcription factor by activation of Akt in glomerular mesangial cells.

    PubMed

    Ghosh Choudhury, Goutam; Lenin, Mahimainathan; Calhaun, Cheresa; Zhang, Jian-Hua; Abboud, Hanna E

    2003-02-01

    Regulation of the forkhead domain transcription factors by PDGF has not been studied. In this report, we investigated the role of PDGF-induced Akt in regulating forkhead domain protein FKHRL1 in glomerular mesangial cells. PDGF increased phosphorylation of FKHRL1 in a time- and PI 3 kinase-dependent manner. Expression of dominant negative Akt by adenovirus-mediated gene transfer blocked PDGF-induced FKHRL1 phosphorylation. PDGF inhibited transcription of a forkhead DNA binding element-driven reporter gene. This inhibition was mimicked by constitutively active myristoylated Akt. Moreover, FKHR1-mediated transcription of the reporter gene was completely attenuated by both PDGF and Myr-Akt. One of the targets of forkhead transcription factors is the proapoptotic Fas ligand (FasL) gene. PDGF, as well as Myr-Akt, inhibited transcription of FasL. In contrast, inhibition of PI 3 kinase and dominant negative Akt increased FasL gene transcription, suggesting that suppression of PI 3 kinase/Akt signalling may induce apoptosis in mesangial cells via upregulation of FasL expression. However, expression of dominant negative Akt by adenovirus did not induce apoptosis in mesangial cells, suggesting that Akt-independent antiapoptotic mechanisms also exist. Together, our data demonstrate for the first time that PDGF inactivates forkhead domain transcription factor by Akt-dependent phosphorylation and that suppression of Akt signalling is not sufficient to induce apoptosis in mesangial cells.

  17. The transcription factor RFX5 is a transcriptional activator of the TPP1 gene in hepatocellular carcinoma.

    PubMed

    Zhao, Yangjing; Xie, Xingwang; Liao, Weijia; Zhang, Henghui; Cao, Hui; Fei, Ran; Wang, Xueyan; Wei, Lai; Shao, Qixiang; Chen, Hongsong

    2017-01-01

    Regulatory factor X-5 (RFX5) was previously characterized as an essential and highly specific regulator of major histocompatibility class II (MHCII) gene expression in the immune system. We found that RFX5 is significantly upregulated in hepatocellular carcinoma (HCC) tumors and cell lines compared with non-tumor tissues in mRNA expression levels, but it fails to induce the expression of MHCII. However, RFX5 can strongly bind to the tripeptidyl peptidase 1 (TPP1) promoter region and then increase its transcriptional activity. We also found that manipulation the expression of RFX5 can significantly affect the expression of TPP1 in HepG2, which suggested that RFX5 can transcriptionally activate TPP1 in HCC. Moreover, TPP1 is overexpressed in HCC tissues and significantly correlated with poor prognosis of HCC patients, suggesting that it may have potential biological implications in HCC.

  18. Transcription factor activation following exposure of an intact lung preparation to metallic particulate matter.

    PubMed Central

    Samet, James M; Silbajoris, Robert; Huang, Tony; Jaspers, Ilona

    2002-01-01

    Metallic constituents contained in ambient particulate matter have been associated with adverse effects in a number of epidemiologic, in vitro, and in vivo studies. Residual oil fly ash (ROFA) is a metallic by-product of the combustion of fossil fuel oil, which has been shown to induce a variety of proinflammatory responses in lung cells. We have examined signaling pathways activated in response to ROFA exposure and recently reported that ROFA treatment activates multiple mitogen-activated protein (MAP) kinases in the rat lung. In the present study we extended our investigations on the mechanism of toxicity of ROFA to include transcription factors whose activities are regulated by MAP kinases as well as possible effectors of transcriptional changes that mediate the effects of ROFA. We applied immunohistochemical methods to detect ROFA-induced activation of nuclear factor-kappa B (NF kappa B), activating transcription factor-2 (ATF-2), c-Jun, and cAMP response element binding protein (CREB) in intact lung tissue and confirmed and characterized their functional activation using DNA binding assays. We performed these studies using a perfused rabbit lung model that is devoid of blood elements in order to distinguish between intrinsic lung cell effects and effects that are secondary to inflammatory cell influx. We report here that exposure to ROFA results in a rapid activation of all of the transcription factors studied by exerting direct effects on lung cells. These findings validate the use of immunohistochemistry to detect transcription factor activation in vivo and demonstrate the utility of studying signaling changes in response to environmental exposures. PMID:12361922

  19. Gene deregulation and chronic activation in natural killer cells deficient in the transcription factor ETS1.

    PubMed

    Ramirez, Kevin; Chandler, Katherine J; Spaulding, Christina; Zandi, Sasan; Sigvardsson, Mikael; Graves, Barbara J; Kee, Barbara L

    2012-06-29

    Multiple transcription factors guide the development of mature functional natural killer (NK) cells, yet little is known about their function. We used global gene expression and genome-wide binding analyses combined with developmental and functional studies to unveil three roles for the ETS1 transcription factor in NK cells. ETS1 functions at the earliest stages of NK cell development to promote expression of critical transcriptional regulators including T-BET and ID2, NK cell receptors (NKRs) including NKp46, Ly49H, and Ly49D, and signaling molecules essential for NKR function. As a consequence, Ets1(-/-) NK cells fail to degranulate after stimulation through activating NKRs. Nonetheless, these cells are hyperresponsive to cytokines and have characteristics of chronic stimulation including increased expression of inhibitory NKRs and multiple activation-associated genes. Therefore, ETS1 regulates a broad gene expression program in NK cells that promotes target cell recognition while limiting cytokine-driven activation.

  20. Transcriptional Activation Domains of Human Heat Shock Factor 1 Recruit Human SWI/SNF

    PubMed Central

    Sullivan, E. Kelly; Weirich, Christine S.; Guyon, Jeffrey R.; Sif, Saïd; Kingston, Robert E.

    2001-01-01

    Chromatin remodeling complexes such as SWI/SNF use the energy of ATP hydrolysis to remodel nucleosomal DNA and increase transcription of nucleosomal templates. Human heat shock factor one (hHSF1) is a tightly regulated activator that stimulates transcriptional initiation and elongation using different portions of its activation domains. Here we demonstrate that hHSF1 associates with BRG1, the ATPase subunit of human SWI/SNF (hSWI/SNF) at endogenous protein concentrations. We also show that hHSF1 activation domains recruit hSWI/SNF to a chromatin template in a purified system. Mutation of hHSF1 residues responsible for activation of transcriptional elongation has the most severe effect on recruitment of SWI/SNF and association of hHSF1 with BRG1, suggesting that recruitment of chromatin remodeling activity might play a role in stimulation of elongation. PMID:11486022

  1. 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

  2. Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain.

    PubMed Central

    Kirkpatrick, D T; Fan, Q; Petes, T D

    1999-01-01

    The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot. Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination. We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of recombination hotspots, alpha (transcription factor dependent) and beta (transcription factor independent). PMID:10224246

  3. Chromatin Remodeling Mediated by Drosophila GAGA Factor and ISWI Activates fushi tarazu Gene Transcription In Vitro

    PubMed Central

    Okada, Masahiro; Hirose, Susumu

    1998-01-01

    GAGA factor is known to remodel the chromatin structure in concert with nucleosome-remodeling factor NURF in a Drosophila embryonic S150 extract. The promoter region of the Drosophila fushi tarazu (ftz) gene carries several binding sites for GAGA factor. Both the GAGA factor-binding sites and GAGA factor per se are necessary for the proper expression of ftz in vivo. We observed transcriptional activation of the ftz gene when a preassembled chromatin template was incubated with GAGA factor and the S150 extract. The chromatin structure within the ftz promoter was specifically disrupted by incubation of the preassembled chromatin with GAGA factor and the S150 extract. Both transcriptional activation and chromatin disruption were blocked by an antiserum raised against ISWI or by base substitutions in the GAGA factor-binding sites in the ftz promoter region. These results demonstrate that GAGA factor- and ISWI-mediated disruption of the chromatin structure within the promoter region of ftz activates transcription on the chromatin template. PMID:9566866

  4. TRANSCRIPTION FACTOR ACTIVATION FOLLOWING EXPOSURE OF AN INTACT LUNG PREPARATION TO METALLIC PARTICULATE MATTER

    EPA Science Inventory

    TRANSCRIPTION FACTOR ACTIVATION FOLLOWING EXPOSURE OF AN INTACT LUNG PREPARATION TO METALLIC PARTICULATE MATTER

    James M. Samet1,2, Robert Silbajoris1, Tony Huang1 and Ilona Jaspers3

    1Human Studies Division, National Health and Environmental Effects Research Laborato...

  5. MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

    PubMed Central

    Nezich, Catherine L.; Wang, Chunxin; Fogel, Adam I.

    2015-01-01

    The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats–generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy. PMID:26240184

  6. Inflammatory transcription factors as activation markers and functional readouts in immune-to-brain communication.

    PubMed

    Rummel, Christoph

    2016-05-01

    Immune-to-brain communication pathways involve humoral mediators, including cytokines, central modulation by neuronal afferents and immune cell trafficking to the brain. During systemic inflammation these pathways contribute to mediating brain-controlled sickness symptoms including fever. Experimentally, activation of these signaling pathways can be mimicked and studied when injecting animals with pathogen associated molecular patterns (PAMPS). One central component of the brain inflammatory response, which leads, for example, to fever induction, is transcriptional activation of brain cells via cytokines and PAMPS. We and others have studied the spatiotemporal activation and the physiological significance of transcription factors for the induction of inflammation within the brain and the manifestation of fever. Evidence has revealed a role of nuclear factor (NF)κB in the initiation, signal transducer and activator of transcription (STAT)3 in the maintenance and NF-interleukin (IL)6 in the maintenance or even termination of brain-inflammation and fever. Moreover, psychological stressors, such as exposure to a novel environment, leads to increased body core temperature and genomic NF-IL6-activation, suggesting a potential use of NF-IL6-immunohistochemistry as a multimodal brain cell activation marker and a role for NF-IL6 for differential brain activity. In addition, the nutritional status, as reflected by circulating levels of the cytokine-like hormone leptin, influence immune-to-brain communication and age-dependent changes in LPS-induced fever. Overall, transcription factors remain therapeutically important targets for the treatment of brain-inflammation and fever induction during infectious/non-infectious inflammatory and psychological stress. However, the exact physiological role and significance of these transcription factors requires to be further investigated.

  7. Protein-DNA array-based identification of transcription factor activities differentially regulated in obliterative bronchiolitis

    PubMed Central

    Dong, Ming; Wang, Xin; Zhao, Hong-Lin; Zhao, Yu-Xia; Jing, Ya-Qing; Yuan, Jing-Hua; Guo, Yi-Jiu; Chen, Xing-Long; Li, Ke-Qiu; Li, Guang

    2015-01-01

    Lung transplantation has already become the preferred treatment option for a variety of end-stage pulmonary failure. However the long-term results of lung transplantation are still not compelling and the major death reason is commonly due to obliterative bronchiolitis (OB) which is considered as chronic rejection presenting manifests physiologically as a progressive decline in FEV1. Transcription factors (TFs) play a key role in regulating gene expression and in providing an interconnecting regulatory between related pathway elements. Although the transcription factors are required for expression of the proinflammatory cytokines and immune proteins which are involved in obliterative bronchiolitis following lung transplantation, the alterations of the transcription factors in OB have not yet been revealed. Therefore, to investigate the alteration pattern of the transcription factors in OB, we used protein/DNA arrays. Mice orthotopic tracheal transplantation model was used in this studying. In this study, we explored the activity profiles of TFs in Protein/DNA array data of tracheal tissue in 14 and 28 day after transplanted. From a total of 345 screened TFs, we identified 42 TFs that showed associated with OB progression. Our data indicate that TFs may be potentially involved in the pathogenesis of OB, and can prevent, diagnose and treat OB after lung transplantation. In development of OB, some of the TFs may have ability to modulate the transcription of inflammatory proteins such cytokines, inflammatory enzymes and so on. PMID:26261607

  8. Systems analysis of transcription factor activities in environments with stable and dynamic oxygen concentrations.

    PubMed

    Rolfe, Matthew D; Ocone, Andrea; Stapleton, Melanie R; Hall, Simon; Trotter, Eleanor W; Poole, Robert K; Sanguinetti, Guido; Green, Jeffrey

    2012-07-01

    Understanding gene regulation requires knowledge of changes in transcription factor (TF) activities. Simultaneous direct measurement of numerous TF activities is currently impossible. Nevertheless, statistical approaches to infer TF activities have yielded non-trivial and verifiable predictions for individual TFs. Here, global statistical modelling identifies changes in TF activities from transcript profiles of Escherichia coli growing in stable (fixed oxygen availabilities) and dynamic (changing oxygen availability) environments. A core oxygen-responsive TF network, supplemented by additional TFs acting under specific conditions, was identified. The activities of the cytoplasmic oxygen-responsive TF, FNR, and the membrane-bound terminal oxidases implied that, even on the scale of the bacterial cell, spatial effects significantly influence oxygen-sensing. Several transcripts exhibited asymmetrical patterns of abundance in aerobic to anaerobic and anaerobic to aerobic transitions. One of these transcripts, ndh, encodes a major component of the aerobic respiratory chain and is regulated by oxygen-responsive TFs ArcA and FNR. Kinetic modelling indicated that ArcA and FNR behaviour could not explain the ndh transcript profile, leading to the identification of another TF, PdhR, as the source of the asymmetry. Thus, this approach illustrates how systematic examination of regulatory responses in stable and dynamic environments yields new mechanistic insights into adaptive processes.

  9. Longitudinal in vivo bioimaging of hepatocyte transcription factor activity following cholestatic liver injury in mice

    PubMed Central

    Delhove, Juliette M. K. M.; Buckley, Suzanne M. K.; Perocheau, Dany P.; Karda, Rajvinder; Arbuthnot, Patrick; Henderson, Neil C.; Waddington, Simon N.; McKay, Tristan R.

    2017-01-01

    Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-κB and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-κB and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes. PMID:28157201

  10. Longitudinal in vivo bioimaging of hepatocyte transcription factor activity following cholestatic liver injury in mice.

    PubMed

    Delhove, Juliette M K M; Buckley, Suzanne M K; Perocheau, Dany P; Karda, Rajvinder; Arbuthnot, Patrick; Henderson, Neil C; Waddington, Simon N; McKay, Tristan R

    2017-02-03

    Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-κB and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-κB and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes.

  11. C3 exoenzyme impairs cell proliferation and apoptosis by altering the activity of transcription factors.

    PubMed

    von Elsner, Leonie; Hagemann, Sandra; Just, Ingo; Rohrbeck, Astrid

    2016-09-01

    C3 exoenzyme from C. botulinum is an ADP-ribosyltransferase that inactivates selectively RhoA, B, and C by coupling an ADP-ribose moiety. Rho-GTPases are involved in various cellular processes, such as regulation of actin cytoskeleton, cell proliferation, and apoptosis. Previous studies of our group with the murine hippocampal cell line HT22 revealed a C3-mediated inhibition of cell proliferation after 48 h and a prevention of serum-starved cells from apoptosis. For both effects, alterations of various signaling pathways are already known, including also changes on the transcriptional level. Investigations on the transcriptional activity in HT22 cells treated with C3 for 48 h identified five out of 48 transcription factors namely Sp1, ATF2, E2F-1, CBF, and Stat6 with a significantly regulated activity. For validation of identified transcription factors, studies on the protein level of certain target genes were performed. Western blot analyses exhibited an enhanced abundance of Sp1 target genes p21 and COX-2 as well as an increase in phosphorylation of c-Jun. In contrast, the level of p53 and apoptosis-inducing GADD153, a target gene of ATF2, was decreased. Our results reveal that C3 regulates the transcriptional activity of Sp1 and ATF2 resulting downstream in an altered protein abundance of various target genes. As the affected proteins are involved in the regulation of cell proliferation and apoptosis, thus the C3-mediated anti-proliferative and anti-apoptotic effects are consequences of the Rho-dependent alterations of the activity of certain transcriptional factors.

  12. Multiple transcription factor codes activate epidermal wound-response genes in Drosophila.

    PubMed

    Pearson, Joseph C; Juarez, Michelle T; Kim, Myungjin; Drivenes, Øyvind; McGinnis, William

    2009-02-17

    Wounds in Drosophila and mouse embryos induce similar genetic pathways to repair epidermal barriers. However, the transcription factors that transduce wound signals to repair epidermal barriers are largely unknown. We characterize the transcriptional regulatory enhancers of 4 genes-Ddc, ple, msn, and kkv-that are rapidly activated in epidermal cells surrounding wounds in late Drosophila embryos and early larvae. These epidermal wound enhancers all contain evolutionarily conserved sequences matching binding sites for JUN/FOS and GRH transcription factors, but vary widely in trans- and cis-requirements for these inputs and their binding sites. We propose that the combination of GRH and FOS is part of an ancient wound-response pathway still used in vertebrates and invertebrates, but that other mechanisms have evolved that result in similar transcriptional output. A common, but largely untested assumption of bioinformatic analyses of gene regulatory networks is that transcription units activated in the same spatial and temporal patterns will require the same cis-regulatory codes. Our results indicate that this is an overly simplistic view.

  13. Visualization of positive transcription elongation factor b (P-TEFb) activation in living cells.

    PubMed

    Fujinaga, Koh; Luo, Zeping; Schaufele, Fred; Peterlin, B Matija

    2015-01-16

    Regulation of transcription elongation by positive transcription elongation factor b (P-TEFb) plays a central role in determining the state of cell activation, proliferation, and differentiation. In cells, P-TEFb exists in active and inactive forms. Its release from the inactive 7SK small nuclear ribonucleoprotein complex is a critical step for P-TEFb to activate transcription elongation. However, no good method exists to analyze this P-TEFb equilibrium in living cells. Only inaccurate and labor-intensive cell-free biochemical assays are currently available. In this study, we present the first experimental system to monitor P-TEFb activation in living cells. We created a bimolecular fluorescence complementation assay to detect interactions between P-TEFb and its substrate, the C-terminal domain of RNA polymerase II. When cells were treated with suberoylanilide hydroxamic acid, which releases P-TEFb from the 7SK small nuclear ribonucleoprotein, they turned green. Other known P-TEFb-releasing agents, including histone deacetylase inhibitors, bromodomain and extraterminal bromodomain inhibitors, and protein kinase C agonists, also scored positive in this assay. Finally, we identified 5'-azacytidine as a new P-TEFb-releasing agent. This release of P-TEFb correlated directly with activation of human HIV and HEXIM1 transcription. Thus, our visualization of P-TEFb activation by fluorescent complementation assay could be used to find new P-TEFb-releasing agents, compare different classes of agents, and assess their efficacy singly and/or in combination.

  14. Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8.

    PubMed

    Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L; Rosen, Barry P; Tamás, Markus J

    2015-12-28

    The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

  15. Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

    PubMed Central

    Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L.; Rosen, Barry P.

    2015-01-01

    The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation. PMID:26711267

  16. Stress-Activated Cap’n’collar Transcription Factors in Aging and Human Disease

    PubMed Central

    Sykiotis, Gerasimos P.; Bohmann, Dirk

    2010-01-01

    Cap’n’collar (Cnc) transcription factors are conserved in metazoans and have important developmental and homeostatic functions. The vertebrate Nrf1, Nrf2, and Nrf3, the Caenorhabditis elegans SKN-1, and the Drosophila CncC comprise a subgroup of Cnc factors that mediate adaptive responses to cellular stress. The most studied stress-activated Cnc factor is Nrf2, which orchestrates the transcriptional response of cells to oxidative stressors and electrophilic xenobiotics. In rodent models, signaling by Nrf2 defends against oxidative stress and aging-associated disorders, such as neurodegeneration, respiratory diseases, and cancer. In humans, polymorphisms that decrease Nrf2 abundance have been associated with various pathologies of the skin, respiratory system, and digestive tract. In addition to preventing disease in rodents and humans, Cnc factors have lifespan-extending and anti-aging functions in invertebrates. However, despite the pro-longevity and antioxidant roles of stress-activated Cnc factors, their activity paradoxically declines in aging model organisms and in humans suffering from progressing respiratory disease or neurodegeneration. We review the roles and regulation of stress-activated Cnc factors across species, present all reported instances in which their activity is paradoxically decreased in aging and disease, and discuss the possibility that the pharmacological restoration of Nrf2 signaling may be useful in the prevention and treatment of age-related diseases. PMID:20215646

  17. Transforming growth factor β-activated kinase 1 transcriptionally suppresses hepatitis B virus replication

    PubMed Central

    Pang, Jinke; Zhang, Geng; Lin, Yong; Xie, Zhanglian; Liu, Hongyan; Tang, Libo; Lu, Mengji; Yan, Ran; Guo, Haitao; Sun, Jian; Hou, Jinlin; Zhang, Xiaoyong

    2017-01-01

    Hepatitis B Virus (HBV) replication in hepatocytes is restricted by the host innate immune system and related intracellular signaling pathways. Transforming growth factor β-activated kinase 1 (TAK1) is a key mediator of toll-like receptors and pro-inflammatory cytokine signaling pathways. Here, we report that silencing or inhibition of endogenous TAK1 in hepatoma cell lines leads to an upregulation of HBV replication, transcription, and antigen expression. In contrast, overexpression of TAK1 significantly suppresses HBV replication, while an enzymatically inactive form of TAK1 exerts no effect. By screening TAK1-associated signaling pathways with inhibitors and siRNAs, we found that the MAPK-JNK pathway was involved in TAK1-mediated HBV suppression. Moreover, TAK1 knockdown or JNK pathway inhibition induced the expression of farnesoid X receptor α, a transcription factor that upregulates HBV transcription. Finally, ectopic expression of TAK1 in a HBV hydrodynamic injection mouse model resulted in lower levels of HBV DNA and antigens in both liver and serum. In conclusion, our data suggest that TAK1 inhibits HBV primarily at viral transcription level through activation of MAPK-JNK pathway, thus TAK1 represents an intrinsic host restriction factor for HBV replication in hepatocytes. PMID:28045080

  18. Transcriptional activation of mouse mast cell Protease-7 by activin and transforming growth factor-beta is inhibited by microphthalmia-associated transcription factor.

    PubMed

    Funaba, Masayuki; Ikeda, Teruo; Murakami, Masaru; Ogawa, Kenji; Tsuchida, Kunihiro; Sugino, Hiromu; Abe, Matanobu

    2003-12-26

    Previous studies have revealed that activin A and transforming growth factor-beta1 (TGF-beta1) induced migration and morphological changes toward differentiation in bone marrow-derived cultured mast cell progenitors (BMCMCs). Here we show up-regulation of mouse mast cell protease-7 (mMCP-7), which is expressed in differentiated mast cells, by activin A and TGF-beta1 in BMCMCs, and the molecular mechanism of the gene induction of mmcp-7. Smad3, a signal mediator of the activin/TGF-beta pathway, transcriptionally activated mmcp-7. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, and heart and skeletal muscle, inhibited Smad3-mediated mmcp-7 transcription. MITF associated with Smad3, and the C terminus of MITF and the MH1 and linker region of Smad3 were required for this association. Complex formation between Smad3 and MITF was neither necessary nor sufficient for the inhibition of Smad3 signaling by MITF. MITF inhibited the transcriptional activation induced by the MH2 domain of Smad3. In addition, MITF-truncated N-terminal amino acids could associate with Smad3 but did not inhibit Smad3-mediated transcription. The level of Smad3 was decreased by co-expression of MITF but not of dominant-negative MITF, which resulted from proteasomal protein degradation. The changes in the level of Smad3 protein were paralleled by those in Smad3-mediated signaling activity. These findings suggest that MITF negatively regulates Smad-dependent activin/TGF-beta signaling in a tissue-specific manner.

  19. In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters

    PubMed Central

    Buckley, Suzanne M. K.; Delhove, Juliette M. K. M.; Perocheau, Dany P.; Karda, Rajvinder; Rahim, Ahad A.; Howe, Steven J.; Ward, Natalie J.; Birrell, Mark A.; Belvisi, Maria G.; Arbuthnot, Patrick; Johnson, Mark R.; Waddington, Simon N.; McKay, Tristan R.

    2015-01-01

    The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruses to neonatal rodents as an alternative to the existing technology of generating germline transgenic light producing rodents. At this age, neonates acquire immune tolerance to the conditionally responsive luciferase reporter. This simple and transferrable procedure permits surrogate quantitation of transcription factor activity over the lifetime of the animal. We show principal efficacy by temporally quantifying NFκB activity in the brain, liver and lungs of somatotransgenic reporter mice subjected to lipopolysaccharide (LPS)-induced inflammation. This response is ablated in Tlr4−/− mice or when co-administered with the anti-inflammatory glucocorticoid analogue dexamethasone. Furthermore, we show the malleability of this technology by quantifying NFκB-mediated luciferase expression in outbred rats. Finally, we use somatotransgenic bioimaging to longitudinally quantify LPS- and ActivinA-induced upregulation of liver specific glucocorticoid receptor and Smad2/3 reporter constructs in somatotransgenic mice, respectively. PMID:26138224

  20. Plants contain a novel multi-member class of heat shock factors without transcriptional activator potential.

    PubMed

    Czarnecka-Verner, E; Yuan, C X; Scharf, K D; Englich, G; Gurley, W B

    2000-07-01

    Based on phylogeny of DNA-binding domains and the organization of hydrophobic repeats, two families of heat shock transcription factors (HSFs) exist in plants. Class A HSFs are involved in the activation of the heat shock response, but the role of class B HSFs is not clear. When transcriptional activities of full-length HSFs were monitored in tobacco protoplasts, no class B HSFs from soybean or Arabidopsis showed activity under control or heat stress conditions. Additional assays confirmed the finding that the class B HSFs lacked the capacity to activate transcription. Fusion of a heterologous activation domain from human HSF1 (AD2) to the C-terminus of GmHSFB1-34 gave no evidence of synergistic enhancement of AD2 activity, which would be expected if weak activation domains were present. Furthermore, activity of AtHSFB1-4 (class B) was not rescued by coexpression with AtHSFA4-21 (class A) indicating that the class A HSF was not able to provide a missing function required for class B activity. The transcriptional activation potential of Arabidopsis AtHSFA4-21 was mapped primarily to a 39 amino acid fragment in the C-terminus enriched in bulky hydrophobic and acidic residues. Deletion mutagenesis of the C-terminal activator regions of tomato and Arabidopsis HSFs indicated that these plant HSFs lack heat-inducible regulatory regions analogous to those of mammalian HSF1. These findings suggest that heat shock regulation in plants may differ from metazoans by partitioning negative and positive functional domains onto separate HSF proteins. Class A HSFs are primarily responsible for stress-inducible activation of heat shock genes whereas some of the inert class B HSFs may be specialized for repression, or down-regulation, of the heat shock response.

  1. Transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in Bacillus subtilis.

    PubMed

    Fedorova, Ksenia; Kayumov, Airat; Woyda, Kathrin; Ilinskaja, Olga; Forchhammer, Karl

    2013-05-02

    The Bacillus subtilis glutamine synthetase (GS) plays a dual role in cell metabolism by functioning as catalyst and regulator. GS catalyses the ATP-dependent synthesis of glutamine from glutamate and ammonium. Under nitrogen-rich conditions, GS becomes feedback-inhibited by high intracellular glutamine levels and then binds transcription factors GlnR and TnrA, which control the genes of nitrogen assimilation. While GS-bound TnrA is no longer able to interact with DNA, GlnR-DNA binding is shown to be stimulated by GS complex formation. In this paper we show a new physiological feature of the interaction between glutamine synthetase and TnrA. The transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in vivo and in vitro, while the GlnR protein does not affect the activity of the enzyme.

  2. Copper is required for cobalt-induced transcriptional activity of hypoxia-inducible factor-1.

    PubMed

    Qiu, Liying; Ding, Xueqin; Zhang, Zhen; Kang, Y James

    2012-08-01

    Cobalt inhibits prolyl hydroxylases, leading to the accumulation of hypoxia-inducible factor-1α (HIF-1α) and a concomitant increase in the transcriptional activity of HIF-1. Therefore, cobalt has been under development as a drug for activating HIF-1 under some disease conditions. However, it has been shown that ischemic conditions resulted in the loss of copper, and the activation of HIF-1 would not occur unless copper was supplemented. The present study was undertaken to test the hypothesis that copper is also required for the cobalt activation of HIF-1 transcriptional activity. Human umbilical vein endothelial cells subjected to treatment with cobalt chloride (CoCl(2)) at concentrations above 25 μM for 2 h resulted in an accumulation of HIF-1α, which was determined by Western blot analysis, and an increase in the expression of vascular endothelial growth factor (VEGF), which was determined by real-time reverse transcription-polymerase chain reaction analysis for mRNA levels and enzyme-linked immunosorbent assay analysis for protein levels. The copper chelator tetraethylenepentamine at 25 μM did not significantly affect the accumulation of HIF-1α but blocked increases in VEGF mRNA and protein levels, an effect that could be reversed by the addition of 25 μM copper sulfate (CuSO(4)). In addition, gene silencing of the copper chaperone for Cu,Zn-superoxide dismutase blocked VEGF expression with little effect on cobalt-induced HIF-1α accumulation. The present study thus demonstrates that copper was required for cobalt-activated transcriptional activity of HIF-1, although copper did not affect cobalt-induced accumulation of HIF-1α in the cells.

  3. Caffeic acid phenethyl ester inhibits T-cell activation by targeting both nuclear factor of activated T-cells and NF-kappaB transcription factors.

    PubMed

    Márquez, Nieves; Sancho, Rocío; Macho, Antonio; Calzado, Marco A; Fiebich, Bernd L; Muñoz, Eduardo

    2004-03-01

    Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of honeybee hives, has been shown to reveal anti-inflammatory properties. Since T-cells play a key role in the onset of several inflammatory diseases, we have evaluated the immunosuppressive activity of CAPE in human T-cells, discovering that this phenolic compound is a potent inhibitor of early and late events in T-cell receptor-mediated T-cell activation. Moreover, we found that CAPE specifically inhibited both interleukin (IL)-2 gene transcription and IL-2 synthesis in stimulated T-cells. To further characterize the inhibitory mechanisms of CAPE at the transcriptional level, we examined the DNA binding and transcriptional activities of nuclear factor (NF)-kappaB, nuclear factor of activated cells (NFAT), and activator protein-1 (AP-1) transcription factors in Jurkat cells. We found that CAPE inhibited NF-kappaB-dependent transcriptional activity without affecting the degradation of the cytoplasmic NF-kappaB inhibitory protein, IkappaBalpha. However, both NF-kappaB binding to DNA and transcriptional activity of a Gal4-p65 hybrid protein were clearly prevented in CAPE-treated Jurkat cells. Moreover, CAPE inhibited both the DNA-binding and transcriptional activity of NFAT, a result that correlated with its ability to inhibit phorbol 12-myristate 13-acetate plus ionomycin-induced NFAT1 dephosphorylation. These findings provide new insights into the molecular mechanisms involved in the immunomodulatory and anti-inflammatory activities of this natural compound.

  4. The activating transcription factor 3 protein suppresses the oncogenic function of mutant p53 proteins.

    PubMed

    Wei, Saisai; Wang, Hongbo; Lu, Chunwan; Malmut, Sarah; Zhang, Jianqiao; Ren, Shumei; Yu, Guohua; Wang, Wei; Tang, Dale D; Yan, Chunhong

    2014-03-28

    Mutant p53 proteins (mutp53) often acquire oncogenic activities, conferring drug resistance and/or promoting cancer cell migration and invasion. Although it has been well established that such a gain of function is mainly achieved through interaction with transcriptional regulators, thereby modulating cancer-associated gene expression, how the mutp53 function is regulated remains elusive. Here we report that activating transcription factor 3 (ATF3) bound common mutp53 (e.g. R175H and R273H) and, subsequently, suppressed their oncogenic activities. ATF3 repressed mutp53-induced NFKB2 expression and sensitized R175H-expressing cancer cells to cisplatin and etoposide treatments. Moreover, ATF3 appeared to suppress R175H- and R273H-mediated cancer cell migration and invasion as a consequence of preventing the transcription factor p63 from inactivation by mutp53. Accordingly, ATF3 promoted the expression of the metastasis suppressor SHARP1 in mutp53-expressing cells. An ATF3 mutant devoid of the mutp53-binding domain failed to disrupt the mutp53-p63 binding and, thus, lost the activity to suppress mutp53-mediated migration, suggesting that ATF3 binds to mutp53 to suppress its oncogenic function. In line with these results, we found that down-regulation of ATF3 expression correlated with lymph node metastasis in TP53-mutated human lung cancer. We conclude that ATF3 can suppress mutp53 oncogenic function, thereby contributing to tumor suppression in TP53-mutated cancer.

  5. Brain-derived neurotrophic factor activation of NFAT (nuclear factor of activated T-cells)-dependent transcription: a role for the transcription factor NFATc4 in neurotrophin-mediated gene expression.

    PubMed

    Groth, Rachel D; Mermelstein, Paul G

    2003-09-03

    A member of the neurotrophin family, brain-derived neurotrophic factor (BDNF) regulates neuronal survival and differentiation during development. Within the adult brain, BDNF is also important in neuronal adaptive processes, such as the activity-dependent plasticity that underlies learning and memory. These long-term changes in synaptic strength are mediated through alterations in gene expression. However, many of the mechanisms by which BDNF is linked to transcriptional and translational regulation remain unknown. Recently, the transcription factor NFATc4 (nuclear factor of activated T-cells isoform 4) was discovered in neurons, where it is believed to play an important role in long-term changes in neuronal function. Interestingly, NFATc4 is particularly sensitive to the second messenger systems activated by BDNF. Thus, we hypothesized that NFAT-dependent transcription may be an important mediator of BDNF-induced plasticity. In cultured rat CA3-CA1 hippocampal neurons, BDNF activated NFAT-dependent transcription via TrkB receptors. Inhibition of calcineurin blocked BDNF-induced nuclear translocation of NFATc4, thus preventing transcription. Further, phospholipase C was a critical signaling intermediate between BDNF activation of TrkB and the initiation of NFAT-dependent transcription. Both inositol 1,4,5-triphosphate (IP3)-mediated release of calcium from intracellular stores and activation of protein kinase C were required for BDNF-induced NFAT-dependent transcription. Finally, increased expression of IP3 receptor 1 and BDNF after neuronal exposure to BDNF was linked to NFAT-dependent transcription. These results suggest that NFATc4 plays a crucial role in neurotrophin-mediated synaptic plasticity.

  6. The transcription activity of heat shock factor 4b is regulated by FGF2.

    PubMed

    Hu, Yan-Zhong; Zhang, Jun; Li, Shulian; Wang, Chuan; Chu, Liujie; Zhang, Zhi; Ma, Zengyi; Wang, Mingli; Jiang, Qiying; Liu, Guangchao; Qi, Yijun; Ma, Yuanfang

    2013-02-01

    Heat shock factor 4b has been found to be closely associated with postnatal lens development. It expresses in postnatal lens epithelial and secondary fiber cells and controls the expression of small heat shock proteins which are important for lens homeostasis. However, the signal pathways underlying Hsf4b are still not completely understood. Here we present that Hsf4b transcription activity is regulated by FGF2 a key growth factor that is involved in regulating lens development at multiple stages. FGF2 can promote Hsf4b nuclear-translocation and the expression of Hsp25 and αB-crystallin, the key downstream targets of Hsf4b in the Hsf4b-reconstituted mouse hsf4-/- lens epithelial cells. Further study indicates that FGF2 can induce Hsf4b protein stabilization through ERK1/2-mediated posttranslational phosphorylation or sumoylation. Hsf4b can promote FGF2-induced morphology transition from lens epithelial cell to the fiber cell, and this morphology transition can be inhibited by ERK1/2 inhibitor U0126. Taken together, our data demonstrate that Hsf4b is a novel downstream transcription factor of FGF2, and its transcription activity is associated with FGF2-modulated lens epithelial cell-fiber cell transition.

  7. EMT-activating transcription factors in cancer: beyond EMT and tumor invasiveness.

    PubMed

    Sánchez-Tilló, Ester; Liu, Yongqing; de Barrios, Oriol; Siles, Laura; Fanlo, Lucia; Cuatrecasas, Miriam; Darling, Douglas S; Dean, Douglas C; Castells, Antoni; Postigo, Antonio

    2012-10-01

    Cancer is a complex multistep process involving genetic and epigenetic changes that eventually result in the activation of oncogenic pathways and/or inactivation of tumor suppressor signals. During cancer progression, cancer cells acquire a number of hallmarks that promote tumor growth and invasion. A crucial mechanism by which carcinoma cells enhance their invasive capacity is the dissolution of intercellular adhesions and the acquisition of a more motile mesenchymal phenotype as part of an epithelial-to-mesenchymal transition (EMT). Although many transcription factors can trigger it, the full molecular reprogramming occurring during an EMT is mainly orchestrated by three major groups of transcription factors: the ZEB, Snail and Twist families. Upregulated expression of these EMT-activating transcription factors (EMT-ATFs) promotes tumor invasiveness in cell lines and xenograft mice models and has been associated with poor clinical prognosis in human cancers. Evidence accumulated in the last few years indicates that EMT-ATFs also regulate an expanding set of cancer cell capabilities beyond tumor invasion. Thus, EMT-ATFs have been shown to cooperate in oncogenic transformation, regulate cancer cell stemness, override safeguard programs against cancer like apoptosis and senescence, determine resistance to chemotherapy and promote tumor angiogenesis. This article reviews the expanding portfolio of functions played by EMT-ATFs in cancer progression.

  8. Molecular genetic analysis of activation-tagged transcription factors thought to be involved in photomorphogenesis

    SciTech Connect

    Neff, Michael M.

    2011-06-23

    This is a final report for Department of Energy Grant No. DE-FG02-08ER15927 entitled “Molecular Genetic Analysis of Activation-Tagged Transcription Factors Thought to be Involved in Photomorphogenesis”. Based on our preliminary photobiological and genetic analysis of the sob1-D mutant, we hypothesized that OBP3 is a transcription factor involved in both phytochrome and cryptochrome-mediated signal transduction. In addition, we hypothesized that OBP3 is involved in auxin signaling and root development. Based on our preliminary photobiological and genetic analysis of the sob2-D mutant, we also hypothesized that a related gene, LEP, is involved in hormone signaling and seedling development.

  9. SUMO represses transcriptional activity of the Drosophila SoxNeuro and human Sox3 central nervous system-specific transcription factors.

    PubMed

    Savare, Jean; Bonneaud, Nathalie; Girard, Franck

    2005-06-01

    Sry high mobility group (HMG) box (Sox) transcription factors are involved in the development of central nervous system (CNS) in all metazoans. Little is known on the molecular mechanisms that regulate their transcriptional activity. Covalent posttranslational modification by small ubiquitin-like modifier (SUMO) regulates several nuclear events, including the transcriptional activity of transcription factors. Here, we demonstrate that SoxNeuro, an HMG box-containing transcription factor involved in neuroblast formation in Drosophila, is a substrate for SUMO modification. SUMOylation assays in HeLa cells and Drosophila S2 cells reveal that lysine 439 is the major SUMO acceptor site. The sequence in SoxNeuro targeted for SUMOylation, IKSE, is part of a small inhibitory domain, able to repress in cis the activity of two adjacent transcriptional activation domains. Our data show that SUMO modification represses SoxNeuro transcriptional activity in transfected cells. Overexpression in Drosophila embryos of a SoxN form that cannot be targeted for SUMOylation strongly impairs the development of the CNS, suggesting that SUMO modification of SoxN is crucial for regulating its activity in vivo. Finally, we present evidence that SUMO modification of group B1 Sox factors was conserved during evolution, because Sox3, the human counterpart of SoxN, is also negatively regulated through SUMO modification.

  10. The metal-responsive transcription factor-1 contributes to HIF-1 activation during hypoxic stress

    SciTech Connect

    Murphy, Brian J. . E-mail: brian.murphy@sri.com; Sato, Barbara G.; Dalton, Timothy P.; Laderoute, Keith R.

    2005-11-25

    Hypoxia-inducible factor-1 (HIF-1), the major transcriptional regulator of the mammalian cellular response to low oxygen (hypoxia), is embedded within a complex network of signaling pathways. We have been investigating the importance of another stress-responsive transcription factor, MTF-1, for the adaptation of cells to hypoxia. This article reports that MTF-1 plays a central role in hypoxic cells by contributing to HIF-1 activity. Loss of MTF-1 in transformed Mtf1 null mouse embryonic fibroblasts (MEFs) results in an attenuation of nuclear HIF-1{alpha} protein accumulation, HIF-1 transcriptional activity, and expression of an established HIF-1 target gene, glucose transporter-1 (Glut1). Mtf1 null (Mtf1 KO) MEFs also have constitutively higher levels of both glutathione (GSH) and the rate-limiting enzyme involved in GSH synthesis-glutamate cysteine ligase catalytic subunit-than wild type cells. The altered cellular redox state arising from increased GSH may perturb oxygen-sensing mechanisms in hypoxic Mtf1 KO cells and decrease the accumulation of HIF-1{alpha} protein. Together, these novel findings define a role for MTF-1 in the regulation of HIF-1 activity.

  11. A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3

    PubMed Central

    Dalton, Jutta C.; Bätz, Ulrike; Liu, Jason; Curie, Gemma L.; Quail, Peter H.

    2016-01-01

    Transcriptional activation domains (TADs) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput manner. A plant transcriptional activator, PIF3 (phytochrome interacting factor 3), was fused to the yeast GAL4-DNA-binding Domain (BD), driving expression of the URA3 (Orotidine 5′-phosphate decarboxylase) reporter, and used for negative selection on 5-fluroorotic acid (5FOA). Randomly mutagenized variants of PIF3 were then selected for a loss or reduction in transcriptional activation activity by survival on FOA. In the process, we developed a strategy to eliminate false positives from negative selection that can be used for both reverse-1- and 2-hybrid screens. With this method we were able to identify two distinct regions in PIF3 with transcriptional activation activity, both of which are functionally conserved in PIF1, PIF4, and PIF5. Both are collectively necessary for full PIF3 transcriptional activity, but neither is sufficient to induce transcription autonomously. We also found that the TAD appear to overlap physically with other PIF3 functions, such as phyB binding activity and consequent phosphorylation. Our protocol should provide a valuable tool for identifying, analyzing and characterizing novel TADs in eukaryotic transcription factors, and thus potentially contribute to the unraveling of the mechanism underlying transcriptional activation. PMID:27379152

  12. Genetic factors affecting gene transcription and catalytic activity of UDP-glucuronosyltransferases in human liver.

    PubMed

    Liu, Wanqing; Ramírez, Jacqueline; Gamazon, Eric R; Mirkov, Snezana; Chen, Peixian; Wu, Kehua; Sun, Chang; Cox, Nancy J; Cook, Edwin; Das, Soma; Ratain, Mark J

    2014-10-15

    The aim of this study was to discover cis- and trans-acting factors significantly affecting mRNA expression and catalytic activity of human hepatic UDP-glucuronosyltransferases (UGTs). Transcription levels of five major hepatic UGT1A (UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9) and five UGT2B (UGT2B4, UGT2B7, UGT2B10, UGT2B15 and UGT2B17) genes were quantified in human liver tissue samples (n = 125) using real-time PCR. Glucuronidation activities of 14 substrates were measured in 47 livers. We genotyped 167 tagSNPs (single-nucleotide polymorphisms) in UGT1A (n = 43) and UGT2B (n = 124), as well as the known functional UGT1A1*28 and UGT2B17 CNV (copy number variation) polymorphisms. Transcription levels of 15 transcription factors (TFs) known to regulate these UGTs were quantified. We found that UGT expression and activity were highly variable among the livers (median and range of coefficient of variations: 135%, 74-217% and 52%, 39-105%, respectively). CAR, PXR and ESR1 were found to be the most important trans-regulators of UGT transcription (median and range of correlation coefficients: 46%, 6-58%; 47%, 9-58%; and 52%, 24-75%, respectively). Hepatic UGT activities were mainly determined by UGT gene transcription levels. Twenty-one polymorphisms were significantly (FDR-adjusted P < 0.05) associated with mRNA expression and/or activities of UGT1A1, UGT1A3 and UGT2B17. We found novel SNPs in the UGT2B17 CNV region accounting for variability in UGT2B17 gene transcription and testosterone glucuronidation rate, in addition to that attributable to the UGT2B17 CNV. Our study discovered novel pharmacogenetic markers and provided detailed insight into the genetic network regulating hepatic UGTs.

  13. Activated STAT1 transcription factors conduct distinct saltatory movements in the cell nucleus.

    PubMed

    Speil, Jasmin; Baumgart, Eugen; Siebrasse, Jan-Peter; Veith, Roman; Vinkemeier, Uwe; Kubitscheck, Ulrich

    2011-12-07

    The activation of STAT transcription factors is a critical determinant of their subcellular distribution and their ability to regulate gene expression. Yet, it is not known how activation affects the behavior of individual STAT molecules in the cytoplasm and nucleus. To investigate this issue, we injected fluorescently labeled STAT1 in living HeLa cells and traced them by single-molecule microscopy. We determined that STAT1 moved stochastically in the cytoplasm and nucleus with very short residence times (<0.03 s) before activation. Upon activation, STAT1 mobility in the cytoplasm decreased ∼2.5-fold, indicating reduced movement of STAT1/importinα/β complexes to the nucleus. In the nucleus, activated STAT1 displayed a distinct saltatory mobility, with residence times of up to 5 s and intermittent diffusive motion. In this manner, activated STAT1 factors can occupy their putative chromatin target sites within ∼2 s. These results provide a better understanding of the timescales on which cellular signaling and regulated gene transcription operate at the single-molecule level.

  14. Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors.

    PubMed

    Zhou, Hui; Lin-Wang, Kui; Wang, Huiliang; Gu, Chao; Dare, Andrew P; Espley, Richard V; He, Huaping; Allan, Andrew C; Han, Yuepeng

    2015-04-01

    Anthocyanin pigmentation is an important consumer trait in peach (Prunus persica). In this study, the genetic basis of the blood-flesh trait was investigated using the cultivar Dahongpao, which shows high levels of cyanidin-3-glucoside in the mesocarp. Elevation of anthocyanin levels in the flesh was correlated with the expression of an R2R3 MYB transcription factor, PpMYB10.1. However, PpMYB10.1 did not co-segregate with the blood-flesh trait. The blood-flesh trait was mapped to a 200-kb interval on peach linkage group (LG) 5. Within this interval, a gene encoding a NAC domain transcription factor (TF) was found to be highly up-regulated in blood-fleshed peaches when compared with non-red-fleshed peaches. This NAC TF, designated blood (BL), acts as a heterodimer with PpNAC1 which shows high levels of expression in fruit at late developmental stages. We show that the heterodimer of BL and PpNAC1 can activate the transcription of PpMYB10.1, resulting in anthocyanin pigmentation in tobacco. Furthermore, silencing the BL gene reduces anthocyanin pigmentation in blood-fleshed peaches. The transactivation activity of the BL-PpNAC1 heterodimer is repressed by a SQUAMOSA promoter-binding protein-like TF, PpSPL1. Low levels of PpMYB10.1 expression in fruit at early developmental stages is probably attributable to lower levels of expression of PpNAC1 plus the presence of high levels of repressors such as PpSPL1. We present a mechanism whereby BL is the key gene for the blood-flesh trait in peach via its activation of PpMYB10.1 in maturing fruit. Partner TFs such as basic helix-loop-helix proteins and NAC1 are required, as is the removal of transcriptional repressors.

  15. Interaction of the Transcription Start Site Core Region and Transcription Factor YY1 Determine Ascorbate Transporter SVCT2 Exon 1a Promoter Activity

    PubMed Central

    Qiao, Huan; May, James M.

    2012-01-01

    Transcription of the ascorbate transporter, SVCT2, is driven by two distinct promoters in exon 1 of the transporter sequence. The exon 1a promoter lacks a classical transcription start site and little is known about regulation of promoter activity in the transcription start site core (TSSC) region. Here we present evidence that the TSSC binds the multifunctional initiator-binding protein YY1. Electrophoresis shift assays using YY1 antibody showed that YY1 is present as one of two major complexes that specifically bind to the TSSC. The other complex contains the transcription factor NF-Y. Mutations in the TSSC that decreased YY1 binding also impaired the exon 1a promoter activity despite the presence of an upstream activating NF-Y/USF complex, suggesting that YY1 is involved in the regulation of the exon 1a transcription. Furthermore, YY1 interaction with NF-Y and/or USF synergistically enhanced the exon 1a promoter activity in transient transfections and co-activator p300 enhanced their synergistic activation. We propose that the TSSC plays a vital role in the exon 1a transcription and that this function is partially carried out by the transcription factor YY1. Moreover, co-activator p300 might be able to synergistically enhance the TSSC function via a “bridge” mechanism with upstream sequences. PMID:22532872

  16. Dynamic transcription factor activity and networks during ErbB2 breast oncogenesis and targeted therapy.

    PubMed

    Weiss, M S; Peñalver Bernabé, B; Shin, S; Asztalos, S; Dubbury, S J; Mui, M D; Bellis, A D; Bluver, D; Tonetti, D A; Saez-Rodriguez, J; Broadbelt, L J; Jeruss, J S; Shea, L D

    2014-12-01

    Tissue development and disease progression are multi-stage processes controlled by an evolving set of key regulatory factors, and identifying these factors necessitates a dynamic analysis spanning relevant time scales. Current omics approaches depend on incomplete biological databases to identify critical cellular processes. Herein, we present TRACER (TRanscriptional Activity CEll aRrays), which was employed to quantify the dynamic activity of numerous transcription factor (TFs) simultaneously in 3D and networks for TRACER (NTRACER), a computational algorithm that allows for cellular rewiring to establish dynamic regulatory networks based on activity of TF reporter constructs. We identified major hubs at various stages of culture associated with normal and abnormal tissue growth (i.e., ELK-1 and E2F1, respectively) and the mechanism of action for a targeted therapeutic, lapatinib, through GATA-1, which were confirmed in human ErbB2 positive breast cancer patients and human ErbB2 positive breast cancer cell lines that were either sensitive or resistant to lapatinib.

  17. Transgenic songbirds with suppressed or enhanced activity of CREB transcription factor

    PubMed Central

    Abe, Kentaro; Matsui, Sumiko; Watanabe, Dai

    2015-01-01

    Songbirds postnatally develop their skill to utter and to perceive a vocal signal for communication. How genetic and environmental influences act in concert to regulate the development of such skill is not fully understood. Here, we report the phenotype of transgenic songbirds with altered intrinsic activity of cAMP response element-binding protein (CREB) transcription factor. By viral vector-mediated modification of genomic DNA, we established germ line-transmitted lines of zebra finches, which exhibited enhanced or suppressed activity of CREB. Although intrinsically acquired vocalizations or their hearing ability were not affected, the transgenic birds showed reduced vocal learning quality of their own songs and impaired audio-memory formation against conspecific songs. These results thus demonstrate that appropriate activity of CREB is necessary for the postnatal acquisition of learned behavior in songbirds, and the CREB transgenic birds offer a unique opportunity to separately manipulate both genetic and environmental factors that impinge on the postnatal song learning. PMID:26048905

  18. WRKY6 Transcription Factor Restricts Arsenate Uptake and Transposon Activation in Arabidopsis[W

    PubMed Central

    Castrillo, Gabriel; Sánchez-Bermejo, Eduardo; de Lorenzo, Laura; Crevillén, Pedro; Fraile-Escanciano, Ana; TC, Mohan; Mouriz, Alfonso; Catarecha, Pablo; Sobrino-Plata, Juan; Olsson, Sanna; Leo del Puerto, Yolanda; Mateos, Isabel; Rojo, Enrique; Hernández, Luis E.; Jarillo, Jose A.; Piñeiro, Manuel; Paz-Ares, Javier; Leyva, Antonio

    2013-01-01

    Stress constantly challenges plant adaptation to the environment. Of all stress types, arsenic was a major threat during the early evolution of plants. The most prevalent chemical form of arsenic is arsenate, whose similarity to phosphate renders it easily incorporated into cells via the phosphate transporters. Here, we found that arsenate stress provokes a notable transposon burst in plants, in coordination with arsenate/phosphate transporter repression, which immediately restricts arsenate uptake. This repression was accompanied by delocalization of the phosphate transporter from the plasma membrane. When arsenate was removed, the system rapidly restored transcriptional expression and membrane localization of the transporter. We identify WRKY6 as an arsenate-responsive transcription factor that mediates arsenate/phosphate transporter gene expression and restricts arsenate-induced transposon activation. Plants therefore have a dual WRKY-dependent signaling mechanism that modulates arsenate uptake and transposon expression, providing a coordinated strategy for arsenate tolerance and transposon gene silencing. PMID:23922208

  19. An inducible transcription factor activates expression of human immunodeficiency virus in T cells

    NASA Astrophysics Data System (ADS)

    Nabel, Gary; Baltimore, David

    1987-04-01

    Human immunodeficiency virus (HIV) production from latently infected T lymphocytes can be induced with compounds that activate the cells to secrete lymphokines1,2. The elements in the HIV genome which control activation are not known but expression might be regulated through a variety of DNA elements. The cis-acting control elements of the viral genome are enhancer and promoter regions. The virus also encodes trans-acting factors specified by the tat-III (refs 3-6) and art genes7. We have examined whether products specific to activated T cells might stimulate viral transcription by binding to regions on viral DNA. Activation of T cells, which increases HIV expression up to 50-fold, correlated with induction of a DNA binding protein indistinguishable from a recognized transcription factor, called NF-κB (ref. 8), with binding sites in the viral enhancer. Mutation of these binding sites abolished inducibility. That NF-κB acts in synergy with the viral tat-III gene product to enhance HIV expression in T cells may have implications for the pathogenesis of AIDS (acquired immune deficiency syndrome).

  20. Nerve growth factor enhances the CRE-dependent transcriptional activity activated by nobiletin in PC12 cells.

    PubMed

    Takito, Jiro; Kimura, Junko; Kajima, Koji; Uozumi, Nobuyuki; Watanabe, Makoto; Yokosuka, Akihito; Mimaki, Yoshihiro; Nakamura, Masanori; Ohizumi, Yasushi

    2016-07-01

    Prevention and treatment of Alzheimer disease are urgent problems for elderly people in developed countries. We previously reported that nobiletin, a poly-methoxylated flavone from the citrus peel, improved the symptoms in various types of animal models of memory loss and activated the cAMP responsive element (CRE)-dependent transcription in PC12 cells. Nobiletin activated the cAMP/PKA/MEK/Erk/MAPK signaling pathway without using the TrkA signaling activated by nerve growth factor (NGF). Here, we examined the effect of combination of nobiletin and NGF on the CRE-dependent transcription in PC12 cells. Although NGF alone had little effect on the CRE-dependent transcription, NGF markedly enhanced the CRE-dependent transcription induced by nobiletin. The NGF-induced enhancement was neutralized by a TrkA antagonist, K252a. This effect of NGF was effective on the early signaling event elicited by nobiletin. These results suggested that there was crosstalk between NGF and nobiletin signaling in activating the CRE-dependent transcription in PC12 cells.

  1. Age-associated changes in basal c-fos transcription factor binding activity in rat hearts.

    PubMed

    Tsou, H; Azhar, G; Lu, X G; Kovacs, S; Peacocke, M; Wei, J Y

    1996-12-15

    The early response proto-oncogene c-fos is expressed at very low levels in the mammalian heart at baseline. To further investigate the mechanism of altered c-fos expression with age, we studied in the basal state the binding of five transcription proteins to their cognate sites in the c-fos promoter/enhancer region, in adult and old F344 rats. Our results show a reduced binding of E2F and AP1 proteins to the c-fos promoter in aging hearts. The major calcium/cyclic AMP response element (CRE) and SP1 binding was unchanged. The only increase seen with age was in the serum response element (SRE) binding proteins. SRE is the point of convergence of different signal transduction pathways (via MAP kinases and the Rho family of GTPases) at the c-fos promoter. Increased SRE binding may reflect a compensation for a decreased binding of other transcription proteins to the c-fos promoter, alteration in the phosphorylation status of SRF, or a change in the ternary complex factors Elk 1 or SAP 1. Other possibilities include defects in the signal transduction pathways with aging, which combine to produce an overall negative balance in the function of the c-fos promoter despite the increased SRE binding activity. Both in vitro and in vivo experiments have shown decreased c-fos expression with age. This may be due partly to alterations in the basal levels of transcription factor binding.

  2. Protective upregulation of activating transcription factor-3 against glutamate neurotoxicity in neuronal cells under ischemia.

    PubMed

    Takarada, Takeshi; Kou, Miki; Hida, Miho; Fukumori, Ryo; Nakamura, Saki; Kutsukake, Takaya; Kuramoto, Nobuyuki; Hinoi, Eiichi; Yoneda, Yukio

    2016-05-01

    This study evaluates the pathological role of the stress sensor activating transcription factor-3 (ATF3) in ischemic neurotoxicity. Upregulation of the transcript and protein for ATF3 was seen 2-10 hr after reperfusion in the ipsilateral cerebral hemisphere of mice with transient middle cerebral artery occlusion for 2 hr. Immunohistochemical analysis confirmed the expression of ATF3 by cells immunoreactive for a neuronal marker in neocortex, hippocampus, and striatum within 2 hr after reperfusion. In murine neocortical neurons previously cultured under ischemic conditions for 2 hr, transient upregulation of both Atf3 and ATF3 expression was similarly found during subsequent culture for 2-24 hr under normoxia. Lentiviral overexpression of ATF3 ameliorated the neurotoxicity of glutamate (Glu) in cultured murine neurons along with a slight but statistically significant inhibition of both Fluo-3 and rhodamine-2 fluorescence increases by N-methyl-D-aspartate. Similarly, transient upregulation was seen in Atf3 and ATF3 expression during the culture for 48 hr in neuronal Neuro2A cells previously cultured under ischemic conditions for 2 hr. Luciferase reporter analysis with ATF3 promoter together with immunoblotting revealed the possible involvement of several transcription factors responsive to extracellular and intracellular stressors in the transactivation of the Atf3 gene in Neuro2A cells. ATF3 could be upregulated to play a role in mechanisms underlying mitigation of the neurotoxicity mediated by the endogenous neurotoxin Glu at an early stage after ischemic signal inputs.

  3. Molecular Genetic Analysis of Activation-tagged Transcription Factors Thought to be Involved in Photomorphogenesis

    SciTech Connect

    Neff, Michael

    2011-06-23

    Plants utilize light as a source of information via families of photoreceptors such as the red/far-red absorbing phytochromes (PHY) and the blue/UVA absorbing cryptochromes (CRY). The main goal of the Neff lab is to use molecular-genetic mutant screens to elucidate signaling components downstream of these photoreceptors. Activation-tagging mutagenesis led to the identification of two putative transcription factors that may be involved in both photomorphogenesis and hormone signaling pathways. sob1-D (suppressor of phyB-dominant) mutant phenotypes are caused by the over-expression of a Dof transcription factor previously named OBP3. Our previous studies indicate that OBP3 is a negative regulator of light-mediated cotyledon expansion and may be involved in modulating responsiveness to the growth-regulating hormone auxin. The sob2-D mutant uncovers a role for LEP, a putative AP2/EREBP-like transcription factor, in seed germination, hypocotyl elongation and responsiveness to the hormone abscisic acid. Based on photobiological and genetic analysis of OBP3-knockdown and LEP-null mutations, we hypothesize that these transcription factors are involved in both light-mediated seedling development and hormone signaling. To examine the role that these genes play in photomorphogenesis we will: 1) Further explore the genetic role of OBP3 in cotyledon/leaf expansion and other photomorphogenic processes as well as examine potential physical interactions between OBP3 and CRY1 or other signaling components that genetically interact with this transcription factor 2) Test the hypothesis that OBP3 is genetically involved in auxin signaling and root development as well as examine the affects of this hormone and light on OBP3 protein accumulation. 3) Test the hypothesis that LEP is involved in seed germination, seedling photomorphogenesis and hormone signaling. Together these experiments will lead to a greater understanding of the complexity of interactions between photoreceptors and DNA

  4. On involvement of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells, activator protein-1 and signal transducer and activator of transcription-3 in photodynamic therapy-induced death of crayfish neurons and satellite glial cells

    NASA Astrophysics Data System (ADS)

    Berezhnaya, Elena; Neginskaya, Marya; Kovaleva, Vera; Sharifulina, Svetlana; Ischenko, Irina; Komandirov, Maxim; Rudkovskii, Mikhail; Uzdensky, Anatoly B.

    2015-07-01

    Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT.

  5. Mitochondrial Ca2+ Cycling Facilitates Activation of the Transcription Factor NFAT in Sensory Neurons

    PubMed Central

    Kim, Man-Su; Usachev, Yuriy M.

    2009-01-01

    Ca2+-dependent gene regulation controls many aspects of neuronal plasticity. Significant progress has been made toward understanding the roles of voltage- and ligand-gated Ca2+ channels in triggering specific transcriptional responses. In contrast, the functional importance of Ca2+ buffers and Ca2+ transporters in neuronal gene regulation is less clear despite their critical contribution to the spatio-temporal control of Ca2+ signals. Here we examined the role of mitochondrial Ca2+ uptake and release in regulating the Ca2+-dependent transcription factor NFAT that has been implicated in synaptic plasticity, axonal growth and neuronal survival. Intense stimulation of sensory neurons by action potentials or TRPV1 agonists induced rapid activation and nuclear import of NFAT. Nuclear translocation of NFAT was associated with a characteristic prolonged [Ca2+]i elevation (plateau) that resulted from Ca2+ uptake by, and its subsequent release from mitochondria. Measurements using a mitochondrial Ca2+ indicator, mtPericam, showed that this process recruited mitochondria throughout the cell body, including the perinuclear region. [Ca2+]i levels attained during the plateau phase were similar to or higher than those required for NFAT activation (200–300 nM). The elimination of the [Ca2+]i plateau by blocking either mitochondrial Ca2+ uptake via the uniporter or Ca2+ release via the mitochondrial Na+/Ca2+ exchanger strongly reduced nuclear import of NFAT. Furthermore, preventing Ca2+ mobilization via the mitochondrial Na+/Ca2+ exchanger diminished NFAT-mediated transcription. Collectively, these data implicate activity-induced Ca2+ uptake and prolonged release from mitochondria as a novel regulatory mechanism in neuronal excitation-transcription coupling. PMID:19793968

  6. The protein level and transcription activity of activating transcription factor 1 is regulated by prolyl isomerase Pin1 in nasopharyngeal carcinoma progression.

    PubMed

    Huang, Guo-Liang; Liao, Dan; Chen, Hua; Lu, Yan; Chen, Liyong; Li, Huahui; Li, Binbin; Liu, Weilong; Ye, Caiguo; Li, Tong; Zhu, Zhu; Wang, Jian; Uchida, Takafumi; Zou, Ying; Dong, Zigang; He, Zhiwei

    2016-12-29

    The function of activating transcription factor 1 (ATF1) and the mechanism about why ATF1 was over-phosphorylated in nasopharyngeal carcinoma (NPC) progression is completely undiscovered. In this study, a series of experiments both in vitro and in vivo were used to characterize a promotive function of ATF1 in NPC tumorigenesis and identify prolyl isomerase Pin1 as a novel regulator of ATF1 at post-transcription. First, we found that overexpression of ATF1 promoted colony formation in NPC. However, the high protein level of ATF1 in NPC was not resulted from high mRNA level. Then, a direct interaction between Pin1 and ATF1 at Thr184 was demonstrated using mammalian two-hybrid assay and coimmunoprecipitation. Cycloheximide (CHX) treatment indicated Pin1 stabilized the expression of ATF1 at post-transcription level. We confirmed that Pin1 upregulated ATF1 transcriptional activity of Bcl-2 using luciferase reporter assay, quantitative RT-PCR and western blot. Furthermore, the newly identified phosphorylation of ATF1 at Thr184 was suggested to have an important role in ATF1 function of transcription and tumor promotion. Finally, high expression of Pin1 in NPC tissue was found to be positively correlated with ATF1. The ATF1 promoted NPC tumorigenesis was regulated by Pin1 both in vitro and in vivo. All these findings clearly state that Pin1 is a novel regulator of ATF1 at Thr184 and thereby enhances ATF1 transcription activity and tumorigenesis promotive function in NPC.

  7. The protein level and transcription activity of activating transcription factor 1 is regulated by prolyl isomerase Pin1 in nasopharyngeal carcinoma progression

    PubMed Central

    Huang, Guo-Liang; Liao, Dan; Chen, Hua; Lu, Yan; Chen, Liyong; Li, Huahui; Li, Binbin; Liu, Weilong; Ye, Caiguo; Li, Tong; Zhu, Zhu; Wang, Jian; Uchida, Takafumi; Zou, Ying; Dong, Zigang; He, Zhiwei

    2016-01-01

    The function of activating transcription factor 1 (ATF1) and the mechanism about why ATF1 was over-phosphorylated in nasopharyngeal carcinoma (NPC) progression is completely undiscovered. In this study, a series of experiments both in vitro and in vivo were used to characterize a promotive function of ATF1 in NPC tumorigenesis and identify prolyl isomerase Pin1 as a novel regulator of ATF1 at post-transcription. First, we found that overexpression of ATF1 promoted colony formation in NPC. However, the high protein level of ATF1 in NPC was not resulted from high mRNA level. Then, a direct interaction between Pin1 and ATF1 at Thr184 was demonstrated using mammalian two-hybrid assay and coimmunoprecipitation. Cycloheximide (CHX) treatment indicated Pin1 stabilized the expression of ATF1 at post-transcription level. We confirmed that Pin1 upregulated ATF1 transcriptional activity of Bcl-2 using luciferase reporter assay, quantitative RT-PCR and western blot. Furthermore, the newly identified phosphorylation of ATF1 at Thr184 was suggested to have an important role in ATF1 function of transcription and tumor promotion. Finally, high expression of Pin1 in NPC tissue was found to be positively correlated with ATF1. The ATF1 promoted NPC tumorigenesis was regulated by Pin1 both in vitro and in vivo. All these findings clearly state that Pin1 is a novel regulator of ATF1 at Thr184 and thereby enhances ATF1 transcription activity and tumorigenesis promotive function in NPC. PMID:28032861

  8. The Transcriptional Activator Krüppel-like Factor-6 Is Required for CNS Myelination.

    PubMed

    Laitman, Benjamin M; Asp, Linnéa; Mariani, John N; Zhang, Jingya; Liu, Jia; Sawai, Setsu; Chapouly, Candice; Horng, Sam; Kramer, Elisabeth G; Mitiku, Nesanet; Loo, Hannah; Burlant, Natalie; Pedre, Xiomara; Hara, Yuko; Nudelman, German; Zaslavsky, Elena; Lee, Young-Min; Braun, David A; Lu, Q Richard; Narla, Goutham; Raine, Cedric S; Friedman, Scott L; Casaccia, Patrizia; John, Gareth R

    2016-05-01

    Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination.

  9. The Transcriptional Activator Krüppel-like Factor-6 Is Required for CNS Myelination

    PubMed Central

    Mariani, John N.; Zhang, Jingya; Liu, Jia; Sawai, Setsu; Chapouly, Candice; Horng, Sam; Kramer, Elisabeth G.; Loo, Hannah; Burlant, Natalie; Nudelman, German; Lee, Young-Min; Braun, David A.; Lu, Q. Richard; Narla, Goutham; Raine, Cedric S.; Friedman, Scott L.; Casaccia, Patrizia; John, Gareth R.

    2016-01-01

    Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination. PMID:27213272

  10. Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novel sigma factor.

    PubMed

    Dupuy, Bruno; Mani, Nagraj; Katayama, Seiichi; Sonenshein, Abraham L

    2005-02-01

    Expression of the plasmid-encoded Clostridium perfringens gene for bacteriocin BCN5 was shown to depend in vivo and in vitro on the activity of UviA protein. UviA, also plasmid-encoded, proved to be an RNA polymerase sigma factor and was also partly autoregulatory. The uviA gene has two promoters; one provided a UviA-independent, basal level of gene expression while the stronger, UviA-dependent promoter was only utilized after the cell experienced DNA damage. As a result, BCN5 synthesis is induced by treatment with UV light or mitomycin C. UviA is related to a special class of sigma factors found to date only in Clostridium species and responsible for activating transcription of toxin genes in Clostridium difficile, Clostridium tetani, and Clostridium botulinum.

  11. 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.

  12. 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

  13. Conserved and Diverged Functions of the Calcineurin-Activated Prz1 Transcription Factor in Fission Yeast.

    PubMed

    Chatfield-Reed, Kate; Vachon, Lianne; Kwon, Eun-Joo Gina; Chua, Gordon

    2016-04-01

    Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeast Schizosaccharomyces pombe Genome-wide studies of the Crz1 and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This article identifies an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2 and tunicamycin treatment, as well as a ∆pmr1 genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of the Saccharomyces cerevisiae ortholog Crz1 These genes were functionally enriched for Crz1-conserved processes such as cell-wall biosynthesis. Overexpression of prz1(+)increased resistance to the cell-wall degradation enzyme zymolyase, likely from upregulation of theO-mannosyltransferase encoding gene omh1(+) Loss of omh1(+)abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss of prz1(+)resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the ∆prz1 strain was abrogated by the loss of gsf2(+) or cbf12(+) This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell-wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes between Crz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional regulatory network.

  14. Conserved and Diverged Functions of the Calcineurin-Activated Prz1 Transcription Factor in Fission Yeast

    PubMed Central

    Chatfield-Reed, Kate; Vachon, Lianne; Kwon, Eun-Joo Gina; Chua, Gordon

    2016-01-01

    Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeast Schizosaccharomyces pombe. Genome-wide studies of the Crz1 and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This article identifies an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2 and tunicamycin treatment, as well as a ∆pmr1 genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of the Saccharomyces cerevisiae ortholog Crz1. These genes were functionally enriched for Crz1-conserved processes such as cell-wall biosynthesis. Overexpression of prz1+ increased resistance to the cell-wall degradation enzyme zymolyase, likely from upregulation of the O-mannosyltransferase encoding gene omh1+. Loss of omh1+ abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss of prz1+ resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the ∆prz1 strain was abrogated by the loss of gsf2+ or cbf12+. This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell-wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes between Crz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional regulatory network. PMID:26896331

  15. Transcriptional activation of the herpes simplex virus type 1 UL38 promoter conferred by the cis-acting downstream activation sequence is mediated by a cellular transcription factor.

    PubMed

    Guzowski, J F; Singh, J; Wagner, E K

    1994-12-01

    The herpes simplex virus (HSV) type 1 strict late (gamma) UL38 promoter contains three cis-acting transcriptional elements: a TATA box, a specific initiator element, and the downstream activation sequence (DAS). DAS is located between positions +20 and +33 within the 5' untranslated leader region and strongly influences transcript levels during productive infection. In this communication, we further characterize DAS and investigate its mechanism of action. DAS function has a strict spacing requirement, and DAS contains an essential 6-bp core element. A similarly positioned element from the gamma gC gene (UL44) has partial DAS function within the UL38 promoter context, and the promoter controlling expression of the gamma US11 transcript contains an identically located element with functional and sequence similarity to UL38 DAS. These data suggest that downstream elements are a common feature of many HSV gamma promoters. Results with recombinant viruses containing modifications of the TATA box or initiator element of the UL38 promoter suggest that DAS functions to increase transcription initiation and not the efficiency of transcription elongation. In vitro transcription assays using uninfected HeLa nuclear extracts show that, as in productive infection with recombinant viruses, the deletion of DAS from the UL38 promoter dramatically decreases RNA expression. Finally, electrophoretic mobility shift assays and UV cross-linking experiments show that DAS DNA forms a specific, stable complex with a cellular protein (the DAS-binding factor) of approximately 35 kDa. These data strongly suggest that the interaction of cellular DAS-binding factor with DAS is required for efficient expression of UL38 and other HSV late genes.

  16. A modified reverse one-hybrid screen identifies transcriptional activation in Phyochrome-Interacting Factor 3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcriptional activation domains (TAD) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput...

  17. The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells

    SciTech Connect

    Zhang, Yingyi; Zhao, Yu; Li, Leilei; Shen, Yu; Cai, Xiaoli; Zhang, Xiaodong; Ye, Lihong

    2013-05-03

    Highlights: •HBXIP is able to upregulate the expression of PDGFB in breast cancer cells. •HBXIP serves as a coactivator of activating transcription factor Sp1. •HBXIP stimulates the PDGFB promoter via activating transcription factor Sp1. •HBXIP promotes the proliferation of breast cancer cell via upregulating PDGFB. -- Abstract: We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-κB (NF-κB) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB) plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-κB through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells.

  18. Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana

    PubMed Central

    Sheikh, Arsheed H.; Eschen-Lippold, Lennart; Pecher, Pascal; Hoehenwarter, Wolfgang; Sinha, Alok K.; Scheel, Dierk; Lee, Justin

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense. PMID:26870073

  19. Activity of a C. elegans GATA transcription factor, ELT-1, expressed in yeast.

    PubMed

    Shim, Y H; Bonner, J J; Blumenthal, T

    1995-11-10

    The GATA motif (WGATAR) is found in the promoter regions of numerous Caenorhabditis elegans genes, including two intestine-specific genes, vit-2 and ges-1, in which it has been shown to be required for promoter function. The protein ELT-1, encoded by a single-copy gene homologous to the GATA family of vertebrate transcription factors, is potentially capable of interacting with this element. In order to determine whether ELT-1 is a transcriptional activator that recognizes this sequence, we have expressed it under the control of the GAL1 promoter in yeast. lacZ driven by the CYC1 promoter lacking an upstream activation sequence (UAS) but containing GATA sequences was used as a reporter. beta-Galactosidase was expressed upon induction only when GATA sequences were present, and expression was increased dramatically by additional binding sites. Deletion analysis demonstrated that the C terminus, containing only one of the two zinc fingers, is sufficient for activation. In addition, the DNA-binding domain and two transactivation regions were identified by fusing these isolated domains to previously defined domains of heterologous transcription factors. While most single base alterations in the GATA core sequence eliminated activity, an A to C change in position four, creating a GATC core, was found to increase activity significantly. The deleted ELT-1 protein containing only the C-terminal Zn finger was sufficient for activation in response to GATA, but both fingers were required for activation at GATC. A variety of sites with non-optimal sequences surrounding the GATA core also were found to be excluded better by the protein containing both Zn fingers. Furthermore, a fusion protein containing the entire ELT-1 DNA binding domain fused to the VP16 activation domain was found to have an even greater preference for the GATC core, as well as the optimal flanking bases. We conclude that, although ELT-1 having only its C-terminal finger is capable of activation in response to

  20. ATP-dependent motor activity of the transcription termination factor Rho from Mycobacterium tuberculosis.

    PubMed

    D'Heygère, François; Schwartz, Annie; Coste, Franck; Castaing, Bertrand; Boudvillain, Marc

    2015-07-13

    The bacterial transcription termination factor Rho-a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity-is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate transcriptional complexes is not conserved throughout the bacterial kingdom. Here, however, we demonstrate that MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or transcription elongation complexes. Moreover, we show that idiosyncratic features of the MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal 'RNA binding' domain and by a non-canonical R-loop residue in its C-terminal 'motor' domain. We also show that the 'motor' domain of MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors.

  1. Statistical reconstruction of transcription factor activity using Michaelis-Menten kinetics.

    PubMed

    Khanin, R; Vinciotti, V; Mersinias, V; Smith, C P; Wit, E

    2007-09-01

    The basic building block of a gene regulatory network consists of a gene encoding a transcription factor (TF) and the gene(s) it regulates. Considerable efforts have been directed recently at devising experiments and algorithms to determine TFs and their corresponding target genes using gene expression and other types of data. The underlying problem is that the expression of a gene coding for the TF provides only limited information about the activity of the TF, which can also be controlled posttranscriptionally. In the absence of a reliable technology to routinely measure the activity of regulators, it is of great importance to understand whether this activity can be inferred from gene expression data. We here develop a statistical framework to reconstruct the activity of a TF from gene expression data of the target genes in its regulatory module. The novelty of our approach is that we embed the deterministic Michaelis-Menten model of gene regulation in this statistical framework. The kinetic parameters of the gene regulation model are inferred together with the profile of the TF regulator. We also obtain a goodness-of-fit test to verify the fit of the model. The model is applied to a time series involving the Streptomyces coelicolor bacterium. We focus on the transcriptional activator cdaR, which is partly responsible for the production of a particular type of antibiotic. The aim is to reconstruct the activity profile of this regulator. Our approach can be extended to include more complex regulatory relationships, such as multiple regulatory factors, competition, and cooperativity.

  2. [Techniques for assaying the activity of transcription factor NF-κB].

    PubMed

    Ling, Xiao-Qian; Wang, Jin-Ke

    2013-05-01

    NF-κB is a stimulatory transcription factor that is ubiquitous in almost all kinds of cells. When cells are under various stimuli, NF-κB is activated and regulates large numbers of target genes, and thus controls important cellular processes, ranging from cell growth and differentiation to apoptosis and cancer. Therefore, NF-κB is a forefront hotspot transcription factor that is intensively studied in virtually all fields of biomedical sciences, and becomes a promising target for disease therapy and drug screening. The activity detection is the first and inevitable step for the studies of NF-κB activation and function.Therefore, the techniques for detection of NF-κB activity have always been paid more attention and continuously developed. Especially in recent year, along with the development of each disciplines, various new techniques have been developed, including ELISA-like assays based on dsDNA-coupled plate, filter binding assays, FRET assays, fluorescence reporting and nucleic acids amplification assays based on exonuclease and endonuclease, MS and flow cytometry assays based on immunomicrobeads, and other biophysical and electrochemical assays. Some of these techniques have already played important roles in NF-κB studies. This paper reviewed new techniques developed in recent years by classification, in order to provide an overview of NF-κB activity assays, which may be helpful for researchers to select appropriate techniques used in their studies. Moreover, the learning and understanding of these techniques may inspire researchers to improve currently existing techniques and develop novel methods for the studies of NF-κB.

  3. Hypoxia-Inducible Factor 1 Is an Inductor of Transcription Factor Activating Protein 2 Epsilon Expression during Chondrogenic Differentiation.

    PubMed

    Niebler, Stephan; Angele, Peter; Kujat, Richard; Bosserhoff, Anja K

    2015-01-01

    The transcription factor AP-2ε (activating enhancer-binding protein epsilon) is expressed in cartilage of humans and mice. However, knowledge about regulatory mechanisms influencing AP-2ε expression is limited. Using quantitative real time PCR, we detected a significant increase in AP-2ε mRNA expression comparing initial and late stages of chondrogenic differentiation processes in vitro and in vivo. Interestingly, in these samples the expression pattern of the prominent hypoxia marker gene angiopoietin-like 4 (Angptl4) strongly correlated with that of AP-2ε suggesting that hypoxia might represent an external regulator of AP-2ε expression in mammals. In order to show this, experiments directly targeting the activity of hypoxia-inducible factor-1 (HIF1), the complex mediating responses to oxygen deprivation, were performed. While the HIF1-activating compounds 2,2'-dipyridyl and desferrioxamine resulted in significantly enhanced mRNA concentration of AP-2ε, siRNA against HIF1α led to a significantly reduced expression rate of AP-2ε. Additionally, we detected a significant upregulation of the AP-2ε mRNA level after oxygen deprivation. In sum, these different experimental approaches revealed a novel role for the HIF1 complex in the regulation of the AP-2ε gene in cartilaginous cells and underlined the important role of hypoxia as an important external regulatory stimulus during chondrogenic differentiation modulating the expression of downstream transcription factors.

  4. A Global Genomic and Genetic Strategy to Predict Pathway Activation of Xenobiotic Responsive Transcription Factors in the Mouse Liver

    EPA Science Inventory

    Many drugs and environmentally-relevant chemicals activate xenobiotic-responsive transcription factors(TF). Identification of target genes of these factors would be useful in predicting pathway activation in in vitro chemical screening. Starting with a large compendium of Affymet...

  5. The purple cauliflower arises from activation of a MYB transcription factor.

    PubMed

    Chiu, Li-Wei; Zhou, Xiangjun; Burke, Sarah; Wu, Xianli; Prior, Ronald L; Li, Li

    2010-11-01

    Anthocyanins are responsible for the color of many flowers, fruits, and vegetables. An interesting and unique Purple (Pr) gene mutation in cauliflower (Brassica oleracea var botrytis) confers an abnormal pattern of anthocyanin accumulation, giving the striking mutant phenotype of intense purple color in curds and a few other tissues. To unravel the nature of the Pr mutation in cauliflower, we isolated the Pr gene via a combination of candidate gene analysis and fine mapping. Pr encoded a R2R3 MYB transcription factor that exhibited tissue-specific expression, consistent with an abnormal anthocyanin accumulation pattern in the mutant. Transgenic Arabidopsis (Arabidopsis thaliana) and cauliflower plants expressing the Pr-D allele recapitulated the mutant phenotype, confirming the isolation of the Pr gene. Up-regulation of Pr specifically activated a basic helix-loop-helix transcription factor and a subset of anthocyanin structural genes encoding flavonoid 3'-hydroxylase, dihydroflavonol 4-reductase, and leucoanthocyanidin dioxygenase to confer ectopic accumulation of pigments in the purple cauliflower. Our results indicate that the genetic variation including a Harbinger DNA transposon insertion in the upstream regulatory region of the Pr-D allele is responsible for the up-regulation of the Pr gene in inducing phenotypic change in the plant. The successful isolation of Pr provides important information on the regulatory control of anthocyanin biosynthesis in Brassica vegetables, and offers a genetic resource for development of new varieties with enhanced health-promoting properties and visual appeal.

  6. HBXIP up-regulates ACSL1 through activating transcriptional factor Sp1 in breast cancer.

    PubMed

    Wang, Yue; Cai, Xiaoli; Zhang, Shuqin; Cui, Ming; Liu, Fabao; Sun, Baodi; Zhang, Weiying; Zhang, Xiaodong; Ye, Lihong

    2017-03-11

    The oncoprotein hepatitis B X-interacting protein (HBXIP) results in the dysregulation of lipid metabolism to enhance the development of breast cancer. Acyl-CoA synthetase long-chain family member 1 (ACSL1) is required for thioesterification of long-chain fatty acids into their acyl-CoA derivatives. In this study, we present a hypothesis that HBXIP might be involved in the regulation of ACSL1 in breast cancer. Interestingly, we found that the overexpression of HBXIP was able to up-regulate ACSL1 at the levels of mRNA and protein in a dose-dependent manner in breast cancer cells. Conversely, silencing of HBXIP led to the opposite results. Mechanistically, HBXIP as a coactivator interacted with transcriptional factor Sp1 through binding to the promoter of ACSL1 by ChIP assays analysis, leading to the transcription of ACSL1 in breast cancer cells. Immunohistochemistry staining revealed that the positive rate of ACSL1 was 71.4% (35/49) in clinical breast cancer tissues, HBXIP 79.6% (39/49), in which the positive rate of ACSL1 was 76.9% (30/39) in the HBXIP-positive specimens. But, few positive rate of ACSL1 10% (1/10) was observed in normal breast tissues. The mRNA levels of ACSL1 were significantly higher in clinical breast cancer tissues than those in their corresponding peritumor tissues. The mRNA levels of ACSL1 were positively associated with those of HBXIP in clinical breast cancer tissues. Thus, we conclude that the oncoprotein HBXIP is able to up-regulate ACSL1 through activating the transcriptional factor Sp1 in breast cancer.

  7. Aldosterone Activates Transcription Factor Nrf2 in Kidney Cells Both In Vitro and In Vivo

    PubMed Central

    Oteiza, Patricia I.; Link, Samuel; Hey, Valentin; Stopper, Helga; Schupp, Nicole

    2014-01-01

    Abstract Aims: An increased kidney cancer risk was found in hypertensive patients, who frequently exhibit hyperaldosteronism, known to contribute to kidney injury, with oxidative stress playing an important role. The capacity of kidney cells to up-regulate transcription factor nuclear factor-erythroid-2-related factor 2 (Nrf2), a key regulator of the cellular antioxidative defense, as a prevention of aldosterone-induced oxidative damage was investigated both in vitro and in vivo. Results: Aldosterone activated Nrf2 and increased the expression of enzymes involved in glutathione (GSH) synthesis and detoxification. This activation depended on the mineralocorticoid receptor (MR) and oxidative stress. In vitro, Nrf2 activation, GSH amounts, and target gene levels decreased after 24 h, while oxidant levels remained high. Nrf2 activation could not protect cells against oxidative DNA damage, as aldosterone-induced double-strand breaks and 7,8-dihydro-8-oxo-guanine (8-oxodG) lesions steadily rose. The Nrf2 activator sulforaphane enhanced the Nrf2 response both in vitro and in vivo, thereby preventing aldosterone-induced DNA damage. In vivo, Nrf2 activation further had beneficial effects on the aldosterone-caused blood pressure increase and loss of kidney function. Innovation: This is the first study showing the activation of Nrf2 by aldosterone. Moreover, the results identify sulforaphane as a substance that is capable of preventing aldosterone-induced damage both in vivo and in vitro. Conclusion: Aldosterone-induced Nrf2 adaptive response cannot neutralize oxidative actions of chronically increased aldosterone, which, therefore could be causally involved in the increased cancer incidence of hypertensive individuals. Enhancing the cellular antioxidative defense with sulforaphane might exhibit beneficial effects. Antioxid. Redox Signal. 21, 2126–2142. PMID:24512358

  8. Activation of transcription factor genes in striatum by cocaine: role of both serotonin and dopamine systems.

    PubMed

    Bhat, R V; Baraban, J M

    1993-10-01

    Acute administration of cocaine increases expression of the transcription factor genes c-fos and zif268 in the striatum. This response is thought to be mediated via D1 dopamine (DA) receptors, as it is blocked by the selective D1 receptor antagonist SCH 23390. However, the directly acting D1 receptor agonists, apomorphine and SKF 38393, do not mimic cocaine's activation of these genes raising the possibility that D1 receptor activation is necessary, but not sufficient, to trigger transcription factor expression. Because cocaine blocks uptake of norepinephrine (NE) and serotonin (5-HT), as well as DA, we examined whether cocaine's ability to inhibit NE and 5-HT uptake may contribute to its induction of c-fos and zif268 expression in striatum. In examining the effects of selective monoamine uptake inhibitors, we observed that fluoxetine or citalopram, selective inhibitors of 5-HT uptake, potentiated the ability of mazindol, a DA and NE uptake inhibitor, to induce zif268 and c-fos expression, even though these 5-HT uptake inhibitors had no effect when administered alone. In contrast, the selective NE uptake inhibitor, desipramine, administered alone, or in combination with fluoxetine, did not increase expression of zif268 or c-fos. Furthermore, selective denervation of 5-HT projections by p-chloroamphetamine treatment attenuated the increase in zif268 and c-fos expression induced by cocaine in the striatum. In contrast, selective lesions of NE projections with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride failed to block cocaine's activation of these genes in the striatum. Taken together, these findings indicate that cocaine's ability to induce striatal expression of c-fos and zif268 is mediated by its effects on both the 5-HT and DA systems.

  9. The ERF11 Transcription Factor Promotes Internode Elongation by Activating Gibberellin Biosynthesis and Signaling1[OPEN

    PubMed Central

    Zhou, Xin; Zhang, Zhong-Lin; Tyler, Ludmila; Yusuke, Jikumaru; Qiu, Kai; Lumba, Shelley; Desveaux, Darrell; McCourt, Peter; Sun, Tai-ping

    2016-01-01

    The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6. AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways. PMID:27255484

  10. The ETS Factor Myeloid Elf-1-Like Factor (MEF)/Elf4 Is Transcriptionally and Functionally Activated by Hypoxia.

    PubMed

    Suico, Mary Ann; Taura, Manabu; Kudo, Eriko; Gotoh, Kumiko; Shuto, Tsuyoshi; Okada, Seiji; Kai, Hirofumi

    2016-01-01

    Hypoxia-inducible factor (HIF)-1α is a transcription factor belonging to the HIF family that is activated in mammalian cells during conditions of low oxygen tension or hypoxia to induce an adaptive response and promote cell survival. Some of the genes targeted by HIF-1α are important for angiogenesis and proliferation. Here, we found that the E26 transformation-specific (ETS) transcription factor myeloid elf-1-like factor (MEF)/Elf4 is activated by HIF-1α. MEF induces genes such as human beta-defensin 2 (HβD2) and perforin (PRF1), and is known to affect the cell cycle. Treatment with hypoxia mimetic CoCl2 or low O2 incubation up-regulated MEF mRNA and protein levels in various cell lines. HIF-1α overexpression in HEK293 cells also increased MEF mRNA and protein levels. In contrast, HIF-1α knockdown by small interfering RNA (siRNA) suppressed the induction of MEF in response to hypoxia. HIF-1α binds to the hypoxia response element in the MEF promoter region (-200 bp) and activates MEF promoter under hypoxia condition. The induction of MEF by hypoxia/HIF-1α correlated with the increase of MEF target genes HβD2 and PRF1. Intriguingly, the hypoxia-induced expression of HIF-1α target gene vascular endothelial growth factor (VEGF) was enhanced by the exogenous addition of MEF. Overall, these data indicate that hypoxia or HIF-1α positively regulates MEF expression and function.

  11. Transcriptional activation of human CDCA8 gene regulated by transcription factor NF-Y in embryonic stem cells and cancer cells.

    PubMed

    Dai, Can; Miao, Cong-Xiu; Xu, Xiao-Ming; Liu, Lv-Jun; Gu, Yi-Fan; Zhou, Di; Chen, Lian-Sheng; Lin, Ge; Lu, Guang-Xiu

    2015-09-11

    The cell division cycle associated 8 (CDCA8) gene plays an important role in mitosis. Overexpression of CDCA8 was reported in some human cancers and is required for cancer growth and progression. We found CDCA8 expression was also high in human ES cells (hESCs) but dropped significantly upon hESC differentiation. However, the regulation of CDCA8 expression has not yet been studied. Here, we characterized the CDCA8 promoter and identified its cis-elements and transcription factors. Three transcription start sites were identified. Reporter gene assays revealed that the CDCA8 promoter was activated in hESCs and cancer cell lines. The promoter drove the reporter expression specifically to pluripotent cells during early mouse embryo development and to tumor tissues in tumor-bearing mice. These results indicate that CDCA8 is transcriptionally activated in hESCs and cancer cells. Mechanistically, two key activation elements, bound by transcription factor NF-Y and CREB1, respectively, were identified in the CDCA8 basic promoter by mutation analyses and electrophoretic motility shift assays. NF-Y binding is positively correlated with promoter activities in different cell types. Interestingly, the NF-YA subunit, binding to the promoter, is primarily a short isoform in hESCs and a long isoform in cancer cells, indicating a different activation mechanism of the CDCA8 transcription between hESCs and cancer cells. Finally, enhanced CDCA8 promoter activities by NF-Y overexpression and reduced CDCA8 transcription by NF-Y knockdown further verified that NF-Y is a positive regulator of CDCA8 transcription. Our study unearths the molecular mechanisms underlying the activation of CDCA8 expression in hESCs and cancer cells, which provides a better understanding of its biological functions.

  12. Inhibition of Phosphatase Activity Follows Decline in Sulfatase Activity and Leads to Transcriptional Effects through Sustained Phosphorylation of Transcription Factor MITF

    PubMed Central

    Bhattacharyya, Sumit; Feferman, Leo; Tobacman, Joanne K.

    2016-01-01

    Arylsulfatase B (B-acetylgalactosamine 4-sulfatase; ARSB) is the enzyme that removes 4-sulfate groups from the non-reducing end of the glycosaminoglycans chondroitin 4-sulfate and dermatan sulfate. Decline in ARSB has been shown in malignant prostate, colonic, and mammary cells and tissues, and decline in ARSB leads to transcriptional events mediated by galectin-3 with AP-1 and Sp1. Increased mRNA expression of GPNMB (transmembrane glycoprotein NMB) in HepG2 cells and in hepatic tissue from ARSB-deficient mice followed decline in expression of ARSB and was mediated by the microphthalmia-associated transcription factor (MITF), but was unaffected by silencing galectin-3. Since GPNMB is increased in multiple malignancies, studies were performed to determine how decline in ARSB increased GPNMB expression. The mechanism by which decline in ARSB increased nuclear phospho-MITF was due to reduced activity of SHP2, a protein tyrosine phosphatase with Src homology (SH2) domains that regulates multiple cellular processes. SHP2 activity declined due to increased binding with chondroitin 4-sulfate when ARSB was reduced. When SHP2 activity was inhibited, phosphorylations of p38 mitogen-associated phosphokinase (MAPK) and of MITF increased, leading to GPNMB promoter activation. A dominant negative SHP2 construct, the SHP2 inhibitor PHSP1, and silencing of ARSB increased phospho-p38, nuclear MITF, and GPNMB. In contrast, constitutively active SHP2 and overexpression of ARSB inhibited GPNMB expression. The interaction between chondroitin 4-sulfate and SHP2 is a novel intersection between sulfation and phosphorylation, by which decline in ARSB and increased chondroitin 4-sulfation can inhibit SHP2, thereby regulating downstream tyrosine phosphorylations by sustained phosphorylations with associated activation of signaling and transcriptional events. PMID:27078017

  13. Transcription factor-based biosensor

    DOEpatents

    Dietrich, Jeffrey A; Keasling, Jay D

    2013-10-08

    The present invention provides for a system comprising a BmoR transcription factor, a .sigma..sup.54-RNA polymerase, and a pBMO promoter operatively linked to a reporter gene, wherein the pBMO promoter is capable of expression of the reporter gene with an activated form of the BmoR and the .sigma..sup.54-RNA polymerase.

  14. Limits on information transduction through amplitude and frequency regulation of transcription factor activity.

    PubMed

    Hansen, Anders S; O'Shea, Erin K

    2015-05-18

    Signaling pathways often transmit multiple signals through a single shared transcription factor (TF) and encode signal information by differentially regulating TF dynamics. However, signal information will be lost unless it can be reliably decoded by downstream genes. To understand the limits on dynamic information transduction, we apply information theory to quantify how much gene expression information the yeast TF Msn2 can transduce to target genes in the amplitude or frequency of its activation dynamics. We find that although the amount of information transmitted by Msn2 to single target genes is limited, information transduction can be increased by modulating promoter cis-elements or by integrating information from multiple genes. By correcting for extrinsic noise, we estimate an upper bound on information transduction. Overall, we find that information transduction through amplitude and frequency regulation of Msn2 is limited to error-free transduction of signal identity, but not signal intensity information.

  15. Intermedin/adrenomedullin 2 is a stress-inducible gene controlled by activating transcription factor 4.

    PubMed

    Kovaleva, Irina E; Garaeva, Alisa A; Chumakov, Peter M; Evstafieva, Alexandra G

    2016-09-15

    Intermedin or adrenomedullin 2 is a set of calcitonin-related peptides with a putative tumor angiogenesis promoting activity that are formed by proteolytic processing of the ADM2 gene product. It has been proposed that the ADM2 gene is regulated by the estrogen response element (ERE) and hypoxia response elements (HRE) found within its promoter region. In the present study we reveal a functional mechanism by which ADM2 participates in the unfolded protein response (UPR) and in responses to the mitochondrial respiration chain inhibition. We show that the ADM2 gene is controlled by activating transcription factor 4 (ATF4), the principal regulator of the integrated stress response (ISR). The upregulation of ADM2 mRNA could be prevented by the pharmacological ISR inhibitor ISRIB and by the downregulation of ATF4 with specific shRNA, while ectopic expression of ATF4 cDNA resulted in a notable increase in ADM2 gene transcription. A potential ATF4-binding site was identified in the coding region of the ADM2 gene and the requirement of this site during the ATF4-mediated ADM2 gene promoter activation was validated by the luciferase reporter assay. Mutagenesis of the putative ATF4-response element prevented the induction of luciferase activity in response to ATF4 overproduction, as well as in response to mitochondrial electron transfer chain inhibition by piericidin A and ER stress induction by tunicamycin and brefeldin A. Since ADM2 was shown to inhibit ATF4 expression during myocardial ER stress, a feedback mechanism could be proposed for the ADM2 regulation under ER stress conditions.

  16. SparseNCA: Sparse Network Component Analysis for Recovering Transcription Factor Activities with Incomplete Prior Information.

    PubMed

    Noor, Amina; Ahmad, Aitzaz; Serpedin, Erchin

    2015-10-27

    Network component analysis (NCA) is an important method for inferring transcriptional regulatory networks (TRNs) and recovering transcription factor activities (TFAs) using gene expression data, and the prior information about the connectivity matrix. The algorithms currently available crucially depend on the completeness of this prior information. However, inaccuracies in the measurement process may render incompleteness in the available knowledge about the connectivity matrix. Hence, computationally efficient algorithms are needed to overcome the possible incompleteness in the available data. We present a sparse network component analysis algorithm (sparseNCA), which incorporates the effect of incompleteness in the estimation of TRNs by imposing an additional sparsity constraint using the `1 norm, which results in a greater estimation accuracy. In order to improve the computational efficiency, an iterative re-weighted `2 method is proposed for the NCA problem which not only promotes sparsity but is hundreds of times faster than the `1 norm based solution. The performance of sparseNCA is rigorously compared to that of FastNCA and NINCA using synthetic data as well as real data. It is shown that sparseNCA outperforms the existing state-of-the-art algorithms both in terms of estimation accuracy and consistency with the added advantage of low computational complexity. The performance of sparseNCA compared to its predecessors is particularly pronounced in case of incomplete prior information about the sparsity of the network. Subnetwork analysis is performed on the E.coli data which reiterates the superior consistency of the proposed algorithm.

  17. TATA-box DNA binding activity and subunit composition for RNA polymerase III transcription factor IIIB from Xenopus laevis.

    PubMed Central

    McBryant, S J; Meier, E; Leresche, A; Sharp, S J; Wolf, V J; Gottesfeld, J M

    1996-01-01

    The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-associated factors (TAFs). Previous studies have shown that DNA oligonucleotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exposed in TFIIIB. We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription inhibition assays and a gel mobility shift assay. Gel shift competition assays with mutant and nonspecific DNAs demonstrate the specificity of the TFIIIB-TATA box DNA complex. The apparent dissociation constant for this protein-DNA interaction is approximately 0.4 nM, similar to the affinity of yeast TBP for the same sequence. TFIIIB transcriptional activity and TATA-box binding activity cofractionate during a series of four ion-exchange chromatographic steps, and reconstituted transcription reactions demonstrate that the TATA-box DNA-protein complex contains TFIIIB TAF activity. Polypeptides with apparent molecular masses of 75 and 92 kDa are associated with TBP in this complex. These polypeptides were renatured after elution from sodium dodecyl sulfate-gels and tested individually and in combination for TFIIIB TAF activity. Recombinant TBP along with protein fractions containing the 75- and 92-kDa polypeptides were sufficient to reconstitute TFIIIB transcriptional activity and DNA binding activity, suggesting that Xenopus TFIIIB is composed of TBP along with these polypeptides. PMID:8756620

  18. Colocalization recognition-activated cascade signal amplification strategy for ultrasensitive detection of transcription factors.

    PubMed

    Zhu, Desong; Wang, Lei; Xu, Xiaowen; Jiang, Wei

    2017-03-15

    Transcription factors (TFs) bind to specific double-stranded DNA (dsDNA) sequences in the regulatory regions of genes to regulate the process of gene transcription. Their expression levels sensitively reflect cell developmental situation and disease state. TFs have become potential diagnostic markers and therapeutic targets of cancers and some other diseases. Hence, high sensitive detection of TFs is of vital importance for early diagnosis of diseases and drugs development. The traditional exonucleases-assisted signal amplification methods suffered from the false positives caused by incomplete digestion of excess recognition probes. Herein, based on a new recognition way-colocalization recognition (CR)-activated dual signal amplification, an ultrasensitive fluorescent detection strategy for TFs was developed. TFs-induced the colocalization of three split recognition components resulted in noticeable increases of local effective concentrations and hybridization of three split components, which activated the subsequent cascade signal amplification including strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). This strategy eliminated the false positive influence and achieved ultra-high sensitivity towards the purified NF-κB p50 with detection limit of 2.0×10(-13)M. Moreover, NF-κB p50 can be detected in as low as 0.21ngμL(-1) HeLa cell nuclear extracts. In addition, this proposed strategy could be used for the screening of NF-κB p50 activity inhibitors and potential anti-NF-κB p50 drugs. Finally, our proposed strategy offered a potential method for reliable detection of TFs in medical diagnosis and treatment research of cancers and other related diseases.

  19. RAI1 transcription factor activity is impaired in mutants associated with Smith-Magenis Syndrome.

    PubMed

    Carmona-Mora, Paulina; Canales, Cesar P; Cao, Lei; Perez, Irene C; Srivastava, Anand K; Young, Juan I; Walz, Katherina

    2012-01-01

    Smith-Magenis Syndrome (SMS) is a complex genomic disorder mostly caused by the haploinsufficiency of the Retinoic Acid Induced 1 gene (RAI1), located in the chromosomal region 17p11.2. In a subset of SMS patients, heterozygous mutations in RAI1 are found. Here we investigate the molecular properties of these mutated forms and their relationship with the resulting phenotype. We compared the clinical phenotype of SMS patients carrying a mutation in RAI1 coding region either in the N-terminal or the C-terminal half of the protein and no significant differences were found. In order to study the molecular mechanism related to these two groups of RAI1 mutations first we analyzed those mutations that result in the truncated protein corresponding to the N-terminal half of RAI1 finding that they have cytoplasmic localization (in contrast to full length RAI1) and no ability to activate the transcription through an endogenous target: the BDNF enhancer. Similar results were found in lymphoblastoid cells derived from a SMS patient carrying RAI1 c.3103insC, where both mutant and wild type products of RAI1 were detected. The wild type form of RAI1 was found in the chromatin bound and nuclear matrix subcellular fractions while the mutant product was mainly cytoplasmic. In addition, missense mutations at the C-terminal half of RAI1 presented a correct nuclear localization but no activation of the endogenous target. Our results showed for the first time a correlation between RAI1 mutations and abnormal protein function plus they suggest that a reduction of total RAI1 transcription factor activity is at the heart of the SMS clinical presentation.

  20. The AP-1 transcription factor homolog Pf-AP-1 activates transcription of multiple biomineral proteins and potentially participates in Pinctada fucata biomineralization.

    PubMed

    Zheng, Xiangnan; Cheng, Minzhang; Xiang, Liang; Liang, Jian; Xie, Liping; Zhang, Rongqing

    2015-09-25

    Activator protein-1 (AP-1) is an important bZIP transcription factor that regulates a series of physiological processes by specifically activating transcription of several genes, and one of its well-chartered functions in mammals is participating in bone mineralization. We isolated and cloned the complete cDNA of a Jun/AP-1 homolog from Pinctada fucata and called it Pf-AP-1. Pf-AP-1 had a highly conserved bZIP region and phosphorylation sites compared with those from mammals. A tissue distribution analysis showed that Pf-AP-1 was ubiquitously expressed in P. fucata and the mRNA level of Pf-AP-1 is extremely high in mantle. Pf-AP-1 expression was positively associated with multiple biomineral proteins in the mantle. The luciferase reporter assay in a mammalian cell line showed that Pf-AP-1 significantly up-regulates the transcriptional activity of the promoters of KRMP, Pearlin, and Prisilkin39. Inhibiting the activity of Pf-AP-1 depressed the expression of multiple matrix proteins. Pf-AP-1 showed a unique expression pattern during shell regeneration and pearl sac development, which was similar to the pattern observed for biomineral proteins. These results suggest that the Pf-AP-1 AP-1 homolog is an important transcription factor that regulates transcription of several biomineral proteins simultaneously and plays a role in P. fucata biomineralization, particularly during pearl and shell formation.

  1. Microglial phenotype is regulated by activity of the transcription factor, NFAT

    PubMed Central

    Nagamoto-Combs, Kumi

    2010-01-01

    The transcription factor family, nuclear factor of activated T cells (NFAT), regulates immune cell phenotype. Four different calcium/calmodulin-regulated isoforms have been identified in the periphery, but isoform expression in microglia, the resident immune cells of the central nervous system, has not been fully defined. In this study microglial NFAT isoform expression and involvement in regulating inflammatory responses in murine primary microglia culture was examined. Western blot analysis demonstrated robust detection of NFATc1 and c2 isoforms in microglia. Electrophoretic mobility shift assays demonstrated increased NFAT-DNA binding from nuclear extracts of lipopolysaccharide (LPS) stimulated microglia. Moreover, LPS-stimulated microglia behaved similarly to T cell receptor agonist antibody-stimulated Jurkat cells demonstrating a transient increase in NFAT-driven luciferase reporter gene expression. LPS-induced NFAT-luciferase activity in microglia was attenuated by pretreatment with tat-VIVIT, a cell-permeable NFAT inhibitory peptide. Furthermore, LPS-mediated secretion of microglial cytokines, TNF-α and MCP-1, was decreased by treatment with tat-VIVIT but not with tat-VEET, a negative control peptide. These results demonstrate that NFAT plays a role in regulating proinflammatory responses in cultured murine microglia. PMID:20631193

  2. Protein kinase A activation of the surfactant protein B gene is mediated by phosphorylation of thyroid transcription factor 1.

    PubMed

    Yan, C; Whitsett, J A

    1997-07-11

    Thyroid transcription factor 1 (TTF-1) is a homeodomain-containing nuclear transcription factor expressed in epithelial cells of the lung and thyroid. TTF-1 binds to and activates the transcription of genes expressed selectively in the respiratory epithelium including pulmonary surfactant A, B, C and Clara cell secretory protein. Transfection with a plasmid encoding the cyclic AMP-dependent protein kinase (protein kinase A; PKA) catalytic subunit, Cat-beta, stimulated the phosphorylation of a TTF-1-flag fusion protein 6-7-fold in H441 pulmonary adenocarcinoma cells. Recombinant TTF-1 was phosphorylated by purified PKA catalytic subunit in the presence of [gamma-32P]ATP. PKA catalytic subunit family members, Cat-alpha and Cat-beta, markedly enhanced the transcriptional activation of surfactant B gene promoters by TTF-1 in vitro. Peptide mapping was used to identify a PKA phosphorylation site at the NH2 terminus of TTF-1. A 17-amino acid synthetic peptide comprising this site completely inhibited the PKA-dependent phosphorylation of TTF-1 in vitro. A substitution mutation of TTF-1 (Thr9 two head right arrow Ala) abolished phosphorylation by PKA and reduced transactivation of the surfactant B gene promoter. Transfection with a plasmid encoding the cAMP regulatory element binding factor inhibited transcriptional activity of the surfactant protein B gene promoter. Phosphorylation of TTF-1 mediates PKA-dependent activation of surfactant protein B gene transcription.

  3. MicroRNA-214 suppresses gluconeogenesis by targeting activating transcriptional factor 4.

    PubMed

    Li, Kai; Zhang, Jin; Yu, Junjie; Liu, Bin; Guo, Yajie; Deng, Jiali; Chen, Shanghai; Wang, Chunxia; Guo, Feifan

    2015-03-27

    Although the gluconeogenesis pathway is already a target for the treatment of type 2 diabetes, the potential role of microRNAs (miRNAs) in gluconeogenesis remains unclear. Here, we investigated the physiological functions of miR-214 in gluconeogenesis. The expression of miR-214 was suppressed by glucagon via protein kinase A signaling in primary hepatocytes, and miR-214 was down-regulated in the livers of fasted, high fat diet-induced diabetic and leptin receptor-mutated (db/db) mice. The overexpression of miR-214 in primary hepatocytes suppressed glucose production, and silencing miR-214 reversed this effect. Gluconeogenesis was suppressed in the livers of mice injected with an adenovirus expressing miR-214 (Ad-miR-214). Additionally, Ad-miR-214 alleviated high fat diet-induced elevation of gluconeogenesis and hyperglycemia. Furthermore, we found that activating transcription factor 4 (ATF4), a reported target of miR-214, can reverse the suppressive effect of miR-214 on gluconeogenesis in primary hepatocytes, and this suppressive effect was blocked in liver-specific ATF4 knock-out mice. ATF4 regulated gluconeogenesis via affecting forkhead box protein O1 (FOXO1) transcriptional activity. Finally, liver-specific miR-214 transgenic mice exhibited suppressed gluconeogenesis and reduced expression of ATF4, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase in liver. Taken together, our results suggest that the miR-214-ATF4 axis is a novel pathway for the regulation of hepatic gluconeogenesis.

  4. MicroRNA-214 Suppresses Gluconeogenesis by Targeting Activating Transcriptional Factor 4*

    PubMed Central

    Li, Kai; Zhang, Jin; Yu, Junjie; Liu, Bin; Guo, Yajie; Deng, Jiali; Chen, Shanghai; Wang, Chunxia; Guo, Feifan

    2015-01-01

    Although the gluconeogenesis pathway is already a target for the treatment of type 2 diabetes, the potential role of microRNAs (miRNAs) in gluconeogenesis remains unclear. Here, we investigated the physiological functions of miR-214 in gluconeogenesis. The expression of miR-214 was suppressed by glucagon via protein kinase A signaling in primary hepatocytes, and miR-214 was down-regulated in the livers of fasted, high fat diet-induced diabetic and leptin receptor-mutated (db/db) mice. The overexpression of miR-214 in primary hepatocytes suppressed glucose production, and silencing miR-214 reversed this effect. Gluconeogenesis was suppressed in the livers of mice injected with an adenovirus expressing miR-214 (Ad-miR-214). Additionally, Ad-miR-214 alleviated high fat diet-induced elevation of gluconeogenesis and hyperglycemia. Furthermore, we found that activating transcription factor 4 (ATF4), a reported target of miR-214, can reverse the suppressive effect of miR-214 on gluconeogenesis in primary hepatocytes, and this suppressive effect was blocked in liver-specific ATF4 knock-out mice. ATF4 regulated gluconeogenesis via affecting forkhead box protein O1 (FOXO1) transcriptional activity. Finally, liver-specific miR-214 transgenic mice exhibited suppressed gluconeogenesis and reduced expression of ATF4, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase in liver. Taken together, our results suggest that the miR-214-ATF4 axis is a novel pathway for the regulation of hepatic gluconeogenesis. PMID:25657009

  5. The Brn-2 transcription factor links activated BRAF to melanoma proliferation.

    PubMed

    Goodall, Jane; Wellbrock, Claudia; Dexter, Timothy J; Roberts, Karen; Marais, Richard; Goding, Colin R

    2004-04-01

    Malignant melanoma, an aggressive and increasingly common cancer, is characterized by a strikingly high rate (70%) of mutations in BRAF, a key component of the mitogen-activated protein (MAP) kinase signaling pathway. How signaling events downstream from BRAF affect the underlying program of gene expression is poorly understood. We show that the Brn-2 POU domain transcription factor is highly expressed in melanoma cell lines but not in melanocytes or melanoblasts and that overexpression of Brn-2 in melanocytes results in increased proliferation. Expression of Brn-2 is strongly upregulated by Ras and MAP kinase signaling. Importantly, the Brn-2 promoter is stimulated by kinase-activating BRAF mutants and endogenous Brn-2 expression is inhibited by RNA interference-mediated downregulation of BRAF. Moreover, silent interfering RNA-mediated depletion of Brn-2 in melanoma cells expressing activated BRAF leads to decreased proliferation. The results suggest that the high levels of Brn-2 expression observed in melanomas link BRAF signaling to increased proliferation.

  6. Expression of the leukocyte early activation antigen CD69 is regulated by the transcription factor AP-1.

    PubMed

    Castellanos, M C; Muñoz, C; Montoya, M C; Lara-Pezzi, E; López-Cabrera, M; de Landázuri, M O

    1997-12-01

    The leukocyte Ag CD69, one of the earliest cell surface activation Ags, is up-regulated at the transcriptional level by proinflammatory stimuli involving the NF-kappaB/Rel family of transcription factors. However, promoter fragments lacking a critical kappaB motif respond to other stimuli such as phorbol esters and triggering Abs against TCR/CD3. Since the 5' promoter flanking region of the CD69 gene contains several putative binding sequences for transcription factor activating protein-1 (AP-1), we explored its role in the inducible expression of CD69. Stimuli that induce AP-1, but not NF-kappaB, such as pyrrolidine dithiocarbamate, augmented the cell surface expression of CD69 as well as its mRNA levels, and the promoter activity of the CD69 gene. This up-regulation is accompanied by an increased binding of jun and fos family members to a consensus AP-1 binding site of the proximal (-16) CD69 promoter region, which seems to be functionally responsive to different activation signals and is trans activated by c-jun expression vectors. Furthermore, cotransfection of a dominant negative version of c-jun, but not IkappaB, abolished the inducible transcriptional activity of the CD69 promoter. In conclusion, the inducible expression of the CD69 gene by mitogenic signals is regulated by the transcription factor AP-1.

  7. Activating Transcription Factor 4 and X Box Binding Protein 1 of Litopenaeus vannamei Transcriptional Regulated White Spot Syndrome Virus Genes Wsv023 and Wsv083

    PubMed Central

    Li, Xiao-Yun; Pang, Li-Ran; Chen, Yong-Gui; Weng, Shao-Ping; Yue, Hai-Tao; Zhang, Ze-Zhi; Chen, Yi-Hong; He, Jian-Guo

    2013-01-01

    In response to endoplasmic reticulum (ER) stress, the signaling pathway termed unfolded protein response (UPR) is activated. To investigate the role of UPR in Litopenaeus vannamei immunity, the activating transcription factor 4 (designated as LvATF4) which belonged to a branch of the UPR, the [protein kinase RNA (PKR)-like ER kinase, (PERK)]-[eukaryotic initiation factor 2 subunit alpha (eIF2α)] pathway, was identified and characterized. The full-length cDNA of LvATF4 was 1972 bp long, with an open reading frame of 1299 bp long that encoded a 432 amino acid protein. LvATF4 was highly expressed in gills, intestines and stomach. For the white spot syndrome virus (WSSV) challenge, LvATF4 was upregulated in the gills after 3 hpi and increased by 1.9-fold (96 hpi) compared to the mock-treated group. The LvATF4 knock-down by RNA interference resulted in a lower cumulative mortality of L. vannamei under WSSV infection. Reporter gene assays show that LvATF4 could upregulate the expression of the WSSV gene wsv023 based on the activating transcription factor/cyclic adenosine 3′, 5′-monophosphate response element (ATF/CRE). Another transcription factor of L. vannamei, X box binding protein 1 (designated as LvXBP1), has a significant function in [inositol-requiring enzyme-1(IRE1) – (XBP1)] pathway. This transcription factor upregulated the expression of the WSSV gene wsv083 based on the UPR element (UPRE). These results suggest that in L. vannamei UPR signaling pathway transcription factors are important for WSSV and might facilitate WSSV infection. PMID:23638122

  8. Resveratrol Enhances Antitumor Activity of TRAIL in Prostate Cancer Xenografts through Activation of FOXO Transcription Factor

    PubMed Central

    Ganapathy, Suthakar; Chen, Qinghe; Singh, Karan P.; Shankar, Sharmila; Srivastava, Rakesh K.

    2010-01-01

    Background Resveratrol (3, 4′, 5 tri-hydroxystilbene), a naturally occurring polyphenol, exhibits anti-inflammatory, antioxidant, cardioprotective and antitumor activities. We have recently shown that resveratrol can enhance the apoptosis-inducing potential of TRAIL in prostate cancer cells through multiple mechanisms in vitro. Therefore, the present study was designed to validate whether resveratrol can enhance the apoptosis-inducing potential of TRAIL in a xenograft model of prostate cancer. Methodology/Principal Findings Resveratrol and TRAIL alone inhibited growth of PC-3 xenografts in nude mice by inhibiting tumor cell proliferation (PCNA and Ki67 staining) and inducing apoptosis (TUNEL staining). The combination of resveratrol and TRAIL was more effective in inhibiting tumor growth than single agent alone. In xenografted tumors, resveratrol upregulated the expressions of TRAIL-R1/DR4, TRAIL-R2/DR5, Bax and p27/K IP1, and inhibited the expression of Bcl-2 and cyclin D1. Treatment of mice with resveratrol and TRAIL alone inhibited angiogenesis (as demonstrated by reduced number of blood vessels, and VEGF and VEGFR2 positive cells) and markers of metastasis (MMP-2 and MMP-9). The combination of resveratrol with TRAIL further inhibited number of blood vessels in tumors, and circulating endothelial growth factor receptor 2-positive endothelial cells than single agent alone. Furthermore, resveratrol inhibited the cytoplasmic phosphorylation of FKHRL1 resulting in its enhanced activation as demonstrated by increased DNA binding activity. Conclusions/Significance These data suggest that resveratrol can enhance the apoptosis-inducing potential of TRAIL by activating FKHRL1 and its target genes. The ability of resveratrol to inhibit tumor growth, metastasis and angiogenesis, and enhance the therapeutic potential of TRAIL suggests that resveratrol alone or in combination with TRAIL can be used for the management of prostate cancer. PMID:21209944

  9. Transcriptional Regulation by Hypoxia Inducible Factors

    PubMed Central

    Espinosa, Joaquín M.

    2015-01-01

    The cellular response to oxygen deprivation is governed largely by a family of transcription factors known as Hypoxia Inducible Factors (HIFs). This review focuses on the molecular mechanisms by which HIFs regulate the transcriptional apparatus to enable the cellular and organismal response to hypoxia. We discuss here how the various HIF polypeptides, their post-translational modifications, binding partners and transcriptional cofactors affect RNA polymerase II activity to drive context-dependent transcriptional programs during hypoxia. PMID:24099156

  10. Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency

    NASA Technical Reports Server (NTRS)

    Mukhopadhyay, C. K.; Mazumder, B.; Fox, P. L.

    2000-01-01

    A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these

  11. Identification and characterization a novel transcription factor activator protein-1 in the sea cucumber Apostichopus japonicus.

    PubMed

    Yang, Limeng; Li, Chenghua; Chang, Yaqing; Gao, Yinxue; Wang, Yi; Wei, Jing; Song, Jian; Sun, Ping

    2015-08-01

    The transcription factor activator protein-1 (AP-1) is an important gene expression regulator with typical Jun and region-leucine zipper (bZIP) domains and can respond to a plethora of physiological and pathological stimulus. In this study, we identified a novel AP-1 gene in Apostichopus japonicus by transcriptome sequencing and RACE approaches (designated as AjAP-1). The full-length of AjAP-1 was of 2944 bp including a 5' untranslated region (UTR) of 201 bp, a 3' UTR of 1753 bp and a putative open reading frame of 990 bp encoding a polypeptide of 329 amino acid residues. Two representative domains of Jun and bZIP as well as two nuclear localization signals (NLSs) were also detected in deduced amino acid of AjAP-1. Spatial distribution expression indicated that AjAP-1 was ubiquitously expressed in all examined tissues with predominant expression in the body wall, moderate in the tube feet, respiratory tree and colemocytes and slightly weak in the intestine and longitudinal muscle. Time-course expression analysis in intestine and coelomocytes revealed that AjAP-1 both reached its peak expression at 4 h after Vibrio splendidus challenge with a 2.6 and 8.2-fold increase compared to their control groups, respectively. Taken together, all these results suggested that AjAP-1 was a novel immune factor and might be involved in the processes of anti-bacteria response in sea cucumber.

  12. Enhanced oxidative stress resistance through activation of a zinc deficiency transcription factor in Brachypodium distachyon.

    PubMed

    Glover-Cutter, Kira M; Alderman, Stephen; Dombrowski, James E; Martin, Ruth C

    2014-11-01

    Identification of viable strategies to increase stress resistance of crops will become increasingly important for the goal of global food security as our population increases and our climate changes. Considering that resistance to oxidative stress is oftentimes an indicator of health and longevity in animal systems, characterizing conserved pathways known to increase oxidative stress resistance could prove fruitful for crop improvement strategies. This report argues for the usefulness and practicality of the model organism Brachypodium distachyon for identifying and validating stress resistance factors. Specifically, we focus on a zinc deficiency B. distachyon basic leucine zipper transcription factor, BdbZIP10, and its role in oxidative stress in the model organism B. distachyon. When overexpressed, BdbZIP10 protects plants and callus tissue from oxidative stress insults, most likely through distinct and direct activation of protective oxidative stress genes. Increased oxidative stress resistance and cell viability through the overexpression of BdbZIP10 highlight the utility of investigating conserved stress responses between plant and animal systems.

  13. The loss of activating transcription factor 4 (ATF4) reduces bone toughness and fracture toughness.

    PubMed

    Makowski, Alexander J; Uppuganti, Sasidhar; Wadeer, Sandra A; Whitehead, Jack M; Rowland, Barbara J; Granke, Mathilde; Mahadevan-Jansen, Anita; Yang, Xiangli; Nyman, Jeffry S

    2014-05-01

    Even though age-related changes to bone tissue affecting fracture risk are well characterized, only a few matrix-related factors have been identified as important to maintaining fracture resistance. As a gene critical to osteoblast differentiation, activating transcription factor 4 (ATF4) is possibly one of these important factors. To test the hypothesis that the loss of ATF4 affects the fracture resistance of bone beyond bone mass and structure, we harvested bones from Atf4+/+ and Atf4-/- littermates at 8 and 20 weeks of age (n≥9 per group) for bone assessment across several length scales. From whole bone mechanical tests in bending, femurs from Atf4-/- mice were found to be brittle with reduced toughness and fracture toughness compared to femurs from Atf4+/+ mice. However, there were no differences in material strength and in tissue hardness, as determined by nanoindentation, between the genotypes, irrespective of age. Tissue mineral density of the cortex at the point of loading as determined by micro-computed tomography was also not significantly different. However, by analyzing local composition by Raman Spectroscopy (RS), bone tissue of Atf4-/- mice was found to have higher mineral to collagen ratio compared to wild-type tissue, primarily at 20 weeks of age. From RS analysis of intact femurs at 2 orthogonal orientations relative to the polarization axis of the laser, we also found that the organizational-sensitive peak ratio, ν1Phosphate per Amide I, changed to a greater extent upon bone rotation for Atf4-deficient tissue, implying bone matrix organization may contribute to the brittleness phenotype. Target genes of ATF4 activity are not only important to osteoblast differentiation but also in maintaining bone toughness and fracture toughness.

  14. The Loss of Activating Transcription Factor 4 (ATF4) Reduces Bone Toughness and Fracture Toughness

    PubMed Central

    Makowski, Alexander J.; Uppuganti, Sasidhar; Waader, Sandra A.; Whitehead, Jack M.; Rowland, Barbara J.; Granke, Mathilde; Mahadevan-Jansen, Anita; Yang, Xiangli; Nyman, Jeffry S.

    2014-01-01

    Even though age-related changes to bone tissue affecting fracture risk are well characterized, only a few matrix-related factors have been identified as important to maintaining fracture resistance. As a gene critical to osteoblast differentiation, activating transcription factor 4 (ATF4) is possibly one of the seimportant factors. To test the hypothesis that the loss of ATF4 affects the fracture resistance of bone beyond bone mass and structure, we harvested bones from Atf4+/+ and Atf4−/− littermates at 8 and 20 weeks of age (n≥9 per group) for bone assessment across several length scales. From whole bone mechanical tests in bending, femurs from Atf4−/− mice were found to be brittle with reduced toughness and fracture toughness compared to femurs from Atf4+/+ mice. However, there were no differences in material strength and in tissue hardness, as determined by nanoindentation, between the genotypes, irrespective age. Tissue mineral density of the cortex at the point of loading as determined by micro-computed tomography was also not significantly different. However, by analyzing local composition by Raman Spectroscopy (RS), bone tissue of Atf4−/− mice was found to have higher mineral to collagen ratio compared to wild-type tissue, primarily at 20 weeks of age. From RS analysis of intact femurs at 2 orthogonal orientations relative to the polarization axis of the laser, we also found that the organizational-sensitive peak ratio, ν1 Phosphate per Amide I, changed to a greater extent upon bone rotation for Atf4-deficient tissue, implying bone matrix organization may contribute to the brittleness phenotype. Target genes of ATF4 activity are not only important to osteoblast differentiation but also maintaining bone toughness and fracture toughness. PMID:24509412

  15. POZ domain transcription factor, FBI-1, represses transcription of ADH5/FDH by interacting with the zinc finger and interfering with DNA binding activity of Sp1.

    PubMed

    Lee, Dong-Kee; Suh, Dongchul; Edenberg, Howard J; Hur, Man-Wook

    2002-07-26

    The POZ domain is a protein-protein interaction motif that is found in many transcription factors, which are important for development, oncogenesis, apoptosis, and transcription repression. We cloned the POZ domain transcription factor, FBI-1, that recognizes the cis-element (bp -38 to -22) located just upstream of the core Sp1 binding sites (bp -22 to +22) of the ADH5/FDH minimal promoter (bp -38 to +61) in vitro and in vivo, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ADH5/FDH minimal promoter is potently repressed by the FBI-1. Glutathione S-transferase fusion protein pull-down showed that the POZ domains of FBI-1, Plzf, and Bcl-6 directly interact with the zinc finger DNA binding domain of Sp1. DNase I footprinting assays showed that the interaction prevents binding of Sp1 to the GC boxes of the ADH5/FDH promoter. Gal4-POZ domain fusions targeted proximal to the GC boxes repress transcription of the Gal4 upstream activator sequence-Sp1-adenovirus major late promoter. Our data suggest that POZ domain represses transcription by interacting with Sp1 zinc fingers and by interfering with the DNA binding activity of Sp1.

  16. Arabidopsis Sigma Factor Binding Proteins Are Activators of the WRKY33 Transcription Factor in Plant Defense[W

    PubMed Central

    Lai, Zhibing; Li, Ying; Wang, Fei; Cheng, Yuan; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2011-01-01

    Necrotrophic pathogens are important plant pathogens that cause many devastating plant diseases. Despite their impact, our understanding of the plant defense response to necrotrophic pathogens is limited. The WRKY33 transcription factor is important for plant resistance to necrotrophic pathogens; therefore, elucidation of its functions will enhance our understanding of plant immunity to necrotrophic pathogens. Here, we report the identification of two WRKY33-interacting proteins, nuclear-encoded SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2, which also interact with plastid-encoded plastid RNA polymerase SIGMA FACTOR1. Both SIB1 and SIB2 contain an N-terminal chloroplast targeting signal and a putative nuclear localization signal, suggesting that they are dual targeted. Bimolecular fluorescence complementation indicates that WRKY33 interacts with SIBs in the nucleus of plant cells. Both SIB1 and SIB2 contain a short VQ motif that is important for interaction with WRKY33. The two VQ motif–containing proteins recognize the C-terminal WRKY domain and stimulate the DNA binding activity of WRKY33. Like WRKY33, both SIB1 and SIB2 are rapidly and strongly induced by the necrotrophic pathogen Botrytis cinerea. Resistance to B. cinerea is compromised in the sib1 and sib2 mutants but enhanced in SIB1-overexpressing transgenic plants. These results suggest that dual-targeted SIB1 and SIB2 function as activators of WRKY33 in plant defense against necrotrophic pathogens. PMID:21990940

  17. Lycopene activates antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers.

    PubMed

    Sahin, K; Orhan, C; Tuzcu, M; Sahin, N; Hayirli, A; Bilgili, S; Kucuk, O

    2016-05-01

    This study was conducted to evaluate the effects of dietary lycopene supplementation on growth performance, antioxidant status, and muscle nuclear transcription factor [Kelch like-ECH-associated protein 1 (Keap1) and (erythroid-derived 2)-like 2 (Nrf2)] expressions in broiler chickens exposed to heat stress (HS). A total of 180 one-day-old male broiler chicks (Ross 308) were assigned randomly to one of 2×3 factorially arranged treatments: two housing temperatures (22°C for 24 h/d; thermoneutral, TN or 34°C for 8 h/d HS) and three dietary lycopene levels (0, 200, or 400 mg/kg). Each treatment consisted of three replicates of 10 birds. Birds were reared to 42 d of age. Heat stress caused reductions in feed intake and weight gain by 12.2 and 20.7% and increased feed efficiency by 10.8% (P<0.0001 for all). Increasing dietary lycopene level improved performance in both environments. Birds reared under the HS environment had lower serum and muscle lycopene concentration (0.34 vs. 0.50 μg/mL and 2.80 vs. 2.13 μg/g), activities of superoxide dismutase (151 vs. 126 U/mL and 131 vs. 155 U/mg protein), glutathione peroxidase (184 vs. 154 U/mL and 1.39 vs. 1.74 U/mg protein), and higher malondialdehyde (MDA) concentration (0.53 vs. 0.83 μg/mL and 0.78 vs. 0.45 μg/ mg protein) than birds reared under the TN environment. Changes in levels of lycopene and MDA and activities of enzymes in serum and muscle varied by the environmental temperature as dietary lycopene level increased. Moreover, increasing dietary lycopene level suppressed muscle Keap1 expression and enhanced muscle Nrf2 expression, which had increased by 150% and decreased by 40%, respectively in response to HS. In conclusion, lycopene supplementation alleviates adverse effects of HS on performance through modulating expressions of stress-related nuclear transcription factors.

  18. Transcription factors in alkaloid biosynthesis.

    PubMed

    Yamada, Yasuyuki; Sato, Fumihiko

    2013-01-01

    Higher plants produce a large variety of low-molecular weight secondary compounds. Among them, nitrogen-containing alkaloids are the most biologically active and are often used pharmaceutically. Whereas alkaloid chemistry has been intensively investigated, alkaloid biosynthesis, including the relevant biosynthetic enzymes, genes and their regulation, and especially transcription factors, is largely unknown, as only a limited number of plant species produce certain types of alkaloids and they are difficult to study. Recently, however, several groups have succeeded in isolating the transcription factors that are involved in the biosynthesis of several types of alkaloids, including bHLH, ERF, and WRKY. Most of them show Jasmonate (JA) responsiveness, which suggests that the JA signaling cascade plays an important role in alkaloid biosynthesis. Here, we summarize the types and functions of transcription factors that have been isolated in alkaloid biosynthesis, and characterize their similarities and differences compared to those in other secondary metabolite pathways, such as phenylpropanoid and terpenoid biosyntheses. The evolution of this biosynthetic pathway and regulatory network, as well as the application of these transcription factors to metabolic engineering, is discussed.

  19. Evaluating Transcription Factor Activity Changes by Scoring Unexplained Target Genes in Expression Data

    PubMed Central

    Berchtold, Evi; Csaba, Gergely; Zimmer, Ralf

    2016-01-01

    Several methods predict activity changes of transcription factors (TFs) from a given regulatory network and measured expression data. But available gene regulatory networks are incomplete and contain many condition-dependent regulations that are not relevant for the specific expression measurement. It is not known which combination of active TFs is needed to cause a change in the expression of a target gene. A method to systematically evaluate the inferred activity changes is missing. We present such an evaluation strategy that indicates for how many target genes the observed expression changes can be explained by a given set of active TFs. To overcome the problem that the exact combination of active TFs needed to activate a gene is typically not known, we assume a gene to be explained if there exists any combination for which the predicted active TFs can possibly explain the observed change of the gene. We introduce the i-score (inconsistency score), which quantifies how many genes could not be explained by the set of activity changes of TFs. We observe that, even for these minimal requirements, published methods yield many unexplained target genes, i.e. large i-scores. This holds for all methods and all expression datasets we evaluated. We provide new optimization methods to calculate the best possible (minimal) i-score given the network and measured expression data. The evaluation of this optimized i-score on a large data compendium yields many unexplained target genes for almost every case. This indicates that currently available regulatory networks are still far from being complete. Both the presented Act-SAT and Act-A* methods produce optimal sets of TF activity changes, which can be used to investigate the difficult interplay of expression and network data. A web server and a command line tool to calculate our i-score and to find the active TFs associated with the minimal i-score is available from https://services.bio.ifi.lmu.de/i-score. PMID:27723775

  20. mTOR-dependent activation of the transcription factor TIF-IA links rRNA synthesis to nutrient availability.

    PubMed

    Mayer, Christine; Zhao, Jian; Yuan, Xuejun; Grummt, Ingrid

    2004-02-15

    In cycling cells, transcription of ribosomal RNA genes by RNA polymerase I (Pol I) is tightly coordinated with cell growth. Here, we show that the mammalian target of rapamycin (mTOR) regulates Pol I transcription by modulating the activity of TIF-IA, a regulatory factor that senses nutrient and growth-factor availability. Inhibition of mTOR signaling by rapamycin inactivates TIF-IA and impairs transcription-initiation complex formation. Moreover, rapamycin treatment leads to translocation of TIF-IA into the cytoplasm. Rapamycin-mediated inactivation of TIF-IA is caused by hypophosphorylation of Se 44 (S44) and hyperphosphorylation of Se 199 (S199). Phosphorylation at these sites affects TIF-IA activity in opposite ways, for example, phosphorylation of S44 activates and S199 inactivates TIF-IA. The results identify a new target formTOR-signaling pathways and elucidate the molecular mechanism underlying mTOR-dependent regulation of RNA synthesis.

  1. Expression of the RelB transcription factor correlates with the activation of human dendritic cells

    PubMed Central

    Clark, G J; Gunningham, S; Troy, A; Vuckovic, S; Hart, D N J

    1999-01-01

    The RelB gene product is a member of the nuclear factor (NF)-κB family of transcription factors. It has been identified recently within mouse antigen-presenting cells and human monocyte-derived dendritic cells (DC). Disruption of the mouse RelB gene is accompanied, amongst other phenotypes, by abnormalities in the antigen-presenting cell lineages. In order to define RelB expression during human DC differentiation, we have analysed RelB mRNA by reverse transcriptase–polymerase chain reaction and RelB protein by intracellular staining in CD34+ precursors and different types of DC preparations. RelB mRNA was not detected in CD34+ precursor populations. Fresh blood DC (lineage−human leucocyte antigen-DR+ (lin−HLA-DR+)) lacked RelB mRNA and cytoplasmic RelB protein but a period of in vitro culture induced RelB expression in blood DC. Purified Langerhans’ cells (LC) (CD1a+ HLA-DR+) failed to express RelB mRNA. Immunocytochemical staining identified RelB protein in human skin epithelium. RelB protein was expressed in a very few CD1a+, CD83+ or CMRF-44+ dermal DC but was not present in CD1a+ LC. Tonsil DC (lin−HLA-DR+ CMRF-44+) were positive for RelB mRNA and RelB protein. Intestinal DC (HLA-DR+) also lacked immunoreactive RelB protein. The majority of interdigitating CD83+, CMRF-44+, CMRF-56+ or p55+ DC located in paracortical T-lymphocyte areas of lymph node and tonsil contained RelB protein. The expression of RelB mRNA and RelB protein correlates with the activated phase of blood DC and the postmigration cell (activated) stage of tissue DC development. PMID:10540217

  2. Simple enzymatic assays for the in vitro motor activity of transcription termination factor Rho from Escherichia coli.

    PubMed

    Boudvillain, Marc; Walmacq, Céline; Schwartz, Annie; Jacquinot, Frédérique

    2010-01-01

    The transcription termination factor Rho from Escherichia coli is a ring-shaped homo-hexameric protein that preferentially interacts with naked cytosine-rich Rut (Rho utilization) regions of nascent RNA transcripts. Once bound to the RNA chain, Rho uses ATP as an energy source to produce mechanical work and disruptive forces that ultimately lead to the dissociation of the ternary transcription complex. Although transcription termination assays have been useful to study Rho activity in various experimental contexts, they do not report directly on Rho mechanisms and kinetics. Here, we describe complementary ATP-dependent RNA-DNA helicase and streptavidin displacement assays that can be used to monitor in vitro Rho's motor activity in a more direct and quantitative manner.

  3. Activation of the Ig Iα1 promoter by the transcription factor Ets-1 triggers Ig Iα1-Cα1 germline transcription in epithelial cancer cells.

    PubMed

    Duan, Zhi; Zheng, Hui; Xu, San; Jiang, Yiqun; Liu, Haidan; Li, Ming; Hu, Duosha; Li, Wei; Bode, Ann M; Dong, Zigang; Cao, Ya

    2014-03-01

    Immunoglobulins (Igs) are known to be synthesized and secreted only by B lymphocytes. Class switch recombination (CSR) is a key event that enables B cells to express Igs, and one of the crucial steps for CSR initiation is the germline transcription of Ig genes. Surprisingly, recent studies have demonstrated that the Ig genes are also expressed in some epithelial cancer cells; however, the mechanisms underlying how cancer cells initiate CSR and express Igs are still unknown. In this study, we confirmed that the Ig Iα1 promoter in cancer cell lines was activated by the Ets-1 transcription factor, and the activity of the Ig Iα1 promoter and Ig Iα1-Cα1 germline transcription were attenuated after knockdown of Ets-1 by specific small interfering RNAs (siRNA). Furthermore, the expression of Ets-1 and Igα heavy chain in cancer cells was dose dependently upregulated by TGF-β1. These results indicate that activation of the Ig Iα1 promoter by the transcription factor Ets-1 is a critical pathway and provides a novel mechanism for Ig expression in non-B cell cancers.

  4. Band-pass processing in a GPCR signaling pathway selects for NFAT transcription factor activation.

    PubMed

    Sumit, M; Neubig, R R; Takayama, S; Linderman, J J

    2015-11-01

    Many biological processes are rhythmic and proper timing is increasingly appreciated as being critical for development and maintenance of physiological functions. To understand how temporal modulation of an input signal influences downstream responses, we employ microfluidic pulsatile stimulation of a G-protein coupled receptor, the muscarinic M3 receptor, in single cells with simultaneous real-time imaging of both intracellular calcium and NFAT nuclear localization. Interestingly, we find that reduced stimulation with pulses of ligand can give more efficient transcription factor activation, if stimuli are timed appropriately. Our experiments and computational analyses show that M3 receptor-induced calcium oscillations form a low pass filter while calcium-induced NFAT translocation forms a high pass filter. The combination acts as a band-pass filter optimized for intermediate frequencies of stimulation. We demonstrate that receptor desensitization and NFAT translocation rates determine critical features of the band-pass filter and that the band-pass may be shifted for different receptors or NFAT dynamics. As an example, we show that the two NFAT isoforms (NFAT4 and NFAT1) have shifted band-pass windows for the same receptor. While we focus specifically on the M3 muscarinic receptor and NFAT translocation, band-pass processing is expected to be a general theme that applies to multiple signaling pathways.

  5. The ETS-5 transcription factor regulates activity states in Caenorhabditis elegans by controlling satiety

    PubMed Central

    Juozaityte, Vaida; Pladevall-Morera, David; Podolska, Agnieszka; Nørgaard, Steffen; Pocock, Roger

    2017-01-01

    Animal behavior is shaped through interplay among genes, the environment, and previous experience. As in mammals, satiety signals induce quiescence in Caenorhabditis elegans. Here we report that the C. elegans transcription factor ETS-5, an ortholog of mammalian FEV/Pet1, controls satiety-induced quiescence. Nutritional status has a major influence on C. elegans behavior. When foraging, food availability controls behavioral state switching between active (roaming) and sedentary (dwelling) states; however, when provided with high-quality food, C. elegans become sated and enter quiescence. We show that ETS-5 acts to promote roaming and inhibit quiescence by setting the internal “satiety quotient” through fat regulation. Acting from the ASG and BAG sensory neurons, we show that ETS-5 functions in a complex network with serotonergic and neuropeptide signaling pathways to control food-regulated behavioral state switching. Taken together, our results identify a neuronal mechanism for controlling intestinal fat stores and organismal behavioral states in C. elegans, and establish a paradigm for the elucidation of obesity-relevant mechanisms. PMID:28193866

  6. The phytoestrogen genistein modulates lysosomal metabolism and transcription factor EB (TFEB) activation.

    PubMed

    Moskot, Marta; Montefusco, Sandro; Jakóbkiewicz-Banecka, Joanna; Mozolewski, Paweł; Węgrzyn, Alicja; Di Bernardo, Diego; Węgrzyn, Grzegorz; Medina, Diego L; Ballabio, Andrea; Gabig-Cimińska, Magdalena

    2014-06-13

    Genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) has been previously proposed as a potential drug for use in substrate reduction therapy for mucopolysaccharidoses, a group of inherited metabolic diseases caused by mutations leading to inefficient degradation of glycosaminoglycans (GAGs) in lysosomes. It was demonstrated that this isoflavone can cross the blood-brain barrier, making it an especially desirable potential drug for the treatment of neurological symptoms present in most lysosomal storage diseases. So far, no comprehensive genomic analyses have been performed to elucidate the molecular mechanisms underlying the effect elicited by genistein. Therefore, the aim of this work was to identify the genistein-modulated gene network regulating GAG biosynthesis and degradation, taking into consideration the entire lysosomal metabolism. Our analyses identified over 60 genes with known roles in lysosomal biogenesis and/or function whose expression was enhanced by genistein. Moreover, 19 genes whose products are involved in both GAG synthesis and degradation pathways were found to be remarkably differentially regulated by genistein treatment. We found a regulatory network linking genistein-mediated control of transcription factor EB (TFEB) gene expression, TFEB nuclear translocation, and activation of TFEB-dependent lysosome biogenesis to lysosomal metabolism. Our data indicate that the molecular mechanism of genistein action involves not only impairment of GAG synthesis but more importantly lysosomal enhancement via TFEB. These findings contribute to explaining the beneficial effects of genistein in lysosomal storage diseases as well as envisage new therapeutic approaches to treat these devastating diseases.

  7. Autophagy Activation by Transcription Factor EB (TFEB) in Striatum of HDQ175/Q7 Mice

    PubMed Central

    Vodicka, Petr; Chase, Kathryn; Iuliano, Maria; Tousley, Adelaide; Valentine, Dana T.; Sapp, Ellen; Kegel-Gleason, Kimberly B.; Sena-Esteves, Miguel; Aronin, Neil; DiFiglia, Marian

    2016-01-01

    Background: Mutant huntingtin (mHTT) is encoded by the Huntington’s disease (HD) gene and its accumulation in the brain contributes to HD pathogenesis. Reducing mHTT levels through activation of the autophagosome-lysosomal pathway may have therapeutic benefit. Transcription factor EB (TFEB) regulates lysosome biogenesis and autophagy. Objective: To examine if increasing TFEB protein levels in HD mouse striatum induces autophagy and influences mHTT levels. Methods: We introduced cDNA encoding TFEB with an HA tag (TFEB-HA) under the control of neuron specific synapsin 1 promoter into the striatum of 3 month old HDQ175/Q7 mice using adeno-associated virus AAV2/9. The levels of exogenous TFEB were analyzed using qPCR and Western blot. Proteins involved in autophagy, levels of huntingtin, and striatal-enriched proteins were examined using biochemical and/or immunohistochemical methods. Results: In HD mice expressing TFEB-HA, HA immunoreactivity distributed throughout the striatum in neuronal cell bodies and processes and preferentially in neuronal nuclei and overlapped with a loss of DARPP32 immunoreactivity. TFEB-HA mRNA and protein were detected in striatal lysates. There were increased levels of proteins involved with autophagosome/lysosome activity including LAMP-2A, LC3II, and cathepsin D and reduced levels of mutant HTT and the striatal enriched proteins DARPP32 and PDE10A. Compared to WT mice, HDQ175/Q7 mice had elevated levels of the ER stress protein GRP78/BiP and with TFEB-HA expression, increased levels of the astrocyte marker GFAP and pro-caspase 3. Conclusion: These results suggest that TFEB expression in the striatum of HDQ175/Q7 mice stimulates autophagy and lysosome activity, and lowers mHTT, but may also increase a neuronal stress response. PMID:27689619

  8. Transcription factors GAF and HSF act at distinct regulatory steps to modulate stress-induced gene activation.

    PubMed

    Duarte, Fabiana M; Fuda, Nicholas J; Mahat, Dig B; Core, Leighton J; Guertin, Michael J; Lis, John T

    2016-08-01

    The coordinated regulation of gene expression at the transcriptional level is fundamental to development and homeostasis. Inducible systems are invaluable when studying transcription because the regulatory process can be triggered instantaneously, allowing the tracking of ordered mechanistic events. Here, we use precision run-on sequencing (PRO-seq) to examine the genome-wide heat shock (HS) response in Drosophila and the function of two key transcription factors on the immediate transcription activation or repression of all genes regulated by HS. We identify the primary HS response genes and the rate-limiting steps in the transcription cycle that GAGA-associated factor (GAF) and HS factor (HSF) regulate. We demonstrate that GAF acts upstream of promoter-proximally paused RNA polymerase II (Pol II) formation (likely at the step of chromatin opening) and that GAF-facilitated Pol II pausing is critical for HS activation. In contrast, HSF is dispensable for establishing or maintaining Pol II pausing but is critical for the release of paused Pol II into the gene body at a subset of highly activated genes. Additionally, HSF has no detectable role in the rapid HS repression of thousands of genes.

  9. Induction of the DNA-binding and transcriptional activities of heat shock factor 1 is uncoupled in Xenopus oocytes.

    PubMed

    Bharadwaj, S; Hnatov, A; Ali, A; Ovsenek, N

    1998-03-12

    The DNA-binding and transcriptional activities of the heat shock transcription factor 1 (HSF1) are repressed under normal conditions and rapidly upregulated by heat stress. Here, we tested for the ability of various stress agents to activate HSF1 in the Xenopus oocyte model system. The HSE-binding activity of HSF1 was induced by a number of chemical stresses including cadmium, aluminum, iron, mercury, arsenite, ethanol, methanol, and salicylate. HSE-binding was not induced by several stresses known to induce the synthesis of hsps in other cell types in different organisms including zinc, copper, cobalt, manganese, recovery from anoxia, UV-irradiation, and increased pH. The inability of several known inducers of the stress response to activate the HSE-binding ability of HSF1 suggests that certain stress activation pathways may be absent or inactive in oocytes. The transcriptional activity of oocyte HSF1 was induced by heat, cadmium, and arsenite, but many of the agents that induced HSE-binding failed to stimulate HSF1-mediated transcription. The apparent uncoupling of inducible HSE-binding and transcriptional activities of HSF1 under a variety of stress regimes indicates that these events are regulated by independent mechanisms in the oocyte.

  10. Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3.

    PubMed Central

    Wang, X W; Forrester, K; Yeh, H; Feitelson, M A; Gu, J R; Harris, C C

    1994-01-01

    Chronic active hepatitis caused by infection with hepatitis B virus, a DNA virus, is a major risk factor for human hepatocellular carcinoma. Since the oncogenicity of several DNA viruses is dependent on the interaction of their viral oncoproteins with cellular tumor-suppressor gene products, we investigated the interaction between hepatitis B virus X protein (HBX) and human wild-type p53 protein. HBX complexes with the wild-type p53 protein and inhibits its sequence-specific DNA binding in vitro. HBX expression also inhibits p53-mediated transcriptional activation in vivo and the in vitro association of p53 and ERCC3, a general transcription factor involved in nucleotide excision repair. Therefore, HBX may affect a wide range of p53 functions and contribute to the molecular pathogenesis of human hepatocellular carcinoma. Images PMID:8134379

  11. Transcription Factors Bind Thousands of Active and InactiveRegions in the Drosophila Blastoderm

    SciTech Connect

    Li, Xiao-Yong; MacArthur, Stewart; Bourgon, Richard; Nix, David; Pollard, Daniel A.; Iyer, Venky N.; Hechmer, Aaron; Simirenko, Lisa; Stapleton, Mark; Luengo Hendriks, Cris L.; Chu, Hou Cheng; Ogawa, Nobuo; Inwood, William; Sementchenko, Victor; Beaton, Amy; Weiszmann, Richard; Celniker, Susan E.; Knowles, David W.; Gingeras, Tom; Speed, Terence P.; Eisen, Michael B.; Biggin, Mark D.

    2008-01-10

    Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. Here, we use whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior-posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched in bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over forty well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs) transcribed in the blastoderm, as well as all major zygotically transcribed dorsal-ventral patterning genes, whose expression we show to be quantitatively modulated by anterior-posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly-bound regions are not involved in early

  12. Simulated binding of transcription factors to active and inactive regions folds human chromosomes into loops, rosettes and topological domains.

    PubMed

    Brackley, Chris A; Johnson, James; Kelly, Steven; Cook, Peter R; Marenduzzo, Davide

    2016-05-05

    Biophysicists are modeling conformations of interphase chromosomes, often basing the strengths of interactions between segments distant on the genetic map on contact frequencies determined experimentally. Here, instead, we develop a fitting-free, minimal model: bivalent or multivalent red and green 'transcription factors' bind to cognate sites in strings of beads ('chromatin') to form molecular bridges stabilizing loops. In the absence of additional explicit forces, molecular dynamic simulations reveal that bound factors spontaneously cluster-red with red, green with green, but rarely red with green-to give structures reminiscent of transcription factories. Binding of just two transcription factors (or proteins) to active and inactive regions of human chromosomes yields rosettes, topological domains and contact maps much like those seen experimentally. This emergent 'bridging-induced attraction' proves to be a robust, simple and generic force able to organize interphase chromosomes at all scales.

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

    PubMed

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

    2016-02-01

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

  14. Cartography of hematopoietic stem cell commitment dependent upon a reporter for transcription factor activation.

    PubMed

    Akashi, Koichi

    2007-06-01

    A hierarchical hematopoietic developmental tree has been proposed based on the result of prospective purification of lineage-restricted progenitors. For more detailed mapping for hematopoietic stem cell (HSC) commitment, we tracked the expression of PU.1, a major granulocyte/monocyte (GM)- and lymphoid-related transcription factor, from the HSC to the myelolymphoid progenitor stages by using a mouse line harboring a knockin reporter for PU.1. This approach enabled us to find a new progenitor population committed to GM and lymphoid lineages within the HSC fraction. This result suggests that there should be another developmental pathway independent of the conventional one with myeloid versus lymphoid bifurcation, represented by common myeloid progenitors and common lymphoid progenitors, respectively. The utilization of the transcription factor expression as a functional marker might be useful to obtain cartography of the hematopoietic development at a higher resolution.

  15. Characterization of a novel transcriptionally active domain in the transforming growth factor beta-regulated Smad3 protein.

    PubMed

    Prokova, Vassiliki; Mavridou, Sofia; Papakosta, Paraskevi; Kardassis, Dimitris

    2005-01-01

    Transforming growth factor beta (TGFbeta) regulates transcriptional responses via activation of cytoplasmic effector proteins termed Smads. Following their phosphorylation by the type I TGFbeta receptor, Smads form oligomers and translocate to the nucleus where they activate the transcription of TGFbeta target genes in cooperation with nuclear cofactors and coactivators. In the present study, we have undertaken a deletion analysis of human Smad3 protein in order to characterize domains that are essential for transcriptional activation in mammalian cells. With this analysis, we showed that Smad3 contains two domains with transcriptional activation function: the MH2 domain and a second middle domain that includes the linker region and the first two beta strands of the MH2 domain. Using a protein-protein interaction assay based on biotinylation in vivo, we were able to show that a Smad3 protein bearing an internal deletion in the middle transactivation domain is characterized by normal oligomerization and receptor activation properties. However, this mutant has reduced transactivation capacity on synthetic or natural promoters and is unable to interact physically and functionally with the histone acetyltransferase p/CAF. The loss of interaction with p/CAF or other coactivators could account, at least in part, for the reduced transactivation capacity of this Smad3 mutant. Our data support an essential role of the previously uncharacterized middle region of Smad3 for nuclear functions, such as transcriptional activation and interaction with coactivators.

  16. Functional diversification of FD transcription factors in rice, components of florigen activation complexes.

    PubMed

    Tsuji, Hiroyuki; Nakamura, Hiroyuki; Taoka, Ken-ichiro; Shimamoto, Ko

    2013-03-01

    Florigen, a protein encoded by the FLOWERING LOCUS T (FT) in Arabidopsis and Heading date 3a (Hd3a) in rice, is the universal flowering hormone in plants. Florigen is transported from leaves to the shoot apical meristem and initiates floral evocation. In shoot apical cells, conserved cytoplasmic 14-3-3 proteins act as florigen receptors. A hexameric florigen activation complex (FAC) composed of Hd3a, 14-3-3 proteins, and OsFD1, a transcription factor, activates OsMADS15, a rice homolog of Arabidopsis APETALA1, leading to flowering. Because FD is a key component of the FAC, we characterized the FD gene family and their functions. Phylogenetic analysis of FD genes indicated that this family is divided into two groups: (i) canonical FD genes that are conserved among eudicots and non-Poaceae monocots; and (ii) Poaceae-specific FD genes that are organized into three subgroups: Poaceae FD1, FD2 and FD3. The Poaceae FD1 group shares a small sequence motif, T(A/V)LSLNS, with FDs of eudicots and non-Poaceae monocots. Overexpression of OsFD2, a member of the Poaceae FD2 group, produced smaller leaves with shorter plastochrons, suggesting that OsFD2 controls leaf development. In vivo subcellular localization of Hd3a, 14-3-3 and OsFD2 suggested that in contrast to OsFD1, OsFD2 is restricted to the cytoplasm through its interaction with the cytoplasmic 14-3-3 proteins, and interaction of Hd3a with 14-3-3 facilitates nuclear translocation of the FAC containing OsFD2. These results suggest that FD function has diverged between OsFD1 and OsFD2, but formation of a FAC is essential for their function.

  17. Functional Diversification of FD Transcription Factors in Rice, Components of Florigen Activation Complexes

    PubMed Central

    Tsuji, Hiroyuki; Nakamura, Hiroyuki; Taoka, Ken-ichiro; Shimamoto, Ko

    2013-01-01

    Florigen, a protein encoded by the FLOWERING LOCUS T (FT) in Arabidopsis and Heading date 3a (Hd3a) in rice, is the universal flowering hormone in plants. Florigen is transported from leaves to the shoot apical meristem and initiates floral evocation. In shoot apical cells, conserved cytoplasmic 14-3-3 proteins act as florigen receptors. A hexameric florigen activation complex (FAC) composed of Hd3a, 14-3-3 proteins, and OsFD1, a transcription factor, activates OsMADS15, a rice homolog of Arabidopsis APETALA1, leading to flowering. Because FD is a key component of the FAC, we characterized the FD gene family and their functions. Phylogenetic analysis of FD genes indicated that this family is divided into two groups: (i) canonical FD genes that are conserved among eudicots and non-Poaceae monocots; and (ii) Poaceae-specific FD genes that are organized into three subgroups: Poaceae FD1, FD2 and FD3. The Poaceae FD1 group shares a small sequence motif, T(A/V)LSLNS, with FDs of eudicots and non-Poaceae monocots. Overexpression of OsFD2, a member of the Poaceae FD2 group, produced smaller leaves with shorter plastochrons, suggesting that OsFD2 controls leaf development. In vivo subcellular localization of Hd3a, 14-3-3 and OsFD2 suggested that in contrast to OsFD1, OsFD2 is restricted to the cytoplasm through its interaction with the cytoplasmic 14-3-3 proteins, and interaction of Hd3a with 14-3-3 facilitates nuclear translocation of the FAC containing OsFD2. These results suggest that FD function has diverged between OsFD1 and OsFD2, but formation of a FAC is essential for their function. PMID:23324168

  18. Transforming growth factor β signaling upregulates the expression of human GDP-fucose transporter by activating transcription factor Sp1.

    PubMed

    Xu, Yu-Xin; Ma, Anna; Liu, Li

    2013-01-01

    GDP-fucose transporter plays a crucial role in fucosylation of glycoproteins by providing activated fucose donor, GDP-fucose, for fucosyltransferases in the lumen of the Golgi apparatus. Fucose-containing glycans are involved in many biological processes, which are essential for growth and development. Mutations in the GDP-fucose transporter gene cause leukocyte adhesion deficiency syndrome II, a disease characterized by slow growth, mental retardation and immunodeficiency. However, no information is available regarding its transcriptional regulation. Here, by using human cells, we show that TGF-β1 specifically induces the GDP-fucose transporter expression, but not other transporters tested such as CMP-sialic acid transporter, suggesting a diversity of regulatory pathways for the expression of these transporters. The regulatory elements that are responsive to the TGF-β1 stimulation are present in the region between bp -330 and -268 in the GDP-fucose transporter promoter. We found that this region contains two identical octamer GC-rich motifs (GGGGCGTG) that were demonstrated to be essential for the transporter expression. We also show that the transcription factor Sp1 specifically binds to the GC-rich motifs in vitro and Sp1 coupled with phospho-Smad2 is associated with the promoter region covering the Sp1-binding motifs in vivo using chromatin immunoprecipitation (ChIP) assays. In addition, we further confirmed that Sp1 is essential for the GDP-fucose transporter expression stimulated by TGF-β1 using a luciferase reporter system. These results highlight the role of TGF-β signaling in regulation of the GDP-fucose transporter expression via activating Sp1. This is the first transcriptional study for any nucleotide sugar transporters that have been identified so far. Notably, TGF-β1 receptor itself is known to be modified by fucosylation. Given the essential role of GDP-fucose transporter in fucosylation, the finding that TGF-β1 stimulates the expression of

  19. Activator and repressor functions of the Mot3 transcription factor in the osmostress response of Saccharomyces cerevisiae.

    PubMed

    Martínez-Montañés, Fernando; Rienzo, Alessandro; Poveda-Huertes, Daniel; Pascual-Ahuir, Amparo; Proft, Markus

    2013-05-01

    Mot3 and Rox1 are transcriptional repressors of hypoxic genes. Both factors recently have been found to be involved in the adaptive response to hyperosmotic stress, with an important function in the adjustment of ergosterol biosynthesis. Here, we determine the gene expression profile of a mot3 rox1 double mutant under acute osmostress at the genomic scale in order to identify the target genes affected by both transcription factors upon stress. Unexpectedly, we find a specific subgroup of osmostress-inducible genes to be under positive control of Mot3. These Mot3-activated stress genes also depend on the general stress activators Msn2 and Msn4. We confirm that both Mot3 and Msn4 bind directly to some promoter regions of this gene group. Furthermore, osmostress-induced binding of the Msn2 and Msn4 factors to these target promoters is severely affected by the loss of Mot3 function. The genes repressed by Mot3 and Rox1 preferentially encode proteins of the cell wall and plasma membrane. Cell conjugation was the most significantly enriched biological process which was negatively regulated by both factors and by osmotic stress. The mating response was repressed by salt stress dependent on Mot3 and Rox1 function. Taking our findings together, the Mot3 transcriptional regulator has unanticipated diverse functions in the cellular adjustment to osmotic stress, including transcriptional activation and modulation of mating efficiency.

  20. Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility

    SciTech Connect

    Dogan, Nergiz; Wu, Weisheng; Morrissey, Christapher S.; Chen, Kuan-Bei; Stonestrom, Aaron; Long, Maria; Keller, Cheryl A.; Cheng, Yong; Jain, Deepti; Visel, Axel; Pennacchio, Len A.; Weiss, Mitchell J.; Blobel, Gerd A.; Hardison, Ross C.

    2015-04-23

    Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the results of these and other studies. Results: TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included. Conclusions: Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.

  1. Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility

    DOE PAGES

    Dogan, Nergiz; Wu, Weisheng; Morrissey, Christapher S.; ...

    2015-04-23

    Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the resultsmore » of these and other studies. Results: TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included. Conclusions: Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.« less

  2. Nuclear respiratory factors 1 and 2 utilize similar glutamine-containing clusters of hydrophobic residues to activate transcription.

    PubMed Central

    Gugneja, S; Virbasius, C M; Scarpulla, R C

    1996-01-01

    Nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) are ubiquitous transcription factors that have been implicated in the control of nuclear genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication. Recently, both factors have been found to be major transcriptional determinants for a subset of these genes that define a class of simple promoters involved in respiratory chain expression. Here, functional domains required for transactivation by NRF-1 have been defined. An atypical nuclear localization signal resides in a conserved amino-terminal region adjacent to the DNA binding domain and consists of functionally redundant clusters of basic residues. A second domain in the carboxy-terminal half of the molecule is necessary for transcriptional activation. The activation domains of both NRF-1 and NRF-2 were extensively characterized by both deletion and alanine substitution mutagenesis. The results show that these domains do not fall into known classes defined by a preponderance of amino acid residues, including glutamines, prolines, or isoleucines, as found in other eukaryotic activators. Rather, in both factors, a series of tandemly arranged clusters of hydrophobic amino acids were required for activation. Although all of the functional clusters contain glutamines, the glutamines differ from the hydrophobic residues in that they are inconsequential for activation. Unlike the NRF-2 domain, which contains its essential hydrophobic motifs within 40 residues, the NRF-1 domain spans about 40% of the molecule and appears to have a bipartite structure. The findings indicate that NRF-1 and NRF-2 utilize similar hydrophobic structural motifs for activating transcription. PMID:8816484

  3. A Global Genomic and Genetic Strategy to Identify, Validate and Use Gene Signatures of Xenobiotic-Responsive Transcription Factors in Prediction of Pathway Activation in the Mouse Liver

    EPA Science Inventory

    Many drugs and environmentally-relevant chemicals activate xenobiotic-responsive transcription factors. Identification of target genes of these factors would be useful in predicting pathway activation in in vitro chemical screening as well as their involvement in disease states. ...

  4. Activation of transcription factors in human bronchial epithelial cells exposed to aqueous extracts of mainstream cigarette smoke in vitro.

    PubMed

    Sekine, Takashi; Hirata, Tadashi; Mine, Toshiki; Fukano, Yasuo

    2016-01-01

    This study aimed to identify the most sensitive transcription factor activated by cigarette smoke extract (CSE) and to explore cigarette smoke components that have high biological activities in a cell-base assay. Previously, we found evidence that implicated 10 different transcription factors as having a high biological activity to CSE in vitro, based on the results of a comprehensive gene expression profile. For this study, luciferase reporter assays for each transcription factor were developed in two types of human bronchial epithelial cells: NCI-H292 and BEAS-2B cells. The results demonstrated that the nuclear factor erythroid 2-related factor 2 (NRF2)/anti-oxidant response element (ARE) pathway was the most sensitive in response to CSE. Consistently, hemo oxygenase-1 (HO-1), a downstream target gene of NRF2, was effectively up-regulated in BEAS-2B cells exposed to CSE. Moreover, among 1395 cigarette smoke components, naphthoquinones including 9,10-phenaotrenquinone, quinones, benzenediols and α, β-unsaturated carbonyls, were identified as major smoke components that contribute to activating the NRF2/ARE pathway, as indicated by the ARE-reporter assay in BEAS-2B cells. Taken together, NRF2 appears to be a key molecule in the CSE-induced cellular response, and the employed methodology is helpful for the analysis of molecular and cellular effects by CSE.

  5. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

    PubMed

    Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

    2014-04-01

    The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

  6. Tcf1 and Lef1 transcription factors establish CD8(+) T cell identity through intrinsic HDAC activity.

    PubMed

    Xing, Shaojun; Li, Fengyin; Zeng, Zhouhao; Zhao, Yunjie; Yu, Shuyang; Shan, Qiang; Li, Yalan; Phillips, Farrah C; Maina, Peterson K; Qi, Hank H; Liu, Chengyu; Zhu, Jun; Pope, R Marshall; Musselman, Catherine A; Zeng, Chen; Peng, Weiqun; Xue, Hai-Hui

    2016-06-01

    The CD4(+) and CD8(+) T cell dichotomy is essential for effective cellular immunity. How individual T cell identity is established remains poorly understood. Here we show that the high-mobility group (HMG) transcription factors Tcf1 and Lef1 are essential for repressing CD4(+) lineage-associated genes including Cd4, Foxp3 and Rorc in CD8(+) T cells. Tcf1- and Lef1-deficient CD8(+) T cells exhibit histone hyperacetylation, which can be ascribed to intrinsic histone deacetylase (HDAC) activity in Tcf1 and Lef1. Mutation of five conserved amino acids in the Tcf1 HDAC domain diminishes HDAC activity and the ability to suppress CD4(+) lineage genes in CD8(+) T cells. These findings reveal that sequence-specific transcription factors can utilize intrinsic HDAC activity to guard cell identity by repressing lineage-inappropriate genes.

  7. Controlling the motor activity of a transcription-repair coupling factor: autoinhibition and the role of RNA polymerase.

    PubMed

    Smith, Abigail J; Szczelkun, Mark D; Savery, Nigel J

    2007-01-01

    Motor proteins that couple ATP hydrolysis to movement along nucleic acids play a variety of essential roles in DNA metabolism. Often these enzymes function as components of macromolecular complexes, and DNA translocation by the motor protein drives movement of other components of the complex. In order to understand how the activity of motor proteins is regulated within multi-protein complexes we have studied the bacterial transcription-repair coupling factor, Mfd, which is a helicase superfamily 2 member that binds to RNA polymerase (RNAP) and removes stalled transcription complexes from DNA. Using an oligonucleotide displacement assay that monitors protein movement on double-stranded DNA we show that Mfd has little motor activity in isolation, but exhibits efficient oligonucleotide displacement activity when bound to a stalled transcription complex. Deletion of the C-terminal domain of Mfd increases the ATPase activity of the protein and allows efficient oligo-displacement in the absence of RNAP. Our results suggest that an autoinhibitory domain ensures the motor activity of Mfd is only functional within the correct macromolecular context: recruitment of Mfd to a stalled transcription complex relieves the autoinhibition and unmasks the motor activity.

  8. ATP-dependent motor activity of the transcription termination factor Rho from Mycobacterium tuberculosis

    PubMed Central

    D'Heygère, François; Schwartz, Annie; Coste, Franck; Castaing, Bertrand; Boudvillain, Marc

    2015-01-01

    The bacterial transcription termination factor Rho—a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity—is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate transcriptional complexes is not conserved throughout the bacterial kingdom. Here, however, we demonstrate that MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or transcription elongation complexes. Moreover, we show that idiosyncratic features of the MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal ‘RNA binding’ domain and by a non-canonical R-loop residue in its C-terminal ‘motor’ domain. We also show that the ‘motor’ domain of MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors. PMID:25999346

  9. Nuclear factors that bind two regions important to transcriptional activity of the simian immunodeficiency virus long terminal repeat.

    PubMed Central

    Winandy, S; Renjifo, B; Li, Y; Hopkins, N

    1992-01-01

    Previous studies identified two regions in the U3 region of a molecular clone of simian immunodeficiency virus, SIVmac142, that are important to transcriptional activity under conditions of induction as well as basal-level expression (B. Renjifo, N. A. Speck, S. Winandy, N. Hopkins, and Y. Li, J. Virol. 64:3130-3134, 1990). One region includes the NF-kappa B binding site, while the other lies just 5' of this site between nucleotides -162 and -114 (the -162 to -114 region). The fact that the NF-kappa B site mutation attenuated transcriptional activity in uninduced T cells and fibroblasts where activated NF-kappa B would not be present suggested that a factor(s) other than NF-kappa B could be acting through this site. In this study, we have identified a factor which binds to a cis element overlapping the NF-kappa B site. This factor, which we call simian factor 3 (SF3), would play a role in regulation under conditions of basal level expression, whereas under conditions of induction, NF-kappa B would act via this region. SF3 may also bind to an element in the -162 to -114 region. In addition, we have identified two other factors that bind the -162 to -114 region. One, which we designated SF1, is a ubiquitous basal factor, and the other, SF2, is a T-cell-predominant phorbol myristate acetate-inducible factor. Through identification of nuclear factors that interact with the U3 region of the SIVmac142 long terminal repeat, we can gain insight into how this virus is transcriptionally regulated under conditions of basal-level expression as well as conditions of T-cell activation. Images PMID:1501272

  10. Release of positive transcription elongation factor b (P-TEFb) from 7SK small nuclear ribonucleoprotein (snRNP) activates hexamethylene bisacetamide-inducible protein (HEXIM1) transcription.

    PubMed

    Liu, Pingyang; Xiang, Yanhui; Fujinaga, Koh; Bartholomeeusen, Koen; Nilson, Kyle A; Price, David H; Peterlin, B Matija

    2014-04-04

    By phosphorylating negative elongation factors and the C-terminal domain of RNA polymerase II (RNAPII), positive transcription elongation factor b (P-TEFb), which is composed of CycT1 or CycT2 and CDK9, activates eukaryotic transcription elongation. In growing cells, it is found in active and inactive forms. In the former, free P-TEFb is a potent transcriptional coactivator. In the latter, it is inhibited by HEXIM1 or HEXIM2 in the 7SK small nuclear ribonucleoprotein (snRNP), which contains, additionally, 7SK snRNA, methyl phosphate-capping enzyme (MePCE), and La-related protein 7 (LARP7). This P-TEFb equilibrium determines the state of growth and proliferation of the cell. In this study, the release of P-TEFb from the 7SK snRNP led to increased synthesis of HEXIM1 but not HEXIM2 in HeLa cells, and this occurred only from an unannotated, proximal promoter. ChIP with sequencing revealed P-TEFb-sensitive poised RNA polymerase II at this proximal but not the previously annotated distal HEXIM1 promoter. Its immediate upstream sequences were fused to luciferase reporters and were found to be responsive to many P-TEFb-releasing compounds. The superelongation complex subunits AF4/FMR2 family member 4 (AFF4) and elongation factor RNA polymerase II 2 (ELL2) were recruited to this proximal promoter after P-TEFb release and were required for its transcriptional effects. Thus, P-TEFb regulates its own equilibrium in cells, most likely to maintain optimal cellular homeostasis.

  11. Histone deacetylase inhibitors decrease Toll-like receptor-mediated activation of proinflammatory gene expression by impairing transcription factor recruitment

    PubMed Central

    Bode, Konrad A; Schroder, Kate; Hume, David A; Ravasi, Timothy; Heeg, Klaus; Sweet, Matthew J; Dalpke, Alexander H

    2007-01-01

    Post-translational modifications of histone proteins are major mechanisms that modify chromatin structure and regulate gene expression in eukaryotes. Activation of histone acetyltransferases or inhibition of histone deacetylases (HDACs) is generally believed to allow chromatin to assume a more open state, permitting transcriptional activity. We report here the surprising observation that treatment of murine dendritic cells with the HDAC inhibitors trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) in non-apoptotic concentrations strongly inhibited induction of both interleukin-12 protein p40 (IL-12p40) mRNA and protein upon stimulation of Toll-like receptors (TLRs). Moreover, TLR-mediated up-regulation of costimulatory molecules was also inhibited. Up-regulation of tumour necrosis factor-α mRNA and protein in response to TLR agonists was only affected upon prolonged exposure to HDAC inhibitors and regulation of IL-1β was not affected. Similar effects were apparent in murine and human macrophages. Regarding the mode of action, HDAC inhibition increased the acetylation status at the IL-12p40 locus. Nevertheless, IL-12p40 chromatin remodelling, binding of Rel-A and IRF1 to the IL-12p40 promoter and transcriptional activation were abrogated. In contrast, HDAC inhibitors had no effects on upstream nuclear factor-κB and mitogen-activated protein kinase activation. Thus HDACs positively regulate the expression of a subset of cytokine genes by enabling transcription factor recruitment. PMID:17635610

  12. Activating transcription factor 3, a useful marker for regenerative response after nerve root injury.

    PubMed

    Lindå, Hans; Sköld, Mattias K; Ochsmann, Thomas

    2011-01-01

    Activating transcription factor 3 (ATF3) is induced in various tissues in response to stress. In this experiment, ATF3 expression was studied in adult rats subjected either to a dorsal or ventral root avulsion (VRA; L4-6), or sciatic nerve transection (SNT). Post-operative survival times varied between 1.5 h and 3 weeks. In additional experiments an avulsed ventral root was directly replanted to the spinal cord. Dorsal root ganglias (DRGs) from humans exposed to traumatic dorsal root avulsions were also examined. After SNT ATF3 immunoreactivity (ATF3 IR) was detected in a few DRG neurons already 6 h after the lesion. After 24 h the number had clearly increased and still at 3 weeks DRG neurons remained labeled. In the ventral horn, ATF3 IR in motoneurons (MN) was first detected 24 h after the SNT, and still 3 weeks post-operatively lesioned MN showed ATF3 labeling. After a VRA many spinal MN showed ATF3 IR already after 3 h, and after 6 h all MN were labeled. At 3 weeks a majority of the lesioned MN had died, but all the remaining ones were labeled. When an avulsed ventral root was directly replanted, MN survived and were still labeled at 5 weeks. In DRG, a few neurons were labeled already at 1.5 h after a dorsal root avulsion. At 24 h the number had increased but still only a minority of the neurons were labeled. At 3 days the number of labeled neurons was reduced, and a further reduction was at hand at 7 days and 3 weeks. In parallel, in humans, 3 days after a traumatic dorsal root avulsion, only a few DRG neurons showed ATF3 IR. At 6 weeks no labeled neurons could be detected. These facts imply that ATF3 response to axotomy involves a distance-dependent mechanism. ATF3 also appears to be a useful and reliable neuronal marker of nerve lesions even in humans. In addition, ATF3 up-regulation in both motor and sensory neurons seems to be linked to regenerative competence.

  13. Signal Transducer and Activator of Transcription (STAT)-3 Activates Nuclear Factor (NF)-κB in Chronic Lymphocytic Leukemia Cells

    PubMed Central

    Liu, Zhiming; Hazan-Halevy, Inbal; Harris, David M.; Li, Ping; Ferrajoli, Alessandra; Faderl, Stefan; Keating, Michael J.; Estrov, Zeev

    2014-01-01

    Nuclear factor (NF)-κB plays a major role in the pathogenesis of B-cell neoplasms. A broad array of mostly extracellular stimuli has been reported to activate NF-κB, to various degrees, in chronic lymphocytic leukemia (CLL) cells. Because CLL cells harbor high levels of unphosphorylated (U) signal transducer and activator of transcription (STAT)-3 protein and U-STAT3 was reported to activate NF-κB, we sought to determine whether U-STAT3 activates NF-κB in CLL. Using the electrophoretic mobility shift assay (EMSA) we studied peripheral blood low-density cells from 15 patients with CLL and found that CLL cell nuclear extracts from all the samples bound to an NF-κB DNA probe, suggesting that NF-κB is constitutively activated in CLL. Immunoprecipitation studies showed that STAT3 bound NF-κB p65, and confocal microscopy studies detected U-STAT3/NF-κB complexes in the nuclei of CLL cells, thereby confirming these findings. Furthermore, infection of CLL cells with retroviral STAT3-shRNA attenuated the binding of NF-κB to DNA, as assessed by EMSA, and downregulated mRNA levels of NF-κB-regulated genes, as assessed by quantitative polymerase chain reaction. Taken together, our data suggest that U-STAT3 binds to the NF-κB p50/p65 dimers and that the U-STAT3/NF-κB complexes bind to DNA and activate NF-κB-regulated genes in CLL cells. PMID:21364020

  14. Early nuclear exclusion of the transcription factor max is associated with retinal ganglion cell death independent of caspase activity.

    PubMed

    Petrs-Silva, Hilda; de Freitas, Fabíola G; Linden, Rafael; Chiarini, Luciana B

    2004-02-01

    We examined the behavior of the transcription factor Max during retrograde neuronal degeneration of retinal ganglion cells. Using immunohistochemistry, we found a progressive redistribution of full-length Max from the nucleus to the cytoplasm and dendrites of the ganglion cells following axon damage. Then, the axotomized cells lose all their content of Max, while undergoing nuclear pyknosis and apoptotic cell death. After treatment of retinal explants with either anisomycin or thapsigargin, the rate of nuclear exclusion of Max accompanied the rate of cell death as modulated by either drug. Treatment with a pan-caspase inhibitor abolished both TUNEL staining and immunoreactivity for activated caspase-3, but did not affect the subcellular redistribution of Max immunoreactivity after axotomy. The data show that nuclear exclusion of the transcription factor Max is an early event, which precedes and is independent of the activation of caspases, during apoptotic cell death in the central nervous system.

  15. Ancient default activators of terminal photoreceptor differentiation in the pancrustacean compound eye: The homeodomain transcription factors Otd and Pph13

    PubMed Central

    Friedrich, Markus; Cook, Tiffany; Zelhof, Andrew C.

    2015-01-01

    The origin of the Drosophila compound eye predates the ancestor of Pancrustacea, the arthropod clade that includes insects and Crustaceans. Recent studies in emerging model systems for pancrustacean development - the red flour beetle Tribolium castaneum and water flea Daphnia pulex - have begun to shed light on the evolutionary conservation of transcriptional mechanisms found for the Drosophila compound eye. Here, we discuss the conserved roles of the transcription factors Otd and Pph13, which complement each other in two terminal events of photoreceptor differentiation: rhabdomere morphogenesis and transcriptional default activation of opsin gene expression. The synthesis of these data allows us to frame an evolutionary developmental model of the earliest events that generated the wavelength-specific photoreceptor subtypes of pancrustacean compound eyes. PMID:27436551

  16. Induction of metallothionein I by phenolic antioxidants requires metal-activated transcription factor 1 (MTF-1) and zinc.

    PubMed Central

    Bi, Yongyi; Palmiter, Richard D; Wood, Kristi M; Ma, Qiang

    2004-01-01

    Phenolic antioxidants, such as tBHQ [2,5-di-(t-butyl)-1,4-hydroquinone], induce Mt1 (metallothionein 1) gene expression and accumulation of MT protein. Induction of Mt1 mRNA does not depend on protein synthesis, and correlates with oxidation-reduction functions of the antioxidants. In the present study, we analysed the biochemical pathway of the induction. Induction depends on the presence of MTF-1 (metal-activated transcription factor 1), a transcription factor that is required for metal-induced transcription of Mt1, but does not require nuclear factor erythroid 2-related factor 2, a tBHQ-activated CNC bZip (cap 'n' collar basic leucine zipper) protein, that is responsible for regulating genes encoding phase II drug-metabolizing enzymes. Moreover, tBHQ induces the expression of MRE-beta Geo, a reporter gene driven by five metal response elements that constitute an optimal MTF-1 binding site. Reconstitution of Mtf1 -null cells with MTF-1 restores induction by both zinc and tBHQ. Unlike activation of phase II genes by tBHQ, induction of Mt1 expression does not occur in the presence of EDTA, when cells are cultured in zinc-depleted medium, or in cells with reduced intracellular 'free' zinc due to overexpression of ZnT1, a zinc-efflux transporter, indicating that induction requires zinc. In addition, fluorescence imaging reveals that tBHQ increases cytoplasmic free zinc concentration by mobilizing intracellular zinc pools. These findings establish that phenolic antioxidants activate Mt1 transcription by a zinc-dependent mechanism, which involves MTF-1 binding to metal regulator elements in the Mt1 gene promoter. PMID:14998373

  17. Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors: a potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis.

    PubMed Central

    Virbasius, J V; Scarpulla, R C

    1994-01-01

    Mitochondrial transcription factor A (mtTFA), the product of a nuclear gene, stimulates transcription from the two divergent mitochondrial promoters and is likely the principal activator of mitochondrial gene expression in vertebrates. Here we establish that the proximal promoter of the human mtTFA gene is highly dependent upon recognition sites for the nuclear respiratory factors, NRF-1 and NRF-2, for activity. These factors have been previously implicated in the activation of numerous nuclear genes that contribute to mitochondrial respiratory function. The affinity-purified factors from HeLa cells specifically bind to the mtTFA NRF-1 and NRF-2 sites through guanine nucleotide contacts that are characteristic for each site. Mutations in these contacts eliminate NRF-1 and NRF-2 binding and also dramatically reduce promoter activity in transfected cells. Although both factors contribute, NRF-1 binding appears to be the major determinant of promoter function. This dependence on NRF-1 activation is confirmed by in vitro transcription using highly purified recombinant proteins that display the same binding specificities as the HeLa cell factors. The activation of the mtTFA promoter by both NRF-1 and NRF-2 therefore provides a link between the expression of nuclear and mitochondrial genes and suggests a mechanism for their coordinate regulation during organelle biogenesis. Images PMID:8108407

  18. Activating transcription factor-4 promotes mineralization in vascular smooth muscle cells

    PubMed Central

    Masuda, Masashi; Miyazaki-Anzai, Shinobu; Keenan, Audrey L.; Shiozaki, Yuji; Okamura, Kayo; Chick, Wallace S.; Williams, Kristina; Zhao, Xiaoyun; Rahman, Shaikh Mizanoor; Tintut, Yin; Adams, Christopher M.

    2016-01-01

    Emerging evidence indicates that upregulation of the ER stress–induced pro-osteogenic transcription factor ATF4 plays an important role in vascular calcification, a common complication in patients with aging, diabetes, and chronic kidney disease (CKD). In this study, we demonstrated the pathophysiological role of ATF4 in vascular calcification using global Atf4 KO, smooth muscle cell–specific (SMC-specific) Atf4 KO, and transgenic (TG) mouse models. Reduced expression of ATF4 in global ATF4-haplodeficient and SMC-specific Atf4 KO mice reduced medial and atherosclerotic calcification under normal kidney and CKD conditions. In contrast, increased expression of ATF4 in SMC-specific Atf4 TG mice caused severe medial and atherosclerotic calcification. We further demonstrated that ATF4 transcriptionally upregulates the expression of type III sodium-dependent phosphate cotransporters (PiT1 and PiT2) by interacting with C/EBPβ. These results demonstrate that the ER stress effector ATF4 plays a critical role in the pathogenesis of vascular calcification through increased phosphate uptake in vascular SMCs. PMID:27812542

  19. 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

  20. Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells

    PubMed Central

    Lepsch, Lucilia B; Munhoz, Carolina D; Kawamoto, Elisa M; Yshii, Lidia M; Lima, Larissa S; Curi-Boaventura, Maria F; Salgado, Thais ML; Curi, Rui; Planeta, Cleopatra S; Scavone, Cristoforo

    2009-01-01

    Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells. PMID:19183502

  1. Cocaine induces cell death and activates the transcription nuclear factor kappa-B in PC12 cells.

    PubMed

    Lepsch, Lucilia B; Munhoz, Carolina D; Kawamoto, Elisa M; Yshii, Lidia M; Lima, Larissa S; Curi-Boaventura, Maria F; Salgado, Thais M L; Curi, Rui; Planeta, Cleopatra S; Scavone, Cristoforo

    2009-02-01

    Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-kappaB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-kappaB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-kappaB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-kappaB activation. Inhibition of NF-kappaB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-kappaB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

  2. Reconstitution of glucotoxic HIT-T15 cells with somatostatin transcription factor-1 partially restores insulin promoter activity.

    PubMed

    Harmon, J S; Tanaka, Y; Olson, L K; Robertson, R P

    1998-06-01

    We have reported that chronic culture of HIT-T15 cells in medium containing supraphysiologic glucose concentrations (11.1 mmol/l) causes a decrease in insulin mRNA levels, insulin content, and insulin release. Furthermore, decreases in insulin gene transcription and binding activity of two essential beta-cell transcription factors, somatostatin transcription factor-1 (STF-1; also known as GSTF, IDX-1, IPF-1, PDX-1, and GSF) and RIPE-3b1 activator, are associated with this glucotoxic effect. In this study, we observed that the loss of RIPE-3b1 occurs much earlier (79% decrease at passage [p]81) than the loss of STF-1 (65% decrease at p104), with abolishment of both factors by p122. Since the STF-1, but not the RIPE-3b1 activator, gene has been cloned, we examined its restorative effects on insulin gene promoter activity after reconstitution with STF-1 cDNA. Basal insulin promoter activities normalized to early (p71-74) passage cells (1.000 +/- 0.069) were 0.4066 +/- 0.093 and 0.142 +/- 0.034 for intermediate (p102-106) and late (p118-122) passage cells, respectively. Early, intermediate, and late passage cells, all chronically cultured in medium containing 11.1 mmol/l glucose, were transfected with STF-1 alone or cotransfected with E2-5, an E-box factor known to be synergistically associated with STF-1. Compared with basal levels, we observed a trend toward an increase in insulin promoter activity in intermediate passage cells with STF-1 transfection (1.43-fold) that became a significant increase when E2-5 was cotransfected (1.78-fold). In late passage cells, transfection of STF-1 alone significantly stimulated a 2.2-fold increase in the insulin promoter activity. Cotransfection of STF-1 and E2-5 in late passage cells stimulated insulin promoter activity 2.8-fold, which was 40% of the activity observed in early passage cells. Control studies in glucotoxic betaTC-6 cells deficient in RIPE-3b1 activator but not STF-1 did not demonstrate an increase in insulin promoter

  3. RNA polymerase I transcription factors in active yeast rRNA gene promoters enhance UV damage formation and inhibit repair.

    PubMed

    Meier, Andreas; Thoma, Fritz

    2005-03-01

    UV photofootprinting and repair of pyrimidine dimers by photolyase was used to investigate chromatin structure, protein-DNA interactions, and DNA repair in the spacer and promoter of Saccharomyces cerevisiae rRNA genes. Saccharomyces cerevisiae contains about 150 copies of rRNA genes separated by nontranscribed spacers. Under exponential growth conditions about half of the genes are transcribed by RNA polymerase I (RNAP-I). Initiation of transcription requires the assembly of the upstream activating factor (UAF), the core factor (CF), TATA binding protein, and RNAP-I with Rrn3p on the upstream element and core promoter. We show that UV irradiation of wild-type cells and transcription factor mutants generates photofootprints in the promoter elements. The core footprint depends on UAF, while the UAF footprint was also detected in absence of the CFs. Fractionation of active and inactive promoters showed the core footprint mainly in the active fraction and similar UAF footprints in both fractions. DNA repair by photolyase was strongly inhibited in active promoters but efficient in inactive promoters. The data suggest that UAF is present in vivo in active and inactive promoters and that recruitment of CF and RNAP-I to active promoters generates a stable complex which inhibits repair.

  4. Dexamethasone rapidly suppresses IL-33-stimulated mast cell function by blocking transcription factor activity.

    PubMed

    Paranjape, Anuya; Chernushevich, Oksana; Qayum, Amina Abdul; Spence, Andrew J; Taruselli, Marcela T; Abebayehu, Daniel; Barnstein, Brian O; McLeod, Jamie Josephine Avila; Baker, Bianca; Bajaj, Gurjas S; Chumanevich, Alena P; Oskeritzian, Carole A; Ryan, John J

    2016-12-01

    Mast cells are critical effectors of allergic disease and can be activated by IL-33, a proinflammatory member of the IL-1 cytokine family. IL-33 worsens the pathology of mast cell-mediated diseases, but therapies to antagonize IL-33 are still forthcoming. Because steroids are the mainstay of allergic disease treatment and are well known to suppress mast cell activation by other stimuli, we examined the effects of the steroid dexamethasone on IL-33-mediated mast cell function. We found that dexamethasone potently and rapidly suppressed cytokine production elicited by IL-33 from murine bone marrow-derived and peritoneal mast cells. IL-33 enhances IgE-mediated mast cell cytokine production, an activity that was also antagonized by dexamethasone. These effects were consistent in human mast cells. We additionally observed that IL-33 augmented migration of IgE-sensitized mast cells toward antigen. This enhancing effect was similarly reversed by dexamethasone. Simultaneous addition of dexamethasone with IL-33 had no effect on the phosphorylation of MAP kinases or NFκB p65 subunit; however, dexamethasone antagonized AP-1- and NFκB-mediated transcriptional activity. Intraperitoneal administration of dexamethasone completely abrogated IL-33-mediated peritoneal neutrophil recruitment and prevented plasma IL-6 elevation. These data demonstrate that steroid therapy may be an effective means of antagonizing the effects of IL-33 on mast cells in vitro and in vivo, acting partly by suppressing IL-33-induced NFκB and AP-1 activity.

  5. New Role for Kruppel-like Factor 14 as a Transcriptional Activator Involved in the Generation of Signaling Lipids*

    PubMed Central

    de Assuncao, Thiago M.; Lomberk, Gwen; Cao, Sheng; Yaqoob, Usman; Mathison, Angela; Simonetto, Douglas A.; Huebert, Robert C.; Urrutia, Raul A.; Shah, Vijay H.

    2014-01-01

    Sphingosine kinase 1 (SK1) is an FGF-inducible gene responsible for generation of sphingosine-1-phosphate, a critical lipid signaling molecule implicated in diverse endothelial cell functions. In this study, we identified SK1 as a target of the canonical FGF2/FGF receptor 1 activation pathway in endothelial cells and sought to identify novel transcriptional pathways that mediate lipid signaling. Studies using the 1.9-kb SK1 promoter and deletion mutants revealed that basal and FGF2-stimulated promoter activity occurred through two GC-rich regions located within 633 bp of the transcription start site. Screening for GC-rich binding transcription factors that could activate this site demonstrated that KLF14, a gene implicated in obesity and the metabolic syndrome, binds to this region. Congruently, overexpression of KLF14 increased basal and FGF2-stimulated SK1 promoter activity by 3-fold, and this effect was abrogated after mutation of the GC-rich sites. In addition, KLF14 siRNA transfection decreased SK1 mRNA and protein levels by 3-fold. Congruently, SK1 mRNA and protein levels were decreased in livers from KLF14 knock-out mice. Combined, luciferase, gel shift, and chromatin immunoprecipitation assays showed that KLF14 couples to p300 to increase the levels of histone marks associated with transcriptional activation (H4K8ac and H3K14ac), while decreasing repressive marks (H3K9me3 and H3K27me3). Collectively, the results demonstrate a novel mechanism whereby SK1 lipid signaling is regulated by epigenetic modifications conferred by KLF14 and p300. Thus, this is the first description of the activity and mechanisms underlying the function of KLF14 as an activator protein and novel regulator of lipid signaling. PMID:24759103

  6. Transcription factor CP2 is involved in activating mBMP4 in mouse mesenchymal stem cells.

    PubMed

    Kang, Ho Chul; Chae, Ji Hyung; Kim, Beom Sue; Han, Su Youne; Kim, Sung-Hyun; Auh, Chung-Kyoon; Yang, Sung-Il; Kim, Chul Geun

    2004-06-30

    CP2 is a member of a family of transcription factors that regulate genes involved in events from early development to terminal differentiation. In an effort to understand how it selects its target genes we carried out a database search, and located several CP2 binding motifs in the promoter region of bone morphogenetic protein-4 (BMP4). BMP4 is a key regulator of cell fate and body patterning throughout development. For the CP2 binding motifs in BMP4 promoter region to be relevant in vivo, CP2 and BMP4 should be expressed together. We found that CP2b and CP2c, two potent transcriptional activators, are expressed in a manner similar to BMP4 during osteoblast differentiation of C3H10T1/2 cells. In in vitro assays, the CP2 proteins bound to two CP2 binding motifs (-715 to -676 and -147 to -118) in the BMP4 promoter, and luciferase reporter assays indicated that this binding was essential for transcription of BMP4 during osteoblast differentiation. Taken together, our data indicate that CP2b and CP2c play important roles during bone development by activating BMP4 transcription.

  7. Cynaropicrin from Cynara scolymus L. suppresses photoaging of skin by inhibiting the transcription activity of nuclear factor-kappa B.

    PubMed

    Tanaka, Yuka Tsuda; Tanaka, Kiyotaka; Kojima, Hiroyuki; Hamada, Tomoji; Masutani, Teruaki; Tsuboi, Makoto; Akao, Yukihiro

    2013-01-15

    Aging of skin is characterized by skin wrinkling, laxity, and pigmentation induced by several environmental stress factors. Histological changes during the photoaging of skin include hyperproliferation of keratinocytes and melanocytes causing skin wrinkles and pigmentation. Nuclear factor kappa B (NF-κB) is one of the representative transcription factors active in conjunction with inflammation. NF-κB is activated by stimulation such as ultraviolet rays and inflammatory cytokines and induces the expression of various genes such as those of basic fibroblast growth factor (bFGF) and matrix metalloprotease-1 (MMP-1). We screened several plant extracts for their possible inhibitory effect on the transcriptional activity of NF-κB. One of them, an extract from Cynara scolymus L., showed a greatest effect on the suppression of NF-κB transactivation. As a result, we found that cynaropicrin, which is a sesquiterpene lactone, inhibited the NF-κB-mediated transactivation of bFGF and MMP-1. Furthermore, it was confirmed that in an in vivo mouse model cynaropicrin prevented skin photoaging processes leading to the hyperproliferation of keratinocytes and melanocytes. These findings taken together indicate that cynaropicrin is an effective antiphotoaging agent that acts by inhibiting NF-κB-mediated transactivation.

  8. Fisetin stimulates autophagic degradation of phosphorylated tau via the activation of TFEB and Nrf2 transcription factors.

    PubMed

    Kim, Sunhyo; Choi, Ki Ju; Cho, Sun-Jung; Yun, Sang-Moon; Jeon, Jae-Pil; Koh, Young Ho; Song, Jihyun; Johnson, Gail V W; Jo, Chulman

    2016-04-26

    The neuronal accumulation of phosphorylated tau plays a critical role in the pathogenesis of Alzheimer's disease (AD). Here, we examined the effect of fisetin, a flavonol, on tau levels. Treatment of cortical cells or primary neurons with fisetin resulted in significant decreases in the levels of phosphorylated tau. In addition, fisetin decreased the levels of sarkosyl-insoluble tau in an active GSK-3β-induced tau aggregation model. However, there was no difference in activities of tau kinases and phosphatases such as protein phosphatase 2A, irrespective of fisetin treatment. Fisetin activated autophagy together with the activation of transcription factor EB (TFEB) and Nrf2 transcriptional factors. The activation of autophagy including TFEB is likely due to fisetin-mediated mammalian target of rapamycin complex 1 (mTORC1) inhibition, since the phosphorylation levels of p70S6 kinase and 4E-BP1 were decreased in the presence of fisetin. Indeed, fisetin-induced phosphorylated tau degradation was attenuated by chemical inhibitors of the autophagy-lysosome pathway. Together the results indicate that fisetin reduces levels of phosphorylated tau through the autophagy pathway activated by TFEB and Nrf2. Our result suggests fisetin should be evaluated further as a potential preventive and therapeutic drug candidate for AD.

  9. Fisetin stimulates autophagic degradation of phosphorylated tau via the activation of TFEB and Nrf2 transcription factors

    PubMed Central

    Kim, Sunhyo; Choi, Ki Ju; Cho, Sun-Jung; Yun, Sang-Moon; Jeon, Jae-Pil; Koh, Young Ho; Song, Jihyun; Johnson, Gail V. W.; Jo, Chulman

    2016-01-01

    The neuronal accumulation of phosphorylated tau plays a critical role in the pathogenesis of Alzheimer’s disease (AD). Here, we examined the effect of fisetin, a flavonol, on tau levels. Treatment of cortical cells or primary neurons with fisetin resulted in significant decreases in the levels of phosphorylated tau. In addition, fisetin decreased the levels of sarkosyl-insoluble tau in an active GSK-3β-induced tau aggregation model. However, there was no difference in activities of tau kinases and phosphatases such as protein phosphatase 2A, irrespective of fisetin treatment. Fisetin activated autophagy together with the activation of transcription factor EB (TFEB) and Nrf2 transcriptional factors. The activation of autophagy including TFEB is likely due to fisetin-mediated mammalian target of rapamycin complex 1 (mTORC1) inhibition, since the phosphorylation levels of p70S6 kinase and 4E-BP1 were decreased in the presence of fisetin. Indeed, fisetin-induced phosphorylated tau degradation was attenuated by chemical inhibitors of the autophagy-lysosome pathway. Together the results indicate that fisetin reduces levels of phosphorylated tau through the autophagy pathway activated by TFEB and Nrf2. Our result suggests fisetin should be evaluated further as a potential preventive and therapeutic drug candidate for AD. PMID:27112200

  10. Phosphoinositide 3-Kinases Upregulate System xc− via Eukaryotic Initiation Factor 2α and Activating Transcription Factor 4 – A Pathway Active in Glioblastomas and Epilepsy

    PubMed Central

    Baxter, Paul; Kassubek, Rebecca; Albrecht, Philipp; Van Liefferinge, Joeri; Westhoff, Mike-Andrew; Halatsch, Marc-Eric; Karpel-Massler, Georg; Meakin, Paul J.; Hayes, John D.; Aronica, Eleonora; Smolders, Ilse; Ludolph, Albert C.; Methner, Axel; Conrad, Marcus; Massie, Ann; Hardingham, Giles E.

    2014-01-01

    Abstract Aims: Phosphoinositide 3-kinases (PI3Ks) relay growth factor signaling and mediate cytoprotection and cell growth. The cystine/glutamate antiporter system xc− imports cystine while exporting glutamate, thereby promoting glutathione synthesis while increasing extracellular cerebral glutamate. The aim of this study was to analyze the pathway through which growth factor and PI3K signaling induce the cystine/glutamate antiporter system xc− and to demonstrate its biological significance for neuroprotection, cell growth, and epilepsy. Results: PI3Ks induce system xc− through glycogen synthase kinase 3β (GSK-3β) inhibition, general control non-derepressible-2-mediated eukaryotic initiation factor 2α phosphorylation, and the subsequent translational up-regulation of activating transcription factor 4. This pathway is essential for PI3Ks to modulate oxidative stress resistance of nerve cells and insulin-induced growth in fibroblasts. Moreover, the pathway is active in human glioblastoma cells. In addition, it is induced in primary cortical neurons in response to robust neuronal activity and in hippocampi from patients with temporal lobe epilepsy. Innovation: Our findings further extend the concepts of how growth factors and PI3Ks induce neuroprotection and cell growth by adding a new branch to the signaling network downstream of GSK-3β, which, ultimately, leads to the induction of the cystine/glutamate antiporter system xc−. Importantly, the induction of this pathway by neuronal activity and in epileptic hippocampi points to a potential role in epilepsy. Conclusion: PI3K-regulated system xc− activity is not only involved in the stress resistance of neuronal cells and in cell growth by increasing the cysteine supply and glutathione synthesis, but also plays a role in the pathophysiology of tumor- and non-tumor-associated epilepsy by up-regulating extracellular cerebral glutamate. Antioxid. Redox Signal. 20: 2907–2922. PMID:24219064

  11. NFAT activation by membrane potential follows a calcium pathway distinct from other activity-related transcription factors in skeletal muscle cells.

    PubMed

    Valdés, Juan Antonio; Gaggero, Eduardo; Hidalgo, Jorge; Leal, Nancy; Jaimovich, Enrique; Carrasco, M Angélica

    2008-03-01

    Depolarization of skeletal muscle cells triggers intracellular Ca2+ signals mediated by ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors. Previously, we have reported that K+-induced depolarization activates transcriptional regulators ERK, cAMP response element-binding protein, c-fos, c-jun, and egr-1 through IP3-dependent Ca2+ release, whereas NF-kappa B activation is elicited by both ryanodine and IP3 receptor-mediated Ca2+ signals. We have further shown that field stimulation with electrical pulses results in an NF-kappa B activation increase dependent of the amount of pulses and independent of their frequency. In this work, we report the results obtained for nuclear factor of activated T cells (NFAT)-mediated transcription and translocation generated by both K+ and electrical stimulation protocols in primary skeletal muscle cells and C2C12 cells. The Ca2+ source for NFAT activation is through release by ryanodine receptors and extracellular Ca2+ entry. We found this activation to be independent of the number of pulses within a physiological range of stimulus frequency and enhanced by long-lasting low-frequency stimulation. Therefore, activation of the NFAT signaling pathway differs from that of NF-kappa B and other transcription factors. Calcineurin enzyme activity correlated well with the relative activation of NFAT translocation and transcription using different stimulation protocols. Furthermore, both K+-induced depolarization and electrical stimulation increased mRNA levels of the type 1 IP3 receptor mediated by calcineurin activity, which suggests that depolarization may regulate IP3 receptor transcription. These results confirm the presence of at least two independent pathways for excitation-transcription coupling in skeletal muscle cells, both dependent on Ca2+ release and triggered by the same voltage sensor but activating different intracellular release channels.

  12. Host-cell positive transcription elongation factor b kinase activity is essential and limiting for HIV type 1 replication.

    PubMed

    Flores, O; Lee, G; Kessler, J; Miller, M; Schlief, W; Tomassini, J; Hazuda, D

    1999-06-22

    HIV-1 gene expression and viral replication require the viral transactivator protein Tat. The RNA polymerase II transcriptional elongation factor P-TEFb (cyclin-dependent kinase 9/cyclin T) is a cellular protein kinase that has recently been shown to be a key component of the Tat-transactivation process. For this report, we studied the requirement for P-TEFb in HIV-1 infection, and we now show that P-TEFb is both essential and limiting for HIV-1 replication. Attenuation of P-TEFb kinase activity either by expression of a dominant-negative cyclin-dependent kinase 9 transgene or through the use of small-molecule inhibitors suppresses HIV-1 gene expression and HIV-1 replication. Inhibition of HIV-1 replication is affected in a manner consistent with a direct and specific effect on P-TEFb and the known functional role of P-TEFb in Tat-activated transcription. Tat-activated expression of HIV-1 genes seems uniquely dependent on P-TEFb, as inhibition of P-TEFb activity and HIV-1 replication can be achieved without compromising cell viability or RNA polymerase II-dependent cellular gene transcription. Selective inhibition of the P-TEFb kinase may therefore provide a novel approach for developing chemotherapeutic agents against HIV-1.

  13. Intracellular ascorbate enhances hypoxia-inducible factor (HIF)-hydroxylase activity and preferentially suppresses the HIF-1 transcriptional response.

    PubMed

    Kuiper, Caroline; Dachs, Gabi U; Currie, Margaret J; Vissers, Margreet C M

    2014-04-01

    Hypoxia-inducible factor (HIF)-1 drives the transcription of hundreds of genes to support cell survival under conditions of microenvironmental and metabolic stress. HIF-1 is downregulated by iron-containing 2-oxoglutarate-dependent enzymes that require ascorbate as a cofactor. The HIF hydroxylases control both protein stability and the formation of an active transcription complex and, consequently, ascorbate could affect HIF-1α stabilization and/or gene expression, but the relative effect of ascorbate on these separate processes has not been well characterized. In this study we examined the effects of known intracellular ascorbate concentrations on both processes in response to various means of hydroxylase inhibition, including CoCl2, NiCl2, desferrioxamine, dimethyloxalylglycine, and hypoxia. Ascorbate inhibited HIF-1 activity most dramatically with all mechanisms of iron competition. In addition, HIF-1-dependent gene expression was effectively prevented by ascorbate and was inhibited even under conditions that allowed HIF-1α protein stabilization. This suggests that (1) ascorbate acts primarily to stabilize and reduce the iron atom in the hydroxylase active site and (2) the asparagine hydroxylase controlling HIF-1 transcriptional activity is particularly susceptible to fluctuations in intracellular ascorbate. These findings suggest that ascorbate plays a significant role in supporting HIF-hydroxylase function and that it could thereby modulate the cell survival response.

  14. S-nitrosylation influences the structure and DNA binding activity of AtMYB30 transcription factor from Arabidopsis thaliana.

    PubMed

    Tavares, Carolina Pereira; Vernal, Javier; Delena, Ricardo Alexandre; Lamattina, Lorenzo; Cassia, Raul; Terenzi, Hernán

    2014-04-01

    MYB proteins are a family of transcription factors that play an important role in plant development and regulatory defense processes. Arabidopsis thaliana MYB30 (AtMYB30), a member of this protein family, is involved in cell death processes during the hypersensitive response (HR) of plants. HR is characterized by a vast production of reactive oxygen species (ROS) and nitric oxide (NO). NO may thus influence the binding of AtMYB30 to DNA. In this work we evaluated the effect of NO on AtMYB30 DNA binding activity, and also in the protein structural properties. A fully active minimal DNA-binding domain (DBD) of AtMYB30 (residues 11-116) containing two cysteine residues (C49 and C53) was overexpressed and purified. Site-directed mutagenesis was used to obtain AtMYB30 DBD mutants C49A and C53A. The DNA binding activity of AtMYB30 DBD, and Cys single mutants is clearly inhibited upon incubation with a NO donor, and S-nitrosylation was confirmed by the biotin switch assay. Finally, in order to understand the mechanism of NO effect on AtMYB30 DNA binding activity we performed circular dichroism analysis, to correlate the observed protein function inhibition and a potential structural impairment on AtMYB30 DBD. Indeed, NO modification of C49 and C53 residues promotes a subtle modification on the secondary structure of this transcription factor. We thus demonstrated, using various techniques, the in vitro effect of NO on AtMYB30 DBD, and thus the potential consequences of NO activity on plant metabolism influenced by this transcription factor.

  15. Carboxy terminus of GATA4 transcription factor is required for its cardiogenic activity and interaction with CDK4

    PubMed Central

    Gallagher, Joseph M.; Yamak, Abir; Kirilenko, Pavel; Black, Sarah; Bochtler, Matthias; Lefebvre, Chantal; Nemer, Mona; Latinkić, Branko V.

    2014-01-01

    GATA4-6 transcription factors regulate numerous aspects of development and homeostasis in multiple tissues of mesodermal and endodermal origin. In the heart, the best studied of these factors, GATA4, has multiple distinct roles in cardiac specification, differentiation, morphogenesis, hypertrophy and survival. To improve understanding of how GATA4 achieves its numerous roles in the heart, here we have focused on the carboxy-terminal domain and the residues required for interaction with cofactors FOG2 and Tbx5. We present evidence that the carboxy terminal region composed of amino acids 362–400 is essential for mediating cardiogenesis in Xenopus pluripotent explants and embryos. In contrast, the same region is not required for endoderm-inducing activity of GATA4. Further evidence is presented that the carboxy terminal cardiogenic region of GATA4 does not operate as a generic transcriptional activator. Potential mechanism of action of the carboxy terminal end of GATA4 is provided by the results showing physical and functional interaction with CDK4, including the enhancement of cardiogenic activity of GATA4 by CDK4. These results establish CDK4 as a GATA4 partner in cardiogenesis. The interactions of GATA4 with its other well described cofactors Tbx5 and FOG2 are known to be involved in heart morphogenesis, but their requirement for cardiac differentiation is unknown. We report that the mutations that disrupt interactions of GATA4 with Tbx5 and FOG2, G295S and V217G, respectively, do not impair cardiogenic activity of GATA4. These findings add support to the view that distinct roles of GATA4 in the heart are mediated by different determinants of the protein. Finally, we show that the rat GATA4 likely induces cardiogenesis cell autonomously or directly as it does not require activity of endodermal transcription factor Sox17, a GATA4 target gene that induces cardiogenesis non-cell autonomously. PMID:25241353

  16. Role of activating transcription factor 3 in the synthesis of latency-associated transcript and maintenance of herpes simplex virus 1 in latent state in ganglia

    PubMed Central

    Shu, Minfeng; Du, Te; Zhou, Grace; Roizman, Bernard

    2015-01-01

    A key property of herpes simplex viruses (HSVs) is their ability to establish latent infection in sensory or autonomic ganglia and to reactivate on physical, hormonal, or emotional stress. In latently infected ganglia, HSVs express a long noncoding RNA, a latency-associated transcript (LAT), which plays a key role in maintaining latently infected neurons, but not viral proteins. To investigate the events leading to reactivation, we examined the use of ganglionic organ cultures that enable rapid reactivation in medium containing antibody to nerve growth factor (NGF) or delayed reactivation in medium containing NGF and epidermal growth factor (EGF). Here we report the discovery that activating transcription factor 3 (ATF3), a stress response protein, profoundly affects the interaction of HSV with its host. Specifically, (i) ATF3 is induced by stress, such as inhibition of protein synthesis or infection; (ii) in infected cells, ATF3 enhances the accumulation of LAT by acting on the response elements in the promoter of the LAT precursor RNA; (iii) ATF3 is induced nearly 100-fold in ganglionic organ cultures; and (iv) ATF3 plays a key role in the maintenance of the latent state, inasmuch as expression of ATF3 bereft of the C-terminal activation domain acts as a dominant negative factor, inducing HSV gene expression in ganglionic organ cultures harboring latent virus and incubated in medium containing NGF and EGF. Thus, ATF3 is a component of a cluster of cellular proteins that together with LAT maintain the integrity of the neurons harboring latent virus. PMID:26305977

  17. Tumor STAT1 transcription factor activity enhances breast tumor growth and immune suppression mediated by myeloid-derived suppressor cells.

    PubMed

    Hix, Laura M; Karavitis, John; Khan, Mohammad W; Shi, Yihui H; Khazaie, Khashayarsha; Zhang, Ming

    2013-04-26

    Previous studies had implicated the IFN-γ transcription factor signal transducer and activator of transcription 1 (STAT1) as a tumor suppressor. However, accumulating evidence has correlated increased STAT1 activation with increased tumor progression in multiple types of cancer, including breast cancer. Indeed, we present evidence that tumor up-regulation of STAT1 activity in human and mouse mammary tumors correlates with increasing disease progression to invasive carcinoma. A microarray analysis comparing low aggressive TM40D and highly aggressive TM40D-MB mouse mammary carcinoma cells revealed significantly higher STAT1 activity in the TM40D-MB cells. Ectopic overexpression of constitutively active STAT1 in TM40D cells promoted mobilization of myeloid-derived suppressor cells (MDSCs) and inhibition of antitumor T cells, resulting in aggressive tumor growth in tumor-transplanted, immunocompetent mice. Conversely, gene knockdown of STAT1 in the metastatic TM40D-MB cells reversed these events and attenuated tumor progression. Importantly, we demonstrate that in human breast cancer, the presence of tumor STAT1 activity and tumor-recruited CD33(+) myeloid cells correlates with increasing disease progression from ductal carcinoma in situ to invasive carcinoma. We conclude that STAT1 activity in breast cancer cells is responsible for shaping an immunosuppressive tumor microenvironment, and inhibiting STAT1 activity is a promising immune therapeutic approach.

  18. Drosophila melanogaster activating transcription factor 4 regulates glycolysis during endoplasmic reticulum stress.

    PubMed

    Lee, Ji Eun; Oney, McKenna; Frizzell, Kimberly; Phadnis, Nitin; Hollien, Julie

    2015-02-13

    Endoplasmic reticulum (ER) stress results from an imbalance between the load of proteins entering the secretory pathway and the ability of the ER to fold and process them. The response to ER stress is mediated by a collection of signaling pathways termed the unfolded protein response, which plays important roles in development and disease. Here we show that in Drosophila melanogaster S2 cells, ER stress induces a coordinated change in the expression of genes involved in carbon metabolism. Genes encoding enzymes that carry out glycolysis were up-regulated, whereas genes encoding proteins in the tricarboxylic acid cycle and respiratory chain complexes were down-regulated. The unfolded protein response transcription factor Atf4 was necessary for the up-regulation of glycolytic enzymes and Lactate dehydrogenase (Ldh). Furthermore, Atf4 binding motifs in promoters for these genes could partially account for their regulation during ER stress. Finally, flies up-regulated Ldh and produced more lactate when subjected to ER stress. Together, these results suggest that Atf4 mediates a shift from a metabolism based on oxidative phosphorylation to one more heavily reliant on glycolysis, reminiscent of aerobic glycolysis or the Warburg effect observed in cancer and other proliferative cells.

  19. Upregulation of the Coagulation Factor VII Gene during Glucose Deprivation Is Mediated by Activating Transcription Factor 4

    PubMed Central

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

    2012-01-01

    Background Constitutive production of blood coagulation proteins by hepatocytes is necessary for hemostasis. Stressful conditions trigger adaptive cellular responses and delay processing of most proteins, potentially affecting plasma levels of proteins secreted exclusively by hepatocytes. We examined the effect of glucose deprivation on expression of coagulation proteins by the human hepatoma cell line, HepG2. Methodology/Principal Findings Expression of coagulation factor VII, which is required for initiation of blood coagulation, was elevated by glucose deprivation, while expression of other coagulation proteins decreased. Realtime PCR and ELISA demonstrated that the relative percentage expression +/− SD of steady-state F7 mRNA and secreted factor VII antigen were significantly increased (from 100+/−15% to 188+/−27% and 100+/−8.8% to 176.3+/−17.3% respectively, p<0.001) at 24 hr of treatment. The integrated stress response was induced, as indicated by upregulation of transcription factor ATF4 and of additional stress-responsive genes. Small interfering RNAs directed against ATF4 potently reduced basal F7 expression, and prevented F7 upregulation by glucose deprivation. The response of the endogenous F7 gene was replicated in reporter gene assays, which further indicated that ATF4 effects were mediated via interaction with an amino acid response element in the F7 promoter. Conclusions/Significance Our data indicated that glucose deprivation enhanced F7 expression in a mechanism reliant on prior ATF4 upregulation primarily due to increased transcription from the ATF4 gene. Of five coagulation protein genes examined, only F7 was upregulated, suggesting that its functions may be important in a systemic response to glucose deprivation stress. PMID:22848420

  20. Activation of the STAT6 transcription factor in Jurkat T-cells by the herpesvirus saimiri Tip protein

    PubMed Central

    Kim, Yuri; Kwon, Eun-Kyung; Jeon, Ju-Hong; So, Insuk; Kim, In-Gyu; Choi, Myung-Sik; Kim, Ik-Sang; Choi, Joong-Kook; Jung, Jae Ung

    2012-01-01

    Herpesvirus saimiri (HVS), a T-lymphotropic monkey herpesvirus, induces fulminant T-cell lymphoma in non-natural primate hosts. In addition, it can immortalize human T-cells in vitro. HVS tyrosine kinase-interacting protein (Tip) is an essential viral gene required for T-cell transformation both in vitro and in vivo. In this study, we found that Tip interacts with the STAT6 transcription factor and induces phosphorylation of STAT6 in T-cells. The interaction with STAT6 requires the Tyr127 residue and Lck-binding domain of Tip, which are indispensable for interleukin (IL)-2-independent T-cell transformation by HVS. It was also demonstrated that Tip induces nuclear translocation of STAT6, as well as activation of STAT6-dependent transcription in Jurkat T-cells. Interestingly, the phosphorylated STAT6 mainly colocalized with vesicles containing Tip within T-cells, but was barely detectable in the nucleus. However, nuclear translocation of phospho-STAT6 and transcriptional activation of STAT6 by IL-4 stimulation were not affected significantly in T-cells expressing Tip. Collectively, these findings suggest that the constitutive activation of STAT6 by Tip in T-cells may contribute to IL-2-independent T-cell transformation by HVS. PMID:22012462

  1. CD45 Phosphatase Inhibits STAT3 Transcription Factor Activity in Myeloid Cells and Promotes Tumor-Associated Macrophage Differentiation.

    PubMed

    Kumar, Vinit; Cheng, Pingyan; Condamine, Thomas; Mony, Sridevi; Languino, Lucia R; McCaffrey, Judith C; Hockstein, Neil; Guarino, Michael; Masters, Gregory; Penman, Emily; Denstman, Fred; Xu, Xiaowei; Altieri, Dario C; Du, Hong; Yan, Cong; Gabrilovich, Dmitry I

    2016-02-16

    Recruitment of monocytic myeloid-derived suppressor cells (MDSCs) and differentiation of tumor-associated macrophages (TAMs) are the major factors contributing to tumor progression and metastasis. We demonstrated that differentiation of TAMs in tumor site from monocytic precursors was controlled by downregulation of the activity of the transcription factor STAT3. Decreased STAT3 activity was caused by hypoxia and affected all myeloid cells but was not observed in tumor cells. Upregulation of CD45 tyrosine phosphatase activity in MDSCs exposed to hypoxia in tumor site was responsible for downregulation of STAT3. This effect was mediated by the disruption of CD45 protein dimerization regulated by sialic acid. Thus, STAT3 has a unique function in the tumor environment in controlling the differentiation of MDSC into TAM, and its regulatory pathway could be a potential target for therapy.

  2. Functional characterization of NAC55 transcription factor from oilseed rape (Brassica napus L.) as a novel transcriptional activator modulating reactive oxygen species accumulation and cell death.

    PubMed

    Niu, Fangfang; Wang, Chen; Yan, Jingli; Guo, Xiaohua; Wu, Feifei; Yang, Bo; Deyholos, Michael K; Jiang, Yuan-Qing

    2016-09-01

    NAC transcription factors (TFs) are plant-specific and play important roles in development, responses to biotic and abiotic cues and hormone signaling. So far, only a few NAC genes have been reported to regulate cell death. In this study, we identified and characterized a NAC55 gene isolated from oilseed rape (Brassica napus L.). BnaNAC55 responds to multiple stresses, including cold, heat, abscisic acid (ABA), jasmonic acid (JA) and a necrotrophic fungal pathogen Sclerotinia sclerotiorum. BnaNAC55 has transactivation activity and is located in the nucleus. BnaNAC55 is able to form homodimers in planta. Unlike ANAC055, full-length BnaNAC55, but not either the N-terminal NAC domain or C-terminal regulatory domain, induces ROS accumulation and hypersensitive response (HR)-like cell death when expressed both in oilseed rape protoplasts and Nicotiana benthamiana. Furthermore, BnaNAC55 expression causes obvious nuclear DNA fragmentation. Moreover, quantitative reverse transcription PCR (qRT-PCR) analysis identified that the expression levels of multiple genes regulating ROS production and scavenging, defense response as well as senescence are significantly induced. Using a dual luciferase reporter assay, we further confirm that BnaNAC55 could activate the expression of a few ROS and defense-related gene expression. Taken together, our work has identified a novel NAC TF from oilseed rape that modulates ROS accumulation and cell death.

  3. The RNA-binding protein FPA regulates flg22-triggered defense responses and transcription factor activity by alternative polyadenylation.

    PubMed

    Lyons, Rebecca; Iwase, Akira; Gänsewig, Thomas; Sherstnev, Alexander; Duc, Céline; Barton, Geoffrey J; Hanada, Kousuke; Higuchi-Takeuchi, Mieko; Matsui, Minami; Sugimoto, Keiko; Kazan, Kemal; Simpson, Gordon G; Shirasu, Ken

    2013-10-09

    RNA-binding proteins (RBPs) play an important role in plant host-microbe interactions. In this study, we show that the plant RBP known as FPA, which regulates 3'-end mRNA polyadenylation, negatively regulates basal resistance to bacterial pathogen Pseudomonas syringae in Arabidopsis. A custom microarray analysis reveals that flg22, a peptide derived from bacterial flagellins, induces expression of alternatively polyadenylated isoforms of mRNA encoding the defence-related transcriptional repressor ETHYLENE RESPONSE FACTOR 4 (ERF4), which is regulated by FPA. Flg22 induces expression of a novel isoform of ERF4 that lacks the ERF-associated amphiphilic repression (EAR) motif, while FPA inhibits this induction. The EAR-lacking isoform of ERF4 acts as a transcriptional activator in vivo and suppresses the flg22-dependent reactive oxygen species burst. We propose that FPA controls use of proximal polyadenylation sites of ERF4, which quantitatively limit the defence response output.

  4. The BCL11A transcription factor directly activates RAG gene expression and V(D)J recombination.

    PubMed

    Lee, Baeck-seung; Dekker, Joseph D; Lee, Bum-kyu; Iyer, Vishwanath R; Sleckman, Barry P; Shaffer, Arthur L; Ippolito, Gregory C; Tucker, Philip W

    2013-05-01

    Recombination-activating gene 1 protein (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining (VDJ) segment recombination, an essential process for antigen receptor expression and lymphocyte development. The transcription factor BCL11A is required for B cell development, but its molecular function(s) in B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds the RAG1 promoter and Erag enhancer to activate RAG1 and RAG2 transcription in pre-B cells. We employed BCL11A overexpression with recombination substrates in a cultured pre-B cell line as well as Cre recombinase-mediated Bcl11a(lox/lox) deletion in explanted murine pre-B cells to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.

  5. The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation

    PubMed Central

    Linares, Anthony J; Lin, Chia-Ho; Damianov, Andrey; Adams, Katrina L; Novitch, Bennett G; Black, Douglas L

    2015-01-01

    The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development. DOI: http://dx.doi.org/10.7554/eLife.09268.001 PMID:26705333

  6. Specific inhibition of transcriptional activity of the constitutive androstane receptor (CAR) by the splicing factor SF3a3.

    PubMed

    Yun, Hye Jin; Kwon, Jungsun; Seol, Wongi

    2008-10-01

    The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily and plays an important role in the degradation of xenobiotics in the liver. Using yeast two-hybrid screening, we identified SF3a3, a 60-kDa subunit of the splicing factor 3a complex, as a specific CAR-interacting protein. We further confirmed their interaction by both co-immunoprecipitation and GST pull-down assay. Functional studies showed that overexpression of SF3a3 inhibited the reporter activity driven by a promoter containing CAR binding sequences by up to 50%, whereas reduced expression of SF3a3 activated the same reporter activity by approximately three-fold. The inhibitory function of SF3a3 is independent of the presence of TCPOBOP, a CAR ligand. These data suggest that SF3a3 functions as a co-repressor of CAR transcriptional activity, in addition to its canonical function.

  7. Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding

    PubMed Central

    Weissbach, Julia; Schikora, Franziska; Weber, Anja; Kessels, Michael

    2016-01-01

    The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin–MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro. The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin–RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation. PMID:26976641

  8. The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2.

    PubMed

    Vető, Borbála; Bojcsuk, Dóra; Bacquet, Caroline; Kiss, Judit; Sipeki, Szabolcs; Martin, Ludovic; Buday, László; Bálint, Bálint L; Arányi, Tamás

    2017-01-01

    Hepatocyte nuclear factor 4 alpha (HNF4α) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4α is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4α. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4α. However, based on our previous results we hypothesized that HNF4α is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4α in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4α leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4α. Accordingly, we have observed decreasing but not disappearing binding of HNF4α to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4α chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4a gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4α-dependent hepatic gene expression.

  9. The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2

    PubMed Central

    Bacquet, Caroline; Kiss, Judit; Sipeki, Szabolcs; Martin, Ludovic; Buday, László; Bálint, Bálint L.; Arányi, Tamás

    2017-01-01

    Hepatocyte nuclear factor 4 alpha (HNF4α) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4α is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4α. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4α. However, based on our previous results we hypothesized that HNF4α is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4α in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4α leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4α. Accordingly, we have observed decreasing but not disappearing binding of HNF4α to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4α chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4a gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4α-dependent hepatic gene expression. PMID:28196117

  10. 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.

  11. Erythroid Krüppel-like factor (EKLF) contains a multifunctional transcriptional activation domain important for inter- and intramolecular interactions.

    PubMed Central

    Chen, X; Bieker, J J

    1996-01-01

    Erythroid Krüppel-like factor (EKLF) is a red cell-restricted transcriptional activator that plays a dominant role in establishing high levels of beta-globin gene expression during erythroid ontogeny. Although its DNA binding domain belongs to the well-studied class of Krüppel-like zinc fingers, its proline-rich activation region has not been thoroughly examined. We have analyzed this region by monitoring the functional effects of its mutagenesis upon EKLF activity in vivo and in vitro. First, using co-transfection assays, we find that the transactivation region contains discrete stimulatory and inhibitory subdomains. Second, in vitro binding assays indicate that the inhibitory domain exerts its effect in cis by interfering with DNA binding. Third, in vivo competition assays demonstrate that EKLF interacts with a positive-acting cellular factor, and that the domain responsible for this trans interaction lies within a 40 amino acid sequence that is coincident with the EKLF minimal transactivation domain. Finally, site-directed mutagenesis of this domain implies that conformation and/or phosphorylation status of its central core may be critical for such interactions. These results point towards post-translational steric and/or allosteric control of EKLF function that may be important not just for its DNA binding ability, but also for its potential to interact with other proteins that fully establish the correct stereospecific array leading to efficient switching of beta-globin transcription during development. Images PMID:8918466

  12. Antioxidant effects of Lactobacillus plantarum via activation of transcription factor Nrf2.

    PubMed

    Gao, Dawei; Gao, Zhengrong; Zhu, Guanghua

    2013-06-01

    The present study was undertaken to investigate antioxidant and hypolipidemic effects, as well as its molecular mechanism of wild Lactobacillus plantarum FC225 isolated from fermented cabbages. The scavenging activities of superoxide anion radical, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and hydroxyl radical were enhanced by FC225 treatment. The strain FC225 also attenuated hyperlipidemic status, decreased lipid peroxidation, plasma cholesterol, triglyceride and low-density lipoprotein cholesterol levels in high fat diet-fed mice. Meanwhile, FC225 therapy could significantly elevate the activities of superoxidase dismutase and glutathione peroxidase, and decrease the content of malondialdehyde (MDA) in liver homogenates, whereas there was no change in catalase activity in high fat diet-fed mice. In addition, compared with the control group, FC225 markedly elevated the gene expression of nuclear factor erythroid 2-related factor 2 (Nrf2), which was in parallel with the increased value of CD4+/CD8+ ratio in the FC225-treated hyperlipidemic mice. The results demonstrated that the strain FC225 confers hypolipidemic and antioxidant protective effects which may be attributable to Nrf2 signal pathway mediated antioxidant enzyme expression.

  13. The transcription factor Sox11 promotes nerve regeneration through activation of the regeneration-associated gene Sprr1a.

    PubMed

    Jing, Xiaotang; Wang, Ting; Huang, Shaohua; Glorioso, Joseph C; Albers, Kathryn M

    2012-01-01

    Factors that enhance the intrinsic growth potential of adult neurons are key players in the successful repair and regeneration of neurons following injury. Injury-induced activation of transcription factors has a central role in this process because they regulate expression of regeneration-associated genes. Sox11 is a developmentally expressed transcription factor that is significantly induced in adult neurons in response to injury. Its function in injured neurons is however undefined. Here, we report studies that use herpes simplex virus (HSV)-vector-mediated expression of Sox11 in adult sensory neurons to assess the effect of Sox11 overexpression on neuron regeneration. Cultured mouse dorsal root ganglia (DRG) neurons transfected with HSV-Sox11 exhibited increased neurite elongation and branching relative to naïve and HSV-vector control treated neurons. Neurons from mice injected in foot skin with HSV-Sox11 exhibited accelerated regeneration of crushed saphenous nerves as indicated by faster regrowth of axons and nerve fibers to the skin, increased myelin thickness and faster return of nerve and skin sensitivity. Downstream targets of HSV-Sox11 were examined by analyzing changes in gene expression of known regeneration-associated genes. This analysis in combination with mutational and chromatin immunoprecipitation assays indicates that the ability of Sox11 to accelerate in vivo nerve regeneration is dependent on its transcriptional activation of the regeneration-associated gene, small proline rich protein 1a (Sprr1a). This finding reveals a new functional linkage between Sox11 and Sprr1a in adult peripheral neuron regeneration.

  14. Tomato Transcription Factors Pti4, Pti5, and Pti6 Activate Defense Responses When Expressed in Arabidopsis

    PubMed Central

    Gu, Yong-Qiang; Wildermuth, Mary C.; Chakravarthy, Suma; Loh, Ying-Tsu; Yang, Caimei; He, Xiaohua; Han, Yu; Martin, Gregory B.

    2002-01-01

    The Pti4, Pti5, and Pti6 proteins from tomato were identified based on their interaction with the product of the Pto disease resistance gene, a Ser-Thr protein kinase. They belong to the ethylene-response factor (ERF) family of plant-unique transcription factors and bind specifically to the GCC-box cis element present in the promoters of many pathogenesis-related (PR) genes. Here, we show that these tomato ERFs are localized to the nucleus and function in vivo as transcription activators that regulate the expression of GCC box–containing PR genes. Expression of Pti4, Pti5, or Pti6 in Arabidopsis activated the expression of the salicylic acid–regulated genes PR1 and PR2. Expression of jasmonic acid– and ethylene-regulated genes, such as PR3, PR4, PDF1.2, and Thi2.1, was affected differently by each of the three tomato ERFs, with Arabidopsis-Pti4 plants having very high levels of PDF1.2 transcripts. Exogenous application of salicylic acid to Arabidopsis-Pti4 plants suppressed the increased expression of PDF1.2 but further stimulated PR1 expression. Arabidopsis plants expressing Pti4 displayed increased resistance to the fungal pathogen Erysiphe orontii and increased tolerance to the bacterial pathogen Pseudomonas syringae pv tomato. These results indicate that Pti4, Pti5, and Pti6 activate the expression of a wide array of PR genes and play important and distinct roles in plant defense. PMID:11971137

  15. The Transcription Factor IRF8 Activates Integrin-mediated TGFβ Signaling and Promotes Neuroinflammation

    PubMed Central

    Yoshida, Yuko; Yoshimi, Ryusuke; Yoshii, Hiroaki; Kim, Daniel; Dey, Anup; Xiong, Huabao; Munasinghe, Jeeva; Yazawa, Itaru; O’Donovan, Michael J.; Maximova, Olga A.; Sharma, Suveena; Zhu, Jinfang; Wang, Hongsheng; Morse, Herbert C.; Ozato, Keiko

    2014-01-01

    SUMMARY Recent epidemiological studies have identified interferon regulatory factor 8 (IRF8) as a susceptibility factor for multiple sclerosis (MS). However, how IRF8 influences the neuroinflammatory disease has remained unknown. By studying the role of IRF8 in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we found that Irf8−/− mice are resistant to EAE. Furthermore, expression of IRF8 in antigen presenting cells (APCs, such as macrophages, dendritic cells and microglia), but not in T cells, facilitated disease onset and progression through multiple pathways. IRF8 enhanced αvβ8 integrin expression in APCs and activated TGFβ signaling leading to T helper 17 (Th17) cell differentiation. IRF8 induced a cytokine milieu that favored growth and maintenance of Th1 and Th17 cells, by stimulating interleukin-12 (IL12) and IL23 production, but inhibiting IL27 during EAE. Finally, IRF8 activated microglia and exacerbated neuroinflammation. Together, this work provides mechanistic bases by which IRF8 contributes to the pathogenesis of MS. PMID:24485804

  16. The transcription factor IRF8 activates integrin-mediated TGF-β signaling and promotes neuroinflammation.

    PubMed

    Yoshida, Yuko; Yoshimi, Ryusuke; Yoshii, Hiroaki; Kim, Daniel; Dey, Anup; Xiong, Huabao; Munasinghe, Jeeva; Yazawa, Itaru; O'Donovan, Michael J; Maximova, Olga A; Sharma, Suveena; Zhu, Jinfang; Wang, Hongsheng; Morse, Herbert C; Ozato, Keiko

    2014-02-20

    Recent epidemiological studies have identified interferon regulatory factor 8 (IRF8) as a susceptibility factor for multiple sclerosis (MS). However, how IRF8 influences the neuroinflammatory disease has remained unknown. By studying the role of IRF8 in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we found that Irf8(-/-) mice are resistant to EAE. Furthermore, expression of IRF8 in antigen-presenting cells (APCs, such as macrophages, dendritic cells, and microglia), but not in T cells, facilitated disease onset and progression through multiple pathways. IRF8 enhanced αvβ8 integrin expression in APCs and activated TGF-β signaling leading to T helper 17 (Th17) cell differentiation. IRF8 induced a cytokine milieu that favored growth and maintenance of Th1 and Th17 cells, by stimulating interleukin-12 (IL-12) and IL-23 production, but inhibiting IL-27 during EAE. Finally, IRF8 activated microglia and exacerbated neuroinflammation. Together, this work provides mechanistic bases by which IRF8 contributes to the pathogenesis of MS.

  17. Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex.

    PubMed

    Sharma, Sonia; Findlay, Gregory M; Bandukwala, Hozefa S; Oberdoerffer, Shalini; Baust, Beate; Li, Zhigang; Schmidt, Valentina; Hogan, Patrick G; Sacks, David B; Rao, Anjana

    2011-07-12

    Nuclear factor of activated T cells (NFAT) proteins are Ca(2+)-regulated transcription factors that control gene expression in many cell types. NFAT proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular Ca(2+), NFAT proteins are dephosphorylated by the Ca(2+)/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. Here we show that phosphorylated NFAT1 is present in a large cytoplasmic RNA-protein scaffold complex that contains a long intergenic noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of NFAT]; a scaffold protein, IQ motif containing GTPase activating protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. Combined knockdown of NRON and IQGAP1 increased NFAT dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of NFAT rephosphorylation and nuclear export; and both NRON-depleted T cells and T cells from IQGAP1-deficient mice showed increased production of NFAT-dependent cytokines. Our results provide evidence that a complex of lincRNA and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincRNAs that bind transcriptional regulators have a similar scaffold function.

  18. Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex

    PubMed Central

    Sharma, Sonia; Findlay, Gregory M.; Bandukwala, Hozefa S.; Oberdoerffer, Shalini; Baust, Beate; Li, Zhigang; Schmidt, Valentina; Hogan, Patrick G.; Sacks, David B.; Rao, Anjana

    2011-01-01

    Nuclear factor of activated T cells (NFAT) proteins are Ca2+-regulated transcription factors that control gene expression in many cell types. NFAT proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular Ca2+, NFAT proteins are dephosphorylated by the Ca2+/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. Here we show that phosphorylated NFAT1 is present in a large cytoplasmic RNA-protein scaffold complex that contains a long intergenic noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of NFAT]; a scaffold protein, IQ motif containing GTPase activating protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. Combined knockdown of NRON and IQGAP1 increased NFAT dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of NFAT rephosphorylation and nuclear export; and both NRON-depleted T cells and T cells from IQGAP1-deficient mice showed increased production of NFAT-dependent cytokines. Our results provide evidence that a complex of lincRNA and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincRNAs that bind transcriptional regulators have a similar scaffold function. PMID:21709260

  19. Activation of the transcription factor NF-κB by retrieval is required for long-term memory reconsolidation

    PubMed Central

    Merlo, Emiliano; Freudenthal, Ramiro; Maldonado, Héctor; Romano, Arturo

    2005-01-01

    Several studies support that stored memories undergo a new period of consolidation after retrieval. It is not known whether this process, termed reconsolidation, requires the same transcriptional mechanisms involved in consolidation. Increasing evidence supports the participation of the transcription factor NF-κB in memory. This was initially demonstrated in the crab Chasmagnathus model of associative contextual memory, in which re-exposure to the training context induces a well characterized reconsolidation process. Here we studied the role of NF-κB in reconsolidation. NF-κB was specifically activated in trained animals re-exposed to the training context but not to a different context. NF-κB was not activated when animals were re-exposed to the context after a weak training protocol insufficient to induce long-term memory. A specific inhibitor of the NF-κB pathway, sulfasalazine, impaired reconsolidation when administered 20 min before re-exposure to the training context but was not effective when a different context was used. These findings indicate for the first time that NF-κB is activated specifically by retrieval and that this activation is required for memory reconsolidation, supporting the view that this molecular mechanism is required in both consolidation and reconsolidation. PMID:15687229

  20. Overexpression of the Transcription Factor Sp1 Activates the OAS-RNAse L-RIG-I Pathway

    PubMed Central

    Dupuis-Maurin, Valéryane; Brinza, Lilia; Baguet, Joël; Plantamura, Emilie; Schicklin, Stéphane; Chambion, Solène; Macari, Claire; Tomkowiak, Martine; Deniaud, Emmanuelle; Leverrier, Yann

    2015-01-01

    Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1) is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells in vitro and in vivo. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen. PMID:25738304

  1. Overexpression of the transcription factor Sp1 activates the OAS-RNAse L-RIG-I pathway.

    PubMed

    Dupuis-Maurin, Valéryane; Brinza, Lilia; Baguet, Joël; Plantamura, Emilie; Schicklin, Stéphane; Chambion, Solène; Macari, Claire; Tomkowiak, Martine; Deniaud, Emmanuelle; Leverrier, Yann; Marvel, Jacqueline; Michallet, Marie-Cécile

    2015-01-01

    Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1) is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells in vitro and in vivo. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen.

  2. Cdk2-dependent phosphorylation of Id2 modulates activity of E2A-related transcription factors.

    PubMed Central

    Hara, E; Hall, M; Peters, G

    1997-01-01

    The helix-loop-helix (HLH) protein Id2 is thought to affect the balance between cell growth and differentiation by negatively regulating the function of basic-helix-loop-helix (bHLH) transcription factors. Id2 acts by forming heterodimers that are unable to bind to specific (E-box) DNA sequences. Here we show that this activity can be overcome by phosphorylation of a serine residue within a consensus target site for cyclin-dependent kinases (Cdks). In vitro, Id2 can be phosphorylated by either cyclin E-Cdk2 or cyclin A-Cdk2 but not by cyclin D-dependent kinases. Analogous phosphorylation occurs in serum-stimulated human diploid fibroblasts at a time in late G1 consistent with the appearance of active cyclin E-Cdk2. The phosphorylation of Id2 in these cells correlates with the restoration of a distinct E-box-dependent DNA-binding complex, suggesting that the levels of this complex are modulated by both the abundance and phosphorylation status of Id2. These data provide a link between cyclin-dependent kinases and bHLH transcription factors that may be critical for the regulation of cell proliferation and differentiation. PMID:9029153

  3. hTERT promotes tumor angiogenesis by activating VEGF via interactions with the Sp1 transcription factor

    PubMed Central

    Liu, Ning; Ding, Deqiang; Hao, Wanyu; Yang, Fan; Wu, Xiaoying; Wang, Miao; Xu, Xiaoling; Ju, Zhenyu; Liu, Jun-Ping; Song, Zhangfa; Shay, Jerry W.; Guo, Yunliang; Cong, Yu-Sheng

    2016-01-01

    Angiogenesis is recognized as an important hallmark of cancer. Although telomerase is thought to be involved in tumor angiogenesis, the evidence and underlying mechanism remain elusive. Here, we demonstrate that human telomerase reverse transcriptase (hTERT) activates vascular epithelial growth factor (VEGF) gene expression through interactions with the VEGF promoter and the transcription factor Sp1. hTERT binds to Sp1 in vitro and in vivo and stimulates angiogenesis in a manner dependent on Sp1. Deletion of the mTert gene in the first generation of Tert null mice compromised tumor growth, with reduced VEGF expression. In addition, we show that hTERT expression levels are positively correlated with those of VEGF in human gastric tumor samples. Together, our results demonstrate that hTERT facilitates tumor angiogenesis by up-regulating VEGF expression through direct interactions with the VEGF gene and the Sp1 transcription factor. These results provide novel insights into hTERT function in tumor progression in addition to its role in telomere maintenance. PMID:27325744

  4. Activation of human papillomavirus type 18 E6-E7 oncogene expression by transcription factor Sp1.

    PubMed Central

    Hoppe-Seyler, F; Butz, K

    1992-01-01

    The human papillomavirus 18 (HPV18) E6 and E7 proteins are considered to be primarily responsive for the transforming activity of the virus. In order to analyse the molecular mechanisms resulting in viral oncoprotein expression, it is necessary to identify the factors involved in the transcriptional regulation of the E6/E7 genes. Here we define by gel retardation experiments a sequence aberrant Sp1 binding site present in the promoter proximal part of the viral transcriptional control region (Upstream Regulatory Region, URR). Functional analyses employing transient reporter assays reveal that this Sp1 element is required for an efficient stimulation of the HPV18 E6/E7-promoter. Mutation of the Sp1 element in the natural context of the HPV18 URR leads to a strong decrease in the activity of the E6/E7-promoter in several cell lines. The magnitude of reduction varies between different cell types and is higher in cell lines of epithelial origin when compared with nonepithelial cells. Cotransfection assays using Sp1 expression vector systems further define the promoter proximal HPV18 Sp1 binding motif as a functional Sp1 element in vivo and show that its integrity is essential for the stimulation of the E6/E7-promoter by augmented levels of Sp1. These results indicate, that the cellular transcription factor Sp1 plays an important role for the stimulation of the E6/E7-promoter by the viral URR and represents a major determinant for the expression of HPV18 transforming genes E6 and E7. Images PMID:1336181

  5. Epigenome analysis reveals TBX5 as a novel transcription factor involved in the activation of rheumatoid arthritis synovial fibroblasts.

    PubMed

    Karouzakis, Emmanuel; Trenkmann, Michelle; Gay, Renate E; Michel, Beat A; Gay, Steffen; Neidhart, Michel

    2014-11-15

    In this study, we analyzed the methylation status of human promoters in rheumatoid arthritis synovial fibroblasts (RASF). Differentially methylated genes between RASF and osteoarthritis synovial fibroblasts (OASF) were identified by methylated DNA immunoprecipitation and hybridization to human promoter tiling arrays. The methylation status was confirmed by pyrosequencing. Gene and protein expression of differentially methylated genes was evaluated with real-time PCR, Western blot, and immunohistochemistry. Chromatin immunoprecipitation was used to measure the gene promoter-associated acetylation and methylation of histones. Transcription factor-specific targets were identified with microarray and luciferase assays. We found that the transcription factor T-box transcription factor 5 (TBX5) was less methylated in rheumatoid arthritis (RA) synovium and RASF than in osteoarthritis (OA) samples. Demethylation of the TBX5 promoter in RASF and RA synovium was accompanied by higher TBX5 expression than in OASF and OA synovium. In RA synovium, TBX5 expression was primarily localized to the synovial lining. In addition, the TBX5 locus was enriched in activating chromatin marks, such as histone 4 lysine 4 trimethylation and histone acetylation, in RASF. In our functional studies, we observed that 790 genes were differentially expressed by 2-6-fold after overexpression of TBX5 in OASF. Bioinformatic analysis of these genes revealed that the chemokines IL-8, CXCL12, and CCL20 were common targets of TBX5 in OASF. Taken together, our data show that TBX5 is a novel inducer of important chemokines in RASF. Thus, we conclude that RASF contribute to the inflammatory processes operating in the pathogenesis of RA via epigenetic control of TBX5.

  6. An activating transcription factor of Litopenaeus vannamei involved in WSSV genes Wsv059 and Wsv166 regulation.

    PubMed

    Li, Xiao-Yun; Yue, Hai-Tao; Zhang, Ze-Zhi; Bi, Hai-Tao; Chen, Yong-Gui; Weng, Shao-Ping; Chan, Siuming; He, Jian-Guo; Chen, Yi-Hong

    2014-12-01

    Members of activating transcription factor/cyclic adenosine 3', 5'-monophosphate response element binding protein (ATF/CREB) family are induced by various stress signals and function as effector molecules. Consequently, cellular changes occur in response to discrete sets of instructions. In this work, we found an ATF transcription factor in Litopenaeus vannamei designated as LvATFβ. The full-length cDNA of LvATFβ was 1388 bp long with an open reading frame of 939 bp that encoded a putative 313 amino acid protein. The protein contained a basic region-leucine zipper (bZip) domain that was a common feature among ATF/CREB transcription factors. LvATFβ was highly expressed in intestines, gills, and heart. LvATFβ expression was dramatically upregulated by white spot syndrome virus (WSSV) infection. Pull-down assay revealed that LvATFβ had strong affinity to promoters of WSSV genes, namely, wsv059 and wsv166. Dual-luciferase reporter assay showed that LvATFβ could upregulate the expression of wsv059 and wsv166. Knocked down LvATFβ resulted in decreased expression of wsv059 and wsv166 in WSSV-challenged L. vannamei. Knocked down expression of wsv059 and wsv166 by RNA interference inhibited the replication and reduce the mortality of L. vannamei during WSSV challenge inoculation. The copy numbers of WSSV in wsv059 and wsv166 knocked down group were significant lower than in the control. These results suggested that LvATFβ may be involved in WSSV replication by regulating the expression of wsv059 and wsv166.

  7. Interactions of transcription factors with chromatin.

    PubMed

    van Bakel, Harm

    2011-01-01

    Sequence-specific transcription factors (TFs) play a central role in regulating transcription initiation by directing the recruitment and activity of the general transcription machinery and accessory factors. It is now well established that many of the effects exerted by TFs in eukaryotes are mediated through interactions with a host of coregulators that modify the chromatin state, resulting in a more open (in case of activation) or closed conformation (in case of repression). The relationship between TFs and chromatin is a two-way street, however, as chromatin can in turn influence the recognition and binding of target sequences by TFs. The aim of this chapter is to highlight how this dynamic interplay between TF-directed remodelling of chromatin and chromatin-adjusted targeting of TF binding determines where and how transcription is initiated, and to what degree it is productive.

  8. Activation of the Transcription Factor NF-[Kappa]B by Retrieval Is Required for Long-Term Memory Reconsolidation

    ERIC Educational Resources Information Center

    Maldonado, Hector; Romano, Arturo; Merlo, Emiliano; Freudenthal, Ramiro

    2005-01-01

    Several studies support that stored memories undergo a new period of consolidation after retrieval. It is not known whether this process, termed reconsolidation, requires the same transcriptional mechanisms involved in consolidation. Increasing evidence supports the participation of the transcription factor NF-[Kappa]B in memory. This was…

  9. Induction of the POU domain transcription factor Oct-2 during T-cell activation by cognate antigen.

    PubMed Central

    Kang, S M; Tsang, W; Doll, S; Scherle, P; Ko, H S; Tran, A C; Lenardo, M J; Staudt, L M

    1992-01-01

    Oct-2 is a transcription factor that binds specifically to octamer DNA motifs in the promoters of immunoglobulin and interleukin-2 genes. All tumor cell lines from the B-cell lineage and a few from the T-cell lineage express Oct-2. To address the role of Oct-2 in the T-cell lineage, we studied the expression of Oct-2 mRNA and protein in nontransformed human and mouse T cells. Oct-2 was found in CD4+ and CD8+ T cells prepared from human peripheral blood and in mouse lymph node T cells. In a T-cell clone specific for pigeon cytochrome c in the context of I-Ek, Oct-2 was induced by antigen stimulation, with the increase in Oct-2 protein seen first at 3 h after activation and continuing for at least 24 h. Oct-2 mRNA induction during antigen-driven T-cell activation was blocked by cyclosporin A, as well as by protein synthesis inhibitors. These results suggest that Oct-2 participates in transcriptional regulation during T-cell activation. The relatively delayed kinetics of Oct-2 induction suggests that Oct-2 mediates the changes in gene expression which occur many hours or days following antigen stimulation of T lymphocytes. Images PMID:1620122

  10. Casticin inhibits the activity of transcription factor Sp1 and the methylation of RECK in MGC803 gastric cancer cells

    PubMed Central

    Yang, Fan; He, Kefei; Huang, Li; Zhang, Lingyan; Liu, Aixue; Zhang, Jiren

    2017-01-01

    The present study investigated the effect of casticin on reversion-inducing-cysteine-rich protein with kazal motifs (RECK) gene expression and intracellular methylation levels in MGC803 gastric cancer cells. Cells were treated with 1, 10 and 30 µmol/l casticin. Western blotting and reverse transcription-quantitative polymerase chain reaction assays were performed to determine the protein expression and mRNA levels of RECK and DNA methyltransferase 1 (DNMT1), respectively. High-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry was used to detect RECK methylation. In addition, MGC803 cell proliferation was measured by an MTT assay and the DNA-binding activity of transcription factor Sp1 was determined using an enzyme-linked immunosorbent assay. The results demonstrated that treatment with 1, 10 and 30 µmol/l casticin significantly increased RECK protein expression and mRNA levels. In addition, casticin (30 µmol/l) decreased RECK promoter methylation levels by 31%, global DNA methylation levels by 39% and nuclear methylation activity by 71.6%. Furthermore, casticin downregulated the mRNA levels and protein expression of DNMT1. The MTT assay demonstrated that MGC803 cell proliferation was inhibited by casticin treatment and DNA binding assays indicated that casticin reduced the DNA-binding activity of Sp1. The present study therefore indicated that casticin inhibits the proliferation of gastric cancer MGC803 cells by upregulating RECK gene expression and reducing intracellular methylation levels. PMID:28352361

  11. Enhancing succinic acid biosynthesis in Escherichia coli by engineering its global transcription factor, catabolite repressor/activator (Cra)

    PubMed Central

    Zhu, Li-Wen; Xia, Shi-Tao; Wei, Li-Na; Li, Hong-Mei; Yuan, Zhan-Peng; Tang, Ya-Jie

    2016-01-01

    This study was initiated to improve E. coli succinate production by engineering the E. coli global transcription factor, Cra (catabolite repressor/activator). Random mutagenesis libraries were generated through error-prone PCR of cra. After re-screening and mutation site integration, the best mutant strain was Tang1541, which provided a final succinate concentration of 79.8 ± 3.1 g/L: i.e., 22.8% greater than that obtained using an empty vector control. The genes and enzymes involved in phosphoenolpyruvate (PEP) carboxylation and the glyoxylate pathway were activated, either directly or indirectly, through the mutation of Cra. The parameters for interaction of Cra and DNA indicated that the Cra mutant was bound to aceBAK, thereby activating the genes involved in glyoxylate pathway and further improving succinate production even in the presence of its effector fructose-1,6-bisphosphate (FBP). It suggested that some of the negative effect of FBP on Cra might have been counteracted through the enhanced binding affinity of the Cra mutant for FBP or the change of Cra structure. This work provides useful information about understanding the transcriptional regulation of succinate biosynthesis. PMID:27811970

  12. Transcription Factors in Xylem Development. Final report

    SciTech Connect

    Sederoff, Ronald; Whetten, Ross; O'Malley, David; Campbell, Malcolm

    1999-07-01

    Answers to the following questions are answered in this report. do the two pine Byb proteins previously identified as candidate transcription factors bind to DNA and activate transcription? In what cell types are tehse Myb proteins expressed? Are these proteins localized to the nucleus? Do other proteins in pine xylem interact with these Myb proteins? Does altered expression of these genes have an impact on xylogenesis, specifically the expression of monolignol biosynthetic genes?

  13. 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.

  14. The transcription factor IRF1 and guanylate-binding proteins target activation of the AIM2 inflammasome by Francisella infection.

    PubMed

    Man, Si Ming; Karki, Rajendra; Malireddi, R K Subbarao; Neale, Geoffrey; Vogel, Peter; Yamamoto, Masahiro; Lamkanfi, Mohamed; Kanneganti, Thirumala-Devi

    2015-05-01

    Inflammasomes are critical for mounting host defense against pathogens. The molecular mechanisms that control activation of the AIM2 inflammasome in response to different cytosolic pathogens remain unclear. Here we found that the transcription factor IRF1 was required for activation of the AIM2 inflammasome during infection with the Francisella tularensis subspecies novicida (F. novicida), whereas engagement of the AIM2 inflammasome by mouse cytomegalovirus (MCMV) or transfected double-stranded DNA did not require IRF1. Infection of F. novicida detected by the DNA sensor cGAS and its adaptor STING induced type I interferon-dependent expression of IRF1, which drove the expression of guanylate-binding proteins (GBPs); this led to intracellular killing of bacteria and DNA release. Our results reveal a specific requirement for IRF1 and GBPs in the liberation of DNA for sensing by AIM2 depending on the pathogen encountered by the cell.

  15. Common and distinct DNA-binding and regulatory activities of the BEN-solo transcription factor family

    PubMed Central

    Dai, Qi; Ren, Aiming; Westholm, Jakub O.; Duan, Hong; Patel, Dinshaw J.

    2015-01-01

    Recently, the BEN (BANP, E5R, and NAC1) domain was recognized as a new class of conserved DNA-binding domain. The fly genome encodes three proteins that bear only a single BEN domain (“BEN-solo” factors); namely, Insensitive (Insv), Bsg25A (Elba1), and CG9883 (Elba2). Insv homodimers preferentially bind CCAATTGG palindromes throughout the genome to mediate transcriptional repression, whereas Bsg25A and Elba2 heterotrimerize with their obligate adaptor, Elba3 (i.e., the ELBA complex), to recognize a CCAATAAG motif in the Fab-7 insulator. While these data suggest distinct DNA-binding properties of BEN-solo proteins, we performed reporter assays that indicate that both Bsg25A and Elba2 can individually recognize Insv consensus sites efficiently. We confirmed this by solving the structure of Bsg25A complexed to the Insv site, which showed that key aspects of the BEN:DNA recognition strategy are similar between these proteins. We next show that both Insv and ELBA proteins are competent to mediate transcriptional repression via Insv consensus sequences but that the ELBA complex appears to be selective for the ELBA site. Reciprocally, genome-wide analysis reveals that Insv exhibits significant cobinding to class I insulator elements, indicating that it may also contribute to insulator function. Indeed, we observed abundant Insv binding within the Hox complexes with substantial overlaps with class I insulators, many of which bear Insv consensus sites. Moreover, Insv coimmunoprecipitates with the class I insulator factor CP190. Finally, we observed that Insv harbors exclusive activity among fly BEN-solo factors with respect to regulation of Notch-mediated cell fate choices in the peripheral nervous system. This in vivo activity is recapitulated by BEND6, a mammalian BEN-solo factor that conserves the Notch corepressor function of Insv but not its capacity to bind Insv consensus sites. Altogether, our data define an array of common and distinct biochemical and functional

  16. G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor.

    PubMed

    Morita, Tsuyoshi; Hayashi, Ken'ichiro

    2013-08-02

    Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin-MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF-SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin-MRTFs interaction.

  17. A Homeodomain Transcription Factor Gene, PfMSX, Activates Expression of Pif Gene in the Pearl Oyster Pinctada fucata

    PubMed Central

    Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

    2014-01-01

    We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5′ flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster. PMID:25099698

  18. Targeting the DNA-binding activity of the human ERG transcription factor using new heterocyclic dithiophene diamidines

    PubMed Central

    Nhili, Raja; Peixoto, Paul; Depauw, Sabine; Flajollet, Sébastien; Dezitter, Xavier; Munde, Manoj M.; Ismail, Mohamed A.; Kumar, Arvind; Farahat, Abdelbasset A.; Stephens, Chad E.; Duterque-Coquillaud, Martine; David Wilson, W.; Boykin, David W.; David-Cordonnier, Marie-Hélène

    2013-01-01

    Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. In this work, we selected the di-(thiophene-phenyl-amidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We further highlighted the structure activity relationships from comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site. PMID:23093599

  19. RNA helicase HEL-1 promotes longevity by specifically activating DAF-16/FOXO transcription factor signaling in Caenorhabditis elegans.

    PubMed

    Seo, Mihwa; Seo, Keunhee; Hwang, Wooseon; Koo, Hee Jung; Hahm, Jeong-Hoon; Yang, Jae-Seong; Han, Seong Kyu; Hwang, Daehee; Kim, Sanguk; Jang, Sung Key; Lee, Yoontae; Nam, Hong Gil; Lee, Seung-Jae V

    2015-08-04

    The homeostatic maintenance of the genomic DNA is crucial for regulating aging processes. However, the role of RNA homeostasis in aging processes remains unknown. RNA helicases are a large family of enzymes that regulate the biogenesis and homeostasis of RNA. However, the functional significance of RNA helicases in aging has not been explored. Here, we report that a large fraction of RNA helicases regulate the lifespan of Caenorhabditis elegans. In particular, we show that a DEAD-box RNA helicase, helicase 1 (HEL-1), promotes longevity by specifically activating the DAF-16/forkhead box O (FOXO) transcription factor signaling pathway. We find that HEL-1 is required for the longevity conferred by reduced insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) and is sufficient for extending lifespan. We further show that the expression of HEL-1 in the intestine and neurons contributes to longevity. HEL-1 enhances the induction of a large fraction of DAF-16 target genes. Thus, the RNA helicase HEL-1 appears to promote longevity in response to decreased IIS as a transcription coregulator of DAF-16. Because HEL-1 and IIS are evolutionarily well conserved, a similar mechanism for longevity regulation via an RNA helicase-dependent regulation of FOXO signaling may operate in mammals, including humans.

  20. TRANSLUCENT GREEN, an ERF family transcription factor, controls water balance in Arabidopsis by activating the expression of aquaporin genes.

    PubMed

    Zhu, Danling; Wu, Zhe; Cao, Guangyu; Li, Jigang; Wei, Jia; Tsuge, Tomohiko; Gu, Hongya; Aoyama, Takashi; Qu, Li-Jia

    2014-04-01

    Water is the most abundant molecule in almost all living organisms. Aquaporins are channel proteins that play critical roles in controlling the water content of cells. Here, we report the identification of an AP2/EREBP family transcription factor in Arabidopsis thaliana, TRANSLUCENT GREEN (TG), whose overexpression in transgenic plants gave enhanced drought tolerance and vitrified leaves. TG protein is localized in the nucleus, binds DRE and GCC elements in vitro, and acts as a transcriptional activator in yeast cells. Microarray analysis revealed a total of 330 genes regulated by TG, among which five genes encode aquaporins. A transient expression assay showed that TG directly binds to the promoters of three aquaporin genes, such as AtTIP1;1, AtTIP2;3, and AtPIP2;2, indicating that TG directly regulates the expression of these genes. Moreover, overexpression of AtTIP1;1 resulted in vitrified phenotypes in transgenic Arabidopsis plants, similar to those observed in TG overexpression lines. Water injection into wild-type leaves recapitulated the vitrified leaf phenotypes, which was reversed by cutting off the water supply from vascular bundles. Taken together, our data support that TG controls water balance in Arabidopsis through directly activating the expression of aquaporin genes.

  1. Reconstructing genome-wide regulatory network of E. coli using transcriptome data and predicted transcription factor activities

    PubMed Central

    2011-01-01

    Background Gene regulatory networks play essential roles in living organisms to control growth, keep internal metabolism running and respond to external environmental changes. Understanding the connections and the activity levels of regulators is important for the research of gene regulatory networks. While relevance score based algorithms that reconstruct gene regulatory networks from transcriptome data can infer genome-wide gene regulatory networks, they are unfortunately prone to false positive results. Transcription factor activities (TFAs) quantitatively reflect the ability of the transcription factor to regulate target genes. However, classic relevance score based gene regulatory network reconstruction algorithms use models do not include the TFA layer, thus missing a key regulatory element. Results This work integrates TFA prediction algorithms with relevance score based network reconstruction algorithms to reconstruct gene regulatory networks with improved accuracy over classic relevance score based algorithms. This method is called Gene expression and Transcription factor activity based Relevance Network (GTRNetwork). Different combinations of TFA prediction algorithms and relevance score functions have been applied to find the most efficient combination. When the integrated GTRNetwork method was applied to E. coli data, the reconstructed genome-wide gene regulatory network predicted 381 new regulatory links. This reconstructed gene regulatory network including the predicted new regulatory links show promising biological significances. Many of the new links are verified by known TF binding site information, and many other links can be verified from the literature and databases such as EcoCyc. The reconstructed gene regulatory network is applied to a recent transcriptome analysis of E. coli during isobutanol stress. In addition to the 16 significantly changed TFAs detected in the original paper, another 7 significantly changed TFAs have been detected by

  2. Two different factors act separately or together to specify functionally distinct activities at a single transcriptional enhancer.

    PubMed Central

    DeFranco, D; Yamamoto, K R

    1986-01-01

    The expression of genes fused downstream of the Moloney murine sarcoma virus (MoMSV) long terminal repeat is stimulated by glucocorticoids. We mapped the glucocorticoid response element that conferred this hormonal regulation and found that it is a hormone-dependent transcriptional enhancer, designated Sg; it resides within DNA fragments that also carry a previously described enhancer element (B. Levinson, G. Khoury, G. Vande Woude, and P. Gruss, Nature [London] 295:568-572, 1982), here termed Sa, whose activity is independent of the hormone. Nuclease footprinting revealed that purified glucocorticoid receptor bound at multiple discrete sites within and at the borders of the tandemly repeated sequence motif that defines Sa. The Sa and Sg activities stimulated the apparent efficiency of cognate or heterologous promoter utilization, individually providing modest enhancement and in concert yielding higher levels of activity. A deletion mutant lacking most of the tandem repeat but retaining a single receptor footprint sequence lost Sa activity but still conferred Sg activity. The two enhancer components could also be distinguished physiologically: both were operative within cultured rat fibroblasts, but only Sg activity was detectable in rat exocrine pancreas cells. Therefore, the sequence determinants of Sa and Sg activity may be interdigitated, and when both components are active, the receptor and a putative Sa factor can apparently bind and act simultaneously. We concluded that MoMSV enhancer activity is effected by at least two distinct binding factors, suggesting that combinatorial regulation of promoter function can be mediated even from a single genetic element. Images PMID:3023887

  3. Dual regulation of heat-shock transcription factor (HSF) activation and DNA-binding activity by H2O2: role of thioredoxin.

    PubMed Central

    Jacquier-Sarlin, M R; Polla, B S

    1996-01-01

    The heat-shock (HS) response is a ubiquitous cellular response to stress, involving the transcriptional activation of HS genes. Reactive oxygen species (ROS) have been shown to regulate the activity of a number of transcription factors. We investigated the redox regulation of the stress response and report here that in the human pre-monocytic line U937 cells, H2O2 induced a concentration-dependent transactivation and DNA-binding activity of heat-shock factor-1 (HSF-1). DNA-binding activity was, however, lower with H2O2 than with HS. We thus hypothesized a dual regulation of HSF by oxidants. We found that oxidizing agents, such as H2O2 and diamide, as well as alkylating agents, such as iodoacetic acid, abolished, in vitro, the HSF-DNA-binding activity induced by HS in vivo. The effects of H2O2 in vitro were reversed by the sulphydryl reducing agent dithiothreitol and the endogenous reductor thioredoxin (TRX), while the effects of iodoacetic acid were irreversible. In addition, TRX also restored the DNA-binding activity of HSF oxidized in vivo, while it was found to be itself induced in vivo by both HS and H2O2. Thus, H2O2 exerts dual effects on the activation and the DNA-binding activity of HSF: on the one hand, H2O2 favours the nuclear translocation of HSF, while on the other, it alters HSF-DNA-binding activity, most likely by oxidizing critical cysteine residues within the DNA-binding domain. HSF thus belongs to the group of ROS-modulated transcription factors. We propose that the time required for TRX induction, which may restore the DNA-binding activity of oxidized HSF, provides an explanation for the delay in heat-shock protein synthesis upon exposure of cells to ROS. PMID:8761470

  4. Expression of peroxisome proliferator-activated receptor and CCAAT/enhancer binding protein transcription factors in cultured human sebocytes.

    PubMed

    Chen, WenChieh; Yang, Chao-Chun; Sheu, Hamm-Ming; Seltmann, Holger; Zouboulis, Christos C

    2003-09-01

    Lipid synthesis and accumulation represent a major step in sebocyte differentiation and it may be of importance for sebocytes to express two families of transcription factors, CCAAT/enhancer binding proteins (c/EBPs) and peroxisome proliferator-activated receptors (PPARs), which were found to play a crucial role in the differentiation of adipocytes. Using the immortalized human sebaceous gland cell line SZ95 we examined the expression of the molecules before and after treatment with testosterone, 5alpha-dihydrotestosterone, dexamethasone, 17beta-estradiol and genistein, at 6, 12, 24, and 48 h, respectively. Reverse transcription-PCR analysis showed expression of peroxisome proliferator-activated receptors -alpha, -delta, -gamma1, -gamma2 and CCAAT/enhancer binding proteins-alpha, -beta, -gamma-delta in native SZ95 sebocytes. In western blot studies, high levels of CCAAT/enhancer binding proteins-alpha and -beta, and peroxisome proliferator-activated receptors-gamma were expressed at 6, 24, and 12 h, respectively. Immunostaining of the cultured sebocytes showed the CCAAT/enhancer binding proteins-alpha and -beta mainly localized within nuclei, whereas peroxisome proliferator-activated receptors-gamma in the cytoplasm. Strong staining of sebocytes was immunohistochemically revealed in the basal layer of sebaceous glands in human scalp and sebaceous nevus. Genistein down-regulated the expression of CCAAT/enhancer binding proteins-alpha and -beta, and peroxisome proliferator-activated receptors-gamma on the protein level. Treatment with linoleic acid for 48 h induced further differentiation of sebocytes leading to abundant lipid synthesis.

  5. Linking Smads and transcriptional activation.

    PubMed

    Inman, Gareth J

    2005-02-15

    TGF-beta1 (transforming growth factor-beta1) is the prototypical member of a large family of pleiotropic cytokines that regulate diverse biological processes during development and adult tissue homoeostasis. TGF-beta signals via membrane bound serine/threonine kinase receptors which transmit their signals via the intracellular signalling molecules Smad2, Smad3 and Smad4. These Smads contain conserved MH1 and MH2 domains separated by a flexible linker domain. Smad2 and Smad3 act as kinase substrates for the receptors, and, following phosphorylation, they form complexes with Smad4 and translocate to the nucleus. These Smad complexes regulate gene expression and ultimately determine the biological response to TGF-beta. In this issue of the Biochemical Journal, Wang et al. have shown that, like Smad4, the linker domain of Smad3 contains a Smad transcriptional activation domain. This is capable of recruiting the p300 transcriptional co-activator and is required for Smad3-dependent transcriptional activation. This study raises interesting questions about the nature and regulation of Smad-regulated gene activation and elevates the status of the linker domain to rival that of the much-lauded MH1 and MH2 domains.

  6. Pioneer transcription factors in cell reprogramming.

    PubMed

    Iwafuchi-Doi, Makiko; Zaret, Kenneth S

    2014-12-15

    A subset of eukaryotic transcription factors possesses the remarkable ability to reprogram one type of cell into another. The transcription factors that reprogram cell fate are invariably those that are crucial for the initial cell programming in embryonic development. To elicit cell programming or reprogramming, transcription factors must be able to engage genes that are developmentally silenced and inappropriate for expression in the original cell. Developmentally silenced genes are typically embedded in "closed" chromatin that is covered by nucleosomes and not hypersensitive to nuclease probes such as DNase I. Biochemical and genomic studies have shown that transcription factors with the highest reprogramming activity often have the special ability to engage their target sites on nucleosomal DNA, thus behaving as "pioneer factors" to initiate events in closed chromatin. Other reprogramming factors appear dependent on pioneer factors for engaging nucleosomes and closed chromatin. However, certain genomic domains in which nucleosomes are occluded by higher-order chromatin structures, such as in heterochromatin, are resistant to pioneer factor binding. Understanding the means by which pioneer factors can engage closed chromatin and how heterochromatin can prevent such binding promises to advance our ability to reprogram cell fates at will and is the topic of this review.

  7. Berberine regulates peroxisome proliferator-activated receptors and positive transcription elongation factor b expression in diabetic adipocytes.

    PubMed

    Zhou, Jiyin; Zhou, Shiwen

    2010-12-15

    Berberine has hypoglycemic and hypolipidemic effects on diabetic rats. This study investigated the relationship between hypoglycemic and hypolipidemic effects of berberine and peroxisome proliferator-activated receptors (PPARs) and positive transcription elongation factor b (P-TEFb) (including cyclin-dependent kinase 9 (CDK9) and cyclin T1) in white adipose tissue of diabetic rats and RNA interference-treated 3T3-L1 cells. Berberine promoted differentiation and inhibited lipid accumulation of 3T3-L1 cells, further decreased PPARα/δ/γ, CDK9 and cyclin T1 mRNA and protein expression and decreased tumor necrosis factor α content in supernatants of both control and RNA interference-treated 3T3-L1 cells. After a 16-week induction with 35 mg/kg streptozotocin (i.p.) and high-carbohydrate/high-fat diet, diabetic rats were treated with 75, 150 and 300 mg/kg berberine and 100 mg/kg fenofibrate or 4 mg/kg rosiglitazone for another 16 weeks. Berberine decreased white adipose tissue to body weight ratio and adipocyte size and increased adipocyte number. Berberine upregulated PPARα/δ/γ, CDK9 and cyclin T1 mRNA and protein expression in adipose tissue, decreased tumor necrosis factor α and free fatty acid content and increased lipoprotein lipase activity in serum and adipose tissue. Berberine modulated metabolic related PPARs expression and differentiation related P-TEFb expression in adipocytes, which are associated with its hypoglycemic and hypolipidemic effects.

  8. A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin.

    PubMed

    Sowd, Gregory A; Serrao, Erik; Wang, Hao; Wang, Weifeng; Fadel, Hind J; Poeschla, Eric M; Engelman, Alan N

    2016-02-23

    Integration is vital to retroviral replication and influences the establishment of the latent HIV reservoir. HIV-1 integration favors active genes, which is in part determined by the interaction between integrase and lens epithelium-derived growth factor (LEDGF)/p75. Because gene targeting remains significantly enriched, relative to random in LEDGF/p75 deficient cells, other host factors likely contribute to gene-tropic integration. Nucleoporins 153 and 358, which bind HIV-1 capsid, play comparatively minor roles in integration targeting, but the influence of another capsid binding protein, cleavage and polyadenylation specificity factor 6 (CPSF6), has not been reported. In this study we knocked down or knocked out CPSF6 in parallel or in tandem with LEDGF/p75. CPSF6 knockout changed viral infectivity kinetics, decreased proviral formation, and preferentially decreased integration into transcriptionally active genes, spliced genes, and regions of chromatin enriched in genes and activating histone modifications. LEDGF/p75 depletion by contrast preferentially altered positional integration targeting within gene bodies. Dual factor knockout reduced integration into genes to below the levels observed with either single knockout and revealed that CPSF6 played a more dominant role than LEDGF/p75 in directing integration to euchromatin. CPSF6 complementation rescued HIV-1 integration site distribution in CPSF6 knockout cells, but complementation with a capsid binding mutant of CPSF6 did not. We conclude that integration targeting proceeds via two distinct mechanisms: capsid-CPSF6 binding directs HIV-1 to actively transcribed euchromatin, where the integrase-LEDGF/p75 interaction drives integration into gene bodies.

  9. Nitric Oxide-Mediated Antioxidative Mechanism in Yeast through the Activation of the Transcription Factor Mac1

    PubMed Central

    Nasuno, Ryo; Aitoku, Miho; Manago, Yuki; Nishimura, Akira; Sasano, Yu; Takagi, Hiroshi

    2014-01-01

    The budding yeast Saccharomyces cerevisiae possesses various defense mechanisms against environmental stresses that generate reactive oxygen species, leading to growth inhibition or cell death. Our recent study showed a novel antioxidative mechanism mediated by nitric oxide (NO) in yeast cells, but the mechanism underlying the oxidative stress tolerance remained unclear. We report here one of the downstream pathways of NO involved in stress-tolerance mechanism in yeast. Our microarray and real-time quantitative PCR analyses revealed that exogenous NO treatment induced the expression of genes responsible for copper metabolism under the control of the transcription factor Mac1, including the CTR1 gene encoding high-affinity copper transporter. Our ChIP analysis also demonstrated that exogenous NO enhances the binding of Mac1 to the promoter region of target genes. Interestingly, we found that NO produced under high-temperature stress conditions increased the transcription level of the CTR1 gene. Furthermore, NO produced during exposure to high temperature also increased intracellular copper content, the activity of Cu,Zn-superoxide dismutase Sod1, and cell viability after exposure to high-temperature in a manner dependent on Mac1. NO did not affect the expression of the MAC1 gene, indicating that NO activates Mac1 through its post-translational modification. Based on the results shown here, we propose a novel NO-mediated antioxidative mechanism that Mac1 activated by NO induces the CTR1 gene, leading to an increase in cellular copper level, and then Cu(I) activates Sod1. This is the first report to unveil the mechanism of NO-dependent antioxidative system in yeast. PMID:25423296

  10. Unbiased identification of signal-activated transcription factors by barcoded synthetic tandem repeat promoter screening (BC-STAR-PROM)

    PubMed Central

    Gosselin, Pauline; Rando, Gianpaolo; Fleury-Olela, Fabienne; Schibler, Ueli

    2016-01-01

    The discovery of transcription factors (TFs) controlling pathways in health and disease is of paramount interest. We designed a widely applicable method, dubbed barcorded synthetic tandem repeat promoter screening (BC-STAR-PROM), to identify signal-activated TFs without any a priori knowledge about their properties. The BC-STAR-PROM library consists of ∼3000 luciferase expression vectors, each harboring a promoter (composed of six tandem repeats of synthetic random DNA) and an associated barcode of 20 base pairs (bp) within the 3′ untranslated mRNA region. Together, the promoter sequences encompass >400,000 bp of random DNA, a sequence complexity sufficient to capture most TFs. Cells transfected with the library are exposed to a signal, and the mRNAs that it encodes are counted by next-generation sequencing of the barcodes. This allows the simultaneous activity tracking of each of the ∼3000 synthetic promoters in a single experiment. Here we establish proof of concept for BC-STAR-PROM by applying it to the identification of TFs induced by drugs affecting actin and tubulin cytoskeleton dynamics. BC-STAR-PROM revealed that serum response factor (SRF) is the only immediate early TF induced by both actin polymerization and microtubule depolymerization. Such changes in cytoskeleton dynamics are known to occur during the cell division cycle, and real-time bioluminescence microscopy indeed revealed cell-autonomous SRF–myocardin-related TF (MRTF) activity bouts in proliferating cells. PMID:27601530

  11. Fragile X mental retardation protein is required for synapse elimination by the activity-dependent transcription factor MEF2.

    PubMed

    Pfeiffer, Brad E; Zang, Tong; Wilkerson, Julia R; Taniguchi, Makoto; Maksimova, Marina A; Smith, Laura N; Cowan, Christopher W; Huber, Kimberly M

    2010-04-29

    Fragile X syndrome (FXS), the most common genetic form of mental retardation and autism, is caused by loss-of-function mutations in an RNA-binding protein, Fragile X Mental Retardation Protein (FMRP). Neurons from patients and the mouse Fmr1 knockout (KO) model are characterized by an excess of dendritic spines, suggesting a deficit in excitatory synapse elimination. In response to neuronal activity, myocyte enhancer factor 2 (MEF2) transcription factors induce robust synapse elimination. Here, we demonstrate that MEF2 activation fails to eliminate functional or structural excitatory synapses in hippocampal neurons from Fmr1 KO mice. Similarly, inhibition of endogenous MEF2 increases synapse number in wild-type but not Fmr1 KO neurons. MEF2-dependent synapse elimination is rescued in Fmr1 KO neurons by acute postsynaptic expression of wild-type but not RNA-binding mutants of FMRP. Our results reveal that active MEF2 and FMRP function together in an acute, cell-autonomous mechanism to eliminate excitatory synapses.

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

    PubMed

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

    2016-10-01

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

  13. Cloning and expression of a transcription factor activator protein-1 (AP-1) member identified from manila clam Venerupis philippinarum.

    PubMed

    Wu, Luning; Zhang, Lei; Zhao, Jianmin; Ning, Xuanxuan; Mu, Changkao; Wang, Chunlin

    2015-02-15

    The transcription factor activator protein-1 (AP-1) proteins are implicated to play a major role in the regulation of numerous genes involved in the function and development of the immune system, cell differentiation, proliferation, apoptosis, etc. It can bind to promoter of its target genes in a sequence-specific manner to transactivate or repress them. In this study, the full-length cDNA of an AP-1 was identified from Venerupis philippinarum (denoted as VpAP-1) by EST analysis and RACE approaches. Phylogenetic analysis indicated that VpAP-1 had higher evolutional conservation to invertebrate than vertebrate counterparts and should be a new member of the AP-1 protein family. Spatial expression analysis found that VpAP-1 transcript was most abundantly expressed in the hemocytes and hepatopancreas, weakly expressed in the tissues of the gills, mantle and muscle. After Vibrio anguillarum challenge, the expression of VpAP-1 transcript in overall hemocyte population was up-regulated in the first 6h, and then decreased to 1.5-fold of the control group at 12h. As time progressed, a second peak of VpAP-1 expression was detected at 24h post-infection, which was 5-fold compared with that of the control group (P<0.01). After that, the expression level was sharply decreased and dropped to 0.5-fold of the control at 96h. The above results indicated that VpAP-1 was perhaps involved in the immune responses against microbe infection and might be contributed to the clearance of bacterial pathogens.

  14. Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts

    NASA Astrophysics Data System (ADS)

    Huang, Yong-Zhen; Zhang, Liang-Zhi; Lai, Xin-Sheng; Li, Ming-Xun; Sun, Yu-Jia; Li, Cong-Jun; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

    2014-04-01

    Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were upregulated during C2C12 differentiation. The IGF2 expression levels were negatively associated with the methylation status in beef cattle (P < 0.05). A luciferase assay for the IGF2 intron 3 and P3 promoter showed that the mutant-type 439 A-SNP-pGL3 in driving reporter gene transcription is significantly higher than that of the wild-type 439 G-SNP-pGL3 construct (P < 0.05). An over-expression assay revealed that ZBED6 regulate IGF2 expression and promote myoblast differentiation. Furthermore, knockdown of ZBED6 led to IGF2 expression change in vitro. Taken together, these results suggest that ZBED6 inhibits IGF2 activity and expression via a G to A transition disrupts the interaction. Thus, we propose that ZBED6 plays a critical role in myogenic differentiation.

  15. JMJD3 aids in reprogramming of bone marrow progenitor cells to hepatic phenotype through epigenetic activation of hepatic transcription factors

    PubMed Central

    Kochat, Veena; Equbal, Zaffar; Baligar, Prakash; Kumar, Vikash; Srivastava, Madhulika; Mukhopadhyay, Asok

    2017-01-01

    The strictly regulated unidirectional differentiation program in some somatic stem/progenitor cells has been found to be modified in the ectopic site (tissue) undergoing regeneration. In these cases, the lineage barrier is crossed by either heterotypic cell fusion or direct differentiation. Though studies have shown the role of coordinated genetic and epigenetic mechanisms in cellular development and differentiation, how the lineage fate of adult bone marrow progenitor cells (BMPCs) is reprogrammed during liver regeneration and whether this lineage switch is stably maintained are not clearly understood. In the present study, we wanted to decipher genetic and epigenetic mechanisms that involve in lineage reprogramming of BMPCs into hepatocyte-like cells. Here we report dynamic transcriptional change during cellular reprogramming of BMPCs to hepatocytes and dissect the epigenetic switch mechanism of BM cell-mediated liver regeneration after acute injury. Genome-wide gene expression analysis in BM-derived hepatocytes, isolated after 1 month and 5 months of transplantation, showed induction of hepatic transcriptional program and diminishing of donor signatures over the time. The transcriptional reprogramming of BM-derived cells was found to be the result of enrichment of activating marks (H3K4me3 and H3K9Ac) and loss of repressive marks (H3K27me3 and H3K9me3) at the promoters of hepatic transcription factors (HTFs). Further analyses showed that BMPCs possess bivalent histone marks (H3K4me3 and H3K27me3) at the promoters of crucial HTFs. H3K27 methylation dynamics at the HTFs was antagonistically regulated by EZH2 and JMJD3. Preliminary evidence suggests a role of JMJD3 in removal of H3K27me3 mark from promoters of HTFs, thus activating epigenetically poised hepatic genes in BMPCs prior to partial nuclear reprogramming. The importance of JMJD3 in reprogramming of BMPCs to hepatic phenotype was confirmed by inhibiting catalytic function of the enzyme using small molecule

  16. Tailor-Made Zinc-Finger Transcription Factors Activate FLO11 Gene Expression with Phenotypic Consequences in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Shieh, Jia-Ching; Cheng, Yu-Che; Su, Mao-Chang; Moore, Michael; Choo, Yen; Klug, Aaron

    2007-01-01

    Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5′ UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity. PMID:17710146

  17. The forkhead transcription factor FOXO3a controls microglial inflammatory activation and eventual apoptotic injury through caspase 3.

    PubMed

    Shang, Yan Chen; Chong, Zhao Zhong; Hou, Jinling; Maiese, Kenneth

    2009-02-01

    Memory loss and cognitive failure are increasingly being identified as potential risks with the recognized increase in life expectancy of the general population. As a result, the development of novel therapeutic strategies for disorders such as Alzheimer's disease have garnered increased attention. The etiologies that can lead to Alzheimer's disease are extremely varied, but a number of therapeutic options are directed against amyloid-beta peptide and inflammatory cell regulation to prevent or halt progressive cognitive loss. In particular, inflammatory microglial cells may have disparate functions that in some scenarios lead to disability through the removal of functional neurovascular cells and in other circumstances foster tissue repair. Given the significance microglial cells hold for neurodegenerative disorders, we therefore examined the function that amyloid (Abeta(1-42)) has upon the microglial cell line EOC 2 and identified a novel role for the forkhead transcription factor FoxO3a and caspase 3. Here we show that Abeta(1-42) leads to progressive injury and apoptotic cell loss in microglial cells that involves both early phosphatidylserine (PS) externalization and late genomic DNA fragmentation over a 24 hour course. Prior to these injury programs, Abeta(1-42) results in the activation and proliferation of microglia as demonstrated by increased proliferating cell nuclear antigen (PCNA) expression and bromodeoxyuridine (BrdU) uptake. Both apoptotic injury as well as the prior activation and proliferation of microglial cells relies upon the presence of FoxO3a, since specific gene silencing of FoxO3a promotes microglial cell protection and prevents the early activation and proliferation of these cells. Furthermore, Abeta(1-42) exposure maintained FoxO3a in an unphosphorylated "active" state and facilitated the cellular trafficking of FoxO3a from the cytoplasm to the cell nucleus to potentially lead to "pro-apoptotic" programs by this transcription factor. One

  18. A WRKY Transcription Factor Recruits the SYG1-Like Protein SHB1 to Activate Gene Expression and Seed Cavity Enlargement

    PubMed Central

    Kang, Xiaojun; Li, Wei; Zhou, Yun; Ni, Min

    2013-01-01

    Seed development in Arabidopsis and in many dicots involves an early proliferation of the endosperm to form a large embryo sac or seed cavity close to the size of the mature seed, followed by a second phase during which the embryo grows and replaces the endosperm. SHORT HYPOCOTYL UNDER BLUE1 (SHB1) is a member of the SYG1 protein family in fungi, Caenorhabditis elegans, flies, and mammals. SHB1 gain-of-function enhances endosperm proliferation, increases seed size, and up-regulates the expression of the WRKY transcription factor gene MINISEED3 (MINI3) and the LRR receptor kinase gene HAIKU2 (IKU2). Mutations in either IKU2 or MINI3 retard endosperm proliferation and reduce seed size. However, the molecular mechanisms underlying the establishment of the seed cavity and hence the seed size remain largely unknown. Here, we show that the expression of MINI3 and IKU2 is repressed before fertilization and after 4 days after pollination (DAP), but is activated by SHB1 from 2 to 4 DAP prior to the formation of the seed cavity. SHB1 associates with their promoters but without a recognizable DNA binding motif, and this association is abolished in mini3 mutant. MINI3 binds to W-boxes in, and recruits SHB1 to, its own and IKU2 promoters. Interestingly, SHB1, but not MINI3, activates transcription of pMINI3::GUS or pIKU2::GUS. We reveal a critical developmental switch through the activation of MINI3 expression by SHB1. The recruitment of SHB1 by MINI3 to its own and IKU2 promoters represents a novel two-step amplification to counter the low expression level of IKU2, which is a trigger for endosperm proliferation and seed cavity enlargement. PMID:23505389

  19. Fibronectin upregulates cGMP-dependent protein kinase type Iβ through C/EBP transcription factor activation in contractile cells.

    PubMed

    Chamorro-Jorganes, Aranzazu; Calleros, Laura; Griera, Mercedes; Saura, Marta; Luengo, Alicia; Rodriguez-Puyol, D; Rodriguez-Puyol, M

    2011-03-01

    The nitric oxide (NO)-soluble guanylate cyclase (sGC) pathway exerts most of its cellular actions through the activation of the cGMP-dependent protein kinase (PKG). Accumulation of extracellular matrix is one of the main structural changes in pathological conditions characterized by a decreased activity of this pathway, such as hypertension, diabetes, or aging, and it is a well-known fact that extracellular matrix proteins modulate cell phenotype through the interaction with membrane receptors such as integrins. The objectives of this study were 1) to evaluate whether extracellular matrix proteins, particularly fibronectin (FN), modulate PKG expression in contractile cells, 2) to analyze the mechanisms involved, and 3) to evaluate the functional consequences. FN increased type I PKG (PKG-I) protein content in human mesangial cells, an effect dependent on the interaction with β(1)-integrin. The FN upregulation of PKG-I protein content was due to increased mRNA expression, determined by augmented transcriptional activity of the PKG-I promoter region. Akt and the transcription factor CCAAT enhancer-binding protein (C/EBP) mediated the genesis of these changes. FN also increased PKG-I in another type of contractile cell, rat vascular smooth muscle cells (RVSMC). Tirofiban, a pharmacological analog of FN, increased PKG-I protein content in RVSMC and rat aortic walls and magnified the hypotensive effect of dibutyryl cGMP in conscious Wistar rats. The present results provide evidence of a mechanism able to increase PKG-I protein content in contractile cells. Elucidation of this novel mechanism provides a rationale for future pharmacotherapy in certain vascular diseases.

  20. Use of green fluorescent fusion protein to track activation of the transcription factor osterix during early osteoblast differentiation

    SciTech Connect

    Tai Guangping; Christodoulou, Ioannis; Bishop, Anne E.; Polak, Julia M. . E-mail: julia.polak@imperial.ac.uk

    2005-08-12

    Osterix (Osx) is a transcription factor required for the differentiation of preosteoblasts into fully functioning osteoblasts. However, the pattern of Osx activation during preosteoblast differentiation and maturation has not been clearly defined. Our aim was to study Osx activation during these processes in osteoblasts differentiating from murine and human embryonic stem cells (ESC). To do this, we constructed an Osx-GFP fusion protein reporter system to track Osx translocation within the cells. The distribution of Osx-GFP at representative stages of differentiation was also investigated by screening primary osteoblasts, mesenchymal stem cells, synoviocytes, and pre-adipocytes. Our experiments revealed that Osx-GFP protein was detectable in the cytoplasm of cultured, differentiated ESC 4 days after plating of enzymatically dispersed embryoid bodies. Osterix-GFP protein became translocated into the nucleus on day 7 following transfer of differentiated ESC to osteogenic medium. After 14 days of differentiation, cells showing nuclear translocation of Osx-GFP formed rudimentary bone nodules that continued to increase in number over the following weeks (through day 21). We also found that Osx translocated into the nuclei of mesenchymal stem cells (C3H10T1/2) and pre-osteoblasts (MC3T3-E1) and showed partial activation in pre-adipocytes (MC3T3-L1). These data suggest that Osx activation occurs at a very early point in the differentiation of the mesenchymal-osteoblastic lineage.

  1. The AtMYB12 activation domain maps to a short C-terminal region of the transcription factor.

    PubMed

    Stracke, Ralf; Turgut-Kara, Neslihan; Weisshaar, Bernd

    2017-03-11

    The Arabidopsis thaliana R2R3-MYB transcription factor MYB12 is a light-inducible, flavonol-specific activator of flavonoid biosynthesis. The transactivation activity of the AtMYB12 protein was analyzed using a C-terminal deletion series in a transient A. thaliana protoplast assay with the goal of mapping the activation domain (AD). Although the deletion of the last 46 C-terminal amino acids did not affect the activation capacity, the deletion of the last 98 amino acids almost totally abolished transactivation of two different target promoters. A domain swap experiment using the yeast GAL4 DNA-binding domain revealed that the region from positions 282 to 328 of AtMYB12 was sufficient for transactivation. In contrast to the R2R3-MYB ADs known thus far, that of AtMYB12 is not located at the rearmost C-terminal end of the protein. The AtMYB12 AD is conserved in other experimentally proven R2R3-MYB flavonol regulators from different species.

  2. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    SciTech Connect

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  3. Mapping of transcription factor motifs in active chromatin identifies IRF5 as key regulator in classical Hodgkin lymphoma

    PubMed Central

    Kreher, Stephan; Bouhlel, M. Amine; Cauchy, Pierre; Lamprecht, Björn; Li, Shuang; Grau, Michael; Hummel, Franziska; Köchert, Karl; Anagnostopoulos, Ioannis; Jöhrens, Korinna; Hummel, Michael; Hiscott, John; Wenzel, Sören-Sebastian; Lenz, Peter; Schneider, Markus; Küppers, Ralf; Scheidereit, Claus; Giefing, Maciej; Siebert, Reiner; Rajewsky, Klaus; Lenz, Georg; Cockerill, Peter N.; Janz, Martin; Dörken, Bernd; Bonifer, Constanze; Mathas, Stephan

    2014-01-01

    Deregulated transcription factor (TF) activities are commonly observed in hematopoietic malignancies. Understanding tumorigenesis therefore requires determining the function and hierarchical role of individual TFs. To identify TFs central to lymphomagenesis, we identified lymphoma type-specific accessible chromatin by global mapping of DNaseI hypersensitive sites and analyzed enriched TF-binding motifs in these regions. Applying this unbiased approach to classical Hodgkin lymphoma (HL), a common B-cell–derived lymphoma with a complex pattern of deregulated TFs, we discovered interferon regulatory factor (IRF) sites among the top enriched motifs. High-level expression of the proinflammatory TF IRF5 was specific to HL cells and crucial for their survival. Furthermore, IRF5 initiated a regulatory cascade in human non-Hodgkin B-cell lines and primary murine B cells by inducing the TF AP-1 and cooperating with NF-κB to activate essential characteristic features of HL. Our strategy efficiently identified a lymphoma type-specific key regulator and uncovered a tumor promoting role of IRF5. PMID:25288773

  4. Activation of nuclear transcription factor-kappaB in mouse brain induced by a simulated microgravity environment

    NASA Technical Reports Server (NTRS)

    Wise, Kimberly C.; Manna, Sunil K.; Yamauchi, Keiko; Ramesh, Vani; Wilson, Bobby L.; Thomas, Renard L.; Sarkar, Shubhashish; Kulkarni, Anil D.; Pellis, Neil R.; Ramesh, Govindarajan T.

    2005-01-01

    Microgravity induces inflammatory responses and modulates immune functions that may increase oxidative stress. Exposure to a microgravity environment induces adverse neurological effects; however, there is little research exploring the etiology of these effects resulting from exposure to such an environment. It is also known that spaceflight is associated with increase in oxidative stress; however, this phenomenon has not been reproduced in land-based simulated microgravity models. In this study, an attempt has been made to show the induction of reactive oxygen species (ROS) in mice brain, using ground-based microgravity simulator. Increased ROS was observed in brain stem and frontal cortex with concomitant decrease in glutathione, on exposing mice to simulated microgravity for 7 d. Oxidative stress-induced activation of nuclear factor-kappaB was observed in all the regions of the brain. Moreover, mitogen-activated protein kinase kinase was phosphorylated equally in all regions of the brain exposed to simulated microgravity. These results suggest that exposure of brain to simulated microgravity can induce expression of certain transcription factors, and these have been earlier argued to be oxidative stress dependent.

  5. Mapping of transcription factor motifs in active chromatin identifies IRF5 as key regulator in classical Hodgkin lymphoma.

    PubMed

    Kreher, Stephan; Bouhlel, M Amine; Cauchy, Pierre; Lamprecht, Björn; Li, Shuang; Grau, Michael; Hummel, Franziska; Köchert, Karl; Anagnostopoulos, Ioannis; Jöhrens, Korinna; Hummel, Michael; Hiscott, John; Wenzel, Sören-Sebastian; Lenz, Peter; Schneider, Markus; Küppers, Ralf; Scheidereit, Claus; Giefing, Maciej; Siebert, Reiner; Rajewsky, Klaus; Lenz, Georg; Cockerill, Peter N; Janz, Martin; Dörken, Bernd; Bonifer, Constanze; Mathas, Stephan

    2014-10-21

    Deregulated transcription factor (TF) activities are commonly observed in hematopoietic malignancies. Understanding tumorigenesis therefore requires determining the function and hierarchical role of individual TFs. To identify TFs central to lymphomagenesis, we identified lymphoma type-specific accessible chromatin by global mapping of DNaseI hypersensitive sites and analyzed enriched TF-binding motifs in these regions. Applying this unbiased approach to classical Hodgkin lymphoma (HL), a common B-cell-derived lymphoma with a complex pattern of deregulated TFs, we discovered interferon regulatory factor (IRF) sites among the top enriched motifs. High-level expression of the proinflammatory TF IRF5 was specific to HL cells and crucial for their survival. Furthermore, IRF5 initiated a regulatory cascade in human non-Hodgkin B-cell lines and primary murine B cells by inducing the TF AP-1 and cooperating with NF-κB to activate essential characteristic features of HL. Our strategy efficiently identified a lymphoma type-specific key regulator and uncovered a tumor promoting role of IRF5.

  6. Onecut transcription factors in development and disease

    PubMed Central

    Kropp, Peter A.; Gannon, Maureen

    2016-01-01

    Developmental processes are remarkably well conserved among species, and among the most highly conserved developmental regulators are transcription factor families. The Onecut transcription factor family consists of three members known for their single “cut” DNA-binding domain and an aberrant homeodomain. The three members of the Onecut family are highly conserved from Drosophila to humans and have significant roles in regulating the development of diverse tissues derived from the ectoderm or endoderm, where they activate a number of gene families. Of note, the genetic interaction between Onecut family members and Neurogenin genes appears to be essential in multiple tissues for proper specification and development of unique cell types. This review highlights the importance of the Onecut factors in cell fate specification and organogenesis, highlighting their role in vertebrates, and discusses their role in the maintenance of cell fate and prevention of disease. We cover the essential spatial and temporal control of Onecut factor expression and how this tight regulation is required for proper specification and subsequent terminal differentiation of multiple tissue types including those within the retina, central nervous system, liver and pancreas. Beyond development, Onecut factors perform necessary functions in mature cell types; their misregulation can contribute to diseases such as pancreatic cancer. Given the importance of this family of transcription factors in development and disease, their consideration in essential transcription factor networks is underappreciated. PMID:28018056

  7. Polyphenol Compound as a Transcription Factor Inhibitor

    PubMed Central

    Park, Seyeon

    2015-01-01

    A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor–DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein–protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)). PMID:26529010

  8. HIGH-DIMENSIONAL PROFILING OF TRANSCRIPTION FACTOR ACTIVITY DIFFERENTIATES TOXCAST CHEMICAL GROUPS

    EPA Science Inventory

    The ToxCast™ project at the U.S. EPA uses a diverse battery of high throughput screening assays and informatics models to rapidly characterize the activity of chemicals. A central goal of the project is to provide empirical evidence to aid in the prioritization of chemicals for a...

  9. Activation of the transcription factor c-Jun in acute cellular and antibody-mediated rejection after kidney transplantation.

    PubMed

    Kobayashi, Akimitsu; Takahashi, Takamune; Horita, Shigeru; Yamamoto, Izumi; Yamamoto, Hiroyasu; Teraoka, Satoshi; Tanabe, Kazunari; Hosoya, Tatsuo; Yamaguchi, Yutaka

    2010-12-01

    c-Jun is a transcription factor that belongs to the activator protein-1 family of proteins. In human kidney disease, c-Jun is activated in glomerular and tubular cells and plays a major role in renal pathophysiology. However, the contribution of this pathway to renal allograft rejection has not been determined. We investigated whether c-Jun is activated in acute allograft rejection. c-Jun activation was assessed with immunohistochemistry using phospho-specific c-Jun antibodies in control human renal tissue and renal tissue from patients with acute cellular rejection, acute antibody-mediated rejection, and no rejection in the month after transplantation. In patients with acute cellular rejection, c-Jun activation was observed primarily in infiltrated T cells associated with tubulitis, interstitial cell infiltration, and endarteritis. The number of infiltrated phosphorylated c-Jun-positive cells in the tubules and interstitium was correlated with the Banff classification "t" and "i" scores. In patients with acute antibody-mediated rejection, c-Jun activation was observed in injured endothelial cells as well as in infiltrated cells, including macrophages, in the glomerular and peritubular capillaries. Furthermore, the serum creatinine levels and changes in serum creatinine from the previous year were significantly correlated with the total tubulointerstitial phosphorylated c-Jun-positive score (representing the number of positive nuclei in the tubules, interstitium, and peritubular capillaries). In conclusion, c-Jun was activated in acute antibody-mediated rejection and acute cellular rejection and was associated with reduced graft function. These findings suggest that c-Jun plays a key role in pathological events and may represent a novel therapeutic target in acute renal allograft rejection.

  10. Protein Inhibitor of Activated STAT Y (PIASy) Regulates Insulin Secretion by Interacting with LIM Homeodomain Transcription Factor Isl1

    PubMed Central

    Yan, Chengzhi; Yu, Chulin; Zhang, Di; Cui, Yan; Zhou, Jinlian; Cui, Sheng

    2016-01-01

    It is known that the LIM homeodomain transcription factor Isl1 is highly expressed in all pancreatic endocrine cells and functions in regulating pancreatic development and insulin secretion. The Isl1 mutation has been found to be associated with type 2 diabetes, but the mechanism responsible for Isl1 regulation of insulin synthesis and secretion still needs to be elucidated. In the present study, the protein inhibitor of activated STAT Y (PIASy) was identified as a novel Isl1-interacting protein with a yeast two-hybrid system, and its interaction with Isl1 was further confirmed by a co-immunoprecipitation experiment. PIASy and Isl1 colocalize in human and mouse pancreas and NIT beta cells. Furthermore, PIASy and Isl1 upregulate insulin gene expression and insulin secretion in a dose-dependent manner by activating the insulin promoter. PIASy and Isl1 mRNA expression levels were also increased in type 2 diabetic db/db mice. In addition, our results demonstrate that PIASy and Isl1 cooperate to activate the insulin promoter through the Isl1 homeodomain and PIASy ring domain. These data suggest that that PIASy regulates insulin synthesis and secretion by interacting with Isl1 and provide new insight into insulin regulation, although the detailed molecular mechanism needs to be clarified in future studies. PMID:28000708

  11. Activation of hypodermal differentiation in the Caenorhabditis elegans embryo by GATA transcription factors ELT-1 and ELT-3.

    PubMed

    Gilleard, J S; McGhee, J D

    2001-04-01

    The Caenorhabditis elegans GATA transcription factor genes elt-1 and elt-3 are expressed in the embryonic hypodermis (also called the epidermis). elt-1 is expressed in precursor cells and is essential for the production of most hypodermal cells (22). elt-3 is expressed in all of the major hypodermal cells except the lateral seam cells, and expression is initiated immediately after the terminal division of precursor lineages (13). Although this expression pattern suggests a role for ELT-3 in hypodermal development, no functional studies have yet been performed. In the present paper, we show that either elt-3 or elt-1 is sufficient, when force expressed in early embryonic blastomeres, to activate a program of hypodermal differentiation even in blastomeres that are not hypodermal precursors in wild-type embryos. We have deleted the elt-3 gene and shown that ELT-3 is not essential for either hypodermal cell differentiation or the viability of the organism. We showed that ELT-3 can activate hypodermal gene expression in the absence of ELT-1 and that, conversely, ELT-1 can activate hypodermal gene expression in the absence of ELT-3. Overall, the combined results of the mutant phenotypes, initial expression times, and our forced-expression experiments suggest that ELT-3 acts downstream of ELT-1 in a redundant pathway controlling hypodermal cell differentiation.

  12. Activation of Transcription Factor MEF2D by Bis(3)-cognitin Protects Dopaminergic Neurons and Ameliorates Parkinsonian Motor Defects*

    PubMed Central

    Yao, Lu; Li, Wenming; She, Hua; Dou, Juan; Jia, Leili; He, Yingli; Yang, Qian; Zhu, Jinqiu; Cápiro, Natalie L.; Walker, Douglas I.; Pennell, Kurt D.; Pang, Yuanping; Liu, Yong; Han, Yifan; Mao, Zixu

    2012-01-01

    Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP+) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP+-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3β (GSK3β) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP+-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP+-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo. PMID:22891246

  13. Activation of transcription factor MEF2D by bis(3)-cognitin protects dopaminergic neurons and ameliorates Parkinsonian motor defects.

    PubMed

    Yao, Lu; Li, Wenming; She, Hua; Dou, Juan; Jia, Leili; He, Yingli; Yang, Qian; Zhu, Jinqiu; Cápiro, Natalie L; Walker, Douglas I; Pennell, Kurt D; Pang, Yuanping; Liu, Yong; Han, Yifan; Mao, Zixu

    2012-10-05

    Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP(+)-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3β (GSK3β) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP(+)-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP(+)-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.

  14. Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

    PubMed

    Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

    2015-11-05

    Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.

  15. Protein Kinases and Transcription Factors Activation in Response to UV-Radiation of Skin: Implications for Carcinogenesis

    PubMed Central

    López-Camarillo, César; Ocampo, Elena Aréchaga; Casamichana, Mavil López; Pérez-Plasencia, Carlos; Álvarez-Sánchez, Elizbeth; Marchat, Laurence A.

    2012-01-01

    Solar ultraviolet (UV) radiation is an important environmental factor that leads to immune suppression, inflammation, photoaging, and skin carcinogenesis. Here, we reviewed the specific signal transduction pathways and transcription factors involved in the cellular response to UV-irradiation. Increasing experimental data supporting a role for p38, MAPK, JNK, ERK1/2, and ATM kinases in the response network to UV exposure is discussed. We also reviewed the participation of NF-κB, AP-1, and NRF2 transcription factors in the control of gene expression after UV-irradiation. In addition, we discussed the promising chemotherapeutic intervention of transcription factors signaling by natural compounds. Finally, we focused on the review of data emerging from the use of DNA microarray technology to determine changes in global gene expression in keratinocytes and melanocytes in response to UV treatment. Efforts to obtain a comprehensive portrait of the transcriptional events regulating photodamage of intact human epidermis after UV exposure reveals the existence of novel factors participating in UV-induced cell death. Progress in understanding the multitude of mechanisms induced by UV-irradiation could lead to the potential use of protein kinases and novel proteins as specific targets for the prevention and control of skin cancer. PMID:22312244

  16. Protein kinases and transcription factors activation in response to UV-radiation of skin: implications for carcinogenesis.

    PubMed

    López-Camarillo, César; Ocampo, Elena Aréchaga; Casamichana, Mavil López; Pérez-Plasencia, Carlos; Alvarez-Sánchez, Elizbeth; Marchat, Laurence A

    2012-01-01

    Solar ultraviolet (UV) radiation is an important environmental factor that leads to immune suppression, inflammation, photoaging, and skin carcinogenesis. Here, we reviewed the specific signal transduction pathways and transcription factors involved in the cellular response to UV-irradiation. Increasing experimental data supporting a role for p38, MAPK, JNK, ERK1/2, and ATM kinases in the response network to UV exposure is discussed. We also reviewed the participation of NF-κB, AP-1, and NRF2 transcription factors in the control of gene expression after UV-irradiation. In addition, we discussed the promising chemotherapeutic intervention of transcription factors signaling by natural compounds. Finally, we focused on the review of data emerging from the use of DNA microarray technology to determine changes in global gene expression in keratinocytes and melanocytes in response to UV treatment. Efforts to obtain a comprehensive portrait of the transcriptional events regulating photodamage of intact human epidermis after UV exposure reveals the existence of novel factors participating in UV-induced cell death. Progress in understanding the multitude of mechanisms induced by UV-irradiation could lead to the potential use of protein kinases and novel proteins as specific targets for the prevention and control of skin cancer.

  17. FGF19 (fibroblast growth factor 19) as a novel target gene for activating transcription factor 4 in response to endoplasmic reticulum stress.

    PubMed

    Shimizu, Makoto; Li, Juan; Maruyama, Ryuto; Inoue, Jun; Sato, Ryuichiro

    2013-02-15

    FGF19 (fibroblast growth factor 19), expressed in the small intestine, acts as an enterohepatic hormone by mediating inhibitory effects on the bile acid synthetic pathway and regulating carbohydrate and lipid metabolism. In an attempt to identify novel agents other than bile acids that induce increased FGF19 expression, we found that some ER (endoplasmic reticulum) stress inducers were effective. When intestinal epithelial Caco-2 cells were incubated with thapsigargin, marked increases were observed in the mRNA and secreted protein levels of FGF19. This was not associated with the farnesoid X receptor. Reporter gene analyses using the 5'-promoter region of FGF19 revealed that a functional AARE (amino-acid-response element) was localized in this region, and this site was responsible for inducing its transcription through ATF4 (activating transcription factor 4), which is activated in response to ER stress. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays showed that ATF4 bound to this site and enhanced FGF19 expression. Overexpression of ATF4 in Caco-2 cells induced increased FGF19 mRNA expression, whereas shRNA (short hairpin RNA)-mediated depletion of ATF4 significantly attenuated a thapsigargin-induced increase in FGF19 mRNA.

  18. Hey bHLH transcription factors.

    PubMed

    Weber, David; Wiese, Cornelia; Gessler, Manfred

    2014-01-01

    Hey bHLH transcription factors are direct targets of canonical Notch signaling. The three mammalian Hey proteins are closely related to Hes proteins and they primarily repress target genes by either directly binding to core promoters or by inhibiting other transcriptional activators. Individual candidate gene approaches and systematic screens identified a number of Hey target genes, which often encode other transcription factors involved in various developmental processes. Here, we review data on interaction partners and target genes and conclude with a model for Hey target gene regulation. Furthermore, we discuss how expression of Hey proteins affects processes like cell fate decisions and differentiation, e.g., in cardiovascular, skeletal, and neural development or oncogenesis and how this relates to the observed developmental defects and phenotypes observed in various knockout mice.

  19. Leukemia inhibitory factor and NGF regulate signal transducers and activators of transcription activation in sympathetic ganglia: convergence of cytokine- and neurotrophin-signaling pathways.

    PubMed

    Rajan, P; Gearan, T; Fink, J S

    1998-08-17

    We have used the response of the superior cervical ganglia (SCG) to axotomy to investigate interactions between neuropoietic cytokines and neurotrophins. Postganglionic sympathetic axotomy leads to a prolonged leukemia inhibitory factor (LIF)-dependent activation of signal transducers and activators of transcription (STAT) factors. To study regulation of LIF-dependent activation of STAT proteins and to mimic the loss of target-derived NGF resulting from postganglionic axotomy in vivo, SCG were explanted into media lacking NGF and activation of STAT proteins was assessed by electrophoretic mobility shift assay. Like postganglionic axotomy in vivo. STAT proteins were activated for up to 8 days after explantation of SCG in vitro. SCG cultured in the presence of NGF showed decreased STAT binding when compared to ganglia cultured in NGF-free media. This inhibition of STAT activation by NGF was only present in ganglia cultured for more than 5 days and was mimicked by brain-derived neurotrophic factor (BDNF). The serine kinase inhibitor H7 augmented the increase of STAT binding produced by explantation, suggesting the presence of a labile repressor of STAT activation in the SCG. These data indicated that the neuropoietic cytokine-signaling pathway interacts with neurotrophin and H7-sensitive-signaling pathways to regulate activation of STAT proteins in sympathetic neurons. Moreover, these data suggest that one of the mechanisms leading to prolonged activation of STAT proteins after postganglionic axotomy in vivo is loss of target-derived neurotrophins.

  20. Transcription Factors Regulating Inflammatory Cytokine Production Are Differentially Expressed in Peripheral Blood Mononuclear Cells of Behçet Disease Depending on Disease Activity

    PubMed Central

    Woo, Min-Yeong; Yun, Su Jin; Lee, Mi Jin; Kim, Kyongmin

    2017-01-01

    Background Behçet disease (BD) is a relapsing inflammatory disease with increased production of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs); however, the underlying molecular mechanisms are not well known. Objective To analyze whether the differential expression of transcription factors is involved in the increased tumor necrosis factor (TNF)-α and interleukin (IL)-6 production by PBMCs of BD patients compared to healthy controls (HCs). Methods Expression of transcription factors was examined by real-time reverse transcriptase-polymerase chain reaction and western blotting. Cytokine production by CD11b+ cells transfected with siRNAs against transcription factors was measured by enzyme-linked immunosorbent assay. Results In the absence of lipopolysaccharide stimulation, the transcript level of CCAAT-enhancer-binding proteins (C/EBP) β was increased in PBMCs from patients with active BD compared to that in PBMCs from patients with stable BD. The C/EBPδ transcript level was higher in PBMCs from patients with active BD than in those from HCs. The activating transcription factor 3 (ATF3) transcript level was increased in PBMCs from patients with stable BD compared to that in PBMCs from HCs. siRNAs targeting C/EBPβ and C/EBPδ significantly reduced the production of IL-6 and TNF-α in lipopolysaccharide-stimulated CD11b+ cells from patients with BD as well as from HCs. Conclusion We found differential expression of C/EBPβ, C/EBPδ, and ATF3 in PBMCs from patients with BD depending on disease activity, indicating the involvement of these molecules in BD pathogenesis.

  1. Lipid metabolism enzyme 5-LOX and its metabolite LTB4 are capable of activating transcription factor NF-{kappa}B in hepatoma cells

    SciTech Connect

    Zhao, Yu; Wang, Wenhui; Wang, Qi; Zhang, Xiaodong; Ye, Lihong

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer 5-LOX is able to upregulate expression of NF-{kappa}B p65. Black-Right-Pointing-Pointer 5-LOX enhances nuclear translocation of NF-{kappa}B p65 via increasing p-I{kappa}B-{alpha} level. Black-Right-Pointing-Pointer 5-LOX stimulates transcriptional activity of NF-{kappa}B in hepatoma cells. Black-Right-Pointing-Pointer LTB4 activates transcriptional activity of NF-{kappa}B in hepatoma cells. -- Abstract: The issue that lipid metabolism enzyme and its metabolites regulate transcription factors in cancer cell is not fully understood. In this study, we first report that the lipid metabolism enzyme 5-Lipoxygenase (5-LOX) and its metabolite leukotriene B4 (LTB4) are capable of activating nuclear factor-{kappa}B (NF-{kappa}B) in hepatoma cells. We found that the treatment of MK886 (an inhibitor of 5-LOX) or knockdown of 5-LOX was able to downregulate the expression of NF-{kappa}B p65 at the mRNA level and decreased the phosphorylation level of inhibitor {kappa}B{alpha} (I{kappa}B{alpha}) in the cytoplasm of hepatoma HepG2 or H7402 cells, which resulted in the decrease of the level of nuclear NF-{kappa}B p65. These were confirmed by immunofluorescence staining in HepG2 cell. Moreover, the above treatments were able to decrease the transcriptional activity of NF-{kappa}B in the cells. The LTB4, one of metabolites of 5-LOX, is responsible for 5-LOX-activated NF-{kappa}B in a dose-dependent manner. Thus, we conclude that the lipid metabolism enzyme 5-LOX and its metabolite LTB4 are capable of activating transcription factor NF-{kappa}B in hepatoma cells. Our finding provides new insight into the significance of lipid metabolism in activation of transcription factors in cancer.

  2. Targeting Transcription Factors in Cancer

    PubMed Central

    Bhagwat, Anand S.; Vakoc, Christopher R.

    2015-01-01

    Transcription factors (TFs) are commonly deregulated in the pathogenesis of human cancer and are a major class of cancer cell dependencies. Consequently, targeting of TFs can be highly effective in treating particular malignancies, as highlighted by the clinical efficacy of agents that target nuclear hormone receptors. In this review we discuss recent advances in our understanding of TFs as drug targets in oncology, with an emphasis on the emerging chemical approaches to modulate TF function. The remarkable diversity and potency of TFs as drivers of cell transformation justifies a continued pursuit of TFs as therapeutic targets for drug discovery. PMID:26645049

  3. Impact of short-term systemic hypoxia on phagocytosis, cytokine production, and transcription factor activation in peripheral blood cells.

    PubMed

    Fritzenwanger, Michael; Jung, Christian; Goebel, Bjoern; Lauten, Alexander; Figulla, Hans R

    2011-01-01

    Hypoxia frequently associated with certain physiologic and pathologic conditions influences numerous cellular functions. Because the effects of short-term hypoxia are incompletely understood, we examined phagocytosis and cytokine production as well as the activation of the transcription factors HIF-1 and NFκB in peripheral blood cells of healthy volunteers exposed to an oxygen concentration equivalent to that found at a height of 5500 m. Furthermore, we analysed plasma HIF-1 and serum concentrations of various HIF-1-dependent genes. Results showed that short-term hypoxia increased phagocytosis in neutrophils without affecting monocyte phagocytosis. Hypoxia decreased basal TNFα concentration in monocytes and basal interferon γ concentration in CD4(+) T lymphocytes. In contrast, plasma HIF and serum VEGF concentrations were not affected by hypoxia, although serum EPO concentration was raised. In PBMC, hypoxia increased cytosolic HIF-1 concentration without affecting nuclear HIF-1 concentration and led to a rise in the nuclear NFκB in PBMC. Our results show that short-term hypoxia affects immune functions in healthy individuals. Furthermore, we speculate that the effects of hypoxia are not due to HIF-1, but are caused by the activation of NFκB .

  4. α-Lipoic acid (α-LA) inhibits the transcriptional activity of interferon regulatory factor 1 (IRF-1) via SUMOylation.

    PubMed

    Sun, Tao; Gao, Fuyu; Lin, Xiaoyan; Yu, Ruixiang; Zhao, Yong; Luan, Jingjie; Li, Hongyan; Song, Mingzhu

    2014-10-01

    Osteoarthritis (OA), one of the most common joint disorders, is one of the leading causes of disability among the elderly. Proinflammatory cytokines, such as interleukin (IL)-1β, which is synthesized locally by synovial cells and chondrocytes, have been shown to play a critical role in sustaining cartilage damage in arthritis by creating an imbalance between cartilage degradation and the repair process. Alpha-lipoic acid (α-LA), which is synthesized in animal and plant tissues, has demonstrated its protective effects in a variety of diseases. However, whether or not LA has a protective effect in OA is still unknown. In this study, we found that α-LA inhibits the IL-1β-induced increase in matrix metallopeptidase 3 (MMP-3) and matrix metallopeptidase 13 (MMP-13) expression and activity. Our data also demonstrate that interferon regulatory factor 1 (IRF-1) participates in the induction of MMP-3 and MMP-13. However, α-LA treatment did not change IRF-1 levels. Importantly, we found that α-LA increases SUMOylation of IRF-1, which attenuates IRF-1's transcriptional activity. Finally, we found that α-LA treatment leads to an increase in SUMO-1, but not in SUMO-2 or SUMO-3. Taken together, this study shows that α-LA exerts anti-inflammatory effects in an IL-1β-stimulated chondrocyte model, thereby suggesting a potential protective effect of α-LA in OA.

  5. Transcription factor ZBED6 mediates IGF2 gene expression by regulating promoter activity and DNA methylation in myoblasts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were up regulated during C2C12 differentiation. The IGF2 expression levels wer...

  6. The transcription factor GATA3 actively represses RUNX3 protein-regulated production of interferon-gamma

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The transcription factor GATA3 is crucial for the differentiation of naive CD4+ T cells into T helper 2 (Th2) cells. Here, we show that deletion of Gata3 allowed the appearance of interferon-g (IFN-g)-producing cells in the absence of interleukin-12 (IL-12) and IFN-g. Such IFN-g production was tra...

  7. Genome-Wide Chromosomal Targets of Oncogenic Transcription Factors

    DTIC Science & Technology

    2005-04-01

    cancer. Cancer involves, at least in part, aberrant programs of gene expression often mediated by oncogenic transcription factors activating downstream...networks that underlie complex gene expression programs that are activated in cancer. Indeed, transcription factors have been proposed as targets of...some of the limitations of ChIP-chip analysis and can be applied to transcription factors important in breast cancer such as c-myc and ER ( estrogen

  8. Effects of Cigarette Smoke on the Activation of Oxidative Stress-Related Transcription Factors in Female A/J Mouse Lung

    PubMed Central

    Tharappel, Job C.; Cholewa, Jill; Espandiari, Parvaneh; Spear, Brett T.; Gairola, C. Gary; Glauert, Howard P.

    2010-01-01

    Cigarette smoke contains a high concentration of free radicals and induces oxidative stress in the lung and other tissues. Several transcription factors are known to be activated by oxidative stress, including nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and hypoxia-inducible factor (HIF). Studies were therefore undertaken to examine if cigarette smoke could activate these transcription factors, as well as other transcription factors that may be important in lung carcinogenesis. Female A/J mice were exposed to cigarette smoke for 2, 5, 10, 15, 20, 42, or 56 days (6 hr/day, 5 days/wk). Cigarette smoke did not increase NF-κB activation at any of these times, but NF-κB DNA binding activity was lower after 15 days and 56 days of smoke exposure. The DNA binding activity of AP-1 was lower after 10 days and 56 days but was not changed after 42 days of smoke exposure. The DNA binding activity of HIF was quantitatively increased after 42 days of smoke exposure but decreased after 56 days. Whether the activation of other transcription factors in the lung could be altered after exposure to cigarette smoke was subsequently examined. The DNA binding activities of FoxF2, myc-CF1, RORE, and p53 were examined after 10 days of smoke exposure. The DNA binding activities of FoxF2 and p53 were quantitatively increased, but those of myc-CF1 and RORE were unaffected. These studies show that cigarette smoke exposure leads to quantitative increases in DNA binding activities of FoxF2 and p53, while the activations of NF-κB, AP-1, and HIF are largely unaffected or reduced. PMID:20711931

  9. Transcriptional activation of human CYP17 in H295R adrenocortical cells depends on complex formation among p54(nrb)/NonO, protein-associated splicing factor, and SF-1, a complex that also participates in repression of transcription.

    PubMed

    Sewer, Marion B; Nguyen, Viet Q; Huang, Ching-Jung; Tucker, Philip W; Kagawa, Norio; Waterman, Michael R

    2002-04-01

    The first 57 bp upstream of the transcription initiation site of the human CYP17 (hCYP17) gene are essential for both basal and cAMP-dependent transcription. EMSA carried out by incubating H295R adrenocortical cell nuclear extracts with radiolabeled -57/-38 probe from the hCYP17 promoter showed the formation of three DNA-protein complexes. The fastest complex contained steroidogenic factor (SF-1) and p54(nrb)/NonO, the intermediate complex contained p54(nrb)/NonO and polypyrimidine tract-binding protein-associated splicing factor (PSF), and the slowest complex contained an SF-1/PSF/p54(nrb)/NonO complex. (Bu)(2)cAMP treatment resulted in a cAMP-inducible increase in the binding intensity of only the upper complex and also activated hCYP17 gene transcription. SF-1 coimmunoprecipitated with p54(nrb)/NonO, indicating direct interaction between these proteins. Functional assays revealed that PSF represses basal transcription. Further, the repression of hCYP17 promoter-reporter construct luciferase activity resulted from PSF interacting with the corepressor mSin3A. Trichostatin A attenuated the inhibition of basal transcription, suggesting that a histone deacetylase interacts with the SF-1/PSF/p54(nrb)/NonO/mSin3A complex. Our studies lend support to the idea that the balance between transcriptional activation and repression is essential in the control of adrenocortical steroid hormone biosynthesis.

  10. Transforming growth factor beta 1-responsive element: closely associated binding sites for USF and CCAAT-binding transcription factor-nuclear factor I in the type 1 plasminogen activator inhibitor gene.

    PubMed Central

    Riccio, A; Pedone, P V; Lund, L R; Olesen, T; Olsen, H S; Andreasen, P A

    1992-01-01

    Transforming growth factor beta (TGF-beta) is the name of a group of closely related polypeptides characterized by a multiplicity of effects, including regulation of extracellular proteolysis and turnover of the extracellular matrix. Its cellular mechanism of action is largely unknown. TGF-beta 1 is a strong and fast inducer of type 1 plasminogen activator inhibitor gene transcription. We have identified a TGF-beta 1-responsive element in the 5'-flanking region of the human type 1 plasminogen activator inhibitor gene and shown that it is functional both in its natural context and when fused to a heterologous nonresponsive promoter. Footprinting and gel retardation experiments showed that two different nuclear factors, present in extracts from both TGF-beta 1-treated and nontreated cells, bind to adjacent sequences contained in the responsive unit. A palindromic sequence binds a trans-acting factor(s) of the CCAAT-binding transcription factor-nuclear factor I family. A partially overlapping dyad symmetry interacts with a second protein that much evidence indicates to be USF. USF is a transactivator belonging to the basic helix-loop-helix family of transcription factors. Mutations which abolish the binding of either CCAAT-binding transcription factor-nuclear factor I or USF result in reduction of transcriptional activation upon exposure to TGF-beta 1, thus showing that both elements of the unit are necessary for the TGF-beta 1 response. We discuss the possible relationship of these findings to the complexity of the TGF-beta action. Images PMID:1549130

  11. Heat shock transcription factor 1 is activated as a consequence of lymphocyte activation and regulates a major proteostasis network in T cells critical for cell division during stress.

    PubMed

    Gandhapudi, Siva K; Murapa, Patience; Threlkeld, Zachary D; Ward, Martin; Sarge, Kevin D; Snow, Charles; Woodward, Jerold G

    2013-10-15

    Heat shock transcription factor 1 (HSF1) is a major transcriptional regulator of the heat shock response in eukaryotic cells. HSF1 is evoked in response to a variety of cellular stressors, including elevated temperatures, oxidative stress, and other proteotoxic stressors. Previously, we demonstrated that HSF1 is activated in naive T cells at fever range temperatures (39.5°C) and is critical for in vitro T cell proliferation at fever temperatures. In this study, we demonstrated that murine HSF1 became activated to the DNA-binding form and transactivated a large number of genes in lymphoid cells strictly as a consequence of receptor activation in the absence of apparent cellular stress. Microarray analysis comparing HSF1(+/+) and HSF1(-/-) gene expression in T cells activated at 37°C revealed a diverse set of 323 genes significantly regulated by HSF1 in nonstressed T cells. In vivo proliferation studies revealed a significant impairment of HSF1(-/-) T cell expansion under conditions mimicking a robust immune response (staphylococcal enterotoxin B-induced T cell activation). This proliferation defect due to loss of HSF1 is observed even under nonfebrile temperatures. HSF1(-/-) T cells activated at fever temperatures show a dramatic reduction in cyclin E and cyclin A proteins during the cell cycle, although the transcription of these genes was modestly affected. Finally, B cell and hematopoietic stem cell proliferation from HSF1(-/-) mice, but not HSF1(+/+) mice, were also attenuated under stressful conditions, indicating that HSF1 is critical for the cell cycle progression of lymphoid cells activated under stressful conditions.

  12. Differential expression of brain-derived neurotrophic factor transcripts after pilocarpine-induced seizure-like activity is related to mode of Ca2+ entry.

    PubMed

    Poulsen, F R; Lauterborn, J; Zimmer, J; Gall, C M

    2004-01-01

    Activity-dependent brain-derived neurotrophic factor (BDNF) expression is Ca2+-dependent, yet little is known about the Ca2+ channel contributions that might direct selective expression of the multiple BDNF transcripts. Here, effects of pilocarpine-induced seizure activity on total BDNF expression and on the individual sensitivity of BDNF transcripts to glutamate receptor and Ca2+ channel blockers were evaluated using hippocampal slice cultures and in situ hybridization of transcript-specific cRNA probes directed against mRNAs for the four 5' exons (I-IV) of the BDNF gene. mRNAs for nerve growth factor (NGF) and tyrosine kinase B (trkB) also were studied. Pilocarpine (5 mM) induced a dose- and time-dependent increase in total BDNF (exon V) mRNA expression in the dentate granule cells and CA3-CA1 pyramidal cells with maximal effects at 6 and 24 h, respectively. Increases were blocked by co-treatment with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX: 25 microM) and the N-methyl-d-aspartic acid receptor antagonist 2-amino-5-phosphonovaleric acid (APV; 25 microM), whereas the L-type voltage sensitive Ca2+ channel blocker nifedipine (20 microM) was without detectable effect. Maximal NGF and trkB mRNA expression was induced by pilocarpine at 4 and 12 h, respectively. For the individual BDNF transcripts, APV blocked pilocarpine-induced increases in transcript II, whereas nifedipine blocked increases in transcripts I and III. Transcript IV levels were not altered by treatment. These results indicate that transcript II makes the greatest contribution to pilocarpine effects on total BDNF mRNA content in this model and provides evidence for regional and Ca2+ channel-specific differences in activity-dependent regulation of the different BDNF transcripts in hippocampus.

  13. 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 is widely used in traditional Indian medicine to cure biliary disorders, anorexia, cough, diabetic wounds, hepatic disorders, rheumatism, and sinusitis. Extensive investigation over the last five decades has indicated that curcumin reduces blood cholesterol, prevents low-density lipoprotein oxidation, inhibits platelet aggregation, suppresses thrombosis and myocardial infarction, suppresses symptoms associated with type II diabetes, rheumatoid arthritis, multiple sclerosis, and Alzheimer's disease, inhibits HIV replication, enhances wound healing, protects from liver injury, increases bile secretion, protects from cataract formation, and protects from pulmonary toxicity and fibrosis. Evidence indicates that the divergent effects of curcumin are dependent on its pleiotropic molecular effects. These include the regulation of signal transduction pathways and direct modulation of several enzymatic activities. Most of these signaling cascades lead to the activation of transcription factors. Curcumin has been found to modulate the activity of several key transcription factors and, in turn, the cellular expression profiles. Curcumin has been shown to elicit vital cellular responses such as cell cycle arrest, apoptosis, and differentiation by activating a cascade of molecular events. In this chapter, we briefly review the effects of curcumin on transcription factors NF-KB, AP-1, Egr-1, STATs, PPAR-gamma, beta-catenin, nrf2, EpRE, p53, CBP, and androgen receptor (AR) and AR-related cofactors giving major emphasis to the molecular mechanisms of its action.

  14. The Transcriptional Factors MurR and Catabolite Activator Protein Regulate N-Acetylmuramic Acid Catabolism in Escherichia coli▿ †

    PubMed Central

    Jaeger, Tina; Mayer, Christoph

    2008-01-01

    The MurNAc etherase MurQ of Escherichia coli is essential for the catabolism of the bacterial cell wall sugar N-acetylmuramic acid (MurNAc) obtained either from the environment or from the endogenous cell wall (i.e., recycling). High-level expression of murQ is required for growth on MurNAc as the sole source of carbon and energy, whereas constitutive low-level expression of murQ is sufficient for the recycling of peptidoglycan fragments continuously released from the cell wall during growth of the bacteria. Here we characterize for the first time the expression of murQ and its regulation by MurR, a member of the poorly characterized RpiR/AlsR family of transcriptional regulators. Deleting murR abolished the extensive lag phase observed for E. coli grown on MurNAc and enhanced murQ transcription some 20-fold. MurR forms a stable multimer (most likely a tetramer) and binds to two adjacent inverted repeats within an operator region. In this way MurR represses transcription from the murQ promoter and also interferes with its own transcription. MurNAc-6-phosphate, the substrate of MurQ, was identified as a specific inducer that weakens binding of MurR to the operator. Moreover, murQ transcription depends on the activation by cyclic AMP (cAMP)-catabolite activator protein (CAP) bound to a class I site upstream of the murQ promoter. murR and murQ are divergently orientated and expressed from nonoverlapping face-to-face (convergent) promoters, yielding transcripts that are complementary at their 5′ ends. As a consequence of this unusual promoter arrangement, cAMP-CAP also affects murR transcription, presumably by acting as a roadblock for RNA polymerase. PMID:18723630

  15. Pregnenolone sulfate activates basic region leucine zipper transcription factors in insulinoma cells: role of voltage-gated Ca2+ channels and transient receptor potential melastatin 3 channels.

    PubMed

    Müller, Isabelle; Rössler, Oliver G; Thiel, Gerald

    2011-12-01

    The neurosteroid pregnenolone sulfate activates a signaling cascade in insulinoma cells involving activation of extracellular signal-regulated protein kinase and enhanced expression of the transcription factor Egr-1. Here, we show that pregnenolone sulfate stimulation leads to a significant elevation of activator protein-1 (AP-1) activity in insulinoma cells. Expression of the basic region leucine zipper (bZIP) transcription factors c-Jun and c-Fos is up-regulated in insulinoma cells and pancreatic β-cells in primary culture after pregnenolone sulfate stimulation. Up-regulation of a chromatin-embedded c-Jun promoter/luciferase reporter gene transcription in pregnenolone sulfate-stimulated insulinoma cells was impaired when the AP-1 binding sites were mutated, indicating that these motifs function as pregnenolone sulfate response elements. In addition, phosphorylation of cAMP response element (CRE)-binding protein is induced and transcription of a CRE-controlled reporter gene is stimulated after pregnenolone sulfate treatment, indicating that the CRE functions as a pregnenolone sulfate response element as well. Pharmacological and genetic experiments revealed that both L-type Ca(2+) channels and transient receptor potential melastatin 3 (TRPM3) channels are essential for connecting pregnenolone sulfate stimulation with enhanced AP-1 activity and bZIP-mediated transcription in insulinoma cells. In contrast, pregnenolone sulfate stimulation did not enhance AP-1 activity or c-Jun and c-Fos expression in pituitary corticotrophs that express functional L-type Ca(2+) channels but only trace amounts of TRPM3. We conclude that expression of L-type Ca(2+) channels is not sufficient to activate bZIP-mediated gene transcription by pregnenolone sulfate. Rather, additional expression of TRPM3 or depolarization of the cells is required to connect pregnenolone sulfate stimulation with enhanced gene transcription.

  16. Transcriptional Regulation of the Sodium-activated Potassium Channel SLICK (KCNT2) Promoter by Nuclear Factor-κB*

    PubMed Central

    Tomasello, Danielle L.; Gancarz-Kausch, Amy M.; Dietz, David M.; Bhattacharjee, Arin

    2015-01-01

    Although recent studies have shown the sodium-activated potassium channel SLACK (KCNT1) can contribute to neuronal excitability, there remains little information on the physiological role of the closely related SLICK (KCNT2) channel. Activation of SLICK channels may be important during pathological states such as ischemia, in which an increase in intracellular sodium and chloride can perturb membrane potential and ion homeostasis. We have identified two NFκB-binding sites within the promoter region of the human SLICK (KCNT2) and orthologous rat Slick (Kcnt2) genes, suggesting that conditions in which NFκB transcriptional activity is elevated promote expression of this channel. NFκB binding to the rat Slick promoter was confirmed in vivo by ChIP analyses, and NFκB was found differentially bound to the two sites. We verified NFκB transcriptional regulation of SLICK/Slick by mutational analyses and studying gene expression by luciferase assay in P19 cells, where NFκB is constitutively active. For the rat gene, activation of the Slick promoter was found to be additive in single NFκB mutations and synergistic in double mutations. Unexpectedly, for the human gene, NFκB exhibited cooperativity in activating the SLICK promoter. The human SLICK promoter constructs were then tested under hypoxic conditions in PC-12 cells, where NFκB is not active. Only under hypoxic conditions could luciferase activity be detected; the double NFκB mutant construct failed to exhibit activity. Transcriptional regulation of Slick by NFκB was verified in primary neurons. The Slick transcript decreased 24 h after NFκB inhibition. Our data show SLICK expression is predominantly under the control of NFκB. Because neuronal NFκB activation occurs during stressful stimuli such as hypoxia and injury, our findings suggest that SLICK is a neuroprotective gene. PMID:26100633

  17. Transcriptional Regulation of the Sodium-activated Potassium Channel SLICK (KCNT2) Promoter by Nuclear Factor-κB.

    PubMed

    Tomasello, Danielle L; Gancarz-Kausch, Amy M; Dietz, David M; Bhattacharjee, Arin

    2015-07-24

    Although recent studies have shown the sodium-activated potassium channel SLACK (KCNT1) can contribute to neuronal excitability, there remains little information on the physiological role of the closely related SLICK (KCNT2) channel. Activation of SLICK channels may be important during pathological states such as ischemia, in which an increase in intracellular sodium and chloride can perturb membrane potential and ion homeostasis. We have identified two NFκB-binding sites within the promoter region of the human SLICK (KCNT2) and orthologous rat Slick (Kcnt2) genes, suggesting that conditions in which NFκB transcriptional activity is elevated promote expression of this channel. NFκB binding to the rat Slick promoter was confirmed in vivo by ChIP analyses, and NFκB was found differentially bound to the two sites. We verified NFκB transcriptional regulation of SLICK/Slick by mutational analyses and studying gene expression by luciferase assay in P19 cells, where NFκB is constitutively active. For the rat gene, activation of the Slick promoter was found to be additive in single NFκB mutations and synergistic in double mutations. Unexpectedly, for the human gene, NFκB exhibited cooperativity in activating the SLICK promoter. The human SLICK promoter constructs were then tested under hypoxic conditions in PC-12 cells, where NFκB is not active. Only under hypoxic conditions could luciferase activity be detected; the double NFκB mutant construct failed to exhibit activity. Transcriptional regulation of Slick by NFκB was verified in primary neurons. The Slick transcript decreased 24 h after NFκB inhibition. Our data show SLICK expression is predominantly under the control of NFκB. Because neuronal NFκB activation occurs during stressful stimuli such as hypoxia and injury, our findings suggest that SLICK is a neuroprotective gene.

  18. Dax-1 and Steroid Receptor RNA Activator (SRA) Function as Transcriptional Coactivators for Steroidogenic Factor 1 in Steroidogenesis▿

    PubMed Central

    Xu, Bin; Yang, Wei-Hsiung; Gerin, Isabelle; Hu, Chang-Deng; Hammer, Gary D.; Koenig, Ronald J.

    2009-01-01

    The nuclear receptor steroidogenic factor 1 (SF-1) is essential for adrenal development and steroidogenesis. The atypical orphan nuclear receptor Dax-1 binds to SF-1 and represses SF-1 target genes. Paradoxically, however, loss-of-function mutations of Dax-1 also cause adrenal hypoplasia, suggesting that Dax-1 may function as an SF-1 coactivator under some circumstances. Indeed, we found that Dax-1 can function as a dosage-dependent SF-1 coactivator. Both SF-1 and Dax-1 bind to steroid receptor RNA activator (SRA), a coactivator that functions as an RNA. The coactivator TIF2 also associates with Dax-1 and synergistically coactivates SF-1 target gene transcription. A naturally occurring Dax-1 mutation inhibits this transactivation, and the mutant Dax-1-TIF2 complex mislocalizes in living cells. Coactivation by Dax-1 is abolished by SRA knockdown. The expression of the steroidogenic gene products steroidogenic acute regulatory protein (StAR) and melanocortin 2 receptor is reduced in adrenal Y1 cells following the knockdown of endogenous SRA. Similarly, the knockdown of endogenous Dax-1 downregulates the expression of the steroidogenic gene products CYP11A1 and StAR in both H295R adrenal and MA-10 Leydig cells. These findings reveal novel functions of SRA and Dax-1 in steroidogenesis and adrenal biology. PMID:19188450

  19. The human neuroendocrine thyrotropin-releasing hormone receptor promoter is activated by the haematopoietic transcription factor c-Myb.

    PubMed Central

    Matre, Vilborg; Høvring, Per I; Fjeldheim, Ase-Karine; Helgeland, Lars; Orvain, Christophe; Andersson, Kristin B; Gautvik, Kaare M; Gabrielsen, Odd S

    2003-01-01

    Thyrotropin-releasing hormone (TRH) receptor (TRHR) is a G-protein-coupled receptor playing a crucial role in the anterior pituitary where it controls the synthesis and secretion of thyroid-stimulating hormone and prolactin. Its widespread presence not only in the central nervous system, but also in peripheral tissues, including thymus, indicates other important, but unknown, functions. One hypothesis is that the neuropeptide TRH could play a role in the immune system. We report here that the human TRHR promoter contains 11 putative response elements for the haematopoietic transcription factor c-Myb and is highly Myb-responsive in transfection assays. Analysis of Myb binding to putative response elements revealed one preferred binding site in intron 1 of the receptor gene. Transfection studies of promoter deletions confirmed that this high-affinity element is necessary for efficient Myb-dependent transactivation of reporter plasmids in CV-1 cells. The Myb-dependent activation of the TRHR promoter was strongly suppressed by expression of a dominant negative Myb-Engrailed fusion. In line with these observations, reverse transcriptase PCR analysis of rat tissues showed that the TRHR gene is expressed both in thymocytes and bone marrow. Furthermore, specific, high-affinity TRH agonist binding to cell-surface receptors was demonstrated in thymocytes and a haematopoietic cell line. Our findings imply a novel functional link between the neuroendocrine and the immune systems at the level of promoter regulation. PMID:12628004

  20. High-Yield Expression in E. coli and Refolding of the bZIP Domain of Activating Transcription Factor 5

    PubMed Central

    Ciaccio, Natalie A.; Moreno, Matthew L.; Bauer, Rachel L.; Laurence, Jennifer S.

    2008-01-01

    Activating Transcription Factor 5 (ATF5) recently has been demonstrated to play a critical role in promoting the survival of human glioblastoma cells. Interference with the function of ATF5 in an in vivo rat model caused glioma cell death in primary tumors but did not affect the status of normal cells surrounding the tumor, suggesting ATF5 may prove an ideal target for anti-cancer therapy. In order to examine ATF5 as a pharmaceutical target, the protein must be produced and purified to sufficient quantity to begin analyses. Here, a procedure for expressing and refolding the bZIP domain of ATF5 in sufficient yield and final concentration to permit assay development and structural characterization of this target using solution NMR is reported. Two-dimensional NMR and circular dichrosim analyses indicate the protein exists in the partially α-helical, monomeric x-form conformation with only a small fraction of ATF5 participating in formation of higher-order structure, presumably coiled-coil homodimerization. Despite the persistence of monomers in solution even at high concentration, an electrophoretic mobility shift assay showed that ATF5 is able to bind to the cAMP response element (CRE) DNA motif. Polyacrylamide gel electrophoresis and mass spectrometry were used to confirm that ATF5 can participate in homodimer formation and that this dimerization is mediated by disulfide bond formation. PMID:18718539

  1. The Unfolded Protein Response and the Phosphorylations of Activating Transcription Factor 2 in the trans-Activation of il23a Promoter Produced by β-Glucans*

    PubMed Central

    Rodríguez, Mario; Domingo, Esther; Alonso, Sara; Frade, Javier García; Eiros, José; Crespo, Mariano Sánchez; Fernández, Nieves

    2014-01-01

    Current views on the control of IL-23 production focus on the regulation of il23a, the gene encoding IL-23 p19, by NF-κB in combination with other transcription factors. C/EBP homologous protein (CHOP), X2-Box-binding protein 1 (XBP1), activator protein 1 (AP1), SMAD, CCAAT/enhancer-binding protein (C/EBPβ), and cAMP-response element-binding protein (CREB) have been involved in response to LPS, but no data are available regarding the mechanism triggered by the fungal mimic and β-glucan-containing stimulus zymosan, which produces IL-23 and to a low extent the related cytokine IL-12 p70. Zymosan induced the mobilization of CHOP from the nuclear fractions to phagocytic vesicles. Hypha-forming Candida also induced the nuclear disappearance of CHOP. Assay of transcription factor binding to the il23a promoter showed an increase of Thr(P)-71–Thr(P)-69-activating transcription factor 2 (ATF2) binding in response to zymosan. PKC and PKA/mitogen- and stress-activated kinase inhibitors down-regulated Thr(P)-71–ATF2 binding to the il23a promoter and il23a mRNA expression. Consistent with the current concept of complementary phosphorylations on N-terminal Thr-71 and Thr-69 of ATF2 by ERK and p38 MAPK, MEK, and p38 MAPK inhibitors blunted Thr(P)-69–ATF2 binding. Knockdown of atf2 mRNA with siRNA correlated with inhibition of il23a mRNA, but it did not affect the expression of il12/23b and il10 mRNA. These data indicate the following: (i) zymosan decreases nuclear proapoptotic CHOP, most likely by promoting its accumulation in phagocytic vesicles; (ii) zymosan-induced il23a mRNA expression is best explained through coordinated κB- and ATF2-dependent transcription; and (iii) il23a expression relies on complementary phosphorylation of ATF2 on Thr-69 and Thr-71 dependent on PKC and MAPK activities. PMID:24982422

  2. Elevated SP-1 Transcription Factor Expression and Activity Drives Basal and Hypoxia-induced Vascular Endothelial Growth Factor (VEGF) Expression in Non-Small Cell Lung Cancer

    PubMed Central

    Deacon, Karl; Onion, David; Kumari, Rajendra; Watson, Susan A.; Knox, Alan J.

    2012-01-01

    VEGF plays a central role in angiogenesis in cancer. Non-small cell lung cancer (NSCLC) tumors have increased microvascular density, localized hypoxia, and high VEGF expression levels; however, there is a lack of understanding of how oncogenic and tumor microenvironment changes such as hypoxia lead to greater VEGF expression in lung and other cancers. We show that NSCLC cells secreted higher levels of VEGF than normal airway epithelial cells. Actinomycin D inhibited all NSCLC VEGF secretion, and VEGF minimal promoter-luciferase reporter constructs were constitutively active until the last 85 base pairs before the transcription start site containing three SP-1 transcription factor-binding sites; mutation of these VEGF promoter SP-1-binding sites eliminated VEGF promoter activity. Furthermore, dominant negative SP-1, mithramycin A, and SP-1 shRNA decreased VEGF promoter activity, whereas overexpression of SP-1 increased VEGF promoter activity. Chromatin immunoprecipitation assays demonstrated SP-1, p300, and PCA/F histone acetyltransferase binding and histone H4 hyperacetylation at the VEGF promoter in NSCLC cells. Cultured NSCLC cells expressed higher levels of SP-1 protein than normal airway epithelial cells, and double-fluorescence immunohistochemistry showed a strong correlation between SP-1 and VEGF in human NSCLC tumors. In addition, hypoxia-driven VEGF expression in NSCLC cells was SP-1-dependent, with hypoxia increasing SP-1 activity and binding to the VEGF promoter. These studies are the first to demonstrate that overexpression of SP-1 plays a central role in hypoxia-induced VEGF secretion. PMID:22992725

  3. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10.

    PubMed

    Espley, Richard V; Hellens, Roger P; Putterill, Jo; Stevenson, David E; Kutty-Amma, Sumathi; Allan, Andrew C

    2007-02-01

    Anthocyanin concentration is an important determinant of the colour of many fruits. In apple (Malus x domestica), centuries of breeding have produced numerous varieties in which levels of anthocyanin pigment vary widely and change in response to environmental and developmental stimuli. The apple fruit cortex is usually colourless, although germplasm does exist where the cortex is highly pigmented due to the accumulation of either anthocyanins or carotenoids. From studies in a diverse array of plant species, it is apparent that anthocyanin biosynthesis is controlled at the level of transcription. Here we report the transcript levels of the anthocyanin biosynthetic genes in a red-fleshed apple compared with a white-fleshed cultivar. We also describe an apple MYB transcription factor, MdMYB10, that is similar in sequence to known anthocyanin regulators in other species. We further show that this transcription factor can induce anthocyanin accumulation in both heterologous and homologous systems, generating pigmented patches in transient assays in tobacco leaves and highly pigmented apple plants following stable transformation with constitutively expressed MdMYB10. Efficient induction of anthocyanin biosynthesis in transient assays by MdMYB10 was dependent on the co-expression of two distinct bHLH proteins from apple, MdbHLH3 and MdbHLH33. The strong correlation between the expression of MdMYB10 and apple anthocyanin levels during fruit development suggests that this transcription factor is responsible for controlling anthocyanin biosynthesis in apple fruit; in the red-fleshed cultivar and in the skin of other varieties, there is an induction of MdMYB10 expression concurrent with colour formation during development. Characterization of MdMYB10 has implications for the development of new varieties through classical breeding or a biotechnological approach.

  4. Transcription factor activity of estrogen receptor α activation upon nonylphenol or bisphenol A treatment enhances the in vitro proliferation, invasion, and migration of neuroblastoma cells

    PubMed Central

    Ma, Hongda; Yao, Yao; Wang, Changli; Zhang, Liyu; Cheng, Long; Wang, Yiren; Wang, Tao; Liang, Erguang; Jia, Hui; Ye, Qinong; Hou, Mingxiao; Feng, Fan

    2016-01-01

    Many kinds of endocrine-disrupting chemicals (EDCs), for example, the environmental estrogens bisphenol A and nonylphenol, may regulate the activity of estrogen receptor α (ERα) and therefore induce potential disruption of normal endocrine function. However, the involvement of EDCs in human cancers, especially in endocrine-related cancer neuroblastoma regulation, is not very clear. In this work, results showed that upon bisphenol A or nonylphenol treatment, the transcription factor activity of ERα was significantly increased in neuroblastoma cell line SH-SY5Y. Bisphenol A and nonylphenol could enhance ERα activity via recruiting it to the target gene promoter. Furthermore, treatment of bisphenol A and nonylphenol enhanced the in vitro proliferation, invasion, and migration ability of neuroblastoma cells. By investigating the role of EDC-induced ERα upregulation, our data extend the understanding of the function of EDCs and further suggest that ERα might be a potential therapeutic target in human neuroblastoma treatment. PMID:27366082

  5. Core promoter-specific gene regulation: TATA box selectivity and Initiator-dependent bi-directionality of serum response factor-activated transcription.

    PubMed

    Xu, Muyu; Gonzalez-Hurtado, Elsie; Martinez, Ernest

    2016-04-01

    Gene-specific activation by enhancers involves their communication with the basal RNA polymerase II transcription machinery at the core promoter. Core promoters are diverse and may contain a variety of sequence elements such as the TATA box, the Initiator (INR), and the downstream promoter element (DPE) recognized, respectively, by the TATA-binding protein (TBP) and TBP-associated factors of the TFIID complex. Core promoter elements contribute to the gene selectivity of enhancers, and INR/DPE-specific enhancers and activators have been identified. Here, we identify a TATA box-selective activating sequence upstream of the human β-actin (ACTB) gene that mediates serum response factor (SRF)-induced transcription from TATA-dependent but not INR-dependent promoters and requires the TATA-binding/bending activity of TBP, which is otherwise dispensable for transcription from a TATA-less promoter. The SRF-dependent ACTB sequence is stereospecific on TATA promoters but activates in an orientation-independent manner a composite TATA/INR-containing promoter. More generally, we show that SRF-regulated genes of the actin/cytoskeleton/contractile family tend to have a TATA box. These results suggest distinct TATA-dependent and INR-dependent mechanisms of TFIID-mediated transcription in mammalian cells that are compatible with only certain stereospecific combinations of activators, and that a TBP-TATA binding mechanism is important for SRF activation of the actin/cytoskeleton-related gene family.

  6. The LIM homeobox transcription factor Lhx2 is required to specify the retina field and synergistically cooperates with Pax6 for Six6 trans-activation.

    PubMed

    Tétreault, Nicolas; Champagne, Marie-Pier; Bernier, Gilbert

    2009-03-15

    In mammals, a limited set of homeobox-containing transcription factors are expressed in the presumptive eye field and required to initiate eye development. How these factors interact together at the genetic and molecular level to coordinate this developmental process is poorly understood. We found that the Lhx2 and Pax6 transcription factors operate in a concerted manner during retinal development to promote transcriptional activation of the Six6 homeobox-gene in primitive and mature retinal progenitors. Lhx2 demarcates the presumptive retina field at the neural plate stage and Lhx2 inactivation delays initiation of Rx, Six3 and Pax6 expression in this domain. The later expressed Six6 is properly activated in the pituitary/hypothalamic axis of Lhx2(-/-) embryos, but expression fails to be initiated in the optic vesicle. Lhx2 and Pax6 associate with the chromatin at several regions of Six6 in vivo and cooperate for trans-activation of Six6 regulatory elements in vitro. In retinal progenitor/stem cells, both Lhx2 and Pax6 are genetically required for proper Six6 expression and forced co-expression of Lhx2 and Pax6 can synergistically trans-activate the Six6 locus. Our work reveals how two master regulators of eye development coordinate their action to sequentially promote tissue-specific transcriptional initiation and full activation of a retinal determinant gene.

  7. DBD: a transcription factor prediction database.

    PubMed

    Kummerfeld, Sarah K; Teichmann, Sarah A

    2006-01-01

    Regulation of gene expression influences almost all biological processes in an organism; sequence-specific DNA-binding transcription factors are critical to this control. For most genomes, the repertoire of transcription factors is only partially known. Hitherto transcription factor identification has been largely based on genome annotation pipelines that use pairwise sequence comparisons, which detect only those factors similar to known genes, or on functional classification schemes that amalgamate many types of proteins into the category of 'transcription factor'. Using a novel transcription factor identification method, the DBD transcription factor database fills this void, providing genome-wide transcription factor predictions for organisms from across the tree of life. The prediction method behind DBD identifies sequence-specific DNA-binding transcription factors through homology using profile hidden Markov models (HMMs) of domains. Thus, it is limited to factors that are homologus to those HMMs. The collection of HMMs is taken from two existing databases (Pfam and SUPERFAMILY), and is limited to models that exclusively detect transcription factors that specifically recognize DNA sequences. It does not include basal transcription factors or chromatin-associated proteins, for instance. Based on comparison with experimentally verified annotation, the prediction procedure is between 95% and 99% accurate. Between one quarter and one-half of our genome-wide predicted transcription factors represent previously uncharacterized proteins. The DBD (www.transcriptionfactor.org) consists of predicted transcription factor repertoires for 150 completely sequenced genomes, their domain assignments and the hand curated list of DNA-binding domain HMMs. Users can browse, search or download the predictions by genome, domain family or sequence identifier, view families of transcription factors based on domain architecture and receive predictions for a protein sequence.

  8. 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.

  9. The Crz1/Sp1 Transcription Factor of Cryptococcus neoformans Is Activated by Calcineurin and Regulates Cell Wall Integrity

    PubMed Central

    Lev, Sophie; Desmarini, Desmarini; Chayakulkeeree, Methee; Sorrell, Tania C.; Djordjevic, Julianne T.

    2012-01-01

    Cryptococcus neoformans survives host temperature and regulates cell wall integrity via a calcium-dependent phosphatase, calcineurin. However, downstream effectors of C. neoformans calcineurin are largely unknown. In S. cerevisiae and other fungal species, a calcineurin-dependent transcription factor Crz1, translocates to nuclei upon activation and triggers expression of target genes. We now show that the C. neoformans Crz1 ortholog (Crz1/Sp1), previously identified as a protein kinase C target during starvation, is a bona fide target of calcineurin under non-starvation conditions, during cell wall stress and growth at high temperature. Both the calcineurin-defective mutant, Δcna1, and a CRZ1/SP1 mutant (Δcrz1) were susceptible to cell wall perturbing agents. Furthermore, expression of the chitin synthase encoding gene, CHS6, was reduced in both mutants. We tracked the subcellular localization of Crz1-GFP in WT C. neoformans and Δcna1 in response to different stimuli, in the presence and absence of the calcineurin inhibitor, FK506. Exposure to elevated temperature (30–37°C vs 25°C) and extracellular calcium caused calcineurin-dependent nuclear accumulation of Crz1-GFP. Unexpectedly, 1M salt and heat shock triggered calcineurin-independent Crz1-GFP sequestration within cytosolic and nuclear puncta. To our knowledge, punctate cytosolic distribution, as opposed to nuclear targeting, is a unique feature of C. neoformans Crz1. We conclude that Crz1 is selectively activated by calcium/calcineurin-dependent and independent signals depending on the environmental conditions. PMID:23251520

  10. Multiple functions of nucleosomes and regulatory factors in transcription.

    PubMed

    Workman, J L; Buchman, A R

    1993-03-01

    The in vivo packaging of DNA with histone proteins to form chromatin makes its transcription a difficult process. Biochemical and genetic studies are beginning to reveal mechanistic details of how transcriptional regulatory factors confront at least two hurdles created by nucleosomes, the primary structural unit of chromatin. Regulatory factors must gain access to their respective binding sites and activate the formation of transcription complexes at core promoter elements. Distinct regulatory factors may be specialized to perform these functions.

  11. The activation of peroxisome proliferator-activated receptor γ is regulated by Krüppel-like transcription factors 6 & 9 under steatotic conditions

    SciTech Connect

    Escalona-Nandez, Ivonne; Guerrero-Escalera, Dafne; Estanes-Hernández, Alma; Ortíz-Ortega, Victor; Tovar, Armando R.; Pérez-Monter, Carlos

    2015-03-20

    Liver steatosis is characterised by lipid droplet deposition in hepatocytes that can leads to an inflammatory and fibrotic phenotype. Peroxisome proliferator-activated receptors (PPARs) play key roles in energetic homeostasis by regulating lipid metabolism in hepatic tissue. In adipose tissue PPARγ regulates the adipocyte differentiation by promoting the expression of lipid-associated genes. Within the liver PPARγ is up-regulated under steatotic conditions; however, which transcription factors participate in its expression is not completely understood. Krüppel-like transcription factors (KLFs) regulate various cellular mechanisms, such as cell proliferation and differentiation. KLFs are key components of adipogenesis by regulating the expression of PPARγ and other proteins such as the C-terminal enhancer binding protein (C/EBP). Here, we demonstrate that the transcript levels of Klf6, Klf9 and Pparγ are increased in response to a steatotic insult in vitro. Chromatin immunoprecipitation (ChIp) experiments showed that klf6 and klf9 are actively recruited to the Pparγ promoter region under these conditions. Accordingly, the loss-of-function experiments reduced cytoplasmic triglyceride accumulation. Here, we demonstrated that KLF6 and KLF9 proteins directly regulate PPARγ expression under steatotic conditions. - Highlights: • Palmitic acid promotes expression of KlF6 & KLF9 in HepG2 cells. • KLF6 and KLF9 promote the expression of PPARγ in response to palmitic acid. • Binding of KLF6 and KLF9 to the PPARγ promoter promotes steatosis in HepG2 cells. • KLF6 and KLF9 loss-of function diminishes the steatosis in HepG2 cells.

  12. Trm1p, a Zn(II)₂Cys₆-type transcription factor, is essential for the transcriptional activation of genes of methanol utilization pathway, in Pichia pastoris.

    PubMed

    Sahu, Umakant; Krishna Rao, Kamisetty; Rangarajan, Pundi N

    2014-08-15

    The zinc finger transcription factors Mxr1p and Rop are key regulators of methanol metabolism in the methylotrophic yeast, Pichia pastoris, while Trm1p and Trm2p regulate methanol metabolism in Candida boidinii. Here, we demonstrate that Trm1p is essential for the expression of genes of methanol utilization (mut) pathway in P. pastoris as well. Expression of AOXI and other genes of mut pathway is severely compromised in P. pastoris ΔTrm1 strain resulting in impaired growth on media containing methanol as the sole source of carbon. Trm1p localizes to the nucleus of cells cultured on glucose or methanol. The zinc finger domain of Mxr1p but not Trm1p binds to AOXI promoter sequences in vitro, indicating that these two positive regulators act by different mechanisms. We conclude that both Trm1p and Mxr1p are essential for the expression of genes of mut pathway in P. pastoris and the mechanism of transcriptional regulation of mut pathway may be similar in P. pastoris and C. boidinii.

  13. Rapid changes in heat-shock cognate 70 levels, heat-shock cognate phosphorylation state, heat-shock transcription factor, and metal transcription factor activity levels in response to heavy metal exposure during sea urchin embryonic development.

    PubMed

    Pinsino, Annalisa; Turturici, Giuseppina; Sconzo, Gabriella; Geraci, Fabiana

    2011-01-01

    The aim of the present study was to analyze and compare the effects of several metals on the embryos of the sea urchin Paracentrotus lividus, a key species within the Mediterranean Sea ecosystem. Embryos were continuously exposed from fertilization to the following metals: 0.6 mg/l copper, 3 mg/l lead, and 6 mg/l nickel. The embryos were then monitored for metal responses at the gastrula stage, which occurred 24 h after exposure. A biochemical multi-experimental approach was taken and involved the investigation of the levels of HSC70 expression and the involvement of heat shock factor (HSF) and/or metal transcription factor (MTF) in the response. Immunoblotting assays and electrophoretic mobility shift assays (EMSA) were used to detect stress protein levels and to study the interaction between DNA and specific transcription factors, respectively. In the 1 h during exposure to heavy metals, changes in HSC70 levels and HSC70 a phosphorylation state were observed. Rapid changes in HSF and MTF DNA-binding activity also occurred during the early stages of heavy metal exposure. In contrast, few developmental abnormalities were observed at the gastrula stage but more abnormalities were observed 48 h after metal exposure. These data demonstrate that changes in HSC70 levels and phosphorylation state as well as in HSF and MTF binding activities may be used to rapidly detect responses to heavy metal exposure. Detection of biochemical and molecular changes in response to metal exposure before manifestation of morpho-pathological effects are important for the prediction of morbidity, and these markers will be useful for determining the response to exposure as part of a toxicological exposure-response experiment and for determining responses for an impact assessment.

  14. Learning, memory, and transcription factors.

    PubMed

    Johnston, Michael V; Alemi, Lily; Harum, Karen H

    2003-03-01

    Cognitive disorders in children have traditionally been described in terms of clinical phenotypes or syndromes, chromosomal lesions, metabolic disorders, or neuropathology. Relatively little is known about how these disorders affect the chemical reactions involved in learning and memory. Experiments in fruit flies, snails, and mice have revealed some highly conserved pathways that are involved in learning, memory, and synaptic plasticity, which is the primary substrate for memory storage. These can be divided into short-term memory storage through local changes in synapses, and long-term storage mediated by activation of transcription to translate new proteins that modify synaptic function. This review summarizes evidence that disruptions in these pathways are involved in human cognitive disorders, including neurofibromatosis type I, Coffin-Lowry syndrome, Rubinstein-Taybi syndrome, Rett syndrome, tuberous sclerosis-2, Down syndrome, X-linked alpha-thalassemia/mental retardation, cretinism, Huntington disease, and lead poisoning.

  15. Synthesis and biological evaluation of diaryl-substituted carboranes as inhibitors of hypoxia inducible factor (HIF)-1 transcriptional activity.

    PubMed

    Minegishi, Hidemitsu; Matsukawa, Takuya; Nakamura, Hiroyuki

    2013-02-01

    Diaromatic-substituted ortho- and meta-carboranes were synthesized as mimics of manassantin A. Among the carboranes synthesized, compounds 1 and 2 showed significant inhibition of hypoxia-induced HIF-1 transcriptional activity, with IC(50) values of 3.2 and 2.2 μM, respectively. Compounds 1 and 2 similarly suppressed hypoxia-induced HIF-1α accumulation in a concentration-dependent manner without affecting the expression level of HIF-1α mRNA. The hypoxia-induced accumulation and translocation of HIF-1α into nuclei were not observed in HeLa cells treated with compounds 1 and 2 by immunofluorescence analysis, revealing that the inhibition of hypoxia-induced HIF-1 transcriptional activity is induced by compounds 1 and 2 through a degradation pathway of the HIF-1α protein under hypoxic conditions.

  16. Extending the functions of the homeotic transcription factor Cdx2 in the digestive system through nontranscriptional activities.

    PubMed

    Freund, Jean-Noël; Duluc, Isabelle; Reimund, Jean-Marie; Gross, Isabelle; Domon-Dell, Claire

    2015-02-07

    The homeoprotein encoded by the intestinal-specific Cdx2 gene is a major regulator of gut development and homeostasis, also involved in colon cancer as well as in intestinal-type metaplasias when it is abnormally expressed outside the gut. At the molecular level, structure/function studies have demonstrated that the Cdx2 protein is a transcription factor containing a conserved homeotic DNA-binding domain made of three alpha helixes arranged in a helix-turn-helix motif, preceded by a transcriptional domain and followed by a regulatory domain. The protein interacts with several thousand sites on the chromatin and widely regulates intestinal functions in stem/progenitor cells as well as in mature differentiated cells. Yet, this transcription factor also acts trough original nontranscriptional mechanisms. Indeed, the identification of novel protein partners of Cdx2 and also of a splicing variant revealed unexpected functions in the control of signaling pathways like the Wnt and NF-κB pathways, in double-strand break DNA repair and in premessenger RNA splicing. These novel functions of Cdx2 must be considered to fully understand the complexity of the role of Cdx2 in the healthy intestine and in diseases.

  17. Fungal-specific transcription factor AbPf2 activates pathogenicity in Alternaria brassicicola

    SciTech Connect

    Cho, Yangrae; Ohm, Robin A.; Grigoriev, Igor V.; Srivastava, Akhil

    2013-05-24

    Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen. To identify molecular determinants of pathogenicity, we created non-pathogenic mutants of a transcription factor-encoding gene, AbPf2. The frequency and timing of germination and appressorium formation on host plants were similar between the non-pathogenic abpf2 mutants and wild-type A. brassicicola. The mutants were also similar in vitro to wild-type A. brassicicola in terms of vegetative growth, conidium production, and responses to a phytoalexin, reactive oxygen species and osmolites. The hyphae of the mutants grew slowly but did not cause disease symptoms on the surface of host plants. Transcripts of the AbPf2 gene increased exponentially soon after wild-type conidia contacted their host plants . A small amount of AbPf2 protein, as monitored using GFP fusions, was present in young, mature conidia. The protein level decreased during saprophytic growth, but increased and was located primarily in fungal nuclei during pathogenesis. Levels of the proteins and transcripts sharply decreased following colonization of host tissues beyond the initial infection site. When expression of the transcription factor was induced in the wild-type during early pathogenesis, 106 fungal genes were also induced in the wild-type but not in the abpf2 mutants. Notably, 33 of the 106 genes encoded secreted proteins, including eight putative effector proteins. Plants inoculated with abpf2 mutants expressed higher levels of genes associated with photosynthesis, the pentose phosphate pathway and primary metabolism, but lower levels of defense-related genes. Our results suggest that AbPf2 is an important regulator of pathogenesis, but does not affect other cellular processes in A. brassicicola.

  18. Fox transcription factors: from development to disease.

    PubMed

    Golson, Maria L; Kaestner, Klaus H

    2016-12-15

    Forkhead box (Fox) transcription factors are evolutionarily conserved in organisms ranging from yeast to humans. They regulate diverse biological processes both during development and throughout adult life. Mutations in many Fox genes are associated with human disease and, as such, various animal models have been generated to study the function of these transcription factors in mechanistic detail. In many cases, the absence of even a single Fox transcription factor is lethal. In this Primer, we provide an overview of the Fox family, highlighting several key Fox transcription factor families that are important for mammalian development.

  19. INSIGHTS FROM GENOMIC PROFILING OF TRANSCRIPTION FACTORS

    PubMed Central

    Farnham, Peggy

    2010-01-01

    A crucial question in the field of gene regulation is whether the location at which a transcription factor binds influences its effectiveness or the mechanism by which it regulates transcription. Comprehensive transcription factor binding maps are needed to address these issues, and genome-wide mapping is now possible thanks to the technological advances of ChIP-chip and ChIP-Seq. This review discusses how recent genomic profiling of transcription factors gives insight into how binding specificity is achieved and what features of chromatin influence the ability of transcription factors to interact with the genome, and also suggests future experiments to further our understanding of the causes and consequences of transcription factor-genome interactions. PMID:19668247

  20. Purification & Characterization of Transcription Factors

    PubMed Central

    Nagore, LI; Nadeau, RJ; Guo, Q; Jadhav, YLA; Jarrett, HW; Haskins, WE

    2013-01-01

    Transcription factors (TFs) are essential for the expression of all proteins, including those involved in human health and disease. However, TFs are resistant to proteomic characterization because they are frequently masked by more abundant proteins due to the limited dynamic range of capillary liquid chromatography-tandem mass spectrometry and protein database searching. Purification methods, particularly strategies that exploit the high affinity of TFs for DNA response elements on gene promoters, can enrich TFs prior to proteomic analysis to improve dynamic range and penetrance of the TF proteome. For example, trapping of TF complexes specific for particular response elements has been achieved by recovering the element DNA-protein complex on solid supports. Additional methods for improving dynamic range include two- and three-dimensional gel electrophoresis incorporating electrophoretic mobility shift assays and Southwestern blotting for detection. Here we review methods for TF purification and characterization. We fully expect that future investigations will apply these and other methods to illuminate this important but challenging proteome. PMID:23832591

  1. Nrf2 Transcription Factor Can Directly Regulate mTOR: LINKING CYTOPROTECTIVE GENE EXPRESSION TO A MAJOR METABOLIC REGULATOR THAT GENERATES REDOX ACTIVITY.

    PubMed

    Bendavit, Gabriel; Aboulkassim, Tahar; Hilmi, Khalid; Shah, Sujay; Batist, Gerald

    2016-12-02

    Nrf2 is a master transcription factor that regulates a wide variety of cellular proteins by recognizing and binding to antioxidant response elements (AREs) in their gene promoter regions. In this study we show that increasing cellular Nrf2 results in transcriptional activation of the gene for mTOR, which is central to the PI3K signaling pathway. This is the case in cells with normal physiological PI3K. However, in cells with abnormally active PI3K increased cellular Nrf2 levels have no effect on mTOR. ChIP assays results show that increased Nrf2 binding is associated with decreased p65 binding and H3-K27me3 signal (marker of gene repression) as well as increased H3-K4me3 signal (marker of gene activation). However, in cells with PI3K activation, no effect of cellular Nrf2 increase on mTOR transcription was observed. In these cells, increasing Nrf2 levels increases Nrf2 promoter binding marginally, whereas p65 binding and H3-K27me3 mark were significantly increased, and H3-K4me3 signal is reduced. Together, these data show for the first time that Nrf2 directly regulates mTOR transcription when the PI3K pathway is intact, whereas this function is lost when PI3K is activated. We have identified a link between the Nrf2 system of sensing environmental stress and mTOR, which is a key cellular protein in metabolism. Studies in cells with activating mutations in the PI3K pathway suggest that Nrf2 transcriptional regulation of mTOR is related to promoter binding of p65 and of methylation of histone residues permissive of transcription.

  2. 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.

  3. The weak, fine-tuned binding of ubiquitous transcription factors to the Il-2 enhancer contributes to its T cell-restricted activity.

    PubMed Central

    Hentsch, B; Mouzaki, A; Pfeuffer, I; Rungger, D; Serfling, E

    1992-01-01

    The T lymphocyte-specific enhancers of the murine and human Interleukin 2 (Il-2) genes harbour several binding sites for ubiquitous transcription factors. All these sites for the binding of AP-1, NF-kB or Oct-1 are non-canonical sites, i.e. they differ in one or a few base pairs from consensus sequences for the optimal binding of these factors. Although the factors bind weakly to these sites, the latter are functionally important because their mutation to non-binding sites results in a decrease of inducible activity of the Il-2 enhancer. Conversion of three sites to canonical binding sites of Octamer factors, AP-1 and NF-kB results in a drastic increase in enhancer activity and the induction of the Il-2 enhancer in non-T cells, such as B cell lines, murine L cells and human HeLa cells. The introduction of two or three canonical sites into the enhancer leads to a further increase of its activity. Il-2 enhancer induction is also observed in B cells when the concentration of AP-1 and Oct factors increases as a result of cotransfections with FosB and Octamer expression plasmids. When Il-2 enhancer constructs carrying canonical factor binding sites were injected into Xenopus oocytes the strong binding of ubiquitous factors substantially overcomes the silencing effect of negatively acting factors present in resting primary T lymphocytes. These results suggest a fine-tuned interplay between ubiquitous and lymphoid-specific factors binding to and transactivating the Il-2 enhancer and show that the binding affinity of ubiquitous factors to the enhancer contributes to its cell-type specific activity. Moreover, we believe that a dramatic increase of transcriptional activity brought about by single point mutations at strategic important factor binding sites may also have relevance to the activation of nuclear oncogenes. Images PMID:1614851

  4. The yeast transcription factor Crz1 is activated by light in a Ca2+/calcineurin-dependent and PKA-independent manner.

    PubMed

    Bodvard, Kristofer; Jörhov, Anna; Blomberg, Anders; Molin, Mikael; Käll, Mikael

    2013-01-01

    Light in the visible range can be stressful to non-photosynthetic organisms. The yeast Saccharomyces cerevisiae has earlier been reported to respond to blue light via activation of the stress-regulated transcription factor Msn2p. Environmental changes also induce activation of calcineurin, a Ca(2+)/calmodulin dependent phosphatase, which in turn controls gene transcription by dephosphorylating the transcription factor Crz1p. We investigated the connection between cellular stress caused by blue light and Ca(2+) signalling in yeast by monitoring the nuclear localization dynamics of Crz1p, Msn2p and Msn4p. The three proteins exhibit distinctly different stress responses in relation to light exposure. Msn2p, and to a lesser degree Msn4p, oscillate rapidly between the nucleus and the cytoplasm in an apparently stochastic fashion. Crz1p, in contrast, displays a rapid and permanent nuclear localization induced by illumination, which triggers Crz1p-dependent transcription of its target gene CMK2. Moreover, increased extracellular Ca(2+) levels stimulates the light-induced responses of all three transcription factors, e.g. Crz1p localizes much quicker to the nucleus and a larger fraction of cells exhibits permanent Msn2p nuclear localization at higher Ca(2+) concentration. Studies in mutants lacking Ca(2+) transporters indicate that influx of extracellular Ca(2+) is crucial for the initial stages of light-induced Crz1p nuclear localization, while mobilization of intracellular Ca(2+) stores appears necessary for a sustained response. Importantly, we found that Crz1p nuclear localization is dependent on calcineurin and the carrier protein Nmd5p, while not being affected by increased protein kinase A activity (PKA), which strongly inhibits light-induced nuclear localization of Msn2/4p. We conclude that the two central signalling pathways, cAMP-PKA-Msn2/4 and Ca(2+)-calcineurin-Crz1, are both activated by blue light illumination.

  5. Scaling factors: transcription factors regulating subcellular domains.

    PubMed

    Mills, Jason C; Taghert, Paul H

    2012-01-01

    Developing cells acquire mature fates in part by selective (i.e. qualitatively different) expression of a few cell-specific genes. However, all cells share the same basic repertoire of molecular and subcellular building blocks. Therefore, cells must also specialize according to quantitative differences in cell-specific distributions of those common molecular resources. Here we propose the novel hypothesis that evolutionarily-conserved transcription factors called scaling factors (SFs) regulate quantitative differences among mature cell types. SFs: (1) are induced during late stages of cell maturation; (2) are dedicated to specific subcellular domains; and, thus, (3) allow cells to emphasize specific subcellular features. We identify candidate SFs and discuss one in detail: MIST1 (BHLHA15, vertebrates)/DIMM (CG8667, Drosophila); professional secretory cells use this SF to scale up regulated secretion. Because cells use SFs to develop their mature properties and also to adapt them to ever-changing environmental conditions, SF aberrations likely contribute to diseases of adult onset.

  6. TALE-induced bHLH transcription factors that activate a pectate lyase contribute to water soaking in bacterial spot of tomato

    PubMed Central

    Schwartz, Allison R.; Morbitzer, Robert; Lahaye, Thomas; Staskawicz, Brian J.

    2017-01-01

    AvrHah1 [avirulence (avr) gene homologous to avrBs3 and hax2, no. 1] is a transcription activator-like (TAL) effector (TALE) in Xanthomonas gardneri that induces water-soaked disease lesions on fruits and leaves during bacterial spot of tomato. We observe that water from outside the leaf is drawn into the apoplast in X. gardneri-infected, but not X. gardneriΔavrHah1 (XgΔavrHah1)-infected, plants, conferring a dark, water-soaked appearance. The pull of water can facilitate entry of additional bacterial cells into the apoplast. Comparing the transcriptomes of tomato infected with X. gardneri vs. XgΔavrHah1 revealed the differential up-regulation of two basic helix–loop–helix (bHLH) transcription factors with predicted effector binding elements (EBEs) for AvrHah1. We mined our RNA-sequencing data for differentially up-regulated genes that could be direct targets of the bHLH transcription factors and therefore indirect targets of AvrHah1. We show that two pectin modification genes, a pectate lyase and pectinesterase, are targets of both bHLH transcription factors. Designer TALEs (dTALEs) for the bHLH transcription factors and the pectate lyase, but not for the pectinesterase, complement water soaking when delivered by XgΔavrHah1. By perturbing transcriptional networks and/or modifying the plant cell wall, AvrHah1 may promote water uptake to enhance tissue damage and eventual bacterial egression from the apoplast to the leaf surface. Understanding how disease symptoms develop may be a useful tool for improving the tolerance of crops from damaging disease lesions. PMID:28100489

  7. RNA Activation of the Vascular Endothelial Growth Factor Gene (VEGF) Promoter by Double-Stranded RNA and Hypoxia: Role of Noncoding VEGF Promoter Transcripts

    PubMed Central

    Wagner, Kay-Dietrich; Hofman, Paul; Van Obberghen, Emmanuel

    2016-01-01

    RNA activation (RNAa) is a gene regulation process in which promoter-targeted short double-stranded RNAs (dsRNAs) or microRNAs (miRs) induce target gene expression at the transcriptional level. Here, we investigate the presence of cryptic promoter transcripts within the VEGF promoter. Single-strand sense and antisense noncoding vascular endothelial growth factor (NcVEGF) promoter transcripts are identified, and their respective expression is studied in cells transfected with a VEGF promoter targeted dsRNA, namely, dsVEGF706, in hypoxic cells and in human malignant lung tissues. Interestingly, in dsVEGF706-transfected, as well as in hypoxic cells, NcVEGF expression levels increase coordinately with coding VEGF expression. Ago2 interaction with both sense and antisense NcVEGFs is increased in hypoxic cells, whereas in dsVEGF706-transfected cells, Ago2 and the antisense strand of the dsRNA interact specifically with the sense NcVEGF transcript. Furthermore, both dsVEGF706 and ectopic NcVEGF transcripts are able to activate the VEGF promoter endogenously present or in a reporter construct. Finally, using small interfering RNA targeting Ago2, we show that RNAa plays a role in the maintenance of increased VEGF and NcVEGF expression after hypoxia. Given the central role of VEGF in major human diseases, including cancer, this novel molecular mechanism is poised to reveal promising possibilities for therapeutic interventions. PMID:26976645

  8. Effect of transcription factor GATA-2 on phagocytic activity of alveolar macrophages from Pneumocystis carinii-infected hosts.

    PubMed

    Lasbury, Mark E; Tang, Xing; Durant, Pamela J; Lee, Chao-Hung

    2003-09-01

    Alveolar macrophages from Pneumocystis carinii-infected hosts are defective in phagocytosis (W. Chen, J. W. Mills, and A. G. Harmsen, Int. J. Exp. Pathol. 73:709-720, 1992; H. Koziel et al., J. Clin. Investig. 102:1332-1344, 1998). Experiments were performed to determine whether this defect is specific for P. carinii organisms. The results showed that these macrophages were unable to phagocytose both P. carinii organisms and fluorescein isothiocyanate (FITC)-conjugated latex beads, indicating that alveolar macrophages from P. carinii-infected hosts have a general defect in phagocytosis. To determine whether this defect correlates with the recently discovered down-regulation of the GATA-2 transcription factor gene during P. carinii infection, alveolar macrophages from dexamethasone-suppressed or healthy rats were treated with anti-GATA-2 oligonucleotides and then assayed for phagocytosis. Aliquots of the alveolar macrophages were also treated with the sense oligonucleotides as the control. Cells treated with the antisense oligonucleotides were found to have a 46% reduction in phagocytosis of P. carinii organisms and a 65% reduction in phagocytosis of FITC-latex beads compared to those treated with the sense oligonucleotides. To determine whether the defect in phagocytosis in alveolar macrophages from P. carinii-infected hosts can be corrected by overexpression of GATA-2, a plasmid containing the rat GATA-2 gene in the sense orientation driven by the cytomegalovirus (CMV) promoter was introduced into alveolar macrophages from P. carinii-infected rats. Aliquots of the same cells transfected with a plasmid containing GATA-2 in the antisense orientation relative to the CMV promoter served as the control. Alveolar macrophages treated with the sense GATA-2 expression construct were found to increase their phagocytic activity by 66% in phagocytosis of P. carinii organisms and by 280% in phagocytosis of FITC-latex beads compared to those that received the antisense GATA-2

  9. Prunus transcription factors: breeding perspectives

    PubMed Central

    Bianchi, Valmor J.; Rubio, Manuel; Trainotti, Livio; Verde, Ignazio; Bonghi, Claudio; Martínez-Gómez, Pedro

    2015-01-01

    Many plant processes depend on differential gene expression, which is generally controlled by complex proteins called transcription factors (TFs). In peach, 1533 TFs have been identified, accounting for about 5.5% of the 27,852 protein-coding genes. These TFs are the reference for the rest of the Prunus species. TF studies in Prunus have been performed on the gene expression analysis of different agronomic traits, including control of the flowering process, fruit quality, and biotic and abiotic stress resistance. These studies, using quantitative RT-PCR, have mainly been performed in peach, and to a lesser extent in other species, including almond, apricot, black cherry, Fuji cherry, Japanese apricot, plum, and sour and sweet cherry. Other tools have also been used in TF studies, including cDNA-AFLP, LC-ESI-MS, RNA, and DNA blotting or mapping. More recently, new tools assayed include microarray and high-throughput DNA sequencing (DNA-Seq) and RNA sequencing (RNA-Seq). New functional genomics opportunities include genome resequencing and the well-known synteny among Prunus genomes and transcriptomes. These new functional studies should be applied in breeding programs in the development of molecular markers. With the genome sequences available, some strategies that have been used in model systems (such as SNP genotyping assays and genotyping-by-sequencing) may be applicable in the functional analysis of Prunus TFs as well. In addition, the knowledge of the gene functions and position in the peach reference genome of the TFs represents an additional advantage. These facts could greatly facilitate the isolation of genes via QTL (quantitative trait loci) map-based cloning in the different Prunus species, following the association of these TFs with the identified QTLs using the peach reference genome. PMID:26124770

  10. Pb2+ induces gastrin gene expression by extracellular signal-regulated kinases 1/2 and transcription factor activator protein 1 in human gastric carcinoma cells.

    PubMed

    Chan, Chien-Pin; Tsai, Yao-Ting; Chen, Yao-Li; Hsu, Yu-Wen; Tseng, Joseph T; Chuang, Hung-Yi; Shiurba, Robert; Lee, Mei-Hsien; Wang, Jaw-Yuan; Chang, Wei-Chiao

    2015-02-01

    Divalent lead ions (Pb(2+) ) are toxic environmental pollutants known to cause serious health problems in humans and animals. Absorption of Pb(2+) from air, water, and food takes place in the respiratory and digestive tracts. The ways in which absorbed Pb(2+) affects cell physiology are just beginning to be understood at the molecular level. Here, we used reverse transcription PCR and Western blotting to analyze cultures of human gastric carcinoma cells exposed to 10 μM lead nitrate. We found that Pb(2+) induces gastrin hormone gene transcription and translation in a time-dependent manner. Promoter deletion analysis revealed that activator protein 1 (AP1) was necessary for gastrin gene transcription in cells exposed to Pb(2+) . MitogIen-activated protein kinase (MAPK)/ERK kinase inhibitor PD98059 suppressed the Pb(2+) -induced increase in messenger RNA. Epidermal growth factor receptor (EGFR) inhibitors AG1478 and PD153035 reduced both transcription and phosphorylation by extracellular signal-regulated kinase (ERK1/2). Cells exposed to Pb(2+) also increased production of c-Jun protein, a component of AP1, and over-expression of c-Jun enhanced activation of the gastrin promoter. In sum, the findings suggest the EGFR-ERK1/2-AP1 pathway mediates the effects of Pb(2+) on gastrin gene activity in cell culture.

  11. The mycobacterial antibiotic resistance determinant WhiB7 acts as a transcriptional activator by binding the primary sigma factor SigA (RpoV)

    PubMed Central

    Burian, Ján; Yim, Grace; Hsing, Michael; Axerio-Cilies, Peter; Cherkasov, Artem; Spiegelman, George B.; Thompson, Charles J.

    2013-01-01

    Tuberculosis therapeutic options are limited by the high intrinsic antibiotic resistance of Mycobacterium tuberculosis. The putative transcriptional regulator WhiB7 is crucial for the activation of systems that provide resistance to diverse antibiotic classes. Here, we used in vitro run-off, two-hybrid assays, as well as mutagenic, complementation and protein pull-down experiments, to characterize WhiB7 as an auto-regulatory, redox-sensitive transcriptional activator in Mycobacterium smegmatis. We provide the first direct biochemical proof that a WhiB protein promotes transcription and also demonstrate that this activity is sensitive to oxidation (diamide). Its partner protein for transcriptional activation was identified as SigA, the primary sigma factor subunit of RNA polymerase. Residues required for the interaction mapped to region 4 of SigA (including R515H) or adjacent domains of WhiB7 (including E63D). WhiB7’s ability to provide a specific spectrum of antibiotic-resistance was dependent on these residues as well as its C-terminal AT-hook module that binds to an AT-rich motif immediately upstream of the −35 hexamer recognized by SigA. These experimentally established constrains, combined with protein structure predictions, were used to generate a working model of the WhiB7–SigA-promoter complex. Inhibitors preventing WhiB7 interactions could allow the use of previously ineffective antibiotics for treatment of mycobacterial diseases. PMID:23990327

  12. Nox4 is critical for hypoxia-inducible factor 2-alpha transcriptional activity in von Hippel-Lindau-deficient renal cell carcinoma.

    PubMed

    Maranchie, Jodi K; Zhan, Ye

    2005-10-15

    Inactivation of the von Hippel-Lindau tumor suppressor (VHL) is an early event in >60% of sporadic clear cell renal cell carcinoma (RCC). Loss of VHL E3 ubiquitin ligase function results in accumulation of the alpha-subunit of the hypoxia-inducible heterodimeric transcription factor (HIF-alpha) and transcription of an array of genes including vascular endothelial growth factor, transforming growth factor-alpha, and erythropoietin. Studies have shown that HIF-alpha can be alternatively activated by reactive oxygen species. Nox4 is an NADP(H) oxidase that generates signaling levels of superoxide and is found in greatest abundance in the distal renal tubules. To determine if Nox4 contributes to HIF activity in RCC, we examined the impact of Nox4 expression on HIF-alpha expression and transactivation. We report here that small inhibitory RNA (siRNA) knockdown of Nox4 in 786-0 human renal tumor cells expressing empty vector (PRC) or wild-type VHL (WT) results in 50% decrease in intracellular reactive oxygen species as measured by a fluorescent 2',7'-dichlorofluorescin diacetate assay, and >85% reduction in HIF2-alpha mRNA and protein levels by quantitative reverse transcription-PCR and Western blot analysis. Furthermore, expression of the HIF target genes, vascular endothelial growth factor, transforming growth factor-alpha, and Glut-1 was abrogated by 93%, 74%, and 99%, respectively, after stable transfection with Nox4 siRNA relative to nontargeting siRNA, as determined by quantitative reverse transcription-PCR. Thus, renal Nox4 expression is essential for full HIF2-alpha expression and activity in 786-0 renal tumor cells, even in the absence of functional VHL. We propose the use of Nox4 as a target in the treatment of clear cell RCC.

  13. The endoplasmic reticulum stress response is stimulated through the continuous activation of transcription factors ATF6 and XBP1 in Ins2+/Akita pancreatic beta cells.

    PubMed

    Nozaki, Jun ichi; Kubota, Hiroshi; Yoshida, Hiderou; Naitoh, Motoko; Goji, Junko; Yoshinaga, Takeo; Mori, Kazutoshi; Koizumi, Akio; Nagata, Kazuhiro

    2004-03-01

    The dominant C96Y mutation of one of the two murine insulin genes, Ins2, causes diabetes mellitus in 'Akita' mice. Here we established pancreatic islet beta cell lines from heterozygous mice (Ins2+/Akita). Western blot analysis of endoplasmic reticulum (ER) molecular chaperones indicated that Grp78, Grp94 and Orp150 are significantly increased in Ins2+/Akita cells compared with wild-type (Ins2+/+) cells. Reporter gene assays using the human GRP78 promoter with or without the ER stress response element (ERSE) showed that Ins2+/Akita cells exhibit significantly stronger ERSE-dependent transcriptional activity than Ins2+/+ cells. Transient over-expression of the Ins2 C96Y mutant in wild-type beta cells induces a stronger ERSE-dependent stress response than does wild-type Ins2 over-expression. The ERSE-binding transcription factor ATF6 is strongly activated in Ins2+/Akita cells. The activity of a reporter containing the specific binding sequence of another ERSE-binding transcription factor, XBP1, is also enhanced in Ins2+/Akita cells. Levels of active forms of XBP1 mRNA and protein are both markedly elevated in Ins2+/Akita cells. These results indicate that this cell line is subject to continuous ER stress and that the Ins2 C96Y mutation induces the expression of ER chaperones through the activation of ATF6 and XBP1.

  14. The Drosophila transcription factor Adf-1 (nalyot) regulates dendrite growth by controlling FasII and Staufen expression downstream of CaMKII and neural activity.

    PubMed

    Timmerman, Christina; Suppiah, Somu; Gurudatta, Baraka V; Yang, Jingping; Banerjee, Christopher; Sandstrom, David J; Corces, Victor G; Sanyal, Subhabrata

    2013-07-17

    Memory deficits in Drosophila nalyot mutants suggest that the Myb family transcription factor Adf-1 is an important regulator of developmental plasticity in the brain. However, the cellular functions for this transcription factor in neurons or molecular mechanisms by which it regulates plasticity remain unknown. Here, we use in vivo 3D reconstruction of identifiable larval motor neuron dendrites to show that Adf-1 is required cell autonomously for dendritic development and activity-dependent plasticity of motor neurons downstream of CaMKII. Adf-1 inhibition reduces dendrite growth and neuronal excitability, and results in motor deficits and altered transcriptional profiles. Surprisingly, analysis by comparative chromatin immunoprecipitation followed by sequencing (ChIP-Seq) of Adf-1, RNA Polymerase II (Pol II), and histone modifications in Kc cells shows that Adf-1 binding correlates positively with high Pol II-pausing indices and negatively with active chromatin marks such as H3K4me3 and H3K27ac. Consistently, the expression of Adf-1 targets Staufen and Fasciclin II (FasII), identified through larval brain ChIP-Seq for Adf-1, is negatively regulated by Adf-1, and manipulations of these genes predictably modify dendrite growth. Our results imply mechanistic interactions between transcriptional and local translational machinery in neurons as well as conserved neuronal growth mechanisms mediated by cell adhesion molecules, and suggest that CaMKII, Adf-1, FasII, and Staufen influence crucial aspects of dendrite development and plasticity with potential implications for memory formation. Further, our experiments reveal molecular details underlying transcriptional regulation by Adf-1, and indicate active interaction between Adf-1 and epigenetic regulators of gene expression during activity-dependent neuronal plasticity.

  15. Activating Transcription Factor 4 (ATF4) modulates Rho GTPase levels and function via regulation of RhoGDIα

    PubMed Central

    Pasini, Silvia; Liu, Jin; Corona, Carlo; Peze-Heidsieck, Eugenie; Shelanski, Michael; Greene, Lloyd A.

    2016-01-01

    In earlier studies, we showed that ATF4 down-regulation affects post-synaptic development and dendritic spine morphology in neurons through increased turnover of the Rho GTPase Cell Division Cycle 42 (Cdc42) protein. Here, we find that ATF4 down-regulation in both hippocampal and cortical neuron cultures reduces protein and message levels of RhoGDIα, a stabilizer of the Rho GTPases including Cdc42. This effect is rescued by an shATF4-resistant active form of ATF4, but not by a mutant that lacks transcriptional activity. This is, at least in part, due to the fact that Arhgdia, the gene encoding RhoGDIα, is a direct transcriptional target of ATF4 as is shown in ChIP assays. This pathway is not restricted to neurons. This is seen in an impairment of cell migration on ATF4 reduction in non-neuronal cells. In conclusion, we have identified a new cellular pathway in which ATF4 regulates the expression of RhoGDIα that in turn affects Rho GTPase protein levels, and thereby, controls cellular functions as diverse as memory and cell motility. PMID:27841340

  16. The oncoprotein HBXIP up-regulates FGF4 through activating transcriptional factor Sp1 to promote the migration of breast cancer cells.

    PubMed

    Shi, Hui; Li, Yinghui; Feng, Guoxing; Li, Leilei; Fang, Runping; Wang, Zhen; Qu, Jie; Ding, Peijian; Zhang, Xiaodong; Ye, Lihong

    2016-02-26

    We have reported that the oncoprotein hepatitis B X-interacting protein (HBXIP) is able to promote migration of breast cancer cells. Fibroblast growth factor 4 (FGF4) is a multipotent growth factor and is highly expressed in various human cancers. However, the regulatory mechanism of FGF4 in breast cancer remains poorly understood. In the present study, we report that HBXIP is able to up-regulate FGF4 to enhance the migration of breast cancer cells. Immunohistochemistry staining showed that HBXIP and FGF4 were highly expressed in clinical metastatic lymph nodes of breast tumor. The expression levels of HBXIP were positively related to those of FGF4 in clinical breast cancer tissues. Then, we validated that HBXIP up-regulated the expression of FGF4 at the levels of promoter, mRNA and protein by luciferase reporter gene assays, reverse transcription-polymerase chain reaction and Western blot analysis. Moreover, we found that HBXIP was able to activate FGF4 promoter through transcriptional factor Sp1 by luciferase reporter gene assays. Chromatin immunoprecipitation assays confirmed that HBXIP coactivated Sp1 to stimulate FGF4 promoter. In function, we showed that HBXIP promoted breast cancer cell migration through FGF4 by wound healing and transwell cell migration assays. Thus, we conclude that the oncoprotein HBXIP up-regulates FGF4 through activating transcriptional factor Sp1 to promote the migration of breast cancer cells. Therapeutically, HBXIP may serve as a novel target in breast cancer.

  17. Duplicate Maize Wrinkled1 Transcription Factors Activate Target Genes Involved in Seed Oil Biosynthesis1[C][W

    PubMed Central

    Pouvreau, Benjamin; Baud, Sébastien; Vernoud, Vanessa; Morin, Valérie; Py, Cyrille; Gendrot, Ghislaine; Pichon, Jean-Philippe; Rouster, Jacques; Paul, Wyatt; Rogowsky, Peter M.

    2011-01-01

    WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs. PMID:21474435

  18. First molluscan transcription factor activator protein-1 (Ap-1) member from disk abalone and its expression profiling against immune challenge and tissue injury.

    PubMed

    De Zoysa, Mahanama; Nikapitiya, Chamilani; Lee, Youngdeuk; Lee, Sukkyoung; Oh, Chulhong; Whang, Ilson; Yeo, Sang-Yeop; Choi, Cheol Young; Lee, Jehee

    2010-12-01

    The regulation of transcriptional activation is an essential and critical point in gene expression. In this study, we describe a novel transcription factor activator protein-1 (Ap-1) gene from disk abalone Haliotis discus discus (AbAp-1) for the first time in mollusk. It was identified by homology screening of an abalone normalized cDNA library. The cloned AbAp-1 consists of a 945 bp coding region that encodes a putative protein containing 315 amino acids. The AbAp-1 gene is composed of a characteristic Jun transcription factor domain and a highly conserved basic leucine zipper (bZIP) signature similar to known Ap-1 genes. The AbAp-1 shares 46, 43 and, 40% amino acid identities with fish (Takifugu rubripes), human and insect (Ixodes scapularis) Ap-1, respectively. Quantitative real time RT-PCR analysis confirmed that AbAp-1 gene expression is constitutive in all selected tissues. AbAp-1 was upregulated in gills after bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes) challenge; and, upregulated in hemocytes and gills by viral hemorrhagic septicemia virus (VHSV) challenge. Shell damage and tissue injury also increased the transcriptional level of Ap-1 in mantle together with other transcription factors (NF-kB, LITAF) and pro-inflammatory TNF-α. All results considered, identification and gene expression data demonstrate that abalone Ap-1 is an important regulator in innate immune response against bacteria and virus, as well as in the inflammatory response during tissue injury. In addition, stimulation of Ap-1 under different external stimuli could be useful to understand the Ap-1 biology and its downstream target genes, especially in abalone-like mollusks.

  19. HIF transcription factors, inflammation, and immunity.

    PubMed

    Palazon, Asis; Goldrath, Ananda W; Nizet, Victor; Johnson, Randall S

    2014-10-16

    The hypoxic response in cells and tissues is mediated by the family of hypoxia-inducible factor (HIF) transcription factors; these play an integral role in the metabolic changes that drive cellular adaptation to low oxygen availability. HIF expression and stabilization in immune cells can be triggered by hypoxia, but also by other factors associated with pathological stress: e.g., inflammation, infectious microorganisms, and cancer. HIF induces a number of aspects of host immune function, from boosting phagocyte microbicidal capacity to driving T cell differentiation and cytotoxic activity. Cellular metabolism is emerging as a key regulator of immunity, and it constitutes another layer of fine-tuned immune control by HIF that can dictate myeloid cell and lymphocyte development, fate, and function. Here we discuss how oxygen sensing in the immune microenvironment shapes immunological response and examine how HIF and the hypoxia pathway control innate and adaptive immunity.

  20. A C-repeat binding factor transcriptional activator (CBF/DREB1) from European bilberry (Vaccinium myrtillus) induces freezing tolerance when expressed in Arabidopsis thaliana.

    PubMed

    Oakenfull, Rachael J; Baxter, Robert; Knight, Marc R

    2013-01-01

    Freezing stress affects all plants from temperate zones to the poles. Global climate change means such freezing events are becoming less predictable. This in turn reduces the ability of plants to predict the approaching low temperatures and cold acclimate. This has consequences for crop yields and distribution of wild plant species. C-repeat binding factors (CBFs) are transcription factors previously shown to play a vital role in the acclimation process of Arabidopsis thaliana, controlling the expression of hundreds of genes whose products are necessary for freezing tolerance. Work in other plant species cements CBFs as key determinants in the trait of freezing tolerance in higher plants. To test the function of CBFs from highly freezing tolerant plants species we cloned and sequenced CBF transcription factors from three Vaccinium species (Vaccinium myrtillus, Vaccinium uliginosum and Vaccinium vitis-idaea) which we collected in the Arctic. We tested the activity of CBF transcription factors from the three Vaccinium species by producing transgenic Arabidopsis lines overexpressing them. Only the Vaccinium myrtillus CBF was able to substantially activate COR (CBF-target) gene expression in the absence of cold. Correspondingly, only the lines expressing the Vaccinium myrtillus CBF were constitutively freezing tolerant. The basis for the differences in potency of the three Vaccinium CBFs was tested by observing cellular localisation and protein levels. All three CBFs were correctly targeted to the nucleus, but Vaccinium uliginosum CBF appeared to be relatively unstable. The reasons for lack of potency for Vaccinium vitis-idaea CBF were not due to stability or targeting, and we speculate that this was due to altered transcription factor function.

  1. 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.

  2. Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR).

    PubMed

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Isaac, Sara; Eden, Amir; Lauria, Ines; Langer, Thomas; Orly, Joseph

    2015-06-15

    High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases.

  3. Transient receptor potential melastatin-3 (TRPM3)-induced activation of AP-1 requires Ca2+ ions and the transcription factors c-Jun, ATF2, and ternary complex factor.

    PubMed

    Lesch, Andrea; Hui, Xin; Lipp, Peter; Thiel, Gerald

    2015-04-01

    The steroid pregnenolone sulfate activates the transcription factor activator protein-1 (AP-1) via stimulation of transient receptor potential melastatin-3 (TRPM3) channels. Here, we show that the signaling pathway requires an influx of Ca(2+) ions into the cells and a rise in the intracellular Ca(2+) levels. The upregulation of AP-1 was attenuated in cells that overexpressed mitogen activated protein kinase phosphatase-1, indicating that Ca(2+) ions prolong the signaling cascade via activation of mitogen activated protein kinases. On the transcriptional level, expression of a dominant-negative mutant of the basic region leucine zipper protein c-Jun, a major constituent of the AP-1 transcription factor complex, or expression of a c-Jun-specific short hairpin RNA attenuated pregnenolone sulfate-induced AP-1 activation. In addition, stimulation of TRPM3 channels increased the transcriptional activation potential of the basic region leucine zipper protein ATF2. Inhibition of ATF2 target gene expression via expression of a dominant-negative mutant of ATF2 or expression of an ATF2-specific short hairpin RNA interfered with TRPM3-mediated stimulation of AP-1. Moreover, we show that a dominant-negative mutant of the ternary complex factor (TCF) Elk-1 attenuated the upregulation of AP-1 following stimulation of TRPM3 channels. Thus, c-Jun, ATF2, and TCFs are required to connect the intracellular signaling cascade elicited by activation of TRPM3 channels with enhanced transcription of AP-1-regulated genes. We conclude that pregnenolone sulfate-induced TRPM3 channel activation changes the gene expression pattern of the cells by activating transcription of c-Jun-, ATF2-, and TCF-controlled genes.

  4. Sry is a transcriptional activator.

    PubMed

    Dubin, R A; Ostrer, H

    1994-09-01

    The SRY gene functions as a genetic switch in gonadal ridge initiating testis determination. The mouse Sry and human SRY open reading frames (ORFs) share a conserved DNA-binding domain (the HMG-box) yet exhibit no additional homology outside this region. As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and mouse SRY ORFs contain a nuclear localization signal. The mouse Sry HMG-box domain selectively binds the sequence NACAAT in vitro when challenged with a random pool of oligonucleotides and binds AACAAT with the highest affinity. When put under the control of a heterologous promotor, the mouse Sry gene activated transcription of a reporter gene containing multiple copies of the AACAAT binding site. Activation was likewise observed for a GAL4-responsive reporter gene, when the mouse Sry gene was linked to the DNA-binding domain of GAL4. Using this system, the activation function was mapped to a glutamine/histidine-rich domain. In addition, LexA-mouse Sry fusion genes activated a LexA-responsive reporter gene in yeast. In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation. These studies suggest that both the human and the mouse SRY ORFs encode nuclear, DNA-binding proteins and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains.

  5. Deoxycholic acid inhibited proliferation and induced apoptosis and necrosis by regulating the activity of transcription factors in rat pancreatic acinar cell line AR42J.

    PubMed

    Zhang, Guixin; Zhang, Jingwen; Shang, Dong; Qi, Bing; Chen, Hailong

    2015-09-01

    The objective of this study is to investigate the effect of deoxycholic acid (DCA) on rat pancreatic acinar cell line AR42J and the functional mechanisms of DCA on AR42J cells. AR42J cells were treated with various concentrations of DCA for 24 h and also treated with 0.4 mmol/L DCA for multiple times, and then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to detect the AR42J cell survival rate. Flow cytometric was used to detect the cell apoptosis and necrosis in AR42J cells treated with 0.4 mmol/L and 0.8 mmol/L DCA. The cells treated with phosphate buffer saline (PBS) were served as control. In addition, the DNA-binding activity assays of transcription factors (TFs) in nuclear proteins of cells treated with DCA were determined using Panomics Procarta Transcription Factor Assay Kit. The relative survival rates were markedly decreased (P < 0.05) in a dose- and time-dependent manner. Compared with control group, the cell apoptosis and necrosis ratio were both significantly elevated in 0.4 mmol/L DCA and 0.8 mmol/L DCA groups (P < 0.01). A significant increase (P < 0.05) in the activity of transcription factor 2 (ATF2), interferon-stimulated response element (ISRE), NKX-2.5, androgen receptor (AR), p53, and hypoxia-inducible factor-1 (HIF-1) was observed, and the activity of peroxisome proliferator-activated receptor (PPAR), activator protein 1 (AP1), and E2F1 was reduced (P < 0.05). In conclusion, DCA inhibited proliferation and induced apoptosis and necrosis in AR42J cells. The expression changes of related genes regulated by TFs might be the molecular mechanism of AR42J cell injury.

  6. The transcription factors Pap1 and Prr1 collaborate to activate antioxidant, but not drug tolerance, genes in response to H2O2.

    PubMed

    Calvo, Isabel A; García, Patricia; Ayté, José; Hidalgo, Elena

    2012-06-01

    In response to hydrogen peroxide (H(2)O(2)), the transcription factor Pap1 from Schizosaccharomyces pombe regulates transcription of genes required for adaptation to oxidative stress and for tolerance to toxic drugs. H(2)O(2) induces oxidation of Pap1, its nuclear accumulation and expression of more than fifty Pap1-dependent genes. Oxidation and nuclear accumulation of Pap1 can also be accomplished by genetic inhibition of thioredoxin reductase. Furthermore, genetic alteration of the nuclear export pathway, or mutations in Pap1 nuclear export signal trigger nuclear accumulation of reduced Pap1. We show here that a subset of Pap1-dependent genes, such as those coding for the efflux pump Caf5, the ubiquitin-like protein Obr1 or the dehydrogenase SPCC663.08c, only require nuclear Pap1 for activation, whereas another subset of genes, those coding for the antioxidants catalase, sulfiredoxin or thioredoxin reductase, do need oxidized Pap1 to form a heterodimer with the constitutively nuclear transcription factor Prr1. The ability of Pap1 to bind and activate drug tolerance promoters is independent on Prr1, whereas its affinity for the antioxidant promoters is significantly enhanced upon association with Prr1. This finding suggests that the activation of both antioxidant and drug resistance genes in response to oxidative stress share a common inducer, H(2)O(2), but alternative effectors.

  7. Cisplatin upregulates Saccharomyces cerevisiae genes involved in iron homeostasis through activation of the iron insufficiency-responsive transcription factor Aft1.

    PubMed

    Kimura, Akiko; Ohashi, Kazuaki; Naganuma, Akira

    2007-02-01

    The response of Saccharomyces cerevisiae to cisplatin was investigated by examining variations in gene expression using cDNA microarrays and confirming the results by reverse transcription polymerase chain reaction (RT-PCR). The mRNA levels of 14 proteins involved in iron homeostasis were shown to be increased by cisplatin. Interestingly, the expression of all 14 genes is known to be regulated by Aft1, a transcription factor activated in response to iron insufficiency. The promoter of one of these genes, FET3, has been relatively well studied, so we performed a reporter assay using the FET3 promoter and showed that an Aft1 binding site in the promoter region is indispensable for induction of transcription by cisplatin. The active domain of Aft1 necessary for activation of the FET3 promoter by cisplatin is identical to the one required for activation by bathophenanthroline sulfonate, an inhibitor of cellular iron uptake. Furthermore, we found that cisplatin inhibits the uptake of (55)Fe(II) into yeast cells. These findings suggest that cisplatin activates Aft1 through the inhibition of iron uptake into the cells, after which the expression of Aft1 target genes involved in iron uptake might be induced.

  8. Yin Yang 1: a multifaceted protein beyond a transcription factor.

    PubMed

    Deng, Zhiyong; Cao, Paul; Wan, Mei Mei; Sui, Guangchao

    2010-01-01

    As a transcription factor, Yin Yang 1 (YY1) regulates the transcription of a dazzling list of genes and the number of its targets still mounts. Recent studies revealed that YY1 possesses functions independent of its DNA binding activity and its regulatory role in tumorigenesis has started to emerge.

  9. Creating cellular diversity through transcription factor competition

    PubMed Central

    Göttgens, Berthold

    2015-01-01

    The development of blood cells has long served as a model system to study the generation of diverse mature cells from multipotent progenitors. The article by Org et al (2015) reveals how transcription factor competition on primed DNA templates may contribute to embryonic blood cell specification during the early stages of mesoderm development. The study not only provides new insights into the functionality of the key haematopoietic transcription factor Scl/Tal1, but also provides a potentially widely applicable framework for transcription factor-mediated cell fate specification. PMID:25680687

  10. Distinct mechanism of activation of two transcription factors, AmyR and MalR, involved in amylolytic enzyme production in Aspergillus oryzae.

    PubMed

    Suzuki, Kuta; Tanaka, Mizuki; Konno, Yui; Ichikawa, Takanori; Ichinose, Sakurako; Hasegawa-Shiro, Sachiko; Shintani, Takahiro; Gomi, Katsuya

    2015-02-01

    The production of amylolytic enzymes in Aspergillus oryzae is induced in the presence of starch or maltose, and two Zn2Cys6-type transcription factors, AmyR and MalR, are involved in this regulation. AmyR directly regulates the expression of amylase genes, and MalR controls the expression of maltose-utilizing (MAL) cluster genes. Deletion of malR gene resulted in poor growth on starch medium and reduction in α-amylase production level. To elucidate the activation mechanisms of these two transcription factors in amylase production, the expression profiles of amylases and MAL cluster genes under carbon catabolite derepression condition and subcellular localization of these transcription factors fused with a green fluorescent protein (GFP) were examined. Glucose, maltose, and isomaltose induced the expression of amylase genes, and GFP-AmyR was translocated from the cytoplasm to nucleus after the addition of these sugars. Rapid induction of amylase gene expression and nuclear localization of GFP-AmyR by isomaltose suggested that this sugar was the strongest inducer for AmyR activation. In contrast, GFP-MalR was constitutively localized in the nucleus and the expression of MAL cluster genes was induced by maltose, but not by glucose or isomaltose. In the presence of maltose, the expression of amylase genes was preceded by MAL cluster gene expression. Furthermore, deletion of the malR gene resulted in a significant decrease in the α-amylase activity induced by maltose, but had apparently no effect on the expression of α-amylase genes in the presence of isomaltose. These results suggested that activation of AmyR and MalR is regulated in a different manner, and the preceding activation of MalR is essential for the utilization of maltose as an inducer for AmyR activation.

  11. The external amino acid signaling pathway promotes activation of Stp1 and Uga35/Dal81 transcription factors for induction of the AGP1 gene in Saccharomyces cerevisiae.

    PubMed Central

    Abdel-Sater, Fadi; Iraqui, Ismaïl; Urrestarazu, Antonio; André, Bruno

    2004-01-01

    Yeast cells respond to the presence of amino acids in their environment by inducing transcription of several amino acid permease genes including AGP1, BAP2, and BAP3. The signaling pathway responsible for this induction involves Ssy1, a permease-like sensor of external amino acids, and culminates with proteolytic cleavage and translocation to the nucleus of the zinc-finger proteins Stp1 and Stp2, the lack of which abolishes induction of BAP2 and BAP3. Here we show that Stp1-but not Stp2-plays an important role in AGP1 induction, although significant induction of AGP1 by amino acids persists in stp1 and stp1 stp2 mutants. This residual induction depends on the Uga35/Dal81 transcription factor, indicating that the external amino acid signaling pathway activates not only Stp1 and Stp2, but also another Uga35/Dal81-dependent transcriptional circuit. Analysis of the AGP1 gene's upstream region revealed that Stp1 and Uga35/Dal81 act synergistically through a 21-bp cis-acting sequence similar to the UAS(AA) element previously found in the BAP2 and BAP3 upstream regions. Although cells growing under poor nitrogen-supply conditions display much higher induction of AGP1 expression than cells growing under good nitrogen-supply conditions, the UAS(AA) itself is totally insensitive to nitrogen availability. Nitrogen-source control of AGP1 induction is mediated by the GATA factor Gln3, likely acting through adjacent 5'-GATA-3' sequences, to amplify the positive effect of UAS(AA). Our data indicate that Stp1 may act in combination with distinct sets of transcription factors, according to the gene context, to promote induction of transcription in response to external amino acids. The data also suggest that Uga35/Dal81 is yet another transcription factor under the control of the external amino acid sensing pathway. Finally, the data show that the TOR pathway mediating global nitrogen control of transcription does not interfere with the external amino acid signaling pathway. PMID

  12. Transcription factor repertoire of homeostatic eosinophilopoiesis

    PubMed Central

    Bouffi, Carine; Kartashov, Andrey V.; Schollaert, Kaila L.; Chen, Xiaoting; Bacon, W. Clark; Weirauch, Matthew T.; Barski, Artem; Fulkerson, Patricia C.

    2015-01-01

    The production of mature eosinophils is a tightly orchestrated process with the aim to sustain normal eosinophil levels in tissues while also maintaining low numbers of these complex and sensitive cells in the blood. To identify regulators of homeostatic eosinophilopoiesis in mice, we took a global approach to identify genome-wide transcriptome and epigenome changes that occur during homeostasis at critical developmental stages, including eosinophil-lineage commitment and lineage maturation. Our analyses revealed a markedly greater number of transcriptome alterations associated with eosinophil maturation (1199 genes) than with eosinophil-lineage commitment (490 genes), highlighting the greater transcriptional investment necessary for differentiation. Eosinophil progenitors (EoPs) were noted to express high levels of granule proteins and contain granules with an ultrastructure distinct from that of mature resting eosinophils. Our analyses also delineated a 976-gene eosinophil-lineage transcriptome that included a repertoire of 56 transcription factors, many of which have never previously been associated with eosinophils. EoPs and eosinophils, but not granulocyte-monocyte progenitors (GMPs) or neutrophils, expressed Helios and Aiolos, members of the Ikaros family of transcription factors, which regulate gene expression via modulation of chromatin structure and DNA accessibility. Epigenetic studies revealed a distinct distribution of active chromatin marks between genes induced with lineage commitment and genes induced with cell maturation during eosinophil development. In addition, Aiolos and Helios binding sites were significantly enriched in genes expressed by EoPs and eosinophils with active chromatin, highlighting a potential novel role for Helios and Aiolos in regulating gene expression during eosinophil development. PMID:26268651

  13. Identification of human autoantibodies to transcription factor IIB.

    PubMed Central

    Abendroth, F D; Peterson, S R; Galman, M; Suwa, A; Hardin, J A; Dynan, W S

    1995-01-01

    We have characterized the ability of various human autoimmune sera to react with RNA polymerase II transcription factors. One serum, which strongly inhibited transcription in a cell-free system, was shown to contain antibodies directed against human TFIIB. The serum did not show reactivity against the other general transcription factors, including human TBP, TFIIE and TFIIF. The inhibition of transcription was directly attributable to depletion of TFIIB activity, as demonstrated by reconstitution of activity with recombinant TFIIB. It has long been recognized that components of the RNA processing machinery are major human autoantigens. The present results show that at least one general transcription factor required for messenger RNA synthesis is an autoantigen as well. Images PMID:7651839

  14. Synergistic trans-activation of the human C-reactive protein promoter by transcription factor HNF-1 binding at two distinct sites.

    PubMed Central

    Toniatti, C; Demartis, A; Monaci, P; Nicosia, A; Ciliberto, G

    1990-01-01

    The promoter region of the human C-reactive protein (CRP) gene comprises two distinct regions (APREs, for Acute Phase Responsive Elements) each one containing information necessary and sufficient for liver specific and IL-6 inducible expression in human hepatoma Hep3B cells. In this paper we show that both APREs contain a low affinity binding site for the liver specific transcription factor HNF-1/LF-B1. The two sites are separated by approximately 80 bp. Mutations in either of the two sites abolish inducible expression. The same effect is specifically obtained in cotransfection competition experiments when the human albumin HNF-1 site is used as competitor. However, HNF-1 is not the intranuclear mediator of IL-6 because synthetic promoters formed by multimerized copies of different HNF-1 binding sites are not transcriptionally activated by this cytokine. An expression vector encoding full length HNF-1 is capable of trans-activating transcription from the wild-type CRP promoter but not from mutants which have lost the ability to bind HNF-1. Moreover, the level of trans-activation observed with the natural promoter containing both HNF-1 binding sites is far greater than the level of mutated variants containing only one of the two sites. This result strongly suggests that two HNF-1 molecules bound simultaneously to sites distant from each other can act synergistically to activate gene expression. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 7. PMID:2265613

  15. 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.

  16. Macrophage cell death and transcriptional response are actively triggered by the fungal virulence factor Cbp1 during H. capsulatum infection

    PubMed Central

    English, Bevin C.; Murray, Davina Hocking; Lee, Young Nam; Coady, Alison; Sil, Anita

    2016-01-01

    Summary Microbial pathogens induce or inhibit death of host cells during infection, with significant consequences for virulence and disease progression. Death of an infected host cell can either facilitate release and dissemination of intracellular pathogens or promote pathogen clearance. Histoplasma capsulatum is an intracellular fungal pathogen that replicates robustly within macrophages and triggers macrophage lysis by unknown means. To identify H. capsulatum effectors of macrophage lysis, we performed a genetic screen and discovered three mutants that grew to wild-type levels within macrophages but failed to elicit host-cell death. Each mutant was defective in production of the previously identified secreted protein Cbp1 (calcium-binding protein 1), whose role in intracellular growth had not been fully investigated. We found that Cbp1 was dispensable for high levels of intracellular growth, but required to elicit a unique transcriptional signature in macrophages, including genes whose induction was previously associated with endoplasmic reticulum stress and host-cell death. Additionally Cbp1 was required for activation of cell-death caspases-3/7, and macrophage death during H. capsulatum infection was dependent on the pro-apoptotic proteins Bax and Bak. Taken together, these findings strongly suggest that the ability of Cbp1 to actively program host-cell death is an essential step in H. capsulatum pathogenesis. PMID:26288377

  17. Macrophage cell death and transcriptional response are actively triggered by the fungal virulence factor Cbp1 during H. capsulatum infection.

    PubMed

    Isaac, Dervla T; Berkes, Charlotte A; English, Bevin C; Hocking Murray, Davina; Lee, Young Nam; Coady, Alison; Sil, Anita

    2015-12-01

    Microbial pathogens induce or inhibit death of host cells during infection, with significant consequences for virulence and disease progression. Death of an infected host cell can either facilitate release and dissemination of intracellular pathogens or promote pathogen clearance. Histoplasma capsulatum is an intracellular fungal pathogen that replicates robustly within macrophages and triggers macrophage lysis by unknown means. To identify H. capsulatum effectors of macrophage lysis, we performed a genetic screen and discovered three mutants that grew to wild-type levels within macrophages but failed to elicit host-cell death. Each mutant was defective in production of the previously identified secreted protein Cbp1 (calcium-binding protein 1), whose role in intracellular growth had not been fully investigated. We found that Cbp1 was dispensable for high levels of intracellular growth but required to elicit a unique transcriptional signature in macrophages, including genes whose induction was previously associated with endoplasmic reticulum stress and host-cell death. Additionally, Cbp1 was required for activation of cell-death caspases-3/7, and macrophage death during H. capsulatum infection was dependent on the pro-apoptotic proteins Bax and Bak. Taken together, these findings strongly suggest that the ability of Cbp1 to actively program host-cell death is an essential step in H. capsulatum pathogenesis.

  18. Transcriptional Control of Synaptic Plasticity by Transcription Factor NF-κB.

    PubMed

    Engelmann, Christian; Haenold, Ronny

    2016-01-01

    Activation of nuclear factor kappa B (NF-κB) transcription factors is required for the induction of synaptic plasticity and memory formation. All components of this signaling pathway are localized at synapses, and transcriptionally active NF-κB dimers move to the nucleus to translate synaptic signals into altered gene expression. Neuron-specific inhibition results in altered connectivity of excitatory and inhibitory synapses and functionally in selective learning deficits. Recent research on transgenic mice with impaired or hyperactivated NF-κB gave important insights into plasticity-related target gene expression that is regulated by NF-κB. In this minireview, we update the available data on the role of this transcription factor for learning and memory formation and comment on cross-sectional activation of NF-κB in the aged and diseased brain that may directly or indirectly affect κB-dependent transcription of synaptic genes.

  19. Ultraviolet irradiation induces CYR61/CCN1, a mediator of collagen homeostasis, through activation of transcription factor AP-1 in human skin fibroblasts.

    PubMed

    Quan, Taihao; Qin, Zhaoping; Xu, Yiru; He, Tianyuan; Kang, Sewon; Voorhees, John J; Fisher, Gary J

    2010-06-01

    UV irradiation from the sun elevates the production of collagen-degrading matrix metalloproteinases (MMPs) and reduces the production of new collagen. This imbalance of collagen homeostasis impairs the structure and function of the dermal collagenous extracellular matrix (ECM), thereby promoting premature skin aging (photoaging). We report here that aberrant dermal collagen homeostasis in UV-irradiated human skin is mediated in part by a CCN-family member, cysteine-rich protein-61 (CYR61/CCN1). CYR61 is significantly elevated in acutely UV-irradiated human skin in vivo, and UV-irradiated human skin fibroblasts. Knockdown of CYR61 significantly attenuates UV irradiation-induced inhibition of type-I procollagen and upregulation of MMP-1. Determination of CYR61 mRNA and protein indicates that the primary mechanism of CYR61 induction by UV irradiation is transcriptional. Analysis of CYR61 proximal promoter showed that a sequence conforming to the consensus binding site for transcription factor activator protein-1 (AP-1) is required for promoter activity. UV irradiation increased the binding of AP-1-family members c-Jun and c-Fos to this AP-1 site. Furthermore, functional blockade of c-Jun or knockdown of c-Jun significantly reduced the UV irradiation-induced activation of CYR61 promoter and CYR61 gene expression. These data show that CYR61 is transcriptionally regulated by UV irradiation through transcription factor AP-1, and mediates altered collagen homeostasis that occurs in response to UV irradiation in human skin fibroblasts.

  20. How Intrinsic Molecular Dynamics Control Intramolecular Communication in Signal Transducers and Activators of Transcription Factor STAT5

    PubMed Central

    Langenfeld, Florent; Guarracino, Yann; Arock, Michel; Trouvé, Alain; Tchertanov, Luba

    2015-01-01

    Signal Transducer and Activator of Transcription STAT5 is a key mediator of cell proliferation, differentiation and survival. While STAT5 activity is tightly regulated in normal cells, its constitutive activation directly contributes to oncogenesis and is associated with a broad range of hematological and solid tumor cancers. Therefore the development of compounds able to modulate pathogenic activation of this protein is a very challenging endeavor. A crucial step of drug design is the understanding of the protein conformational features and the definition of putative binding site(s) for such modulators. Currently, there is no structural data available for human STAT5 and our study is the first footprint towards the description of structure and dynamics of this protein. We investigated structural and dynamical features of the two STAT5 isoforms, STAT5a and STAT5b, taken into account their phosphorylation status. The study was based on the exploration of molecular dynamics simulations by different analytical methods. Despite the overall folding similarity of STAT5 proteins, the MD conformations display specific structural and dynamical features for each protein, indicating first, sequence-encoded structural properties and second, phosphorylation-induced effects which contribute to local and long-distance structural rearrangements interpreted as allosteric event. Further examination of the dynamical coupling between distant sites provides evidence for alternative profiles of the communication pathways inside and between the STAT5 domains. These results add a new insight to the understanding of the crucial role of intrinsic molecular dynamics in mediating intramolecular signaling in STAT5. Two pockets, localized in close proximity to the phosphotyrosine-binding site and adjacent to the channel for communication pathways across STAT5, may constitute valid targets to develop inhibitors able to modulate the function-related communication properties of this signaling

  1. Changes in the Expression of Transcription Factors Involved in Modulating the Expression of EPO-R in Activated Human CD4-Positive Lymphocytes.

    PubMed

    Lisowska, Katarzyna A; Frackowiak, Joanna E; Mikosik, Anna; Witkowski, Jacek M

    2013-01-01

    We have recently described the presence of the erythropoietin receptor (EPO-R) on CD4(+) lymphocytes and demonstrated that its expression increases during their activation, reaching a level reported to be typical for erythroid progenitors. This observation suggests that EPO-R expression is modulated during lymphocyte activation, which may be important for the cells' function. Here we investigated whether the expression of GATA1, GATA3 and Sp1 transcription factors is correlated with the expression of EPO-R in human CD4(+) lymphocytes stimulated with monoclonal anti-CD3 antibody. The expression of GATA1, GATA3 and Sp1 transcription factors in CD4(+) cells was estimated before and after stimulation with anti-CD3 antibody by Western Blot and flow cytometry. The expression of EPO-R was measured using real-time PCR and flow cytometry. There was no change in the expression of GATA1 and GATA3 in CD4(+) lymphocytes after stimulation with anti-CD3 antibody. However, stimulation resulted in the significantly increased expression of the Sp1 factor. CD4(+) lymphocytes stimulated with anti-CD3 antibody exhibited an increase in both the expression level of EPOR gene and the number of EPO-R molecules on the cells' surface, the latter being significantly correlated with the increased expression of Sp1. Sp1 is noted to be the single transcription factor among the ones studied whose level changes as a result of CD4(+) lymphocyte stimulation. It seems that Sp1 may significantly affect the number of EPO-R molecules present on the surface of activated CD4(+) lymphocytes.

  2. Prostaglandin F2a activates stress response signaling and induces expression of activating transcription factor 3 (ATF3) in bovine large luteal cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pulsatile uterine secretion of prostaglandin F2 alpha (PGF) triggers the regression of the corpus luteum (CL). Recent studies have explored global changes in gene expression in response to PGF that may contribute to structural and functional regression of the CL. Activating transcription facto...

  3. From tissue mechanics to transcription factors

    PubMed Central

    Janmey, Paul A.; Wells, Rebecca G.; Assoian, Richard K.; McCulloch, Christopher A.

    2015-01-01

    Changes in tissue stiffness are frequently associated with diseases such as cancer, fibrosis, and atherosclerosis. Several recent studies suggest that, in addition to resulting from pathology, mechanical changes may play a role akin to soluble factors in causing the progression of disease, and similar mechanical control might be essential for normal tissue development and homeostasis. Many cell types alter their structure and function in response to exogenous forces or as a function of the mechanical properties of the materials to which they adhere. This review summarizes recent progress in identifying intracellular signaling pathways, and especially transcriptional programs, that are differentially activated when cells adhere to materials with different mechanical properties or when they are subject to tension arising from external forces. Several cytoplasmic or cytoskeletal signaling pathways involving small GTPases, focal adhesion kinase and transforming growth factor beta as well as the transcriptional regulators MRTF-A, NFκB, and Yap/Taz have emerged as important mediators of mechanical signaling. PMID:23969122

  4. Activation of transcription factor NF-kappaB by the adenovirus E3/19K protein requires its ER retention

    PubMed Central

    1996-01-01

    We have recently shown that the accumulation of diverse viral and cellular membrane proteins in the ER activates the higher eukaryotic transcription factor NF-kappaB. This defined a novel ER-nuclear signal transduction pathway, which is distinct from the previously described unfolded protein response (UPR). The well characterized UPR pathway is activated by the presence of un- or malfolded proteins in the ER. In contrast, the ER stress signal which activates the NF-kappaB pathway is not known. Here we used the adenovirus early region protein E3/19K as a model to investigate the nature of the NF-kappaB-activating signal emitted by the ER. E3/19K resides in the endoplasmic reticulum where it binds to MHC class I molecules, thereby preventing their transport to the cell surface. It is maintained in the ER by a retention signal sequence in its carboxy terminus, which causes the protein to be continuously retrieved to the ER from post-ER compartments. Mutation of this sequence allows E3/19K to reach the cell surface. We show here that expression of E3/19K potently activates a functional NF-kappaB transcription factor. The activated NF-kappaB complexes contained p50/p65 and p50/c-rel heterodimers. E3/19K interaction with MHC class I was not important for NF-kappaB activation since mutant proteins which no longer bind MHC molecules remained fully capable of inducing NF- kappaB. However, activation of both NF-kappaB DNA binding and kappaB- dependent transactivation relied on E3/19K ER retention: mutants, which were expressed on the cell surface, could no longer activate the transcription factor. This identifies the NF-kappaB-activating signal as the accumulation of proteins in the ER membrane, a condition we have termed "ER overload." We show that ER overload-mediated NF-kappaB activation but not TNF-stimulated NF-kappaB induction can be inhibited by the intracellular Ca2+ chelator TMB-8. Moreover, treatment of cells with two inhibitors of the ER-resident Ca(2+) -dependent

  5. 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.

  6. Enhancing lysosomal biogenesis and autophagic flux by activating the transcription factor EB protects against cadmium-induced neurotoxicity

    PubMed Central

    Pi, Huifeng; Li, Min; Tian, Li; Yang, Zhiqi; Yu, Zhengping; Zhou, Zhou

    2017-01-01

    Cadmium (Cd), a highly ubiquitous heavy metal, is a well-known inducer of neurotoxicity. However, the mechanism underlying cadmium-induced neurotoxicity remains unclear. In this study, we found that Cd inhibits autophagosome-lysosome fusion and impairs lysosomal function by reducing the levels of lysosomal-associated membrane proteins, inhibiting lysosomal proteolysis and altering lysosomal pH, contributing to defects in autophagic clearance and subsequently leading to nerve cell death. In addition, Cd decreases transcription factor EB (TFEB) expression at both the mRNA and protein levels. Furthermore, Cd induces the nuclear translocation of TFEB and TFEB target-gene expression, associated with compromised lysosomal function or a compensatory effect after the impairment of the autophagic flux. Notably, restoration of the levels of lysosomal-associated membrane protein, lysosomal proteolysis, lysosomal pH and autophagic flux through Tfeb overexpression protects against Cd-induced neurotoxicity, and this protective effect is incompletely dependent on TFEB nuclear translocation. Moreover, gene transfer of the master autophagy regulator TFEB results in the clearance of toxic proteins and the correction of Cd-induced neurotoxicity in vivo. Our study is the first to demonstrate that Cd disrupts lysosomal function and autophagic flux and manipulation of TFEB signalling may be a therapeutic approach for antagonizing Cd-induced neurotoxicity. PMID:28240313

  7. hrpL activates Erwinia amylovora hrp gene transcription and is a member of the ECF subfamily of sigma factors.

    PubMed Central

    Wei, Z M; Beer, S V

    1995-01-01

    hrpL of Erwinia amylovora Ea321 encodes a 21.7-kDa regulatory protein, similar to members of the ECF (extra cytoplasmic functions) subfamily of eubacterial RNA polymerase sigma factors. hrpL is a single-gene operon in complementation group VI of the E. amylovora hrp gene cluster. Its product is required by Ea321 to elicit the hypersensitive response (HR) and to cause disease. HrpL controls the expression of five independent hrp loci, including hrpN, which encodes harpin, a proteinaceous elicitor of the HR. hrpL is environmentally regulated, and its expression is affected by hrpS, another regulatory gene of the hrp gene cluster of E. amylovora. pCPP1078, a multicopy plasmid carrying hrpL, is able to restore HR-eliciting ability to hrpS mutants. A conserved motif was identified upstream of the hrpI and hrpN operons, which are transcriptionally regulated by hrpL. This conserved motif shares a high degree of similarity with other biochemically defined or putative ECF-dependent promoter sequences, including sequences upstream of Streptomyces coelicolor dagA P2, Pseudomonas aeruginosa algD, Pseudomonas syringae pv. syringae 61 hrpZ, and P. syringae pv. tomato avrD. In spite of the similarity between the hrpL genes of E. amylovora and P. syringae 61, no functional cross-complementation was observed. PMID:7592386

  8. Activation of the Oryza sativa non-symbiotic haemoglobin-2 promoter by the cytokinin-regulated transcription factor, ARR1.

    PubMed

    Ross, Emily J H; Stone, Julie M; Elowsky, Christian G; Arredondo-Peter, Raul; Klucas, Robert V; Sarath, Gautam

    2004-08-01

    Using in silico methods, several putative phytohormone-responsive cis-elements in the Oryza sativa non-symbiotic haemoglobin (NSHB) 1-4 and Arabidopsis thaliana NSHB1-2 promoters have been identified. An OsNSHB2 promoter::GUS reporter gene fusion shows tissue-specific expression in A. thaliana. GUS expression was observed in roots, the vasculature of young leaves, in flowers, and in the pedicel/stem junction. In transient assays, activity of the OsNSHB2 promoter was significantly up-regulated in the presence of the cytokinin, 6-benzylaminopurine (BA). Deletion analyses indicated that the full-length promoter was required for maximal trans-activation in the presence of cytokinin. Mutation of the single cytokinin-regulated ARR1-binding element abolished promoter activation in response to cytokinin. Constitutive expression of ARR1 under the control of the 35S cauliflower mosaic virus promoter enhanced wild-type OsNSHB2 promoter activity, but had no effect on the activity of the mutated promoter in the absence of cytokinin. However, overexpression of ARR1 in the presence of cytokinin resulted in super-activation of the wild-type promoter. The mutated promoter was only moderately activated in the presence of cytokinin and ARR1, indicating that the OsNSHB2 promoter can be regulated by the ARR1 protein, but requires other cytokinin-induced factors for optimal activation. This is the first report that identifies a trans-acting factor involved in the activation of a NSHB gene.

  9. Loss of Ribosomal Protein L11 Blocks Stress Activation of the Bacillus subtilis Transcription Factor ςB

    PubMed Central

    Zhang, Shuyu; Scott, Janelle M.; Haldenwang, W. G.

    2001-01-01

    ςB, the general stress response sigma factor of Bacillus subtilis, is activated when the cell's energy levels decline or the bacterium is exposed to environmental stress (e.g., heat shock, ethanol). Physical stress activates ςB through a collection of regulatory kinases and phosphatases (the Rsb proteins) which catalyze the release of ςB from an anti-ςB factor inhibitor. The means by which diverse stresses communicate with the Rsb proteins is unknown; however, a role for the ribosome in this process was suggested when several of the upstream members of the ςB stress activation cascade (RsbR, -S, and -T) were found to cofractionate with ribosomes in crude B. subtilis extracts. We now present evidence for the involvement of a ribosome-mediated process in the stress activation of ςB. B. subtilis strains resistant to the antibiotic thiostrepton, due to the loss of ribosomal protein L11 (RplK), were found to be blocked in the stress activation of ςB. Neither the energy-responsive activation of ςB nor stress-dependent chaperone gene induction (a ςB-independent stress response) was inhibited by the loss of L11. The Rsb proteins required for stress activation of ςB are shown to be active in the RplK− strain but fail to be triggered by stress. The data demonstrate that the B. subtilis ribosomes provide an essential input for the stress activation of ςB and suggest that the ribosomes may themselves be the sensors for stress in this system. PMID:11244072

  10. Promoter occupancy of the basal class I transcription factor A differs strongly between active and silent VSG expression sites in Trypanosoma brucei.

    PubMed

    Nguyen, Tu N; Müller, Laura S M; Park, Sung Hee; Siegel, T Nicolai; Günzl, Arthur

    2014-03-01

    Monoallelic expression within a gene family is found in pathogens exhibiting antigenic variation and in mammalian olfactory neurons. Trypanosoma brucei, a lethal parasite living in the human bloodstream, expresses variant surface glycoprotein (VSG) from 1 of 15 bloodstream expression sites (BESs) by virtue of a multifunctional RNA polymerase I. The active BES is transcribed in an extranucleolar compartment termed the expression site body (ESB), whereas silent BESs, located elsewhere within the nucleus, are repressed epigenetically. The regulatory mechanisms, however, are poorly understood. Here we show that two essential subunits of the basal class I transcription factor A (CITFA) predominantly occupied the promoter of the active BES relative to that of a silent BES, a phenotype that was maintained after switching BESs in situ. In these experiments, high promoter occupancy of CITFA was coupled to high levels of both promoter-proximal RNA abundance and RNA polymerase I occupancy. Accordingly, fluorescently tagged CITFA-7 was concentrated in the nucleolus and the ESB. Because a ChIP-seq analysis found that along the entire BES, CITFA-7 is specifically enriched only at the promoter, our data strongly indicate that monoallelic BES transcription is activated by a mechanism that functions at the level of transcription initiation.

  11. Novel interactions between human T-cell leukemia virus type I Tax and activating transcription factor 3 at a cyclic AMP-responsive element.

    PubMed Central

    Low, K G; Chu, H M; Schwartz, P M; Daniels, G M; Melner, M H; Comb, M J

    1994-01-01

    Human proenkephalin gene transcription is transactivated by human T-cell leukemia virus type I (HTLV-I) Tax in human Jurkat T lymphocytes. This transactivation was further enhanced in Jurkat cells treated with concanavalin A, cyclic AMP, or 12-O-tetradecanoylphorbol-13-acetate. Deletion and cis-element transfer analyses of the human proenkephalin promoter identified a cyclic AMP-responsive AP-1 element (-92 to -86) as both necessary and sufficient to confer Tax-dependent transactivation. Different AP-1 or cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) proteins which bind this element were expressed in murine teratocarcinoma F9 cells to identify those capable of mediating Tax-dependent transactivation of human proenkephalin gene transcription. Although CREB, c-Fos, c-Jun, and JunD did not have significant effects, JunB inhibited the Tax-dependent transactivation. In contrast, ATF3 dramatically induced Tax-dependent transactivation, which was further enhanced by protein kinase A. Electrophoretic mobility shift assays with recombinant fusion proteins expressed and purified from bacteria indicate that the DNA-binding activity of ATF3 is also dramatically enhanced by Tax. Chimeric fusion proteins consisting of the DNA-binding domain of the yeast transcription factor Gal4 and the amino-terminal domain (residues 1 to 66) of ATF3 were able to mediate Tax-dependent transactivation of a Gal4-responsive promoter, which suggests a direct involvement of this region of ATF3. Recombinant fusion proteins of glutathione S-transferase with either the amino- or carboxy-terminal (residues 139 to 181) domain of ATF3 were able to specifically interact with Tax. Furthermore, specific antisera directed against Tax coimmunoprecipitated ATF3 only in the presence of Tax. Images PMID:8007991

  12. Cyclin-dependent kinase 5 modulates the transcriptional activity of the mineralocorticoid receptor and regulates expression of brain-derived neurotrophic factor.

    PubMed

    Kino, Tomoshige; Jaffe, Howard; Amin, Niranjana D; Chakrabarti, Mayukh; Zheng, Ya-Li; Chrousos, George P; Pant, Harish C

    2010-05-01

    Glucocorticoids, major end effectors of the stress response, play an essential role in the homeostasis of the central nervous system (CNS) and contribute to memory consolidation and emotional control through their intracellular receptors, the glucocorticoid and mineralocorticoid receptors. Cyclin-dependent kinase 5 (CDK5), on the other hand, plays important roles in the morphogenesis and functions of the central nervous system, and its aberrant activation has been associated with development of neurodegenerative disorders. We previously reported that CDK5 phosphorylated the glucocorticoid receptor and modulated its transcriptional activity. Here we found that CDK5 also regulated mineralocorticoid receptor-induced transcriptional activity by phosphorylating multiple serine and threonine residues located in its N-terminal domain through physical interaction. Aldosterone and dexamethasone, respectively, increased and suppressed mRNA/protein expression of brain-derived neurotrophic factor (BDNF) in rat cortical neuronal cells, whereas the endogenous glucocorticoid corticosterone showed a biphasic effect. CDK5 enhanced the effect of aldosterone and dexamethasone on BDNF expression. Because this neurotrophic factor plays critical roles in neuronal viability, synaptic plasticity, consolidation of memory, and emotional changes, we suggest that aberrant activation of CDK5 might influence these functions through corticosteroid receptors/BDNF.

  13. Single-prolonged stress activates the transcription factor ATF6α branch of the unfolded protein response in rat neurons of dorsal raphe nucleus.

    PubMed

    Xie, Juhua; Han, Fang; Shi, Yuxiu

    2015-01-01

    In our previous studies, we have found that endoplasmic reticulum (ER) stress is associated with post-traumatic stress disorder (PTSD), however, the activation of ER stress sensors in PTSD remains unclear. ATF6 alpha (ATF6α) is an ER-membrane-bound transcription factor and functions as a critical sensor and regulator of ER stress in mammalian cells. The goal of this study is to detect whether there is activation of the transcription factor ATF6α branch of the unfolded protein response in the dorsal raphe nucleus neurons of the rats exposed to single-prolonged stress (SPS), which is a model employed extensively in PTSD study. Our results have demonstrated that SPS activated the ER transmembrane protein ATF6α via its cleavage; and induced the up-regulation of the downstream targets of ATF6α, the mRNA of XBP1 and GRP94. To the best of our knowledge, this is the first study to investigate the relationship between the ATF6α pathways and PTSD, and our results show that SPS activates the ATF6α branch of the ER stress response, which may be contributed to the pathogenesis of PTSD.

  14. The human papillomavirus type 16 E7 gene product interacts with and trans-activates the AP1 family of transcription factors.

    PubMed Central

    Antinore, M J; Birrer, M J; Patel, D; Nader, L; McCance, D J

    1996-01-01

    The E7 gene product of human papillomavirus type 16 (HPV16) binds to the retinoblastoma gene product (pRb) and dissociates pRb-E2F complexes. However, the observation that the ability of E7 to bind pRb is not required for the HPV16-induced immortalization of primary keratinocytes prompted a search for other cellular factors bound by E7. Using a glutathione-S-transferase (GST) fusion protein system, we show that E7 complexes with AP1 transcription factors including c-Jun, JunB, JunD and c-Fos. The ability of E7 to complex with c-Jun in vivo is demonstrated by co-immunoprecipitation and the yeast two-hybrid system. An analysis of E7 point mutants in the GST system indicates that the E7 zinc-finger motif, but not the pRb binding domain, is involved in these interactions. Using c-Jun deletion mutants, E7 binding maps between amino acids 224 and 286 of c-Jun. E7 trans-activates c-Jun-induced transcription from a Jun responsive promoter, and this activity correlates with the ability of E7 mutants to bind Jun proteins. Finally, a transcriptionally inactive c-Jun deletion, which can bind E7, interferes with the E7-induced transformation of rat embryo fibroblasts in cooperation with an activated ras, indicating that the Jun-E7 interaction is physiologically relevant and that Jun factors may be targeted in the E7 transformation pathway. Images PMID:8617242

  15. 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.

  16. Entinostat up-regulates the CAMP gene encoding LL-37 via activation of STAT3 and HIF-1α transcription factors

    PubMed Central

    Miraglia, Erica; Nylén, Frank; Johansson, Katarina; Arnér, Elias; Cebula, Marcus; Farmand, Susan; Ottosson, Håkan; Strömberg, Roger; Gudmundsson, Gudmundur H.; Agerberth, Birgitta; Bergman, Peter

    2016-01-01

    Bacterial resistance against classical antibiotics is a growing problem and the development of new antibiotics is limited. Thus, novel alternatives to antibiotics are warranted. Antimicrobial peptides (AMPs) are effector molecules of innate immunity that can be induced by several compounds, including vitamin D and phenyl-butyrate (PBA). Utilizing a luciferase based assay, we recently discovered that the histone deacetylase inhibitor Entinostat is a potent inducer of the CAMP gene encoding the human cathelicidin LL-37. Here we investigate a mechanism for the induction and also find that Entinostat up-regulates human β-defensin 1. Analysis of the CAMP promoter sequence revealed binding sites for the transcription factors STAT3 and HIF-1α. By using short hairpin RNA and selective inhibitors, we found that both transcription factors are involved in Entinostat-induced expression of LL-37. However, only HIF-1α was found to be recruited to the CAMP promoter, suggesting that Entinostat activates STAT3, which promotes transcription of CAMP by increasing the expression of HIF-1α. Finally, we provide in vivo relevance to our findings by showing that Entinostat-elicited LL-37 expression was impaired in macrophages from a patient with a STAT3-mutation. Combined, our findings support a role for STAT3 and HIF-1α in the regulation of LL-37 expression. PMID:27633343

  17. The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis.

    PubMed

    Roczniak-Ferguson, Agnes; Petit, Constance S; Froehlich, Florian; Qian, Sharon; Ky, Jennifer; Angarola, Brittany; Walther, Tobias C; Ferguson, Shawn M

    2012-06-12

    Lysosomes are the major cellular site for clearance of defective organelles and digestion of internalized material. Demand on lysosomal capacity can vary greatly, and lysosomal function must be adjusted to maintain cellular homeostasis. Here, we identified an interaction between the lysosome-localized mechanistic target of rapamycin complex 1 (mTORC1) and the transcription factor TFEB (transcription factor EB), which promotes lysosome biogenesis. When lysosomal activity was adequate, mTOR-dependent phosphorylation of TFEB on Ser(211) triggered the binding of 14-3-3 proteins to TFEB, resulting in retention of the transcription factor in the cytoplasm. Inhibition of lysosomal function reduced the mTOR-dependent phosphorylation of TFEB, resulting in diminished interactions between TFEB and 14-3-3 proteins and the translocation of TFEB into the nucleus, where it could stimulate genes involved in lysosomal biogenesis. These results identify TFEB as a target of mTOR and suggest a mechanism for matching the transcriptional regulation of genes encoding proteins of autophagosomes and lysosomes to cellular need. The closely related transcription factors MITF (microphthalmia transcription factor) and TFE3 (transcription factor E3) also localized to lysosomes and accumulated in the nucleus when lysosome function was inhibited, thus broadening the range of physiological contexts under which this regulatory mechanism may prove important.

  18. Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1.

    PubMed Central

    Baler, R; Dahl, G; Voellmy, R

    1993-01-01

    Transcriptional activity of heat shock (hsp) genes is controlled by a heat-activated, group-specific transcription factor(s) recognizing arrays of inverted repeats of the element NGAAN. To date genes for two human factors, HSF1 and HSF2, have been isolated. To define their properties as well as the changes they undergo during heat stress activation, we prepared polyclonal antibodies to these factors. Using these tools, we have shown that human HeLa cells constitutively synthesize HSF1, but we were unable to detect HSF2. In unstressed cells HSF1 is present mainly in complexes with an apparent molecular mass of about 200 kDa, unable to bind to DNA. Heat treatment induces a shift in the apparent molecular mass of HSF1 to about 700 kDa, concomitant with the acquisition of DNA-binding ability. Cross-linking experiments suggest that this change in complex size may reflect the trimerization of monomeric HSF1. Human HSF1 expressed in Xenopus oocytes does not bind DNA, but derepression of DNA-binding activity, as well as oligomerization of HSF1, occurs during heat treatment at the same temperature at which hsp gene expression is induced in this organism, suggesting that a conserved Xenopus protein(s) plays a role in this regulation. Inactive HSF1 resides in the cytoplasm of human cells; on activation it rapidly translocates to a soluble nuclear fraction, and shortly thereafter it becomes associated with the nuclear pellet. On heat shock, activatable HSF1, which might already have been posttranslationally modified in the unstressed cell, undergoes further modification. These different process provide multiple points of regulation of hsp gene expression. Images PMID:8455624

  19. The Activity of the Pseudomonas aeruginosa Virulence Regulator σ(VreI) Is Modulated by the Anti-σ Factor VreR and the Transcription Factor PhoB.

    PubMed

    Quesada, Jose M; Otero-Asman, Joaquín R; Bastiaansen, Karlijn C; Civantos, Cristina; Llamas, María A

    2016-01-01

    Gene regulation in bacteria is primarily controlled at the level of transcription initiation by modifying the affinity of the RNA polymerase (RNAP) for the promoter. This control often occurs through the substitution of the RNAP sigma (σ) subunit. Next to the primary σ factor, most bacteria contain a variable number of alternative σ factors of which the extracytoplasmic function group (σ(ECF)) is predominant. Pseudomonas aeruginosa contains nineteen σ(ECF), including the virulence regulator σ(VreI). σ(VreI) is encoded by the vreAIR operon, which also encodes a receptor-like protein (VreA) and an anti-σ factor (VreR). These three proteins form a signal transduction pathway known as PUMA3, which controls expression of P. aeruginosa virulence functions. Expression of the vreAIR operon occurs under inorganic phosphate (Pi) limitation and requires the PhoB transcription factor. Intriguingly, the genes of the σ(VreI) regulon are also expressed in low Pi despite the fact that the σ(VreI) repressor, the anti-σ factor VreR, is also produced in this condition. Here we show that although σ(VreI) is partially active under Pi starvation, maximal transcription of the σ(VreI) regulon genes requires the removal of VreR. This strongly suggests that an extra signal, probably host-derived, is required in vivo for full σ(VreI) activation. Furthermore, we demonstrate that the activity of σ(VreI) is modulated not only by VreR but also by the transcription factor PhoB. Presence of this regulator is an absolute requirement for σ(VreI) to complex the DNA and initiate transcription of the PUMA3 regulon. The potential DNA binding sites of these two proteins, which include a pho box and -10 and -35 elements, are proposed.

  20. The Activity of the Pseudomonas aeruginosa Virulence Regulator σVreI Is Modulated by the Anti-σ Factor VreR and the Transcription Factor PhoB

    PubMed Central

    Quesada, Jose M.; Otero-Asman, Joaquín R.; Bastiaansen, Karlijn C.; Civantos, Cristina; Llamas, María A.

    2016-01-01

    Gene regulation in bacteria is primarily controlled at the level of transcription initiation by modifying the affinity of the RNA polymerase (RNAP) for the promoter. This control often occurs through the substitution of the RNAP sigma (σ) subunit. Next to the primary σ factor, most bacteria contain a variable number of alternative σ factors of which the extracytoplasmic function group (σECF) is predominant. Pseudomonas aeruginosa contains nineteen σECF, including the virulence regulator σVreI. σVreI is encoded by the vreAIR operon, which also encodes a receptor-like protein (VreA) and an anti-σ factor (VreR). These three proteins form a signal transduction pathway known as PUMA3, which controls expression of P. aeruginosa virulence functions. Expression of the vreAIR operon occurs under inorganic phosphate (Pi) limitation and requires the PhoB transcription factor. Intriguingly, the genes of the σVreI regulon are also expressed in low Pi despite the fact that the σVreI repressor, the anti-σ factor VreR, is also produced in this condition. Here we show that although σVreI is partially active under Pi starvation, maximal transcription of the σVreI regulon genes requires the removal of VreR. This strongly suggests that an extra signal, probably host-derived, is required in vivo for full σVreI activation. Furthermore, we demonstrate that the activity of σVreI is modulated not only by VreR but also by the transcription factor PhoB. Presence of this regulator is an absolute requirement for σVreI to complex the DNA and initiate transcription of the PUMA3 regulon. The potential DNA binding sites of these two proteins, which include a pho box and −10 and −35 elements, are proposed. PMID:27536271

  1. Transcription factor-induced activation of cardiac gene expression in human c-kit+ cardiac progenitor cells

    PubMed Central

    Vajravelu, Bathri N.; Moktar, Afsoon; Cao, Pengxiao; Moore, Joseph B.; Bolli, Roberto

    2017-01-01

    Although transplantation of c-kit+ cardiac progenitor cells (CPCs) significantly alleviates post-myocardial infarction left ventricular dysfunction, generation of cardiomyocytes by exogenous CPCs in the recipient heart has often been limited. Inducing robust differentiation would be necessary for improving the efficacy of the regenerative cardiac cell therapy. We assessed the hypothesis that differentiation of human c-kit+ CPCs can be enhanced by priming them with cardiac transcription factors (TFs). We introduced five different TFs (Gata4, MEF2C, NKX2.5, TBX5, and BAF60C) into CPCs, either alone or in combination, and then examined the expression of marker genes associated with the major cardiac cell types using quantitative RT-PCR. When introduced individually, Gata4 and TBX5 induced a subset of myocyte markers. Moreover, Gata4 alone significantly induced smooth muscle cell and fibroblast markers. Interestingly, these gene expression changes brought by Gata4 were also accompanied by morphological changes. In contrast, MEF2C and NKX2.5 were largely ineffective in initiating cardiac gene expression in CPCs. Surprisingly, introduction of multiple TFs in different combinations mostly failed to act synergistically. Likewise, addition of BAF60C to Gata4 and/or TBX5 did not further potentiate their effects on cardiac gene expression. Based on our results, it appears that GATA4 is able to potentiate gene expression programs associated with multiple cardiovascular lineages in CPCs, suggesting that GATA4 may be effective in priming CPCs for enhanced differentiation in the setting of stem cell therapy. PMID:28355297

  2. Transcription factor-induced activation of cardiac gene expression in human c-kit+ cardiac progenitor cells.

    PubMed

    Al-Maqtari, Tareq; Hong, Kyung U; Vajravelu, Bathri N; Moktar, Afsoon; Cao, Pengxiao; Moore, Joseph B; Bolli, Roberto

    2017-01-01

    Although transplantation of c-kit+ cardiac progenitor cells (CPCs) significantly alleviates post-myocardial infarction left ventricular dysfunction, generation of cardiomyocytes by exogenous CPCs in the recipient heart has often been limited. Inducing robust differentiation would be necessary for improving the efficacy of the regenerative cardiac cell therapy. We assessed the hypothesis that differentiation of human c-kit+ CPCs can be enhanced by priming them with cardiac transcription factors (TFs). We introduced five different TFs (Gata4, MEF2C, NKX2.5, TBX5, and BAF60C) into CPCs, either alone or in combination, and then examined the expression of marker genes associated with the major cardiac cell types using quantitative RT-PCR. When introduced individually, Gata4 and TBX5 induced a subset of myocyte markers. Moreover, Gata4 alone significantly induced smooth muscle cell and fibroblast markers. Interestingly, these gene expression changes brought by Gata4 were also accompanied by morphological changes. In contrast, MEF2C and NKX2.5 were largely ineffective in initiating cardiac gene expression in CPCs. Surprisingly, introduction of multiple TFs in different combinations mostly failed to act synergistically. Likewise, addition of BAF60C to Gata4 and/or TBX5 did not further potentiate their effects on cardiac gene expression. Based on our results, it appears that GATA4 is able to potentiate gene expression programs associated with multiple cardiovascular lineages in CPCs, suggesting that GATA4 may be effective in priming CPCs for enhanced differentiation in the setting of stem cell therapy.

  3. Erythropoietin-mediated expression of placenta growth factor is regulated via activation of hypoxia-inducible factor-1α and post-transcriptionally by miR-214 in sickle cell disease.

    PubMed

    Gonsalves, Caryn S; Li, Chen; Mpollo, Marthe-Sandrine Eiymo Mwa; Pullarkat, Vinod; Malik, Punam; Tahara, Stanley M; Kalra, Vijay K

    2015-06-15

    Placental growth factor (PlGF) plays an important role in various pathological conditions and diseases such as inflammation, cancer, atherosclerosis and sickle cell disease (SCD). Abnormally high PlGF levels in SCD patients are associated with increased inflammation and pulmonary hypertension (PHT) and reactive airway disease; however, the transcriptional and post-transcriptional mechanisms regulating PlGF expression are not well defined. Herein, we show that treatment of human erythroid cells and colony forming units with erythropoietin (EPO) increased PlGF expression. Our studies showed EPO-mediated activation of HIF-1α led to subsequent binding of HIF-1α to hypoxia response elements (HREs) within the PlGF promoter, as demonstrated by luciferase transcription reporter assays and ChIP analysis of the endogenous gene. Additionally, we showed miR-214 post-transcriptionally regulated the expression of PlGF as demonstrated by luciferase reporter assays using wild-type (wt) and mutant PlGF-3'-UTR constructs. Furthermore, synthesis of miR-214, located in an intron of DNM3 (dynamin 3), was transcriptionally regulated by transcription factors, peroxisome proliferator-activated receptor-α (PPARα) and hypoxia-inducible factor-1α (HIF-1α). These results were corroborated in vivo wherein plasma from SCD patients and lung tissues from sickle mice showed an inverse correlation between PlGF and miR-214 levels. Finally, we observed that miR-214 expression could be induced by fenofibrate, a Food and Drug Administration (FDA) approved PPARα agonist, thus revealing a potential therapeutic approach for reduction in PlGF levels by increasing miR-214 transcription. This strategy has potential clinical implications for several pathological conditions including SCD.

  4. Induction of sensitivity to the cytotoxic action of tumor necrosis factor alpha by adenovirus E1A is independent of transformation and transcriptional activation.

    PubMed Central

    Ames, R S; Holskin, B; Mitcho, M; Shalloway, D; Chen, M J

    1990-01-01

    We have previously shown that expression of the adenovirus E1A 12S or 13S products in NIH 3T3 fibroblasts induces susceptibility to the cytotoxic actions of tumor necrosis factor alpha (TNF alpha). A large number of studies have mapped the multiple biological functions of the 12S and 13S products to three highly conserved regions (CR) within the E1A sequence. Here we used plasmids coding for E1A deletion and point mutants in these regions to generate target cell lines for TNF alpha cytotoxicity assays to determine which regions and functions are necessary for the induction of TNF alpha sensitivity. Expression of CR1 was required for the induction of TNF alpha sensitivity. This finding did not reflect a requirement for transforming or transcriptional repression activity, since some mutants that were defective in both of these properties were able to induce TNF alpha sensitivity. CR2 transformation-defective point mutants, but not a CR2/3 region deletion mutant, were also able to induce sensitivity. In addition, NIH 3T3 cells expressing the retroviral transcription activators tat from human immunodeficiency virus type 1 and tax from human T-lymphotropic virus type I were not sensitive to TNF alpha. However, the possibility that E1A-mediated transcriptional activation can augment the induction of TNF alpha sensitivity is not excluded. Comparison of data from previous biological studies with the TNF alpha cytotoxicity assays presented here suggested that the mechanism by which E1A induces sensitivity to TNF alpha in NIH 3T3 cells is independent of many of the known E1A biological functions, including transformation in cooperation with ras, immortalization, induction of DNA synthesis in quiescent cells, and transcriptional repression. A novel E1A-mediated effect may be involved, although our data do not exclude the possibility that sensitization to TNF alpha is mediated through E1A binding to cellular proteins. Images PMID:2143540

  5. The Chromatin Regulator DMAP1 Modulates Activity of the Nuclear Factor κB (NF-κB) Transcription Factor Relish in the Drosophila Innate Immune Response*

    PubMed Central

    Goto, Akira; Fukuyama, Hidehiro; Imler, Jean-Luc; Hoffmann, Jules A.

    2014-01-01

    The host defense of the model organism Drosophila is under the control of two major signaling cascades controlling transcription factors of the NF-κB family, the Toll and the immune deficiency (IMD) pathways. The latter shares extensive similarities with the mammalian TNF-R pathway and was initially discovered for its role in anti-Gram-negative bacterial reactions. A previous interactome study from this laboratory reported that an unexpectedly large number of proteins are binding to the canonical components of the IMD pathway. Here, we focus on DNA methyltransferase-associated protein 1 (DMAP1), which this study identified as an interactant of Relish, a Drosophila transcription factor reminiscent of the mammalian p105 NF-κB protein. We show that silencing of DMAP1 expression both in S2 cells and in flies results in a significant reduction of Escherichia coli-induced expression of antimicrobial peptides. Epistatic analysis indicates that DMAP1 acts in parallel or downstream of Relish. Co-immunoprecipitation experiments further reveal that, in addition to Relish, DMAP1 also interacts with Akirin and the Brahma-associated protein 55 kDa (BAP55). Taken together, these results reveal that DMAP1 is a novel nuclear modulator of the IMD pathway, possibly acting at the level of chromatin remodeling. PMID:24947515

  6. Dehydroepiandrosterone sulfate mediates activation of transcription factors CREB and ATF-1 via a Gα11-coupled receptor in the spermatogenic cell line GC-2.

    PubMed

    Shihan, Mazen; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2013-12-01

    Dehydroepiandrosterone sulfate (DHEAS) is a circulating steroid produced in the adrenal cortex, brain, and gonads. Whereas a series of investigations attest to neuroprotective effects of the steroid in the brain, surprisingly little is known about the physiological effects of DHEAS on cells of the reproductive system. Here we demonstrate that DHEAS acting on the spermatogenic cell line GC-2 induces a time- and concentration-dependent phosphorylation of c-Src and Erk1/2 and activates the transcription factors activating transforming factor-1 (ATF-1) and cyclic AMP-responsive element binding protein (CREB). These actions are consistent with the non-classical signaling pathway of testosterone and suggest that DHEAS is a pro-androgen that is converted into testosterone in order to exert its biological activity. The fact, however, that steroid sulfatase mRNA was not detected in the GC-2 cells and the clear demonstration of DHEAS-induced activation of Erk1/2, ATF-1 and CREB after silencing the androgen receptor by small interfering RNA (siRNA) clearly contradict this assumption and make it appear unlikely that DHEAS has to be converted in the cytosol into a different steroid in order to activate the kinases and transcription factors mentioned. Instead, it is likely that the DHEAS-induced signaling is mediated through the interaction of the steroid with a membrane-bound G-protein-coupled receptor, since silencing of Guanine nucleotide-binding protein subunit alpha-11 (Gnα11) leads to the abolition of the DHEAS-induced stimulation of Erk1/2, ATF-1, and CREB. The investigation presented here shows a hormone-like activity of DHEAS on a spermatogenic cell line. Since DHEAS is produced in male and female reproductive organs, these findings could help to define new roles for DHEAS in the physiology of reproduction.

  7. Homeodomain transcription factor Hesx1/Rpx occupies Prop-1 activation sites in porcine follicle stimulating hormone (FSH) beta subunit promoter.

    PubMed

    Susa, Takao; Nakayama, Michie; Kitahara, Kousuke; Kimoto, Fuyuko; Kato, Takako; Kato, Yukio

    2007-06-08

    Homeodomain repressor factor Hesx1/Rpx plays a crucial role in the formation of Rathke's pouch at the start of pituitary organogenesis and represses the Prop-1-dependent expression of Pit-1 gene, which promotes the differentiation of Pit-1-dependent hormone producing cells. Recently, we discovered a novel function of Prop-1 by which it activates the porcine follicle stimulating hormone beta subunit (FSHbeta) gene through Fd2 region (-852/-746). The present study aimed to determine whether Hesx1 exerts its role in the Prop-1-dependent activation of FSHbeta gene. Transient transfection assay for the porcine FSHbeta promoter -985/+10, electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis for Fd2 region were carried out. Transfection assay in GH3 cells demonstrated that expression of Hesx1 alone does not change the promoter activity but the coexpression with Prop-1 represses the Prop-1-dependent activation of FSHbeta promoter. Similar results were obtained for the mutant reporter vector deleting the region -745/-104 indicating that Fd2 region is a target site of Hesx1 as well as Prop-1. EMSA and DNase I footprinting analysis using recombinant Hesx1 and Prop-1 protein demonstrated that Hesx1 and Prop-1 certainly bind to the AT-rich regions in a different manner. These results suggest that Hesx1 blocks the advanced expression of FSHbeta gene in the early stage of pituitary development, and Prop-1 thereafter appears and activates this gene.

  8. Discovery of Orally Available Runt-Related Transcription Factor 3 (RUNX3) Modulators for Anticancer Chemotherapy by Epigenetic Activation and Protein Stabilization.

    PubMed

    Yang, Jee Sun; Lee, Chulho; Cho, Misun; Kim, Hyuntae; Kim, Jae Hyun; Choi, Seonghwi; Oh, Soo Jin; Kang, Jong Soon; Jeong, Jin-Hyun; Kim, Hyun-Jung; Han, Gyoonhee

    2015-04-23

    Recently, we identified a novel strategy for anticancer chemotherapy by restoring runt-related transcription factor 3 (RUNX3) levels via lactam-based histone deacetylase (HDAC) inhibitors that stabilize RUNX3. Described here are the synthesis, biological evaluation, and pharmacokinetic evaluation of new synthetic small molecules based on pyridone-based HDAC inhibitors that specifically stabilize RUNX3 by acetylation and regulate its function. Many of the newly synthesized compounds showed favorable RUNX activities, HDAC inhibitory activities, and inhibitory activities on the growth of human cancer cell lines. Notably, one of these new derivatives, (E)-N-hydroxy-3-(2-oxo-1-(quinolin-2-ylmethyl)-1,2-dihydropyridin-3-yl)acrylamide (4l), significantly restored RUNX3 in a dose-dependent manner and showed high metabolic stability, a good pharmacokinetic profile with high oral bioavailability and long half-life, and strong antitumor activity. This study suggests that pyridone-based analogues modulate RUNX3 activity through epigenetic regulation as well as strong transcriptional and post-translational regulation of RUNX3 and could be potential clinical candidates as orally available RUNX3 modulators for the treatment of cancer.

  9. Functional specialization of transcription elongation factors

    PubMed Central

    Belogurov, Georgiy A; Mooney, Rachel A; Svetlov, Vladimir; Landick, Robert; Artsimovitch, Irina

    2009-01-01

    Elongation factors NusG and RfaH evolved from a common ancestor and utilize the same binding site on RNA polymerase (RNAP) to modulate transcription. However, although NusG associates with RNAP transcribing most Escherichia coli genes, RfaH regulates just a few operons containing ops, a DNA sequence that mediates RfaH recruitment. Here, we describe the mechanism by which this specificity is maintained. We observe that RfaH action is indeed restricted to those several operons that are devoid of NusG in vivo. We also show that RfaH and NusG compete for their effects on transcript elongation and termination in vitro. Our data argue that RfaH recognizes its DNA target even in the presence of NusG. Once recruited, RfaH remains stably associated with RNAP, thereby precluding NusG binding. We envision a pathway by which a specialized regulator has evolved in the background of its ubiquitous paralogue. We propose that RfaH and NusG may have opposite regulatory functions: although NusG appears to function in concert with Rho, RfaH inhibits Rho action and activates the expression of poorly translated, frequently foreign genes. PMID:19096362

  10. Differential requirement of the transcription factor Mcm1 for activation of the Candida albicans multidrug efflux pump MDR1 by its regulators Mrr1 and Cap1.

    PubMed

    Mogavero, Selene; Tavanti, Arianna; Senesi, Sonia; Rogers, P David; Morschhäuser, Joachim

    2011-05-01

    Overexpression of the multidrug efflux pump Mdr1 causes increased fluconazole resistance in the pathogenic yeast Candida albicans. The transcription factors Mrr1 and Cap1 mediate MDR1 upregulation in response to inducing stimuli, and gain-of-function mutations in Mrr1 or Cap1, which render the transcription factors hyperactive, result in constitutive MDR1 overexpression. The essential MADS box transcription factor Mcm1 also binds to the MDR1 promoter, but its role in inducible or constitutive MDR1 upregulation is unknown. Using a conditional mutant in which Mcm1 can be depleted from the cells, we investigated the importance of Mcm1 for MDR1 expression. We found that Mcm1 was dispensable for MDR1 upregulation by H2O2 but was required for full MDR1 induction by benomyl. A C-terminally truncated, hyperactive Cap1 could upregulate MDR1 expression both in the presence and in the absence of Mcm1. In contrast, a hyperactive Mrr1 containing a gain-of-function mutation depended on Mcm1 to cause MDR1 overexpression. These results demonstrate a differential requirement for the coregulator Mcm1 for Cap1- and Mrr1-mediated MDR1 upregulation. When activated by oxidative stress or a gain-of-function mutation, Cap1 can induce MDR1 expression independently of Mcm1, whereas Mrr1 requires either Mcm1 or an active Cap1 to cause overexpression of the MDR1 efflux pump. Our findings provide more detailed insight into the molecular mechanisms of drug resistance in this important human fungal pathogen.

  11. Interactions of purified transcription factors: binding of yeast MAT alpha 1 and PRTF to cell type-specific, upstream activating sequences.

    PubMed Central

    Tan, S; Ammerer, G; Richmond, T J

    1988-01-01

    Pheromone receptor transcription factor (PRTF) and MAT alpha 1 are protein transcription factors that are involved in the regulation of the alpha-specific genes in Saccharomyces cerevisiae. We have expressed MAT alpha 1 as a fusion protein in Escherichia coli and purified it from inclusion bodies in milligram quantities. The MAT alpha 1 protein was obtained after specific cleavage of the fusion protein. Quantitative band shift electrophoresis was used to determine the equilibrium dissociation constants that describe the multicomponent binding equilibrium between the PRTF and MAT alpha 1 proteins, and alpha-specific STE3 upstream activating sequence (UAS) DNA. The dissociation constant for the complex of PRTF and the a-specific UAS of STE2 was also measured and found to be 5.9 X 10(-11) M, only three times less than that for the PRTF-STE3 UAS complex. Analyses of these complexes by DNase I footprinting demonstrate that the PRTF binding site is confined to the palindromic P-box sequence in the case of the STE3 UAS, but extends symmetrically from this central region to cover 28 bp for the STE2 UAS. When MAT alpha 1 is bound to the PRTF-STE3 complex, the region of DNA protected is enlarged to that seen for the PRTF-STE2 complex. Our results using these two purified factors in vitro suggest that PRTF has nearly the same affinity for a- and alpha-specific UAS elements and that transcriptional activation requires a particular conformational state for the PRTF-DNA complex which occurs in the PRTF-STE2 and MAT alpha 1-PRTF-STE3 complexes, but not in the PRTF-STE3 complex. Images PMID:2854061

  12. BjMYB1, a transcription factor implicated in plant defence through activating BjCHI1 chitinase expression by binding to a W-box-like element

    PubMed Central

    Gao, Ying; Jia, Shuangwei; Wang, Chunlian; Wang, Fujun; Wang, Fajun; Zhao, Kaijun

    2016-01-01

    We previously identified the W-box-like-4 (Wbl-4) element (GTAGTGACTCAT), one of six Wbl elements in the BjC-P promoter of the unusual chitinase gene BjCHI1 from Brassica juncea, as the core element responsive to fungal infection. Here, we report the isolation and characterization of the cognate transcription factor interacting with the Wbl-4 element. Using Wbl-4 as a target, we performed yeast one-hybrid screening of a B. juncea cDNA library and isolated an R2R3-MYB transcription factor designated as BjMYB1. BjMYB1 was localized in the nucleus of plant cells. EMSA assays confirmed that BjMYB1 binds to the Wbl-4 element. Transiently expressed BjMYB1 up-regulated the activity of the BjC-P promoter through its binding to the Wbl-4 element in tobacco (Nicotiana benthamiana) leaves. In B. juncea, BjMYB1 displayed a similar induced expression pattern as that of BjCHI1 upon infection by the fungus Botrytis cinerea. Moreover, heterogeneous overexpression of BjMYB1 significantly elevated the resistance of transgenic Arabidopsis thaliana to the fungus B. cinerea. These results suggest that BjMYB1 is potentially involved in host defence against fungal attack through activating the expression of BjCHI1 by binding to the Wbl-4 element in the BjC-P promoter. This finding demonstrates a novel DNA target of plant MYB transcription factors. PMID:27353280

  13. Rad51 activates polyomavirus JC early transcription.

    PubMed

    White, Martyn K; Kaminski, Rafal; Khalili, Kamel; Wollebo, Hassen S

    2014-01-01

    The human neurotropic polyomavirus JC (JCV) causes the fatal CNS demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV infection is very common and after primary infection, the virus is able to persist in an asymptomatic state. Rarely, and usually only under conditions of immune impairment, JCV re-emerges to actively replicate in the astrocytes and oligodendrocytes of the brain causing PML. The regulatory events involved in the reactivation of active viral replication in PML are not well understood but previous studies have implicated the transcription factor NF-κB acting at a well-characterized site in the JCV noncoding control region (NCCR). NF-κB in turn is regulated in a number of ways including activation by cytokines such as TNF-α, interactions with other transcription factors and epigenetic events involving protein acetylation--all of which can regulate the transcriptional activity of JCV. Active JCV infection is marked by the occurrence of rapid and extensive DNA damage in the host cell and the induction of the expression of cellular proteins involved in DNA repair including Rad51, a major component of the homologous recombination-directed double-strand break DNA repair machinery. Here we show that increased Rad51 expression activates the JCV early promoter. This activation is co-operative with the stimulation caused by NF-κB p65, abrogated by mutation of the NF-κB binding site or siRNA to NFκB p65 and enhanced by the histone deacetylase inhibitor sodium butyrate. These data indicate that the induction of Rad51 resulting from infection with JCV acts through NF-κB via its binding site to stimulate JCV early transcription. We suggest that this provides a novel positive feedback mechanism to enhance viral gene expression during the early stage of JCV infection.

  14. A non-bacterial transcription factor inhibits bacterial transcription by a multipronged mechanism.

    PubMed

    Sheppard, Carol; James, Ellen; Barton, Geraint; Matthews, Stephen; Severinov, Konstantin; Wigneshweraraj, Sivaramesh

    2013-04-01

    The process of transcription initiation is the major target for regulation of gene expression in bacteria and is performed by a multi-subunit RNA polymerase enzyme (RNAp). A complex network of regulatory elements controls the activity of the RNAp to fine-tune transcriptional output. Thus, RNAp is a nexus for controlling bacterial gene expression at the transcription level. Many bacteriophages, viruses that infect bacteria, encode transcription factors that specifically target and modulate the activity of the host RNAp and, thereby, facilitate the acquisition of the host bacteria by the phage. Here, we describe the modus operandi of a T7 bacteriophage-encoded small protein called Gp2 and define Gp2 as a non-bacterial regulator of bacterial transcription.

  15. Networks of WRKY transcription factors in defense signaling.

    PubMed

    Eulgem, Thomas; Somssich, Imre E

    2007-08-01

    Members of the complex family of WRKY transcription factors have been implicated in the regulation of transcriptional reprogramming associated with plant immune responses. Recently genetic evidence directly proving their significance as positive and negative regulators of disease resistance has accumulated. WRKY genes were shown to be functionally connected forming a transcriptional network composed of positive and negative feedback loops and feed-forward modules. Within a web of partially redundant elements some WRKY factors hold central positions mediating fast and efficient activation of defense programs. A key mechanism triggering strong immune responses appears to be based on the inactivation of defense-suppressing WRKY proteins.

  16. Transcription factors make a turn into migration

    PubMed Central

    2009-01-01

    The formation of the brain depends on a tightly regulated process of proliferation, neuronal fate specification and migration which eventually leads to the final architecture of the cerebral cortex. The specification of different neuronal subtypes depends on a complex developmental program mastered by several transcription factors. Besides, it was shown that the same transcription factors can subsequently control neural migration. However, the mechanisms of this regulation are still unclear. Two papers recently published by Heng et al.1 and Nóbrega-Pereira et al.2 confirm that these transcription factors are involved in controlling neural migration. In addition, these studies show that these transcription factors can control neural migration via different molecular mechanisms: Heng and coworkers show that Neurogenin 2 controls neural migration by directly regulating the expression of the small GTPase Rnd2 (a modulator of cytoskeletal dynamics); whereas Nóbrega-Pereira and colleagues demonstrate that Nkx2-1 establishes the response to guidance cues, in migrating interneurons, by directly regulating the expression of the semaphorin receptor Neuropilin 2. Taken together, these findings support the idea that transcription factors are reused during development to control neural migration and they shed light on the molecular mechanisms underlying this regulation. PMID:19262164

  17. Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts

    NASA Technical Reports Server (NTRS)

    Thomas, M. J.; Umayahara, Y.; Shu, H.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

    1996-01-01

    Insulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other agents that increase cAMP activated IGF-I gene transcription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction was mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have characterized inducible DNA-protein interactions involving this site. Transient transfections of IGF-I P1 reporter genes into primary rat osteoblasts showed that the 328-base pair untranslated region of exon 1 was required for a full 5.3-fold response to PGE2; mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction by 65%. PGE2 stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extracts from untreated osteoblast cultures, was detected within 2 h of PGE2 treatment, and was maximal by 4 h. This DNA-protein interaction was not observed in cytoplasmic extracts from PGE2-treated cultures, indicating nuclear localization of the protein kinase A-activated factor(s). Activation of this factor was not blocked by cycloheximide (Chx), and Chx did not impair stimulation of IGF-I gene expression by PGE2. In contrast, binding to a consensus cAMP response element (CRE; 5'-TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE2 or Chx. Competition gel mobility shift analysis using mutated DNA probes identified 5'-CGCAATCG-3' as the minimal sequence needed for inducible binding. All modified IGF-I P1 promoterreporter genes with mutations within this CRE sequence also showed a diminished functional response to PGE2. These results identify the CRE within the 5'-untranslated region of IGF-I exon 1 that is required for hormonal

  18. Arabidopsis WRKY45 transcription factor activates PHOSPHATE TRANSPORTER1;1 expression in response to phosphate starvation.

    PubMed

    Wang, Hui; Xu, Qian; Kong, You-Han; Chen, Yun; Duan, Jun-Ye; Wu, Wei-Hua; Chen, Yi-Fang

    2014-04-01

    The WRKY transcription factor family has more than 70 members in the Arabidopsis (Arabidopsis thaliana) genome, and some of them are involved in plant responses to biotic and abiotic stresses. This study evaluated the role of WRKY45 in regulating phosphate (Pi) uptake in Arabidopsis. WRKY45 was localized in the nucleus and mainly expressed in roots. During Pi starvation, WRKY45 expression was markedly induced, typically in roots. WRKY45 overexpression in Arabidopsis increased Pi content and uptake, while RNA interference suppression of WRKY45 decreased Pi content and uptake. Furthermore, the WRKY45-overexpressing lines were more sensitive to arsenate, the analog of Pi, compared with wild-type seedlings. These results indicate that WRKY45 positively regulates Arabidopsis Pi uptake. Quantitative real-time polymerase chain reaction and β-glucuronidase staining assays showed that PHOSPHATE TRANSPORTER1;1 (PHT1;1) expression was enhanced in the WRKY45-overexpressing lines and slightly repressed in the WRKY45 RNA interference line. Chromatin immunoprecipitation and electrophoretic mobility shift assay results indicated that WRKY45 can bind to two W-boxes within the PHT1;1 promoter, confirming the role of WRKY45 in directly up-regulating PHT1;1 expression. The pht1;1 mutant showed decreased Pi content and uptake, and overexpression of PHT1;1 resulted in enhanced Pi content and uptake. Furthermore, the PHT1;1-overexpressing line was much more sensitive to arsenate than WRKY45-overexpressing and wild-type seedlings, indicating that PHT1;1 overexpression can enhance Arabidopsis Pi uptake. Moreover, the enhanced Pi uptake and the increased arsenate sensitivity of the WRKY45-overexpressing line was impaired by pht1;1 (35S:WRKY45-18::pht1;1), demonstrating an epistatic genetic regulation between WRKY45 and PHT1;1. Together, our results demonstrate that WRKY45 is involved in Arabidopsis response to Pi starvation by direct up-regulation of PHT1;1 expression.

  19. Angiotensin II-induced protein kinase D activates the ATF/CREB family of transcription factors and promotes StAR mRNA expression.

    PubMed

    Olala, Lawrence O; Choudhary, Vivek; Johnson, Maribeth H; Bollag, Wendy B

    2014-07-01

    Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

  20. Activation of the Transcription Factor GLI1 by WNT Signaling Underlies the Role of SULFATASE 2 as a Regulator of Tissue Regeneration*

    PubMed Central

    Nakamura, Ikuo; Fernandez-Barrena, Maite G.; Ortiz-Ruiz, Maria C.; Almada, Luciana L.; Hu, Chunling; Elsawa, Sherine F.; Mills, Lisa D.; Romecin, Paola A.; Gulaid, Kadra H.; Moser, Catherine D.; Han, Jing-Jing; Vrabel, Anne; Hanse, Eric A.; Akogyeram, Nicholas A.; Albrecht, Jeffrey H.; Monga, Satdarshan P. S.; Sanderson, Schuyler O.; Prieto, Jesus; Roberts, Lewis R.; Fernandez-Zapico, Martin E.

    2013-01-01

    Tissue regeneration requires the activation of a set of specific growth signaling pathways. The identity of these cascades and their biological roles are known; however, the molecular mechanisms regulating the interplay between these pathways remain poorly understood. Here, we define a new role for SULFATASE 2 (SULF2) in regulating tissue regeneration and define the WNT-GLI1 axis as a novel downstream effector for this sulfatase in a liver model of tissue regeneration. SULF2 is a heparan sulfate 6-O-endosulfatase, which releases growth factors from extracellular storage sites turning active multiple signaling pathways. We demonstrate that SULF2-KO mice display delayed regeneration after partial hepatectomy (PH). Mechanistic analysis of the SULF2-KO phenotype showed a decrease in WNT signaling pathway activity in vivo. In isolated hepatocytes, SULF2 deficiency blocked WNT-induced β-CATENIN nuclear translocation, TCF activation, and proliferation. Furthermore, we identified the transcription factor GLI1 as a novel target of the SULF2-WNT cascade. WNT induces GLI1 expression in a SULF2- and β-CATENIN-dependent manner. GLI1-KO mice phenocopied the SULF2-KO, showing delayed regeneration and decreased hepatocyte proliferation. Moreover, we identified CYCLIN D1, a key mediator of cell growth during tissue regeneration, as a GLI1 transcriptional target. GLI1 binds to the cyclin d1 promoter and regulates its activity and expression. Finally, restoring GLI1 expression in the liver of SULF2-KO mice after PH rescues CYCLIN D1 expression and hepatocyte proliferation to wild-type levels. Thus, together these findings define a novel pathway in which SULF2 regulates tissue regeneration in part via the activation of a novel WNT-GLI1-CYCLIN D1 pathway. PMID:23740243

  1. Activation of the transcription factor GLI1 by WNT signaling underlies the role of SULFATASE 2 as a regulator of tissue regeneration.

    PubMed

    Nakamura, Ikuo; Fernandez-Barrena, Maite G; Ortiz-Ruiz, Maria C; Almada, Luciana L; Hu, Chunling; Elsawa, Sherine F; Mills, Lisa D; Romecin, Paola A; Gulaid, Kadra H; Moser, Catherine D; Han, Jing-Jing; Vrabel, Anne; Hanse, Eric A; Akogyeram, Nicholas A; Albrecht, Jeffrey H; Monga, Satdarshan P S; Sanderson, Schuyler O; Prieto, Jesus; Roberts, Lewis R; Fernandez-Zapico, Martin E

    2013-07-19

    Tissue regeneration requires the activation of a set of specific growth signaling pathways. The identity of these cascades and their biological roles are known; however, the molecular mechanisms regulating the interplay between these pathways remain poorly understood. Here, we define a new role for SULFATASE 2 (SULF2) in regulating tissue regeneration and define the WNT-GLI1 axis as a novel downstream effector for this sulfatase in a liver model of tissue regeneration. SULF2 is a heparan sulfate 6-O-endosulfatase, which releases growth factors from extracellular storage sites turning active multiple signaling pathways. We demonstrate that SULF2-KO mice display delayed regeneration after partial hepatectomy (PH). Mechanistic analysis of the SULF2-KO phenotype showed a decrease in WNT signaling pathway activity in vivo. In isolated hepatocytes, SULF2 deficiency blocked WNT-induced β-CATENIN nuclear translocation, TCF activation, and proliferation. Furthermore, we identified the transcription factor GLI1 as a novel target of the SULF2-WNT cascade. WNT induces GLI1 expression in a SULF2- and β-CATENIN-dependent manner. GLI1-KO mice phenocopied the SULF2-KO, showing delayed regeneration and decreased hepatocyte proliferation. Moreover, we identified CYCLIN D1, a key mediator of cell growth during tissue regeneration, as a GLI1 transcriptional target. GLI1 binds to the cyclin d1 promoter and regulates its activity and expression. Finally, restoring GLI1 expression in the liver of SULF2-KO mice after PH rescues CYCLIN D1 expression and hepatocyte proliferation to wild-type levels. Thus, together these findings define a novel pathway in which SULF2 regulates tissue regeneration in part via the activation of a novel WNT-GLI1-CYCLIN D1 pathway.

  2. Differential effects of Sp cellular transcription factors on viral promoter activation by varicella-zoster virus (VZV) IE62 protein.

    PubMed

    Khalil, Mohamed I; Ruyechan, William T; Hay, John; Arvin, Ann

    2015-11-01

    The immediate early (IE) 62 protein is the major varicella-zoster virus (VZV) regulatory factor. Analysis of the VZV genome revealed 40 predicted GC-rich boxes within 36 promoters. We examined effects of ectopic expression of Sp1-Sp4 on IE62- mediated transactivation of three viral promoters. Ectopic expression of Sp3 and Sp4 enhanced IE62 activation of ORF3 and gI promoters while Sp3 reduced IE62 activation of ORF28/29 promoter and VZV DNA replication. Sp2 reduced IE62 transactivation of gI while Sp1 had no significant influence on IE62 activation with any of these viral promoters. Electrophoretic mobility shift assays (EMSA) confirmed binding of Sp1 and Sp3 but not Sp2 and Sp4 to the gI promoter. Sp1-4 bound to IE62 and amino acids 238-258 of IE62 were important for the interaction with Sp3 and Sp4 as well as Sp1. This work shows that Sp family members have differential effects on IE62-mediated transactivation in a promoter-dependent manner.

  3. A Novel Fluid Resuscitation Strategy Modulates Pulmonary Transcription Factor Activation in a Murine Model of Hemorrhagic Shock

    PubMed Central

    Costantini, Todd W.; Deree, Jessica; Martins, J.O.; Putnam, James G.; de Campos, Tercio; Coimbra, Raul

    2010-01-01

    INTRODUCTION: Combining the hemodynamic and immune benefits of hypertonic saline with the anti-inflammatory effects of the phosphodiesterase inhibitor pentoxifylline (HSPTX) as a hemorrhagic shock resuscitation strategy reduces lung injury when compared with the effects of Ringer’s lactate (RL). We hypothesized that HSPTX exerts its anti-inflammatory effects by interfering with nuclear factor kappa B/cAMP response element-binding protein (NF-κB-CREB) competition for the coactivator CREB-binding protein (CBP) in lung tissue, thus affecting pro-inflammatory mediator production. METHODS: Male Sprague-Dawley rats underwent 60 minutes of hemorrhagic shock to reach a mean arterial blood pressure of 35 mmHg followed by resuscitation with either RL or HSPTX (7.5% HS + 25 mg/kg PTX). After four hours, lung samples were collected. NF-κB activation was assessed by measuring the levels of phosphorylated cytoplasmic inhibitor of kappa B (I-κB) and nuclear NF-κB p65 by western blot. NF-κB and CREB DNA-binding activity were measured by electrophoretic mobility shift assay (EMSA). Competition between NF-κB and CREB for the coactivator CBP was determined by immunoprecipitation. Interleukin-8 (IL-8) levels in the lung were measured by ELISA. RESULTS: RL resuscitation produced significantly higher levels of lung IL-8 levels, I-κB phosphorylation, p65 phosphorylation, and NF-κB DNA binding compared with HSPTX. NF-κB-CBP-binding activity was similar in both groups, whereas CREB-CBP-binding activity was significantly increased with HSPTX. CREB-DNA binding-activity increased to a greater level with HSPTX compared with RL. DISCUSSION: HSPTX decreases lung inflammation following hemorrhagic shock compared with conventional resuscitation using RL through attenuation of NF-κB signaling and increased CREB-DNA binding activity. HSPTX may have therapeutic potential in the attenuation of ischemia-reperfusion injury observed after severe hemorrhagic shock. PMID:20613939

  4. Tumor Necrosis Factor Receptor Associated Factors (TRAFs) 2 and 3 Form a Transcriptional Complex with Phosho-RNA Polymerase II and p65 in CD40 Ligand Activated Neuro2a Cells.

    PubMed

    El Hokayem, Jimmy; Brittain, George C; Nawaz, Zafar; Bethea, John R

    2017-03-01

    The tumor necrosis factor receptor-associated factors (TRAFs) have been classically described as adaptor proteins that function as solely cytosolic signaling intermediates for the TNF receptor superfamily, Toll-like receptors (TLRs), NOD, like receptors (NLRs), cytokine receptors, and others. In this study, we show for the first time that TRAFs are present within the cytoplasm and nucleus of Neuro2a cells and primary cortical neurons, and that TRAF2 and TRAF3 translocate into the nucleus within minutes of CD40L stimulation. Analysis of the transcriptional regulatory potential of TRAFs by luciferase assay revealed that each of the TRAFs differentially functions as a transcriptional activator or repressor in a cell-specific manner. Interestingly, ChIP-qPCR data demonstrate that TRAFs 2/3, p65, and pRNAPol II form part of a transcriptional complex on the Icam-1 gene promoter upon CD40L stimulation. We further determined that TRAF2 recruitment to the nucleus is critical for the ubiquitination of H2b, a transcription permissive epigenetic modification. Our findings demonstrate for the first time that TRAFs 2/3 participate in the formation of a CD40L-induced transcriptional complex in neuronal cells.

  5. Addition and correction: the NF-kappa B-like DNA binding activity observed in Dictyostelium nuclear extracts is due to the GBF transcription factor.

    PubMed

    Traincard, F; Ponte, E; Pun, J; Coukell, B; Veron, M

    2001-10-01

    We have previously reported that a NF-kappa B transduction pathway was likely to be present in the cellular slime mold Dictyostelium discoideum. This conclusion was based on several observations, including the detection of developmentally regulated DNA binding proteins in Dictyostelium nuclear extracts that bound to bona fide kappa B sequences. We have now performed additional experiments which demonstrate that the protein responsible for this NF-kappa B-like DNA binding activity is the Dictyostelium GBF (G box regulatory element binding factor) transcription factor. This result, along with the fact that no sequence with significant similarity to components of the mammalian NF-kappa B pathway can be found in Dictyostelium genome, now almost entirely sequenced, led us to reconsider our previous conclusion on the occurrence of a NF-kappa B signal transduction pathway in Dictyostelium.

  6. Distinctive features of Egr transcription factor regulation and DNA binding activity in CA1 of the hippocampus in synaptic plasticity and consolidation and reconsolidation of fear memory.

    PubMed

    Cheval, Hélène; Chagneau, Carine; Levasseur, Grégoire; Veyrac, Alexandra; Faucon-Biguet, Nicole; Laroche, Serge; Davis, Sabrina

    2012-03-01

    Activity-dependent regulation of Egr1/Zif268, a transcription factor (TF) of the Egr family, is essential for stabilization of dentate gyrus synaptic plasticity and consolidation and reconsolidation of several forms of memory. The gene can be rapidly induced in selective brain circuits after certain types of learning or after recall. Here, we focused on area CA1 and examined regulation of Egr1, Egr2, and Egr3 mRNA and protein, and their DNA binding activity to the Egr response element (ERE) at different times after LTP in vivo and after learning and recall of a fear memory. We found LTP in CA1 leads to rapid induction of the three Egrs, however only Egr1 protein was overexpressed without a co-ordinated change in binding activity, indicating a fundamental difference between CA1 and dentate gyrus LTP. Our investigations in fear memory reveal that both learning and retrieval lead to an increase in binding of constitutively expressed Egr1 and Egr3 to the ERE, but not Egr2. Memory recall was also associated with increased Egr1 protein translation. The nature and temporal dynamics of these changes and tests for interactions between TFs suggest that in addition to ERE-mediated transcription, Egr1 in CA1 may interact with the TF c-Fos to regulate genes via other DNA response elements.

  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. Loss of Interdependent Binding by the FoxO1 and FoxA1/A2 Forkhead Transcription Factors Culminates in Perturbation of Active Chromatin Marks and Binding of Transcriptional Regulators at Insulin-sensitive Genes.

    PubMed

    Yalley, Akua; Schill, Daniel; Hatta, Mitsutoki; Johnson, Nicole; Cirillo, Lisa Ann

    2016-04-15

    FoxO1 binds to insulin response elements located in the promoters of insulin-like growth factor-binding protein 1 (IGFBP1) and glucose-6-phosphatase (G6Pase), activating their expression. Insulin-mediated phosphorylation of FoxO1 promotes cytoplasmic translocation, inhibiting FoxO1-mediated transactivation. We have previously demonstrated that FoxO1 opens and remodels chromatin assembled from the IGFBP1 promoter via a highly conserved winged helix motif. This finding, which established FoxO1 as a "pioneer" factor, suggested a model whereby FoxO1 chromatin remodeling at regulatory targets facilitates binding and recruitment of additional regulatory factors. However, the impact of FoxO1 phosphorylation on its ability to bind chromatin and the effect of FoxO1 loss on recruitment of neighboring transcription factors at its regulatory targets in liver chromatin is unknown. In this study, we demonstrate that an amino acid substitution that mimics insulin-mediated phosphorylation of a serine in the winged helix DNA binding motif curtails FoxO1 nucleosome binding. We also demonstrate that shRNA-mediated loss of FoxO1 binding to the IGFBP1 and G6Pase promoters in HepG2 cells significantly reduces binding of RNA polymerase II and the pioneer factors FoxA1/A2. Knockdown of FoxA1 similarly reduced binding of RNA polymerase II and FoxO1. Reduction in acetylation of histone H3 Lys-27 accompanies loss of FoxO1 and FoxA1/A2 binding. Interdependent binding of FoxO1 and FoxA1/A2 possibly entails cooperative binding because FoxO1 and FoxA1/A2 facilitate one another's binding to IGFPB1 promoter DNA. These results illustrate how transcription factors can nucleate transcriptional events in chromatin in response to signaling events and suggest a model for regulation of hepatic glucose metabolism through interdependent FoxO/FoxA binding.

  9. Keap1 silencing boosts lipopolysaccharide-induced transcription of interleukin 6 via activation of nuclear factor κB in macrophages

    SciTech Connect

    Lv, Peng; Xue, Peng; Dong, Jian; Peng, Hui; Clewell, Rebecca; Wang, Aiping; Wang, Yue; Peng, Shuangqing; Qu, Weidong; Zhang, Qiang; Andersen, Melvin E.; Pi, Jingbo

    2013-11-01

    Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), regulate IL6 transcription. Kelch-like ECH-associated protein 1 (Keap1) is a substrate adaptor protein for the Cullin 3-dependent E3 ubiquitin ligase complex, which regulates the degradation of many proteins, including Nrf2 and IκB kinase β (IKKβ). Here, we found that stable knockdown of Keap1 (Keap1-KD) in RAW 264.7 (RAW) mouse macrophages and human monocyte THP-1 cells significantly increased expression of Il6, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001–0.1 μg/ml)-challenged conditions. However, Nrf2 activation alone, by tert-butylhydroquinone treatment of RAW cells, did not increase expression of Il6. Compared to cells transduced with scrambled non-target negative control shRNA, Keap1-KD RAW cells showed enhanced protein levels of IKKβ and increased expression and phosphorylation of NF-κB p65 under non-stressed and LPS-treated conditions. Because the expression of Il6 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikkβ, but not Nrf2, it appears that stabilized IKKβ is responsible for the enhanced transactivation of Il6 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of Il6 via NF-κB activation. Given the importance of IL6 in the inflammatory response, the Keap1–IKKβ–NF-κB pathway may be a novel target for treatment and prevention of inflammation and associated disorders. - Highlights: • Knockdown of Keap1 increases expression of Il6 in macrophages. • Silencing of Keap1 results in protein accumulation of IKKβ and NF-κB p65. • Induction of Il6 resulting from Keap1 silencing is attributed to NF-κB activation.

  10. CCAAT/enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone

    NASA Technical Reports Server (NTRS)

    Umayahara, Y.; Ji, C.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast

  11. Integrin αvβ3 mediates the synergetic regulation of core-binding factor α1 transcriptional activity by gravity and insulin-like growth factor-1 through phosphoinositide 3-kinase signaling.

    PubMed

    Dai, Zhongquan; Guo, Feima; Wu, Feng; Xu, Hongjie; Yang, Chao; Li, Jinqiao; Liang, Peilong; Zhang, Hongyu; Qu, Lina; Tan, Yingjun; Wan, Yumin; Li, Yinghui

    2014-12-01

    Mechanical stimulation and biological factors coordinately regulate bone development and regeneration; however, the underlying mechanisms are poorly understood. Microgravity induces bone loss, which may be partly related to the development of resistance to local cytokines, including insulin-like growth factor 1 (IGF-1). Here, we report the involvement of integrin αvβ3 in microgravity-associated bone loss. An established OSE-3T3 cell model was stably transfected with a 6OSE2 (Osteoblast-Specific Element 2)-luciferase reporter and cultured under simulated microgravity (SMG) and hypergravity (HG) conditions in the presence or absence of IGF-1, the disintegrin echistatin, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, or combinations of these agents. Activity of core-binding factor α1 (Cbfa1), an essential transcription factor for osteoblastic differentiation and osteogenesis, was reflected by luciferase activity. Different gravity conditions affected the induction of IGF-1 and subsequent effects on Cbfa1 transcription activity. SMG and HG influenced the expression and activity of integrin αvβ3 and phosphorylation level of p85. LY294002 inhibited the effects of HG or IGF-1 on Cbfa1 activity, indicating that HG and IGF-1 could increase Cbfa1 activity via PI3K signaling. Inhibition of integrin αvβ3 by echistatin attenuated the induction of IGF-1 and thus its effect on Cbfa1 activity under normal and HG conditions. Co-immunoprecipitation demonstrated that integrin β3 interacted with insulin receptor substrate 1, and that this interaction was decreased under SMG and increased under HG conditions. These results suggest that integrin αvβ3 mediates the synergetic regulation of Cbfa1 transcription activity by gravity and IGF-1 via PI3K signaling.

  12. Mechanism of metabolic control. Target of rapamycin signaling links nitrogen quality to the activity of the Rtg1 and Rtg3 transcription factors.

    PubMed

    Komeili, A; Wedaman, K P; O'Shea, E K; Powers, T

    2000-11-13

    De novo biosynthesis of amino acids uses intermediates provided by the TCA cycle that must be replenished by anaplerotic reactions to maintain the respiratory competency of the cell. Genome-wide expression analyses in Saccharomyces cerevisiae reveal that many of the genes involved in these reactions are repressed in the presence of the preferred nitrogen sources glutamine or glutamate. Expression of these genes in media containing urea or ammonia as a sole nitrogen source requires the heterodimeric bZip transcription factors Rtg1 and Rtg3 and correlates with a redistribution of the Rtg1p/Rtg3 complex from a predominantly cytoplasmic to a predominantly nuclear location. Nuclear import of the complex requires the cytoplasmic protein Rtg2, a previously identified upstream regulator of Rtg1 and Rtg3, whereas export requires the importin-beta-family member Msn5. Remarkably, nuclear accumulation of Rtg1/Rtg3, as well as expression of their target genes, is induced by addition of rapamycin, a specific inhibitor of the target of rapamycin (TOR) kinases. We demonstrate further that Rtg3 is a phosphoprotein and that its phosphorylation state changes after rapamycin treatment. Taken together, these results demonstrate that target of rapamycin signaling regulates specific anaplerotic reactions by coupling nitrogen quality to the activity and subcellular localization of distinct transcription factors.

  13. Eomesodermin controls interleukin-5 production in memory T helper 2 cells through inhibition of activity of the transcription factor GATA3.

    PubMed

    Endo, Yusuke; Iwamura, Chiaki; Kuwahara, Makoto; Suzuki, Akane; Sugaya, Kaoru; Tumes, Damon J; Tokoyoda, Koji; Hosokawa, Hiroyuki; Yamashita, Masakatsu; Nakayama, Toshinori

    2011-11-23

    The regulation of memory CD4(+) helper T (Th) cell function, such as polarized cytokine production, remains unclear. Here we show that memory T helper 2 (Th2) cells are divided into four subpopulations by CD62L and CXCR3 expression. All four subpopulations produced interleukin-4 (IL-4) and IL-13, whereas only the CD62L(lo)CXCR3(lo) population produced IL-5 accompanied by increased H3-K4 methylation at the Il5 gene locus. The transcription factor Eomesodermin (encoded by Eomes) was highly expressed in memory Th2 cells, whereas its expression was selectively downregulated in the IL-5-producing cells. Il5 expression was enhanced in Eomes-deficient cells, and Eomesodermin was shown to interact with the transcription factor GATA3, preventing GATA3 binding to the Il5 promoter. Memory Th2 cell-dependent