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Sample records for 15d-pgj2 mediates repression

  1. 15-Deoxy-{delta}{sup 12,14}-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-{alpha} by decreasing levels of acetylated histone H3 and H4 at its promoter

    SciTech Connect

    Engdahl, Ryan . E-mail: rengdahl@temple.edu; Monroy, M. Alexandra; Daly, John M.

    2007-07-20

    Prostaglandin metabolite 15-Deoxy-{delta}{sup 12,14}-prostaglandin J2 (15d-PGJ2) is known to inhibit a number of pro-inflammatory cytokines as well as being a ligand for nuclear receptor PPAR{gamma}. We investigated the ability of 15d-PGJ2 to inhibit TNF-{alpha} gene expression through mechanisms that involve histone modification. Pretreatment with 15d-PGJ2 (10 {mu}M) inhibited LPS-stimulated TNF-{alpha} mRNA in THP-1 monocytes or PMA-differentiated cells to nearly basal levels. A specific PPAR{gamma} ligand, GW1929, failed to inhibit LPS-induced TNF-{alpha} mRNA expression nor did a PPAR{gamma} antagonist, GW9662, alter the repression of TNF-{alpha} mRNA in LPS-stimulated cells pretreated with 15d-PGJ2 suggesting a PPAR{gamma}-independent inhibition of TNF-{alpha} mRNA in THP-1 cells. Transfection studies with a reporter construct and subsequent treatment with 15d-PGJ2 demonstrated a dose-dependent inhibition of the TNF-{alpha} promoter. Additional studies demonstrated that inhibition of histone deacetylases with trichostatin A (TSA) or overexpression of histone acetyltransferase CBP could overcome 15d-PGJ2-mediated repression of the TNF-{alpha} promoter, suggesting that an important mechanism whereby 15d-PGJ2 suppresses a cytokine is through factors that regulate histone modifications. To examine the endogenous TNF-{alpha} promoter, chromatin immunoprecipitations (ChIP) were performed. ChIP assays demonstrated that LPS stimulation induced an increase in histone H3 and H4 acetylation at the TNF-{alpha} promoter, which was reduced in cells pretreated with 15d-PGJ2. These results highlight the ability of acetylation and deacetylation factors to affect the TNF-{alpha} promoter and demonstrate that an additional important mechanism whereby 15d-PGJ2 mediates TNF-{alpha} transcriptional repression by altering levels of acetylated histone H3 and H4 at its promoter.

  2. Activation of the MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis protects MG-63 osteosarcoma cells against 15d-PGJ2-mediated cell death.

    PubMed

    Koyani, Chintan N; Kitz, Kerstin; Rossmann, Christine; Bernhart, Eva; Huber, Evelyn; Trummer, Christopher; Windischhofer, Werner; Sattler, Wolfgang; Malle, Ernst

    2016-03-15

    Despite considerable efforts to improve treatment modalities for osteosarcoma (OS), patient survival remains poor mainly due to pro-survival pathways in OS cells. Among others, prostaglandins (PGs) are the potent regulators of bone homoeostasis and OS pathophysiology. Therefore, the present study aimed to elucidate the impact of 15-deoxy-Δ(12,14)-PGJ2 (15d-PGJ2, a stable PGD2 degradation product) on cell death/cell survival pathways in p53-deficient MG-63 OS cells. Our findings show that 15d-PGJ2 induces generation of reactive oxygen species that promote p38 MAPK activation and subsequent Akt phosphorylation. This pathway induced nuclear expression of Nrf2 and Egr1, and increased transcription of haem oxygenase-1 (HO-1) and the catalytic subunit of glutamate cysteine ligase (GCLc), catalysing the first step in GSH synthesis. Silencing of Nrf2, Egr1 and HO-1 significantly elevated 15d-PGJ2-mediated reduction of cellular metabolic activity. Activation of cell survival genes including HO-1 and GCLc inhibited 15d-PGJ2-induced cleavage of pro-caspase-3 and PARP. Annexin V/propidium iodide staining showed an increase in early/late apoptotic cells in response to 15d-PGJ2. The observed 15d-PGJ2-mediated signalling events are independent of PGD2 receptors (DP1 and DP2) and PPARγ. In addition, the electrophilic carbon atom C9 is a prerequisite for the observed activity of 15d-PGJ2. The present data show that the intracellular redox imbalance acted as a node and triggered both death and survival pathways in response to 15d-PGJ2. Pharmacological or genetic interference of the pro-survival pathway, the p38 MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis, sensitizes MG-63 cells towards 15d-PGJ2-mediated apoptosis. PMID:26801686

  3. Activation of the MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis protects MG-63 osteosarcoma cells against 15d-PGJ2-mediated cell death

    PubMed Central

    Koyani, Chintan N.; Kitz, Kerstin; Rossmann, Christine; Bernhart, Eva; Huber, Evelyn; Trummer, Christopher; Windischhofer, Werner; Sattler, Wolfgang; Malle, Ernst

    2016-01-01

    Despite considerable efforts to improve treatment modalities for osteosarcoma (OS), patient survival remains poor mainly due to pro-survival pathways in OS cells. Among others, prostaglandins (PGs) are the potent regulators of bone homoeostasis and OS pathophysiology. Therefore, the present study aimed to elucidate the impact of 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2, a stable PGD2 degradation product) on cell death/cell survival pathways in p53-deficient MG-63 OS cells. Our findings show that 15d-PGJ2 induces generation of reactive oxygen species that promote p38 MAPK activation and subsequent Akt phosphorylation. This pathway induced nuclear expression of Nrf2 and Egr1, and increased transcription of haem oxygenase-1 (HO-1) and the catalytic subunit of glutamate cysteine ligase (GCLc), catalysing the first step in GSH synthesis. Silencing of Nrf2, Egr1 and HO-1 significantly elevated 15d-PGJ2-mediated reduction of cellular metabolic activity. Activation of cell survival genes including HO-1 and GCLc inhibited 15d-PGJ2-induced cleavage of pro-caspase-3 and PARP. Annexin V/propidium iodide staining showed an increase in early/late apoptotic cells in response to 15d-PGJ2. The observed 15d-PGJ2-mediated signalling events are independent of PGD2 receptors (DP1 and DP2) and PPARγ. In addition, the electrophilic carbon atom C9 is a prerequisite for the observed activity of 15d-PGJ2. The present data show that the intracellular redox imbalance acted as a node and triggered both death and survival pathways in response to 15d-PGJ2. Pharmacological or genetic interference of the pro-survival pathway, the p38 MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis, sensitizes MG-63 cells towards 15d-PGJ2-mediated apoptosis. PMID:26801686

  4. Autocrine secretion of 15d-PGJ2 mediates simvastatin-induced apoptotic burst in human metastatic melanoma cells

    PubMed Central

    Wasinger, Christine; Künzl, Martin; Minichsdorfer, Christoph; Höller, Christoph; Zellner, Maria; Hohenegger, Martin

    2014-01-01

    Background and Purpose Despite new therapeutic approaches, metastatic melanomas still have a poor prognosis. Statins reduce low-density lipoprotein cholesterol and exert anti-inflammatory and anti-proliferative actions. We have recently shown that simvastatin triggers an apoptotic burst in human metastatic melanoma cells by the synthesis of an autocrine factor. Experimental Approach The current in vitro study was performed in human metastatic melanoma cell lines (A375, 518a2) and primary human melanocytes and melanoma cells. The secretome of simvastatin-stressed cells was analysed with two-dimensional difference gel electrophoresis and MS. The signalling pathways involved were analysed at the protein and mRNA level using pharmacological approaches and siRNA technology. Key Results Simvastatin was shown to activate a stress cascade, leading to the synthesis of 15-deoxy-12,14-PGJ2 (15d-PGJ2), in a p38- and COX-2-dependent manner. Significant concentrations of 15d-PGJ2 were reached in the medium of melanoma cells, which were sufficient to activate caspase 8 and the mitochondrial pathway of apoptosis. Inhibition of lipocalin-type PGD synthase, a key enzyme for 15d-PGJ2 synthesis, abolished the apoptotic effect of simvastatin. Moreover, 15d-PGJ2 was shown to bind to the fatty acid-binding protein 5 (FABP5), which was up-regulated and predominantly detected in the secretome of simvastatin-stressed cells. Knockdown of FABP5 abolished simvastatin-induced activation of PPAR-γ and amplified the apoptotic response. Conclusions and Implications We characterized simvastatin-induced activation of the 15d-PGJ2/FABP5 signalling cascades, which triggered an apoptotic burst in melanoma cells but did not affect primary human melanocytes. These data support the rationale for the pharmacological targeting of 15d-PGJ2 in metastatic melanoma. PMID:25091578

  5. The cyclopentenone prostaglandin 15d-PGJ2 inhibits the NLRP1 and NLRP3 inflammasomes

    PubMed Central

    Maier, Nolan K.; Leppla, Stephen H.; Moayeri, Mahtab

    2015-01-01

    Inflammasomes are cytosolic protein complexes that respond to diverse danger signals by activating caspase-1. The sensor components of the inflammasome, often proteins of the nucleotide-binding oligomerization domain-like receptor (NLR) family, detect stress, danger stimuli, and pathogen-associated molecular patterns. We report that the eicosanoid 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and related cyclopentenone prostaglandins inhibit caspase-1 activation by the NLRP1 and NLRP3 inflammasomes. This inhibition was independent of 15d-PGJ2’s well characterized role as a peroxisome proliferator receptor-γ agonist, its activation of NRF2, or its anti-inflammatory function as an inhibitor of NF-κB. Instead, 15d-PGJ2 prevents the autoproteolytic activation of caspase-1 and the maturation of IL-1β through induction of a cellular state inhibitory to caspase-1 proteolytic function. The eicosanoid does not directly modify or inactivate the caspase-1 enzyme. Rather, inhibition is dependent on de novo protein synthesis. In a mouse peritonitis model of gout, using monosodium urate crystals to activate NLRP3,15d-PGJ2 caused a significant inhibition of cell recruitment and associated IL-1β release. Furthermore, in a murine anthrax infection model, 15d-PGJ2 reversed anthrax lethal toxin-mediated NLRP1-dependent resistance. The findings reported in this work suggest a novel mechanism for the anti-inflammatory properties of the cyclopentenone prostaglandins through inhibition of caspase-1 and the inflammasome. PMID:25681332

  6. The cyclopentenone prostaglandin 15d-PGJ2 inhibits the NLRP1 and NLRP3 inflammasomes.

    PubMed

    Maier, Nolan K; Leppla, Stephen H; Moayeri, Mahtab

    2015-03-15

    Inflammasomes are cytosolic protein complexes that respond to diverse danger signals by activating caspase-1. The sensor components of the inflammasome, often proteins of the nucleotide-binding oligomerization domain-like receptor (NLR) family, detect stress, danger stimuli, and pathogen-associated molecular patterns. We report that the eicosanoid 15-deoxy-Δ(12,14)-PGJ2 (15d-PGJ2) and related cyclopentenone PGs inhibit caspase-1 activation by the NLR family leucine-rich repeat protein (NLRP)1 and NLRP3 inflammasomes. This inhibition was independent of the well-characterized role of 15d-PGJ2 as a peroxisome proliferator receptor-γ agonist, its activation of NF erythroid 2-related factor 2, or its anti-inflammatory function as an inhibitor of NF-κB. Instead, 15d-PGJ2 prevents the autoproteolytic activation of caspase-1 and the maturation of IL-1β through induction of a cellular state inhibitory to caspase-1 proteolytic function. The eicosanoid does not directly modify or inactivate the caspase-1 enzyme. Rather, inhibition is dependent on de novo protein synthesis. In a mouse peritonitis model of gout, using monosodium urate crystals to activate NLRP3, 15d-PGJ2 caused a significant inhibition of cell recruitment and associated IL-1β release. Furthermore, in a murine anthrax infection model, 15d-PGJ2 reversed anthrax lethal toxin-mediated NLRP1-dependent resistance. The findings reported in this study suggest a novel mechanism for the anti-inflammatory properties of the cyclopentenone PGs through inhibition of caspase-1 and the inflammasome. PMID:25681332

  7. Elimination of the biphasic pharmacodynamics of 15d-PGJ2 by controlling its release from a nanoemulsion

    PubMed Central

    Abbasi, Saed; Kajimoto, Kazuaki; Harashima, Hideyoshi

    2016-01-01

    15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has a dual action of stimulating anti-inflammation and anti-proliferation when exogenously administered at high doses. However, at lower doses, it can be toxic inducing opposite actions, ie, stimulation of both inflammation and cell proliferation. This biphasic phenomenon of 15d-PGJ2 is believed to be due to its multitarget behavior. In this study, we provide a strategy for controlling such biphasic pharmacodynamics by separating its dual actions while retaining the beneficial one by using a nanoemulsion (NE). The 15d-PGJ2 was encapsulated in the NE composed of triolein/distearoyl phosphatidylcholine/Tween 80 at a high encapsulation ratio (>83%). Furthermore, NE enhanced drug retention by slowing down its release rate, which was, unconventionally, inversely dependent on the total surface area of the NE system. Next, focusing on the biphasic effect on cell proliferation, we found that the 15d-PGJ2-loaded slow-release NE showed only a dose-dependent inhibition of the viability of a mouse macrophage cell line, RAW264.7, although a fast-release NE as well as free 15d-PGJ2 exerted a biphasic effect. The observed slow-release kinetics are believed to be responsible for elimination of the biphasic pharmacodynamics of 15d-PGJ2 mainly for two reasons: 1) a high proportion of 15d-PGJ2 that is retained in the NE was delivered to the cytosol, where proapoptotic targets are located and 2) 15d-PGJ2 was able to bypass cell membrane-associated targets that lead to the induction of cellular proliferation. Collectively, our strategy of eliminating the 15d-PGJ2-induced biphasic pharmacodynamics was based on the delivery of 15d-PGJ2 to its desired site of action, excluding undesired sites, on a subcellular level. PMID:27354798

  8. 15d-PGJ2-Loaded Solid Lipid Nanoparticles: Physicochemical Characterization and Evaluation of Pharmacological Effects on Inflammation.

    PubMed

    de Melo, Nathalie Ferreira Silva; de Macedo, Cristina Gomes; Bonfante, Ricardo; Abdalla, Henrique Ballassini; da Silva, Camila Morais Gonçalves; Pasquoto, Tatiane; de Lima, Renata; Fraceto, Leonardo Fernandes; Clemente-Napimoga, Juliana Trindade; Napimoga, Marcelo Henrique

    2016-01-01

    15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, has physiological properties including pronounced anti-inflammatory activity, though it binds strongly to serum albumin. The use of solid lipid nanoparticles (SLN) can improve therapeutic properties increasing drug efficiency and availability. 15d-PGJ2-SLN was therefore developed and investigated in terms of its immunomodulatory potential. 15d-PGJ2-SLN and unloaded SLN were physicochemically characterized and experiments in vivo were performed. Animals were pretreated with 15d-PGJ2-SLN at concentrations of 3, 10 or 30 μg·kg-1 before inflammatory stimulus with carrageenan (Cg), lipopolysaccharide (LPS) or mBSA (immune response). Interleukins (IL-1β, IL-10 and IL-17) levels were also evaluated in exudates. The 15d-PGJ2-SLN system showed good colloidal parameters and encapsulation efficiency of 96%. The results showed that the formulation was stable for up to 120 days with low hemolytic effects. The 15d-PGJ2-SLN formulation was able to reduce neutrophil migration in three inflammation models tested using low concentrations of 15d-PGJ2. Additionally, 15d-PGJ2-SLN increased IL-10 levels and reduced IL-1β as well as IL-17 in peritoneal fluid. The new 15d-PGJ2-SLN formulation highlights perspectives of a potent anti-inflammatory system using low concentrations of 15d-PGJ2. PMID:27575486

  9. 15d-PGJ2-Loaded Solid Lipid Nanoparticles: Physicochemical Characterization and Evaluation of Pharmacological Effects on Inflammation

    PubMed Central

    da Silva, Camila Morais Gonçalves; Pasquoto, Tatiane; de Lima, Renata; Fraceto, Leonardo Fernandes

    2016-01-01

    15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, has physiological properties including pronounced anti-inflammatory activity, though it binds strongly to serum albumin. The use of solid lipid nanoparticles (SLN) can improve therapeutic properties increasing drug efficiency and availability. 15d-PGJ2-SLN was therefore developed and investigated in terms of its immunomodulatory potential. 15d-PGJ2-SLN and unloaded SLN were physicochemically characterized and experiments in vivo were performed. Animals were pretreated with 15d-PGJ2-SLN at concentrations of 3, 10 or 30 μg·kg-1 before inflammatory stimulus with carrageenan (Cg), lipopolysaccharide (LPS) or mBSA (immune response). Interleukins (IL-1β, IL-10 and IL-17) levels were also evaluated in exudates. The 15d-PGJ2-SLN system showed good colloidal parameters and encapsulation efficiency of 96%. The results showed that the formulation was stable for up to 120 days with low hemolytic effects. The 15d-PGJ2-SLN formulation was able to reduce neutrophil migration in three inflammation models tested using low concentrations of 15d-PGJ2. Additionally, 15d-PGJ2-SLN increased IL-10 levels and reduced IL-1β as well as IL-17 in peritoneal fluid. The new 15d-PGJ2-SLN formulation highlights perspectives of a potent anti-inflammatory system using low concentrations of 15d-PGJ2. PMID:27575486

  10. The anti-inflammatory prostaglandin 15d-PGJ2 and its nuclear receptor PPARgamma are decreased in schizophrenia.

    PubMed

    Martínez-Gras, Isabel; Pérez-Nievas, Beatriz G; García-Bueno, Borja; Madrigal, José L M; Andrés-Esteban, Eva; Rodríguez-Jiménez, Roberto; Hoenicka, Janet; Palomo, Tomás; Rubio, Gabriel; Leza, Juan C

    2011-05-01

    A number of findings suggest that inflammation plays a role in the pathophysiology of schizophrenia. Taking into account a physiological balance between pro- and anti-inflammatory mediators, we measured the plasma levels of cyclooxygenase-derived mediators and other key pro- and anti-inflammatory transcription factors in peripheral blood mononuclear cells (PBMC). Forty healthy subjects and 46 treated chronic schizophrenic patients with an acutely exacerbated condition who met DSM-IV criteria were included. COX by-products prostaglandin E2 (PGE2) and 15d-prostaglandin J2 (15d-PGJ2) plasma levels were measured by EIA. Peroxisome proliferator-activated receptor gamma (PPARγ) as well as nuclear factor kappaB (NFκB) activity in nuclear extracts from PBMC and expression of its inhibitory subunit IκBα in cytosolic extracts were determined using ELISA-based kits. Schizophrenic patients showed higher plasma levels of pro-inflammatory PGE2 than age-matched controls (p=0.043). On the contrary, levels of anti-inflammatory 15-d-PGJ2 were lower (p=0.004), correlating with a lower expression of its nuclear target, PPARγ in nuclear extracts from PBMC (p=0.001). Although no changes in NFκB activity were observed between patients and healthy controls, the expression of its inhibitory protein IκBα was lower in the patients compared to the controls (p=0.027). These findings suggest that schizophrenia is associated with a systemic imbalance in the plasma levels of pro-inflammatory/anti-inflammatory prostaglandins in favor of the former. Furthermore, the expression and activity of anti-inflammatory PPARγ are diminished in PBMC, which indicates a state of inflammation and blunted anti-inflammatory counterbalancing mechanisms at systemic level in these patients. PMID:21334179

  11. 15d-PGJ2 and rosiglitazone suppress Janus kinase-STAT inflammatory signaling through induction of suppressor of cytokine signaling 1 (SOCS1) and SOCS3 in glia.

    PubMed

    Park, Eun Jung; Park, Soo Young; Joe, Eun-hye; Jou, Ilo

    2003-04-25

    Peroxisome proliferator-activated receptor (PPAR)-gamma agonists are now emerging as therapeutic drugs for various inflammatory diseases. However, their molecular mechanism of action remains to be elucidated. Here we report a novel mechanism that underlies the PPAR-gamma agonist-mediated suppression of brain inflammation. We show that 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)) and rosiglitazone reduce the phosphorylation of STAT1 and STAT3 as well as Janus kinase 1 (JAK1) and JAK2 in activated astrocytes and microglia. The PPAR-gamma agonist-mediated reduction in phosphorylation leads to the suppression of JAK-STAT-dependent inflammatory responses. The effects of 15d-PGJ(2) and rosiglitazone are not mediated by activation of PPAR-gamma. 15d-PGJ(2) and rosiglitazone rapidly induce the transcription of suppressor of cytokine signaling (SOCS) 1 and 3, which in turn inhibit JAK activity in activated glial cells. In addition, Src homology 2 domain-containing protein phosphatase 2 (SHP2), another negative regulator of JAK activity, is also involved in their anti-inflammatory action. Our data suggest that 15d-PGJ(2) and rosiglitazone suppress the initiation of JAK-STAT inflammatory signaling independently of PPAR-gamma, thus attenuating brain inflammation. PMID:12584205

  12. Mitochondrial remodeling following fission inhibition by 15d-PGJ2 involves molecular changes in mitochondrial fusion protein OPA1

    SciTech Connect

    Kar, Rekha; Mishra, Nandita; Singha, Prajjal K.; Venkatachalam, Manjeri A.; Saikumar, Pothana

    2010-09-03

    Research highlights: {yields} Chemical inhibition of fission protein Drp1 leads to mitochondrial fusion. {yields} Increased fusion stimulates molecular changes in mitochondrial fusion protein OPA1. {yields} Proteolysis of larger isoforms, new synthesis and ubiquitination of OPA1 occur. {yields} Loss of mitochondrial tubular rigidity and disorganization of cristae. {yields} Generation of large swollen dysfunctional mitochondria. -- Abstract: We showed earlier that 15 deoxy {Delta}{sup 12,14} prostaglandin J2 (15d-PGJ2) inactivates Drp1 and induces mitochondrial fusion . However, prolonged incubation of cells with 15d-PGJ2 resulted in remodeling of fused mitochondria into large swollen mitochondria with irregular cristae structure. While initial fusion of mitochondria by 15d-PGJ2 required the presence of both outer (Mfn1 and Mfn2) and inner (OPA1) mitochondrial membrane fusion proteins, later mitochondrial changes involved increased degradation of the fusion protein OPA1 and ubiquitination of newly synthesized OPA1 along with decreased expression of Mfn1 and Mfn2, which likely contributed to the loss of tubular rigidity, disorganization of cristae, and formation of large swollen degenerated dysfunctional mitochondria. Similar to inhibition of Drp1 by 15d-PGJ2, decreased expression of fission protein Drp1 by siRNA also resulted in the loss of fusion proteins. Prevention of 15d-PGJ2 induced mitochondrial elongation by thiol antioxidants prevented not only loss of OPA1 isoforms but also its ubiquitination. These findings provide novel insights into unforeseen complexity of molecular events that modulate mitochondrial plasticity.

  13. Proteome analysis identified the PPARγ ligand 15d-PGJ2 as a novel drug inhibiting melanoma progression and interfering with tumor-stroma interaction.

    PubMed

    Paulitschke, Verena; Gruber, Silke; Hofstätter, Elisabeth; Haudek-Prinz, Verena; Klepeisz, Philipp; Schicher, Nikolaus; Jonak, Constanze; Petzelbauer, Peter; Pehamberger, Hubert; Gerner, Christopher; Kunstfeld, Rainer

    2012-01-01

    Peroxisome proliferator-activated receptors (PPARs) have been originally thought to be restricted to lipid metabolism or glucose homeostasis. Recently, evidence is growing that PPARγ ligands have inhibitory effects on tumor growth. To shed light on the potential therapeutic effects on melanoma we tested a panel of PPAR agonists on their ability to block tumor proliferation in vitro. Whereas ciglitazone, troglitazone and WY14643 showed moderate effects on proliferation, 15d-PGJ2 displayed profound anti-tumor activity on four different melanoma cell lines tested. Additionally, 15d-PGJ2 inhibited proliferation of tumor-associated fibroblasts and tube formation of endothelial cells. 15d-PGJ2 induced the tumor suppressor gene p21, a G(2)/M arrest and inhibited tumor cell migration. Shot gun proteome analysis in addition to 2D-gel electrophoresis and immunoprecipitation of A375 melanoma cells suggested that 15d-PGJ2 might exert its effects via modification and/or downregulation of Hsp-90 (heat shock protein 90) and several chaperones. Applying the recently established CPL/MUW database with a panel of defined classification signatures, we demonstrated a regulation of proteins involved in metastasis, transport or protein synthesis including paxillin, angio-associated migratory cell protein or matrix metalloproteinase-2 as confirmed by zymography. Our data revealed for the first time a profound effect of the single compound 15d-PGJ2 on melanoma cells in addition to the tumor-associated microenvironment suggesting synergistic therapeutic efficiency. PMID:23049949

  14. Proteome Analysis Identified the PPARγ Ligand 15d-PGJ2 as a Novel Drug Inhibiting Melanoma Progression and Interfering with Tumor-Stroma Interaction

    PubMed Central

    Paulitschke, Verena; Gruber, Silke; Hofstätter, Elisabeth; Haudek-Prinz, Verena; Klepeisz, Philipp; Schicher, Nikolaus; Jonak, Constanze; Petzelbauer, Peter; Pehamberger, Hubert; Gerner, Christopher; Kunstfeld, Rainer

    2012-01-01

    Peroxisome proliferator-activated receptors (PPARs) have been originally thought to be restricted to lipid metabolism or glucose homeostasis. Recently, evidence is growing that PPARγ ligands have inhibitory effects on tumor growth. To shed light on the potential therapeutic effects on melanoma we tested a panel of PPAR agonists on their ability to block tumor proliferation in vitro. Whereas ciglitazone, troglitazone and WY14643 showed moderate effects on proliferation, 15d-PGJ2 displayed profound anti-tumor activity on four different melanoma cell lines tested. Additionally, 15d-PGJ2 inhibited proliferation of tumor-associated fibroblasts and tube formation of endothelial cells. 15d-PGJ2 induced the tumor suppressor gene p21, a G2/M arrest and inhibited tumor cell migration. Shot gun proteome analysis in addition to 2D-gel electrophoresis and immunoprecipitation of A375 melanoma cells suggested that 15d-PGJ2 might exert its effects via modification and/or downregulation of Hsp-90 (heat shock protein 90) and several chaperones. Applying the recently established CPL/MUW database with a panel of defined classification signatures, we demonstrated a regulation of proteins involved in metastasis, transport or protein synthesis including paxillin, angio-associated migratory cell protein or matrix metalloproteinase-2 as confirmed by zymography. Our data revealed for the first time a profound effect of the single compound 15d-PGJ2 on melanoma cells in addition to the tumor-associated microenvironment suggesting synergistic therapeutic efficiency. PMID:23049949

  15. The low molecular weight fraction of human serum albumin upregulates production of 15d-PGJ2 in Peripheral Blood Mononuclear Cells.

    PubMed

    Thomas, Gregory W; Rael, Leonard T; Hausburg, Melissa; Frederick, Elizabeth D; Mains, Charles W; Slone, Denetta; Carrick, Matthew M; Bar-Or, David

    2016-05-13

    Activation of the innate immune system involves a series of events designed to counteract the initial insult followed by the clearance of debris and promotion of healing. Aberrant regulation can lead to systemic inflammatory response syndrome, multiple organ failure, and chronic inflammation. A better understanding of the innate immune response may help manage complications while allowing for proper immune progression. In this study, the ability of several classes of anti-inflammatory drugs to affect LPS-induced cytokine and prostaglandin release from peripheral blood mononuclear cells (PBMC) was evaluated. PBMC were cultured in the presence of dexamethasone (DEX), ibuprofen (IBU), and the low molecular weight fraction of 5% albumin (LMWF5A) followed by stimulation with LPS. After 24 h, TNFα, PGE2, and 15d-PGJ2 release was determined by ELISA. Distinct immunomodulation patterns emerged following LPS stimulation of PBMC in the presence of said compounds. DEX, a steroid with strong immunosuppressive properties, reduced TNFα, PGE2, and 15d-PGJ2 release. IBU caused significant reduction in prostaglandin release while TNFα release was unchanged. An emerging biologic with known anti-inflammatory properties, LMWF5A, significantly reduced TNFα release while enhancing PGE2 and 15d-PGJ2 release. Incubating LMWF5A together with IBU negated this observed increased prostaglandin release without affecting the suppression of TNFα release. Additionally, LMWF5A caused an increase in COX-2 transcription and translation. LMWF5A exhibited a unique immune modulation pattern in PBMC, disparate from steroid or NSAID administration. This enhancement of prostaglandin release (specifically 15d-PGJ2), in conjunction with a decrease in TNFα release, suggests a switch that favors resolution and decreased inflammation. PMID:27095392

  16. Total Synthesis of Prostaglandin 15d-PGJ(2) and Investigation of its Effect on the Secretion of IL-6 and IL-12.

    PubMed

    Egger, Julian; Fischer, Stefan; Bretscher, Peter; Freigang, Stefan; Kopf, Manfred; Carreira, Erick M

    2015-09-01

    An efficient synthesis of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2, 1) is reported. The route described allows for diversification of the parent structure to prepare seven analogues of 1 in which the positioning of electrophilic sites is varied. These analogues were tested in SAR studies for their ability to reduce the secretion of proinflammatory cytokines. It was shown that the endocyclic enone is crucial for the bioactivity investigated and that the conjugated ω-side chain serves in a reinforcing manner. PMID:26301727

  17. The role of aldo-keto reductase 1C3 (AKR1C3)-mediated prostaglandin D2 (PGD2) metabolism in keloids.

    PubMed

    Mantel, Alon; Newsome, Austin; Thekkudan, Theresa; Frazier, Robert; Katdare, Meena

    2016-01-01

    Keloids are progressively expanding scars, mostly prevalent in individuals of African descent. Previous data identified increased mast cell number and activation state in keloids suggesting a role in disease progression. The major eicosanoid secreted by mast cells is prostaglandin D2 (PGD2), a relatively unstable pro-inflammatory mediator which can be spontaneously converted to 15-deoxy-(Delta12,14)-prostaglandin J2(15d-PGJ2) or enzymatically metabolized to 9α,11β-PGF2 by aldo-keto reductase 1C3 (AKR1C3). In this work, we investigated the possible role of PGD2 and its metabolites in keloids using CRL1762 keloid fibroblasts (KF) and immunohistochemical staining. Our data suggested approximately 3-fold increase of tryptase-positive mast cell count in keloids compared with normal skin. Furthermore, AKR1C3 was overexpressed in the fibrotic area of keloids while relatively weak staining detected in normal skin. Metabolism of PGD2 to 9α,11β-PGF2 by both, KF and normal fibroblasts, was dependent on AKR1C3 as this reaction was attenuated in the presence of the AKR1C3 inhibitor, 2'-hydroxyflavanone, or in cells with decreased AKR1C3 expression. 15d-PGJ2, but not the other tested PGs, inhibited KF proliferation, attenuated KF-mediated collagen gel contraction and increased caspase-3 activation. In addition, treatment with 15d-PGJ2 activated P38-MAPK, induced reactive oxygen species and upregulated superoxide dismutase-1 (SOD-1). Finally, inhibition of P38-MAPK further augmented 15d-PGJ2-induced caspase-3 cleavage and attenuated its effect on SOD-1 transcription. This work suggests that localized dual inhibition of AKR1C3 and P38-MAPK may inhibit keloid progression. Inhibiting AKR1C3 activity may generate oxidative environment due to redirection of PGD2 metabolism towards 15d-PGJ2 while inhibition of P38-MAPK will sensitize keloid cells to ROS-induced apoptosis. PMID:26308156

  18. BEND3 mediates transcriptional repression and heterochromatin organization.

    PubMed

    Khan, Abid; Prasanth, Supriya G

    2015-01-01

    Transcription repression plays a central role in gene regulation. Transcription repressors utilize diverse strategies to mediate transcriptional repression. We have recently demonstrated that BEND3 (BANP, E5R and Nac1 domain) protein represses rDNA transcription by stabilizing a NoRC component. We discuss the role of BEND3 as a global regulator of gene expression and propose a model whereby BEND3 associates with chromatin remodeling complexes to modulate gene expression and heterochromatin organization. PMID:26507581

  19. Molecular mechanisms of COUP-TF-mediated transcriptional repression: evidence for transrepression and active repression.

    PubMed Central

    Leng, X; Cooney, A J; Tsai, S Y; Tsai, M J

    1996-01-01

    COUP-TF, an orphan member of the nuclear receptor superfamily, has been proposed to play a key role in regulating organogenesis, neurogenesis, and cellular differentiation during embryonic development. Since heterodimierization is a common theme within the nuclear receptor superfamily and has been demonstrated to modulate transcriptional properties of heterodimeric partners via allosteric interactions, we have devised a strategy to examine the silencing function of COUP-TF in a heterodimeric context. We find that the intrinsic active repression function of COUP-TF is not affected by heterodimerization. Moreover, COUP-TF can transrepress the ligand-dependent activation of its heterodimeric partners without its own DNA binding site. Using receptor deletion mutants in transfection assays, we show that the region necessary for COUP-TF silencing function is not sufficient for its transrepression activity. Furthermore, our studies indicate that in addition to its active repression function, COUP-TF can repress several different types of activator-dependent transactivation. However, this active repression function of COUP-TF may be differentially regulated by some other activator(s). These studies provide new insights into the molecular mechanism(s) of COUP-TF-mediated repression. PMID:8628300

  20. MicroRNA-mediated repression of nonsense mRNAs

    PubMed Central

    Zhao, Ya; Lin, Jimin; Xu, Beiying; Hu, Sida; Zhang, Xue; Wu, Ligang

    2014-01-01

    Numerous studies have established important roles for microRNAs (miRNAs) in regulating gene expression. Here, we report that miRNAs also serve as a surveillance system to repress the expression of nonsense mRNAs that may produce harmful truncated proteins. Upon recognition of the premature termination codon by the translating ribosome, the downstream portion of the coding region of an mRNA is redefined as part of the 3′ untranslated region; as a result, the miRNA-responsive elements embedded in this region can be detected by miRNAs, triggering accelerated mRNA deadenylation and translational inhibition. We demonstrate that naturally occurring cancer-causing APC (adenomatous polyposis coli) nonsense mutants which escape nonsense-mediated mRNA decay (NMD) are repressed by miRNA-mediated surveillance. In addition, we show that miRNA-mediated surveillance and exon–exon junction complex-mediated NMD are not mutually exclusive and act additively to enhance the repressive activity. Therefore, we have uncovered a new role for miRNAs in repressing nonsense mutant mRNAs. DOI: http://dx.doi.org/10.7554/eLife.03032.001 PMID:25107276

  1. SMRT isoforms mediate repression and anti-repression of nuclear receptor heterodimers.

    PubMed Central

    Chen, J D; Umesono, K; Evans, R M

    1996-01-01

    Transcriptional repression represents an important component in the regulation of cell differentiation and oncogenesis mediated by nuclear hormone receptors. Hormones act to relieve repression, thus allowing receptors to function as transcriptional activators. The transcriptional corepressor SMRT was identified as a silencing mediator for retinoid and thyroid hormone receptors. SMRT is highly related to another corepressor, N-CoR, suggesting the existence of a new family of receptor-interacting proteins. We demonstrate that SMRT is a ubiquitous nuclear protein that interacts with unliganded receptor heterodimers in mammalian cells. Furthermore, expression of the receptor-interacting domain of SMRT acts as an antirepressor, suggesting the potential importance of splicing variants as modulators of thyroid hormone and retinoic acid signaling. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:8755515

  2. Modes of TAL effector-mediated repression

    PubMed Central

    Werner, Jeannette; Gossen, Manfred

    2014-01-01

    Engineered transcription activator-like effectors, or TALEs, have emerged as a new class of designer DNA-binding proteins. Their DNA recognition sites can be specified with great flexibility. When fused to appropriate transcriptional regulatory domains, they can serve as designer transcription factors, modulating the activity of targeted promoters. We created tet operator (tetO)-specific TALEs (tetTALEs), with an identical DNA-binding site as the Tet repressor (TetR) and the TetR-based transcription factors that are extensively used in eukaryotic transcriptional control systems. Different constellations of tetTALEs and tetO modified chromosomal transcription units were analyzed for their efficacy in mammalian cells. We find that tetTALE-silencers can entirely abrogate expression from the strong human EF1α promoter when binding upstream of the transcriptional control sequence. Remarkably, the DNA-binding domain of tetTALE alone can effectively counteract trans-activation mediated by the potent tettrans-activator and also directly interfere with RNA polymerase II transcription initiation from the strong CMV promoter. Our results demonstrate that TALEs can act as highly versatile tools in genetic engineering, serving as trans-activators, trans-silencers and also competitive repressors. PMID:25389273

  3. 15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits Homing of Bone Marrow-Derived Mesenchymal Stem Cells Triggered by Chronic Liver Injury via Redox Pathway

    PubMed Central

    Liu, Xin; Jia, Shuangshuang; Li, Weiyang; Yang, Le; Yang, Lin; Wang, Lin; Li, Liying

    2015-01-01

    It has been reported that bone marrow-derived mesenchymal stem cells (BMSCs) have capacity to migrate to the damaged liver and contribute to fibrogenesis in chronic liver diseases. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand for peroxisome proliferator-activated receptor gamma (PPARγ), is considered a new inhibitor of cell migration. However, the actions of 15d-PGJ2 on BMSC migration remain unknown. In this study, we investigated the effects of 15d-PGJ2 on the migration of BMSCs using a mouse model of chronic liver fibrosis and primary mouse BMSCs. Our results demonstrated that in vivo, 15d-PGJ2 administration inhibited the homing of BMSCs to injured liver by flow cytometric analysis and, in vitro, 15d-PGJ2 suppressed primary BMSC migration in a dose-dependent manner determined by Boyden chamber assay. Furthermore, the repressive effect of 15d-PGJ2 was blocked by reactive oxygen species (ROS) inhibitor, but not PPARγ antagonist, and action of 15d-PGJ2 was not reproduced by PPARγ synthetic ligands. In addition, 15d-PGJ2 triggered a significant ROS production and cytoskeletal remodeling in BMSCs. In conclusion, our results suggest that 15d-PGJ2 plays a crucial role in homing of BMSCs to the injured liver dependent on ROS production, independently of PPARγ, which may represent a new strategy in the treatment of liver fibrosis. PMID:26457076

  4. 15-Deoxy-delta 12,14-prostaglandin J2 biphasically regulates the proliferation of mouse hippocampal neural progenitor cells by modulating the redox state.

    PubMed

    Katura, Takashi; Moriya, Takahiro; Nakahata, Norimichi

    2010-04-01

    The activity of neural progenitor cells (NPCs) is regulated by various humoral factors. Although prostaglandin (PG) D(2) is known to mediate various physiological brain functions such as sleep, its actions on NPCs have not been fully understood. In the process of investigating the effects of PGD(2) on NPCs, we found that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), an endogenous metabolite of PGD(2), exhibits a novel regulation of the proliferation of NPCs derived from mouse hippocampus. 15d-PGJ(2) showed biphasic effects on epidermal growth factor-induced proliferation of NPCs; facilitation at low concentrations ( approximately 0.3 muM) and suppression at higher concentrations (0.5-10 microM) in vitro. 2-Chloro-5-nitrobenzanilide (GW9662), an inhibitor of peroxisome proliferator-activated receptor gamma, known to be a molecular target for 15d-PGJ(2), failed to abolish the effects of 15d-PGJ(2). 9,10-dihydro-15d-PGJ(2) (CAY10410), a structural analog of 15d-PGJ(2) lacking the electrophilic carbon in the cyclopentenone ring, did not show 15d-PGJ(2)-like actions. Treatment with 15d-PGJ(2) increased the levels of reactive oxygen species and decreased endogenous GSH levels. Furthermore, supplementation with a membrane-permeable analog of glutathione, GSH ethyl ester (2 mM), diminished the biphasic effects of 15d-PGJ(2). Finally, cell division in the dentate gyrus of postnatal mice was increased by injection of low-dose (1 ng i.c.v.) 15d-PGJ(2) and suppressed by high-dose (30 ng) 15d-PGJ(2). These results suggest that 15d-PGJ(2) regulates the proliferation of NPCs via its electrophilic nature, which enables covalent binding to molecules such as GSH. PMID:20086036

  5. Resveratrol-Mediated Repression and Reversion of Prostatic Myofibroblast Phenoconversion

    PubMed Central

    Gharaee-Kermani, Mehrnaz; Moore, Bethany B.; Macoska, Jill A.

    2016-01-01

    Background Resveratrol, a phytoalexin found in berries, peanuts, grapes, and red wine, inhibits oxidation, inflammation, and cell proliferation and collagen synthesis in multiple cell types and or animal models. It represses collagen deposition in the vasculature, heart, lung, kidney, liver, and esophagus in animal models and may have some utility as an anti-fibrotic. Recent studies have shown that increased collagen deposition and tissue stiffness in the peri-urethral area of the prostate are associated with lower urinary tract dysfunction (LUTD) and urinary obstructive symptoms. The aim of this study was to determine whether Resveratrol might be useful to inhibit or revert TGFβ- and/or CXCL12-mediated myofibroblast phenoconversion of prostate fibroblasts in vitro, and therefore whether the use of anti-fibrotic therapeutics might be efficacious for the treatment of LUTD. Methods Primary prostate and lung tissues were explanted and fibroblast monolayers expanded in vitro. Primary and N1 immortalized prostate stromal fibroblasts, as well as primary fibroblasts cultured from a normal lung and one affected by idiopathic pulmonary fibrosis (IPF) for comparison, were grown in serum–free defined media supplemented with vehicle, TGFβ or CXCL12, pre- or post-treatment with Resveratrol, and were evaluated using immunofluorescence for alpha smooth muscle actin (αSMA) and collagen I (COL1) protein expression and assessed for cell proliferation, apoptosis, and COL1 and EGR1 transcript expression. Results This study showed that low concentrations of Resveratrol (≤50 μM) had no effect on N1 or primary prostate fibroblast cell proliferation, apoptosis, or COL1 or EGR1 gene transcription but repressed and reversed myofibroblast phenoconversion. As expected, these same effects were observed for IPF lung fibroblasts though higher levels of Resveratrol (≥100uM) were required. Taken together, these data suggest that, like lung fibroblasts, prostate fibroblast to

  6. BRCA1-mediated repression of select X chromosome genes.

    PubMed

    Jazaeri, Amir A; Chandramouli, Gadisetti VR; Aprelikova, Olga; Nuber, Ulrike A; Sotiriou, Christos; Liu, Edison T; Ropers, H Hilger; Yee, Cindy J; Boyd, Jeff; Barrett, J Carl

    2004-09-21

    Recently BRCA1 has been implicated in the regulation of gene expression from the X chromosome. In this study the influence of BRCA1 on expression of X chromosome genes was investigated. Complementary DNA microarrays were used to compare the expression levels of X chromosome genes in 18 BRCA1-associated ovarian cancers to those of the 13 "BRCA1-like" and 14 "BRCA2-like" sporadic tumors (as defined by previously reported expression profiling). Significance was determined using parametric statistics with P < 0.005 as a cutoff. Forty of 178 total X-chromosome transcripts were differentially expressed between the BRCA1-associated tumors and sporadic cancers with a BRCA2-like molecular profile. Thirty of these 40 genes showed higher mean expression in the BRCA1-associated samples including all 11 transcripts that mapped to Xp11. In contrast, four of 178 total X chromosome transcripts showed significant differential expression between BRCA1-associated and sporadic tumors with a BRCA1-like molecular profile. All four mapped to Xp11 and showed higher mean expression in BRCA1-associated tumors. Re-expression of BRCA1 in HCC1937 BRCA1-deficient breast cancer cell resulted in the repression of 21 transcripts. Eleven of the 21 (54.5%) transcripts mapped to Xp11. However, there was no significant overlap between these Xp11 genes and those found to be differentially expressed between BRCA1-associated and sporadic ovarian cancer samples. These results demonstrate that BRCA1 mediates the repression of several X chromosome genes, many of which map to the Xp11 locus. PMID:15383145

  7. HES-Mediated Repression of Pten in Caenorhabditis elegans

    PubMed Central

    Chou, Han Ting; Vazquez, Raymarie Gomez; Wang, Kun; Campbell, Richard; Milledge, Gaolin Zheng; Walthall, Walter W.; Johnson, Casonya M.

    2015-01-01

    The hairy/enhancer-of-split (HES) group of transcription factors controls embryonic development, often by acting downstream of the Notch signaling pathway; however, little is known about postembryonic roles of these proteins. In Caenorhabditis elegans, the six proteins that make up the REF-1 family are considered to be HES orthologs that act in both Notch-dependent and Notch-independent pathways to regulate embryonic events. To further our understanding of how the REF-1 family works to coordinate postembryonic cellular events, we performed a functional characterization of the REF-1 family member, HLH-25. We show that, after embryogenesis, hlh-25 expression persists throughout every developmental stage, including dauer, into adulthood. Like animals that carry loss-of-function alleles in genes required for normal cell-cycle progression, the phenotypes of hlh-25 animals include reduced brood size, unfertilized oocytes, and abnormal gonad morphology. Using gene expression microarray, we show that the HLH-25 transcriptional network correlates with the phenotypes of hlh-25 animals and that the C. elegans Pten ortholog, daf-18, is one major hub in the network. Finally, we show that HLH-25 regulates C. elegans lifespan and dauer recovery, which correlates with a role in the transcriptional repression of daf-18 activity. Collectively, these data provide the first genetic evidence that HLH-25 may be a functional ortholog of mammalian HES1, which represses PTEN activity in mice and human cells. PMID:26438299

  8. The MOX promoter in Hansenula polymorpha is ultrasensitive to glucose-mediated carbon catabolite repression.

    PubMed

    Dusny, Christian; Schmid, Andreas

    2016-09-01

    Redesigning biology towards specific purposes requires a functional understanding of genetic circuits. We present a quantitative in-depth study on the regulation of the methanol-specific MOX promoter system (PMOX) at the single-cell level. We investigated PMOX regulation in the methylotrophic yeast Hansenula (Ogataea) polymorpha with respect to glucose-mediated carbon catabolite repression. This promoter system is particularly delicate as the glucose as carbon and energy source in turn represses MOX promoter activity. Decoupling single cells from population activity revealed a hitherto underrated ultrasensitivity of the MOX promoter to glucose repression. Environmental control with single-cell technologies enabled quantitative insights into the balance between activation and repression of PMOX with respect to extracellular glucose concentrations. While population-based studies suggested full MOX promoter derepression at extracellular glucose concentrations of ∼1 g L(-1), we showed that glucose-mediated catabolite repression already occurs at concentrations as low as 5 × 10(-4) g L(-1) These findings demonstrate the importance of uncoupling single cells from populations for understanding the mechanisms of promoter regulation in a quantitative manner. PMID:27527102

  9. Hairy and Groucho mediate the action of juvenile hormone receptor Methoprene-tolerant in gene repression.

    PubMed

    Saha, Tusar T; Shin, Sang Woon; Dou, Wei; Roy, Sourav; Zhao, Bo; Hou, Yuan; Wang, Xue-Li; Zou, Zhen; Girke, Thomas; Raikhel, Alexander S

    2016-02-01

    The arthropod-specific juvenile hormone (JH) controls numerous essential functions. Its involvement in gene activation is known to be mediated by the transcription factor Methoprene-tolerant (Met), which turns on JH-controlled genes by directly binding to E-box-like motifs in their regulatory regions. However, it remains unclear how JH represses genes. We used the Aedes aegypti female mosquito, in which JH is necessary for reproductive maturation, to show that a repressor, Hairy, is required for the gene-repressive action of JH and Met. The RNA interference (RNAi) screen for Met and Hairy in the Aedes female fat body revealed a large cohort of Met- and Hairy-corepressed genes. Analysis of selected genes from this cohort demonstrated that they are repressed by JH, but RNAi of either Met or Hairy renders JH ineffective in repressing these genes in an in vitro fat-body culture assay. Moreover, this JH action was prevented by the addition of the translational inhibitor cycloheximide (CHX) to the culture, indicating the existence of an indirect regulatory hierarchy. The lack of Hairy protein in the CHX-treated tissue was verified using immunoblot analysis, and the upstream regions of Met/Hairy-corepressed genes were shown to contain common binding motifs that interact with Hairy. Groucho (gro) RNAi silencing phenocopied the effect of Hairy RNAi knockdown, indicating that it is involved in the JH/Met/Hairy hierarchy. Finally, the requirement of Hairy and Gro for gene repression was confirmed in a cell transfection assay. Thus, our study has established that Hairy and its cofactor Gro mediate the repressive function of JH and Met. PMID:26744312

  10. Hairy and Groucho mediate the action of juvenile hormone receptor Methoprene-tolerant in gene repression

    PubMed Central

    Saha, Tusar T.; Shin, Sang Woon; Dou, Wei; Roy, Sourav; Zhao, Bo; Hou, Yuan; Wang, Xue-Li; Zou, Zhen; Girke, Thomas; Raikhel, Alexander S.

    2016-01-01

    The arthropod-specific juvenile hormone (JH) controls numerous essential functions. Its involvement in gene activation is known to be mediated by the transcription factor Methoprene-tolerant (Met), which turns on JH-controlled genes by directly binding to E-box–like motifs in their regulatory regions. However, it remains unclear how JH represses genes. We used the Aedes aegypti female mosquito, in which JH is necessary for reproductive maturation, to show that a repressor, Hairy, is required for the gene-repressive action of JH and Met. The RNA interference (RNAi) screen for Met and Hairy in the Aedes female fat body revealed a large cohort of Met- and Hairy-corepressed genes. Analysis of selected genes from this cohort demonstrated that they are repressed by JH, but RNAi of either Met or Hairy renders JH ineffective in repressing these genes in an in vitro fat-body culture assay. Moreover, this JH action was prevented by the addition of the translational inhibitor cycloheximide (CHX) to the culture, indicating the existence of an indirect regulatory hierarchy. The lack of Hairy protein in the CHX-treated tissue was verified using immunoblot analysis, and the upstream regions of Met/Hairy-corepressed genes were shown to contain common binding motifs that interact with Hairy. Groucho (gro) RNAi silencing phenocopied the effect of Hairy RNAi knockdown, indicating that it is involved in the JH/Met/Hairy hierarchy. Finally, the requirement of Hairy and Gro for gene repression was confirmed in a cell transfection assay. Thus, our study has established that Hairy and its cofactor Gro mediate the repressive function of JH and Met. PMID:26744312

  11. FOG-1 recruits the NuRD repressor complex to mediate transcriptional repression by GATA-1

    PubMed Central

    Hong, Wei; Nakazawa, Minako; Chen, Ying-Yu; Kori, Rajashree; Vakoc, Christopher R; Rakowski, Carrie; Blobel, Gerd A

    2005-01-01

    Transcription factor GATA-1 and its cofactor FOG-1 coordinate erythroid cell maturation by activating erythroid-specific genes and repressing genes associated with the undifferentiated state. Here we show that FOG-1 binds to the NuRD corepressor complex in vitro and in vivo. The interaction is mediated by a small conserved domain at the extreme N-terminus of FOG-1 that is necessary and sufficient for NuRD binding. This domain defines a novel repression module found in diverse transcriptional repressors. NuRD is present at GATA-1/FOG-1-repressed genes in erythroid cells in vivo. Point mutations near the N-terminus of FOG-1 that abrogate NuRD binding block gene repression by FOG-1. Finally, the ability of GATA-1 to repress transcription was impaired in erythroid cells expressing mutant forms of FOG-1 that are defective for NuRD binding. Together, these studies show that FOG-1 and likely other FOG-like proteins are corepressors that link GATA factors to histone deacetylation and nucleosome remodeling. PMID:15920470

  12. Helix-loop-helix transcription factors mediate activation and repression of the p75LNGFR gene.

    PubMed Central

    Chiaramello, A; Neuman, K; Palm, K; Metsis, M; Neuman, T

    1995-01-01

    Sequence analysis of rat and human low-affinity nerve growth factor receptor p75LNGFR gene promoter regions revealed a single E-box cis-acting element, located upstream of the major transcription start sites. Deletion analysis of the E-box sequence demonstrated that it significantly contributes to p75LNGFR promoter activity. This E box has a dual function; it mediates either activation or repression of the p75LNGFR promoter activity, depending on the interacting transcription factors. We showed that the two isoforms of the class A basic helix-loop-helix (bHLH) transcription factor ME1 (ME1a and ME1b), the murine homolog of the human HEB transcription factor, specifically repress p75LNGFR promoter activity. This repression can be released by coexpression of the HLH Id2 transcriptional regulator. In vitro analyses demonstrated that ME1a forms a stable complex with the p75LNGFR E box and likely competes with activating E-box-binding proteins. By using ME1a-overexpressing PC12 cells, we showed that the endogenous p75LNGFR gene is a target of ME1a repression. Together, these data demonstrate that the p75LNGFR E box and the interacting bHLH transcription factors are involved in the regulation of p75LNGFR gene expression. These results also show that class A bHLH transcription factors can repress and Id-like negative regulators can stimulate gene expression. PMID:7565756

  13. Wnt-mediated repression via bipartite DNA recognition by TCF in the Drosophila hematopoietic system.

    PubMed

    Zhang, Chen U; Blauwkamp, Timothy A; Burby, Peter E; Cadigan, Ken M

    2014-08-01

    The Wnt/β-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previously, we demonstrated that for activated targets in Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. Here, we report that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/β-catenin signaling controls prohemocytic differentiation. Our study provides a paradigm for direct repression of target gene expression by Wnt/β-catenin signaling and allosteric regulation of a transcription factor by DNA. PMID:25144371

  14. Wnt-Mediated Repression via Bipartite DNA Recognition by TCF in the Drosophila Hematopoietic System

    PubMed Central

    Zhang, Chen U.; Blauwkamp, Timothy A.; Burby, Peter E.; Cadigan, Ken M.

    2014-01-01

    The Wnt/β-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previously, we demonstrated that for activated targets in Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. Here, we report that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/β-catenin signaling controls prohemocytic differentiation. Our study provides a paradigm for direct repression of target gene expression by Wnt/β-catenin signaling and allosteric regulation of a transcription factor by DNA. PMID:25144371

  15. Histone methyltransferase Ash1L mediates activity-dependent repression of neurexin-1α

    PubMed Central

    Zhu, Τao; Liang, Chen; Li, Dongdong; Tian, Miaomiao; Liu, Sanxiong; Gao, Guanjun; Guan, Ji-Song

    2016-01-01

    Activity-dependent transcription is critical for the regulation of long-term synaptic plasticity and plastic rewiring in the brain. Here, we report that the transcription of neurexin1α (nrxn1α), a presynaptic adhesion molecule for synaptic formation, is regulated by transient neuronal activation. We showed that 10 minutes of firing at 50 Hz in neurons repressed the expression of nrxn1α for 24 hours in a primary cortical neuron culture through a transcriptional repression mechanism. By performing a screening assay using a synthetic zinc finger protein (ZFP) to pull down the proteins enriched near the nrxn1α promoter region in vivo, we identified that Ash1L, a histone methyltransferase, is enriched in the nrxn1α promoter. Neuronal activity triggered binding of Ash1L to the promoter and enriched the histone marker H3K36me2 at the nrxn1α promoter region. Knockout of Ash1L in mice completely abolished the activity-dependent repression of nrxn1α. Taken together, our results reveal that a novel process of activity-dependent transcriptional repression exists in neurons and that Ash1L mediates the long-term repression of nrxn1α, thus implicating an important role for epigenetic modification in brain functioning. PMID:27229316

  16. Reduced expression of ribosomal proteins relieves microRNA-mediated repression.

    PubMed

    Janas, Maja M; Wang, Eric; Love, Tara; Harris, Abigail S; Stevenson, Kristen; Semmelmann, Karlheinz; Shaffer, Jonathan M; Chen, Po-Hao; Doench, John G; Yerramilli, Subrahmanyam V B K; Neuberg, Donna S; Iliopoulos, Dimitrios; Housman, David E; Burge, Christopher B; Novina, Carl D

    2012-04-27

    MicroRNAs (miRNAs) regulate physiological and pathological processes by inducing posttranscriptional repression of target messenger RNAs (mRNAs) via incompletely understood mechanisms. To discover factors required for human miRNA activity, we performed an RNAi screen using a reporter cell line of miRNA-mediated repression of translation initiation. We report that reduced expression of ribosomal protein genes (RPGs) dissociated miRNA complexes from target mRNAs, leading to increased polysome association, translation, and stability of miRNA-targeted mRNAs relative to untargeted mRNAs. RNA sequencing of polysomes indicated substantial overlap in sets of genes exhibiting increased or decreased polysomal association after Argonaute or RPG knockdowns, suggesting similarity in affected pathways. miRNA profiling of monosomes and polysomes demonstrated that miRNAs cosediment with ribosomes. RPG knockdowns decreased miRNAs in monosomes and increased their target mRNAs in polysomes. Our data show that most miRNAs repress translation and that the levels of RPGs modulate miRNA-mediated repression of translation initiation. PMID:22541556

  17. Biosynthesis of 15-deoxy-delta12,14-PGJ2 and the ligation of PPARgamma.

    PubMed

    Bell-Parikh, L Chastine; Ide, Tomomi; Lawson, John A; McNamara, Peter; Reilly, Muredach; FitzGerald, Garret A

    2003-09-01

    15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) has been identified as an endogenous ligand for PPARgamma, inducing adipogenesis in vitro. Additional roles for this molecule in the propagation and resolution of inflammation, ligation of NF-kappaB, and mediation of apoptosis have been proposed. However, quantitative, physiochemical evidence for the formation of 15d-PGJ2 in vivo is lacking. We report that 15d-PGJ2 is detectable using liquid chromatography-mass spectrometry-mass spectrometry at low picomolar concentrations in the medium of 3T3-L1 preadipocytes. However, despite induction of COX-2, production of PGs, including 15d-PGJ2, does not increase during adipocyte differentiation, a process unaltered by COX inhibition. 15d-PGJ2 is detectable as a minor product of COX-2 in human urine. However, its biosynthesis is unaltered during or after COX activation in vivo by LPS. Furthermore, the biosynthesis of 15d-PGJ2 is not augmented in the joint fluid of patients with arthritis, nor is its urinary excretion increased in patients with diabetes or obesity. 15d-PGJ2 is not the endogenous mediator of PPARgamma-dependent adipocyte activation and is unaltered in clinical settings in which PPARgamma activation has been implicated. PMID:12975479

  18. FLC-mediated flowering repression is positively regulated by sumoylation

    PubMed Central

    Seo, Hak Soo

    2014-01-01

    Flowering locus C (FLC), a floral repressor, is a critical factor for the transition from the vegetative to the reproductive phase. Here, the mechanisms regulating the activity and stability of the FLC protein were investigated. Bimolecular fluorescence complementation and in vitro pull-down analyses showed that FLC interacts with the E3 small ubiquitin-like modifier (SUMO) ligase AtSIZ1, suggesting that AtSIZ1 is an E3 SUMO ligase for FLC. In vitro sumoylation assays showed that FLC is modified by SUMO in the presence of SUMO-activating enzyme E1 and conjugating enzyme E2, but its sumoylation is inhibited by AtSIZ1. In transgenic plants, inducible AtSIZ1 overexpression led to an increase in the concentration of FLC and delayed the post-translational decay of FLC, indicating that AtSIZ1 stabilizes FLC through direct binding. Also, the flowering time in mutant FLC (K154R, a mutation of the sumoylation site)-overexpressing plants was comparable with that in the wild type, whereas flowering was considerably delayed in FLC-overexpressing plants, supporting the notion that sumoylation is an important mechanism for FLC function. The data indicate that the sumoylation of FLC is critical for its role in the control of flowering time and that AtSIZ1 positively regulates FLC-mediated floral suppression. PMID:24218331

  19. Inhibition of tumor cell growth by Sigma1 ligand mediated translational repression

    SciTech Connect

    Kim, Felix J.; Schrock, Joel M.; Spino, Christina M.; Marino, Jacqueline C.; Pasternak, Gavril W.

    2012-09-21

    Highlights: Black-Right-Pointing-Pointer Sigma1 ligand treatment mediates decrease in tumor cell mass. Black-Right-Pointing-Pointer Identification of a Sigma1 ligand with reversible translational repressor actions. Black-Right-Pointing-Pointer Demonstration of a role for Sigma1 in cellular protein synthesis. -- Abstract: Treatment with sigma1 receptor (Sigma1) ligands can inhibit cell proliferation in vitro and tumor growth in vivo. However, the cellular pathways engaged in response to Sigma1 ligand treatment that contribute to these outcomes remain largely undefined. Here, we show that treatment with putative antagonists of Sigma1 decreases cell mass. This effect corresponds with repressed cap-dependent translation initiation in multiple breast and prostate cancer cell lines. Sigma1 antagonist treatment suppresses phosphorylation of translational regulator proteins p70S6K, S6, and 4E-BP1. RNAi-mediated knockdown of Sigma1 also results in translational repression, consistent with the effects of antagonist treatment. Sigma1 antagonist mediated translational repression and decreased cell size are both reversible. Together, these data reveal a role for Sigma1 in tumor cell protein synthesis, and demonstrate that small molecule Sigma1 ligands can be used as modulators of protein translation.

  20. Insights into GATA-1 Mediated Gene Activation versus Repression via Genome-wide Chromatin Occupancy Analysis

    PubMed Central

    Yu, Ming; Riva, Laura; Xie, Huafeng; Schindler, Yocheved; Moran, Tyler B.; Cheng, Yong; Yu, Duonan; Hardison, Ross; Weiss, Mitchell J; Orkin, Stuart H.; Bernstein, Bradley E.; Fraenkel, Ernest; Cantor, Alan B.

    2009-01-01

    Summary The transcription factor GATA-1 is required for terminal erythroid maturation and functions as an activator or repressor depending on gene context. Yet its in vivo site selectivity and ability to distinguish between activated versus repressed genes remain incompletely understood. In this study, we performed GATA-1 ChIP-seq in erythroid cells and compared it to GATA-1 induced gene expression changes. Bound and differentially expressed genes contain a greater number of GATA binding motifs, a higher frequency of palindromic GATA sites, and closer occupancy to the transcriptional start site versus non-differentially expressed genes. Moreover, we show that the transcription factor Zbtb7a occupies GATA-1 bound regions of some direct GATA-1 target genes, that the presence of SCL/TAL1 helps distinguish transcriptional activation versus repression, and that Polycomb Repressive Complex 2 (PRC2) is involved in epigenetic silencing of a subset of GATA-1 repressed genes. These data provide insights into GATA-1 mediated gene regulation in vivo. PMID:19941827

  1. Probes of chromatin accessibility in the Drosophila bithorax complex respond differently to Polycomb-mediated repression.

    PubMed Central

    McCall, K; Bender, W

    1996-01-01

    The Polycomb group (PcG) of genes are required for maintenance of the repressed state of the homeotic genes in Drosophila. There are similarities between the PcG repression and mating-type silencing in yeast or heterochromatic position effect in Drosophila, which suggest that PcG repression may involve a highly compacted chromatin structure. To test for such a structure, heterologous DNA- binding proteins were used as probes for DNA accessibility in Drosophila embryos. Binding sites for the yeast transcriptional activator GAL4 and for bacteriophage T7 RNA polymerase were inserted into the bithorax (bx) regulatory region of the endogenous Ultrabithorax (Ubx) gene, which is regulated by the PcG. Ubiquitously expressed GAL4 protein directs transcription through its binding sites only in the posterior segments where the bx region is active. The block to GAL4 activation in the more anterior segments is dependent on Polycomb (Pc) function. In contrast, T7 RNA polymerase can transcribe from its target promoter in all segments of the embryo. Thus, Pc-mediated repression blocks activated polymerase II transcription, but does not simply exclude all proteins. Images PMID:8599940

  2. Catabolite repression in Lactobacillus casei ATCC 393 is mediated by CcpA.

    PubMed Central

    Monedero, V; Gosalbes, M J; Pérez-Martínez, G

    1997-01-01

    The chromosomal ccpA gene from Lactobacillus casei ATCC 393 has been cloned and sequenced. It encodes the CcpA protein, a central catabolite regulator belonging to the LacI-GalR family of bacterial repressors, and shows 54% identity with CcpA proteins from Bacillus subtilis and Bacillus megaterium. The L. casei ccpA gene was able to complement a B. subtilis ccpA mutant. An L. casei ccpA mutant showed increased doubling times and a relief of the catabolite repression of some enzymatic activities, such as N-acetylglucosaminidase and phospho-beta-galactosidase. Detailed analysis of CcpA activity was performed by using the promoter region of the L. casei chromosomal lacTEGF operon which is subject to catabolite repression and contains a catabolite responsive element (cre) consensus sequence. Deletion of this cre site or the presence of the ccpA mutation abolished the catabolite repression of a lacp::gusA fusion. These data support the role of CcpA as a common regulatory element mediating catabolite repression in low-GC-content gram-positive bacteria. PMID:9352913

  3. 15-Deoxy-Δ12,14-prostaglandin J2 Induces Apoptosis and Upregulates SOCS3 in Human Thyroid Cancer Cells

    PubMed Central

    Trindade-da-Silva, Carlos Antônio; Reis, Carolina Fernandes; Vecchi, Lara; Napimoga, Marcelo Henrique; Sperandio, Marcelo; Matias Colombo, Bruna França; Alves, Patrícia Terra; Ward, Laura Sterian; Ueira-Vieira, Carlos; Goulart, Luiz Ricardo

    2016-01-01

    The cyclopentenone prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a natural ligand of peroxisome proliferator-activated receptor gamma (PPAR-γ) and a potential mediator of apoptosis in cancer cells. In the present study, we evaluated the effect of 15d-PGJ2 in human thyroid papillary carcinoma cells (TPC-1) using different doses of 15d-PGJ2 (0.6 to 20 μM) to determine IC50 (9.3 μM) via the MTT assay. The supernatant culture medium of the TPC-1 cells that was treated either with 15d-PGJ2 or with vehicle (control) for 24 hours was assessed for IL-6 secretion via CBA assay. RT-qPCR was used to evaluate mRNA expression of IL-6, SOCS1, SOCS3, and STAT3. TPC-1 cells treated with 15d-PGJ2 decreased the secretion and expression of IL-6 and STAT3, while it increased SOCS1 and SOCS3. Overall, we demonstrated that 15d-PGJ2 downregulated IL-6 signaling pathway and led TPC-1 cells into apoptosis. In conclusion, 15d-PGJ2 shows the potential to become a new therapeutic approach for thyroid tumors. PMID:27190500

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

    SciTech Connect

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

    2014-02-07

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

  5. Hypoxia-derived oxidative stress mediates epigenetic repression of PKCɛ gene in foetal rat hearts

    PubMed Central

    Patterson, Andrew J.; Xiao, Daliao; Xiong, Fuxia; Dixon, Brandon; Zhang, Lubo

    2012-01-01

    Aims Hypoxia causes protein kinase C epsilon (PKCɛ) gene repression in foetal hearts, resulting in heightened cardiac susceptibility to ischaemic injury in offspring. We tested the hypothesis that hypoxia inducible factor 1 (HIF-1) and/or reactive oxygen species (ROS) mediate hypoxia-induced PKCɛ gene repression. Methods and results Hypoxia induced in vivo to pregnant rats, ex vivo to isolated foetal rat hearts, and in vitro in the rat embryonic ventricular myocyte cell line H9c2 resulted in a comparable decrease in PKCɛ protein and mRNA abundance in foetal hearts and H9c2 cells, which was associated with a significant increase in CpG methylation of the SP1-binding sites at the PKCɛ promoter. In H9c2 cells and foetal hearts, hypoxia caused nuclear accumulation of HIF-1α, which was inhibited by 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole and 2-methoxy estradiol. The HIF-1α inhibitors had no significant effect on hypoxia-induced PKCɛ mRNA repression. Hypoxia produced a time-dependent increase in ROS production in H9c2 cells and foetal hearts that was blocked by ROS scavengers N-acetyl-cysteine or tempol. In accordance, N-acetyl-cysteine and tempol, but not apocynin, inhibited the hypoxic effect and restored PKCɛ protein and mRNA expression to the control values in foetal hearts and H9c2 cells. The ROS scavengers blocked hypoxia-induced CpG methylation of the SP1-binding sites, restored SP1 binding to the PKCɛ promoter, and abrogated the hypoxia-induced increase in the susceptibility of the heart to ischaemic injury in offspring. Conclusions The results demonstrate that hypoxia induces epigenetic repression of the PKCɛ gene through a NADPH oxidase-independent ROS-mediated pathway in the foetal heart, leading to heightened heart vulnerability to ischaemic injury in offspring. PMID:22139554

  6. Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

    PubMed Central

    Wang, Kai; Sengupta, Satyaki; Magnani, Luca; Wilson, Catherine A.; Henry, R. William; Knott, Jason G.

    2010-01-01

    During blastocyst formation the segregation of the inner cell mass (ICM) and trophectoderm is governed by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2. Evidence indicates that suppression of Oct4 expression in the trophectoderm is mediated by Cdx2. Nonetheless, the underlying epigenetic modifiers required for Cdx2-dependent repression of Oct4 are largely unknown. Here we show that the chromatin remodeling protein Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. By employing a combination of RNA interference (RNAi) and gene expression analysis we found that both Brg1 Knockdown (KD) and Cdx2 KD blastocysts exhibit widespread expression of Oct4 in the trophectoderm. Interestingly, in Brg1 KD blastocysts and Cdx2 KD blastocysts, the expression of Cdx2 and Brg1 is unchanged, respectively. To address whether Brg1 cooperates with Cdx2 to repress Oct4 transcription in the developing trophectoderm, we utilized preimplantation embryos, trophoblast stem (TS) cells and Cdx2-inducible embryonic stem (ES) cells as model systems. We found that: (1) combined knockdown (KD) of Brg1 and Cdx2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of Brg1 or Cdx2 alone, (2) endogenous Brg1 co-immunoprecipitated with Cdx2 in TS cell extracts, (3) in blastocysts Brg1 and Cdx2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells Brg1 and Cdx2 are recruited to the Oct4 promoter. Lastly, to determine how Brg1 may induce epigenetic silencing of the Oct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of Brg1 KD blastocysts. This analysis revealed that Brg1-dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. In toto, these results demonstrate that Brg1 cooperates with Cdx2 to repress Oct4 expression in the developing trophectoderm to ensure normal development. PMID:20485553

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

    SciTech Connect

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

    2011-01-07

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

  8. Evolution of VRN2/Ghd7-Like Genes in Vernalization-Mediated Repression of Grass Flowering.

    PubMed

    Woods, Daniel P; McKeown, Meghan A; Dong, Yinxin; Preston, Jill C; Amasino, Richard M

    2016-04-01

    Flowering of many plant species is coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization provides competence to flower after prolonged cold exposure, and a vernalization requirement prevents flowering from occurring prior to winter. In winter wheat (Triticum aestivum) and barley (Hordeum vulgare), three genes VRN1, VRN2, and FT form a regulatory loop that regulates the initiation of flowering. Prior to cold exposure, VRN2 represses FT. During cold, VRN1 expression increases, resulting in the repression of VRN2, which in turn allows activation of FT during long days to induce flowering. Here, we test whether the circuitry of this regulatory loop is conserved across Pooideae, consistent with their niche transition from the tropics to the temperate zone. Our phylogenetic analyses of VRN2-like genes reveal a duplication event occurred before the diversification of the grasses that gave rise to a CO9 and VRN2/Ghd7 clade and support orthology between wheat/barley VRN2 and rice (Oryza sativa) Ghd7 Our Brachypodium distachyon VRN1 and VRN2 knockdown and overexpression experiments demonstrate functional conservation of grass VRN1 and VRN2 in the promotion and repression of flowering, respectively. However, expression analyses in a range of pooids demonstrate that the cold repression of VRN2 is unique to core Pooideae such as wheat and barley. Furthermore, VRN1 knockdown in B. distachyon demonstrates that the VRN1-mediated suppression of VRN2 is not conserved. Thus, the VRN1-VRN2 feature of the regulatory loop appears to have evolved late in the diversification of temperate grasses. PMID:26848096

  9. DEWAX-mediated transcriptional repression of cuticular wax biosynthesis in Arabidopsis thaliana

    PubMed Central

    Suh, Mi Chung; Go, Young Sam

    2014-01-01

    The aerial parts of plants are covered with a cuticular wax layer, which is the first barrier between a plant and its environment. Although cuticular wax deposition increases more in the light than in the dark, little is known about the molecular mechanisms underlying the regulation of cuticular wax biosynthesis. Recently DEWAX (Decrease Wax Biosynthesis) encoding an AP2/ERF transcription factor was found to be preferentially expressed in the epidermis and induced by darkness. Wax analysis of the dewax knockout mutant, wild type, and DEWAX overexpression lines (OX) indicates that DEWAX is a negative regulator of cuticular wax biosynthesis. DEWAX represses the expression of wax biosynthetic genes CER1, LACS2, ACLA2, and ECR via direct interaction with their promoters. Cuticular wax biosynthesis is negatively regulated twice a day by the expression of DEWAX; throughout the night and another for stomata closing. Taken together, it is evident that DEWAX-mediated negative regulation of the wax biosynthetic genes plays role in determining the total wax loads produced in Arabidopsis during daily dark and light cycles. In addition, significantly higher levels of DEWAX transcripts in leaves than stems suggest that DEWAX-mediated transcriptional repression might be involved in the organ-specific regulation of total wax amounts on plant surfaces. PMID:25763625

  10. 15d-prostaglandin J2 enhancement of nerve growth factor-induced neurite outgrowth is blocked by the chemoattractant receptor- homologous molecule expressed on T-helper type 2 cells (CRTH2) antagonist CAY10471 in PC12 cells.

    PubMed

    Hatanaka, Michiyoshi; Shibata, Norihiro; Shintani, Norihito; Haba, Ryota; Hayata, Atsuko; Hashimoto, Hitoshi; Baba, Akemichi

    2010-01-01

    The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTH2) is the most recently identified prostaglandin (PG) receptor for both PGD(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). We examined the mechanism by which 15d-PGJ(2) enhances nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. CAY10471 (CRTH2 antagonist) inhibited both the neurite-promotion and p38 mitogen-activated protein (MAP) kinase phosphorylation induced by 15d-PGJ(2). In contrast, 13,14-dihydro-15-keto-PGD(2 )(DK-PGD(2)) (selective CRTH2 agonist) stimulated its phosphorylation but failed to produce neurite-promoting effects. These suggest, for the first time, the action of 15d-PGJ(2) is mediated by CRTH2, although the CRTH2 activation alone is insufficient for the underlying action. PMID:20424389

  11. Trbp regulates heart function through miRNA-mediated Sox6 repression

    PubMed Central

    Ding, Jian; Chen, Jinghai; Wang, Yanqun; Kataoka, Masaharu; Ma, Lixin; Zhou, Pingzhu; Hu, Xiaoyun; Lin, Zhiqiang; Nie, Mao; Deng, Zhong-Liang; Pu, William T; Wang, Da-Zhi

    2015-01-01

    Cardiomyopathy is associated with altered expression of genes encoding contractile proteins. Here we show that Trbp (Tarbp2), an RNA binding protein, is required for normal heart function. Cardiac-specific inactivation of Trbp (TrbpcKO) caused progressive cardiomyopathy and lethal heart failure. Trbp loss of function resulted in upregulation of Sox6, repression of genes encoding normal cardiac slow-twitch myofiber proteins, and pathologically increased expression of skeletal fast-twitch myofiber genes. Remarkably, knockdown of Sox6 fully rescued the Trbp mutant phenotype, whereas Sox6 overexpression phenocopied the TrbpcKO phenotype. Trbp inactivation was mechanistically linked to Sox6 upregulation through altered processing of miR-208a, which is a direct inhibitor of Sox6. Transgenic overexpression of miR-208a sufficiently repressed Sox6, restored the balance of fast- and slow- twitch myofiber gene expression, and rescued cardiac function in TrbpcKO mice. Together, our studies reveal a novel Trbp-mediated microRNA processing mechanism in regulating a linear genetic cascade essential for normal heart function. PMID:26029872

  12. Sequestration and Inhibition of Daxx-Mediated Transcriptional Repression by PML

    PubMed Central

    Li, Hui; Leo, Christopher; Zhu, Jiang; Wu, Xiaoyang; O'Neil, Jennifer; Park, Eun-Ju; Chen, J. Don

    2000-01-01

    PML fuses with retinoic acid receptor α (RARα) in the t(15;17) translocation that causes acute promyelocytic leukemia (APL). In addition to localizing diffusely throughout the nucleoplasm, PML mainly resides in discrete nuclear structures known as PML oncogenic domains (PODs), which are disrupted in APL and spinocellular ataxia cells. We isolated the Fas-binding protein Daxx as a PML-interacting protein in a yeast two-hybrid screen. Biochemical and immunofluorescence analyses reveal that Daxx is a nuclear protein that interacts and colocalizes with PML in the PODs. Reporter gene assay shows that Daxx drastically represses basal transcription, likely by recruiting histone deacetylases. PML, but not its oncogenic fusion PML-RARα, inhibits the repressor function of Daxx. In addition, SUMO-1 modification of PML is required for sequestration of Daxx to the PODs and for efficient inhibition of Daxx-mediated transcriptional repression. Consistently, Daxx is found at condensed chromatin in cells that lack PML. These data suggest that Daxx is a novel nuclear protein bearing transcriptional repressor activity that may be regulated by interaction with PML. PMID:10669754

  13. Pax6 represses androgen receptor-mediated transactivation by inhibiting recruitment of the coactivator SPBP.

    PubMed

    Elvenes, Julianne; Thomassen, Ernst Ivan Simon; Johnsen, Sylvia Sagen; Kaino, Katrine; Sjøttem, Eva; Johansen, Terje

    2011-01-01

    The androgen receptor (AR) has a central role in development and maintenance of the male reproductive system and in the etiology of prostate cancer. The transcription factor Pax6 has recently been reported to act as a repressor of AR and to be hypermethylated in prostate cancer cells. SPBP is a transcriptional regulator that previously has been shown to enhance the activity of Pax6. In this study we have identified SPBP to act as a transcriptional coactivator of AR. We also show that Pax6 inhibits SPBP-mediated enhancement of AR activity on the AR target gene probasin promoter, a repression that was partly reversed by increased expression of SPBP. Enhanced expression of Pax6 reduced the amount of SPBP associated with the probasin promoter when assayed by ChIP in HeLa cells. We mapped the interaction between both AR and SPBP, and AR and Pax6 to the DNA-binding domains of the involved proteins. Further binding studies revealed that Pax6 and SPBP compete for binding to AR. These results suggest that Pax6 represses AR activity by displacing and/or inhibiting recruitment of coactivators to AR target promoters. Understanding the mechanism for inhibition of AR coactivators can give rise to molecular targeted drugs for treatment of prostate cancer. PMID:21935435

  14. Pax6 Represses Androgen Receptor-Mediated Transactivation by Inhibiting Recruitment of the Coactivator SPBP

    PubMed Central

    Johnsen, Sylvia Sagen; Kaino, Katrine; Sjøttem, Eva; Johansen, Terje

    2011-01-01

    The androgen receptor (AR) has a central role in development and maintenance of the male reproductive system and in the etiology of prostate cancer. The transcription factor Pax6 has recently been reported to act as a repressor of AR and to be hypermethylated in prostate cancer cells. SPBP is a transcriptional regulator that previously has been shown to enhance the activity of Pax6. In this study we have identified SPBP to act as a transcriptional coactivator of AR. We also show that Pax6 inhibits SPBP-mediated enhancement of AR activity on the AR target gene probasin promoter, a repression that was partly reversed by increased expression of SPBP. Enhanced expression of Pax6 reduced the amount of SPBP associated with the probasin promoter when assayed by ChIP in HeLa cells. We mapped the interaction between both AR and SPBP, and AR and Pax6 to the DNA-binding domains of the involved proteins. Further binding studies revealed that Pax6 and SPBP compete for binding to AR. These results suggest that Pax6 represses AR activity by displacing and/or inhibiting recruitment of coactivators to AR target promoters. Understanding the mechanism for inhibition of AR coactivators can give rise to molecular targeted drugs for treatment of prostate cancer. PMID:21935435

  15. Functional Association between Eyegone and HP1a Mediates wingless Transcriptional Repression during Development

    PubMed Central

    Salvany, Lara; Requena, David

    2012-01-01

    The eyegone (eyg) gene encodes Eyg, a transcription factor of the Pax family with multiple roles during Drosophila development. Although Eyg has been shown to act as a repressor, nothing is known about the mechanism by which it represses its target genes. Here, we show that Eyg forms a protein complex with heterochromatin protein 1a (HP1a). Both proteins bind to the same chromatin regions on polytene chromosomes and act cooperatively to suppress variegation and mediate gene silencing. In addition, Eyg binds to a wingless (wg) enhancer region, recruiting HP1a to assemble a closed, heterochromatin-like conformation that represses transcription of the wg gene. We describe here the evidence that suggests that Eyg, encoded by eyegone (eyg), represses wingless (wg) during eye development by association with HP1a. We show that Eyg forms a protein complex with HP1a and both proteins colocalize on salivary gland polytene chromosomes. Using position effect variegation (PEV) experiments, we demonstrated that eyg has a dose-dependent effect on heterochromatin gene silencing and identified a genetic interaction with HP1a in this process. We further demonstrated that HP1a binds to the same wg enhancer element as Eyg. DNase I sensitivity assays indicated that this enhancer region has a closed heterochromatin-like conformation, which becomes open in eyg mutants. In these mutants, much less HP1a binds to the wg enhancer region, as shown by ChIP experiments. Furthermore, as previously described for Eyg, a reduction in the amount of HP1a in the eye imaginal disc derepresses wg. Together, our results suggest a model in which Eyg specifically binds to the wg enhancer region, recruiting HP1a to that site. The recruitment of HP1a prevents transcription by favoring a closed, heterochromatin-like structure. Thus, for the first time, we show that HP1a plays a direct role in the repression of a developmentally regulated gene, wg, during Drosophila eye development. PMID:22547675

  16. Targeting 15d-Prostaglandin J2 to Hepatic Stellate Cells: Two Options Evaluated

    PubMed Central

    Hagens, Werner I.; Mattos, Adriana; Greupink, Rick; de Jager-Krikken, Alie; Reker-Smit, Catharina; van Loenen-Weemaes, AnneMiek; Gouw, Annette S. H.; Poelstra, Klaas

    2007-01-01

    Purpose Delivery of apoptosis-inducing compounds to hepatic stellate cells (HSC) may be an effective strategy to reverse liver fibrosis. The aim of this study was therefore to examine the selective targeting of the apoptosis-inducing drug 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) with two different HSC-carriers: human serum albumin modified with the sugar mannose-6-phosphate (M6PHSA) or albumin modified with PDGF-receptor recognizing peptides (pPBHSA). Methods and Results After chemical conjugation of 15dPGJ2 to the carriers, the constructs displayed pharmacological activity and specific receptor-mediated binding to HSC in vitro. Unlike 15dPGJ2-pPBHSA, the cellular binding of 15dPGJ2-M6PHSA was reduced by a scavenger receptor antagonist. In vivo, both conjugates rapidly accumulated in fibrotic livers. Intrahepatic analysis revealed that 15dPGJ2-M6PHSA mainly accumulated in HSC, and to a lesser extent in Kupffer cells. 15dPGJ2-pPBHSA also predominantly accumulated in HSC with additional uptake in hepatocytes. Assessment of target receptors in human cirrhotic livers revealed that M6P/IGFII-receptor expression was present in fibrotic areas. PDGF-β receptor expression was abundantly expressed on human fibroblasts. Conclusions These studies show that 15dPGJ2 coupled to either M6PHSA or pPBHSA is specifically taken up by HSC and is highly effective within these cells. Both carriers differ with respect to receptor specificity, leading to differences in intrahepatic distribution. Nevertheless, both carriers can be used to deliver the apoptosis-inducing drug 15dPGJ2 to HSC in vivo. PMID:17245650

  17. GLI3-dependent repression of DR4 mediates hedgehog antagonism of TRAIL-induced apoptosis.

    PubMed

    Kurita, S; Mott, J L; Almada, L L; Bronk, S F; Werneburg, N W; Sun, S-Y; Roberts, L R; Fernandez-Zapico, M E; Gores, G J

    2010-08-26

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through its cognate receptors death receptor 4 (DR4) and death receptor 5 (DR5), preferentially in malignant cells. However, many malignant cells remain resistant to TRAIL cytotoxicity by poorly characterized mechanisms. Here, using cholangiocarcinoma cells, as a model for TRAIL resistance, we identified a role for the oncogenic Hedgehog (Hh)-GLI pathway in the regulation of TRAIL cytotoxicity. Blockade of Hh using pharmacological and genetic tools sensitizes the cells to TRAIL cytotoxicity. Restoration of apoptosis sensitivity coincided with upregulation of DR4 expression, while expression of other death effector proteins remained unaltered. Knockdown of DR4 mimics Hh-mediated resistance to TRAIL cytotoxicity. Hh regulates the expression of DR4 by modulating the activity of its promoter. Luciferase, chromatin immunoprecipitation and expression assays show that the transcription factor GLI3 binds to the DR4 promoter and Hh requires an intact GLI3-repression activity to silence DR4 expression. Finally, small interfering RNA (siRNA)-targeted knockdown of GLI3, but not GLI1 or GLI2, restores DR4 expression and TRAIL sensitivity, indicating that the Hh effect is exclusively mediated by this transcription factor. In conclusion, these data provide evidence of a regulatory mechanism, which modulates TRAIL signaling in cancer cells and suggest new therapeutic approaches for TRAIL-resistant neoplasms. PMID:20562908

  18. GLI3-dependent repression of DR4 mediates hedgehog antagonism of TRAIL-induced apoptosis

    PubMed Central

    Kurita, S; Mott, JL; Almada, LL; Bronk, SF; Werneburg, NW; Sun, S-Y; Roberts, LR; Fernandez-Zapico, ME; Gores, GJ

    2010-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through its cognate receptors death receptor 4 (DR4) and death receptor 5 (DR5), preferentially in malignant cells. However, many malignant cells remain resistant to TRAIL cytotoxicity by poorly characterized mechanisms. Here, using cholangiocarcinoma cells, as a model for TRAIL resistance, we identified a role for the oncogenic Hedgehog (Hh)-GLI pathway in the regulation of TRAIL cytotoxicity. Blockade of Hh using pharmacological and genetic tools sensitizes the cells to TRAIL cytotoxicity. Restoration of apoptosis sensitivity coincided with upregulation of DR4 expression, while expression of other death effector proteins remained unaltered. Knockdown of DR4 mimics Hh-mediated resistance to TRAIL cytotoxicity. Hh regulates the expression of DR4 by modulating the activity of its promoter. Luciferase, chromatin immunoprecipitation and expression assays show that the transcription factor GLI3 binds to the DR4 promoter and Hh requires an intact GLI3-repression activity to silence DR4 expression. Finally, small interfering RNA (siRNA)-targeted knockdown of GLI3, but not GLI1 or GLI2, restores DR4 expression and TRAIL sensitivity, indicating that the Hh effect is exclusively mediated by this transcription factor. In conclusion, these data provide evidence of a regulatory mechanism, which modulates TRAIL signaling in cancer cells and suggest new therapeutic approaches for TRAIL-resistant neoplasms. PMID:20562908

  19. miRNA-mediated auxin signalling repression during Vat-mediated aphid resistance in Cucumis melo.

    PubMed

    Sattar, Sampurna; Addo-Quaye, Charles; Thompson, Gary A

    2016-06-01

    Resistance to Aphis gossypii in melon is attributed to the presence of the single dominant R gene virus aphid transmission (Vat), which is biologically expressed as antibiosis, antixenosis and tolerance. However, the mechanism of resistance is poorly understood at the molecular level. Aphid-induced transcriptional changes, including differentially expressed miRNA profiles that correspond to resistance interaction have been reported in melon. The potential regulatory roles of miRNAs in Vat-mediated aphid resistance were further revealed by identifying the specific miRNA degradation targets. A total of 70 miRNA:target pairs, including 28 novel miRNA:target pairs, for the differentially expressed miRNAs were identified: 11 were associated with phytohormone regulation, including six miRNAs that potentially regulate auxin interactions. A model for a redundant regulatory system of miRNA-mediated auxin insensitivity is proposed that incorporates auxin perception, auxin modification and auxin-regulated transcription. Chemically inhibiting the transport inhibitor response-1 (TIR-1) auxin receptor in susceptible melon tissues provides in vivo support for the model of auxin-mediated impacts on A. gossypii resistance. PMID:26437210

  20. Modulation of p53-mediated transcriptional repression and apoptosis by the adenovirus E1B 19K protein.

    PubMed Central

    Sabbatini, P; Chiou, S K; Rao, L; White, E

    1995-01-01

    BRK cell lines that stably express adenovirus E1A and a murine temperature-sensitive p53 undergo apoptosis when p53 assumes the wild-type conformation. Expression of the E1B 19,000-molecular-weight (19K) protein rescues cells from this p53-mediated apoptosis and diverts cells to a growth-arrested state. As p53 likely functions as a tumor suppressor by regulating transcription, the ability of the E1B 19K protein to regulate p53-mediated transactivation and transcriptional repression was investigated. In promoter-reporter assays the E1B 19K did not block p53-mediated transactivation but did alleviate p53-mediated transcriptional repression. E1B 19K expression permitted efficient transcriptional activation of the p21/WAF-1/cip-1 mRNA by p53, consistent with maintenance of the growth arrest function of p53. The E1B 19K protein is thereby unique among DNA virus-transforming proteins that target p53 for inactivation in that it selectively modulates the transcriptional properties of p53. The E1B 19K protein also rescued cells from apoptosis induced by inhibitors of transcription and protein synthesis. This suggests that cell death may result from the inhibition of expression of survival factors which function to maintain cell viability. p53 may induce apoptosis through generalized transcriptional repression. In turn, the E1B 19K protein may prevent p53-mediated apoptosis by alleviating p53-mediated transcriptional repression. PMID:7823921

  1. Cross-talk between TLR4 and PPARγ pathways in the arachidonic acid-induced inflammatory response in pancreatic acini.

    PubMed

    Mateu, A; Ramudo, L; Manso, M A; De Dios, I

    2015-12-01

    Arachidonic acid (AA) is generally associated with inflammation in different settings. We assess the molecular mechanisms involved in the inflammatory response exerted by AA on pancreatic acini as an approach to acute pancreatitis (AP). Celecoxib (COX-2 inhibitor), TAK-242 (TLR4 inhibitor) and 15d-PGJ2 (PPARγ agonist) were used to ascertain the signaling pathways. In addition, we examine the effects of TAK-242 and 15d-PGJ2 on AP induced in rats by bile-pancreatic duct obstruction (BPDO). To carry out in vitro studies, acini were isolated from pancreas of control rats. Generation of PGE2 and TXB2, activation of pro-inflammatory pathways (MAPKs, NF-κB, and JAK/STAT3) and overexpression of CCL2 and P-selectin was found in AA-treated acini. In addition, AA up-regulated TLR4 and down-regulated PPARγ expression. Celecoxib prevented the up-regulation of CCL2 and P-selectin but did not show any effect on the AA-mediated changes in TLR4 and PPARγ expression. TAK-242, reduced the generation of AA metabolites and repressed both the cascade of pro-inflammatory events which led to CCL2 and P-selectin overexpression as well as the AA-induced PPARγ down-regulation. Thus, TLR4 acts as upstream activating pro-inflammatory and inhibiting anti-inflammatory pathways. 15d-PGJ2 down-regulated TLR4 expression and hence prevented the synthesis of AA metabolites and the inflammatory response mediated by them. Reciprocal negative cross-talk between TLR4 and PPARγ pathways is evidenced. In vivo experiments showed that TAK-242 and 15d-PGJ2 treatments reduced the inflammatory response in BPDO-induced AP. We conclude that through TLR4-dependent mechanisms, AA up-regulated CCL2 and P-selectin in pancreatic acini, partly mediated by the generation of PGE2 and TXB2, which activated pro-inflammatory pathways, but also directly by down-regulating PPARγ expression with anti-inflammatory activity. In vitro and in vivo studies support the role of TLR4 in AP and the use of TLR4 inhibitors and

  2. Hepatocyte-Ductal Transdifferentiation Is Mediated by Reciprocal Repression of SOX9 and C/EBPα

    PubMed Central

    O'Neill, Kathy E.; Thowfeequ, Shifaan; Li, Wan-Chun; Eberhard, Daniel; Dutton, James R.; Slack, Jonathan M.W.

    2014-01-01

    Abstract Primary hepatocytes rapidly dedifferentiate when cultured in vitro. We have studied the mechanism of hepatocyte dedifferentiation by using two culture media: one that maintains hepatocytes in a differentiated state and another that allows dedifferentiation. We show that dedifferentiation involves partial transformation of hepatocytes into cells that resemble biliary epithelial cells. Lineage labeling and time-lapse filming confirm that the dedifferentiated cells are derived from hepatocytes and not from contaminating ductal or fibroblastic cells in the original culture. Furthermore, we establish that the conversion of hepatocytes to biliary-like cells is regulated by mutual antagonism of CCAAT/enhancer binding protein alpha (C/EBPα) and SOX9, which have opposing effects on the expression of hepatocyte and ductal genes. Thus, hepatocyte dedifferentiation induces the biliary gene expression program by alleviating C/EBPα-mediated repression of Sox9. We propose that reciprocal antagonism of C/EBPα and SOX9 also operates in the formation of hepatocytes and biliary ducts from hepatoblasts during normal embryonic development. These data demonstrate that reprogramming of differentiated cells can be used to model the acquisition and maintenance of cell fate in vivo. PMID:25153359

  3. New protein kinase and protein phosphatase families mediate signal transduction in bacterial catabolite repression.

    PubMed

    Galinier, A; Kravanja, M; Engelmann, R; Hengstenberg, W; Kilhoffer, M C; Deutscher, J; Haiech, J

    1998-02-17

    Carbon catabolite repression (CCR) is the prototype of a signal transduction mechanism. In enteric bacteria, cAMP was considered to be the second messenger in CCR by playing a role reminiscent of its actions in eukaryotic cells. However, recent results suggest that CCR in Escherichia coli is mediated mainly by an inducer exclusion mechanism. In many Gram-positive bacteria, CCR is triggered by fructose-1,6-bisphosphate, which activates HPr kinase, presumed to be one of the most ancient serine protein kinases. We here report cloning of the Bacillus subtilis hprK and hprP genes and characterization of the encoded HPr kinase and P-Ser-HPr phosphatase. P-Ser-HPr phosphatase forms a new family of phosphatases together with bacterial phosphoglycolate phosphatase, yeast glycerol-3-phosphatase, and 2-deoxyglucose-6-phosphate phosphatase whereas HPr kinase represents a new family of protein kinases on its own. It does not contain the domain structure typical for eukaryotic protein kinases. Although up to now the HPr modifying/demodifying enzymes were thought to exist only in Gram-positive bacteria, a sequence comparison revealed that they also are present in several Gram-negative pathogenic bacteria. PMID:9465101

  4. Human involucrin promoter mediates repression-resistant and compartment-specific LEKTI expression.

    PubMed

    Di, Wei-Li; Semenova, Ekaterina; Larcher, Fernando; Del Rio, Marcela; Harper, John I; Thrasher, Adrian J; Qasim, Waseem

    2012-01-01

    Gene-modified skin grafts, produced through gene transfer to human keratinocyte stem cells, offer the possibility of therapeutic benefit for inherited skin diseases. We have previously described efficient lentiviral vector-mediated gene transfer to keratinocyte stem cells and the generation of human skin grafts for the inherited skin disease, Netherton syndrome, which arises due to mutations in serine protease inhibitor Kazal-type 5 (SPINK5). Vectors incorporating an internal murine retroviral-derived promoter [spleen focus-forming virus (SFFV)] in combination with a codon-optimized SPINK5 transgene supported high levels of reconstitution and robust correction of skin architecture. Subsequent longer-term experiments have uncovered unanticipated silencing phenomena, with loss of SPINK5 gene expression over time. The inadvertent introduction of CpG sites during codon optimization appears to have rendered vectors susceptible to silencing due to methylation across the promoter-transgene boundary. Substitution of the methylation-susceptible SFFV promoter with a 572-bp minimal human involucrin promoter (INVOp), which encodes very few CpG sites, prevented repression of the SPINK5 transgene and resulted in durable and highly compartment-specific reconstitution of lympho-epithelial Kazal-type-related inhibitor (LEKTI) in human skin grafted onto immunodeficient mice. We conclude that skin grafts modified with lentiviral vectors encoding INVOp offer a suitable platform for therapeutic gene therapy in Netherton syndrome, and our experience highlights unanticipated effects of transgene codon optimization. PMID:21895535

  5. Repressed PKCδ activation in glycodelin-expressing cells mediates resistance to phorbol ester and TGFβ.

    PubMed

    Hautala, Laura C; Koistinen, Riitta; Koistinen, Hannu

    2016-10-01

    Glycodelin is a glycoprotein mainly expressed in well-differentiated epithelial cells in reproductive tissues. In normal secretory endometrium, the expression of glycodelin is abundant and regulated by progesterone. In hormone-related cancers glycodelin expression is associated with well-differentiated tumors. We have previously found that glycodelin drives epithelial differentiation of HEC-1B endometrial adenocarcinoma cells, resulting in reduced tumor growth in a preclinical mouse model. Here we show that glycodelin-transfected HEC-1B cells have repressed protein kinase C delta (PKCδ) activation, likely due to downregulation of PDK1, and are resistant to phenotypic change and enhanced migration induced by phorbol 12-myristate 13-acetate (PMA). In control cells, which do not express glycodelin, the effects of PMA were abolished by using PKCδ and PDK1 inhibitors, and knockdown of PKCδ, MEK1 and 2, or ERK1 and 2 by siRNAs. Similarly, transforming growth factor β (TGFβ)-induced phenotypic change was only seen in control cells, not in glycodelin-producing cells, and it was mediated by PKCδ. Taken together, these results strongly suggest that PKCδ, via MAPK pathway, is involved in the glycodelin-driven cell differentiation rendering the cells resistant to stimulation by PMA and TGFβ. PMID:27373413

  6. CRISPR-ERA: a comprehensive design tool for CRISPR-mediated gene editing, repression and activation

    PubMed Central

    Liu, Honglei; Wei, Zheng; Dominguez, Antonia; Li, Yanda; Wang, Xiaowo; Qi, Lei S.

    2015-01-01

    Summary: The CRISPR/Cas9 system was recently developed as a powerful and flexible technology for targeted genome engineering, including genome editing (altering the genetic sequence) and gene regulation (without altering the genetic sequence). These applications require the design of single guide RNAs (sgRNAs) that are efficient and specific. However, this remains challenging, as it requires the consideration of many criteria. Several sgRNA design tools have been developed for gene editing, but currently there is no tool for the design of sgRNAs for gene regulation. With accumulating experimental data on the use of CRISPR/Cas9 for gene editing and regulation, we implement a comprehensive computational tool based on a set of sgRNA design rules summarized from these published reports. We report a genome-wide sgRNA design tool and provide an online website for predicting sgRNAs that are efficient and specific. We name the tool CRISPR-ERA, for clustered regularly interspaced short palindromic repeat-mediated editing, repression, and activation (ERA). Availability and implementation: http://CRISPR-ERA.stanford.edu. Contact: stanley.qi@stanford.edu or xwwang@tsinghua.edu.cn Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26209430

  7. Phosphotransferase system-mediated glucose uptake is repressed in phosphoglucoisomerase-deficient Corynebacterium glutamicum strains.

    PubMed

    Lindner, Steffen N; Petrov, Dimitar P; Hagmann, Christian T; Henrich, Alexander; Krämer, Reinhard; Eikmanns, Bernhard J; Wendisch, Volker F; Seibold, Gerd M

    2013-04-01

    Corynebacterium glutamicum is particularly known for its industrial application in the production of amino acids. Amino acid overproduction comes along with a high NADPH demand, which is covered mainly by the oxidative part of the pentose phosphate pathway (PPP). In previous studies, the complete redirection of the carbon flux toward the PPP by chromosomal inactivation of the pgi gene, encoding the phosphoglucoisomerase, has been applied for the improvement of C. glutamicum amino acid production strains, but this was accompanied by severe negative effects on the growth characteristics. To investigate these effects in a genetically defined background, we deleted the pgi gene in the type strain C. glutamicum ATCC 13032. The resulting strain, C. glutamicum Δpgi, lacked detectable phosphoglucoisomerase activity and grew poorly with glucose as the sole substrate. Apart from the already reported inhibition of the PPP by NADPH accumulation, we detected a drastic reduction of the phosphotransferase system (PTS)-mediated glucose uptake in C. glutamicum Δpgi. Furthermore, Northern blot analyses revealed that expression of ptsG, which encodes the glucose-specific EII permease of the PTS, was abolished in this mutant. Applying our findings, we optimized l-lysine production in the model strain C. glutamicum DM1729 by deletion of pgi and overexpression of plasmid-encoded ptsG. l-Lysine yields and productivity with C. glutamicum Δpgi(pBB1-ptsG) were significantly higher than those with C. glutamicum Δpgi(pBB1). These results show that ptsG overexpression is required to overcome the repressed activity of PTS-mediated glucose uptake in pgi-deficient C. glutamicum strains, thus enabling efficient as well as fast l-lysine production. PMID:23396334

  8. Different mechanisms contribute to the E2-mediated transcriptional repression of human papillomavirus type 18 viral oncogenes.

    PubMed

    Demeret, C; Desaintes, C; Yaniv, M; Thierry, F

    1997-12-01

    Transcription of the human papillomavirus type 18 (HPV18) E6 and E7 oncogenes is repressed by the viral E2 protein. In C33 cells, we have previously shown that of the four E2 binding sites (E2 BS) present in the HPV18 long control region (LCR), only the binding site adjacent to the TATA box (E2 BS 1) was involved in E2-mediated repression. In the present study, we sought to determine whether this phenomenon was conserved in other cell lines. We first showed that all three E2 BS proximal to the P105 promoter were required for full repression of its activity in HeLa and HaCaT cells. Repression by E2 at E2 BS 2 occurred through the displacement of Sp1. Second, a truncated E2 product, lacking the N-terminal transactivation domain, repressed transcription more efficiently than the full-length protein. Repression was abolished when the N-terminal domain of E2 was replaced by the activation domain of VP16. The VP16-E2 chimeric protein could activate transcription from an LCR mutated in its TATA box. DNA-protein binding studies showed that E2 associates with its four binding sites in the LCR with similar affinities. However, challenge of such complexes with excess binding sites demonstrated that interaction with E2 BS 4 was the most stable while interaction with E2 BS 1 was the least stable. Furthermore, complexes with the full-length E2 were less stable than those formed with the N-terminally truncated protein. PMID:9371593

  9. 15-deoxy prostaglandin J2, the nonenzymatic metabolite of prostaglandin D2, induces apoptosis in keratinocytes of human hair follicles: a possible explanation for prostaglandin D2-mediated inhibition of hair growth.

    PubMed

    Joo, Hyun Woo; Kang, Yoo Ri; Kwack, Mi Hee; Sung, Young Kwan

    2016-07-01

    Recent studies have shown that prostaglandin D2 (PGD2) and its nonenzymatic metabolite, 15-deoxy-Δ(12,14)-prostaglandin J2 (15-dPGJ2), inhibit in vitro growth of explanted human hair follicles and inhibit hair growth in mice through the GPR44 (DP2). However, the underlying mechanism is still unclear. In this study, we first investigated the expression of DP2 in human hair follicles and in cultured follicular cells. We found that DP2 is strongly expressed in the outer root sheath (ORS) cells and weakly expressed in the dermal papilla (DP) cells. We observed slight growth stimulation when ORS and DP cells were treated with PGD2. We also observed slight growth stimulation when DP and ORS cells were treated with low concentrations (0.5 and 1 μM) of 15-dPGJ2. However, 5 μM 15-dPGJ2 inhibited the viability and caused apoptosis of both cell types. Exposure of cultured human hair follicles to 15-dPGJ2 resulted in significant apoptosis in follicular keratinocytes. Altogether, our data provide an evidence that 15-dPGJ2 promotes apoptosis in follicular keratinocytes and provide rationale for developing remedies for the prevention and treatment of hair loss based on DP2 antagonism. PMID:27185495

  10. Recruitment of histone methyltransferase G9a mediates transcriptional repression of Fgf21 gene by E4BP4 protein.

    PubMed

    Tong, Xin; Zhang, Deqiang; Buelow, Katie; Guha, Anirvan; Arthurs, Blake; Brady, Hugh J M; Yin, Lei

    2013-02-22

    The liver responds to fasting-refeeding cycles by reprogramming expression of metabolic genes. Fasting potently induces one of the key hepatic hormones, fibroblast growth factor 21 (FGF21), to promote lipolysis, fatty acid oxidation, and ketogenesis, whereas refeeding suppresses its expression. We previously reported that the basic leucine zipper transcription factor E4BP4 (E4 binding protein 4) represses Fgf21 expression and disrupts its circadian oscillations in cultured hepatocytes. However, the epigenetic mechanism for E4BP4-dependent suppression of Fgf21 has not yet been addressed. Here we present evidence that histone methyltransferase G9a mediates E4BP4-dependent repression of Fgf21 during refeeding by promoting repressive histone modification. We find that Fgf21 expression is up-regulated in E4bp4 knock-out mouse liver. We demonstrate that the G9a-specific inhibitor BIX01294 abolishes suppression of the Fgf21 promoter activity by E4BP4, whereas overexpression of E4bp4 leads to increased levels of dimethylation of histone 3 lysine 9 (H3K9me2) around the Fgf21 promoter region. Furthermore, we also show that E4BP4 interacts with G9a, and knockdown of G9a blocks repression of Fgf21 promoter activity and expression in cells overexpressing E4bp4. A G9a mutant lacking catalytic activity, due to deletion of the SET domain, fails to inhibit the Fgf21 promoter activity. Importantly, acute hepatic knockdown by adenoviral shRNA targeting G9a abolishes Fgf21 repression by refeeding, concomitant with decreased levels of H3K9me2 around the Fgf21 promoter region. In summary, we show that G9a mediates E4BP4-dependent suppression of hepatic Fgf21 by enhancing histone methylation (H3K9me2) of the Fgf21 promoter. PMID:23283977

  11. The gap protein knirps mediates both quenching and direct repression in the Drosophila embryo.

    PubMed Central

    Arnosti, D N; Gray, S; Barolo, S; Zhou, J; Levine, M

    1996-01-01

    Transcriptional repression is essential for establishing localized patterns of gene expression during Drosophila embryogenesis. Several mechanisms of repression have been proposed, including competition, quenching and direct repression of the transcription complex. Previous studies suggest that the knirps orphan receptor (kni) may repress transcription via competition, and exclude the binding of the bicoid (bcd) activator to an overlapping site in a target promoter. Here we present evidence that kni can quench, or locally inhibit, upstream activators within a heterologous enhancer in transgenic embryos. The range of kni repression is approximately 50-100 bp, so that neighboring enhancers in a modular promoter are free to interact with the transcription complex (enhancer autonomy). However, kni can also repress the transcription complex when bound in promoter-proximal regions. In this position, kni functions as a dominant repressor and blocks multiple enhancers in a modular promoter. Our studies suggest that short-range repression represents a flexible form of gene regulation, exhibiting enhancer- or promoter-specific effects depending on the location of repressor binding sites. Images PMID:8670869

  12. The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans.

    PubMed

    Grants, Jennifer M; Ying, Lisa T L; Yoda, Akinori; You, Charlotte C; Okano, Hideyuki; Sawa, Hitoshi; Taubert, Stefan

    2016-02-01

    Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinase-dependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals. PMID:26715664

  13. 15-Deoxy-Δ12,14-prostaglandin J2 inhibits macrophage colonization by Salmonella enterica serovar Typhimurium.

    PubMed

    Buckner, Michelle M C; Antunes, L Caetano M; Gill, Navkiran; Russell, Shannon L; Shames, Stephanie R; Finlay, B Brett

    2013-01-01

    15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) is an anti-inflammatory downstream product of the cyclooxygenase enzymes. It has been implicated to play a protective role in a variety of inflammatory mediated diseases, including rheumatoid arthritis, neural damage, and myocardial infarctions. Here we show that 15d-PGJ2 also plays a role in Salmonella infection. Salmonella enterica Typhimurium is a Gram-negative facultative intracellular pathogen that is able to survive and replicate inside phagocytic immune cells, allowing for bacterial dissemination to systemic sites. Salmonella species cause a wide range of morbidity and mortality due to gastroenteritis and typhoid fever. Previously we have shown that in mouse models of typhoid fever, Salmonella infection causes a major perturbation in the prostaglandin pathway. Specifically, we saw that 15d-PGJ2 production was significantly increased in both liver and feces. In this work we show that 15d-PGJ2 production is also significantly increased in macrophages infected with Salmonella. Furthermore, we show that the addition of 15d-PGJ2 to Salmonella infected RAW264.7, J774, and bone marrow derived macrophages is sufficient to significantly reduce bacterial colonization. We also show evidence that 15d-PGJ2 is reducing bacterial uptake by macrophages. 15d-PGJ2 reduces the inflammatory response of these infected macrophages, as evidenced by a reduction in the production of cytokines and reactive nitrogen species. The inflammatory response of the macrophage is important for full Salmonella virulence, as it can give the bacteria cues for virulence. The reduction in bacterial colonization is independent of the expression of Salmonella virulence genes SPI1 and SPI2, and is independent of the 15d-PGJ2 ligand PPAR-γ. 15d-PGJ2 also causes an increase in ERK1/2 phosphorylation in infected macrophages. In conclusion, we show here that 15d-PGJ2 mediates the outcome of bacterial infection, a previously unidentified role for this

  14. Sustained translational repression by eIF2α–P mediates prion neurodegeneration

    PubMed Central

    Moreno, Julie A.; Radford, Helois; Peretti, Diego; Steinert, Joern R.; Verity, Nicholas; Martin, Maria Guerra; Halliday, Mark; Morgan, Jason; Dinsdale, David; Ortori, Catherine A.; Barrett, David A.; Tsaytler, Pavel; Bertolotti, Anne; Willis, Anne E.; Bushell, Martin; Mallucci, Giovanna R.

    2012-01-01

    The mechanisms leading to neuronal death in neurodegenerative disease are poorly understood. Many of these disorders, including Alzheimer’s (AD), Parkinson’s (PD) and prion diseases, are associated with the accumulation of misfolded disease-specific proteins. The unfolded protein response (UPR) is a protective cellular mechanism triggered by rising levels of misfolded proteins. One arm of this pathway results in the transient shutdown of protein translation, through phosphorylation of the alpha subunit of eukaryotic translation initiation factor, eIF2α. UPR activation and/or increased eIF2α–P levels are seen in patients with AD, PD and prion disease 1-4, but how this links to neurodegeneration is unknown. Here we show that accumulation of prion protein (PrP) during prion replication causes persistent translational repression of global protein synthesis by eIF2α–P, associated with synaptic failure and neuronal loss in prion-diseased mice. Further, we show that promoting translational recovery in hippocampi of prion-infected mice is neuroprotective. Over-expression of GADD34, a specific eIF2α–P phosphatase, as well as reduction of PrP levels by lentivirally-mediated RNAi, reduced eIF2α–P levels. As a result, both approaches restored vital translation rates during prion disease, rescuing synaptic deficits and neuronal loss, and thereby significantly increasing survival. In contrast, salubrinal, an inhibitor of eIF2α-P dephosphorylation5 increased eIF2α-P levels, exacerbating neurotoxicity and significantly reducing survival in prion diseased mice. Given the prevalence of protein misfolding and UPR activation in several neurodegenerative diseases, our results suggest that manipulation of common pathways such as translational control, rather than disease-specific approaches, may lead to new therapies preventing synaptic failure and neuronal loss across the spectrum of these disorders. PMID:22622579

  15. GATA4 mediates gene repression in the mature mouse small intestine through interactions with Friend of GATA (FOG) cofactors

    PubMed Central

    Beuling, Eva; Bosse, Tjalling; aan de Kerk, Daniel J.; Piaseckyj, Christina M.; Fujiwara, Yuko; Katz, Samuel G.; Orkin, Stuart H.; Grand, Richard J.; Krasinski, Stephen D.

    2008-01-01

    GATA4, a transcription factor expressed in the proximal small intestine but not in the distal ileum, maintains proximal-distal distinctions by multiple processes involving gene repression, gene activation, and cell fate determination. Friend of GATA (FOG) is an evolutionarily conserved family of cofactors whose members physically associate with GATA factors and mediate GATA-regulated repression in multiple tissues. Using a novel, inducible, intestine-specific Gata4 knock-in model in mice, in which wild-type GATA4 is specifically inactivated in the small intestine, but a GATA4 mutant that does not bind FOG cofactors (GATA4ki) continues to be expressed, we found that ileal-specific genes were significantly induced in the proximal small intestine (P<0.01); in contrast, genes restricted to proximal small intestine and cell lineage markers were unaffected, indicating that GATA4-FOG interactions contribute specifically to the repression function of GATA4 within this organ. Fog1 mRNA displayed a proximal-distal pattern that parallels that of Gata4, and FOG1 protein was co-expressed with GATA4 in intestinal epithelial cells, implicating FOG1 as the likely mediator of GATA4 function in the small intestine. Our data are the first to indicate FOG function and expression in the mammalian small intestine. PMID:18692040

  16. The DDX6-4E-T interaction mediates translational repression and P-body assembly.

    PubMed

    Kamenska, Anastasiia; Simpson, Clare; Vindry, Caroline; Broomhead, Helen; Bénard, Marianne; Ernoult-Lange, Michèle; Lee, Benjamin P; Harries, Lorna W; Weil, Dominique; Standart, Nancy

    2016-07-27

    4E-Transporter binds eIF4E via its consensus sequence YXXXXLΦ, shared with eIF4G, and is a nucleocytoplasmic shuttling protein found enriched in P-(rocessing) bodies. 4E-T inhibits general protein synthesis by reducing available eIF4E levels. Recently, we showed that 4E-T bound to mRNA however represses its translation in an eIF4E-independent manner, and contributes to silencing of mRNAs targeted by miRNAs. Here, we address further the mechanism of translational repression by 4E-T by first identifying and delineating the interacting sites of its major partners by mass spectrometry and western blotting, including DDX6, UNR, unrip, PAT1B, LSM14A and CNOT4. Furthermore, we document novel binding between 4E-T partners including UNR-CNOT4 and unrip-LSM14A, altogether suggesting 4E-T nucleates a complex network of RNA-binding protein interactions. In functional assays, we demonstrate that joint deletion of two short conserved motifs that bind UNR and DDX6 relieves repression of 4E-T-bound mRNA, in part reliant on the 4E-T-DDX6-CNOT1 axis. We also show that the DDX6-4E-T interaction mediates miRNA-dependent translational repression and de novo P-body assembly, implying that translational repression and formation of new P-bodies are coupled processes. Altogether these findings considerably extend our understanding of the role of 4E-T in gene regulation, important in development and neurogenesis. PMID:27342281

  17. Dual role of Med12 in PRC1-dependent gene repression and ncRNA-mediated transcriptional activation.

    PubMed

    Papadopoulou, Thaleia; Kaymak, Aysegül; Sayols, Sergi; Richly, Holger

    2016-06-01

    Mediator is considered an enhancer of RNA-Polymerase II dependent transcription but its function and regulation in pluripotent mouse embryonic stem cells (mESCs) remains unresolved. One means of controlling the function of Mediator is provided by the binding of the Cdk8 module (Med12, Cdk8, Ccnc and Med13) to the core Mediator. Here we report that Med12 operates together with PRC1 to silence key developmental genes in pluripotency. At the molecular level, while PRC1 represses genes it is also required to assemble ncRNA containing Med12-Mediator complexes. In the course of cellular differentiation the H2A ubiquitin binding protein Zrf1 abrogates PRC1-Med12 binding and facilitates the association of Cdk8 with Mediator. This remodeling of Mediator-associated protein complexes converts Mediator from a transcriptional repressor to a transcriptional enhancer, which then mediates ncRNA-dependent activation of Polycomb target genes. Altogether, our data reveal how the interplay of PRC1, ncRNA and Mediator complexes controls pluripotency and cellular differentiation. PMID:27096886

  18. Lactose-mediated carbon catabolite repression of putrescine production in dairy Lactococcus lactis is strain dependent.

    PubMed

    del Rio, Beatriz; Ladero, Victor; Redruello, Begoña; Linares, Daniel M; Fernández, Maria; Martín, Maria Cruz; Alvarez, Miguel A

    2015-06-01

    Lactococcus lactis is the lactic acid bacterial (LAB) species most widely used as a primary starter in the dairy industry. However, several strains of L. lactis produce the biogenic amine putrescine via the agmatine deiminase (AGDI) pathway. We previously reported the putrescine biosynthesis pathway in L. lactis subsp. cremoris GE2-14 to be regulated by carbon catabolic repression (CCR) via glucose but not lactose (Linares et al., 2013). The present study shows that both these sugars repress putrescine biosynthesis in L. lactis subsp. lactis T3/33, a strain isolated from a Spanish artisanal cheese. Furthermore, we demonstrated that both glucose and lactose repressed the transcriptional activity of the aguBDAC catabolic genes of the AGDI route. Finally, a screening performed in putrescine-producing dairy L. lactis strains determined that putrescine biosynthesis was repressed by lactose in all the L. lactis subsp. lactis strains tested, but in only one L. lactis subsp. cremoris strain. Given the obvious importance of the lactose-repression in cheese putrescine accumulation, it is advisable to consider the diversity of L. lactis in this sense and characterize consequently the starter cultures to select the safest strains. PMID:25791004

  19. Transcriptional repression of the testis-specific histone H1t gene mediated by an element upstream of the H1/AC box.

    PubMed

    Wolfe, Steven A; Grimes, Sidney R

    2003-04-10

    The testis-specific histone H1t gene is transcribed exclusively in primary spermatocytes and may be important for chromatin structure, transcription, and DNA repair during this stage of spermatogenesis. Transcriptional repression of the gene in other cell types is mediated in part by specific proximal and distal promoter elements and in some cell types by methylation of CpG dinucleotides within the promoter. Our laboratory identified a distal promoter element located between 948 and 780 bp upstream from the transcription initiation site and another laboratory identified a GC-rich region between the TATA box and transcription initiation site that contribute to repression. In this article we address transcriptional repression of the histone H1t gene by an element within the proximal promoter. We report discovery of an element designated H1t promoter repressor element (RE) located between -130 and -106 bp that contributes to repression. The findings support the hypothesis that multiple mechanisms are involved in transcriptional repression of the H1t gene. Transcriptional repression mediated by the RE element in NIH 3T3 cells appears to differ significantly from the mechanism mediated by the GC-rich region. Furthermore, binding proteins that form the RE complex are not present in rat testis where the gene is actively transcribed. Our findings provide a molecular basis for histone H1t gene repression. PMID:12711397

  20. JAZ8 Lacks a Canonical Degron and Has an EAR Motif That Mediates Transcriptional Repression of Jasmonate Responses in Arabidopsis[C][W

    PubMed Central

    Shyu, Christine; Figueroa, Pablo; DePew, Cody L.; Cooke, Thomas F.; Sheard, Laura B.; Moreno, Javier E.; Katsir, Leron; Zheng, Ning; Browse, John; Howe, Gregg A.

    2012-01-01

    The lipid-derived hormone jasmonoyl-l-Ile (JA-Ile) initiates large-scale changes in gene expression by stabilizing the interaction of JASMONATE ZIM domain (JAZ) repressors with the F-box protein CORONATINE INSENSITIVE1 (COI1), which results in JAZ degradation by the ubiquitin-proteasome pathway. Recent structural studies show that the JAZ1 degradation signal (degron) includes a short conserved LPIAR motif that seals JA-Ile in its binding pocket at the COI1-JAZ interface. Here, we show that Arabidopsis thaliana JAZ8 lacks this motif and thus is unable to associate strongly with COI1 in the presence of JA-Ile. As a consequence, JAZ8 is stabilized against jasmonate (JA)-mediated degradation and, when ectopically expressed in Arabidopsis, represses JA-regulated growth and defense responses. These findings indicate that sequence variation in a hypervariable region of the degron affects JAZ stability and JA-regulated physiological responses. We also show that JAZ8-mediated repression depends on an LxLxL-type EAR (for ERF-associated amphiphilic repression) motif at the JAZ8 N terminus that binds the corepressor TOPLESS and represses transcriptional activation. JAZ8-mediated repression does not require the ZIM domain, which, in other JAZ proteins, recruits TOPLESS through the EAR motif–containing adaptor protein NINJA. These findings show that EAR repression domains in a subgroup of JAZ proteins repress gene expression through direct recruitment of corepressors to cognate transcription factors. PMID:22327740

  1. Homeodomain-interacting protein kinase 2-dependent repression of myogenic differentiation is relieved by its caspase-mediated cleavage.

    PubMed

    de la Vega, Laureano; Hornung, Juliane; Kremmer, Elisabeth; Milanovic, Maja; Schmitz, M Lienhard

    2013-06-01

    Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes. HIPK2 is constitutively associated with a multi-protein complex containing histone deacetylase (HDAC)3 and HDAC4 that serves to silence MEF2C-dependent transcription in undifferentiated myoblasts. HIPK2 interferes with gene expression on phosphorylation and HDAC3-dependent deacetylation of MEF2C. Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein. The short form of the kinase loses its affinity to the repressive multi-protein complex and its ability to bind HDAC3 and HDAC4, thus alleviating its repressive function for expression of muscle genes. This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis. PMID:23620283

  2. Homeodomain-interacting protein kinase 2-dependent repression of myogenic differentiation is relieved by its caspase-mediated cleavage

    PubMed Central

    de la Vega, Laureano; Hornung, Juliane; Kremmer, Elisabeth; Milanovic, Maja; Schmitz, M. Lienhard

    2013-01-01

    Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes. HIPK2 is constitutively associated with a multi-protein complex containing histone deacetylase (HDAC)3 and HDAC4 that serves to silence MEF2C-dependent transcription in undifferentiated myoblasts. HIPK2 interferes with gene expression on phosphorylation and HDAC3-dependent deacetylation of MEF2C. Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein. The short form of the kinase loses its affinity to the repressive multi-protein complex and its ability to bind HDAC3 and HDAC4, thus alleviating its repressive function for expression of muscle genes. This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis. PMID:23620283

  3. FER-1, an enhancer of the ferritin H gene and a target of E1A-mediated transcriptional repression.

    PubMed Central

    Tsuji, Y; Akebi, N; Lam, T K; Nakabeppu, Y; Torti, S V; Torti, F M

    1995-01-01

    Ferritin, the major intracellular iron storage protein of eucaryotic cells, is regulated during inflammation and malignancy. We previously reported that transcription of the H subunit of ferritin (ferritin H) is negatively regulated by the adenovirus E1A oncogene in mouse NIH 3T3 fibroblasts (Y. Tsuji, E. Kwak, T. Saika, S. V. Torti, and F. M. Torti, J. Biol. Chem. 268:7270-7275, 1993). To elucidate the mechanism of transcriptional repression of the ferritin H gene by E1A, a series of deletions in the 5' flanking region of the mouse ferritin H gene were constructed, fused to the chloramphenicol acetyltransferase (CAT) gene, and transiently cotransfected into NIH 3T3 cells with an E1A expression plasmid. The results indicate that the E1A-responsive region is located approximately 4.1 kb 5' to the transcription initiation site of the ferritin H gene. Further analyses revealed that a 37-bp region, termed FER-1, is the target of E1A-mediated repression. This region also serves as an enhancer, augmenting ferritin H transcription independently of position and orientation. FER-1 was dissected into two component elements, i.e., a 22-bp dyad symmetry element and a 7-bp AP1-like sequence. Insertion of these DNA sequences into a ferritin H-CAT chimeric gene lacking an E1A-responsive region indicated that (i) the 22-bp dyad symmetry sequence by itself has no enhancer activity, (ii) the AP1-like sequence has moderate enhancer activity which is repressed by E1A, and (iii) the combination of the dyad symmetry element and the AP1-like sequence is required for maximal enhancer activity and repression by E1A. Gel retardation assays and cotransfection experiments with c-fos and c-jun expression vectors suggested that members of the Fos and Jun families bind to the AP1-like element of FER-1 and contribute to its regulation. In addition, gel retardation assays showed that E1A reduces the ability of nuclear proteins to bind to the AP1-like sequence without affecting the levels of

  4. Evolution of VRN2/Ghd7-Like Genes in Vernalization-Mediated Repression of Grass Flowering1[OPEN

    PubMed Central

    McKeown, Meghan A.

    2016-01-01

    Flowering of many plant species is coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization provides competence to flower after prolonged cold exposure, and a vernalization requirement prevents flowering from occurring prior to winter. In winter wheat (Triticum aestivum) and barley (Hordeum vulgare), three genes VRN1, VRN2, and FT form a regulatory loop that regulates the initiation of flowering. Prior to cold exposure, VRN2 represses FT. During cold, VRN1 expression increases, resulting in the repression of VRN2, which in turn allows activation of FT during long days to induce flowering. Here, we test whether the circuitry of this regulatory loop is conserved across Pooideae, consistent with their niche transition from the tropics to the temperate zone. Our phylogenetic analyses of VRN2-like genes reveal a duplication event occurred before the diversification of the grasses that gave rise to a CO9 and VRN2/Ghd7 clade and support orthology between wheat/barley VRN2 and rice (Oryza sativa) Ghd7. Our Brachypodium distachyon VRN1 and VRN2 knockdown and overexpression experiments demonstrate functional conservation of grass VRN1 and VRN2 in the promotion and repression of flowering, respectively. However, expression analyses in a range of pooids demonstrate that the cold repression of VRN2 is unique to core Pooideae such as wheat and barley. Furthermore, VRN1 knockdown in B. distachyon demonstrates that the VRN1-mediated suppression of VRN2 is not conserved. Thus, the VRN1-VRN2 feature of the regulatory loop appears to have evolved late in the diversification of temperate grasses. PMID:26848096

  5. Identification of a prostaglandin D2 metabolite as a neuritogenesis enhancer targeting the TRPV1 ion channel

    PubMed Central

    Shibata, Takahiro; Takahashi, Katsuhiro; Matsubara, Yui; Inuzuka, Emi; Nakashima, Fumie; Takahashi, Nobuaki; Kozai, Daisuke; Mori, Yasuo; Uchida, Koji

    2016-01-01

    Mast cells play important roles in allergic inflammation by secreting various mediators. In the present study, based on the finding that the medium conditioned by activated RBL-2H3 mast cells enhanced the nerve growth factor (NGF)-induced neuritogenesis of PC12 cells, we attempted to isolate an active compound from the mast cell conditioned culture medium. Our experiment identified 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), one of the PGD2 metabolites, as a potential enhancer of neuritogenesis. 15d-PGJ2 strongly enhanced the neuritogenesis elicited by a low-concentration of NGF that alone was insufficient to induce the neuronal differentiation. This 15d-PGJ2 effect was exerted in a Ca2+-dependent manner, but independently of the NGF receptor TrkA. Importantly, 15d-PGJ2 activated the transient receptor potential vanilloid-type 1 (TRPV1), a non-selective cation channel, leading to the Ca2+ influx. In addition, we observed that (i) NGF promoted the insertion of TRPV1 into the cell surface membrane and (ii) 15d-PGJ2 covalently bound to TRPV1. These findings suggest that the NGF/15d-PGJ2-induced neuritogenesis may be regulated by two sets of mechanisms, one for the translocation of TRPV1 into the cell surface by NGF and one for the activation of TRPV1 by 15d-PGJ2. Thus, there is most likely a link between allergic inflammation and activation of the neuronal differentiation. PMID:26879669

  6. CcpA-mediated repression of Clostridium difficile toxin gene expression.

    PubMed

    Antunes, Ana; Martin-Verstraete, Isabelle; Dupuy, Bruno

    2011-02-01

    The presence of glucose or other rapidly metabolizable carbon sources in the bacterial growth medium strongly represses Clostridium difficile toxin synthesis independently of strain origin. In Gram-positive bacteria, carbon catabolite repression (CCR) is generally regarded as a regulatory mechanism that responds to carbohydrate availability. In the C. difficile genome all elements involved in CCR are present. To elucidate in vivo the role of CCR in C. difficile toxin synthesis, we used the ClosTron gene knockout system to construct mutants of strain JIR8094 that were unable to produce the major components of the CCR signal transduction pathway: the phosphotransferase system (PTS) proteins (Enzyme I and HPr), the HPr kinase/phosphorylase (HprK/P) and the catabolite control protein A, CcpA. Inactivation of the ptsI, ptsH and ccpA genes resulted in derepression of toxin gene expression in the presence of glucose, whereas repression of toxin production was still observed in the hprK mutant, indicating that uptake of glucose is required for repression but that phosphorylation of HPr by HprK is not. C. difficile CcpA was found to bind to the regulatory regions of the tcdA and tcdB genes but not through a consensus cre site motif. Moreover in vivo and in vitro results confirmed that HPr-Ser45-P does not stimulate CcpA-dependent binding to DNA targets. However, fructose-1,6-biphosphate (FBP) alone did increase CcpA binding affinity in the absence of HPr-Ser45-P. These results showed that CcpA represses toxin expression in response to PTS sugar availability, thus linking carbon source utilization to virulence gene expression in C. difficile. PMID:21299645

  7. CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli.

    PubMed

    Cress, Brady F; Toparlak, Ö Duhan; Guleria, Sanjay; Lebovich, Matthew; Stieglitz, Jessica T; Englaender, Jacob A; Jones, J Andrew; Linhardt, Robert J; Koffas, Mattheos A G

    2015-09-18

    Programmable control over an addressable global regulator would enable simultaneous repression of multiple genes and would have tremendous impact on the field of synthetic biology. It has recently been established that CRISPR/Cas systems can be engineered to repress gene transcription at nearly any desired location in a sequence-specific manner, but there remain only a handful of applications described to date. In this work, we report development of a vector possessing a CRISPathBrick feature, enabling rapid modular assembly of natural type II-A CRISPR arrays capable of simultaneously repressing multiple target genes in Escherichia coli. Iterative incorporation of spacers into this CRISPathBrick feature facilitates the combinatorial construction of arrays, from a small number of DNA parts, which can be utilized to generate a suite of complex phenotypes corresponding to an encoded genetic program. We show that CRISPathBrick can be used to tune expression of plasmid-based genes and repress chromosomal targets in probiotic, virulent, and commonly engineered E. coli strains. Furthermore, we describe development of pCRISPReporter, a fluorescent reporter plasmid utilized to quantify dCas9-mediated repression from endogenous promoters. Finally, we demonstrate that dCas9-mediated repression can be harnessed to assess the effect of downregulating both novel and computationally predicted metabolic engineering targets, improving the yield of a heterologous phytochemical through repression of endogenous genes. These tools provide a platform for rapid evaluation of multiplex metabolic engineering interventions. PMID:25822415

  8. The Zinc Finger and C-terminal Domains of MTA Proteins are Required for FOG-2 Mediated Transcriptional Repression via the NuRD Complex

    PubMed Central

    Roche, Andrea E.; Bassett, Brett J.; Samant, Sadhana A.; Hong, Wei; Blobel, Gerd A.; Svensson, Eric C.

    2008-01-01

    FOG-2 is a transcriptional co-regulator that is required for cardiac morphogenesis as mice deficient in this factor die during mid-gestation of cardiac malformations. FOG-2 interacts with GATA4 to attenuate GATA4-dependent gene expression. The first 12 amino acids of FOG-2 (the FOG Repression Motif) are necessary to mediate this repression. To determine the mechanism by which the FOG Repression Motif functions, we identified 7 polypeptides from rat cardiac nuclear extracts that co-purified with a GST-FOG-2 fusion protein. All proteins identified are members of the NuRD nucleosome-remodeling complex. Using in vitro binding and co-immunoprecipitation assays, we demonstrate that Metastasis-Associated proteins (MTA)-1, 2 and 3 and Retinoblastoma binding proteins RbAp46 and RbAp48 interact with FOG-2, but not with a mutant form of FOG-2 that is unable to repress transcription. Further, we define a novel domain located in the C-terminal portion of MTA-1 that mediates the FOG-2/MTA-1 interaction. We also demonstrate that knockdown of MTA protein expression dramatically impairs the ability of FOG-2 to repress GATA4 activity. Finally, we show that the zinc finger domain of MTA-1 is required for FOG-2 mediated transcriptional repression and that this domain interacts with RbAp46 and RbAp48 subunits of the NuRD complex. Together, these results demonstrate the importance of FOG-2/MTA/RbAp interactions for FOG-2 mediated transcriptional repression and further define the molecular interactions between the FOG Repression Motif and the NuRD complex. PMID:18067919

  9. Trichoderma reesei CRE1-mediated Carbon Catabolite Repression in Re-sponse to Sophorose Through RNA Sequencing Analysis.

    PubMed

    Antoniêto, Amanda Cristina Campos; de Paula, Renato Graciano; Castro, Lílian Dos Santos; Silva-Rocha, Rafael; Persinoti, Gabriela Felix; Silva, Roberto Nascimento

    2016-04-01

    Carbon catabolite repression (CCR) mediated by CRE1 in Trichoderma reesei emerged as a mechanism by which the fungus could adapt to new environments. In the presence of readily available carbon sources such as glucose, the fungus activates this mechanism and inhibits the production of cellulolytic complex enzymes to avoid unnecessary energy expenditure. CCR has been well described for the growth of T. reesei in cellulose and glucose, however, little is known about this process when the carbon source is sophorose, one of the most potent inducers of cellulase production. Thus, we performed high-throughput RNA sequencing to better understand CCR during cellulase formation in the presence of sophorose, by comparing the mutant ∆cre1 with its parental strain, QM9414. Of the 9129 genes present in the genome of T. reesei, 184 were upregulated and 344 downregulated in the mutant strain ∆cre1 compared to QM9414. Genes belonging to the CAZy database, and those encoding transcription factors and transporters are among the gene classes that were repressed by CRE1 in the presence of sophorose; most were possible indirectly regulated by CRE1. We also observed that CRE1 activity is carbon-dependent. A recent study from our group showed that in cellulose, CRE1 repress different groups of genes when compared to sophorose. CCR differences between these carbon sources may be due to the release of cellodextrins in the cellulose polymer, resulting in different targets of CRE1 in both carbon sources. These results contribute to a better understanding of CRE1-mediated CCR in T. reesei when glucose comes from a potent inducer of cellulase production such as sophorose, which could prove useful in improving cellulase production by the biotechnology sector. PMID:27226768

  10. Cyclic Di-GMP-Mediated Repression of Swarming Motility by Pseudomonas aeruginosa PA14 Requires the MotAB Stator

    PubMed Central

    Kuchma, S. L.; Delalez, N. J.; Filkins, L. M.; Snavely, E. A.; Armitage, J. P.

    2014-01-01

    The second messenger cyclic diguanylate (c-di-GMP) plays a critical role in the regulation of motility. In Pseudomonas aeruginosa PA14, c-di-GMP inversely controls biofilm formation and surface swarming motility, with high levels of this dinucleotide signal stimulating biofilm formation and repressing swarming. P. aeruginosa encodes two stator complexes, MotAB and MotCD, that participate in the function of its single polar flagellum. Here we show that the repression of swarming motility requires a functional MotAB stator complex. Mutating the motAB genes restores swarming motility to a strain with artificially elevated levels of c-di-GMP as well as stimulates swarming in the wild-type strain, while overexpression of MotA from a plasmid represses swarming motility. Using point mutations in MotA and the FliG rotor protein of the motor supports the conclusion that MotA-FliG interactions are critical for c-di-GMP-mediated swarming inhibition. Finally, we show that high c-di-GMP levels affect the localization of a green fluorescent protein (GFP)-MotD fusion, indicating a mechanism whereby this second messenger has an impact on MotCD function. We propose that when c-di-GMP level is high, the MotAB stator can displace MotCD from the motor, thereby affecting motor function. Our data suggest a newly identified means of c-di-GMP-mediated control of surface motility, perhaps conserved among Pseudomonas, Xanthomonas, and other organisms that encode two stator systems. PMID:25349157

  11. Phosphoribulokinase mediates nitrogenase-induced carbon dioxide fixation gene repression in Rhodobacter sphaeroides.

    PubMed

    Farmer, Ryan M; Tabita, F Robert

    2015-11-01

    In many organisms there is a balance between carbon and nitrogen metabolism. These observations extend to the nitrogen-fixing, nonsulfur purple bacteria, which have the classic family of P(II) regulators that coordinate signals of carbon and nitrogen status to regulate nitrogen metabolism. Curiously, these organisms also possess a reverse mechanism to regulate carbon metabolism based on cellular nitrogen status. In this work, studies in Rhodobacter sphaeroides firmly established that the activity of the enzyme that catalyses nitrogen fixation, nitrogenase, induces a signal that leads to repression of genes encoding enzymes of the Calvin-Benson-Bassham (CBB) CO2 fixation pathway. Additionally, genetic and metabolomic experiments revealed that NADH-activated phosphoribulokinase is an intermediate in the signalling pathway. Thus, nitrogenase activity appears to be linked to cbb gene repression through phosphoribulokinase. PMID:26306848

  12. Spalt-mediated dve repression is a critical regulatory motif and coordinates with Iroquois complex in Drosophila vein formation.

    PubMed

    Sugimori, Seiko; Hasegawa, Aya; Nakagoshi, Hideki

    2016-08-01

    Veins are longitudinal cuticular structures that maintain shape of the wing. Drosophila melanogaster has six longitudinal veins (L1-L6) and two cross veins. The Zn-finger transcription factors of Spalt-complex (Sal) are required for positioning of the L2 and L5, and the homeodomain transcription factors of Iroquois complex (Iro-C) are required for formation of the L3 and L5 veins. The homeodomain transcriptional repressor Defective proventriculus (Dve) is uniformly expressed in the wing pouch of the larval imaginal disc. However, dve mutant wings showed loss of the L2 and L5, but not of the L3 and L4 veins. Temporal dve knockdown experiments indicate that the Dve activity is required for vein formation from late third larval instar to the prepupal stage. In the prepupal wing, Dve expression becomes nearly complementary to that of Sal through the Sal-mediated dve repression. Furthermore, coexpression of Dve and Iro-C relieved of Sal-mediated repression is required for the L5 formation in a dose-dependent manner. The relationship between Sal, Dve, and Iro-C in wing vein specification is quite similar to that in ommatidial cell-type specification. Our results provide information about the conserved function of dve regulatory motifs in cell differentiation. PMID:27349585

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

    PubMed Central

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

    2015-01-01

    In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty. PMID:26671628

  14. Blue light-mediated transcriptional activation and repression of gene expression in bacteria.

    PubMed

    Jayaraman, Premkumar; Devarajan, Kavya; Chua, Tze Kwang; Zhang, Hanzhong; Gunawan, Erry; Poh, Chueh Loo

    2016-08-19

    Light-regulated modules offer unprecedented new ways to control cellular behavior in precise spatial and temporal resolution. The availability of such tools may dramatically accelerate the progression of synthetic biology applications. Nonetheless, current optogenetic toolbox of prokaryotes has potential issues such as lack of rapid and switchable control, less portable, low dynamic expression and limited parts. To address these shortcomings, we have engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light dependent DNA-binding protein EL222. We demonstrated that by modulating the dosage of light pulses or intensity we could control the level of gene expression precisely. We show that both light-inducible and repressible system can function in parallel with high spatial precision in a single cell and can be switched stably between ON- and OFF-states by repetitive pulses of blue light. In addition, the light-inducible and repressible expression kinetics were quantitatively analysed using a mathematical model. We further apply the system, for the first time, to optogenetically synchronize two receiver cells performing different logic behaviors over time using blue light as a molecular clock signal. Overall, our modular approach layers a transformative platform for next-generation light-controllable synthetic biology systems in prokaryotes. PMID:27353329

  15. A Conserved Upstream Open Reading Frame Mediates Sucrose-Induced Repression of TranslationW⃞

    PubMed Central

    Wiese, Anika; Elzinga, Nico; Wobbes, Barry; Smeekens, Sjef

    2004-01-01

    Sugars have been shown to regulate transcription of numerous genes in plants. Sucrose controls translation of the group S basic region leucine zipper (bZIP)-type transcription factor ATB2/AtbZIP11 (Rook et al., 1998a). This control requires the unusually long 5′ untranslated region (UTR) of the gene. Point mutations and deletions of the 5′UTR have uncovered the sequences involved. A highly conserved upstream open reading frame (uORF) coding for 42 amino acids is essential for the repression mechanism. It is conserved in 5′UTRs of bZIP transcription factors from other Arabidopsis thaliana genes and many other plants. ATB2/AtbZIP11 is normally expressed in association with vascular tissues. Ectopic expression of a 5′UTR construct shows that the sucrose repression system is functional in all tissues. AtbZIP2 is another Arabidopsis bZIP transcription factor gene harboring the conserved uORF, which is regulated similarly via sucrose-induced repression of translation. This suggests a general function of the conserved uORF in sucrose-controlled regulation of expression. Our findings imply the operation of a sucrose-sensing pathway that controls translation of several plant bZIP transcription factor genes harboring the conserved uORF in their 5′UTRs. Target genes of such transcription factors will then be regulated in sucrose-dependent way. PMID:15208401

  16. Estrogen receptor α can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation.

    PubMed

    Marques, Maud; Laflamme, Liette; Gaudreau, Luc

    2013-09-01

    Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells. PMID:23828038

  17. Estrogen receptor α can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation

    PubMed Central

    Marques, Maud; Laflamme, Liette; Gaudreau, Luc

    2013-01-01

    Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells. PMID:23828038

  18. GR SUMOylation and formation of an SUMO-SMRT/NCoR1-HDAC3 repressing complex is mandatory for GC-induced IR nGRE-mediated transrepression.

    PubMed

    Hua, Guoqiang; Paulen, Laetitia; Chambon, Pierre

    2016-02-01

    Unique among the nuclear receptor superfamily, the glucocorticoid (GC) receptor (GR) can exert three distinct transcriptional regulatory functions on binding of a single natural (cortisol in human and corticosterone in mice) and synthetic [e.g., dexamethasone (Dex)] hormone. The molecular mechanisms underlying GC-induced positive GC response element [(+)GRE]-mediated activation of transcription are partially understood. In contrast, these mechanisms remain elusive for GC-induced evolutionary conserved inverted repeated negative GC response element (IR nGRE)-mediated direct transrepression and for tethered indirect transrepression that is mediated by DNA-bound NF-κB/activator protein 1 (AP1)/STAT3 activators and instrumental in GC-induced anti-inflammatory activity. We demonstrate here that SUMOylation of lysine K293 (mouse K310) located within an evolutionary conserved sequence in the human GR N-terminal domain allows the formation of a GR-small ubiquitin-related modifiers (SUMOs)-NCoR1/SMRT-HDAC3 repressing complex mandatory for GC-induced IR nGRE-mediated direct repression in vitro, but does not affect transactivation. Importantly, these results were validated in vivo: in K310R mutant mice and in mice ablated selectively for nuclear receptor corepressor 1 (NCoR1)/silencing mediator for retinoid or thyroid-hormone receptors (SMRT) corepressors in skin keratinocytes, Dex-induced direct repression and the formation of repressing complexes on IR nGREs were impaired, whereas transactivation was unaffected. In mice selectively ablated for histone deacetylase 3 (HDAC3) in skin keratinocytes, GC-induced direct repression, but not bindings of GR and of corepressors NCoR1/SMRT, was abolished, indicating that HDAC3 is instrumental in IR nGRE-mediated repression. Moreover, we demonstrate that the binding of HDAC3 to IR nGREs in vivo is mediated through interaction with SMRT/NCoR1. We also show that the GR ligand binding domain (LBD) is not required for SMRT-mediated

  19. c-Fos mediates repression of the apical sodium-dependent bile acid transporter by fibroblast growth factor-19 in mice

    PubMed Central

    Ghosh, Ayantika; Chen, Frank; Banerjee, Swati; Xu, Ming

    2013-01-01

    Fibroblast growth factor-19 (FGF-19), a bile acid-responsive enterokine, is secreted by the ileum and regulates a variety of metabolic processes. These studies examined the signal transduction pathways operant in FGF-19-mediated repression of the apical sodium-dependent bile acid transporter (ASBT). Responses to FGF-19 were assessed in Caco-2 and CT-26 cells and in mice where c-fos was conditionally silenced in the intestine by a cre-lox strategy. FGF-19 treatment of Caco-2 cells or wild-type mice led to a significant reduction in ASBT protein expression and enhanced phosphorylation of extracellular signaling kinase 1/2 (ERK1/2), c-Fos, and c-Jun. FGF-19 treatment of Caco-2 cells led to a reduction in activity of the human ASBT promoter and this repression could be blocked by treatment with a mitogen-activated protein kinase/ERK kinase (MEK1/2) inhibitor or by silencing jun kinase 1, jun kinase 2, c-fos, or c-jun. Site directed mutagenesis of a c-fos binding element in the ASBT promoter blocked FGF-19-mediated repression in luciferase reporter constructs. ASBT promoter activity was repressed by FGF-19 in CT-26 cells and this repression could be reduced by MEK1/2 inhibition or silencing c-fos. FGF-19-mediated repression of ASBT protein expression was abrogated in mice where c-fos was conditionally silenced in the intestine. In contrast, ASBT was repressed in the c-Fos expressing gallbladders of the same mice. The studies demonstrate that FGF-19 represses the expression of ASBT in the ileum and gallbladder via a signal transduction pathway involving MEK1/2, ERK1/2, JNK1, JNK2, and c-Fos. PMID:24309182

  20. MTF-1-Mediated Repression of the Zinc Transporter Zip10 Is Alleviated by Zinc Restriction

    PubMed Central

    Lichten, Louis A.; Ryu, Moon-Suhn; Guo, Liang; Embury, Jennifer; Cousins, Robert J.

    2011-01-01

    The regulation of cellular zinc uptake is a key process in the overall mechanism governing mammalian zinc homeostasis and how zinc participates in cellular functions. We analyzed the zinc transporters of the Zip family in both the brain and liver of zinc-deficient animals and found a large, significant increase in Zip10 expression. Additionally, Zip10 expression decreased in response to zinc repletion. Moreover, isolated mouse hepatocytes, AML12 hepatocytes, and Neuro 2A cells also respond differentially to zinc availability in vitro. Measurement of Zip10 hnRNA and actinomycin D inhibition studies indicate that Zip10 was transcriptionally regulated by zinc deficiency. Through luciferase promoter constructs and ChIP analysis, binding of MTF-1 to a metal response element located 17 bp downstream of the transcription start site was shown to be necessary for zinc-induced repression of Zip10. Furthermore, zinc-activated MTF-1 causes down-regulation of Zip10 transcription by physically blocking Pol II movement through the gene. Lastly, ZIP10 is localized to the plasma membrane of hepatocytes and neuro 2A cells. Collectively, these results reveal a novel repressive role for MTF-1 in the regulation of the Zip10 zinc transporter expression by pausing Pol II transcription. ZIP10 may have roles in control of zinc homeostasis in specific sites particularly those of the brain and liver. Within that context ZIP10 may act as an important survival mechanism during periods of zinc inadequacy. PMID:21738690

  1. Autophagy-regulating TP53INP2 mediates muscle wasting and is repressed in diabetes

    PubMed Central

    Sala, David; Ivanova, Saška; Plana, Natàlia; Ribas, Vicent; Duran, Jordi; Bach, Daniel; Turkseven, Saadet; Laville, Martine; Vidal, Hubert; Karczewska-Kupczewska, Monika; Kowalska, Irina; Straczkowski, Marek; Testar, Xavier; Palacín, Manuel; Sandri, Marco; Serrano, Antonio L.; Zorzano, Antonio

    2014-01-01

    A precise balance between protein degradation and synthesis is essential to preserve skeletal muscle mass. Here, we found that TP53INP2, a homolog of the Drosophila melanogaster DOR protein that regulates autophagy in cellular models, has a direct impact on skeletal muscle mass in vivo. Using different transgenic mouse models, we demonstrated that muscle-specific overexpression of Tp53inp2 reduced muscle mass, while deletion of Tp53inp2 resulted in muscle hypertrophy. TP53INP2 activated basal autophagy in skeletal muscle and sustained p62-independent autophagic degradation of ubiquitinated proteins. Animals with muscle-specific overexpression of Tp53inp2 exhibited enhanced muscle wasting in streptozotocin-induced diabetes that was dependent on autophagy; however, TP53INP2 ablation mitigated experimental diabetes-associated muscle loss. The overexpression or absence of TP53INP2 did not affect muscle wasting in response to denervation, a condition in which autophagy is blocked, further indicating that TP53INP2 alters muscle mass by activating autophagy. Moreover, TP53INP2 expression was markedly repressed in muscle from patients with type 2 diabetes and in murine models of diabetes. Our results indicate that TP53INP2 negatively regulates skeletal muscle mass through activation of autophagy. Furthermore, we propose that TP53INP2 repression is part of an adaptive mechanism aimed at preserving muscle mass under conditions in which insulin action is deficient. PMID:24713655

  2. Receptor interconversion model of hormone action. 3. Estrogen receptor mediated repression of reporter gene activity in A431 cells.

    PubMed

    Nag, A; Park, I; Krust, A; Smith, R G

    1990-03-20

    The chicken estrogen receptor exists in three interconvertible forms, two of which bind estradiol with high affinity and one which lacks the capacity to bind estradiol. Interconversion is regulated by reactions involving ATP/Mg2+. By cotransfecting into A431 cells estrogen receptor cDNA in an expression vector together with the pA2 (-821/-87) tk-CAT vitellogenin construct, we demonstrate that constitutive expression of chloramphenicol acetyltransferase (CAT) activity can be regulated either by selection of ligand or by modifying phosphorylation reactions in the recipient cells. In the presence of estrogen receptors, constitutive expression of CAT activity is inhibited in three situations: (i) in the absence of an estrogenic ligand; (ii) in the presence of an anti-estrogen; and (iii) in the presence of an estrogenic ligand together with 12-O-tetradecanoylphorbol 13-acetate (TPA). Estrogen receptor mediated repression of constitutive CAT activity is not observed with the pA2 (-331/-87) tk-CAT construct, indicating that DNA sequences required for repression are located between -821 and -331 base pairs upstream of the transcription initiation site. PMID:2346742

  3. Structural basis for the Nanos-mediated recruitment of the CCR4–NOT complex and translational repression

    PubMed Central

    Bhandari, Dipankar; Raisch, Tobias; Weichenrieder, Oliver; Jonas, Stefanie; Izaurralde, Elisa

    2014-01-01

    The RNA-binding proteins of the Nanos family play an essential role in germ cell development and survival in a wide range of metazoan species. They function by suppressing the expression of target mRNAs through the recruitment of effector complexes, which include the CCR4–NOT deadenylase complex. Here, we show that the three human Nanos paralogs (Nanos1–3) interact with the CNOT1 C-terminal domain and determine the structural basis for the specific molecular recognition. Nanos1–3 bind CNOT1 through a short CNOT1-interacting motif (NIM) that is conserved in all vertebrates and some invertebrate species. The crystal structure of the human Nanos1 NIM peptide bound to CNOT1 reveals that the peptide opens a conserved hydrophobic pocket on the CNOT1 surface by inserting conserved aromatic residues. The substitutions of these aromatic residues in the Nanos1–3 NIMs abolish binding to CNOT1 and abrogate the ability of the proteins to repress translation. Our findings provide the structural basis for the recruitment of the CCR4–NOT complex by vertebrate Nanos, indicate that the NIMs are the major determinants of the translational repression mediated by Nanos, and identify the CCR4–NOT complex as the main effector complex for Nanos function. PMID:24736845

  4. BCL6 induces EMT by promoting the ZEB1-mediated transcription repression of E-cadherin in breast cancer cells.

    PubMed

    Yu, Jin-Mei; Sun, Wei; Hua, Fang; Xie, Jing; Lin, Heng; Zhou, Dan-Dan; Hu, Zhuo-Wei

    2015-09-01

    B-cell CLL/lymphoma 6 (BCL6), a transcriptional repressor, is involved in the development and progression of breast cancers with uncertain mechanism. The purpose of this study is to investigate the potential effect and mechanism of BCL6 in the regulation of epithelial-mesenchymal transition (EMT), a critical cellular process for controlling the development and progression of breast cancers. We found that BCL6 promoted invasion, migration and growth by stimulating EMT in breast cancer cells. BCL6 induced EMT by enhancing the expression of transcriptional repressor ZEB1 which bound to the E-cadherin promoter and repressing the E-cadherin transcription. Deletion of ZEB1 protected against the pro-EMT roles of BCL6 by restoring the expression of E-cadherin in these cells. Moreover, inhibition of BCL6 with BCL6 inhibitor 79-6 suppressed these functions of BCL6 in breast cancer cells. These findings indicate that BCL6 promotes EMT via enhancing the ZEB1-mediated transcriptional repression of E-cadherin in breast cancer cells. Targeting BCL6 has therapeutic potential against the development and progression of breast cancer. PMID:26049022

  5. mRNA Targeting to Endoplasmic Reticulum Precedes Ago Protein Interaction and MicroRNA (miRNA)-mediated Translation Repression in Mammalian Cells*

    PubMed Central

    Barman, Bahnisikha; Bhattacharyya, Suvendra N.

    2015-01-01

    MicroRNA (miRNA) binds to the 3′-UTR of its target mRNAs to repress protein synthesis. Extensive research was done to understand the mechanism of miRNA-mediated repression in animal cells. Considering the progress in understanding the mechanism, information about the subcellular sites of miRNA-mediated repression is surprisingly limited. In this study, using an inducible expression system for an miRNA target message, we have delineated how a target mRNA passes through polysome association and Ago2 interaction steps on rough endoplasmic reticulum (ER) before the miRNA-mediated repression sets in. From this study, de novo formed target mRNA localization to the ER-bound polysomes manifested as the earliest event, which is followed by Ago2 micro-ribonucleoprotein binding, and translation repression of target message. Compartmentalization of this process to rough ER membrane ensures enrichment of miRNA-targeted messages and micro-ribonucleoprotein components on ER upon reaching a steady state. PMID:26304123

  6. The putrescine biosynthesis pathway in Lactococcus lactis is transcriptionally regulated by carbon catabolic repression, mediated by CcpA.

    PubMed

    Linares, Daniel M; del Río, Beatriz; Ladero, Victor; Redruello, Begoña; Martín, María Cruz; Fernández, María; Alvarez, Miguel A

    2013-07-01

    Lactococcus lactis is the lactic acid bacterium most widely used by the dairy industry as a starter for the manufacture of fermented products such as cheese and buttermilk. However, some strains produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The proteins involved in this pathway, including those necessary for agmatine uptake and conversion into putrescine, are encoded by the aguB, aguD, aguA and aguC genes, which together form an operon. This paper reports the mechanism of regulation of putrescine biosynthesis in L. lactis. It is shown that the aguBDAC operon, which contains a cre site at the promoter of aguB (the first gene of the operon), is transcriptionally regulated by carbon catabolic repression (CCR) mediated by the catabolite control protein CcpA. PMID:23688550

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

    SciTech Connect

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

    2009-08-28

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

  8. Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin Development.

    PubMed

    Perdigoto, Carolina N; Dauber, Katherine L; Bar, Carmit; Tsai, Pai-Chi; Valdes, Victor J; Cohen, Idan; Santoriello, Francis J; Zhao, Dejian; Zheng, Deyou; Hsu, Ya-Chieh; Ezhkova, Elena

    2016-07-01

    An increasing amount of evidence indicates that developmental programs are tightly regulated by the complex interplay between signaling pathways, as well as transcriptional and epigenetic processes. Here, we have uncovered coordination between transcriptional and morphogen cues to specify Merkel cells, poorly understood skin cells that mediate light touch sensations. In murine dorsal skin, Merkel cells are part of touch domes, which are skin structures consisting of specialized keratinocytes, Merkel cells, and afferent neurons, and are located exclusively around primary hair follicles. We show that the developing primary hair follicle functions as a niche required for Merkel cell specification. We find that intraepidermal Sonic hedgehog (Shh) signaling, initiated by the production of Shh ligand in the developing hair follicles, is required for Merkel cell specification. The importance of Shh for Merkel cell formation is further reinforced by the fact that Shh overexpression in embryonic epidermal progenitors leads to ectopic Merkel cells. Interestingly, Shh signaling is common to primary, secondary, and tertiary hair follicles, raising the possibility that there are restrictive mechanisms that regulate Merkel cell specification exclusively around primary hair follicles. Indeed, we find that loss of Polycomb repressive complex 2 (PRC2) in the epidermis results in the formation of ectopic Merkel cells that are associated with all hair types. We show that PRC2 loss expands the field of epidermal cells competent to differentiate into Merkel cells through the upregulation of key Merkel-differentiation genes, which are known PRC2 targets. Importantly, PRC2-mediated repression of the Merkel cell differentiation program requires inductive Shh signaling to form mature Merkel cells. Our study exemplifies how the interplay between epigenetic and morphogen cues regulates the complex patterning and formation of the mammalian skin structures. PMID:27414999

  9. Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin Development

    PubMed Central

    Bar, Carmit; Tsai, Pai-Chi; Valdes, Victor J.; Cohen, Idan; Santoriello, Francis J.; Zhao, Dejian; Hsu, Ya-Chieh; Ezhkova, Elena

    2016-01-01

    An increasing amount of evidence indicates that developmental programs are tightly regulated by the complex interplay between signaling pathways, as well as transcriptional and epigenetic processes. Here, we have uncovered coordination between transcriptional and morphogen cues to specify Merkel cells, poorly understood skin cells that mediate light touch sensations. In murine dorsal skin, Merkel cells are part of touch domes, which are skin structures consisting of specialized keratinocytes, Merkel cells, and afferent neurons, and are located exclusively around primary hair follicles. We show that the developing primary hair follicle functions as a niche required for Merkel cell specification. We find that intraepidermal Sonic hedgehog (Shh) signaling, initiated by the production of Shh ligand in the developing hair follicles, is required for Merkel cell specification. The importance of Shh for Merkel cell formation is further reinforced by the fact that Shh overexpression in embryonic epidermal progenitors leads to ectopic Merkel cells. Interestingly, Shh signaling is common to primary, secondary, and tertiary hair follicles, raising the possibility that there are restrictive mechanisms that regulate Merkel cell specification exclusively around primary hair follicles. Indeed, we find that loss of Polycomb repressive complex 2 (PRC2) in the epidermis results in the formation of ectopic Merkel cells that are associated with all hair types. We show that PRC2 loss expands the field of epidermal cells competent to differentiate into Merkel cells through the upregulation of key Merkel-differentiation genes, which are known PRC2 targets. Importantly, PRC2-mediated repression of the Merkel cell differentiation program requires inductive Shh signaling to form mature Merkel cells. Our study exemplifies how the interplay between epigenetic and morphogen cues regulates the complex patterning and formation of the mammalian skin structures. PMID:27414999

  10. Ikaros mediates gene silencing in T cells through Polycomb repressive complex 2

    PubMed Central

    Oravecz, Attila; Apostolov, Apostol; Polak, Katarzyna; Jost, Bernard; Le Gras, Stéphanie; Chan, Susan; Kastner, Philippe

    2015-01-01

    T-cell development is accompanied by epigenetic changes that ensure the silencing of stem cell-related genes and the activation of lymphocyte-specific programmes. How transcription factors influence these changes remains unclear. We show that the Ikaros transcription factor forms a complex with Polycomb repressive complex 2 (PRC2) in CD4−CD8− thymocytes and allows its binding to more than 500 developmentally regulated loci, including those normally activated in haematopoietic stem cells and others induced by the Notch pathway. Loss of Ikaros in CD4−CD8− cells leads to reduced histone H3 lysine 27 trimethylation and ectopic gene expression. Furthermore, Ikaros binding triggers PRC2 recruitment and Ikaros interacts with PRC2 independently of the nucleosome remodelling and deacetylation complex. Our results identify Ikaros as a fundamental regulator of PRC2 function in developing T cells. PMID:26549758

  11. Ikaros mediates gene silencing in T cells through Polycomb repressive complex 2.

    PubMed

    Oravecz, Attila; Apostolov, Apostol; Polak, Katarzyna; Jost, Bernard; Le Gras, Stéphanie; Chan, Susan; Kastner, Philippe

    2015-01-01

    T-cell development is accompanied by epigenetic changes that ensure the silencing of stem cell-related genes and the activation of lymphocyte-specific programmes. How transcription factors influence these changes remains unclear. We show that the Ikaros transcription factor forms a complex with Polycomb repressive complex 2 (PRC2) in CD4(-)CD8(-) thymocytes and allows its binding to more than 500 developmentally regulated loci, including those normally activated in haematopoietic stem cells and others induced by the Notch pathway. Loss of Ikaros in CD4(-)CD8(-) cells leads to reduced histone H3 lysine 27 trimethylation and ectopic gene expression. Furthermore, Ikaros binding triggers PRC2 recruitment and Ikaros interacts with PRC2 independently of the nucleosome remodelling and deacetylation complex. Our results identify Ikaros as a fundamental regulator of PRC2 function in developing T cells. PMID:26549758

  12. Velvet-mediated repression of β-glucan synthesis in Aspergillus nidulans spores

    PubMed Central

    Park, Hee-Soo; Man Yu, Yeong; Lee, Mi-Kyung; Jae Maeng, Pil; Chang Kim, Sun; Yu, Jae-Hyuk

    2015-01-01

    Beta-glucans are a heterologous group of fibrous glucose polymers that are a major constituent of cell walls in Ascomycetes and Basidiomycetes fungi. Synthesis of β (1,3)- and (1,6)-glucans is coordinated with fungal cell growth and development, thus, is under tight genetic regulation. Here, we report that β-glucan synthesis in both asexual and sexual spores is turned off by the NF-kB like fungal regulators VosA and VelB in Aspergillus nidulans. Our genetic and genomic analyses have revealed that both VosA and VelB are necessary for proper down-regulation of cell wall biosynthetic genes including those associated with β-glucan synthesis in both types of spores. The deletion of vosA or velB results in elevated accumulation of β-glucan in asexual spores. Double mutant analyses indicate that VosA and VelB play an inter-dependent role in repressing β-glucan synthesis in asexual spores. In vivo chromatin immuno-precipitation analysis shows that both VelB and VosA bind to the promoter region of the β-glucan synthase gene fksA in asexual spores. Similarly, VosA is required for proper repression of β-glucan synthesis in sexual spores. In summary, the VosA-VelB hetero-complex is a key regulatory unit tightly controlling proper levels of β-glucan synthesis in asexual and sexual spores. PMID:25960370

  13. Velvet-mediated repression of β-glucan synthesis in Aspergillus nidulans spores.

    PubMed

    Park, Hee-Soo; Yu, Yeong Man; Lee, Mi-Kyung; Maeng, Pil Jae; Kim, Sun Chang; Yu, Jae-Hyuk

    2015-01-01

    Beta-glucans are a heterologous group of fibrous glucose polymers that are a major constituent of cell walls in Ascomycetes and Basidiomycetes fungi. Synthesis of β (1,3)- and (1,6)-glucans is coordinated with fungal cell growth and development, thus, is under tight genetic regulation. Here, we report that β-glucan synthesis in both asexual and sexual spores is turned off by the NF-kB like fungal regulators VosA and VelB in Aspergillus nidulans. Our genetic and genomic analyses have revealed that both VosA and VelB are necessary for proper down-regulation of cell wall biosynthetic genes including those associated with β-glucan synthesis in both types of spores. The deletion of vosA or velB results in elevated accumulation of β-glucan in asexual spores. Double mutant analyses indicate that VosA and VelB play an inter-dependent role in repressing β-glucan synthesis in asexual spores. In vivo chromatin immuno-precipitation analysis shows that both VelB and VosA bind to the promoter region of the β-glucan synthase gene fksA in asexual spores. Similarly, VosA is required for proper repression of β-glucan synthesis in sexual spores. In summary, the VosA-VelB hetero-complex is a key regulatory unit tightly controlling proper levels of β-glucan synthesis in asexual and sexual spores. PMID:25960370

  14. Repression of DNA-binding dependent glucocorticoid receptor-mediated gene expression

    PubMed Central

    Muzikar, Katy A.; Nickols, Nicholas G.; Dervan, Peter B.

    2009-01-01

    The glucocorticoid receptor (GR) affects the transcription of genes involved in diverse processes, including energy metabolism and the immune response, through DNA-binding dependent and independent mechanisms. The DNA-binding dependent mechanism occurs by direct binding of GR to glucocorticoid response elements (GREs) at regulatory regions of target genes. The DNA-binding independent mechanism involves binding of GR to transcription factors and coactivators that, in turn, contact DNA. A small molecule that competes with GR for binding to GREs could be expected to affect the DNA-dependent pathway selectively by interfering with the protein-DNA interface. We show that a DNA-binding polyamide that targets the consensus GRE sequence binds the glucocorticoid-induced zipper (GILZ) GRE, inhibits expression of GILZ and several other known GR target genes, and reduces GR occupancy at the GILZ promoter. Genome-wide expression analysis of the effects of this polyamide on a set of glucocorticoid-induced and -repressed genes could help to elucidate the mechanism of GR regulation for these genes. PMID:19805343

  15. An Intronic Flk1 Enhancer Directs Arterial-Specific Expression via RBPJ-Mediated Venous Repression

    PubMed Central

    Becker, Philipp W.; Sacilotto, Natalia; Nornes, Svanhild; Neal, Alice; Thomas, Max O.; Liu, Ke; Preece, Chris; Ratnayaka, Indrika; Davies, Benjamin; Bou-Gharios, George

    2016-01-01

    Objective— The vascular endothelial growth factor (VEGF) receptor Flk1 is essential for vascular development, but the signaling and transcriptional pathways by which its expression is regulated in endothelial cells remain unclear. Although previous studies have identified 2 Flk1 regulatory enhancers, these are dispensable for Flk1 expression, indicating that additional enhancers contribute to Flk1 regulation in endothelial cells. In the present study, we sought to identify Flk1 enhancers contributing to expression in endothelial cells. Approach and Results— A region of the 10th intron of the Flk1 gene (Flk1in10) was identified as a putative enhancer and tested in mouse and zebrafish transgenic models. This region robustly directed reporter gene expression in arterial endothelial cells. Using a combination of targeted mutagenesis of transcription factor–binding sites and gene silencing of transcription factors, we found that Gata and Ets factors are required for Flk1in10 enhancer activity in all endothelial cells. Furthermore, we showed that activity of the Flk1in10 enhancer is restricted to arteries through repression of gene expression in venous endothelial cells by the Notch pathway transcriptional regulator Rbpj. Conclusions— This study demonstrates a novel mechanism of arterial–venous identity acquisition, indicates a direct link between the Notch and VEGF signaling pathways, and illustrates how cis-regulatory diversity permits differential expression outcomes from a limited repertoire of transcriptional regulators. PMID:27079877

  16. PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

    PubMed Central

    González Wusener, Ana E.; González, Ángela; Nakamura, Fumihiko; Arregui, Carlos O.

    2016-01-01

    ABSTRACT Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration. PMID:26700725

  17. EphrinB2 repression through ZEB2 mediates tumour invasion and anti-angiogenic resistance

    PubMed Central

    Depner, C.; zum Buttel, H.; Böğürcü, N.; Cuesta, A. M.; Aburto, M. R.; Seidel, S.; Finkelmeier, F.; Foss, F.; Hofmann, J.; Kaulich, K.; Barbus, S.; Segarra, M.; Reifenberger, G.; Garvalov, B. K.; Acker, T.; Acker-Palmer, A.

    2016-01-01

    Diffuse invasion of the surrounding brain parenchyma is a major obstacle in the treatment of gliomas with various therapeutics, including anti-angiogenic agents. Here we identify the epi-/genetic and microenvironmental downregulation of ephrinB2 as a crucial step that promotes tumour invasion by abrogation of repulsive signals. We demonstrate that ephrinB2 is downregulated in human gliomas as a consequence of promoter hypermethylation and gene deletion. Consistently, genetic deletion of ephrinB2 in a murine high-grade glioma model increases invasion. Importantly, ephrinB2 gene silencing is complemented by a hypoxia-induced transcriptional repression. Mechanistically, hypoxia-inducible factor (HIF)-1α induces the EMT repressor ZEB2, which directly downregulates ephrinB2 through promoter binding to enhance tumour invasiveness. This mechanism is activated following anti-angiogenic treatment of gliomas and is efficiently blocked by disrupting ZEB2 activity. Taken together, our results identify ZEB2 as an attractive therapeutic target to inhibit tumour invasion and counteract tumour resistance mechanisms induced by anti-angiogenic treatment strategies. PMID:27470974

  18. COX5B Regulates MAVS-mediated Antiviral Signaling through Interaction with ATG5 and Repressing ROS Production

    PubMed Central

    Nie, Xuanli; Sun, Liwei; Tang, Tie-shan; Chen, Dahua; Sun, Qinmiao

    2012-01-01

    Innate antiviral immunity is the first line of the host defense system that rapidly detects invading viruses. Mitochondria function as platforms for innate antiviral signal transduction in mammals through the adaptor protein, MAVS. Excessive activation of MAVS-mediated antiviral signaling leads to dysfunction of mitochondria and cell apoptosis that likely causes the pathogenesis of autoimmunity. However, the mechanism of how MAVS is regulated at mitochondria remains unknown. Here we show that the Cytochrome c Oxidase (CcO) complex subunit COX5B physically interacts with MAVS and negatively regulates the MAVS-mediated antiviral pathway. Mechanistically, we find that while activation of MAVS leads to increased ROS production and COX5B expression, COX5B down-regulated MAVS signaling by repressing ROS production. Importantly, our study reveals that COX5B coordinates with the autophagy pathway to control MAVS aggregation, thereby balancing the antiviral signaling activity. Thus, our study provides novel insights into the link between mitochondrial electron transport system and the autophagy pathway in regulating innate antiviral immunity. PMID:23308066

  19. NFATc1 Mediates HDAC-Dependent Transcriptional Repression of Osteocalcin Expression During Osteoblast Differentiation

    PubMed Central

    Choo, Min-Kyung; Yeo, Hyeonju; Zayzafoon, Majd

    2009-01-01

    We previously reported that the in vivo and in vitro suppression of Nuclear Factor of Activated T Cells (NFAT) signaling increases osteoblast differentiation and bone formation. To investigate the mechanism by which NFATc1 regulates osteoblast differentiation, we established an osteoblast cell line that overexpresses a constitutively active NFATc1 (ca-NFATc1). The activation of NFATc1 significantly inhibits osteoblast differentiation and function, demonstrated by inhibition of alkaline phosphatase activity and mineralization as well as a decrease in gene expression of early and late markers of osteoblast differentiation such as osterix and osteocalcin, respectively. By focusing on the specific role of NFATc1 during late differentiation, we discovered that the inhibition of osteocalcin gene expression by NFATc1 was associated with a repression of the osteocalcin promoter activity, and a decrease in TCF/LEF transactivation. Also, overexpression of NFATc1 completely blocked the decrease in total histone deacetylase (HDAC) activity during osteoblast differentiation and prevented the hyperacetylation of histones H3 and H4. Mechanistically, we show by Chromatin Immunoprecipitation (ChIP) assay that the overexpression of NFATc1 sustains the binding of HDAC3 on the proximal region of the osteocalcin promoter, resulting in complete hypoacetylation of histones H3 and H4 when compared to GFP-expressing osteoblasts. In contrast, the inhibition of NFATc1 nuclear translocation either by cyclosporin or by using primary mouse osteoblasts with deleted calcineurin b1 prevents HDAC3 from associating with the proximal regulatory site of the osteocalcin promoter. These preliminary results suggest that NFATc1 acts as a transcriptional co-repressor of osteocalcin promoter possibly in an HDAC-dependent manner. PMID:19463978

  20. Net, a negative Ras-switchable TCF, contains a second inhibition domain, the CID, that mediates repression through interactions with CtBP and de-acetylation.

    PubMed

    Criqui-Filipe, P; Ducret, C; Maira, S M; Wasylyk, B

    1999-06-15

    Signalling cascades are integrated at the transcriptional level by the interplay between factors such as the ternary complex factors (TCFs) that interact with serum response factor (SRF) and the serum response element (SRE) of the fos promoter. Net is a negative TCF that is switched to a positive regulator by the Ras signal. To understand the mechanisms of repression by Net, we used a yeast two-hybrid screen to identify factors that interact with its inhibitory domain. We isolated mCtBP1, the murine homologue of huCtBP1, a factor implicated in negative regulation of transformation by E1A plus Ras. We show that mCtBP1 interacts strongly with Net both in vitro and in vivo. The CtBP interaction domain of Net, the CID, mediates repression independently of the previously identified negative element, the NID. The CID inhibits by recruiting the co-repressor mCtBP1. The CID and mCtBP1 need to use de-acetylase activity for repression, whereas the NID apparently represses by other mechanisms. Finally, we provide evidence that CtBP and de-acetylation repress the c-fos SRE in low serum when it is inactive, but not in high serum when it is active. These results provide insights into the cross-talk between pathways that inhibit and stimulate transformation at the level of Net, a regulator of gene expression. PMID:10369679

  1. Acute TNF-induced repression of cell identity genes is mediated by NFκB-directed redistribution of cofactors from super-enhancers.

    PubMed

    Schmidt, Søren Fisker; Larsen, Bjørk Ditlev; Loft, Anne; Nielsen, Ronni; Madsen, Jesper Grud Skat; Mandrup, Susanne

    2015-09-01

    The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in low-grade adipose tissue inflammation and development of insulin resistance during obesity. In this context, nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) is directly involved and required for the acute activation of the inflammatory gene program. Here, we show that the major transactivating subunit of NFκB, v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), is also required for acute TNF-induced suppression of adipocyte genes. Notably, this repression does not involve RELA binding to the associated enhancers but rather loss of cofactors and enhancer RNA (eRNA) selectively from high-occupancy sites within super-enhancers. Based on these data, we have developed models that, with high accuracy, predict which enhancers and genes are repressed by TNF in adipocytes. We show that these models are applicable to other cell types where TNF represses genes associated with super-enhancers in a highly cell-type-specific manner. Our results propose a novel paradigm for NFκB-mediated repression, whereby NFκB selectively redistributes cofactors from high-occupancy enhancers, thereby specifically repressing super-enhancer-associated cell identity genes. PMID:26113076

  2. Acute TNF-induced repression of cell identity genes is mediated by NFκB-directed redistribution of cofactors from super-enhancers

    PubMed Central

    Schmidt, Søren Fisker; Larsen, Bjørk Ditlev; Loft, Anne; Nielsen, Ronni; Madsen, Jesper Grud Skat; Mandrup, Susanne

    2015-01-01

    The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in low-grade adipose tissue inflammation and development of insulin resistance during obesity. In this context, nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) is directly involved and required for the acute activation of the inflammatory gene program. Here, we show that the major transactivating subunit of NFκB, v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), is also required for acute TNF-induced suppression of adipocyte genes. Notably, this repression does not involve RELA binding to the associated enhancers but rather loss of cofactors and enhancer RNA (eRNA) selectively from high-occupancy sites within super-enhancers. Based on these data, we have developed models that, with high accuracy, predict which enhancers and genes are repressed by TNF in adipocytes. We show that these models are applicable to other cell types where TNF represses genes associated with super-enhancers in a highly cell-type–specific manner. Our results propose a novel paradigm for NFκB-mediated repression, whereby NFκB selectively redistributes cofactors from high-occupancy enhancers, thereby specifically repressing super-enhancer-associated cell identity genes. PMID:26113076

  3. Region-Specific Activation of oskar mRNA Translation by Inhibition of Bruno-Mediated Repression

    PubMed Central

    Kim, Goheun; Pai, Chin-I; Sato, Keiji; Person, Maria D.; Nakamura, Akira; Macdonald, Paul M.

    2015-01-01

    A complex program of translational repression, mRNA localization, and translational activation ensures that Oskar (Osk) protein accumulates only at the posterior pole of the Drosophila oocyte. Inappropriate expression of Osk disrupts embryonic axial patterning, and is lethal. A key factor in translational repression is Bruno (Bru), which binds to regulatory elements in the osk mRNA 3′ UTR. After posterior localization of osk mRNA, repression by Bru must be alleviated. Here we describe an in vivo assay system to monitor the spatial pattern of Bru-dependent repression, separate from the full complexity of osk regulation. This assay reveals a form of translational activation—region-specific activation—which acts regionally in the oocyte, is not mechanistically coupled to mRNA localization, and functions by inhibiting repression by Bru. We also show that Bru dimerizes and identify mutations that disrupt this interaction to test its role in vivo. Loss of dimerization does not disrupt repression, as might have been expected from an existing model for the mechanism of repression. However, loss of dimerization does impair regional activation of translation, suggesting that dimerization may constrain, not promote, repression. Our work provides new insight into the question of how localized mRNAs become translationally active, showing that repression of osk mRNA is locally inactivated by a mechanism acting independent of mRNA localization. PMID:25723530

  4. FIAT represses ATF4-mediated transcription to regulate bone mass in transgenic mice.

    PubMed

    Yu, Vionnie W C; Ambartsoumian, Gourgen; Verlinden, Lieve; Moir, Janet M; Prud'homme, Josée; Gauthier, Claude; Roughley, Peter J; St-Arnaud, René

    2005-05-23

    We report the characterization of factor inhibiting activating transcription factor 4 (ATF4)-mediated transcription (FIAT), a leucine zipper nuclear protein. FIAT interacted with ATF4 to inhibit binding of ATF4 to DNA and block ATF4-mediated transcription of the osteocalcin gene in vitro. Transgenic mice overexpressing FIAT in osteoblasts also had reduced osteocalcin gene expression and decreased bone mineral density, bone volume, mineralized volume, trabecular thickness, trabecular number, and decreased rigidity of long bones. Mineral homeostasis, osteoclast number and activity, and osteoblast proliferation and apoptosis were unchanged in transgenics. Expression of osteoblastic differentiation markers was largely unaffected and type I collagen synthesis was unchanged. Mineral apposition rate was reduced in transgenic mice, suggesting that the lowered bone mass was due to a decline in osteoblast activity. This cell-autonomous decrease in osteoblast activity was confirmed by measuring reduced alkaline phosphatase activity and mineralization in primary osteoblast cultures. These results show that FIAT regulates bone mass accrual and establish FIAT as a novel transcriptional regulator of osteoblastic function. PMID:15911876

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

    SciTech Connect

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

    2008-06-20

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

  6. Tumor suppressor SMAR1 mediates cyclin D1 repression by recruitment of the SIN3/histone deacetylase 1 complex.

    PubMed

    Rampalli, Shravanti; Pavithra, L; Bhatt, Altaf; Kundu, Tapas K; Chattopadhyay, Samit

    2005-10-01

    Matrix attachment region binding proteins have been shown to play an important role in gene regulation by altering chromatin in a stage- and tissue-specific manner. Our previous studies report that SMAR1, a matrix-associated protein, regresses B16-F1-induced tumors in mice. Here we show SMAR1 targets the cyclin D1 promoter, a gene product whose dysregulation is attributed to breast malignancies. Our studies reveal that SMAR1 represses cyclin D1 gene expression, which can be reversed by small interfering RNA specific to SMAR1. We demonstrate that SMAR1 interacts with histone deacetylation complex 1, SIN3, and pocket retinoblastomas to form a multiprotein repressor complex. This interaction is mediated by the SMAR1(160-350) domain. Our data suggest SMAR1 recruits a repressor complex to the cyclin D1 promoter that results in deacetylation of chromatin at that locus, which spreads to a distance of at least the 5 kb studied upstream of the cyclin D1 promoter. Interestingly, we find that the high induction of cyclin D1 in breast cancer cell lines can be correlated to the decreased levels of SMAR1 in these lines. Our results establish the molecular mechanism exhibited by SMAR1 to regulate cyclin D1 by modification of chromatin. PMID:16166625

  7. Nanos-mediated repression of hid protects larval sensory neurons after a global switch in sensitivity to apoptotic signals.

    PubMed

    Bhogal, Balpreet; Plaza-Jennings, Amara; Gavis, Elizabeth R

    2016-06-15

    Dendritic arbor morphology is a key determinant of neuronal function. Once established, dendrite branching patterns must be maintained as the animal develops to ensure receptive field coverage. The translational repressors Nanos (Nos) and Pumilio (Pum) are required to maintain dendrite growth and branching of Drosophila larval class IV dendritic arborization (da) neurons, but their specific regulatory role remains unknown. We show that Nos-Pum-mediated repression of the pro-apoptotic gene head involution defective (hid) is required to maintain a balance of dendritic growth and retraction in class IV da neurons and that upregulation of hid results in decreased branching because of an increase in caspase activity. The temporal requirement for nos correlates with an ecdysone-triggered switch in sensitivity to apoptotic stimuli that occurs during the mid-L3 transition. We find that hid is required during pupariation for caspase-dependent pruning of class IV da neurons and that Nos and Pum delay pruning. Together, these results suggest that Nos and Pum provide a crucial neuroprotective regulatory layer to ensure that neurons behave appropriately in response to developmental cues. PMID:27256879

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

    SciTech Connect

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

    2010-04-25

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

  9. Enteric glia modulate epithelial cell proliferation and differentiation through 15-deoxy-Δ12,14-prostaglandin J2

    PubMed Central

    Bach-Ngohou, Kalyane; Mahé, Maxime M; Aubert, Philippe; Abdo, Hind; Boni, Sébastien; Bourreille, Arnaud; Denis, Marc G; Lardeux, Bernard; Neunlist, Michel; Masson, Damien

    2010-01-01

    The enteric nervous system (ENS) and its major component, enteric glial cells (EGCs), have recently been identified as a major regulator of intestinal epithelial barrier functions. Indeed, EGCs inhibit intestinal epithelial cell (IEC) proliferation and increase barrier resistance and IEC adhesion via the release of EGC-derived soluble factors. Interestingly, EGC regulation of intestinal epithelial barrier functions is reminiscent of previously reported peroxisome proliferator-activated receptor γ (PPARγ)-dependent functional effects. In this context, the present study aimed at identifying whether EGC could synthesize and release the main PPARγ ligand, 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), and regulate IEC functions such as proliferation and differentiation via a PPARγ dependent pathway. First, we demonstrated that the lipocalin but not the haematopoetic form for prostaglandin D synthase (PGDS), the enzyme responsible of 15dPGJ2 synthesis, was expressed in EGCs of the human submucosal plexus and of the subepithelium, as well as in rat primary culture of ENS and EGC lines. Next, 15dPGJ2 was identified in EGC supernatants of various EGC lines. 15dPGJ2 reproduced EGC inhibitory effects upon IEC proliferation, and inhibition of lipocalin PGDS expression by shRNA abrogated these effects. Furthermore, EGCs induced nuclear translocation of PPARγ in IEC, and both EGC and 15dPGJ2 effects upon IEC proliferation were prevented by the PPARγ antagonist GW9662. Finally, EGC induced differentiation-related gene expression in IEC through a PPARγ-dependent pathway. Our results identified 15dPGJ2 as a novel glial-derived mediator involved in the control of IEC proliferation/differentiation through activation of PPARγ. They also suggest that alterations of glial PGDS expression may modify intestinal epithelial barrier functions and be involved in the development of pathologies such as cancer or inflammatory bowel diseases. PMID:20478974

  10. Complementary Quantitative Proteomics Reveals that Transcription Factor AP-4 Mediates E-box-dependent Complex Formation for Transcriptional Repression of HDM2*

    PubMed Central

    Ku, Wei-Chi; Chiu, Sung-Kay; Chen, Yi-Ju; Huang, Hsin-Hung; Wu, Wen-Guey; Chen, Yu-Ju

    2009-01-01

    Transcription factor activating enhancer-binding protein 4 (AP-4) is a basic helix-loop-helix protein that binds to E-box elements. AP-4 has received increasing attention for its regulatory role in cell growth and development, including transcriptional repression of the human homolog of murine double minute 2 (HDM2), an important oncoprotein controlling cell growth and survival, by an unknown mechanism. Here we demonstrate that AP-4 binds to an E-box located in the HDM2-P2 promoter and represses HDM2 transcription in a p53-independent manner. Incremental truncations of AP-4 revealed that the C-terminal Gln/Pro-rich domain was essential for transcriptional repression of HDM2. To further delineate the molecular mechanism(s) of AP-4 transcriptional control and its potential implications, we used DNA-affinity purification followed by complementary quantitative proteomics, cICAT and iTRAQ labeling methods, to identify a previously unknown E-box-bound AP-4 protein complex containing 75 putative components. The two labeling methods complementarily quantified differentially AP-4-enriched proteins, including the most significant recruitment of DNA damage response proteins, followed by transcription factors, transcriptional repressors/corepressors, and histone-modifying proteins. Specific interaction of AP-4 with CCCTC binding factor, stimulatory protein 1, and histone deacetylase 1 (an AP-4 corepressor) was validated using AP-4 truncation mutants. Importantly, inclusion of trichostatin A did not alleviate AP-4-mediated repression of HDM2 transcription, suggesting a previously unidentified histone deacetylase-independent repression mechanism. In contrast, the complementary quantitative proteomics study suggested that transcription repression occurs via coordination of AP-4 with other transcription factors, histone methyltransferases, and/or a nucleosome remodeling SWI·SNF complex. In addition to previously known functions of AP-4, our data suggest that AP-4 participates in

  11. Repression of hypoxia-inducible factor α signaling by Set7-mediated methylation.

    PubMed

    Liu, Xing; Chen, Zhu; Xu, Chenxi; Leng, Xiaoqian; Cao, Hong; Ouyang, Gang; Xiao, Wuhan

    2015-05-26

    Hypoxia-inducible factor (HIF)-1α and HIF-2α are the main regulators of cellular responses to hypoxia. Post-translational modifications of HIF-1α and 2α are necessary to modulate their functions. The methylation of non-histone proteins by Set7, an SET domain-containing lysine methyltransferase, is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. In this study, we show that Set7 methylates HIF-1α at lysine 32 and HIF-2α at lysine K29; this methylation inhibits the expression of HIF-1α/2α targets by impairing the occupancy of HIF-α on hypoxia response element of HIF target gene promoter. Set7-null fibroblasts and the cells with shRNA-knocked down Set7 exhibit upregulated HIF target genes. Set7 inhibitor blocks HIF-1α/2α methylation to enhance HIF target gene expression. Set7-null fibroblasts and the cells with shRNA-knocked down Set7 or inhibition of Set7 by the inhibitor subjected to hypoxia display an increased glucose uptake and intracellular adenosine triphosphate levels. These findings define a novel modification of HIF-1α/2α and demonstrate that Set7-medited lysine methylation negatively regulates HIF-α transcriptional activity and HIF-1α-mediated glucose homeostasis. PMID:25897119

  12. Arabidopsis AINTEGUMENTA mediates salt tolerance by trans-repressing SCABP8.

    PubMed

    Meng, Lai-Sheng; Wang, Yi-Bo; Yao, Shun-Qiao; Liu, Aizhong

    2015-08-01

    The Arabidopsis AINTEGUMENTA (ANT) gene, which encodes an APETALA2 (AP2)-like transcription factor, controls plant organ cell number and organ size throughout shoot development. ANT is thus a key factor in the development of plant shoots. Here, we have found that ANT plays an essential role in conferring salt tolerance in Arabidopsis. ant-knockout mutants presented a salt-tolerant phenotype, whereas transgenic plants expressing ANT under the 35S promoter (35S:ANT) exhibited more sensitive phenotypes under high salt stress. Further analysis indicated that ANT functions mainly in the shoot response to salt toxicity. Target gene analysis revealed that ANT bound to the promoter of SOS3-LIKE CALCIUM BINDING PROTEIN 8 (SCABP8), which encodes a putative Ca(2+) sensor, thereby inhibiting expression of SCABP8 (also known as CBL10). It has been reported that the salt sensitivity of scabp8 is more prominent in shoot tissues. Genetic experiments indicated that the mutation of SCABP8 suppresses the ant-knockout salt-tolerant phenotype, implying that ANT functions as a negative transcriptional regulator of SCABP8 upon salt stress. Taken together, the above results reveal that ANT is a novel regulator of salt stress and that ANT binds to the SCABP8 promoter, mediating salt tolerance. PMID:26054800

  13. Epigenetic involvement of Alien/ESET complex in thyroid hormone-mediated repression of E2F1 gene expression and cell proliferation

    SciTech Connect

    Hong, Wei; Li, Jinru; Wang, Bo; Chen, Linfeng; Niu, Wenyan; Yao, Zhi; Baniahmad, Aria

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Corepressor Alien interacts with histone methyltransferase ESET in vivo. Black-Right-Pointing-Pointer Alien/ESET complex is recruited to nTRE of T3-responsive gene by liganded TR{beta}1. Black-Right-Pointing-Pointer ESET-mediated H3K9 methylation is required for liganded TR{beta}1-repressed transcription. Black-Right-Pointing-Pointer ESET is involved in T3-repressed G1/S phase transition and proliferation. -- Abstract: The ligand-bound thyroid hormone receptor (TR) is known to repress via a negative TRE (nTRE) the expression of E2F1, a key transcription factor that controls the G1/S phase transition. Alien has been identified as a novel interacting factor of E2F1 and acts as a corepressor of E2F1. The detailed molecular mechanism by which Alien inhibits E2F1 gene expression remains unclear. Here, we report that the histone H3 lysine 9 (H3K9) methyltransferase (HMT) ESET is an integral component of the corepressor Alien complex and the Alien/ESET complex is recruited to both sites, the E2F1 and the nTRE site of the E2F1 gene while the recruitment to the negative thyroid hormone response element (nTRE) is induced by the ligand-bound TR{beta}1 within the E2F1 gene promoter. We show that, overexpression of ESET promotes, whereas knockdown of ESET releases, the inhibition of TR{beta}1-regulated gene transcription upon T3 stimulation; and H3K9 methylation is required for TR{beta}1-repressed transcription. Furthermore, depletion of ESET impairs thyroid hormone-repressed proliferation as well as the G1/S transition of the cell cycle. Taken together, our data indicate that ESET is involved in TR{beta}1-mediated transcription repression and provide a molecular basis of thyroid hormone-induced repression of proliferation.

  14. HtrA3 is regulated by 15-deoxy-{Delta}12,14-prostaglandin J2 independently of PPAR{gamma} in clear cell renal cell carcinomas

    SciTech Connect

    Theoleyre, Sandrine; Mottier, Stephanie; Masson, Damien; Denis, Marc G.

    2010-04-09

    Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) ligands have been shown to possess anti-proliferative effects in many types of cancer. In clear cell renal cell carcinoma (CCRCC), the targets involved in these effects are not known. In this study, we demonstrated that, in CCRCC cell lines, the endogenous PPAR{gamma} ligand 15-deoxy-{Delta}12,14-prostaglandin J2 (15dPGJ2) induces the expression, both at the mRNA and the protein levels, of the HtrA3 gene. This gene belongs to the High-Temperature Requirement Factor A family of serine proteases that repress signaling by TGF-{beta} family members and inhibit cell migration. Rosiglitazone or ciglitazone, synthetic PPAR{gamma} agonists, did not induce HtrA3 expression, and the PPAR{gamma} antagonist GW9662 did not prevent 15dPGJ2 induction, suggesting that the up-regulation of HtrA3 by 15dPGJ2 is independent of PPAR{gamma}. The MEK/ERK inhibitor PD98059 dramatically repressed HtrA3 induction. Altogether, these data indicate that 15dPGJ2 is able to stimulate the expression of HtrA3 through an indirect mechanism involving the MEK/ERK pathway but independent of PPAR{gamma}. Our results provide a better understanding of the mechanisms involved in the regulation of HtrA3, a potential tumor suppressor gene.

  15. CHIP stabilizes amyloid precursor protein via proteasomal degradation and p53-mediated trans-repression of β-secretase.

    PubMed

    Singh, Amir Kumar; Pati, Uttam

    2015-08-01

    In patient with Alzheimer's disease (AD), deposition of amyloid-beta Aβ, a proteolytic cleavage of amyloid precursor protein (APP) by β-secretase/BACE1, forms senile plaque in the brain. BACE1 activation is caused due to oxidative stresses and dysfunction of ubiquitin-proteasome system (UPS), which is linked to p53 inactivation. As partial suppression of BACE1 attenuates Aβ generation and AD-related pathology, it might be an ideal target for AD treatment. We have shown that both in neurons and in HEK-APP cells, BACE1 is a new substrate of E3-ligase CHIP and an inverse relation exists between CHIP and BACE1 level. CHIP inhibits ectopic BACE1 level by promoting its ubiquitination and proteasomal degradation, thus reducing APP processing; it stabilizes APP in neurons, thus reducing Aβ. CHIP(U) (box) domain physically interacts with BACE1; however, both U-box and TPR domain are essential for ubiquitination and degradation of BACE1. Further, BACE1 is a downstream target of p53 and overexpression of p53 decreases BACE1 level. In HEK-APP cells, CHIP is shown to negatively regulate BACE1 promoter through stabilization of p53's DNA-binding conformation and its binding upon 5' UTR element (+127 to +150). We have thus discovered that CHIP regulates p53-mediated trans-repression of BACE1 at both transcriptional and post-translational level. We propose that a CHIP-BACE1-p53 feedback loop might control APP stabilization, which could further be utilized for new therapeutic intervention in AD. PMID:25773675

  16. CHIP stabilizes amyloid precursor protein via proteasomal degradation and p53-mediated trans-repression of β-secretase

    PubMed Central

    Singh, Amir Kumar; Pati, Uttam

    2015-01-01

    In patient with Alzheimer’s disease (AD), deposition of amyloid-beta Aβ, a proteolytic cleavage of amyloid precursor protein (APP) by β-secretase/BACE1, forms senile plaque in the brain. BACE1 activation is caused due to oxidative stresses and dysfunction of ubiquitin–proteasome system (UPS), which is linked to p53 inactivation. As partial suppression of BACE1 attenuates Aβ generation and AD-related pathology, it might be an ideal target for AD treatment. We have shown that both in neurons and in HEK-APP cells, BACE1 is a new substrate of E3-ligase CHIP and an inverse relation exists between CHIP and BACE1 level. CHIP inhibits ectopic BACE1 level by promoting its ubiquitination and proteasomal degradation, thus reducing APP processing; it stabilizes APP in neurons, thus reducing Aβ. CHIPUbox domain physically interacts with BACE1; however, both U-box and TPR domain are essential for ubiquitination and degradation of BACE1. Further, BACE1 is a downstream target of p53 and overexpression of p53 decreases BACE1 level. In HEK-APP cells, CHIP is shown to negatively regulate BACE1 promoter through stabilization of p53’s DNA-binding conformation and its binding upon 5′ UTR element (+127 to +150). We have thus discovered that CHIP regulates p53-mediated trans-repression of BACE1 at both transcriptional and post-translational level. We propose that a CHIP–BACE1–p53 feedback loop might control APP stabilization, which could further be utilized for new therapeutic intervention in AD. PMID:25773675

  17. Sp1 mediates repression of the resistin gene by PPAR{gamma} agonists in 3T3-L1 adipocytes

    SciTech Connect

    Chung, S.S.; Choi, H.H.; Cho, Y.M.; Lee, H.K.; Park, K.S. . E-mail: kspark@snu.ac.kr

    2006-09-15

    Resistin is an adipokine related to obesity and insulin resistance. Expression of the resistin gene is repressed by the treatment of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists, thiazolidinediones (TZDs). In this study, we investigated the mechanism by which TZDs inhibit the resistin gene expression. Resistin gene expression was decreased by TZD in fully differentiated 3T3-L1 adipocytes, which was abolished after treatment of cycloheximide (a protein synthesis inhibitor). TZD could not repress the expression of the resistin gene in the presence of mithramycin A (an Sp1 binding inhibitor). Sp1 binding site of the resistin promoter (-122/-114 bp) was necessary for the repression. Further investigation of the effect of TZDs on the modification of Sp1 showed that the level of O-glycosylation of Sp1 was decreased in this process. These results suggest that PPAR{gamma} activation represses the expression of the resistin gene by modulating Sp1 activity.

  18. Hormone-induced repression of genes requires BRG1-mediated H1.2 deposition at target promoters.

    PubMed

    Nacht, Ana Silvina; Pohl, Andy; Zaurin, Roser; Soronellas, Daniel; Quilez, Javier; Sharma, Priyanka; Wright, Roni H; Beato, Miguel; Vicent, Guillermo P

    2016-08-15

    Eukaryotic gene regulation is associated with changes in chromatin compaction that modulate access to DNA regulatory sequences relevant for transcriptional activation or repression. Although much is known about the mechanism of chromatin remodeling in hormonal gene activation, how repression is accomplished is much less understood. Here we report that in breast cancer cells, ligand-activated progesterone receptor (PR) is directly recruited to transcriptionally repressed genes involved in cell proliferation along with the kinases ERK1/2 and MSK1. PR recruits BRG1 associated with the HP1γ-LSD1 complex repressor complex, which is further anchored via binding of HP1γ to the H3K9me3 signal deposited by SUV39H2. In contrast to what is observed during gene activation, only BRG1 and not the BAF complex is recruited to repressed promoters, likely due to local enrichment of the pioneer factor FOXA1. BRG1 participates in gene repression by interacting with H1.2, facilitating its deposition and stabilizing nucleosome positioning around the transcription start site. Our results uncover a mechanism of hormone-dependent transcriptional repression and a novel role for BRG1 in progestin regulation of breast cancer cell growth. PMID:27390128

  19. Fbw7 Repression by Hes5 Creates a Feedback Loop That Modulates Notch-Mediated Intestinal and Neural Stem Cell Fate Decisions

    PubMed Central

    Tendeng, Christian; Clurman, Bruce E.; Lewis, Julian; Behrens, Axel

    2013-01-01

    FBW7 is a crucial component of an SCF-type E3 ubiquitin ligase, which mediates degradation of an array of different target proteins. The Fbw7 locus comprises three different isoforms, each with its own promoter and each suspected to have a distinct set of substrates. Most FBW7 targets have important functions in developmental processes and oncogenesis, including Notch proteins, which are functionally important substrates of SCF(Fbw7). Notch signalling controls a plethora of cell differentiation decisions in a wide range of species. A prominent role of this signalling pathway is that of mediating lateral inhibition, a process where exchange of signals that repress Notch ligand production amplifies initial differences in Notch activation levels between neighbouring cells, resulting in unequal cell differentiation decisions. Here we show that the downstream Notch signalling effector HES5 directly represses transcription of the E3 ligase Fbw7β, thereby directly bearing on the process of lateral inhibition. Fbw7Δ/+ heterozygous mice showed haploinsufficiency for Notch degradation causing impaired intestinal progenitor cell and neural stem cell differentiation. Notably, concomitant inactivation of Hes5 rescued both phenotypes and restored normal stem cell differentiation potential. In silico modelling suggests that the NICD/HES5/FBW7β positive feedback loop underlies Fbw7 haploinsufficiency. Thus repression of Fbw7β transcription by Notch signalling is an essential mechanism that is coupled to and required for the correct specification of cell fates induced by lateral inhibition. PMID:23776410

  20. Zinc-fingers and homeoboxes 1 (ZHX1) binds DNA methyltransferase (DNMT) 3B to enhance DNMT3B-mediated transcriptional repression

    SciTech Connect

    Kim, Sung-Hak; Park, Jinah; Choi, Moon-Chang; Kim, Hwang-Phill; Park, Jung-Hyun; Jung, Yeonjoo; Lee, Ju-Hee; Oh, Do-Youn; Im, Seock-Ah; Bang, Yung-Jue; Kim, Tae-You; E-mail: kimty@snu.ac.kr

    2007-04-06

    DNA methyltransferases (DNMT) 3B is a de novo DNMT that represses transcription independent of DNMT activity. In order to gain a better insight into DNMT3B-mediated transcriptional repression, we performed a yeast two-hybrid analysis using DNMT3B as a bait. Of the various binding candidates, ZHX1, a member of zinc-finger and homeobox protein, was found to interact with DNMT3B in vivo and in vitro. N-terminal PWWP domain of DNMT3B was required for its interaction with homeobox motifs of ZHX1. ZHX1 contains nuclear localization signal at C-terminal homeobox motif, and both ZHX1 and DNMT3B were co-localized in nucleus. Furthermore, we found that ZHX1 enhanced the transcriptional repression mediated by DNMT3B when DNMT3B is directly targeted to DNA. These results showed for First the direct linkage between DNMT and zinc-fingers homeoboxes protein, leading to enhanced gene silencing by DNMT3B.

  1. Unliganded progesterone receptor-mediated targeting of an RNA-containing repressive complex silences a subset of hormone-inducible genes

    PubMed Central

    Vicent, Guillermo Pablo; Nacht, A. Silvina; Zaurin, Roser; Font-Mateu, Jofre; Soronellas, Daniel; Le Dily, Francois; Reyes, Diana; Beato, Miguel

    2013-01-01

    A close chromatin conformation precludes gene expression in eukaryotic cells. Genes activated by external cues have to overcome this repressive state by locally changing chromatin structure to a more open state. Although much is known about hormonal gene activation, how basal repression of regulated genes is targeted to the correct sites throughout the genome is not well understood. Here we report that in breast cancer cells, the unliganded progesterone receptor (PR) binds genomic sites and targets a repressive complex containing HP1γ (heterochromatin protein 1γ), LSD1 (lysine-specific demethylase 1), HDAC1/2, CoREST (corepressor for REST [RE1 {neuronal repressor element 1} silencing transcription factor]), KDM5B, and the RNA SRA (steroid receptor RNA activator) to 20% of hormone-inducible genes, keeping these genes silenced prior to hormone treatment. The complex is anchored via binding of HP1γ to H3K9me3 (histone H3 tails trimethylated on Lys 9). SRA interacts with PR, HP1γ, and LSD1, and its depletion compromises the loading of the repressive complex to target chromatin-promoting aberrant gene derepression. Upon hormonal treatment, the HP1γ–LSD1 complex is displaced from these constitutively poorly expressed genes as a result of rapid phosphorylation of histone H3 at Ser 10 mediated by MSK1, which is recruited to the target sites by the activated PR. Displacement of the repressive complex enables the loading of coactivators needed for chromatin remodeling and activation of this set of genes, including genes involved in apoptosis and cell proliferation. These results highlight the importance of the unliganded PR in hormonal regulation of breast cancer cells. PMID:23699411

  2. Beyond the classic eicosanoids: Peripherally-acting oxygenated metabolites of polyunsaturated fatty acids mediate pain associated with tissue injury and inflammation.

    PubMed

    Shapiro, Haim; Singer, Pierre; Ariel, Amiram

    2016-08-01

    Pain is a complex sensation that may be protective or cause undue suffering and loss of function, depending on the circumstances. Peripheral nociceptor neurons (PNs) innervate most tissues, and express ion channels, nocisensors, which depolarize the cell in response to intense stimuli and numerous substances. Inflamed tissues manifest inflammatory hyperalgesia in which the threshold for pain and the response to painful stimuli are decreased and increased, respectively. Constituents of the inflammatory milieu sensitize PNs, thereby contributing to hyperalgesia. Polyunsaturated fatty acids undergo enzymatic and free radical-mediated oxygenation into an array of bioactive metabolites, oxygenated polyunsaturated fatty acids (oxy-PUFAs), including the classic eicosanoids. Oxy-PUFA production is enhanced during inflammation. Pioneering studies by Vane and colleagues from the early 1970s first implicated classic eicosanoids in the pain associated with inflammation. Here, we review the production and action of oxy-PUFAs that are not classic eicosanoids, but nevertheless are produced in injured/ inflamed tissues and activate or sensitize PNs. In general, oxy-PUFAs that sensitize PNs may do so directly, by activation of nocisensors, ion channels or GPCRs expressed on the surface of PNs, or indirectly, by increasing the production of inflammatory mediators that activate or sensitize PNs. We focus on oxy-PUFAs that act directly on PNs. Specifically, we discuss the role of arachidonic acid-derived 12S-HpETE, HNE, ONE, PGA2, iso-PGA2 and 15d-PGJ2, 5,6-and 8,9-EET, PGE2-G and 8R,15S-diHETE, as well as the linoleic acid-derived 9-and 13-HODE in inducing acute nocifensive behavior and/or inflammatory hyperalgesia in rodents. The nocisensors TRPV1, TRPV4 and TRPA1, and putative Gαs-type GPCRs are the PN targets of these oxy-PUFAs. PMID:27067460

  3. PTHrP(1-34)-mediated repression of the PHEX gene in osteoblastic cells involves the transcriptional repressor E4BP4.

    PubMed

    Pellicelli, Martin; Taheri, Maryam; St-Louis, Mathieu; Bériault, Véronique; Desgroseillers, Luc; Boileau, Guy; Moreau, Alain

    2012-06-01

    PHosphate-regulating gene with homology to Endopeptidase on the X chromosome (PHEX) has been identified as the gene mutated in X-linked hypophosphatemia (XLH) syndrome, the most prevalent form of rickets in humans. The predominant expression of PHEX in bones and teeth, and the defective mineralization of these tissues in XLH patients indicate that PHEX is an important regulator of mineralization. Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are known to regulate the expression of numerous genes in osteoblastic cells through activation of the protein kinase A pathway, including repression of PHEX. PTH also activates the transcriptional repressor E4BP4 through the same pathway, suggesting that PTH or PTHrP-mediated repression of PHEX expression could involve E4BP4. To evaluate this possibility, we treated UMR-106 osteoblastic cells with PTHrP(1-34), and used RT-PCR and immunoblotting to analyze PHEX and E4BP4 expression. E4BP4 mRNA and protein levels were rapidly increased in cells treated with PTHrP(1-34), with a concomitant decrease in PHEX expression. This downregulation of PHEX could be reproduced by overexpression of E4BP4. Moreover, PTHrP(1-34)-mediated PHEX repression was blocked when cells were transfected with a siRNA targeting E4BP4 mRNA. Finally, DNA pull-down and luciferase assays showed that two E4BP4 response elements located in PHEX promoter were functional. These results underline the important role of E4BP4 in osteoblastic cells and further define the repression mechanism of PHEX gene by PTHrP(1-34). PMID:21826652

  4. NuRD mediates activating and repressive functions of GATA-1 and FOG-1 during blood development

    PubMed Central

    Miccio, Annarita; Wang, Yuhuan; Hong, Wei; Gregory, Gregory D; Wang, Hongxin; Yu, Xiang; Choi, John K; Shelat, Suresh; Tong, Wei; Poncz, Mortimer; Blobel, Gerd A

    2010-01-01

    GATA transcription factors interact with FOG proteins to regulate tissue development by activating and repressing transcription. FOG-1 (ZFPM1), a co-factor for the haematopoietic factor GATA-1, binds to the NuRD co-repressor complex through a conserved N-terminal motif. Surprisingly, we detected NuRD components at both repressed and active GATA-1/FOG-1 target genes in vivo. In addition, while NuRD is required for transcriptional repression in certain contexts, we show a direct requirement of NuRD also for FOG-1-dependent transcriptional activation. Mice in which the FOG-1/NuRD interaction is disrupted display defects similar to germline mutations in the Gata1 and Fog1 genes, including anaemia and macrothrombocytopaenia. Gene expression analysis in primary mutant erythroid cells and megakaryocytes (MKs) revealed an essential function for NuRD during both the repression and activation of select GATA-1/FOG-1 target genes. These results show that NuRD is a critical co-factor for FOG-1 and underscore the versatile use of NuRD by lineage-specific transcription factors to activate and repress gene transcription in the appropriate cellular and genetic context. PMID:19927129

  5. FMRP regulates miR196a-mediated repression of HOXB8 via interaction with the AGO2 MID domain.

    PubMed

    Li, Ying; Tang, Wei; Zhang, Li-rong; Zhang, Chun-yang

    2014-07-01

    Fragile X syndrome (FXS) is caused by the loss of expression of fragile X mental retardation protein (FMRP), a selective RNA-binding protein that negatively regulates mRNA substrates. FMRP can regulate the translation via the cross-talk with the miRNA machinery, but the functional association among FMRP, miRNAs and mutual target mRNAs has rarely been studied. In this research, we find that HOXB8 mRNA is a target of FMRP associated with miR-196a-induced silencing, and discover that phosphorylation of FMRP promotes the miR-196a-mediated repression of HOXB8 without affecting the interaction between FMRP and mRNA. We further identify that the FMRP-binding site involved in the miR-196a-mediated repression of HOXB8 locates in the downstream neighbourhood of the miR-196a recognition element in the 3'UTR of HOXB8. Importantly, we reveal that FMRP faces toward the MID domain of AGO2 and interacts with a specific binding pocket (coordination with T544, K533 and K570) in the domain. Our research might provide new insights into both the cross-talk between FMRP and miRNA-mediated regulation of mRNA translation and the molecular pathogenesis of FXS. PMID:24727796

  6. The catenin p120{sup ctn} inhibits Kaiso-mediated transcriptional repression of the {beta}-catenin/TCF target gene matrilysin

    SciTech Connect

    Spring, Christopher M.; Kelly, Kevin F.; O'Kelly, Ita; Graham, Monica; Crawford, Howard C.; Daniel, Juliet M. . E-mail: danielj@mcmaster.ca

    2005-05-01

    The POZ-zinc finger transcription factor Kaiso was first identified as a specific binding partner for the Armadillo catenin and cell adhesion cofactor, p120{sup ctn}. Kaiso is a unique POZ protein with bi-modal DNA-binding properties; it associates with a sequence-specific DNA consensus Kaiso binding site (KBS) or methylated CpG dinucleotides, and regulates transcription of artificial promoters containing either site. Interestingly, the promoter of the Wnt/{beta}-catenin/TCF target gene matrilysin possesses two conserved copies of the KBS, which suggested that Kaiso might regulate matrilysin expression. In this study, we demonstrate using chromatin immunoprecipitation analysis that Kaiso associates with the matrilysin promoter in vivo. Minimal promoter assays further confirmed that Kaiso specifically repressed transcription of the matrilysin promoter; mutation of the KBS element or RNAi-mediated depletion of Kaiso abrogated this effect. More importantly, Kaiso blocked {beta}-catenin-mediated activation of the matrilysin promoter. Consistent with our previous findings, both Kaiso-DNA binding and Kaiso-mediated transcriptional repression of the matrilysin promoter were inhibited by overexpression of wild-type p120{sup ctn}, but not by a p120{sup ctn} mutant exhibiting impaired nuclear import. Collectively, our data establish Kaiso as a sequence-specific transcriptional repressor of the matrilysin promoter, and suggest that p120{sup ctn} and {beta}-catenin act in a synergistic manner, via distinct mechanisms, to activate matrilysin expression.

  7. Interferon-γ Induces Major Histocompatibility Class II Transactivator (CIITA) That Mediates Collagen Repression and Major Histocompatibility Class II Activation by Human Aortic Smooth Muscle Cells

    PubMed Central

    Butticè, Giovanna; Miller, Janice; Wang, Lin; Smith, Barbara D.

    2006-01-01

    Chronic inflammation in atherosclerosis is responsible for plaque instability through alterations in extracellular matrix. Previously, we demonstrated that major histocompatibility class II (MHC II) transactivator (CIITA) in a complex with regulatory factor for X box 5 (RFX5) is a crucial protein mediating interferon (IFN)-γ–induced repression of collagen type I gene transcription in fibroblasts. This article demonstrates that, in smooth muscle cells (SMCs), IFN-γ dramatically increases the expression of CIITA isoforms III and IV, with no increase in expression of CIITA isoform I. Expression of CIITA III and IV correlates with decreased collagen type I and increased MHC II gene expression. Exogenous expression of CIITA I, III, and IV, in transiently transfected SMCs, represses collagen type I promoters (COL1A1 and COL1A2) and activates MHC II promoter. Levels of CIITA and RFX5 increase in the nucleus of cells treated with IFN-γ. Moreover, simvastatin lowers the IFN-γ–induced expression of RFX5 and MHC II in addition to repressing collagen expression. However, simvastatin does not block the IFN-γ–induced expression of CIITA III and IV, suggesting a CIITA-independent mechanism. This first demonstration that RFX5 and CIITA isoforms are expressed in SMCs after IFN-γ stimulation suggest that CIITA could be a key factor in plaque stability in atherosclerosis. PMID:16439692

  8. Stromelysin-3 induction and interstitial collagenase repression by retinoic acid. Therapeutical implication of receptor-selective retinoids dissociating transactivation and AP-1-mediated transrepression.

    PubMed

    Guérin, E; Ludwig, M G; Basset, P; Anglard, P

    1997-04-25

    Human stromelysin-3 and interstitial collagenase are matrix metalloproteinases whose expression by stromal cells in several types of carcinomas has been associated with cancer progression. We compared here the regulation of the expression of both proteinases by retinoids in human fibroblasts. Physiological concentrations of retinoic acid were found to simultaneously induce stromelysin-3 and repress interstitial collagenase. In both cases, the involvement of a transcriptional mechanism was supported by run-on assays. Furthermore, in transient transfection experiments, the activity of the stromelysin-3 promoter was induced by retinoic acid through endogenous receptors acting on a DR1 retinoic acid-responsive element. The ligand-dependent activation of the receptors was also investigated by using selective synthetic retinoids, and we demonstrated that retinoic acid-retinoid X receptor heterodimers were the most potent functional units controlling both stromelysin-3 induction and interstitial collagenase repression. However, specific retinoids dissociating the transactivation and the AP-1-mediated transrepression functions of the receptors were found to repress interstitial collagenase without inducing stromelysin-3. These findings indicate that such retinoids may represent efficient inhibitors of matrix metalloproteinase expression in the treatment of human carcinomas. PMID:9111003

  9. Small molecule/ML327 mediated transcriptional de-repression of E-cadherin and inhibition of epithelial-to-mesenchymal transition

    PubMed Central

    An, Hanbing; Stoops, Sydney L.; Deane, Natasha G.; Zhu, Jing; Zi, Jinghuan; Weaver, Connie; Waterson, Alex G.; Zijlstra, Andries; Lindsley, Craig W.; Beauchamp, Robert Daniel

    2015-01-01

    Transcriptional repression of E-cadherin is a hallmark of Epithelial-to-Mesenchymal Transition (EMT) and is associated with cancer cell invasion and metastasis. Understanding the mechanisms underlying E-cadherin repression during EMT may provide insights into the development of novel targeted therapeutics for cancer. Here, we report on the chemical probe, ML327, which de-represses E-cadherin transcription, partially reverses EMT, and inhibits cancer cell invasiveness and tumor cell migration in vitro and in vivo. Induction of E-cadherin mRNA expression by ML327 treatment does not require de novo protein synthesis. RNA sequencing analysis revealed that ML327 treatment significantly alters expression of over 2,500 genes within three hours in the presence of the translational inhibitor, cycloheximide. Network analysis reveals Hepatocyte Nuclear Factor 4-alpha (HNF4α) as the most significant upstream transcriptional regulator of multiple genes whose expressions were altered by ML327 treatment. Further, small interfering RNA-mediated depletion of HNF4α markedly attenuates the E-cadherin expression response to ML327. In summary, ML327 represents a valuable tool to understand mechanisms of EMT and may provide the basis for a novel targeted therapeutic strategy for carcinomas. PMID:26082441

  10. Dot1a-AF9 Complex Mediates Histone H3 Lys-79 Hypermethylation and Repression of ENaCα in an Aldosterone-sensitive Manner*

    PubMed Central

    Zhang, Wenzheng; Xia, Xuefeng; Reisenauer, Mary Rose; Hemenway, Charles S.; Kone, Bruce C.

    2010-01-01

    Aldosterone is a major regulator of epithelial Na+ absorption and acts in large part through induction of the epithelial Na+ channel (ENaC) gene in the renal collecting duct. We previously identified Dot1a as an aldosterone early repressed gene and a repressor of ENaCα transcription through mediating histone H3 Lys-79 methylation associated with the ENaCα promoter. Here, we report a novel aldosterone-signaling network involving AF9, Dot1a, and ENaCα. AF9 and Dot1a interact in vitro and in vivo as evidenced in multiple assays and colocalize in the nuclei of mIMCD3 renal collecting duct cells. Overexpression of AF9 results in hypermethylation of histone H3 Lys-79 at the endogenous ENaCα promoter at most, but not all subregions examined, repression of endogenous ENaCα mRNA expression and acts synergistically with Dot1a to inhibit ENaCα promoter-luciferase constructs. In contrast, RNA interference-mediated knockdown of AF9 causes the opposite effects. Chromatin immunoprecipitation assays reveal that overexpressed FLAG-AF9, endogenous AF9, and Dot1a are each associated with the ENaCα promoter. Aldosterone negatively regulates AF9 expression at both mRNA and protein levels. Thus, Dot1a-AF9 modulates histone H3 Lys-79 methylation at the ENaCα promoter and represses ENaCα transcription in an aldosterone-sensitive manner. This mechanism appears to be more broadly applicable to other aldosterone-regulated genes because overexpression of AF9 alone or in combination with Dot1a inhibited mRNA levels of three other known aldosterone-inducible genes in mIMCD3 cells. PMID:16636056

  11. Analysis of Bacillus subtilis hut operon expression indicates that histidine-dependent induction is mediated primarily by transcriptional antitermination and that amino acid repression is mediated by two mechanisms: regulation of transcription initiation and inhibition of histidine transport.

    PubMed Central

    Wray, L V; Fisher, S H

    1994-01-01

    Expression of the Bacillus subtilis hut operon is induced by histidine and subject to regulation by carbon catabolite repression and amino acid repression. A set of hut-lacZ transcriptional fusions was constructed and used to identify the cis-acting sites required for histidine induction and amino acid repression. Histidine induction was found to be primarily mediated by transcriptional antitermination at a palindromic sequence located immediately downstream of the first structural gene in the hut operon, hutP. High levels of histidine induction were observed only in hut-lacZ fusions which contained this palindromic sequence. The hutC1 mutation, which results in constitutive expression of the hut operon, was sequenced and found to contain a GC to TA transversion located within the stem-loop structure. Transcription of hut DNA in vitro revealed that the palindromic structure functions as a transcriptional terminator with wild-type hut DNA but not with hutC1 DNA. Two sites were found to be involved in amino acid repression of hut expression: (i) an operator, hutOA, which lies downstream of the hut promoter, and (ii) the hut terminator. The rate of [14C]histidine uptake in amino acid-grown cells was sixfold lower than that seen in cells grown without amino acids. Thus, inhibition of histidine transport in amino acid-grown cells indirectly regulates hut expression by interfering with histidine induction at the hut terminator. Images PMID:8071225

  12. Positive Regulatory Domain I-Binding Factor 1 mediates repression of the MHC Class II Transactivator (CIITA) type IV promoter

    PubMed Central

    Chen, Han; Gilbert, Carolyn A.; Hudson, John A.; Bolick, Sophia C.; Wright, Kenneth L.; Piskurich, Janet F.

    2006-01-01

    MHC class II transactivator (CIITA), a co-activator that controls MHC class II (MHC II) transcription, functions as the master regulator of MHC II expression. Persistent activity of the CIITA type III promoter (pIII), one of the four potential promoters of this gene, is responsible for constitutive expression of MHC II by B lymphocytes. In addition, IFN-γ induces expression of CIITA in these cells through the type IV promoter (pIV). Positive regulatory domain 1-binding factor 1 (PRDI-BF1), called B lymphocyte-induced maturation protein 1 (Blimp-1) in mice, represses the expression of CIITA pIII in plasma and multiple myeloma cells. To investigate regulation of CIITA pIV expression by PRDI-BF1 in the B lymphocyte lineage, protein/DNA binding studies, and functional promoter analyses were performed. PRDI-BF1 bound to the IRF-E site in CIITA pIV. Ectopic expression of either PRDI-BF1 or Blimp-1 repressed this promoter in B lymphocytes. In vitro binding and functional analyses of CIITA pIV demonstrated that the IFN regulatory factor-element (IRF-E) is the target of this repression. In vivo genomic footprint analysis demonstrated protein binding at the IRF-E site of CIITA pIV in U266 myeloma cells, which express PRDI-BF1. PRDI-BF1β, a truncated form of PRDI-BF1 that is co-expressed in myeloma cells, also bound to the IRF-E site and repressed CIITA pIV. These findings demonstrate for the first time that, in addition to silencing expression of CIITA pIII in B lymphocytes, PRDI-BF1 is capable of binding and suppressing CIITA pIV. PMID:16765445

  13. MeCP2-Mediated Transcription Repression in the Basolateral Amygdala May Underlie Heightened Anxiety in a Mouse Model of Rett Syndrome

    PubMed Central

    Adachi, Megumi; Autry, Anita E.; Covington, Herb E.; Monteggia, Lisa M.

    2010-01-01

    Rett syndrome (RTT) is an X-linked neurodevelopmental disorder that results from loss of function mutations in the methyl-CpG binding protein 2 (MECP2) gene. Using viral-mediated basolateral amygdala (BLA)-specific deletion of Mecp2 in mice, we show that intact Mecp2 function is required for normal anxiety behavior as well as some types of learning and memory. To examine whether these behavioral deficits are the result of impaired transcriptional repression, because Mecp2 is believed to act as a transcriptional repressor in complex with histone deacetylases (HDACs), we infused a HDAC inhibitor chronically into the BLA of wild-type mice. We found that HDAC inhibition produces behavioral deficits similar to those observed after the deletion of Mecp2 in the BLA. These results suggest a key role for Mecp2 as a transcriptional repressor in the BLA in mediating behavioral features of RTT. PMID:19339616

  14. Myc Inhibits p27-Induced Erythroid Differentiation of Leukemia Cells by Repressing Erythroid Master Genes without Reversing p27-Mediated Cell Cycle Arrest▿ ‡

    PubMed Central

    Acosta, Juan C.; Ferrándiz, Nuria; Bretones, Gabriel; Torrano, Verónica; Blanco, Rosa; Richard, Carlos; O'Connell, Brenda; Sedivy, John; Delgado, M. Dolores; León, Javier

    2008-01-01

    Inhibition of differentiation has been proposed as an important mechanism for Myc-induced tumorigenesis, but the mechanisms involved are unclear. We have established a genetically defined differentiation model in human leukemia K562 cells by conditional expression of the cyclin-dependent kinase (Cdk) inhibitor p27 (inducible by Zn2+) and Myc (activatable by 4-hydroxy-tamoxifen). Induction of p27 resulted in erythroid differentiation, accompanied by Cdk inhibition and G1 arrest. Interestingly, activation of Myc inhibited p27-mediated erythroid differentiation without affecting p27-mediated proliferation arrest. Microarray-based gene expression indicated that, in the presence of p27, Myc blocked the upregulation of several erythroid-cell-specific genes, including NFE2, JUNB, and GATA1 (transcription factors with a pivotal role in erythropoiesis). Moreover, Myc also blocked the upregulation of Mad1, a transcriptional antagonist of Myc that is able to induce erythroid differentiation. Cotransfection experiments demonstrated that Myc-mediated inhibition of differentiation is partly dependent on the repression of Mad1 and GATA1. In conclusion, this model demonstrates that Myc-mediated inhibition of differentiation depends on the regulation of a specific gene program, whereas it is independent of p27-mediated cell cycle arrest. Our results support the hypothesis that differentiation inhibition is an important Myc tumorigenic mechanism that is independent of cell proliferation. PMID:18838534

  15. Myc inhibits p27-induced erythroid differentiation of leukemia cells by repressing erythroid master genes without reversing p27-mediated cell cycle arrest.

    PubMed

    Acosta, Juan C; Ferrándiz, Nuria; Bretones, Gabriel; Torrano, Verónica; Blanco, Rosa; Richard, Carlos; O'Connell, Brenda; Sedivy, John; Delgado, M Dolores; León, Javier

    2008-12-01

    Inhibition of differentiation has been proposed as an important mechanism for Myc-induced tumorigenesis, but the mechanisms involved are unclear. We have established a genetically defined differentiation model in human leukemia K562 cells by conditional expression of the cyclin-dependent kinase (Cdk) inhibitor p27 (inducible by Zn(2+)) and Myc (activatable by 4-hydroxy-tamoxifen). Induction of p27 resulted in erythroid differentiation, accompanied by Cdk inhibition and G(1) arrest. Interestingly, activation of Myc inhibited p27-mediated erythroid differentiation without affecting p27-mediated proliferation arrest. Microarray-based gene expression indicated that, in the presence of p27, Myc blocked the upregulation of several erythroid-cell-specific genes, including NFE2, JUNB, and GATA1 (transcription factors with a pivotal role in erythropoiesis). Moreover, Myc also blocked the upregulation of Mad1, a transcriptional antagonist of Myc that is able to induce erythroid differentiation. Cotransfection experiments demonstrated that Myc-mediated inhibition of differentiation is partly dependent on the repression of Mad1 and GATA1. In conclusion, this model demonstrates that Myc-mediated inhibition of differentiation depends on the regulation of a specific gene program, whereas it is independent of p27-mediated cell cycle arrest. Our results support the hypothesis that differentiation inhibition is an important Myc tumorigenic mechanism that is independent of cell proliferation. PMID:18838534

  16. HESX1- and TCF3-mediated repression of Wnt/β-catenin targets is required for normal development of the anterior forebrain

    PubMed Central

    Andoniadou, Cynthia L.; Signore, Massimo; Young, Rodrigo M.; Gaston-Massuet, Carles; Wilson, Stephen W.; Fuchs, Elaine; Martinez-Barbera, Juan Pedro

    2011-01-01

    The Wnt/β-catenin pathway plays an essential role during regionalisation of the vertebrate neural plate and its inhibition in the most anterior neural ectoderm is required for normal forebrain development. Hesx1 is a conserved vertebrate-specific transcription factor that is required for forebrain development in Xenopus, mice and humans. Mouse embryos deficient for Hesx1 exhibit a variable degree of forebrain defects, but the molecular mechanisms underlying these defects are not fully understood. Here, we show that injection of a hesx1 morpholino into a ‘sensitised’ zygotic headless (tcf3) mutant background leads to severe forebrain and eye defects, suggesting an interaction between Hesx1 and the Wnt pathway during zebrafish forebrain development. Consistent with a requirement for Wnt signalling repression, we highlight a synergistic gene dosage-dependent interaction between Hesx1 and Tcf3, a transcriptional repressor of Wnt target genes, to maintain anterior forebrain identity during mouse embryogenesis. In addition, we reveal that Tcf3 is essential within the neural ectoderm to maintain anterior character and that its interaction with Hesx1 ensures the repression of Wnt targets in the developing forebrain. By employing a conditional loss-of-function approach in mouse, we demonstrate that deletion of β-catenin, and concomitant reduction of Wnt signalling in the developing anterior forebrain of Hesx1-deficient embryos, leads to a significant rescue of the forebrain defects. Finally, transcriptional profiling of anterior forebrain precursors from mouse embryos expressing eGFP from the Hesx1 locus provides molecular evidence supporting a novel function of Hesx1 in mediating repression of Wnt/β-catenin target activation in the developing forebrain. PMID:22007134

  17. Role of GlnR in Acid-Mediated Repression of Genes Encoding Proteins Involved in Glutamine and Glutamate Metabolism in Streptococcus mutans▿ †

    PubMed Central

    Chen , Pei-Min; Chen, Yi-Ywan M.; Yu, Sung-Liang; Sher, Singh; Lai, Chern-Hsiung; Chia, Jean-San

    2010-01-01

    The acid tolerance response (ATR) is one of the major virulence traits of Streptococcus mutans. In this study, the role of GlnR in acid-mediated gene repression that affects the adaptive ATR in S. mutans was investigated. Using a whole-genome microarray and in silico analyses, we demonstrated that GlnR and the GlnR box (ATGTNAN7TNACAT) were involved in the transcriptional repression of clusters of genes encoding proteins involved in glutamine and glutamate metabolism under acidic challenge. Reverse transcription-PCR (RT-PCR) analysis revealed that the coordinated regulation of the GlnR regulon occurred 5 min after acid treatment and that prolonged acid exposure (30 min) resulted in further reduction in expression. A lower level but consistent reduction in response to acidic pH was also observed in chemostat-grown cells, confirming the negative regulation of GlnR. The repression by GlnR through the GlnR box in response to acidic pH was further confirmed in the citBZC operon, containing genes encoding the first three enzymes in the glutamine/glutamate biosynthesis pathway. The survival rate of the GlnR-deficient mutant at pH 2.8 was more than 10-fold lower than that in the wild-type strain 45 min after acid treatment, suggesting that the GlnR regulon participates in S. mutans ATR. It is hypothesized that downregulation of the synthesis of the amino acid precursors in response to acid challenge would promote citrate metabolism to pyruvate, with the consumption of H+ and potential ATP synthesis. Such regulation will ensure an optimal acid adaption in S. mutans. PMID:20173059

  18. RflM mediates target specificity of the RcsCDB phosphorelay system for transcriptional repression of flagellar synthesis in Salmonella enterica.

    PubMed

    Kühne, Caroline; Singer, Hanna M; Grabisch, Eva; Codutti, Luca; Carlomagno, Teresa; Scrima, Andrea; Erhardt, Marc

    2016-09-01

    The bacterial flagellum enables directed movement of Salmonella enterica towards favorable conditions in liquid environments. Regulation of flagellar synthesis is tightly controlled by various environmental signals at transcriptional and post-transcriptional levels. The flagellar master regulator FlhD4 C2 resides on top of the flagellar transcriptional hierarchy and is under autogenous control by FlhD4 C2 -dependent activation of the repressor rflM. The inhibitory activity of RflM depends on the presence of RcsB, the response regulator of the RcsCDB phosphorelay system. In this study, we elucidated the molecular mechanism of RflM-dependent repression of flhDC. We show that RcsB and RflM form a heterodimer that coordinately represses flhDC transcription independent of RcsB phosphorylation. RcsB-RflM complex binds to a RcsB box downstream the P1 transcriptional start site of the flhDC promoter with increased affinity compared to RcsB in the absence of RflM. We propose that RflM stabilizes binding of unphosphorylated RcsB to the flhDC promoter in absence of environmental cues. Thus, RflM is a novel auxiliary regulatory protein that mediates target specificity of RcsB for flhDC repression. The cooperative action of the RcsB-RflM repressor complex allows Salmonella to fine-tune initiation of flagellar gene expression and adds another level to the complex regulation of flagellar synthesis. PMID:27206164

  19. Akt1-mediated Gata3 phosphorylation controls the repression of IFNγ in memory-type Th2 cells

    PubMed Central

    Hosokawa, Hiroyuki; Tanaka, Tomoaki; Endo, Yusuke; Kato, Miki; Shinoda, Kenta; Suzuki, Akane; Motohashi, Shinichiro; Matsumoto, Masaki; Nakayama, Keiichi I.; Nakayama, Toshinori

    2016-01-01

    Th2 cells produce Th2 cytokines such as IL-4, IL-5 and IL-13, but repress Th1 cytokine IFNγ. Recent studies have revealed various distinct memory-type Th2 cell subsets, one of which produces a substantial amount of IFNγ in addition to Th2 cytokines, however it remains unclear precisely how these Th2 cells produce IFNγ. We herein show that phosphorylation of Gata3 at Ser308, Thr315 and Ser316 induces dissociation of a histone deacetylase Hdac2 from the Gata3/Chd4 repressive complex in Th2 cells. We also identify Akt1 as a Gata3-phosphorylating kinase, and the activation of Akt1 induces derepression of Tbx21 and Ifng expression in Th2 cells. Moreover, T-bet-dependent IFNγ expression in IFNγ-producing memory Th2 cells appears to be controlled by the phosphorylation status of Gata3 in human and murine systems. Thus, this study highlights the molecular basis for posttranslational modifications of Gata3 that control the regulation of IFNγ expression in memory Th2 cells. PMID:27053161

  20. Akt1-mediated Gata3 phosphorylation controls the repression of IFNγ in memory-type Th2 cells.

    PubMed

    Hosokawa, Hiroyuki; Tanaka, Tomoaki; Endo, Yusuke; Kato, Miki; Shinoda, Kenta; Suzuki, Akane; Motohashi, Shinichiro; Matsumoto, Masaki; Nakayama, Keiichi I; Nakayama, Toshinori

    2016-01-01

    Th2 cells produce Th2 cytokines such as IL-4, IL-5 and IL-13, but repress Th1 cytokine IFNγ. Recent studies have revealed various distinct memory-type Th2 cell subsets, one of which produces a substantial amount of IFNγ in addition to Th2 cytokines, however it remains unclear precisely how these Th2 cells produce IFNγ. We herein show that phosphorylation of Gata3 at Ser308, Thr315 and Ser316 induces dissociation of a histone deacetylase Hdac2 from the Gata3/Chd4 repressive complex in Th2 cells. We also identify Akt1 as a Gata3-phosphorylating kinase, and the activation of Akt1 induces derepression of Tbx21 and Ifng expression in Th2 cells. Moreover, T-bet-dependent IFNγ expression in IFNγ-producing memory Th2 cells appears to be controlled by the phosphorylation status of Gata3 in human and murine systems. Thus, this study highlights the molecular basis for posttranslational modifications of Gata3 that control the regulation of IFNγ expression in memory Th2 cells. PMID:27053161

  1. Pro Isomerization in MLL1 PHD3-Bromo Cassette Connects H3K4me Readout to CyP33 and HDAC-Mediated Repression

    SciTech Connect

    Wang, Zhanxin; Song, Jikui; Milne, Thomas A.; Wang, Gang G.; Li, Haitao; Allis, C. David; Patel, Dinshaw J.

    2010-09-13

    The MLL1 gene is a frequent target for recurrent chromosomal translocations, resulting in transformation of hematopoietic precursors into leukemia stem cells. Here, we report on structure-function studies that elucidate molecular events in MLL1 binding of histone H3K4me3/2 marks and recruitment of the cyclophilin CyP33. CyP33 contains a PPIase and a RRM domain and regulates MLL1 function through HDAC recruitment. We find that the PPIase domain of CyP33 regulates the conformation of MLL1 through proline isomerization within the PHD3-Bromo linker, thereby disrupting the PHD3-Bromo interface and facilitating binding of the MLL1-PHD3 domain to the CyP33-RRM domain. H3K4me3/2 and CyP33-RRM target different surfaces of MLL1-PHD3 and can bind simultaneously to form a ternary complex. Furthermore, the MLL1-CyP33 interaction is required for repression of HOXA9 and HOXC8 genes in vivo. Our results highlight the role of PHD3-Bromo cassette as a regulatory platform, orchestrating MLL1 binding of H3K4me3/2 marks and cyclophilin-mediated repression through HDAC recruitment.

  2. Specific contacts of the −35 region of the galP1 promoter by RNA polymerase inhibit GalR-mediated DNA looping repression

    PubMed Central

    Csiszovszki, Zsolt; Lewis, Dale E. A.; Le, Phuoc; Sneppen, Kim; Semsey, Szabolcs

    2012-01-01

    The P1 promoter of the galactose operon in Escherichia coli is one of the best studied examples of ‘extended −10’ promoters. Recognition of the P1 promoter does not require specific contacts between RNA polymerase and its poor −35 element. To investigate whether specific recognition of the −35 element would affect the regulation of P1 by GalR, we mutagenized the −35 element of P1, isolated variants of the −35 element and studied the regulation of the mutant promoters by in vitro transcription assays and by mathematical modeling. The results show that the GalR-mediated DNA loop is less efficient in repressing P1 transcription when RNA polymerase binds to the −10 and −35 elements concomitantly. Our results suggest that promoters that lack specific −35 element recognition allow decoupling of local chromosome structure from transcription initiation. PMID:22941635

  3. Specific contacts of the -35 region of the galP1 promoter by RNA polymerase inhibit GalR-mediated DNA looping repression.

    PubMed

    Csiszovszki, Zsolt; Lewis, Dale E A; Le, Phuoc; Sneppen, Kim; Semsey, Szabolcs

    2012-11-01

    The P1 promoter of the galactose operon in Escherichia coli is one of the best studied examples of 'extended -10' promoters. Recognition of the P1 promoter does not require specific contacts between RNA polymerase and its poor -35 element. To investigate whether specific recognition of the -35 element would affect the regulation of P1 by GalR, we mutagenized the -35 element of P1, isolated variants of the -35 element and studied the regulation of the mutant promoters by in vitro transcription assays and by mathematical modeling. The results show that the GalR-mediated DNA loop is less efficient in repressing P1 transcription when RNA polymerase binds to the -10 and -35 elements concomitantly. Our results suggest that promoters that lack specific -35 element recognition allow decoupling of local chromosome structure from transcription initiation. PMID:22941635

  4. Activated Nrf2 Interacts with Kaposi's Sarcoma-Associated Herpesvirus Latency Protein LANA-1 and Host Protein KAP1 To Mediate Global Lytic Gene Repression

    PubMed Central

    Gjyshi, Olsi; Roy, Arunava; Dutta, Sujoy; Veettil, Mohanan Valiya; Dutta, Dipanjan

    2015-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. We have previously shown that KSHV utilizes the host transcription factor Nrf2 to aid in infection of endothelial cells and oncogenesis. Here, we investigate the role of Nrf2 in PEL and PEL-derived cell lines and show that KSHV latency induces Nrf2 protein levels and transcriptional activity through the COX-2/PGE2/EP4/PKCζ axis. Next-generation sequencing of KSHV transcripts in the PEL-derived BCBL-1 cell line revealed that knockdown of this activated Nrf2 results in global elevation of lytic genes. Nrf2 inhibition by the chemical brusatol also induces lytic gene expression. Both Nrf2 knockdown and brusatol-mediated inhibition induced KSHV lytic reactivation in BCBL-1 cells. In a series of follow-up experiments, we characterized the mechanism of Nrf2-mediated regulation of KSHV lytic repression during latency. Biochemical assays showed that Nrf2 interacted with KSHV latency-associated nuclear antigen 1 (LANA-1) and the host transcriptional repressor KAP1, which together have been shown to repress lytic gene expression. Promoter studies showed that although Nrf2 alone induces the open reading frame 50 (ORF50) promoter, its association with LANA-1 and KAP1 abrogates this effect. Interestingly, LANA-1 is crucial for efficient KAP1/Nrf2 association, while Nrf2 is essential for LANA-1 and KAP1 recruitment to the ORF50 promoter and its repression. Overall, these results suggest that activated Nrf2, LANA-1, and KAP1 assemble on the ORF50 promoter in a temporal fashion. Initially, Nrf2 binds to and activates the ORF50 promoter during early de novo infection, an effect that is exploited during latency by LANA-1-mediated recruitment of the host transcriptional repressor KAP1 on Nrf2. Cell death assays further showed that Nrf2 and KAP1 knockdown induce significant cell death in PEL cell lines

  5. miR-146b-5p mediates p16-dependent repression of IL-6 and suppresses paracrine procarcinogenic effects of breast stromal fibroblasts

    PubMed Central

    Al-Ansari, Mysoon M.; Aboussekhra, Abdelilah

    2015-01-01

    Increasing evidence support the critical roles of active stromal fibroblasts in breast cancer development and spread. However, the mediators and the mechanisms of regulation are still not well defined. We have shown here that the tumor suppressor p16INK4A protein inhibits the pro-carcinogenic effects of breast stromal fibroblasts through repressing the expression/secretion of IL-6. Indeed, p16INK4A suppresses IL-6 at the mRNA and protein levels. This effect is mediated trough miR-146b-5p, which inhibits IL-6 expression through a specific sequence at the IL-6 3′UTR. In addition, we present clear evidence that miR-146b-5p inhibition is sufficient to transactivate breast stromal fibroblasts, which promote epithelial-to-mesenchymal-transition in breast cancer cells in a paracrine manner. By contrast, ectopic expression of miR-146b-5p in active fibroblasts abrogated their pro-carcinogenic effects. The physiological importance of miR-146b-5p inhibition was revealed by showing that the levels of pre-miR-146b-5p as well as its mature form are reduced in cancer-associated fibroblasts as compared with their normal adjacent counterparts from cancer-free tissues isolated from the same patients. Interestingly, treatment of active breast stromal fibroblasts with curcumin increased the level of the p16INK4A coding CDKN2A mRNA and miR-146b-5p and suppressed IL-6, which confirms the repressive effect of these two tumor suppressor molecules on IL-6, and shows the possible “normalization” of cancer-related active fibroblasts. These results show that miR-146b-5p has non-cell-autonomous tumor suppressor function through inhibition of IL-6, suggesting that targeting this microRNA in breast stromal fibroblasts could be of great therapeutic value. PMID:26338965

  6. Pronephric tubule morphogenesis in zebrafish depends on Mnx mediated repression of irx1b within the intermediate mesoderm.

    PubMed

    Ott, Elisabeth; Wendik, Björn; Srivastava, Monika; Pacho, Frederic; Töchterle, Sonja; Salvenmoser, Willi; Meyer, Dirk

    2016-03-01

    Mutations in the homeobox transcription factor MNX1 are the major cause of dominantly inherited sacral agenesis. Studies in model organisms revealed conserved mnx gene requirements in neuronal and pancreatic development while Mnx activities that could explain the caudal mesoderm specific agenesis phenotype remain elusive. Here we use the zebrafish pronephros as a simple yet genetically conserved model for kidney formation to uncover a novel role of Mnx factors in nephron morphogenesis. Pronephros formation can formally be divided in four stages, the specification of nephric mesoderm from the intermediate mesoderm (IM), growth and epithelialisation, segmentation and formation of the glomerular capillary tuft. Two of the three mnx genes in zebrafish are dynamically transcribed in caudal IM in a time window that proceeds segmentation. We show that expression of one mnx gene, mnx2b, is restricted to the pronephric lineage and that mnx2b knock-down causes proximal pronephric tubule dilation and impaired pronephric excretion. Using expression profiling of embryos transgenic for conditional activation and repression of Mnx regulated genes, we further identified irx1b as a direct target of Mnx factors. Consistent with a repression of irx1b by Mnx factors, the transcripts of irx1b and mnx genes are found in mutual exclusive regions in the IM, and blocking of Mnx functions results in a caudal expansion of the IM-specific irx1b expression. Finally, we find that knock-down of irx1b is sufficient to rescue proximal pronephric tubule dilation and impaired nephron function in mnx-morpholino injected embryos. Our data revealed a first caudal mesoderm specific requirement of Mnx factors in a non-human system and they demonstrate that Mnx-dependent restriction of IM-specific irx1b activation is required for the morphogenesis and function of the zebrafish pronephros. PMID:26472045

  7. Benzoate Mediates Repression of C4-Dicarboxylate Utilization in “Aromatoleum aromaticum” EbN1

    PubMed Central

    Trautwein, Kathleen; Grundmann, Olav; Wöhlbrand, Lars; Eberlein, Christian; Boll, Matthias

    2012-01-01

    Diauxic growth was observed in anaerobic C4-dicarboxylate-adapted cells of “Aromatoleum aromaticum” EbN1 due to preferred benzoate utilization from a substrate mixture of a C4-dicarboxylate (succinate, fumarate, or malate) and benzoate. Differential protein profiles (two-dimensional difference gel electrophoresis [2D DIGE]) revealed dynamic changes in abundance for proteins involved in anaerobic benzoate catabolism and C4-dicarboxylate uptake. In the first active growth phase, benzoate utilization was paralleled by maximal abundance of proteins involved in anaerobic benzoate degradation (e.g., benzoyl-coenzyme A [CoA] reductase) and minimal abundance of DctP (EbA4158), the periplasmic binding protein of a predicted C4-dicarboxylate tripartite ATP-independent periplasmic (TRAP) transporter (DctPQM). The opposite was observed during subsequent succinate utilization in the second active growth phase. The increased dctP (respectively, dctPQM) transcript and DctP protein abundance following benzoate depletion suggests that repression of C4-dicarboxylate uptake seems to be a main determinant for the observed diauxie. PMID:22081395

  8. Characterization of Cellulase Secretion and Cre1-Mediated Carbon Source Repression in the Potential Lignocellulose-Degrading Strain Trichoderma asperellum T-1

    PubMed Central

    Wang, Qun; Lin, Hui; Shen, Qi; Fan, Xiaoping; Bai, Naling; Zhao, Yuhua

    2015-01-01

    Trichoderma asperellum, a traditional bio-control species, was demonstrated to be an excellent candidate for lignocellulose degradation in this work. Comparing to the representatively industrial strain of Trichoderma reeseiQM6a, T. asperellum T-1 showed more robust growth, stronger spore production, faster secretion of lignocellulose-decomposing enzymes and better pH tolerance. The reducing sugar released by strain T-1 on the second day of fermentation was 87% higher than that of strain QM6a, although the maximum reducing sugar yield and the cellulase production persistence of the strain T-1 were lower. Our experiment found that the cellulase secretion was strongly inhibited by glucose, suggesting the existence of carbon source repression pathway in T. asperellum T-1. The inhibiting effect was enhanced with an increase in glucose concentration and was closely related to mycelium growth. SDS-PAGE and secondary mass-spectrum identification confirmed that the expression of endo-1,4-β-xylanase I in T. asperellum T-1 was down-regulated when glucose was added. The factor Cre1, which plays an important role in the down-regulation of the endo-1,4-β-xylanase I gene, was investigated by bioinformatics methods. The protein structure of Cre1, analyzed using multiple protein sequence alignment, indicates the existence of the Zn-fingers domain. Then, the binding sites of Cre1 on the endo-1,4-β-xylanase I gene promoter were further elucidated. This study is the first report about Cre1-mediated carbon repression in the bio-control strain T. asperellum T-1. All of the above results provided good references for better understanding T. asperellum T-1 and improving its application for lignocellulose degradation. PMID:25741694

  9. CovS simultaneously activates and inhibits the CovR-mediated repression of distinct subsets of group A Streptococcus virulence factor-encoding genes.

    PubMed

    Treviño, Jeanette; Perez, Nataly; Ramirez-Peña, Esmeralda; Liu, Zhuyun; Shelburne, Samuel A; Musser, James M; Sumby, Paul

    2009-08-01

    To colonize and cause disease at distinct anatomical sites, bacterial pathogens must tailor gene expression in a microenvironment-specific manner. The molecular mechanisms that control the ability of the human bacterial pathogen group A Streptococcus (GAS) to transition between infection sites have yet to be fully elucidated. A key regulator of GAS virulence gene expression is the CovR-CovS two-component regulatory system (also known as CsrR-CsrS). covR and covS mutant strains arise spontaneously during invasive infections and, in in vivo models of infection, rapidly become dominant. Here, we compared wild-type GAS with covR, covS, and covRS isogenic mutant strains to investigate the heterogeneity in the types of natural mutations that occur in covR and covS and the phenotypic consequences of covR or covS mutation. We found that the response regulator CovR retains some regulatory function in the absence of CovS and that CovS modulates CovR to significantly enhance repression of one group of genes (e.g., the speA, hasA, and ska genes) while it reduces repression of a second group of genes (e.g., the speB, grab, and spd3 genes). We also found that different in vivo-induced covR mutations can lead to strikingly different transcriptomes. While covS mutant strains show increased virulence in several invasive models of infection, we determined that these mutants are significantly outcompeted by wild-type GAS during growth in human saliva, an ex vivo model of upper respiratory tract infection. We propose that CovS-mediated regulation of CovR activity plays an important role in the ability of GAS to cycle between pharyngeal and invasive infections. PMID:19451242

  10. CovS Simultaneously Activates and Inhibits the CovR-Mediated Repression of Distinct Subsets of Group A Streptococcus Virulence Factor-Encoding Genes▿ †

    PubMed Central

    Treviño, Jeanette; Perez, Nataly; Ramirez-Peña, Esmeralda; Liu, Zhuyun; Shelburne, Samuel A.; Musser, James M.; Sumby, Paul

    2009-01-01

    To colonize and cause disease at distinct anatomical sites, bacterial pathogens must tailor gene expression in a microenvironment-specific manner. The molecular mechanisms that control the ability of the human bacterial pathogen group A Streptococcus (GAS) to transition between infection sites have yet to be fully elucidated. A key regulator of GAS virulence gene expression is the CovR-CovS two-component regulatory system (also known as CsrR-CsrS). covR and covS mutant strains arise spontaneously during invasive infections and, in in vivo models of infection, rapidly become dominant. Here, we compared wild-type GAS with covR, covS, and covRS isogenic mutant strains to investigate the heterogeneity in the types of natural mutations that occur in covR and covS and the phenotypic consequences of covR or covS mutation. We found that the response regulator CovR retains some regulatory function in the absence of CovS and that CovS modulates CovR to significantly enhance repression of one group of genes (e.g., the speA, hasA, and ska genes) while it reduces repression of a second group of genes (e.g., the speB, grab, and spd3 genes). We also found that different in vivo-induced covR mutations can lead to strikingly different transcriptomes. While covS mutant strains show increased virulence in several invasive models of infection, we determined that these mutants are significantly outcompeted by wild-type GAS during growth in human saliva, an ex vivo model of upper respiratory tract infection. We propose that CovS-mediated regulation of CovR activity plays an important role in the ability of GAS to cycle between pharyngeal and invasive infections. PMID:19451242

  11. The Zinc Finger Transcription Factor ZXDC Activates CCL2 Gene Expression by Opposing BCL6-mediated Repression

    PubMed Central

    Ramsey, Jon E.; Fontes, Joseph D.

    2013-01-01

    The zinc finger X-linked duplicated (ZXD) family of transcription factors has been implicated in regulating transcription of major histocompatibility complex class II genes in antigen presenting cells; roles beyond this function are not yet known. The expression of one gene in this family, ZXD family zinc finger C (ZXDC), is enriched in myeloid lineages and therefore we hypothesized that ZXDC may regulate myeloid-specific gene expression. Here we demonstrate that ZXDC regulates genes involved in myeloid cell differentiation and inflammation. Overexpression of the larger isoform of ZXDC, ZXDC1, activates expression of monocyte-specific markers of differentiation and synergizes with phorbol 12-myristate 13-acetate (which causes differentiation) in the human leukemic monoblast cell line U937. To identify additional gene targets of ZXDC1, we performed gene expression profiling which revealed multiple inflammatory gene clusters regulated by ZXDC1. Using a combination of approaches we show that ZXDC1 activates transcription of a gene within one of the regulated clusters, chemokine (C-C motif) ligand 2 (CCL2; monocyte chemoattractant protein 1; MCP1) via a previously defined distal regulatory element. Further, ZXDC1-dependent up-regulation of the gene involves eviction of the transcriptional repressor B-cell CLL/lymphoma 6 (BCL6), a factor known to be important in resolving inflammatory responses, from this region of the promoter. Collectively, our data show that ZXDC1 is a regulator in the process of myeloid function and that ZXDC1 is responsible for Ccl2 gene de-repression by BCL6. PMID:23954399

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

    SciTech Connect

    Kim, Inae; Hur, Jung; Jeong, Sunjoo

    2015-01-30

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

  13. TRIM28 Is an E3 Ligase for ARF-Mediated NPM1/B23 SUMOylation That Represses Centrosome Amplification

    PubMed Central

    Neo, Shu Hui; Itahana, Yoko; Alagu, Jennifer; Kitagawa, Mayumi; Guo, Alvin Kunyao; Lee, Sang Hyun; Tang, Kai

    2015-01-01

    The tumor suppressor ARF enhances the SUMOylation of target proteins; however, the physiological function of ARF-mediated SUMOylation has been unclear due to the lack of a known, associated E3 SUMO ligase. Here we uncover TRIM28/KAP1 as a novel ARF-binding protein and SUMO E3 ligase for NPM1/B23. ARF and TRIM28 cooperate to SUMOylate NPM1, a nucleolar protein that regulates centrosome duplication and genomic stability. ARF-mediated SUMOylation of NPM1 was attenuated by TRIM28 depletion and enhanced by TRIM28 overexpression. Coexpression of ARF and TRIM28 promoted NPM1 centrosomal localization by enhancing its SUMOylation and suppressed centrosome amplification; these functions required the E3 ligase activity of TRIM28. Conversely, depletion of ARF or TRIM28 increased centrosome amplification. ARF also counteracted oncogenic Ras-induced centrosome amplification. Centrosome amplification is often induced by oncogenic insults, leading to genomic instability. However, the mechanisms employed by tumor suppressors to protect the genome are poorly understood. Our findings suggest a novel role for ARF in maintaining genome integrity by facilitating TRIM28-mediated SUMOylation of NPM1, thus preventing centrosome amplification. PMID:26055329

  14. Salvianolic acid A preconditioning confers protection against concanavalin A-induced liver injury through SIRT1-mediated repression of p66shc in mice

    SciTech Connect

    Xu, Xiaomei; Hu, Yan; Zhai, Xiaohan; Lin, Musen; Chen, Zhao; Tian, Xiaofeng; Zhang, Feng; Gao, Dongyan; Ma, Xiaochi; Lv, Li; Yao, Jihong

    2013-11-15

    Salvianolic acid A (SalA) is a phenolic carboxylic acid derivative extracted from Salvia miltiorrhiza. It has many biological and pharmaceutical activities. The purpose of this study was to investigate the effect of SalA on concanavalin A (ConA)-induced acute hepatic injury in Kunming mice and to explore the role of SIRT1 in such an effect. The results showed that in vivo pretreatment with SalA significantly reduced ConA-induced elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and decreased levels of the hepatotoxic cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Moreover, the SalA pretreatment ameliorated the increases in NF-κB and in cleaved caspase-3 caused by ConA exposure. Whereas, the pretreatment completely reversed expression of the B-cell lymphoma-extra large (Bcl-xL). More importantly, the SalA pretreatment significantly increased the expression of SIRT1, a NAD{sup +}-dependent deacetylase, which was known to attenuate acute hypoxia damage and metabolic liver diseases. In our study, the increase in SIRT1 was closely associated with down-regulation of the p66 isoform (p66shc) of growth factor adapter Shc at both protein and mRNA levels. In HepG2 cell culture, SalA pretreatment increased SIRT1 expression in a time and dose-dependent manner and such an increase was abrogated by siRNA knockdown of SIRT1. Additionally, inhibition of SIRT1 significantly reversed the decreased expression of p66shc, and attenuated SalA-induced p66shc down-regulation. Collectively, the present study indicated that SalA may be a potent activator of SIRT and that SalA can alleviate ConA-induced hepatitis through SIRT1-mediated repression of the p66shc pathway. - Highlights: • We report for the first time that SalA protects against ConA-induced hepatitis. • We find that SalA is a potential activator of SIRT1. • SalA's protection against hepatitis involves SIRT1-mediated repression of p66shc.

  15. Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function

    PubMed Central

    Pinz, Sophia; Unser, Samy; Buob, Dominik; Fischer, Philipp; Jobst, Belinda; Rascle, Anne

    2015-01-01

    Signal transducer and activator of transcription STAT5 is essential for the regulation of proliferation and survival genes. Its activity is tightly regulated through cytokine signaling and is often upregulated in cancer. We showed previously that the deacetylase inhibitor trichostatin A (TSA) inhibits STAT5-mediated transcription by preventing recruitment of the transcriptional machinery at a step following STAT5 binding to DNA. The mechanism and factors involved in this inhibition remain unknown. We now show that deacetylase inhibitors do not target STAT5 acetylation, as we initially hypothesized. Instead, they induce a rapid increase in global histone acetylation apparently resulting in the delocalization of the bromodomain and extra-terminal (BET) protein Brd2 and of the Brd2-associated factor TBP to hyperacetylated chromatin. Treatment with the BET inhibitor (+)-JQ1 inhibited expression of STAT5 target genes, supporting a role of BET proteins in the regulation of STAT5 activity. Accordingly, chromatin immunoprecipitation demonstrated that Brd2 is associated with the transcriptionally active STAT5 target gene Cis and is displaced upon TSA treatment. Our data therefore indicate that Brd2 is required for the proper recruitment of the transcriptional machinery at STAT5 target genes and that deacetylase inhibitors suppress STAT5-mediated transcription by interfering with Brd2 function. PMID:25769527

  16. Small Molecule, NSC95397, Inhibits the CtBP1-Protein Partner Interaction and CtBP1-Mediated Transcriptional Repression.

    PubMed

    Blevins, Melanie A; Kouznetsova, Jennifer; Krueger, Aaron B; King, Rebecca; Griner, Lesley Mathews; Hu, Xin; Southall, Noel; Marugan, Juan J; Zhang, Qinghong; Ferrer, Marc; Zhao, Rui

    2015-06-01

    Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor that suppresses multiple proapoptotic and epithelial genes. CtBP is overexpressed in many human cancers, and its overexpression increases stem cell-like features, epithelial-mesenchymal transition, and cancer cell survival. Knockdown of CtBP also increases apoptosis independent of p53 in cell culture. Therefore, targeting CtBP with small molecules that disrupt its interaction with transcription factor partners may be an effective cancer therapy. To elicit its corepressing effect, CtBP binds to a conserved peptide motif in each transcription factor partner. We developed an AlphaScreen high-throughput screening assay to monitor the interaction between CtBP and E1A (which mimics the interaction between CtBP and its transcriptional partners). We screened the LOPAC library of 1280 bioactive compounds and identified NSC95397, which inhibits the CtBP-E1A interaction (IC50 = 2.9 µM). The inhibitory activity of NSC95397 was confirmed using two secondary assays and a counterscreen. NSC95397 also behaved as a weak substrate of CtBP dehydrogenase activity and did not inhibit another dehydrogenase, lactase dehydrogenase. Finally, NSC95397 was able to disrupt CtBP-mediated transcriptional repression of a target gene. These studies present a new possibility for the development of a therapeutic agent targeting tumors through disrupting the CtBP transcriptional complex. PMID:25477201

  17. CD151-α3β1 integrin complexes suppress ovarian tumor growth by repressing slug-mediated EMT and canonical Wnt signaling.

    PubMed

    Baldwin, Lauren A; Hoff, John T; Lefringhouse, Jason; Zhang, Michael; Jia, Changhe; Liu, Zeyi; Erfani, Sonia; Jin, Hongyan; Xu, Mei; She, Qing-Bai; van Nagell, John R; Wang, Chi; Chen, Li; Plattner, Rina; Kaetzel, David M; Luo, Jia; Lu, Michael; West, Dava; Liu, Chunming; Ueland, Fred R; Drapkin, Ronny; Zhou, Binhua P; Yang, Xiuwei H

    2014-12-15

    Human ovarian cancer is diagnosed in the late, metastatic stages but the underlying mechanisms remain poorly understood. We report a surprising functional link between CD151-α3β1 integrin complexes and the malignancy of serous-type ovarian cancer. Analyses of clinical specimens indicate that CD151 expression is significantly reduced or diminished in 90% of metastatic lesions, while it remains detectable in 58% of primary tumors. These observations suggest a putative tumor-suppressing role of CD151 in ovarian cancer. Indeed, our analyses show that knocking down CD151 or α3 integrin enhances tumor cell proliferation, growth and ascites production in nude mice. These changes are accompanied by impaired cell-cell contacts and aberrant expression of E-cadherin, Mucin 5AC and fibronectin, largely reminiscent of an epithelial to mesenchymal transition (EMT)-like change. Importantly, Slug, a master regulator of EMT, is markedly elevated. Knocking down Slug partially restores CD151-α3β1 integrin complex-dependent suppression of cell proliferation. Moreover, disruption of these adhesion protein complexes is accompanied by a concomitant activation of canonical Wnt signaling, including elevated levels of β-catenin and Axin-2 as well as resistance to the inhibition in β-catenin-dependent transcriptional complexes. Together, our study demonstrates that CD151-α3β1 integrin complexes regulate ovarian tumor growth by repressing Slug-mediated EMT and Wnt signaling. PMID:25356755

  18. CD151-α3β1 integrin complexes suppress ovarian tumor growth by repressing slug-mediated EMT and canonical Wnt signaling

    PubMed Central

    Zhang, Michael; Jia, Changhe; Liu, Zeyi; Erfani, Sonia; Jin, Hongyan; Xu, Mei; She, Qing-Bai; van Nagell, John R.; Wang, Chi; Chen, Li; Plattner, Rina; Kaetzel, David M.; Luo, Jia; Lu, Michael; West, Dava; Liu, Chunming; Ueland, Fred R.; Drapkin, Ronny; Zhou, Binhua P.; Yang, Xiuwei H.

    2014-01-01

    Human ovarian cancer is diagnosed in the late, metastatic stages but the underlying mechanisms remain poorly understood. We report a surprising functional link between CD151-α3β1 integrin complexes and the malignancy of serous-type ovarian cancer. Analyses of clinical specimens indicate that CD151 expression is significantly reduced or diminished in 90% of metastatic lesions, while it remains detectable in 58% of primary tumors. These observations suggest a putative tumor-suppressing role of CD151 in ovarian cancer. Indeed, our analyses show that knocking down CD151 or α3 integrin enhances tumor cell proliferation, growth and ascites production in nude mice. These changes are accompanied by impaired cell-cell contacts and aberrant expression of E-cadherin, Mucin 5AC and fibronectin, largely reminiscent of an epithelial to mesenchymal transition (EMT)-like change. Importantly, Slug, a master regulator of EMT, is markedly elevated. Knocking down Slug partially restores CD151-α3β1 integrin complex-dependent suppression of cell proliferation. Moreover, disruption of these adhesion protein complexes is accompanied by a concomitant activation of canonical Wnt signaling, including elevated levels of β-catenin and Axin-2 as well as resistance to the inhibition in β-catenin-dependent transcriptional complexes. Together, our study demonstrates that CD151-α3β1 integrin complexes regulate ovarian tumor growth by repressing Slug-mediated EMT and Wnt signaling. PMID:25356755

  19. Mutational Analysis of AREA, a Transcriptional Activator Mediating Nitrogen Metabolite Repression in Aspergillus nidulans and a Member of the “Streetwise” GATA Family of Transcription Factors

    PubMed Central

    Wilson, Richard A.; Arst, Herbert N.

    1998-01-01

    Summary: The transcriptional activator AREA is a member of the GATA family of transcription factors and mediates nitrogen metabolite repression in the fungus Aspergillus nidulans. The nutritional versatility of A. nidulans and its amenability to classical and reverse genetic manipulations make the AREA DNA binding domain (DBD) a useful model for analyzing GATA family DBDs, particularly as structures of two AREA-DNA complexes have been determined. The 109 extant mutant forms of the AREA DBD surveyed here constitute one of the highest totals of eukaryotic transcription factor DBD mutants, are discussed in light of the roles of individual residues, and are compared to corresponding mutant sequence changes in other fungal GATA factor DBDs. Other topics include delineation of the DBD using both homology and mutational truncation, use of frameshift reversion to detect regions of tolerance to mutational change, the finding that duplication of the DBD can apparently enhance AREA function, and use of the AREA system to analyze a vertebrate GATA factor DBD. Some major points to emerge from work on the AREA DBD are (i) tolerance to sequence change (with retention of function) is surprisingly great, (ii) mutational changes in a transcription factor can have widely differing, even opposing, effects on expression of different structural genes so that monitoring expression of one or even several structural genes can be insufficient and possibly misleading, and (iii) a mutational change altering local hydrophobic packing and DNA binding target specificity can markedly influence the behavior of mutational changes elsewhere in the DBD. PMID:9729601

  20. Poly(ADP-ribose) Polymerase 1 Represses Liver X Receptor-mediated ABCA1 Expression and Cholesterol Efflux in Macrophages.

    PubMed

    Shrestha, Elina; Hussein, Maryem A; Savas, Jeffery N; Ouimet, Mireille; Barrett, Tessa J; Leone, Sarah; Yates, John R; Moore, Kathryn J; Fisher, Edward A; Garabedian, Michael J

    2016-05-20

    Liver X receptors (LXR) are oxysterol-activated nuclear receptors that play a central role in reverse cholesterol transport through up-regulation of ATP-binding cassette transporters (ABCA1 and ABCG1) that mediate cellular cholesterol efflux. Mouse models of atherosclerosis exhibit reduced atherosclerosis and enhanced regression of established plaques upon LXR activation. However, the coregulatory factors that affect LXR-dependent gene activation in macrophages remain to be elucidated. To identify novel regulators of LXR that modulate its activity, we used affinity purification and mass spectrometry to analyze nuclear LXRα complexes and identified poly(ADP-ribose) polymerase-1 (PARP-1) as an LXR-associated factor. In fact, PARP-1 interacted with both LXRα and LXRβ. Both depletion of PARP-1 and inhibition of PARP-1 activity augmented LXR ligand-induced ABCA1 expression in the RAW 264.7 macrophage line and primary bone marrow-derived macrophages but did not affect LXR-dependent expression of other target genes, ABCG1 and SREBP-1c. Chromatin immunoprecipitation experiments confirmed PARP-1 recruitment at the LXR response element in the promoter of the ABCA1 gene. Further, we demonstrated that LXR is poly(ADP-ribosyl)ated by PARP-1, a potential mechanism by which PARP-1 influences LXR function. Importantly, the PARP inhibitor 3-aminobenzamide enhanced macrophage ABCA1-mediated cholesterol efflux to the lipid-poor apolipoprotein AI. These findings shed light on the important role of PARP-1 on LXR-regulated lipid homeostasis. Understanding the interplay between PARP-1 and LXR may provide insights into developing novel therapeutics for treating atherosclerosis. PMID:27026705

  1. Invasive filamentous growth of Candida albicans is promoted by Czf1p-dependent relief of Efg1p-mediated repression.

    PubMed Central

    Giusani, Angela D; Vinces, Marcelo; Kumamoto, Carol A

    2002-01-01

    Filamentation of Candida albicans occurs in response to many environmental cues. During growth within matrix, Efg1p represses filamentation and Czf1p relieves this repression. We propose that Czf1p interacts with Efg1p, altering its function. The complex regulation of filamentation may reflect the versatility of C. albicans as a pathogen. PMID:11973327

  2. The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation

    PubMed Central

    Castelli, Lydia M.; Talavera, David; Kershaw, Christopher J.; Mohammad-Qureshi, Sarah S.; Costello, Joseph L.; Rowe, William; Sims, Paul F. G.; Grant, Christopher M.; Hubbard, Simon J.; Ashe, Mark P.; Pavitt, Graham D.

    2015-01-01

    Translation initiation factor eIF4E mediates mRNA selection for protein synthesis via the mRNA 5’cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control remain poorly understood. Saccharomyces cerevisiae 4E-BPs, Caf20p and Eap1p, each regulate an overlapping set of mRNAs. We undertook global approaches to identify protein and RNA partners of both 4E-BPs by immunoprecipitation of tagged proteins combined with mass spectrometry or next-generation sequencing. Unexpectedly, mass spectrometry indicated that the 4E-BPs associate with many ribosomal proteins. 80S ribosome and polysome association was independently confirmed and was not dependent upon interaction with eIF4E, as mutated forms of both Caf20p and Eap1p with disrupted eIF4E-binding motifs retain ribosome interaction. Whole-cell proteomics revealed Caf20p mutations cause both up and down-regulation of proteins and that many changes were independent of the 4E-binding motif. Investigations into Caf20p mRNA targets by immunoprecipitation followed by RNA sequencing revealed a strong association between Caf20p and mRNAs involved in transcription and cell cycle processes, consistent with observed cell cycle phenotypes of mutant strains. A core set of over 500 Caf20p-interacting mRNAs comprised of both eIF4E-dependent (75%) and eIF4E-independent targets (25%), which differ in sequence attributes. eIF4E-independent mRNAs share a 3’ UTR motif. Caf20p binds all tested motif-containing 3’ UTRs. Caf20p and the 3’UTR combine to influence ERS1 mRNA polysome association consistent with Caf20p contributing to translational control. Finally ERS1 3’UTR confers Caf20-dependent repression of expression to a heterologous reporter gene. Taken together, these data reveal conserved features of eIF4E-dependent Caf20p mRNA targets and uncover a novel eIF4E-independent mode of Caf20p binding to mRNAs that extends the

  3. TNF-mediated inflammation represses GATA1 and activates p38 MAP kinase in RPS19-deficient hematopoietic progenitors.

    PubMed

    Bibikova, Elena; Youn, Min-Young; Danilova, Nadia; Ono-Uruga, Yukako; Konto-Ghiorghi, Yoan; Ochoa, Rachel; Narla, Anupama; Glader, Bertil; Lin, Shuo; Sakamoto, Kathleen M

    2014-12-11

    Diamond-Blackfan anemia (DBA) is an inherited disorder characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. Upregulation of p53 contributes to the pathogenesis of DBA, but the link between ribosomal protein mutations and erythropoietic defects is not well understood. We found that RPS19 deficiency in hematopoietic progenitor cells leads to decreased GATA1 expression in the erythroid progenitor population and p53-dependent upregulation of tumor necrosis factor-α (TNF-α) in nonerythroid cells. The decrease in GATA1 expression was mediated, at least in part, by activation of p38 MAPK in erythroid cells and rescued by inhibition of TNF-α or p53. The anemia phenotype in rps19-deficient zebrafish was reversed by treatment with the TNF-α inhibitor etanercept. Our data reveal that RPS19 deficiency leads to inflammation, p53-dependent increase in TNF-α, activation of p38 MAPK, and decreased GATA1 expression, suggesting a novel mechanism for the erythroid defects observed in DBA. PMID:25270909

  4. The Drosophila esc and E(z) Proteins Are Direct Partners in Polycomb Group-Mediated Repression

    PubMed Central

    Jones, Clark A.; Ng, Joyce; Peterson, Aidan J.; Morgan, Kelly; Simon, Jeffrey; Jones, Richard S.

    1998-01-01

    The extra sex combs (esc) and Enhancer of zeste [E(z)] proteins are members of the Drosophila Polycomb group (Pc-G) of transcriptional repressors. Here we present evidence for direct physical interaction between the esc and E(z) proteins using yeast two-hybrid and in vitro binding assays. In addition, coimmunoprecipitation from embryo extracts demonstrates association of esc and E(z) in vivo. We have delimited the esc-binding domain of E(z) to an N-terminal 33-amino-acid region. Furthermore, we demonstrate that site-directed mutations in the esc protein previously shown to impair esc function in vivo disrupt esc-E(z) interactions in vitro. We also show an in vitro interaction between the heed and EZH1 proteins, which are human homologs of esc and E(z), respectively. These results suggest that the esc-E(z) molecular partnership has been conserved in evolution. Previous studies suggested that esc is primarily involved in the early stages of Pc-G-mediated silencing during embryogenesis. However, E(z) is continuously required in order to maintain chromosome binding by other Pc-G proteins. In light of these earlier observations and the molecular data presented here, we discuss how esc-E(z) protein complexes may contribute to transcriptional silencing by the Pc-G. PMID:9566901

  5. Evolutionarily conserved repressive activity of WOX proteins mediates leaf blade outgrowth and floral organ development in plants

    PubMed Central

    Lin, Hao; Niu, Lifang; McHale, Neil A.; Ohme-Takagi, Masaru; Mysore, Kirankumar S.; Tadege, Million

    2013-01-01

    The WUSCHEL related homeobox (WOX) genes play key roles in stem cell maintenance, embryonic patterning, and lateral organ development. WOX genes have been categorized into three clades—ancient, intermediate, and modern/WUS—based on phylogenetic analysis, but a functional basis for this classification has not been established. Using the classical bladeless lam1 mutant of Nicotiana sylvestris as a genetic tool, we examined the function of the Medicago truncatula WOX gene, STENOFOLIA (STF), in controlling leaf blade outgrowth. STF and LAM1 are functional orthologs. We found that the introduction of mutations into the WUS-box of STF (STFm1) reduces its ability to complement the lam1 mutant. Fusion of an exogenous repressor domain to STFm1 restores complementation, whereas fusion of an exogenous activator domain to STFm1 enhances the narrow leaf phenotype. These results indicate that transcriptional repressor activity mediated by the WUS-box of STF acts to promote blade outgrowth. With the exception of WOX7, the WUS-box is conserved in the modern clade WOX genes, but is not found in members of the intermediate or ancient clades. Consistent with this, all members of the modern clade except WOX7 can complement the lam1 mutant when expressed using the STF promoter, but members of the intermediate and ancient clades cannot. Furthermore, we found that fusion of either the WUS-box or an exogenous repressor domain to WOX7 or to members of intermediate and ancient WOX clades results in a gain-of-function ability to complement lam1 blade outgrowth. These results suggest that modern clade WOX genes have evolved for repressor activity through acquisition of the WUS-box. PMID:23248305

  6. cIAP2 represses IKKα/β-mediated activation of MDM2 to prevent p53 degradation.

    PubMed

    Lau, Rosanna; Niu, Min Ying; Pratt, M A Christine

    2012-11-01

    Cellular inhibitor of apoptosis proteins (cIAP1 and cIAP2) function to prevent apoptosis and are often overexpressed in various cancers. However, mutations in cIAP1/2 can activate the alternative NFκB pathway through IκBα-kinase-α (IKKα) and are associated with hematopoetic malignancies. In the current study, we found that knockdown of cIAP2 in human mammary epithelial cells resulted in activation of MDM2 through increased SUMOylation and profound reduction of the pool of MDM2 not phosphorylated at Ser166. cIAP2 siRNA markedly decreased p53 levels, which were rescued by addition of the MDM2 inhibitor, Nutlin3a. An IAP antagonist, which induces cIAP degradation, transiently increased MDM2 mRNA. Simultaneous transfection of siRNA for cIAP2 and IKKα reduced MDM2 protein, while expression of a kinase-dead IKKβ strongly increased non-Ser166 P-MDM2. Inhibition of either IKKα or -β partially rescued p53 levels, while concomitant IKKα/β inhibition fully rescued p53 after cIAP2 knockdown. Surprisingly, IKKα knockdown alone increased SUMO-MDM2, suggesting that in the absence of activation, IKKα can prevent MDM2 SUMOylation. cIAP2 knockdown disrupted the interaction between the MDM2 SUMO ligase, PIAS1 and IKKα. Partial knockdown of cIAP2 cooperated with (V12) H-ras-transfected mammary epithelial cells to enhance colony formation. In summary, our data identify a novel role for cIAP2 in maintaining wild-type p53 levels by preventing both an NFκB-mediated increase and IKKα/-β-dependent transcriptional and post-translational modifications of MDM2. Thus, mutations or reductions in cIAP2 could contribute to cancer promotion, in part, through downregulation of p53. PMID:23032264

  7. The DNA gyrase inhibitors, nalidixic acid and oxolinic acid, prevent iron-mediated repression of catechol siderophore synthesis in Azotobacter vinelandii.

    PubMed

    Page, W J; Patrick, J

    1988-01-01

    Low concentrations of nalidixic acid and oxolinic acid that were just inhibitory to Azotobacter vinelandii growth promoted the production of the catechol siderophores azotochelin and aminochelin, in the presence of normally repressive concentrations of Fe3+. There was a limited effect on the pyoverdin siderophore, azotobactin, where low concentrations of Fe3+ were rendered less repressive, but the repression by higher concentrations of Fe3+ was normal. These drugs did not induce high-molecular-mass iron-repressible outer-membrane proteins and similar effects on the regulation of catechol siderophore synthesis were not produced by novobiocin, coumermycin, or ethidium bromide. The timing of nalidixic acid and Fe3+ addition to iron-limited cells was critical. Nalidixic acid had to be added before iron-repression of catechol siderophore synthesis and before the onset of iron-sufficient growth. Continued production of the catechol siderophores, however, was not due to interference with normal iron uptake. These data indicated that nalidixic acid prevented normal iron-repression of catechol siderophore synthesis but could not reverse iron repression once it had occurred. The possible roles of DNA gyrase activity in the regulation of catechol siderophore synthesis is discussed. PMID:2856355

  8. The c-rel protooncogene product represses NF-kappa B p65-mediated transcriptional activation of the long terminal repeat of type 1 human immunodeficiency virus.

    PubMed Central

    Doerre, S; Sista, P; Sun, S C; Ballard, D W; Greene, W C

    1993-01-01

    The long terminal repeat (LTR) of the type 1 human immunodeficiency virus (HIV-1) and the 5' regulatory region of the gene encoding the interleukin 2 receptor alpha subunit (IL-2R alpha) share functional kappa B enhancer elements involved in the regulation of these inducible transcription units during T-cell activation. These kappa B enhancer elements are recognized by a structurally related family of interactive proteins that includes p50, p65, and the product of the c-rel protooncogene (c-Rel). Recent biochemical studies have shown that p65 and p50 form the prototypical NF-kappa B complex, which is rapidly translocated from the cytoplasm to the nucleus during T-cell activation. This intracellular signaling complex potently stimulates kappa B-directed transcription from either the HIV-1 LTR or the IL-2R alpha promoter via the strong transactivation domain present in p65. We now demonstrate that nuclear expression of human c-Rel, which is induced by either phorbol ester or tumor necrosis factor alpha with delayed kinetics relative to p65, markedly represses p65-mediated activation of these transcription units. These inhibitory effects of c-Rel correlate with its DNA-binding activity but not with its ability to heterodimerize with p50, suggesting that c-Rel inhibition involves competition with p50/p65 for occupancy of the kappa B enhancer element. Together, these findings suggest that one function of c-Rel is as a physiologic repressor of the HIV-1 LTR and IL-2R alpha promoters, serving to efficiently counter the strong transcriptional activating effects of p65. Images PMID:8430069

  9. Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-kappa B and C/EBP beta.

    PubMed Central

    Stein, B; Yang, M X

    1995-01-01

    Bone metabolism is regulated by a balance between bone resorption caused by osteoclasts and bone formation caused by osteoblasts. This balance is disturbed in postmenopausal women as a result of lower serum estrogen levels. Estrogen, which is used in hormone replacement therapy to prevent postmenopausal osteoporosis, downregulates expression of the interleukin 6 (IL-6) gene in osteoblasts and bone marrow stromal cells. IL-6 is directly involved in bone resorption by activating immature osteoclasts. We show here that NF-kappa B and C/EBP beta are important regulators of IL-6 gene expression in human osteoblasts. Importantly, the IL-6 promoter is inhibited by estrogen in the absence of a functional estrogen receptor (ER) binding site. This inhibition is mediated by the transcription factors NF-kappa B and C/EBP beta. Evidence is presented for a direct interaction between these two factors and ER. We characterized the protein sequence requirements for this association in vitro and in vivo. The physical and functional interaction depends in part on the DNA binding domain and region D of ER and on the Rel homology domain of NF-kappa B and the bZIP region of C/EBP beta. The cross-coupling between ER, NF-kappa B, and C/EBP beta also results in reduced activity of promoters with ER binding sites. We further show that the mechanism of IL-6 gene repression by estrogen is clearly different from that of activation of promoters with ER binding sites. Therefore, drugs that separate the transactivation and transrepression functions of ER will be very helpful for treatment of osteoporosis without causing undesirable side effects. PMID:7651415

  10. Isolation of a Novel Family of C2H2 Zinc Finger Proteins Implicated in Transcriptional Repression Mediated by Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) Orphan Nuclear Receptors*

    PubMed Central

    Avram, Dorina; Fields, Andrew; Top, Karen Pretty On; Nevrivy, Daniel J.; Ishmael, Jane E.; Leid, Mark

    2010-01-01

    Two novel and related C2H2 zinc finger proteins that are highly expressed in the brain, CTIP1 and CTIP2 (COUP TF-interacting proteins 1 and 2, respectively), were isolated and shown to interact with all members of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of orphan nuclear receptors. The interaction of CTIP1 with ARP1 was studied in detail, and CTIP1 was found to harbor two independent ARP1 interaction domains, ID1 and ID2, whereas the putative AF-2 of ARP1 was required for interaction with CTIP1. CTIP1, which exhibited a punctate staining pattern within the nucleus of transfected cells, recruited cotransfected ARP1 to these foci and potentiated ARP1-mediated transcriptional repression of a reporter construct. However, transcriptional repression mediated by ARP1 acting through CTIP1 did not appear to involve recruitment of a trichostatin A-sensitive histone deacetylase(s) to the template, suggesting that this repression pathway may be distinct from that utilized by several other nuclear receptors. PMID:10744719

  11. Repression: A Bibliography.

    ERIC Educational Resources Information Center

    Pedrini, D. T.; Pedrini, Bonnie C.

    Repression was considered by Freud as a key mechanism for everyone, especially for normals and neurotics. His Repression paper, written in 1915, was psychoanalytically definitive. Of course, much had been written before and even more was written after. Anna Freud's book The Ego and the Mechanisms of Defence included repression and was published by…

  12. The octamer/mu E4 region of the immunoglobulin heavy-chain enhancer mediates gene repression in myeloma x T-lymphoma hybrids.

    PubMed Central

    Shen, L; Lieberman, S; Eckhardt, L A

    1993-01-01

    We have shown previously that the immunoglobulin heavy-chain enhancer acts as a repressor of gene transcription in hybrids between immunoglobulin-producing myelomas and a T-lymphoma line. We have now mapped this repressive activity to a 51-bp enhancer subfragment which contains the octamer and mu E4 protein-binding motifs. Even a single copy of this subfragment will repress gene expression in hybrid cells. Mutational analyses of the repressor fragment suggest that in non-B cells, a strong transcriptional repressor(s) functions through the same motifs important for gene activation in B cells. Changes in chromatin structure that accompany reporter gene repression suggest a general mechanism for prohibiting immunoglobulin heavy-chain locus activation in inappropriate cell types. Images PMID:8497268

  13. HIGH NITROGEN INSENSITIVE 9 (HNI9)-mediated systemic repression of root NO3− uptake is associated with changes in histone methylation

    PubMed Central

    Widiez, Thomas; El Kafafi, El Sayed; Girin, Thomas; Berr, Alexandre; Ruffel, Sandrine; Krouk, Gabriel; Vayssières, Alice; Shen, Wen-Hui; Coruzzi, Gloria M.; Gojon, Alain; Lepetit, Marc

    2011-01-01

    In plants, root nitrate uptake systems are under systemic feedback repression by the N satiety of the whole organism, thus adjusting the N acquisition capacity to the N demand for growth; however, the underlying molecular mechanisms are largely unknown. We previously isolated the Arabidopsis high nitrogen-insensitive 9-1 (hni9-1) mutant, impaired in the systemic feedback repression of the root nitrate transporter NRT2.1 by high N supply. Here, we show that HNI9 encodes Arabidopsis INTERACT WITH SPT6 (AtIWS1), an evolutionary conserved component of the RNA polymerase II complex. HNI9/AtIWS1 acts in roots to repress NRT2.1 transcription in response to high N supply. At a genomic level, HNI9/AtIWS1 is shown to play a broader role in N signaling by regulating several hundred N-responsive genes in roots. Repression of NRT2.1 transcription by high N supply is associated with an HNI9/AtIWS1-dependent increase in histone H3 lysine 27 trimethylation at the NRT2.1 locus. Our findings highlight the hypothesis that posttranslational chromatin modifications control nutrient acquisition in plants. PMID:21788519

  14. Studies in mice, hamsters, and rats demonstrate that repression of hepatic apoA-I expression by taurocholic acid in mice is not mediated by the farnesoid-X-receptor

    PubMed Central

    Gardès, Christophe; Blum, Denise; Bleicher, Konrad; Chaput, Evelyne; Ebeling, Martin; Hartman, Peter; Handschin, Corinne; Richter, Hans; Benson, G. Martin

    2011-01-01

    It is claimed that apoA-I expression is repressed in mice by cholic acid (CA) and its taurine conjugate, taurocholic acid (TCA) via farnesoid X receptor (FXR) activation. We measured apoA-I expression in mice, hamsters, and rats treated with highly potent and selective synthetic FXR agonists or with TCA. All of the synthetic agonists bound to FXR with high affinity in a scintillation proximity assay. However, TCA did not compete with the radioligand up to the highest concentration used (100 μM). The C-site regulatory region of apoA-I, through which FXR has been reported to regulate its expression, is completely conserved across the species investigated. In both male and female human apoA-I-transgenic mice, we reproduced the previously reported strong inhibition of human apoA-I expression upon treatment with the typical supraphysiological dose of TCA used in such studies. However, in contrast to some previous reports, TCA did not repress murine apoA-I expression in the same mice. Also, more-potent and -selective FXR agonists did not affect human or murine apoA-I expression in this model. In LDL receptor-deficient mice and Golden Syrian hamsters, selective FXR agonists did not affect apoA-I expression, whereas in Wistar rats, some even increased apoA-I expression. In conclusion, selective FXR agonists do not repress apoA-I expression in rodents. Repression of human apoA-I expression by TCA in transgenic mice is probably mediated through FXR-independent mechanisms. PMID:21464203

  15. Multiple mechanisms of transcriptional repression by YY1.

    PubMed Central

    Galvin, K M; Shi, Y

    1997-01-01

    The four C-terminal GLI-Krüppel type zinc fingers of YY1 have been identified as a transcriptional repression domain. Previous reports have proposed DNA-bending and activator-quenching mechanisms for this zinc finger-mediated repression. In addition, previous work indicated that p300 and CBP might be involved in YY1-mediated repression. We have analyzed these possible models for the zinc finger-mediated repression. The role of each zinc finger in the repression and DNA-binding functions was determined by using a structure-and-function approach. We show that zinc finger 2 of YY1 plays a central role in both DNA binding and transcriptional repression. However, a survey of a panel of YY1 mutants indicates that these two functions can be separated, which argues against the DNA-bending model for repression. We show that the physical interaction between YY1 and p300, a coactivator for CREB, is not sufficient for repression of CREB-mediated transcription. Our studies indicate that YY1 functions as an activator-specific repressor. Repression of CTF-1-directed transcription may be accomplished through direct physical interaction between YY1 and this activator. In contrast, physical interaction is not necessary for YY1 to repress Sp1- and CREB-mediated transcription. Rather, the repression likely reflects an ability of YY1 to interfere with communication between these activators and their targets within the general transcription machinery. Taken together, our results suggest that YY1 employs multiple mechanisms to achieve activator-specific repression. PMID:9199306

  16. The Basic Helix-Loop-Helix Transcription Factor MYC2 Directly Represses PLETHORA Expression during Jasmonate-Mediated Modulation of the Root Stem Cell Niche in Arabidopsis[W][OA

    PubMed Central

    Chen, Qian; Sun, Jiaqiang; Zhai, Qingzhe; Zhou, Wenkun; Qi, Linlin; Xu, Li; Wang, Bao; Chen, Rong; Jiang, Hongling; Qi, Jing; Li, Xugang; Palme, Klaus; Li, Chuanyou

    2011-01-01

    The root stem cell niche, which in the Arabidopsis thaliana root meristem is an area of four mitotically inactive quiescent cells (QCs) and the surrounding mitotically active stem cells, is critical for root development and growth. We report here that during jasmonate-induced inhibition of primary root growth, jasmonate reduces root meristem activity and leads to irregular QC division and columella stem cell differentiation. Consistently, jasmonate reduces the expression levels of the AP2-domain transcription factors PLETHORA1 (PLT1) and PLT2, which form a developmentally instructive protein gradient and mediate auxin-induced regulation of stem cell niche maintenance. Not surprisingly, the effects of jasmonate on root stem cell niche maintenance and PLT expression require the functioning of MYC2/JASMONATE INSENSITIVE1, a basic helix-loop-helix transcription factor that involves versatile aspects of jasmonate-regulated gene expression. Gel shift and chromatin immunoprecipitation experiments reveal that MYC2 directly binds the promoters of PLT1 and PLT2 and represses their expression. We propose that MYC2-mediated repression of PLT expression integrates jasmonate action into the auxin pathway in regulating root meristem activity and stem cell niche maintenance. This study illustrates a molecular framework for jasmonate-induced inhibition of root growth through interaction with the growth regulator auxin. PMID:21954460

  17. Repression of G1/S Transcription Is Mediated via Interaction of the GTB Motifs of Nrm1 and Whi5 with Swi6

    PubMed Central

    Travesa, Anna; Kalashnikova, Tatyana I.; de Bruin, Robertus A. M.; Cass, Sarah Rose; Chahwan, Charly; Lee, David E.; Lowndes, Noel F.

    2013-01-01

    In Saccharomyces cerevisiae, G1/S transcription factors MBF and SBF regulate a large family of genes important for entry to the cell cycle and DNA replication and repair. Their regulation is crucial for cell viability, and it is conserved throughout evolution. MBF and SBF consist of a common component, Swi6, and a DNA-specific binding protein, Mbp1 and Swi4, respectively. Transcriptional repressors bind to and regulate the activity of both transcription factors. Whi5 binds to SBF and represses its activity at the beginning of the G1 phase to prevent early activation. Nrm1 binds to MBF to repress transcription as cells progress through S phase. Here, we describe a protein motif, the GTB motif (for G1/S transcription factor binding), in Nrm1 and Whi5 that is required to bind to the transcription factors. We also identify a region of the carboxy terminus of Swi6 that is required for Nrm1 and Whi5 binding to their target transcription factors and show that mutation of this region overrides the repression of MBF- and SBF-regulated genes by Nrm1 and Whi5. Finally, we show that the GTB motif is the core of a functional module that is necessary and sufficient for targeting of the transcription factors by their cognate repressors. PMID:23382076

  18. A cis-acting element in the promoter of human ether à go-go 1 potassium channel gene mediates repression by calcitriol in human cervical cancer cells.

    PubMed

    Cázares-Ordoñez, V; González-Duarte, R J; Díaz, L; Ishizawa, M; Uno, S; Ortíz, V; Ordoñez-Sánchez, M L; Makishima, M; Larrea, F; Avila, E

    2015-02-01

    The human ether à go-go 1 potassium channel (hEAG1) is required for cell cycle progression and proliferation of cancer cells. Inhibitors of hEAG1 activity and expression represent potential therapeutic drugs in cancer. Previously, we have shown that hEAG1 expression is downregulated by calcitriol in a variety of cancer cells. Herein, we provided evidence on the regulatory mechanism involved in such repressive effect in cells derived from human cervical cancer. Our results indicate that repression by calcitriol occurs at the transcriptional level and involves a functional negative vitamin D response element (nVDRE) E-box type in the hEAG1 promoter. The described mechanism in this work implies that a protein complex formed by the vitamin D receptor-interacting repressor, the vitamin D receptor, the retinoid X receptor, and the Williams syndrome transcription factor interact with the nVDRE in the hEAG1 promoter in the absence of ligand. Interestingly, all of these transcription factors except the vitamin D receptor-interacting repressor are displaced from hEAG1 promoter in the presence of calcitriol. Our results provide novel mechanistic insights into calcitriol mode of action in repressing hEAG1 gene expression. PMID:25495694

  19. CRISPR/dCas9-mediated Transcriptional Inhibition Ameliorates the Epigenetic Dysregulation at D4Z4 and Represses DUX4-fl in FSH Muscular Dystrophy.

    PubMed

    Himeda, Charis L; Jones, Takako I; Jones, Peter L

    2016-03-01

    Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent myopathies, affecting males and females of all ages. Both forms of the disease are linked by epigenetic derepression of the D4Z4 macrosatellite repeat array at chromosome 4q35, leading to aberrant expression of D4Z4-encoded RNAs in skeletal muscle. Production of full-length DUX4 (DUX4-fl) mRNA from the derepressed D4Z4 array results in misexpression of DUX4-FL protein and its transcriptional targets, and apoptosis, ultimately leading to accumulated muscle pathology. Returning the chromatin at the FSHD locus to its nonpathogenic, epigenetically repressed state would simultaneously affect all D4Z4 RNAs, inhibiting downstream pathogenic pathways, and is thus an attractive therapeutic strategy. Advances in CRISPR/Cas9-based genome editing make it possible to target epigenetic modifiers to an endogenous disease locus, although reports to date have focused on more typical genomic regions. Here, we demonstrate that a CRISPR/dCas9 transcriptional inhibitor can be specifically targeted to the highly repetitive FSHD macrosatellite array and alter the chromatin to repress expression of DUX4-fl in primary FSHD myocytes. These results implicate the promoter and exon 1 of DUX4 as potential therapeutic targets and demonstrate the utility of CRISPR technology for correction of the epigenetic dysregulation in FSHD. PMID:26527377

  20. The cell density-dependent expression of stewartan exopolysaccharide in Pantoea stewartii ssp. stewartii is a function of EsaR-mediated repression of the rcsA gene.

    PubMed

    Minogue, Timothy D; Carlier, Aurelien L; Koutsoudis, Maria D; von Bodman, Susanne B

    2005-04-01

    The LuxR-type quorum-sensing transcription factor EsaR functions as a repressor of exopolysaccharide (EPS) synthesis in the phytopathogenic bacterium Pantoea stewartii ssp. stewartii. The cell density-dependent expression of EPS is critical for Stewart's wilt disease development. Strains deficient in the synthesis of a diffusible acyl-homoserine lactone inducer remain repressed for EPS synthesis and are consequently avirulent. In contrast, disruption of the esaR gene leads to hypermucoidy and attenuated disease development. Ligand-free EsaR functions as a negative autoregulator of the esaR gene and responds to exogenous acyl-homoserine lactone for derepression. The focus of this study was to define the mechanism by which EsaR governs the expression of the cps locus, which encodes functions required for stewartan EPS synthesis and membrane translocation. Genetic and biochemical studies show that EsaR directly represses the transcription of the rcsA gene. RcsA encodes an essential coactivator for RcsA/RcsB-mediated transcriptional activation of cps genes. In vitro assays identify an EsaR DNA binding site within the rcsA promoter that is reasonably well conserved with the previously described esaR box. We also describe that RcsA positively controls its own expression. Interestingly, promoter proximal genes within the cps cluster are significantly more acyl-homoserine lactone responsive than genes located towards the middle or 3' end of the gene cluster. We will discuss a possible role of EsaR-mediated quorum sensing in the differential expression of the cps operon. PMID:15773989

  1. The Polycomb Group Protein L3MBTL1 Represses a SMAD5-Mediated Hematopoietic Transcriptional Program in Human Pluripotent Stem Cells

    PubMed Central

    Perna, Fabiana; Vu, Ly P.; Themeli, Maria; Kriks, Sonja; Hoya-Arias, Ruben; Khanin, Raya; Hricik, Todd; Mansilla-Soto, Jorge; Papapetrou, Eirini P.; Levine, Ross L.; Studer, Lorenz; Sadelain, Michel; Nimer, Stephen D.

    2015-01-01

    Summary Epigenetic regulation of key transcriptional programs is a critical mechanism that controls hematopoietic development, and, thus, aberrant expression patterns or mutations in epigenetic regulators occur frequently in hematologic malignancies. We demonstrate that the Polycomb protein L3MBTL1, which is monoallelically deleted in 20q- myeloid malignancies, represses the ability of stem cells to drive hematopoietic-specific transcriptional programs by regulating the expression of SMAD5 and impairing its recruitment to target regulatory regions. Indeed, knockdown of L3MBTL1 promotes the development of hematopoiesis and impairs neural cell fate in human pluripotent stem cells. We also found a role for L3MBTL1 in regulating SMAD5 target gene expression in mature hematopoietic cell populations, thereby affecting erythroid differentiation. Taken together, we have identified epigenetic priming of hematopoietic-specific transcriptional networks, which may assist in the development of therapeutic approaches for patients with anemia. PMID:25754204

  2. MOZ-mediated repression of p16(INK) (4) (a) is critical for the self-renewal of neural and hematopoietic stem cells.

    PubMed

    Perez-Campo, Flor M; Costa, Guilherme; Lie-A-Ling, Michael; Stifani, Stefano; Kouskoff, Valerie; Lacaud, Georges

    2014-06-01

    Although inhibition of p16(INK4a) expression is critical to preserve the proliferative capacity of stem cells, the molecular mechanisms responsible for silencing p16(INK4a) expression remain poorly characterized. Here, we show that the histone acetyltransferase (HAT) monocytic leukemia zinc finger protein (MOZ) controls the proliferation of both hematopoietic and neural stem cells by modulating the transcriptional repression of p16(INK4a) . In the absence of the HAT activity of MOZ, expression of p16(INK4a) is upregulated in progenitor and stem cells, inducing an early entrance into replicative senescence. Genetic deletion of p16(INK4a) reverses the proliferative defect in both Moz(HAT) (-) (/) (-) hematopoietic and neural progenitors. Our results suggest a critical requirement for MOZ HAT activity to silence p16(INK4a) expression and to protect stem cells from early entrance into replicative senescence. PMID:24307508

  3. SPROUTY-2 represses the epithelial phenotype of colon carcinoma cells via upregulation of ZEB1 mediated by ETS1 and miR-200/miR-150.

    PubMed

    Barbáchano, A; Fernández-Barral, A; Pereira, F; Segura, M F; Ordóñez-Morán, P; Carrillo-de Santa Pau, E; González-Sancho, J M; Hanniford, D; Martínez, N; Costales-Carrera, A; Real, F X; Pálmer, H G; Rojas, J M; Hernando, E; Muñoz, A

    2016-06-01

    SPROUTY-2 (SPRY2) is a modulator of tyrosine kinase receptor signaling with receptor- and cell type-dependent inhibitory or enhancing effects. Studies on the action of SPRY2 in major cancers are conflicting and its role remains unclear. Here we have dissected SPRY2 action in human colon cancer. Global transcriptomic analyses show that SPRY2 downregulates genes encoding tight junction proteins such as claudin-7 and occludin and other cell-to-cell and cell-to-matrix adhesion molecules in human SW480-ADH colon carcinoma cells. Moreover, SPRY2 represses LLGL2/HUGL2, PATJ1/INADL and ST14, main regulators of the polarized epithelial phenotype, and ESRP1, an epithelial-to-mesenchymal transition (EMT) inhibitor. A key action of SPRY2 is the upregulation of the major EMT inducer ZEB1, as these effects are reversed by ZEB1 knock-down by means of RNA interference. Consistently, we found an inverse correlation between the expression level of claudin-7 and those of SPRY2 and ZEB1 in human colon tumors. Mechanistically, ZEB1 upregulation by SPRY2 results from the combined induction of ETS1 transcription factor and the repression of microRNAs (miR-200 family, miR-150) that target ZEB1 RNA. Moreover, SPRY2 increased AKT activation by epidermal growth factor, whereas AKT and also Src inhibition reduced the induction of ZEB1. Altogether, these data suggest that AKT and Src are implicated in SPRY2 action. Collectively, these results show a tumorigenic role of SPRY2 in colon cancer that is based on the dysregulation of tight junction and epithelial polarity master genes via upregulation of ZEB1. The dissection of the mechanism of action of SPRY2 in colon cancer cells is important to understand the upregulation of this gene in a subset of patients with this neoplasia that have poor prognosis. PMID:26455323

  4. Yeast carbon catabolite repression.

    PubMed

    Gancedo, J M

    1998-06-01

    Glucose and related sugars repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources; some of these genes are also repressed by other sugars such as galactose, and the process is known as catabolite repression. The different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins, but there are different circuits of repression for different groups of genes. However, the protein kinase Snf1/Cat1 is shared by the various circuits and is therefore a central element in the regulatory process. Snf1 is not operative in the presence of glucose, and preliminary evidence suggests that Snf1 is in a dephosphorylated state under these conditions. However, the enzymes that phosphorylate and dephosphorylate Snf1 have not been identified, and it is not known how the presence of glucose may affect their activity. What has been established is that Snf1 remains active in mutants lacking either the proteins Grr1/Cat80 or Hxk2 or the Glc7 complex, which functions as a protein phosphatase. One of the main roles of Snf1 is to relieve repression by the Mig1 complex, but it is also required for the operation of transcription factors such as Adr1 and possibly other factors that are still unidentified. Although our knowledge of catabolite repression is still very incomplete, it is possible in certain cases to propose a partial model of the way in which the different elements involved in catabolite repression may be integrated. PMID:9618445

  5. Kicking against the PRCs – A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2

    PubMed Central

    Perera, Pumi; Mora-García, Santiago; de Leau, Erica; Thornton, Harry; de Alves, Flavia Lima; Rapsilber, Juri; Yang, Suxin; James, Geo Velikkakam; Schneeberger, Korbinian; Finnegan, E. Jean; Turck, Franziska; Goodrich, Justin

    2015-01-01

    The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits Pc

  6. E2F/Rb Family Proteins Mediate Interferon Induced Repression of Adenovirus Immediate Early Transcription to Promote Persistent Viral Infection.

    PubMed

    Zheng, Yueting; Stamminger, Thomas; Hearing, Patrick

    2016-01-01

    Interferons (IFNs) are cytokines that have pleiotropic effects and play important roles in innate and adaptive immunity. IFNs have broad antiviral properties and function by different mechanisms. IFNs fail to inhibit wild-type Adenovirus (Ad) replication in established cancer cell lines. In this study, we analyzed the effects of IFNs on Ad replication in normal human cells. Our data demonstrate that both IFNα and IFNγ blocked wild-type Ad5 replication in primary human bronchial epithelial cells (NHBEC) and TERT-immortalized normal human diploid fibroblasts (HDF-TERT). IFNs inhibited the replication of divergent adenoviruses. The inhibition of Ad5 replication by IFNα and IFNγ is the consequence of repression of transcription of the E1A immediate early gene product. Both IFNα and IFNγ impede the association of the transactivator GABP with the E1A enhancer region during the early phase of infection. The repression of E1A expression by IFNs requires a conserved E2F binding site in the E1A enhancer, and IFNs increased the enrichment of the E2F-associated pocket proteins, Rb and p107, at the E1A enhancer in vivo. PD0332991 (Pabociclib), a specific CDK4/6 inhibitor, dephosphoryles pocket proteins to promote their interaction with E2Fs and inhibited wild-type Ad5 replication dependent on the conserved E2F binding site. Consistent with this result, expression of the small E1A oncoprotein, which abrogates E2F/pocket protein interactions, rescued Ad replication in the presence of IFNα or IFNγ. Finally, we established a persistent Ad infection model in vitro and demonstrated that IFNγ suppresses productive Ad replication in a manner dependent on the E2F binding site in the E1A enhancer. This is the first study that probes the molecular basis of persistent adenovirus infection and reveals a novel mechanism by which adenoviruses utilize IFN signaling to suppress lytic virus replication and to promote persistent infection. PMID:26809031

  7. E2F/Rb Family Proteins Mediate Interferon Induced Repression of Adenovirus Immediate Early Transcription to Promote Persistent Viral Infection

    PubMed Central

    Zheng, Yueting; Stamminger, Thomas; Hearing, Patrick

    2016-01-01

    Interferons (IFNs) are cytokines that have pleiotropic effects and play important roles in innate and adaptive immunity. IFNs have broad antiviral properties and function by different mechanisms. IFNs fail to inhibit wild-type Adenovirus (Ad) replication in established cancer cell lines. In this study, we analyzed the effects of IFNs on Ad replication in normal human cells. Our data demonstrate that both IFNα and IFNγ blocked wild-type Ad5 replication in primary human bronchial epithelial cells (NHBEC) and TERT-immortalized normal human diploid fibroblasts (HDF-TERT). IFNs inhibited the replication of divergent adenoviruses. The inhibition of Ad5 replication by IFNα and IFNγ is the consequence of repression of transcription of the E1A immediate early gene product. Both IFNα and IFNγ impede the association of the transactivator GABP with the E1A enhancer region during the early phase of infection. The repression of E1A expression by IFNs requires a conserved E2F binding site in the E1A enhancer, and IFNs increased the enrichment of the E2F-associated pocket proteins, Rb and p107, at the E1A enhancer in vivo. PD0332991 (Pabociclib), a specific CDK4/6 inhibitor, dephosphoryles pocket proteins to promote their interaction with E2Fs and inhibited wild-type Ad5 replication dependent on the conserved E2F binding site. Consistent with this result, expression of the small E1A oncoprotein, which abrogates E2F/pocket protein interactions, rescued Ad replication in the presence of IFNα or IFNγ. Finally, we established a persistent Ad infection model in vitro and demonstrated that IFNγ suppresses productive Ad replication in a manner dependent on the E2F binding site in the E1A enhancer. This is the first study that probes the molecular basis of persistent adenovirus infection and reveals a novel mechanism by which adenoviruses utilize IFN signaling to suppress lytic virus replication and to promote persistent infection. PMID:26809031

  8. VdCYC8, Encoding CYC8 Glucose Repression Mediator Protein, Is Required for Microsclerotia Formation and Full Virulence in Verticillium dahliae

    PubMed Central

    Li, Zhi-Fang; Liu, Yi-Jie; Feng, Zi-Li; Feng, Hong-Jie; Klosterman, Steven J.; Zhou, Fang-Fang; Zhao, Li-Hong; Shi, Yong-Qiang; Zhu, He-Qin

    2015-01-01

    Verticillium dahliae is the primary causal agent for Verticillium wilt disease on a diverse array of economically important crops, including cotton. In previous research, we obtained the low-pathogenicity mutant T286 from the T-DNA insertional mutant library of the highly virulent isolate Vd080 derived from cotton. In this study, the target disrupted gene VdCYC8 was identified by TAIL-PCR, encoding a homolog of CYC8 proteins involved in glucose repression. The deletion mutant ΔCYC8 exhibited several developmental deficiencies, including reduced microsclerotia formation, reduced sporulation, and slower growth. Moreover, compared with the wild type strain Vd080, the pathogenicity of strain ΔCYC8 was significantly decreased on cotton seedlings. However, the complementary mutants ΔCYC8-C led to restoration of the wild type phenotype or near wild type levels of virulence on cotton. Interestingly, pathogenicity of the strains was correlated with VdCYC8 gene expression levels in complemented mutants. Gene expression analyses in the wild type strain Vd080, the ΔCYC8-45 strain, and complemented strain ΔCYC8-C26 indicated that VdCYC8 regulates the transcription levels of several genes in V. dahliae that have roles in melanin and production. PMID:26633180

  9. Zeste maintains repression of Ubx transgenes: Support for a new model of polycomb repression

    SciTech Connect

    Hur, Man-Wook; Laney, Jeffrey D.; Jeon, Sang-Hack; Ali, Janann; Biggin, Mark D.

    2001-09-01

    During late embryogenesis, the expression domains of homeotic genes are maintained by two groups of ubiquitously expressed regulators: the Polycomb repressors and the Trithorax activators. It is not known how the activities of the two maintenance systems are initially targeted to the correct genes. Zeste and GAGA are sequence specific DNA binding proteins previously shown to be Trithorax group activators of the homeotic gene Ultrabithorax (Ubx). Here we demonstrate that Zeste and GAGA DNA binding sites at the proximal promoter are also required to maintain, but not to initiate, repression of Ubx. Further, the repression mediated by Zeste DNA binding site is abolished in zeste null embryos. These data imply that Zeste and probably GAGA mediate Polycomb repression. We present a model in which the dual transcriptional activities of Zeste and GAGA are an essential component of the mechanism that chooses which maintenance system is to be targeted to a given promoter.

  10. Dicer1 imparts essential survival cues in Notch-driven T-ALL via miR-21-mediated tumor suppressor Pdcd4 repression.

    PubMed

    Junker, Fabian; Chabloz, Antoine; Koch, Ute; Radtke, Freddy

    2015-08-20

    The modulatory function of individual microRNAs (miRNAs) in Notch-driven T-cell acute lymphoblastic leukemias (T-ALLs) has recently been established. Although protumorigenic and tumor-suppressive miRNAs are implicated in disease onset in murine models of Notch-driven T-cell leukemia, whether Dicer1-processed miRNAs are essential for Notch-driven T-ALL is currently unknown. Here we used conditional and inducible genetic loss-of-function approaches to test whether the development and maintenance of Notch-driven T-ALL was dependent on Dicer1 function. Mice with specific inactivation of both Dicer1 alleles in the T-cell lineage did not develop Notch-driven T-ALL. In contrast, loss of 1 functional Dicer1 allele did not significantly perturb T-ALL onset and tumor progression. Inducible inactivation of Dicer1 in early stage polyclonal T-ALL cells was sufficient to abrogate T-ALL progression in leukemic mice, whereas late-stage monoclonal T-ALL cells were counterselected against loss of Dicer1. Lineage-tracing experiments revealed that Dicer1 deficiency led to the induction of apoptosis in T-ALL cells, whereas cell cycle progression remained unaltered. Through microarray-based miRNA profiling, we identified miR-21 as a previously unrecognized miRNA deregulated in both mouse and human T-ALL. Herein, we demonstrate that miR-21 regulates T-ALL cell survival via repression of the tumor suppressor Pdcd4. PMID:25979949

  11. E2F mediates cell cycle-dependent transcriptional repression in vivo by recruitment of an HDAC1/mSin3B corepressor complex

    PubMed Central

    Rayman, Joseph B.; Takahashi, Yasuhiko; Indjeian, Vahan B.; Dannenberg, Jan-Hermen; Catchpole, Steven; Watson, Roger J.; te Riele, Hein; Dynlacht, Brian David

    2002-01-01

    Despite biochemical and genetic data suggesting that E2F and pRB (pocket protein) families regulate transcription via chromatin-modifying factors, the precise mechanisms underlying gene regulation by these protein families have not yet been defined in a physiological setting. In this study, we have investigated promoter occupancy in wild-type and pocket protein-deficient primary cells. We show that corepressor complexes consisting of histone deacetylase (HDAC1) and mSin3B were specifically recruited to endogenous E2F-regulated promoters in quiescent cells. These complexes dissociated from promoters once cells reached late G1, coincident with gene activation. Interestingly, recruitment of HDAC1 complexes to promoters depended absolutely on p107 and p130, and required an intact E2F-binding site. In contrast, mSin3B recruitment to certain promoters did not require p107 or p130, suggesting that recruitment of this corepressor can occur via E2F-dependent and -independent mechanisms. Remarkably, loss of pRB had no effect on HDAC1 or mSin3B recruitment. p107/p130 deficiency triggered a dramatic loss of E2F4 nuclear localization as well as transcriptional derepression, which is suggested by nucleosome mapping studies to be the result of localized hyperacetylation of nucleosomes proximal to E2F-binding sites. Taken together, these findings show that p130 escorts E2F4 into the nucleus and, together with corepressor complexes that contain mSin3B and/or HDAC1, directly represses transcription from target genes as cells withdraw from the cell cycle. PMID:11959842

  12. Nerve Injury Diminishes Opioid Analgesia through Lysine Methyltransferase-mediated Transcriptional Repression of μ-Opioid Receptors in Primary Sensory Neurons.

    PubMed

    Zhang, Yuhao; Chen, Shao-Rui; Laumet, Geoffroy; Chen, Hong; Pan, Hui-Lin

    2016-04-15

    The μ-opioid receptor (MOR, encoded by Oprm1) agonists are the mainstay analgesics for treating moderate to severe pain. Nerve injury causes down-regulation of MORs in the dorsal root ganglion (DRG) and diminishes the opioid effect on neuropathic pain. However, the epigenetic mechanisms underlying the diminished MOR expression caused by nerve injury are not clear. G9a (encoded by Ehmt2), a histone 3 at lysine 9 methyltransferase, is a key chromatin regulator responsible for gene silencing. In this study, we determined the role of G9a in diminished MOR expression and opioid analgesic effects in animal models of neuropathic pain. We found that nerve injury in rats induced a long-lasting reduction in the expression level of MORs in the DRG but not in the spinal cord. Nerve injury consistently increased the enrichment of the G9a product histone 3 at lysine 9 dimethylation in the promoter of Oprm1 in the DRG. G9a inhibition or siRNA knockdown fully reversed MOR expression in the injured DRG and potentiated the morphine effect on pain hypersensitivity induced by nerve injury. In mice lacking Ehmt2 in DRG neurons, nerve injury failed to reduce the expression level of MORs and the morphine effect. In addition, G9a inhibition or Ehmt2 knockout in DRG neurons normalized nerve injury-induced reduction in the inhibitory effect of the opioid on synaptic glutamate release from primary afferent nerves. Our findings indicate that G9a contributes critically to transcriptional repression of MORs in primary sensory neurons in neuropathic pain. G9a inhibitors may be used to enhance the opioid analgesic effect in the treatment of chronic neuropathic pain. PMID:26917724

  13. Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element-Binding Protein B.

    PubMed

    Rumora, Amy E; Ferris, Lauren A; Wheeler, Tamar R; Kelm, Robert J

    2016-05-17

    Myofibroblast differentiation is characterized by an increased level of expression of cytoskeletal smooth muscle α-actin. In human and murine fibroblasts, the gene encoding smooth muscle α-actin (Acta2) is tightly regulated by a network of transcription factors that either activate or repress the 5' promoter-enhancer in response to environmental cues signaling tissue repair and remodeling. Purine-rich element-binding protein B (Purβ) suppresses the expression of Acta2 by cooperatively interacting with the sense strand of a 5' polypurine sequence containing an inverted MCAT cis element required for gene activation. In this study, we evaluated the chemical basis of nucleoprotein complex formation between the Purβ repressor and the purine-rich strand of the MCAT element in the mouse Acta2 promoter. Quantitative single-stranded DNA (ssDNA) binding assays conducted in the presence of increasing concentrations of monovalent salt or anionic detergent suggested that the assembly of a high-affinity nucleoprotein complex is driven by a combination of electrostatic and hydrophobic interactions. Consistent with the results of pH titration analysis, site-directed mutagenesis revealed several basic amino acid residues in the intermolecular (R267) and intramolecular (K82 and R159) subdomains that are essential for Purβ transcriptional repressor function in Acta2 promoter-reporter assays. In keeping with their diminished Acta2 repressor activity in fibroblasts, purified Purβ variants containing an R267A mutation exhibited reduced binding affinity for purine-rich ssDNA. Moreover, certain double and triple-point mutants were also defective in binding to the Acta2 corepressor protein, Y-box-binding protein 1. Collectively, these findings establish the repertoire of noncovalent interactions that account for the unique structural and functional properties of Purβ. PMID:27064749

  14. Interaction of ApoA-IV with NR4A1 and NR1D1 Represses G6Pase and PEPCK Transcription: Nuclear Receptor-Mediated Downregulation of Hepatic Gluconeogenesis in Mice and a Human Hepatocyte Cell Line

    PubMed Central

    Li, Xiaoming; Xu, Min; Wang, Fei; Ji, Yong; DavidsoN, W. Sean; Li, Zongfang; Tso, Patrick

    2015-01-01

    We have previously shown that the nuclear receptor, NR1D1, is a cofactor in ApoA-IV-mediated downregulation of gluconeogenesis. Nuclear receptor, NR4A1, is involved in the transcriptional regulation of various genes involved in inflammation, apoptosis, and glucose metabolism. We investigated whether NR4A1 influences the effect of ApoA-IV on hepatic glucose metabolism. Our in situ proximity ligation assays and coimmunoprecipitation experiments indicated that ApoA-IV colocalized with NR4A1 in human liver (HepG2) and kidney (HEK-293) cell lines. The chromatin immunoprecipitation experiments and luciferase reporter assays indicated that the ApoA-IV and NR4A1 colocalized at the RORα response element of the human G6Pase promoter, reducing its transcriptional activity. Our RNA interference experiments showed that knocking down the expression of NR4A1 in primary mouse hepatocytes treated with ApoA-IV increased the expression of NR1D1, G6Pase, and PEPCK, and that knocking down NR1D1 expression increased the level of NR4A1. We also found that ApoA-IV induced the expression of endogenous NR4A1 in both cultured primary mouse hepatocytes and in the mouse liver, and decreased glucose production in primary mouse hepatocytes. Our findings showed that ApoA-IV colocalizes with NR4A1, which suppresses G6Pase and PEPCK gene expression at the transcriptional level, reducing hepatic glucose output and lowering blood glucose. The ApoA-IV-induced increase in NR4A1 expression in hepatocytes mediates further repression of gluconeogenesis. Our findings suggest that NR1D1 and NR4A1 serve similar or complementary functions in the ApoA-IV-mediated regulation of gluconeogenesis. PMID:26556724

  15. Six3-mediated auto repression and eye development requires its interaction with members of the Groucho-related family of co-repressors.

    PubMed

    Zhu, Changqi C; Dyer, Michael A; Uchikawa, Masanori; Kondoh, Hisato; Lagutin, Oleg V; Oliver, Guillermo

    2002-06-01

    Recent findings suggest that Six3, a member of the evolutionarily conserved So/Six homeodomain family, plays an important role in vertebrate visual system development. However, little is known about the molecular mechanisms by which this function is accomplished. Although several members of the So/Six gene family interact with members of the eyes absent (Eya) gene family and function as transcriptional activators, Six3 does not interact with any known member of the Eya family. Here, we report that Grg4 and Grg5, mouse counterparts of the Drosophila transcriptional co-repressor Groucho, interact with mouse Six3 and its closely related member Six6, which may also be involved in vertebrate eye development. The specificity of the interaction was validated by co-immunoprecipitation of Six3 and Grg4 complexes from cell lines. We also show that the interaction between Six3 and Grg5 requires the Q domain of Grg5 and a conserved phenylalanine residue present in an eh1-like motif located in the Six domain of Six3. The pattern of Grg5 expression in the mouse ventral forebrain and developing optic vesicles overlapped that previously reported for Six3 and Six6. Using PCR, we identified a specific DNA motif that is bound by Six3 and we demonstrated that Six3 acts as a potent transcriptional repressor upon its interaction with Groucho-related members. We also demonstrated that this interaction is required for Six3 auto repression. The biological significance of this interaction in the retina and lens was assessed by overexpression experiments using either wild type full-length Six3 cDNA or a mutated form of this gene in which the interaction with Groucho proteins was disrupted. Overexpression of wild type Six3 by in vivo retroviral infection of newborn rat retinae led to an altered photoreceptor phenotype, while the in ovo electroporation of chicken embryos resulted in failure of lens placode invagination and production of delta-crystallin-negative cells within the placode. These

  16. Racism and Surplus Repression.

    ERIC Educational Resources Information Center

    Johnson, Howard

    1983-01-01

    Explores the relationship between Herbert Marcuse's theory of "surplus repression" and Freud's theory of the "unconscious" with respect to latent, hidden, covert, or subliminal aspects of racism in the United States. Argues that unconscious racism, manifested in evasion/avoidance, acting out/projection, and attempted justification, perpetuates…

  17. MafG Sumoylation Is Required for Active Transcriptional Repression

    PubMed Central

    Motohashi, Hozumi; Katsuoka, Fumiki; Miyoshi, Chika; Uchimura, Yasuhiro; Saitoh, Hisato; Francastel, Claire; Engel, James Douglas; Yamamoto, Masayuki

    2006-01-01

    A straightforward mechanism for eliciting transcriptional repression would be to simply block the DNA binding site for activators. Such passive repression is often mediated by transcription factors that lack an intrinsic repressor activity. MafG is a bidirectional regulator of transcription, a repressor in its homodimeric state but an activator when heterodimerized with p45. Here, we report that MafG is conjugated to SUMO-2/3 in vivo. To clarify the possible physiological role(s) for sumoylation in regulating MafG activity, we evaluated mutant and wild-type MafG in transgenic mice and cultured cells. Whereas sumoylation-deficient MafG activated p45-dependent transcription normally and did not affect heterodimer activity, repression by the sumoylation-deficient MafG mutant was severely compromised in vivo. Furthermore, the SUMO-dependent repression activity of MafG was sensitive to histone deacetylase inhibition. Thus, repression by MafG is not achieved through simple passive repression by competing for the activator binding site but requires sumoylation, which then mediates transcriptional repression through recruitment of a repressor complex containing histone deacetylase activity. PMID:16738329

  18. bIgG time for large eaters: monocytes and macrophages as effector and target cells of antibody-mediated immune activation and repression.

    PubMed

    Gordan, Sina; Biburger, Markus; Nimmerjahn, Falk

    2015-11-01

    The mononuclear phagocytic system consists of a great variety of cell subsets localized throughout the body in immunological and non-immunological tissues. While one of their prime tasks is to detect, phagocytose, and kill intruding microorganisms, they are also involved in maintaining tissue homeostasis and immune tolerance toward self through removal of dying cells. Furthermore, monocytes and macrophages have been recognized to play a critical role for mediating immunoglobulin G (IgG)-dependent effector functions, including target cell depletion, tissue inflammation, and immunomodulation. For this, monocyte and macrophage populations are equipped with a complex set of Fc-receptors, enabling them to directly interact with pro- or anti-inflammatory IgG preparations. In this review, we will summarize the most recent findings, supporting a central role of monocytes and macrophages for pro- and anti-inflammatory IgG activity. PMID:26497512

  19. PPARγ Represses Apolipoprotein A-I Gene but Impedes TNFα-Mediated ApoA-I Downregulation in HepG2 Cells.

    PubMed

    Shavva, Vladimir S; Mogilenko, Denis A; Bogomolova, Alexandra M; Nikitin, Artemy A; Dizhe, Ella B; Efremov, Alexander M; Oleinikova, Galina N; Perevozchikov, Andrej P; Orlov, Sergey V

    2016-09-01

    Apolipoprotein A-I (ApoA-I) is the main anti-atherogenic component of human high-density lipoproteins (HDL). ApoA-I gene expression is regulated by several nuclear receptors, which are the sensors for metabolic changes during development of cardiovascular diseases. Activation of nuclear receptor PPARγ has been shown to impact lipid metabolism as well as inflammation. Here, we have shown that synthetic PPARγ agonist GW1929 decreases both ApoA-I mRNA and protein levels in HepG2 cells and the effect of GW1929 on apoA-I gene transcription depends on PPARγ. PPARγ binds to the sites A and C within the hepatic enhancer of apoA-I gene and the negative regulation of apoA-I gene transcription by PPARγ appears to be realized via the site C (-134 to -119). Ligand activation of PPARγ leads to an increase of LXRβ and a decrease of PPARα binding to the apoA-I gene hepatic enhancer in HepG2 cells. GW1929 abolishes the TNFα-mediated decrease of ApoA-I mRNA expression in both HepG2 and Caco-2 cells but does not block TNFα-mediated inhibition of ApoA-I protein secretion by HepG2 cells. These data demonstrate that complex of PPARγ with GW1929 is a negative regulator involved in the control of ApoA-I expression and secretion in human hepatocyte- and enterocyte-like cells. J. Cell. Biochem. 117: 2010-2022, 2016. © 2016 Wiley Periodicals, Inc. PMID:26813964

  20. CDK6-mediated repression of CD25 is required for induction and maintenance of Notch1-induced T-cell acute lymphoblastic leukemia.

    PubMed

    Jena, N; Sheng, J; Hu, J K; Li, W; Zhou, W; Lee, G; Tsichlis, N; Pathak, A; Brown, N; Deshpande, A; Luo, C; Hu, G F; Hinds, P W; Van Etten, R A; Hu, M G

    2016-05-01

    T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subset of acute leukemia, characterized by frequent activation of Notch1 or AKT signaling, where new therapeutic approaches are needed. We showed previously that cyclin-dependent kinase 6 (CDK6) is required for thymic lymphoblastic lymphoma induced by activated AKT. Here, we show CDK6 is required for initiation and maintenance of Notch-induced T-ALL. In a mouse retroviral model, hematopoietic stem/progenitor cells lacking CDK6 protein or expressing kinase-inactive (K43M) CDK6 are resistant to induction of T-ALL by activated Notch, whereas those expressing INK4-insensitive (R31C) CDK6 are permissive. Pharmacologic inhibition of CDK6 kinase induces CD25 and RUNX1 expression, cell cycle arrest and apoptosis in mouse and human T-ALL. Ablation of Cd25 in a K43M background restores Notch-induced T leukemogenesis, with disease that is resistant to CDK6 inhibitors in vivo. These data support a model whereby CDK6-mediated suppression of CD25 is required for initiation of T-ALL by activated Notch1, and CD25 induction mediates the therapeutic response to CDK6 inhibition in established T-ALL. These results both validate CDK6 as a molecular target for therapy of this subset of T-ALL and suggest that CD25 expression could serve as a biomarker for responsiveness of T-ALL to CDK4/6 inhibitor therapy. PMID:26707936

  1. CDK6-mediated repression of CD25 is required for induction and maintenance of Notch1- induced T cell acute lymphoblastic leukemia

    PubMed Central

    Jena, Nilamani; Sheng, Jinghao; Hu, Jamie K.; Li, Wei; Zhou, Wenhui; Lee, Gene; Tsichlis, Nicolaos; Pathak, Aparna; Brown, Nelson; Deshpande, Amit; Luo, Chi; Hu, Guo-fu; Hinds, Philip W.; Van Etten, Richard A.; Hu, Miaofen G.

    2015-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subset of acute leukemia, characterized by frequent activation of Notch1 or AKT signaling, where new_therapeutic approaches are needed. We showed previously that Cyclin-dependent kinase 6 (CDK6) is required for thymic lymphoblastic lymphoma induced by activated AKT. Here, we show CDK6 is required for initiation and maintenance of Notch-induced T-ALL. In a mouse retroviral model, hematopoietic stem/progenitor cells lacking CDK6 protein or expressing kinase-inactive (K43M) CDK6 are resistant to induction of T-ALL by activated Notch, whereas those expressing INK4-insensitive (R31C) CDK6 are permissive. Pharmacologic inhibition of CDK6 kinase induces CD25 and RUNX1 expression, cell cycle arrest, and apoptosis in mouse and human T-ALL. Ablation of Cd25 in a K43M background restores Notch-induced T-leukemogenesis, with disease that is resistant to CDK6 inhibitors in vivo. These data support a model whereby CDK6-mediated suppression of CD25 is required for initiation of T-ALL by activated Notch1, and CD25 induction mediates the therapeutic response to CDK6 inhibition in established T-ALL. These results both validate CDK6 as a molecular target for therapy of this subset of T-ALL and suggest that CD25 expression could serve as a biomarker for responsiveness of T-ALL to CDK4/6 inhibitor therapy. PMID:26707936

  2. RNA Interference-Mediated Repression of MtCCD1 in Mycorrhizal Roots of Medicago truncatula Causes Accumulation of C27 Apocarotenoids, Shedding Light on the Functional Role of CCD11[W][OA

    PubMed Central

    Floss, Daniela S.; Schliemann, Willibald; Schmidt, Jürgen; Strack, Dieter; Walter, Michael H.

    2008-01-01

    Tailoring carotenoids by plant carotenoid cleavage dioxygenases (CCDs) generates various bioactive apocarotenoids. Recombinant CCD1 has been shown to catalyze symmetrical cleavage of C40 carotenoid substrates at 9,10 and 9′,10′ positions. The actual substrate(s) of the enzyme in planta, however, is still unknown. In this study, we have carried out RNA interference (RNAi)-mediated repression of a Medicago truncatula CCD1 gene in hairy roots colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. As a consequence, the normal AM-mediated accumulation of apocarotenoids (C13 cyclohexenone and C14 mycorradicin derivatives) was differentially modified. Mycorradicin derivatives were strongly reduced to 3% to 6% of the controls, while the cyclohexenone derivatives were only reduced to 30% to 47%. Concomitantly, a yellow-orange color appeared in RNAi roots. Based on ultraviolet light spectra and mass spectrometry analyses, the new compounds are C27 apocarotenoic acid derivatives. These metabolic alterations did not lead to major changes in molecular markers of the AM symbiosis, although a moderate shift to more degenerating arbuscules was observed in RNAi roots. The unexpected outcome of the RNAi approach suggests C27 apocarotenoids as the major substrates of CCD1 in mycorrhizal root cells. Moreover, literature data implicate C27 apocarotenoid cleavage as the general functional role of CCD1 in planta. A revised scheme of plant carotenoid cleavage in two consecutive steps is proposed, in which CCD1 catalyzes only the second step in the cytosol (C27 → C14 + C13), while the first step (C40 → C27 + C13) may be catalyzed by CCD7 and/or CCD4 inside plastids. PMID:18790999

  3. Pharmacological targeting of IDO-mediated tolerance for treating autoimmune disease.

    PubMed

    Penberthy, W Todd

    2007-04-01

    established mechanisms of necrosis. Chronic elevation of TNFalpha leading to necrotic events by NAD depletion in autoimmune disease likely occurs via combination of persistent IDO activation and iNOS-peroxynitrate activation of PARP1 both of which deplete NAD. Pharmacological doses of NAD precursors repeatedly provide dramatic therapeutic benefit for rheumatoid arthritis, type 1 diabetes, multiple sclerosis, colitis, other autoimmune diseases, and schizophrenia in either the clinic or animal models. Collectively these observations support the idea that autoimmune disease may in part be considered as localized pellagra manifesting symptoms particular to the inflamed target tissues. Thus pharmacological doses of NAD precursors (nicotinic acid/niacin, nicotinamide/niacinamide, or nicotinamide riboside) should be considered as potentially essential to the therapeutic success of any IDO-inducing regimen for treating autoimmune diseases. Distinct among the NAD precursors, nicotinic acid specifically activates the g-protein coupled receptor (GPCR) GPR109a to produce the IDO-inducing tolerogenic prostaglandins PGE(2) and PGD(2). Next, PGD(2) is converted to the anti-inflammatory prostaglandin, 15d-PGJ(2). These prostaglandins exert potent anti-inflammatory activities through endogenous signaling mechanisms involving the GPCRs EP2, EP4, and DP1 along with PPARgamma respectively. Nicotinamide prevents type 1 diabetes and ameliorates multiple sclerosis in animal models, while nothing is known about the therapeutic potential of nicotinamide riboside. Alternatively the direct targeting of the non-redox NAD-dependent proteins using resveratrol to activate SIRT1 or PJ34 in order to inhibit PARP1 and prevent autoimmune pathogenesis are also given consideration. PMID:17430113

  4. NFκB p50-CCAAT/Enhancer-binding Protein β (C/EBPβ)-mediated Transcriptional Repression of MicroRNA let-7i following Microbial Infection*

    PubMed Central

    O'Hara, Steven P.; Splinter, Patrick L.; Gajdos, Gabriella B.; Trussoni, Christy E.; Fernandez-Zapico, Martin E.; Chen, Xian-Ming; LaRusso, Nicholas F.

    2010-01-01

    MicroRNAs, central players of numerous cellular processes, regulate mRNA stability or translational efficiency. Although these molecular events are established, the mechanisms regulating microRNA function and expression remain largely unknown. The microRNA let-7i regulates Toll-like receptor 4 expression. Here, we identify a novel transcriptional mechanism induced by the protozoan parasite Cryptosporidium parvum and Gram(−) bacteria-derived lipopolysaccharide (LPS) mediating let-7i promoter silencing in human biliary epithelial cells (cholangiocytes). Using cultured cholangiocytes, we show that microbial stimulus decreased let-7i expression, and promoter activity. Analysis of the mechanism revealed that microbial infection promotes the formation of a NFκB p50-C/EBPβ silencer complex in the regulatory sequence. Chromatin immunoprecipitation assays (ChIP) demonstrated that the repressor complex binds to the let-7i promoter following microbial stimulus and promotes histone-H3 deacetylation. Our results provide a novel mechanism of transcriptional regulation of cholangiocyte let-7i expression following microbial insult, a process with potential implications for epithelial innate immune responses in general. PMID:19903813

  5. Saikosaponin-D reduces cisplatin-induced nephrotoxicity by repressing ROS-mediated activation of MAPK and NF-κB signalling pathways.

    PubMed

    Ma, Xiaobin; Dang, Chengxue; Kang, Huafeng; Dai, Zhijun; Lin, Shuai; Guan, Haitao; Liu, Xiaoxu; Wang, Xijing; Hui, Wentao

    2015-09-01

    The nephrotoxicity induced by cisplatin (DDP) severely limits the clinical efficacy of this widely used anticancer agent. The observed nephrotoxicity may be the result of DDP-induced inflammation and apoptosis. Saikosaponin-D (SSD), a triterpenoid saponin, has numerous pharmacological properties. The goal of the present study was to investigate whether and how SSD protected against DDP-induced nephrotoxicity. Non-cytotoxic levels of SSD significantly increased the viability rate, improved the nuclear morphology, and attenuated the caspase-3 activation and programmed apoptosis of DDP-treated HK-2 cells. In addition, SSD treatment markedly inhibited the release of tumour necrosis factor (TNF)-α, interleukin-1β (IL-1β), and interleukin-6 (IL-6), as well as the production of nitric oxide and the expression of inducible nitric oxide synthase (iNOS) by these cells. More importantly, SSD effectively blocked the DDP-induced activation of NF-κB, P38, JNK, and MAPKs. Furthermore, we found that U0126 (a specific inhibitor of MAPKs) strongly inhibited the IKK/IκB/NF-κB-dependent release of pro-inflammatory cytokines and iNOS gene expression. Finally, we demonstrated that SSD decreased the level of reactive oxygen species (ROS) accumulation and that the specific ROS scavenger N-acetylcysteine (NAC) markedly inhibited the DDP-induced activation of MAPK and phosphorylation of the downstream signal NF-κB, which in turn reduced the levels of pro-inflammatory cytokine release and iNOS gene expression. Our results suggest that the SSD-mediated alleviation of DDP-induced nephrotoxicity was due to uncoupling of the ROS, P38, and JNK/NF-κB signalling pathways. PMID:26118633

  6. Abscisic Acid-Induced Resistance against the Brown Spot Pathogen Cochliobolus miyabeanus in Rice Involves MAP Kinase-Mediated Repression of Ethylene Signaling1[C][W][OA

    PubMed Central

    De Vleesschauwer, David; Yang, Yinong; Vera Cruz, Casiana; Höfte, Monica

    2010-01-01

    The plant hormone abscisic acid (ABA) is involved in an array of plant processes, including the regulation of gene expression during adaptive responses to various environmental cues. Apart from its well-established role in abiotic stress adaptation, emerging evidence indicates that ABA is also prominently involved in the regulation and integration of pathogen defense responses. Here, we demonstrate that exogenously administered ABA enhances basal resistance of rice (Oryza sativa) against the brown spot-causing ascomycete Cochliobolus miyabeanus. Microscopic analysis of early infection events in control and ABA-treated plants revealed that this ABA-inducible resistance (ABA-IR) is based on restriction of fungal progression in the mesophyll. We also show that ABA-IR does not rely on boosted expression of salicylic acid-, jasmonic acid -, or callose-dependent resistance mechanisms but, instead, requires a functional Gα-protein. In addition, several lines of evidence are presented suggesting that ABA steers its positive effect on brown spot resistance through antagonistic cross talk with the ethylene (ET) response pathway. Exogenous ethephon application enhances susceptibility, whereas genetic disruption of ET signaling renders plants less vulnerable to C. miyabeanus attack, thereby inducing a level of resistance similar to that observed on ABA-treated wild-type plants. Moreover, ABA treatment alleviates C. miyabeanus-induced activation of the ET reporter gene EBP89, while derepression of pathogen-triggered EBP89 transcription via RNA interference-mediated knockdown of OsMPK5, an ABA-primed mitogen-activated protein kinase gene, compromises ABA-IR. Collectively, these data favor a model whereby exogenous ABA enhances resistance against C. miyabeanus at least in part by suppressing pathogen-induced ET action in an OsMPK5-dependent manner. PMID:20130100

  7. Glycerol-3-Phosphate-Induced Catabolite Repression in Escherichia coli

    PubMed Central

    Eppler, Tanja; Postma, Pieter; Schütz, Alexandra; Völker, Uwe; Boos, Winfried

    2002-01-01

    The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIAGlc, as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIAGlc phosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIAGlc. Isopropyl-β-d-thiogalactopyranoside-induced overexpression of EIIAGlc did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIAGlc. A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIAGlc is controlled by G3P and other phosphorylated sugars such as d-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and d-tagatose-1,6-bisphosphate aldolase. PMID:12003946

  8. High Expression of Rab-like 3 (Rabl3) is Associated with Poor Survival of Patients with Non-Small Cell Lung Cancer via Repression of MAPK8/9/10-Mediated Autophagy.

    PubMed

    Zhang, Weihua; Sun, Jian; Luo, Junming

    2016-01-01

    BACKGROUND Rab-like 3 (Rabl3) is a member of the Rab subfamily of small GTPases which are involved in controlling proliferation and vesicular trafficking. Recent studies suggest that Rab proteins might play a critical role in regulating cancer cell survival, but the underlying mechanisms remain largely unknown. MATERIAL AND METHODS We performed a bioinformatics analysis to examine the correlation between the expression level of Rabl3 and survival of non-small cell lung cancer (NSCLC) patients in three independent cohorts containing 484 patients. The function of Rabl3 was examined in NSCLC cell line A549 in vitro. Following Rabl3 knockdown, cells were stained with propidium iodine (PI) and Annexin V, followed by flow cytometry analysis (FACS) for cell death and autophagy induction. The activity of the MAPK signaling pathway was assessed by Western blotting of different MAPK phosphorylations, and modulated with different chemical inhibitors. RESULTS High expression of Rabl3 was significantly correlated with poor survival in all three independent NSCLC cohorts. In line with this result, Rabl3 was frequently overexpressed in lung cancer cell lines as compared with normal lung fibroblast cell lines. Knockdown of Rabl3 in lung cancer cells significantly enhanced cell death accompanied with autophagy induction, as evidenced by an increased level of autophagy marker LC3-II. Interestingly, Rabl3 knockdown was associated with enhanced activation of MAPK8/9/10 but not MAPK11/12/13/14. Treatment of MAPK8/9/10-specific inhibitor SP600125, but not MAPK11/12/13/14-specific inhibitor SB203580, largely abolished Rabl3 knockdown-induced LC3-I/LC3-II conversion and autophagic cell death. CONCLUSIONS Together, these results suggest that high expression of Rabl3 might inhibit cell death in NSCLCs via repression of MAPK8/9/10-mediated autophagy. PMID:27164297

  9. Transcription of Liver X Receptor Is Down-Regulated by 15-Deoxy-Δ12,14-Prostaglandin J2 through Oxidative Stress in Human Neutrophils

    PubMed Central

    Alba, Gonzalo; Reyes, María Edith; Santa-María, Consuelo; Ramírez, Remedios; Geniz, Isabel; Jiménez, Juan; Martín-Nieto, José; Pintado, Elízabeth; Sobrino, Francisco

    2012-01-01

    Liver X receptors (LXRs) are ligand-activated transcription factors of the nuclear receptor superfamily. They play important roles in controlling cholesterol homeostasis and as regulators of inflammatory gene expression and innate immunity, by blunting the induction of classical pro-inflammatory genes. However, opposite data have also been reported on the consequences of LXR activation by oxysterols, resulting in the specific production of potent pro-inflammatory cytokines and reactive oxygen species (ROS). The effect of the inflammatory state on the expression of LXRs has not been studied in human cells, and constitutes the main aim of the present work. Our data show that when human neutrophils are triggered with synthetic ligands, the synthesis of LXRα mRNA became activated together with transcription of the LXR target genes ABCA1, ABCG1 and SREBP1c. An inflammatory mediator, 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), hindered T0901317-promoted induction of LXRα mRNA expression together with transcription of its target genes in both neutrophils and human macrophages. This down-regulatory effect was dependent on the release of reactive oxygen species elicited by 15dPGJ2, since it was enhanced by pro-oxidant treatment and reversed by antioxidants, and was also mediated by ERK1/2 activation. Present data also support that the 15dPGJ2-induced serine phosphorylation of the LXRα molecule is mediated by ERK1/2. These results allow to postulate that down-regulation of LXR cellular levels by pro-inflammatory stimuli might be involved in the development of different vascular diseases, such as atherosclerosis. PMID:23115616

  10. Nuclear AXIN2 represses MYC gene expression

    SciTech Connect

    Rennoll, Sherri A.; Konsavage, Wesley M.; Yochum, Gregory S.

    2014-01-03

    Highlights: •AXIN2 localizes to cytoplasmic and nuclear compartments in colorectal cancer cells. •Nuclear AXIN2 represses the activity of Wnt-responsive luciferase reporters. •β-Catenin bridges AXIN2 to TCF transcription factors. •AXIN2 binds the MYC promoter and represses MYC gene expression. -- Abstract: The β-catenin transcriptional coactivator is the key mediator of the canonical Wnt signaling pathway. In the absence of Wnt, β-catenin associates with a cytosolic and multi-protein destruction complex where it is phosphorylated and targeted for proteasomal degradation. In the presence of Wnt, the destruction complex is inactivated and β-catenin translocates into the nucleus. In the nucleus, β-catenin binds T-cell factor (TCF) transcription factors to activate expression of c-MYC (MYC) and Axis inhibition protein 2 (AXIN2). AXIN2 is a member of the destruction complex and, thus, serves in a negative feedback loop to control Wnt/β-catenin signaling. AXIN2 is also present in the nucleus, but its function within this compartment is unknown. Here, we demonstrate that AXIN2 localizes to the nuclei of epithelial cells within normal and colonic tumor tissues as well as colorectal cancer cell lines. In the nucleus, AXIN2 represses expression of Wnt/β-catenin-responsive luciferase reporters and forms a complex with β-catenin and TCF. We demonstrate that AXIN2 co-occupies β-catenin/TCF complexes at the MYC promoter region. When constitutively localized to the nucleus, AXIN2 alters the chromatin structure at the MYC promoter and directly represses MYC gene expression. These findings suggest that nuclear AXIN2 functions as a rheostat to control MYC expression in response to Wnt/β-catenin signaling.

  11. An Alternative Transcript of the FOG-2 Gene Encodes a FOG-2 Isoform lacking the FOG Repression Motif

    PubMed Central

    Dale, Rodney M.; Remo, Benjamin F.; Svensson, Eric C.

    2007-01-01

    The FOG family of transcriptional co-factors is composed of two members in mammals: FOG-1 and FOG-2. Both have been shown to bind to GATA factors and function as transcriptional co-repressors in specific cell and promoter contexts. We have previously defined a novel repression domain localized to the N-terminus of each FOG family member, the FOG Repression Motif, which is necessary for FOG-mediated transcriptional repression. In this report, we describe the identification and characterization of a novel isoform of FOG-2 lacking the FOG Repression Motif. In contrast to full-length FOG-2, this isoform is expressed predominately in the embryonic and adult heart. It can bind GATA4 avidly, but is unable to repress GATA4-mediated activation of cardiac-restricted gene promoters. Together, these results suggest that FOG-2 repressive activity may be modulated by the generation of isoforms of FOG-2 lacking the FOG repression motif. PMID:17445768

  12. Involvement of a putative cyclic amp receptor protein (CRP)-like binding sequence and a CRP-like protein in glucose-mediated catabolite repression of thn genes in Rhodococcus sp. strain TFB.

    PubMed

    Tomás-Gallardo, Laura; Santero, Eduardo; Floriano, Belén

    2012-08-01

    Glucose catabolite repression of tetralin catabolic genes in Rhodococcus sp. strain TFB was shown to be exerted by a protein homologous to transcriptional regulators of the cyclic AMP receptor (CRP)-FNR family. The protein was detected bound to putative CRP-like boxes localized at the promoters of the thnA1 and thnS genes. PMID:22636000

  13. Technique Selectively Represses Immune System

    MedlinePlus

    ... Research Matters December 3, 2012 Technique Selectively Represses Immune System Myelin (green) encases and protects nerve fibers (brown). A new technique prevents the immune system from attacking myelin in a mouse model of ...

  14. Aldo-keto Reductase 1C3 (AKR1C3) is overexpressed in skin squamous cell carcinoma (SCC) and affects SCC growth via prostaglandin metabolism

    PubMed Central

    Mantel, Alon; Carpenter-Mendini, Amanda; VanBuskirk, JoAnne; Pentland, Alice P.

    2014-01-01

    Aldo-keto reductase 1C3 (AKR1C3) is an enzyme involved in metabolizing prostaglandins (PGs) and sex hormones. It metabolizes PGD2 to 9α11β-PGF2, diverting the spontaneous conversion of PGD2 to the PPARγ agonist, 15-Deoxy-Delta-12,14-prostaglandin J2 (15d-PGJ2). AKR1C3 is overexpressed in various malignancies, suggesting a tumor promoting function. This work investigates AKR1C3 expression in human non-melanoma skin cancers, revealing overexpression in squamous cell carcinoma (SCC). Effects of AKR1C3 overexpression were then evaluated using 3 SCC cell lines. AKR1C3 was detected in all SCC cell lines and its expression was upregulated in response to its substrate, PGD2. Although attenuating AKR1C3 expression in SCC cells by siRNA did not affect growth, treatment with PGD2 and its dehydration metabolite, 15d-PGJ2, decreased SCC proliferation in a PPARγ-dependent manner. In addition, treatment with the PPARγ agonist pioglitazone profoundly inhibited SCC proliferation. Finally, we generated an SCC cell line that stably overexpressed AKR1C3 (SCC-AKR1C3). SCC-AKR1C3 metabolized PGD2 to 9α11β-PGF2 12 fold faster than the parent cell line and was protected from the anti-proliferative effect mediated by PGD2. This work suggests that PGD2 and its metabolite 15d-PGJ2 attenuate SCC proliferation in a PPARγ-dependent manner, therefore activation of PPARγ by agonists such as Pioglitazone may benefit those at high risk of SCC. PMID:24917395

  15. Antimycotics suppress the Malassezia extract-induced production of CXC chemokine ligand 10 in human keratinocytes.

    PubMed

    Hau, Carren S; Kanda, Naoko; Makimura, Koichi; Watanabe, Shinichi

    2014-02-01

    Malassezia, a lipophilic yeast, exacerbates atopic dermatitis. Malassezia products can penetrate the disintegrated stratum corneum and encounter subcorneal keratinocytes in the skin of atopic dermatitis patients. Type 1 helper T (Th1) cells infiltrate chronic lesions with atopic dermatitis, and antimycotic agents improve its symptoms. We aimed to identify Malassezia-induced chemokines in keratinocytes and examine whether antimycotics suppressed this induction. Normal human keratinocytes were incubated with a Malassezia restricta extract and antimycotics. Chemokine expression was analyzed by enzyme-linked immunosorbent assays and real-time polymerase chain reaction. Signal transducer and activator of transcription (STAT)1 activity was examined by luciferase assays. The tyrosine-phosphorylation of STAT1 was analyzed by western blotting. The M. restricta extract increased the mRNA and protein expression of Th1-attracting CXC chemokine ligand (CXCL)10 and STAT1 activity and phosphorylation in keratinocytes, which was suppressed by a Janus kinase inhibitor. The antimycotics itraconazole, ketoconazole, luliconazole, terbinafine, butenafine and amorolfine suppressed M. restricta extract-induced CXCL10 mRNA and protein expression and STAT1 activity and phosphorylation. These effects were similarly induced by 15-deoxy-Δ-(12,14) -prostaglandin J2 (15d-PGJ2 ), a prostaglandin D2 metabolite. Antimycotics increased the release of 15d-PGJ2 from keratinocytes. The antimycotic-induced suppression of CXCL10 production and STAT1 activity was counteracted by a lipocalin-type prostaglandin D synthase inhibitor. The antimycotics itraconazole, ketoconazole, luliconazole, terbinafine, butenafine and amorolfine may suppress the M. restricta-induced production of CXCL10 by inhibiting STAT1 through an increase in 15d-PGJ2 production in keratinocytes. These antimycotics may block the Th1-mediated inflammation triggered by Malassezia in the chronic phase of atopic dermatitis. PMID

  16. The Functions of MicroRNAs: mRNA Decay and Translational Repression.

    PubMed

    Iwakawa, Hiro-oki; Tomari, Yukihide

    2015-11-01

    MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs, which regulate complementary mRNAs by inducing translational repression and mRNA decay. Although this dual repression system seems to operate in both animals and plants, genetic and biochemical studies suggest that the mechanism underlying the miRNA-mediated silencing is different in the two kingdoms. Here, we review the recent progress in our understanding of how miRNAs mediate translational repression and mRNA decay, and discuss the contributions of the two silencing modes to the overall silencing effect in both kingdoms. PMID:26437588

  17. Androgen Receptor Repression of GnRH Gene Transcription

    PubMed Central

    Brayman, Melissa J.; Pepa, Patricia A.; Berdy, Sara E.

    2012-01-01

    Alterations in androgen levels lead to reproductive defects in both males and females, including hypogonadotropic hypogonadism, anovulation, and infertility. Androgens have been shown to down-regulate GnRH mRNA levels through an androgen receptor (AR)-dependent mechanism. Here, we investigate how androgen regulates expression from the GnRH regulatory region in the GT1-7 cell line, a model of GnRH neurons. A synthetic androgen, R1881, repressed transcription from the GnRH promoter (GnRH-P) in an AR-dependent manner, and liganded AR associated with the chromatin at the GnRH-P in live GT1-7 cells. The three known octamer-binding transcription factor-1 (Oct-1) binding sites in GnRH-P were required for AR-mediated repression, although other sequences were also involved. Although a multimer of the consensus Oct-1 binding site was not repressed, a multimer of the cluster of Oct-1, Pre-B cell leukemia transcription factor (Pbx)/Prep, and NK2 homeobox 1 (Nkx2.1) binding sites, found at −106/−91 in GnRH-P, was sufficient for repression. In fact, overexpression of any of these factors disrupted the androgen response, indicating that a balance of factors in this tripartite complex is required for AR repression. AR bound to this region in EMSA, indicating a direct interaction of AR with DNA or with other transcription factors bound to GnRH-P at this sequence. Collectively, our data demonstrate that GnRH transcription is repressed by AR via multiple sequences in GnRH-P, including three Oct-1 binding sites, and that this repression requires the complex interaction of several transcription factors. PMID:22074952

  18. Novel TCF-binding sites specify transcriptional repression by Wnt signalling.

    PubMed

    Blauwkamp, Timothy A; Chang, Mikyung V; Cadigan, Ken M

    2008-05-21

    Both transcriptional activation and repression have essential functions in maintaining proper spatial and temporal control of gene expression. Although Wnt signalling is often associated with gene activation, we have identified several directly repressed targets of Wnt signalling in Drosophila. Here, we explore how individual Wnt target genes are specified for signal-induced activation or repression. Similar to activation, repression required binding of Armadillo (Arm) to the N terminus of TCF. However, TCF/Arm mediated repression by binding to DNA motifs that are markedly different from typical TCF-binding sites. Conversion of the novel motifs to standard TCF-binding sites reversed the mode of regulation, resulting in Wnt-mediated activation instead of repression. A mutant form of Arm defective in activation was still functional for repression, indicating that distinct domains of the protein are required for each activity. This study suggests that the sequence of TCF-binding sites allosterically regulates the TCF/Arm complex to effect either transcriptional activation or repression. PMID:18418383

  19. Histone Deacetylase Inhibitors: The Epigenetic Therapeutics That Repress Hypoxia-Inducible Factors

    PubMed Central

    Chen, Shuyang; Sang, Nianli

    2011-01-01

    Histone deacetylase inhibitors (HDACIs) have been actively explored as a new generation of chemotherapeutics for cancers, generally known as epigenetic therapeutics. Recent findings indicate that several types of HDACIs repress angiogenesis, a process essential for tumor metabolism and progression. Accumulating evidence supports that this repression is mediated by disrupting the function of hypoxia-inducible factors (HIF-1, HIF-2, and collectively, HIF), which are the master regulators of angiogenesis and cellular adaptation to hypoxia. Since HIF also regulate glucose metabolism, cell survival, microenvironment remodeling, and other alterations commonly required for tumor progression, they are considered as novel targets for cancer chemotherapy. Though the precise biochemical mechanism underlying the HDACI-triggered repression of HIF function remains unclear, potential cellular factors that may link the inhibition of deacetylase activity to the repression of HIF function have been proposed. Here we review published data that inhibitors of type I/II HDACs repress HIF function by either reducing functional HIF-1α levels, or repressing HIF-α transactivation activity. In addition, underlying mechanisms and potential proteins involved in the repression will be discussed. A thorough understanding of HDACI-induced repression of HIF function may facilitate the development of future therapies to either repress or promote angiogenesis for cancer or chronic ischemic disorders, respectively. PMID:21151670

  20. Glucose repression in Saccharomyces cerevisiae.

    PubMed

    Kayikci, Ömur; Nielsen, Jens

    2015-09-01

    Glucose is the primary source of energy for the budding yeast Saccharomyces cerevisiae. Although yeast cells can utilize a wide range of carbon sources, presence of glucose suppresses molecular activities involved in the use of alternate carbon sources as well as it represses respiration and gluconeogenesis. This dominant effect of glucose on yeast carbon metabolism is coordinated by several signaling and metabolic interactions that mainly regulate transcriptional activity but are also effective at post-transcriptional and post-translational levels. This review describes effects of glucose repression on yeast carbon metabolism with a focus on roles of the Snf3/Rgt2 glucose-sensing pathway and Snf1 signal transduction in establishment and relief of glucose repression. PMID:26205245

  1. Glucose repression in Saccharomyces cerevisiae

    PubMed Central

    Kayikci, Ömur; Nielsen, Jens

    2015-01-01

    Glucose is the primary source of energy for the budding yeast Saccharomyces cerevisiae. Although yeast cells can utilize a wide range of carbon sources, presence of glucose suppresses molecular activities involved in the use of alternate carbon sources as well as it represses respiration and gluconeogenesis. This dominant effect of glucose on yeast carbon metabolism is coordinated by several signaling and metabolic interactions that mainly regulate transcriptional activity but are also effective at post-transcriptional and post-translational levels. This review describes effects of glucose repression on yeast carbon metabolism with a focus on roles of the Snf3/Rgt2 glucose-sensing pathway and Snf1 signal transduction in establishment and relief of glucose repression. PMID:26205245

  2. The unified theory of repression.

    PubMed

    Erdelyi, Matthew Hugh

    2006-10-01

    Repression has become an empirical fact that is at once obvious and problematic. Fragmented clinical and laboratory traditions and disputed terminology have resulted in a Babel of misunderstandings in which false distinctions are imposed (e.g., between repression and suppression) and necessary distinctions not drawn (e.g., between the mechanism and the use to which it is put, defense being just one). "Repression" was introduced by Herbart to designate the (nondefensive) inhibition of ideas by other ideas in their struggle for consciousness. Freud adapted repression to the defensive inhibition of "unbearable" mental contents. Substantial experimental literatures on attentional biases, thought avoidance, interference, and intentional forgetting exist, the oldest prototype being the work of Ebbinghaus, who showed that intentional avoidance of memories results in their progressive forgetting over time. It has now become clear, as clinicians had claimed, that the inaccessible materials are often available and emerge indirectly (e.g., procedurally, implicitly). It is also now established that the Ebbinghaus retention function can be partly reversed, with resulting increases of conscious memory over time (hypermnesia). Freud's clinical experience revealed early on that exclusion from consciousness was effected not just by simple repression (inhibition) but also by a variety of distorting techniques, some deployed to degrade latent contents (denial), all eventually subsumed under the rubric of defense mechanisms ("repression in the widest sense"). Freudian and Bartlettian distortions are essentially the same, even in name, except for motive (cognitive vs. emotional), and experimentally induced false memories and other "memory illusions" are laboratory analogs of self-induced distortions. PMID:17156548

  3. Nuclear AXIN2 represses MYC gene expression.

    PubMed

    Rennoll, Sherri A; Konsavage, Wesley M; Yochum, Gregory S

    2014-01-01

    The β-catenin transcriptional coactivator is the key mediator of the canonical Wnt signaling pathway. In the absence of Wnt, β-catenin associates with a cytosolic and multi-protein destruction complex where it is phosphorylated and targeted for proteasomal degradation. In the presence of Wnt, the destruction complex is inactivated and β-catenin translocates into the nucleus. In the nucleus, β-catenin binds T-cell factor (TCF) transcription factors to activate expression of c-MYC (MYC) and Axis inhibition protein 2 (AXIN2). AXIN2 is a member of the destruction complex and, thus, serves in a negative feedback loop to control Wnt/β-catenin signaling. AXIN2 is also present in the nucleus, but its function within this compartment is unknown. Here, we demonstrate that AXIN2 localizes to the nuclei of epithelial cells within normal and colonic tumor tissues as well as colorectal cancer cell lines. In the nucleus, AXIN2 represses expression of Wnt/β-catenin-responsive luciferase reporters and forms a complex with β-catenin and TCF. We demonstrate that AXIN2 co-occupies β-catenin/TCF complexes at the MYC promoter region. When constitutively localized to the nucleus, AXIN2 alters the chromatin structure at the MYC promoter and directly represses MYC gene expression. These findings suggest that nuclear AXIN2 functions as a rheostat to control MYC expression in response to Wnt/β-catenin signaling. PMID:24299953

  4. ATRX represses alternative lengthening of telomeres.

    PubMed

    Napier, Christine E; Huschtscha, Lily I; Harvey, Adam; Bower, Kylie; Noble, Jane R; Hendrickson, Eric A; Reddel, Roger R

    2015-06-30

    The unlimited proliferation of cancer cells requires a mechanism to prevent telomere shortening. Alternative Lengthening of Telomeres (ALT) is an homologous recombination-mediated mechanism of telomere elongation used in tumors, including osteosarcomas, soft tissue sarcoma subtypes, and glial brain tumors. Mutations in the ATRX/DAXX chromatin remodeling complex have been reported in tumors and cell lines that use the ALT mechanism, suggesting that ATRX may be an ALT repressor. We show here that knockout or knockdown of ATRX in mortal cells or immortal telomerase-positive cells is insufficient to activate ALT. Notably, however, in SV40-transformed mortal fibroblasts ATRX loss results in either a significant increase in the proportion of cell lines activating ALT (instead of telomerase) or in a significant decrease in the time prior to ALT activation. These data indicate that loss of ATRX function cooperates with one or more as-yet unidentified genetic or epigenetic alterations to activate ALT. Moreover, transient ATRX expression in ALT-positive/ATRX-negative cells represses ALT activity. These data provide the first direct, functional evidence that ATRX represses ALT. PMID:26001292

  5. Functional dissection of the global repressor Tup1 in yeast: dominant role of the C-terminal repression domain.

    PubMed Central

    Zhang, Zhizhou; Varanasi, Ushasri; Trumbly, Robert J

    2002-01-01

    In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general repressor of transcription. Tup1 and Cyc8 are required for repression of diverse families of genes coordinately controlled by glucose repression, mating type, and other mechanisms. This repression is mediated by recruitment of the Cyc8-Tup1 complex to target promoters by sequence-specific DNA-binding proteins. We created a library of XhoI linker insertions and internal in-frame deletion mutations within the TUP1 coding region. Insertion mutations outside of the WD domains were wild type, while insertions within the WD domains induced mutant phenotypes with differential effects on the target genes SUC2, MFA2, RNR2, and HEM13. Deletion mutations confirmed previous findings of two separate repression domains in the N and C termini. The cumulative data suggest that the C-terminal repression domain, located near the first WD repeat, plays the dominant role in repression. Although the N-terminal repression domain is sufficient for partial repression, deletion of this region does not compromise repression. Surprisingly, deletion of the majority of the histone-binding domain of Tup1 also does not significantly reduce repression. The N-terminal region containing potential alpha-helical coiled coils is required for Tup1 oligomerization and association with Cyc8. Association with Cyc8 is required for repression of SUC2, HEM13, and RNR2 but not MFA2 and STE2. PMID:12136003

  6. Pharmacological Repression of PPARγ Promotes Osteogenesis

    PubMed Central

    Marciano, David P.; Kuruvilla, Dana S.; Boregowda, Siddaraju V.; Asteian, Alice; Hughes, Travis S.; Garcia-Ordonez, Ruben; Corzo, Cesar A.; Khan, Tanya M.; Novick, Scott J.; Park, HaJeung; Kojetin, Douglas J.; Phinney, Donald G.; Bruning, John B.; Kamenecka, Theodore M.; Griffin, Patrick R.

    2015-01-01

    The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis and the pharmacological target of the thiazolidinedione (TZD) class of insulin sensitizers. Activation of PPARγ by TZDs promotes adipogenesis at the expense of osteoblast formation, contributing to their associated adverse effects on bone. Recently we reported the development of PPARγ antagonist SR1664, designed to block the obesity induced phosphorylation of serine 273 (S273) in the absence of classical agonism, to derive insulin sensitizing efficacy with improved therapeutic index. Here we identify the structural mechanism by which SR1664 actively antagonizes PPARγ, and extend these findings to develop the inverse agonist SR2595. Treatment of isolated bone marrow derived mesenchymal stem cells (MSCs) with SR2595 promotes induction of osteogenic differentiation. Together these results identify the structural determinants of ligand mediated PPARγ repression, and suggest a therapeutic approach to promote bone formation. PMID:26068133

  7. Rule of Repression in Chile.

    ERIC Educational Resources Information Center

    American Indian Journal, 1979

    1979-01-01

    This report on the current condition of the Mapuche Indians of Chile is edited from a document on the "Situation of Human Rights in Chile" and details the repressive and inhumane treatment of the largest indigenous ethnic minority in the country. (Author/RTS)

  8. Osa-containing Brahma chromatin remodeling complexes are required for the repression of Wingless target genes

    PubMed Central

    Collins, Russell T.; Treisman, Jessica E.

    2000-01-01

    The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific downstream target genes. We report here that the product of the trithorax group gene osa is required to repress such genes in the absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes present and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression. PMID:11124806

  9. Conserved intron elements repress splicing of a neuron-specific c-src exon in vitro.

    PubMed Central

    Chan, R C; Black, D L

    1995-01-01

    The neuron-specific N1 exon of the mouse c-src transcript is normally skipped in nonneuronal cells. In this study, we examined the sequence requirements for the exclusion of this exon in nonneuronal HeLa cell nuclear extracts. We found that the repression of the N1 exon is mediated by specific intron sequences that flank the N1 exon. Mutagenesis experiments identified conserved CUCUCU elements within these intron regions that are required for the repression of N1 splicing. The addition of an RNA competitor containing the upstream regulatory sequence to the HeLa extract induced splicing of the intron downstream of N1, indicating that the competitor sequence binds to splicing repressor proteins. The similarities between this mechanism for src splicing repression and the repression of other regulated exons point to a common role of exon-spanning interactions in splicing repression. PMID:7565790

  10. Repression of CIITA by the Epstein-Barr virus transcription factor Zta is independent of its dimerization and DNA binding.

    PubMed

    Balan, Nicolae; Osborn, Kay; Sinclair, Alison J

    2016-03-01

    Repression of the cellular CIITA gene is part of the immune evasion strategy of the γherpes virus Epstein-Barr virus (EBV) during its lytic replication cycle in B-cells. In part, this is mediated through downregulation of MHC class II gene expression via the targeted repression of CIITA, the cellular master regulator of MHC class II gene expression. This repression is achieved through a reduction in CIITA promoter activity, initiated by the EBV transcription and replication factor, Zta (BZLF1, EB1, ZEBRA). Zta is the earliest gene expressed during the lytic replication cycle. Zta interacts with sequence-specific elements in promoters, enhancers and the replication origin (ZREs), and also modulates gene expression through interaction with cellular transcription factors and co-activators. Here, we explore the requirements for Zta-mediated repression of the CIITA promoter. We find that repression by Zta is specific for the CIITA promoter and can be achieved in the absence of other EBV genes. Surprisingly, we find that the dimerization region of Zta is not required to mediate repression. This contrasts with an obligate requirement of this region to correctly orientate the DNA contact regions of Zta to mediate activation of gene expression through ZREs. Additional support for the model that Zta represses the CIITA promoter without direct DNA binding comes from promoter mapping that shows that repression does not require the presence of a ZRE in the CIITA promoter. PMID:26653871

  11. Two evolutionarily conserved repression domains in the Drosophila Kruppel protein differ in activator specificity.

    PubMed Central

    Hanna-Rose, W; Licht, J D; Hansen, U

    1997-01-01

    To identify biologically functional regions in the product of the Drosophila melanogaster gene Kruppel, we cloned the Kruppel homolog from Drosophila virilis. Both the previously identified amino (N)-terminal repression region and the DNA-binding region of the D. virilis Kruppel protein are greater than 96% identical to those of the D. melanogaster Kruppel protein, demonstrating a selective pressure to maintain the integrity of each region during 60 million to 80 million years of evolution. An additional region in the carboxyl (C) terminus of Kruppel that was most highly conserved was examined further. A 42-amino-acid stretch within the conserved C-terminal region also encoded a transferable repression domain. The short, C-terminal repression region is a composite of three subregions of distinct amino acid composition, each containing a high proportion of either basic, proline, or acidic residues. Mutagenesis experiments demonstrated, unexpectedly, that the acidic residues contribute to repression function. Both the N-terminal and C-terminal repression regions were tested for the ability to affect transcription mediated by a variety of activator proteins. The N-terminal repression region was able to inhibit transcription in the presence of multiple activators. However, the C-terminal repression region inhibited transcription by only a subset of the activator proteins. The different activator specificities of the two regions suggest that they repress transcription by different mechanisms and may play distinct biological roles during Drosophila development. PMID:9234738

  12. Retinoic acid-mediated repression of human papillomavirus 18 transcription and different ligand regulation of the retinoic acid receptor beta gene in non-tumorigenic and tumorigenic HeLa hybrid cells.

    PubMed Central

    Bartsch, D; Boye, B; Baust, C; zur Hausen, H; Schwarz, E

    1992-01-01

    Human papillomavirus type 18 (HPV18) belongs to the group of genital papillomaviruses involved in the development of cervical carcinomas. Since retinoic acid (RA) is a key regulator of epithelial cell differentiation and a growth inhibitor in vitro of HPV18-positive HeLa cervical carcinoma cells, we have used HeLa and HeLa hybrid cells in order to analyse the effects of RA on expression of the HPV18 E6 and E7 oncogenes and of the cellular RA receptor genes RAR-beta and -gamma. We show here that RA down-regulates HPV18 mRNA levels apparently due to transcriptional repression. Transient cotransfection assays indicated that RARs negatively regulate the HPV18 upstream regulatory region and that the central enhancer can confer RA-dependent repression on a heterologous promoter. RA treatment resulted in induction of RAR-beta mRNA levels in non-tumorigenic HeLa hybrid cells, but not in tumorigenic hybrid segregants nor in HeLa cells. No alterations of the RAR-beta gene or of the HeLa RAR-beta promoter could be revealed by Southern and DNA sequence analysis, respectively. As determined by transient transfection assays, however, the RAR-beta control region was activated by RA more strongly in non-tumorigenic hybrid cells than in HeLa cells, thus indicating differences in trans-acting regulatory factors. Our data suggest that the RARs are potential negative regulators of HPV18 E6 and E7 gene expression, and that dysregulation of the RAR-beta gene either causatively contributes to or is an indicator of tumorigenicity in HeLa and HeLa hybrid cells. Images PMID:1318198

  13. Spe3, which encodes spermidine synthase, is required for full repression through NRE(DIT) in Saccharomyces cerevisiae.

    PubMed Central

    Friesen, H; Tanny, J C; Segall, J

    1998-01-01

    We previously identified a transcriptional regulatory element, which we call NRE(DIT), that is required for repression of the sporulation-specific genes, DIT1 and DIT2, during vegetative growth of Saccharomyces cerevisiae. Repression through this element is dependent on the Ssn6-Tup1 corepressor. In this study, we show that SIN4 contributes to NRE(DIT)-mediated repression, suggesting that changes in chromatin structure are, at least in part, responsible for regulation of DIT gene expression. In a screen for additional genes that function in repression of DIT (FRD genes), we recovered alleles of TUP1, SSN6, SIN4, and ROX3 and identified mutations comprising eight complementation groups of FRD genes. Four of these FRD genes appeared to act specifically in NRE(DIT)mediated repression, and four appeared to be general regulators of gene expression. We cloned the gene complementing the frd3-1 phenotype and found that it was identical to SPE3, which encodes spermidine synthase. Mutant spe3 cells not only failed to support complete repression through NRE(DIT) but also had modest defects in repression of some other genes. Addition of spermidine to the medium partially restored repression to spe3 cells, indicating that spermidine may play a role in vivo as a modulator of gene expression. We suggest various mechanisms by which spermidine could act to repress gene expression. PMID:9725830

  14. Bhlhe40 Represses PGC-1α Activity on Metabolic Gene Promoters in Myogenic Cells

    PubMed Central

    Chung, Shih Ying; Kao, Chien Han; Villarroya, Francesc; Chang, Hsin Yu; Chang, Hsuan Chia; Hsiao, Sheng Pin; Liou, Gunn-Guang

    2015-01-01

    PGC-1α is a transcriptional coactivator promoting oxidative metabolism in many tissues. Its expression in skeletal muscle (SKM) is induced by hypoxia and reactive oxidative species (ROS) generated during exercise, suggesting that PGC-1α might mediate the cross talk between oxidative metabolism and cellular responses to hypoxia and ROS. Here we found that PGC-1α directly interacted with Bhlhe40, a basic helix-loop-helix (bHLH) transcriptional repressor induced by hypoxia, and protects SKM from ROS damage, and they cooccupied PGC-1α-targeted gene promoters/enhancers, which in turn repressed PGC-1α transactivational activity. Bhlhe40 repressed PGC-1α activity through recruiting histone deacetylases (HDACs) and preventing the relief of PGC-1α intramolecular repression caused by its own intrinsic suppressor domain. Knockdown of Bhlhe40 mRNA increased levels of ROS, fatty acid oxidation, mitochondrial DNA, and expression of PGC-1α target genes. Similar effects were also observed when the Bhlhe40-mediated repression was rescued by a dominantly active form of the PGC-1α-interacting domain (PID) from Bhlhe40. We further found that Bhlhe40-mediated repression can be largely relieved by exercise, in which its recruitment to PGC-1α-targeted cis elements was significantly reduced. These observations suggest that Bhlhe40 is a novel regulator of PGC-1α activity repressing oxidative metabolism gene expression and mitochondrion biogenesis in sedentary SKM. PMID:25963661

  15. PPARgamma inhibits osteogenesis via the down-regulation of the expression of COX-2 and iNOS in rats.

    PubMed

    Lin, Tzu-Hung; Yang, Rong-Sen; Tang, Chih-Hsin; Lin, Chih-Peng; Fu, Wen-Mei

    2007-10-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-activated transcription factor, is considered as an anti-osteoblastic factor associated with adiposity and the elderly osteoporosis due to a defect in osteoblastogenesis. We have found that oral administration of PPARgamma activator rosiglitazone decreased tibia BMD and serum ALP but left serum calcium and osteoclast marker C-terminal telopeptide unaffected. In addition, we examined the inhibitory mechanisms of PPARgamma on the bone formation by using PPARgamma activators ciglitazone and 15-deoxy-Delta(12,14)-prostaglandin-J2 (15d-PGJ2). Our data indicated that PPARgamma ligands decreased both mineralized bone nodules and alkaline phosphatase (ALP) activities in cultured primary osteoblasts. Reverse transcription polymerase chain reaction (RT-PCR) showed that the expression of bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN) was inhibited by ciglitizone and 15d-PGJ2. Furthermore, PPARgamma ligands inhibited NF-kappaB associated downstream COX-2 and iNOS osteogenic signaling. The ultrasound (US)-induced elevation of COX-2 and iNOS expression and nitric oxide (NO) production were attenuated in the presence of PPARgamma ligands. Furthermore, local administration of PPARgamma ligands into the metaphysis of rat tibia decreased the bone volume in secondary spongiosa. These results suggest that the activation of PPARgamma inhibits osteoblastic differentiation and the expression of several anabolic mediators involved in bone formation. These data may reflect osteoporosis and less bone formation in the aging people and patients treated with thiazolidinediones. PMID:17669705

  16. Polycomb repressive complex 1 controls uterine decidualization

    PubMed Central

    Bian, Fenghua; Gao, Fei; Kartashov, Andrey V.; Jegga, Anil G.; Barski, Artem; Das, Sanjoy K.

    2016-01-01

    Uterine stromal cell decidualization is an essential part of the reproductive process. Decidual tissue development requires a highly regulated control of the extracellular tissue remodeling; however the mechanism of this regulation remains unknown. Through systematic expression studies, we detected that Cbx4/2, Rybp, and Ring1B [components of polycomb repressive complex 1 (PRC1)] are predominantly utilized in antimesometrial decidualization with polyploidy. Immunofluorescence analyses revealed that PRC1 members are co-localized with its functional histone modifier H2AK119ub1 (mono ubiquitination of histone-H2A at lysine-119) in polyploid cell. A potent small-molecule inhibitor of Ring1A/B E3-ubiquitin ligase or siRNA-mediated suppression of Cbx4 caused inhibition of H2AK119ub1, in conjunction with perturbation of decidualization and polyploidy development, suggesting a role for Cbx4/Ring1B-containing PRC1 in these processes. Analyses of genetic signatures by RNA-seq studies showed that the inhibition of PRC1 function affects 238 genes (154 up and 84 down) during decidualization. Functional enrichment analyses identified that about 38% genes primarily involved in extracellular processes are specifically targeted by PRC1. Furthermore, ~15% of upregulated genes exhibited a significant overlap with the upregulated Bmp2 null-induced genes in mice. Overall, Cbx4/Ring1B-containing PRC1 controls decidualization via regulation of extracellular gene remodeling functions and sheds new insights into underlying molecular mechanism(s) through transcriptional repression regulation. PMID:27181215

  17. Polycomb repressive complex 1 controls uterine decidualization.

    PubMed

    Bian, Fenghua; Gao, Fei; Kartashov, Andrey V; Jegga, Anil G; Barski, Artem; Das, Sanjoy K

    2016-01-01

    Uterine stromal cell decidualization is an essential part of the reproductive process. Decidual tissue development requires a highly regulated control of the extracellular tissue remodeling; however the mechanism of this regulation remains unknown. Through systematic expression studies, we detected that Cbx4/2, Rybp, and Ring1B [components of polycomb repressive complex 1 (PRC1)] are predominantly utilized in antimesometrial decidualization with polyploidy. Immunofluorescence analyses revealed that PRC1 members are co-localized with its functional histone modifier H2AK119ub1 (mono ubiquitination of histone-H2A at lysine-119) in polyploid cell. A potent small-molecule inhibitor of Ring1A/B E3-ubiquitin ligase or siRNA-mediated suppression of Cbx4 caused inhibition of H2AK119ub1, in conjunction with perturbation of decidualization and polyploidy development, suggesting a role for Cbx4/Ring1B-containing PRC1 in these processes. Analyses of genetic signatures by RNA-seq studies showed that the inhibition of PRC1 function affects 238 genes (154 up and 84 down) during decidualization. Functional enrichment analyses identified that about 38% genes primarily involved in extracellular processes are specifically targeted by PRC1. Furthermore, ~15% of upregulated genes exhibited a significant overlap with the upregulated Bmp2 null-induced genes in mice. Overall, Cbx4/Ring1B-containing PRC1 controls decidualization via regulation of extracellular gene remodeling functions and sheds new insights into underlying molecular mechanism(s) through transcriptional repression regulation. PMID:27181215

  18. Molecular functions of the TLE tetramerization domain in Wnt target gene repression

    PubMed Central

    Chodaparambil, Jayanth V; Pate, Kira T; Hepler, Margretta R D; Tsai, Becky P; Muthurajan, Uma M; Luger, Karolin; Waterman, Marian L; Weis, William I

    2014-01-01

    Wnt signaling activates target genes by promoting association of the co-activator β-catenin with TCF/LEF transcription factors. In the absence of β-catenin, target genes are silenced by TCF-mediated recruitment of TLE/Groucho proteins, but the molecular basis for TLE/TCF-dependent repression is unclear. We describe the unusual three-dimensional structure of the N-terminal Q domain of TLE1 that mediates tetramerization and binds to TCFs. We find that differences in repression potential of TCF/LEFs correlates with their affinities for TLE-Q, rather than direct competition between β-catenin and TLE for TCFs as part of an activation–repression switch. Structure-based mutation of the TLE tetramer interface shows that dimers cannot mediate repression, even though they bind to TCFs with the same affinity as tetramers. Furthermore, the TLE Q tetramer, not the dimer, binds to chromatin, specifically to K20 methylated histone H4 tails, suggesting that the TCF/TLE tetramer complex promotes structural transitions of chromatin to mediate repression. PMID:24596249

  19. ATF3 represses PPARγ expression and inhibits adipocyte differentiation

    SciTech Connect

    Jang, Min-Kyung; Jung, Myeong Ho

    2014-11-07

    Highlights: • ATF3 decrease the expression of PPARγ and its target gene in 3T3-L1 adipocytes. • ATF3 represses the promoter activity of PPARγ2 gene. • ATF/CRE (−1537/−1530) is critical for ATF3-mediated downregulation of PPARγ. • ATF3 binds to the promoter region containing the ATF/CRE. • ER stress inhibits adipocyte differentiation through downregulation of PPARγ by ATF3. - Abstract: Activating transcription factor 3 (ATF3) is a stress-adaptive transcription factor that mediates cellular stress response signaling. We previously reported that ATF3 represses CCAAT/enhancer binding protein α (C/EBPα) expression and inhibits 3T3-L1 adipocyte differentiation. In this study, we explored potential role of ATF3 in negatively regulating peroxisome proliferator activated receptor-γ (PPARγ). ATF3 decreased the expression of PPARγ and its target gene in 3T3-L1 adipocytes. ATF3 also repressed the activity of −2.6 Kb promoter of mouse PPARγ2. Overexpression of PPARγ significantly prevented the ATF3-mediated inhibition of 3T3-L1 differentiation. Transfection studies with 5′ deleted-reporters showed that ATF3 repressed the activity of −2037 bp promoter, whereas it did not affect the activity of −1458 bp promoter, suggesting that ATF3 responsive element is located between the −2037 and −1458. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds to ATF/CRE site (5′-TGACGTTT-3′) between −1537 and −1530. Mutation of the ATF/CRE site abrogated ATF3-mediated transrepression of the PPARγ2 promoter. Treatment with thapsigargin, endoplasmic reticulum (ER) stress inducer, increased ATF3 expression, whereas it decreased PPARγ expression. ATF3 knockdown significantly blocked the thapsigargin-mediated downregulation of PPARγ expression. Furthermore, overexpression of PPARγ prevented inhibition of 3T3-L1 differentiation by thapsigargin. Collectively, these results suggest that ATF3-mediated

  20. Mechanism and Role of SOX2 Repression in Seminoma: Relevance to Human Germline Specification.

    PubMed

    Kushwaha, Ritu; Jagadish, Nirmala; Kustagi, Manjunath; Mendiratta, Geetu; Seandel, Marco; Soni, Rekha; Korkola, James E; Thodima, Venkata; Califano, Andrea; Bosl, George J; Chaganti, R S K

    2016-05-10

    Human male germ cell tumors (GCTs) are derived from primordial germ cells (PGCs). The master pluripotency regulator and neuroectodermal lineage effector transcription factor SOX2 is repressed in PGCs and the seminoma (SEM) subset of GCTs. The mechanism of SOX2 repression and its significance to GC and GCT development currently are not understood. Here, we show that SOX2 repression in SEM-derived TCam-2 cells is mediated by the Polycomb repressive complex (PcG) and the repressive H3K27me3 chromatin mark that are enriched at its promoter. Furthermore, SOX2 repression in TCam-2 cells can be abrogated by recruitment of the constitutively expressed H3K27 demethylase UTX to the SOX2 promoter through retinoid signaling, leading to expression of neuronal and other lineage genes. SOX17 has been shown to initiate human PGC specification, with its target PRDM1 suppressing mesendodermal genes. Our results are consistent with a role for SOX2 repression in normal germline development by suppressing neuroectodermal genes. PMID:27132888

  1. YY1 represses human papillomavirus type 16 transcription by quenching AP-1 activity.

    PubMed Central

    O'Connor, M J; Tan, S H; Tan, C H; Bernard, H U

    1996-01-01

    YY1 is a multifunctional transcription factor that has been shown to regulate the expression of a number of cellular and viral genes, including the human papillomavirus (HPV) oncogenes E6 and E7. In this study, we have analyzed the YY1-mediated repression of the HPV type 16 (HPV-16) E6-E7 promoter. A systematic analysis to identify YY1 sites present in the HPV-16 long control region showed that of 30 potential YY1 binding motifs, 24 bound purified recombinant YY1 protein, but only 10 of these were able to bind YY1 when nuclear extracts of HeLa cells were used. Of these, only a cluster of five sites, located in the vicinity of an AP-1 motif, were found to be responsible for repressing the HPV-16 P97 promoter. All five sites were required for repression, the mutation of any one site giving rise to a four- to sixfold increase in transcriptional activity. The target for YY1-mediated repression was identified as being a highly conserved AP-1 site, and we propose that AP-1 may represent a common target for YY1 repression. We also provide data demonstrating that YY1 can bind the transcriptional coactivator CREB-binding protein and propose a potentially novel mechanism by which YY1 represses AP-1 activity as a result of this YY1-CREB-binding protein interaction. PMID:8794287

  2. Gibberellins repress photomorphogenesis in darkness.

    PubMed

    Alabadí, David; Gil, Joan; Blázquez, Miguel A; García-Martínez, José L

    2004-03-01

    Plants undergo two different developmental programs depending on whether they are growing in darkness (skotomorphogenesis) or in the presence of light (photomorphogenesis). It has been proposed that the latter is the default pathway followed by many plants after germination and before the seedling emerges from soil. The transition between the two pathways is tightly regulated. The conserved COP1-based complex is central in the light-dependent repression of photomorphogenesis in darkness. Besides this control, hormones such as brassinosteroids (BRs), cytokinins, auxins, or ethylene also have been shown to regulate, to different extents, this developmental switch. In the present work, we show that the hormone gibberellin (GA) widely participates in this regulation. Studies from Arabidopsis show that both chemical and genetic reductions of endogenous GA levels partially derepress photomorphogenesis in darkness. This is based both on morphological phenotypes, such as hypocotyl elongation and hook and cotyledon opening, and on molecular phenotypes, such as misregulation of the light-controlled genes CAB2 and RbcS. Genetic studies indicate that the GA signaling elements GAI and RGA participate in these responses. Our results also suggest that GA regulation of this response partially depends on BRs. This regulation seems to be conserved across species because lowering endogenous GA levels in pea (Pisum sativum) induces full de-etiolation in darkness, which is not reverted by BR application. Our results, therefore, attribute an important role for GAs in the establishment of etiolated growth and in repression of photomorphogenesis. PMID:14963246

  3. The TOPLESS Interactome: A Framework for Gene Repression in Arabidopsis1[W][OA

    PubMed Central

    Causier, Barry; Ashworth, Mary; Guo, Wenjia; Davies, Brendan

    2012-01-01

    Transcription factors activate or repress target gene expression or switch between activation and repression. In animals and yeast, Groucho/Tup1 corepressor proteins are recruited by diverse transcription factors to induce context-specific transcriptional repression. Two groups of Groucho/Tup1-like corepressors have been described in plants. LEUNIG and LEUNIG_HOMOLOG constitute one group and TOPLESS (TPL) and the four TPL-related (TPR) corepressors form the other. To discover the processes in which TPL and the TPR corepressors operate, high-throughput yeast two-hybrid approaches were used to identify interacting proteins. We found that TPL/TPR corepressors predominantly interact directly with specific transcription factors, many of which were previously implicated in transcriptional repression. The interacting transcription factors reveal that the TPL/TPR family has been coopted multiple times to modulate gene expression in diverse processes, including hormone signaling, stress responses, and the control of flowering time, for which we also show biological validation. The interaction data suggest novel mechanisms for the involvement of TPL/TPR corepressors in auxin and jasmonic acid signaling. A number of short repression domain (RD) sequences have previously been identified in Arabidopsis (Arabidopsis thaliana) transcription factors. All known RD sequences were enriched among the TPL/TPR interactors, and novel TPL-RD interactions were identified. We show that the presence of RD sequences is essential for TPL/TPR recruitment. These data provide a framework for TPL/TPR-dependent transcriptional repression. They allow for predictions about new repressive transcription factors, corepressor interactions, and repression mechanisms and identify a wide range of plant processes that utilize TPL/TPR-mediated gene repression. PMID:22065421

  4. Salmonella promotes virulence by repressing cellulose production

    PubMed Central

    Pontes, Mauricio H.; Lee, Eun-Jin; Choi, Jeongjoon; Groisman, Eduardo A.

    2015-01-01

    Cellulose is the most abundant organic polymer on Earth. In bacteria, cellulose confers protection against environmental insults and is a constituent of biofilms typically formed on abiotic surfaces. We report that, surprisingly, Salmonella enterica serovar Typhimurium makes cellulose when inside macrophages. We determine that preventing cellulose synthesis increases virulence, whereas stimulation of cellulose synthesis inside macrophages decreases virulence. An attenuated mutant lacking the mgtC gene exhibited increased cellulose levels due to increased expression of the cellulose synthase gene bcsA and of cyclic diguanylate, the allosteric activator of the BcsA protein. Inactivation of bcsA restored wild-type virulence to the Salmonella mgtC mutant, but not to other attenuated mutants displaying a wild-type phenotype regarding cellulose. Our findings indicate that a virulence determinant can promote pathogenicity by repressing a pathogen's antivirulence trait. Moreover, they suggest that controlling antivirulence traits increases long-term pathogen fitness by mediating a trade-off between acute virulence and transmission. PMID:25848006

  5. Salmonella promotes virulence by repressing cellulose production.

    PubMed

    Pontes, Mauricio H; Lee, Eun-Jin; Choi, Jeongjoon; Groisman, Eduardo A

    2015-04-21

    Cellulose is the most abundant organic polymer on Earth. In bacteria, cellulose confers protection against environmental insults and is a constituent of biofilms typically formed on abiotic surfaces. We report that, surprisingly, Salmonella enterica serovar Typhimurium makes cellulose when inside macrophages. We determine that preventing cellulose synthesis increases virulence, whereas stimulation of cellulose synthesis inside macrophages decreases virulence. An attenuated mutant lacking the mgtC gene exhibited increased cellulose levels due to increased expression of the cellulose synthase gene bcsA and of cyclic diguanylate, the allosteric activator of the BcsA protein. Inactivation of bcsA restored wild-type virulence to the Salmonella mgtC mutant, but not to other attenuated mutants displaying a wild-type phenotype regarding cellulose. Our findings indicate that a virulence determinant can promote pathogenicity by repressing a pathogen's antivirulence trait. Moreover, they suggest that controlling antivirulence traits increases long-term pathogen fitness by mediating a trade-off between acute virulence and transmission. PMID:25848006

  6. Enhancer-bound LDB1 regulates a corticotrope promoter-pausing repression program

    PubMed Central

    Zhang, Feng; Tanasa, Bogdan; Merkurjev, Daria; Lin, Chijen; Song, Xiaoyuan; Li, Wenbo; Tan, Yuliang; Liu, Zhijie; Zhang, Jie; Ohgi, Kenneth A.; Krones, Anna; Skowronska-Krawczyk, Dorota; Rosenfeld, Michael G.

    2015-01-01

    Substantial evidence supports the hypothesis that enhancers are critical regulators of cell-type determination, orchestrating both positive and negative transcriptional programs; however, the basic mechanisms by which enhancers orchestrate interactions with cognate promoters during activation and repression events remain incompletely understood. Here we report the required actions of LIM domain-binding protein 1 (LDB1)/cofactor of LIM homeodomain protein 2/nuclear LIM interactor, interacting with the enhancer-binding protein achaete-scute complex homolog 1, to mediate looping to target gene promoters and target gene regulation in corticotrope cells. LDB1-mediated enhancer:promoter looping appears to be required for both activation and repression of these target genes. Although LDB1-dependent activated genes are regulated at the level of transcriptional initiation, the LDB1-dependent repressed transcription units appear to be regulated primarily at the level of promoter pausing, with LDB1 regulating recruitment of metastasis-associated 1 family, member 2, a component of the nucleosome remodeling deacetylase complex, on these negative enhancers, required for the repressive enhancer function. These results indicate that LDB1-dependent looping events can deliver repressive cargo to cognate promoters to mediate promoter pausing events in a pituitary cell type. PMID:25605944

  7. Identification of genes involved in Ca2+ ionophore A23187-mediated apoptosis and demonstration of a high susceptibility for transcriptional repression of cell cycle genes in B lymphoblasts from a patient with Scott syndrome

    PubMed Central

    Kozian, Detlef; Proulle, Valérie; Nitsche, Almut; Galitzine, Marie; Martinez, Marie-Carmen; Schumann, Beatrice; Meyer, Dominique; Herrmann, Matthias; Freyssinet, Jean-Marie; Kerbiriou-Nabias, Danièle

    2005-01-01

    Background In contrast to other agents able to induce apoptosis of cultured cells, Ca2+ ionophore A23187 was shown to elicit direct activation of intracellular signal(s). The phenotype of the cells derived from patients having the hemorrhagic disease Scott syndrome, is associated with an abnormally high proportion of apoptotic cells, both in basal culture medium and upon addition of low ionophore concentrations in long-term cultures. These features are presumably related to the mutation also responsible for the defective procoagulant plasma membrane remodeling. We analyzed the specific transcriptional re-programming induced by A23187 to get insights into the effect of this agent on gene expression and a defective gene regulation in Scott cells. Results The changes in gene expression upon 48 hours treatment with 200 nM A23187 were measured in Scott B lymphoblasts compared to B lymphoblasts derived from the patient's daughter or unrelated individuals using Affymetrix microarrays. In a similar manner in all of the B cell lines, results showed up-regulation of 55 genes, out of 12,000 represented sequences, involved in various pathways of the cell metabolism. In contrast, a group of 54 down-regulated genes, coding for histones and proteins involved in the cell cycle progression, was more significantly repressed in Scott B lymphoblasts than in the other cell lines. These data correlated with the alterations of the cell cycle phases in treated cells and suggested that the potent effect of A23187 in Scott B lymphoblasts may be the consequence of the underlying molecular defect. Conclusion The data illustrate that the ionophore A23187 exerts its pro-apoptotic effect by promoting a complex pattern of genetic changes. These results also suggest that a subset of genes participating in various steps of the cell cycle progress can be transcriptionally regulated in a coordinated fashion. Furthermore, this research brings a new insight into the defect in cultured Scott B

  8. Multiple Gene Repression in Cyanobacteria Using CRISPRi.

    PubMed

    Yao, Lun; Cengic, Ivana; Anfelt, Josefine; Hudson, Elton P

    2016-03-18

    We describe the application of clustered regularly interspaced short palindromic repeats interference (CRISPRi) for gene repression in the model cyanobacterium Synechcocystis sp. PCC 6803. The nuclease-deficient Cas9 from the type-II CRISPR/Cas of Streptrococcus pyogenes was used to repress green fluorescent protein (GFP) to negligible levels. CRISPRi was also used to repress formation of carbon storage compounds polyhydroxybutryate (PHB) and glycogen during nitrogen starvation. As an example of the potential of CRISPRi for basic and applied cyanobacteria research, we simultaneously knocked down 4 putative aldehyde reductases and dehydrogenases at 50-95% repression. This work also demonstrates that tightly repressed promoters allow for inducible and reversible CRISPRi in cyanobacteria. PMID:26689101

  9. ERalpha suppresses slug expression directly by transcriptional repression.

    PubMed

    Ye, Yin; Xiao, Yi; Wang, Wenting; Yearsley, Kurtis; Gao, Jian-Xin; Barsky, Sanford H

    2008-12-01

    Two of the most common signalling pathways in breast cancer are the ER (oestrogen receptor) ligand activation pathway and the E-cadherin snai1 slug EMT (epithelial-mesenchymal transition) pathway. Although these pathways have been thought to interact indirectly, the present study is the first to observe direct interactions between these pathways that involves the regulation of slug expression. Specifically we report that ligand-activated ERalpha suppressed slug expression directly by repression of transcription and that knockdown of ERalpha with RNA interference increased slug expression. More specifically, slug expression was down-regulated in ERalpha-negative MDA-MB-468 cells transfected with ERalpha after treatment with E2 (17beta-oestradiol). The down-regulation of slug in the ERalpha-positive MCF-7 cell line was mediated by direct repression of slug transcription by the formation of a co-repressor complex involving ligand-activated ERalpha protein, HDAC1 (histone deacetylase 1) and N-CoR (nuclear receptor co-repressor). This finding was confirmed by sequential ChIP (chromatin immunoprecipitation) studies. In the MCF-7 cell line, slug expression normally was low. In addition, knockdown of ERalpha with RNA interference in this cell line increased slug expression. This effect could be partially reversed by treatment of the cells with E2. The efficacy of the effect of ERalpha on slug repression was dependent on the overall level of ERalpha. These observations confirmed that slug was an E2-responsive gene. PMID:18588516

  10. Histone acetylation: a switch between repressive and permissive chromatin

    PubMed Central

    Eberharter, Anton; Becker, Peter B.

    2002-01-01

    The organization of eukaryotic chromatin has a major impact on all nuclear processes involving DNA substrates. Gene expression is affected by the positioning of individual nucleosomes relative to regulatory sequence elements, by the folding of the nucleosomal fiber into higher-order structures and by the compartmentalization of functional domains within the nucleus. Because site-specific acetylation of nucleosomal histones influences all three aspects of chromatin organization, it is central to the switch between permissive and repressive chromatin structure. The targeting of enzymes that modulate the histone acetylation status of chromatin, in synergy with the effects mediated by other chromatin remodeling factors, is central to gene regulation. PMID:11882541

  11. Repressive coping and alexithymia in idiopathic environmental intolerance

    PubMed Central

    Zachariae, Robert; Rasmussen, Alice; Johansen, Jeanne Duus; Elberling, Jesper

    2010-01-01

    Objective To examine if the non-expression of negative emotions (i.e., repressive coping) and differences in the ability to process and regulate emotions (i.e., alexithymia) is associated with idiopathic environmental intolerance (IEI). Methods The study included participants who had previously participated in a general population-based study and reported symptoms of environmental intolerance (n = 787) and patients with IEI (n = 237). The participants completed questionnaires assessing IEI, namely, a measure of repressive coping combining scores on the Marlowe–Crowne Social Desirability Scale (MCSDS) and the Taylor Manifest Anxiety Scale (TMAS), the Toronto Alexithymia Scale (TAS-20), and a negative affectivity scale (NAS). Multiple, hierarchical linear regression analyses were conducted using IEI variables as the dependent variables. Results The TMAS and MCSDS scores were independently associated with the IEI variables, but there was no evidence of a role of the repressive coping construct. While the total alexithymia score was unrelated to IEI, the TAS-20 subscale of difficulties identifying feelings (DIF) was independently associated with symptoms attributed to IEI. Negative affectivity was a strong independent predictor of the IEI variables and a mediator of the association between DIF and IEI. Conclusion Our results provide no evidence for a role of repressive coping in IEI, and our hypothesis of an association with alexithymia was only partly supported. In contrast, strong associations between IEI and negative emotional reactions, defensiveness and difficulties identifying feelings were found, suggesting a need for exploring the influence of these emotional reactions in IEI. PMID:21432559

  12. Cell-Context Dependent TCF/LEF Expression and Function: Alternative Tales of Repression, De-Repression and Activation Potentials

    PubMed Central

    Mao, Catherine D; Byers, Stephen W.

    2012-01-01

    Wnt signaling controls cell specification and fate during development and adult tissue homeostasis by converging on a small family of DNA binding factors, the T-cell factor/lymphoid enhancer factor (TCF/LEF) family. In response to Wnt signals, TCF/LEF members undergo a transcriptional switch from repression to activation mediated in part by nuclear β-catenin binding and recruitment of co-activator complexes. In mammals, the specificity and fine tuning of this transcriptional switch is also achieved by the cell-context-dependent expression of four members (TCF7, TCF7L1, TCF7L2, and LEF1) and numerous variants, which display differential DNA binding affinity and specificity, repression strength, activation potential, and regulators. TCF7/LEF1 variants are generated by alternative promoters, alternative exon cassettes, and alternative donor/acceptor splicing sites, allowing combinatorial insertion/exclusion of modular functional and regulatory domains. In this review we present mounting evidence for the interdependency of TCF7/LEF1 variant expression and functions with cell lineage and cell state. We also illustrate how the p53 and nuclear receptor family of transcription factors, known to control cell fate and to inhibit Wnt signaling, may participate in the fine tuning of TCF7/LEF1 repression/activation potentials. PMID:22111711

  13. Selective repression of light harvesting complex 2 formation in Rhodobacter azotoformans by light under semiaerobic conditions.

    PubMed

    Yue, Huiying; Zhao, Chungui; Li, Kai; Yang, Suping

    2015-11-01

    Photosystem formation in anaerobic anoxygenic phototrophic bacteria (APB) is repressed by oxygen but is de-repressed when oxygen tension decreases. Under semiaerobic conditions, the synthesis of photopigments and pigment protein complexes in Rhodobacter (Rba.) sphaeroides are repressed by light. AppA, a blue-light receptor, mediates this regulation. In the present study, it was showed that the synthesis of bacteriochlorophyll, carotenoid, and pigment protein complexes in Rba. azotoformans 134K20 was significantly repressed by oxygen. Oxygen exposure also led to a conversion of spheroidene to spheroidenone. In semiaerobically growing cells, light irradiation resulted in a decrease in the formation of photosystem, and blue light was found to be the most effective light source. Blue light reduced the contents of bacteriochlorophyll and carotenoid slightly, but had negligible effects on light harvesting complex (LH) 1 content, whereas the content of LH2 was significantly decreased indicating that blue light selectively repressed the synthesis of LH2 in semiaerobically growing 134K20. It was concluded that, similar to Rba. sphaeroides, a blue light receptor presented in strain 134K20 played important roles in its light-dependent repression. A possible mechanism involved in controlling the differential inhibitory of blue light on the synthesis of photosystem was discussed. PMID:26193456

  14. Mechanisms and consequences of ATMIN repression in hypoxic conditions: roles for p53 and HIF-1

    PubMed Central

    Leszczynska, Katarzyna B.; Göttgens, Eva-Leonne; Biasoli, Deborah; Olcina, Monica M.; Ient, Jonathan; Anbalagan, Selvakumar; Bernhardt, Stephan; Giaccia, Amato J.; Hammond, Ester M.

    2016-01-01

    Hypoxia-induced replication stress is one of the most physiologically relevant signals known to activate ATM in tumors. Recently, the ATM interactor (ATMIN) was identified as critical for replication stress-induced activation of ATM in response to aphidicolin and hydroxyurea. This suggests an essential role for ATMIN in ATM regulation during hypoxia, which induces replication stress. However, ATMIN also has a role in base excision repair, a process that has been demonstrated to be repressed and less efficient in hypoxic conditions. Here, we demonstrate that ATMIN is dispensable for ATM activation in hypoxia and in contrast to ATM, does not affect cell survival and radiosensitivity in hypoxia. Instead, we show that in hypoxic conditions ATMIN expression is repressed. Repression of ATMIN in hypoxia is mediated by both p53 and HIF-1α in an oxygen dependent manner. The biological consequence of ATMIN repression in hypoxia is decreased expression of the target gene, DYNLL1. An expression signature associated with p53 activity was negatively correlated with DYNLL1 expression in patient samples further supporting the p53 dependent repression of DYNLL1. Together, these data demonstrate multiple mechanisms of ATMIN repression in hypoxia with consequences including impaired BER and down regulation of the ATMIN transcriptional target, DYNLL1. PMID:26875667

  15. GATA-1 Utilizes Ikaros and Polycomb Repressive Complex 2 To Suppress Hes1 and To Promote Erythropoiesis

    PubMed Central

    Ross, Julie; Mavoungou, Lionel; Bresnick, Emery H.

    2012-01-01

    The transcription factor Hairy Enhancer of Split 1 (HES1), a downstream effector of the Notch signaling pathway, is an important regulator of hematopoiesis. Here, we demonstrate that in primary erythroid cells, Hes1 gene expression is transiently repressed around proerythroblast stage of differentiation. Using mouse erythroleukemia cells, we found that the RNA interference (RNAi)-mediated depletion of HES1 enhances erythroid cell differentiation, suggesting that this protein opposes terminal erythroid differentiation. This is also supported by the decreased primary erythroid cell differentiation upon HES1 upregulation in Ikaros-deficient mice. A comprehensive analysis led us to determine that Ikaros favors Hes1 repression in erythroid cells by facilitating recruitment of the master regulator of erythropoiesis GATA-1 alongside FOG-1, which mediates Hes1 repression. GATA-1 is then necessary for the chromatin binding of the NuRD remodeling complex ATPase MI-2, the transcription factor GFI1B, and the histone H3K27 methyltransferase EZH2 along with Polycomb repressive complex 2. We show that EZH2 is required for the transient repression of Hes1 in erythroid cells. In aggregate, our results describe a mechanism whereby GATA-1 utilizes Ikaros and Polycomb repressive complex 2 to promote Hes1 repression as an important step in erythroid cell differentiation. PMID:22778136

  16. The FBXL10/KDM2B scaffolding protein associates with novel polycomb repressive complex-1 to regulate adipogenesis.

    PubMed

    Inagaki, Takeshi; Iwasaki, Satoshi; Matsumura, Yoshihiro; Kawamura, Takeshi; Tanaka, Toshiya; Abe, Yohei; Yamasaki, Ayumu; Tsurutani, Yuya; Yoshida, Ayano; Chikaoka, Yoko; Nakamura, Kanako; Magoori, Kenta; Nakaki, Ryo; Osborne, Timothy F; Fukami, Kiyoko; Aburatani, Hiroyuki; Kodama, Tatsuhiko; Sakai, Juro

    2015-02-13

    Polycomb repressive complex 1 (PRC1) plays an essential role in the epigenetic repression of gene expression during development and cellular differentiation via multiple effector mechanisms, including ubiquitination of H2A and chromatin compaction. However, whether it regulates the stepwise progression of adipogenesis is unknown. Here, we show that FBXL10/KDM2B is an anti-adipogenic factor that is up-regulated during the early phase of 3T3-L1 preadipocyte differentiation and in adipose tissue in a diet-induced model of obesity. Interestingly, inhibition of adipogenesis does not require the JmjC demethylase domain of FBXL10, but it does require the F-box and leucine-rich repeat domains, which we show recruit a noncanonical polycomb repressive complex 1 (PRC1) containing RING1B, SKP1, PCGF1, and BCOR. Knockdown of either RING1B or SKP1 prevented FBXL10-mediated repression of 3T3-L1 preadipocyte differentiation indicating that PRC1 formation mediates the inhibitory effect of FBXL10 on adipogenesis. Using ChIP-seq, we show that FBXL10 recruits RING1B to key specific genomic loci surrounding the key cell cycle and the adipogenic genes Cdk1, Uhrf1, Pparg1, and Pparg2 to repress adipogenesis. These results suggest that FBXL10 represses adipogenesis by targeting a noncanonical PRC1 complex to repress key genes (e.g. Pparg) that control conversion of pluripotent cells into the adipogenic lineage. PMID:25533466

  17. The FBXL10/KDM2B Scaffolding Protein Associates with Novel Polycomb Repressive Complex-1 to Regulate Adipogenesis*

    PubMed Central

    Inagaki, Takeshi; Iwasaki, Satoshi; Matsumura, Yoshihiro; Kawamura, Takeshi; Tanaka, Toshiya; Abe, Yohei; Yamasaki, Ayumu; Tsurutani, Yuya; Yoshida, Ayano; Chikaoka, Yoko; Nakamura, Kanako; Magoori, Kenta; Nakaki, Ryo; Osborne, Timothy F.; Fukami, Kiyoko; Aburatani, Hiroyuki; Kodama, Tatsuhiko; Sakai, Juro

    2015-01-01

    Polycomb repressive complex 1 (PRC1) plays an essential role in the epigenetic repression of gene expression during development and cellular differentiation via multiple effector mechanisms, including ubiquitination of H2A and chromatin compaction. However, whether it regulates the stepwise progression of adipogenesis is unknown. Here, we show that FBXL10/KDM2B is an anti-adipogenic factor that is up-regulated during the early phase of 3T3-L1 preadipocyte differentiation and in adipose tissue in a diet-induced model of obesity. Interestingly, inhibition of adipogenesis does not require the JmjC demethylase domain of FBXL10, but it does require the F-box and leucine-rich repeat domains, which we show recruit a noncanonical polycomb repressive complex 1 (PRC1) containing RING1B, SKP1, PCGF1, and BCOR. Knockdown of either RING1B or SKP1 prevented FBXL10-mediated repression of 3T3-L1 preadipocyte differentiation indicating that PRC1 formation mediates the inhibitory effect of FBXL10 on adipogenesis. Using ChIP-seq, we show that FBXL10 recruits RING1B to key specific genomic loci surrounding the key cell cycle and the adipogenic genes Cdk1, Uhrf1, Pparg1, and Pparg2 to repress adipogenesis. These results suggest that FBXL10 represses adipogenesis by targeting a noncanonical PRC1 complex to repress key genes (e.g. Pparg) that control conversion of pluripotent cells into the adipogenic lineage. PMID:25533466

  18. Redistribution of H3K27me3 upon DNA hypomethylation results in de-repression of Polycomb target genes

    PubMed Central

    2013-01-01

    Background DNA methylation and the Polycomb repression system are epigenetic mechanisms that play important roles in maintaining transcriptional repression. Recent evidence suggests that DNA methylation can attenuate the binding of Polycomb protein components to chromatin and thus plays a role in determining their genomic targeting. However, whether this role of DNA methylation is important in the context of transcriptional regulation is unclear. Results By genome-wide mapping of the Polycomb Repressive Complex 2-signature histone mark, H3K27me3, in severely DNA hypomethylated mouse somatic cells, we show that hypomethylation leads to widespread H3K27me3 redistribution, in a manner that reflects the local DNA methylation status in wild-type cells. Unexpectedly, we observe striking loss of H3K27me3 and Polycomb Repressive Complex 2 from Polycomb target gene promoters in DNA hypomethylated cells, including Hox gene clusters. Importantly, we show that many of these genes become ectopically expressed in DNA hypomethylated cells, consistent with loss of Polycomb-mediated repression. Conclusions An intact DNA methylome is required for appropriate Polycomb-mediated gene repression by constraining Polycomb Repressive Complex 2 targeting. These observations identify a previously unappreciated role for DNA methylation in gene regulation and therefore influence our understanding of how this epigenetic mechanism contributes to normal development and disease. PMID:23531360

  19. Coordinate post-transcriptional repression of Dpp-dependent transcription factors attenuates signal range during development

    PubMed Central

    Newton, Fay G.; Harris, Robin E.; Sutcliffe, Catherine; Ashe, Hilary L.

    2015-01-01

    Precise control of the range of signalling molecule action is crucial for correct cell fate patterning during development. For example, Drosophila ovarian germline stem cells (GSCs) are maintained by exquisitely short-range BMP signalling from the niche. In the absence of BMP signalling, one GSC daughter differentiates into a cystoblast (CB) and this fate is stabilised by Brain tumour (Brat) and Pumilio (Pum)-mediated post-transcriptional repression of mRNAs, including that encoding the Dpp transducer, Mad. However, the identity of other repressed mRNAs and the mechanism of post-transcriptional repression are currently unknown. Here, we identify the Medea and schnurri mRNAs, which encode transcriptional regulators required for activation and/or repression of Dpp target genes, as additional Pum-Brat targets, suggesting that tripartite repression of the transducers is deployed to desensitise the CB to Dpp. In addition, we show that repression by Pum-Brat requires recruitment of the CCR4 and Pop2 deadenylases, with knockdown of deadenylases in vivo giving rise to ectopic GSCs. Consistent with this, Pum-Brat repression leads to poly(A) tail shortening and mRNA degradation in tissue culture cells, and we detect a reduced number of Mad and shn transcripts in the CB relative to the GSC based on single molecule mRNA quantitation. Finally, we show generality of the mechanism by demonstrating that Brat also attenuates pMad and Dpp signalling range in the early embryo. Together our data serve as a platform for understanding how post-transcriptional repression restricts interpretation of BMPs and other cell signals in order to allow robust cell fate patterning during development. PMID:26293305

  20. Chick Pcl2 regulates the left-right asymmetry by repressing Shh expression in Hensen's node.

    PubMed

    Wang, Shusheng; Yu, Xueyan; Zhang, Tao; Zhang, Xiaoyun; Zhang, Zunyi; Chen, YiPing

    2004-09-01

    Asymmetric expression of sonic hedgehog (Shh) in the left side of Hensen's node, a crucial step for specifying the left-right (LR) axis in the chick embryo, is established by the repression of Shh expression in the right side of the node. The transcriptional regulator that mediates this repression has not been identified. We report the isolation and characterization of a novel chick Polycomblike 2 gene, chick Pcl2, which encodes a transcription repressor and displays an asymmetric expression, downstream from Activin-betaB and Bmp4, in the right side of Hensen's node in the developing embryo. In vitro mapping studies define the transcription repression activity to the PHD finger domain of the chick Pcl2 protein. Repression of chick Pcl2 expression in the early embryo results in randomized heart looping direction, which is accompanied by the ectopic expression of Shh in the right side of the node and Shh downstream genes in the right lateral plate mesoderm (LPM), while overexpression of chick Pcl2 represses Shh expression in the node. The repression of Shh by chick Pcl2 was also supported by studies in which chick Pcl2 was overexpressed in the developing chick limb bud and feather bud. Similarly, transgenic overexpression of chick Pcl2 in the developing mouse limb inhibits Shh expression in the ZPA. In vitro pull-down assays demonstrated a direct interaction of the chick Pcl2 PHD finger with EZH2, a component of the ESC/E(Z) repressive complex. Taken together with the fact that chick Pcl2 was found to directly repress Shh promoter activity in vitro, our results demonstrate a crucial role for chick Pcl2 in regulating LR axis patterning in the chick by silencing Shh in the right side of the node. PMID:15294861

  1. Transcriptional Repression of E-Cadherin by Human Papillomavirus Type 16 E6

    PubMed Central

    D'Costa, Zarina J.; Jolly, Carol; Androphy, Elliot J.; Mercer, Andrew; Matthews, Charles M.; Hibma, Merilyn H.

    2012-01-01

    There is increasing evidence supporting DNA virus regulation of the cell adhesion and tumour suppressor protein, E-cadherin. We previously reported that loss of E-cadherin in human papillomavirus (HPV) type 16-infected epidermis is contributed to by the major viral proto-oncogene E6 and is associated with reduced Langerhans cells density, potentially regulating the immune response. The focus of this study is determining how the HPV16 E6 protein mediates E-cadherin repression. We found that the E-cadherin promoter is repressed in cells expressing E6, resulting in fewer E-cadherin transcripts. On exploring the mechanism for this, repression by increased histone deacetylase activity or by increased binding of trans-repressors to the E-cadherin promoter Epal element was discounted. In contrast, DNA methyltransferase (DNMT) activity was increased in E6 expressing cells. Upon inhibiting DNMT activity using 5-Aza-2′-deoxycytidine, E-cadherin transcription was restored in the presence of HPV16 E6. The E-cadherin promoter was not directly methylated, however a mutational analysis showed general promoter repression and reduced binding of the transactivators Sp1 and AML1 and the repressor Slug. Expression of E7 with E6 resulted in a further reduction in surface E-cadherin levels. This is the first report of HPV16 E6-mediated transcriptional repression of this adhesion molecule and tumour suppressor protein. PMID:23189137

  2. Catabolite-induced repression of sporulation in Bacillus subtilis.

    PubMed

    Shafikhani, Sasha H; Partovi, Amir Ali; Leighton, Terrance

    2003-10-01

    In response to nutrient limitations, Bacillus subtilis cells undergo a series of morphological and genetic changes that culminate in the formation of endospores. Conversely, excess catabolites inhibit sporulation. It has been demonstrated previously that excess catabolites caused a decrease in culture medium pH in a process that required functional AbrB. Culture medium acidification was also shown to inhibit sigmaH-dependent sporulation gene expression. The studies reported here investigate the effects of AbrB-mediated pH sensing on B. subtilis developmental competence. We have found that neither addition of a pH stabilizer, MOPS (pH 7.5), nor null mutations in abrB blocked catabolite repression of sporulation. Moreover, catabolite-induced culture medium acidification was observed in cultures of catabolite-resistant sporulation mutants, crsA47, rvtA11, and hpr-16, despite their efficient sporulation. These results suggest that AbrB-mediated pH sensing is not the only mechanism regulating catabolite repression of sporulation. The AbrB pathway may function to channel cells toward genetic competence, as opposed to other postexponential differentiation pathways. PMID:14629011

  3. Pseudomonas fluorescens WCS374r-Induced Systemic Resistance in Rice against Magnaporthe oryzae Is Based on Pseudobactin-Mediated Priming for a Salicylic Acid-Repressible Multifaceted Defense Response1[C][OA

    PubMed Central

    De Vleesschauwer, David; Djavaheri, Mohammad; Bakker, Peter A.H.M.; Höfte, Monica

    2008-01-01

    Selected strains of nonpathogenic rhizobacteria can reduce disease in foliar tissues through the induction of a defense state known as induced systemic resistance (ISR). Compared with the large body of information on ISR in dicotyledonous plants, little is known about the mechanisms underlying rhizobacteria-induced resistance in cereal crops. Here, we demonstrate the ability of Pseudomonas fluorescens WCS374r to trigger ISR in rice (Oryza sativa) against the leaf blast pathogen Magnaporthe oryzae. Using salicylic acid (SA)-nonaccumulating NahG rice, an ethylene-insensitive OsEIN2 antisense line, and the jasmonate-deficient mutant hebiba, we show that this WCS374r-induced resistance is regulated by an SA-independent but jasmonic acid/ethylene-modulated signal transduction pathway. Bacterial mutant analysis uncovered a pseudobactin-type siderophore as the crucial determinant responsible for ISR elicitation. Root application of WCS374r-derived pseudobactin (Psb374) primed naive leaves for accelerated expression of a pronounced multifaceted defense response, consisting of rapid recruitment of phenolic compounds at sites of pathogen entry, concerted expression of a diverse set of structural defenses, and a timely hyperinduction of hydrogen peroxide formation putatively driving cell wall fortification. Exogenous SA application alleviated this Psb374-modulated defense priming, while Psb374 pretreatment antagonized infection-induced transcription of SA-responsive PR genes, suggesting that the Psb374- and SA-modulated signaling pathways are mutually antagonistic. Interestingly, in sharp contrast to WCS374r-mediated ISR, chemical induction of blast resistance by the SA analog benzothiadiazole was independent of jasmonic acid/ethylene signaling and involved the potentiation of SA-responsive gene expression. Together, these results offer novel insights into the signaling circuitry governing induced resistance against M. oryzae and suggest that rice is endowed with multiple

  4. MLL repression domain interacts with histone deacetylases, the polycomb group proteins HPC2 and BMI-1, and the corepressor C-terminal-binding protein

    PubMed Central

    Xia, Zhen-Biao; Anderson, Melanie; Diaz, Manuel O.; Zeleznik-Le, Nancy J.

    2003-01-01

    The MLL (mixed-lineage leukemia) gene is involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia. We previously identified a transcriptional repression domain in MLL, which contains a region with homology to DNA methyltransferase. In chromosomal translocations, the MLL repression domain is retained in the leukemogenic fusion protein and is required for transforming activity of MLL fusion proteins. We explored the mechanism of action of the MLL repression domain. Histone deacetylase 1 interacts with the MLL repression domain, partially mediating its activity; binding of Cyp33 to the adjacent MLL-PHD domain potentiates this binding. Because the MLL repression domain activity was only partially relieved with the histone deacetylase inhibitor trichostatin A, we explored other protein interactions with this domain. Polycomb group proteins HPC2 and BMI-1 and the corepressor C-terminal-binding protein also bind the MLL repression domain. Expression of exogenous BMI-1 potentiates MLL repression domain activity. Functional antagonism between Mll and Bmi-1 has been shown genetically in murine knockout models for Mll and Bmi-1. Our new data suggest a model whereby recruitment of BMI-1 to the MLL protein may be able to modulate its function. Furthermore, repression mediated by histone deacetylases and that mediated by polycomb group proteins may act either independently or together for MLL function in vivo. PMID:12829790

  5. Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition.

    PubMed

    Lizama, Carlos O; Hawkins, John S; Schmitt, Christopher E; Bos, Frank L; Zape, Joan P; Cautivo, Kelly M; Borges Pinto, Hugo; Rhyner, Alexander M; Yu, Hui; Donohoe, Mary E; Wythe, Joshua D; Zovein, Ann C

    2015-01-01

    Changes in cell fate and identity are essential for endothelial-to-haematopoietic transition (EHT), an embryonic process that generates the first adult populations of haematopoietic stem cells (HSCs) from hemogenic endothelial cells. Dissecting EHT regulation is a critical step towards the production of in vitro derived HSCs. Yet, we do not know how distinct endothelial and haematopoietic fates are parsed during the transition. Here we show that genes required for arterial identity function later to repress haematopoietic fate. Tissue-specific, temporally controlled, genetic loss of arterial genes (Sox17 and Notch1) during EHT results in increased production of haematopoietic cells due to loss of Sox17-mediated repression of haematopoietic transcription factors (Runx1 and Gata2). However, the increase in EHT can be abrogated by increased Notch signalling. These findings demonstrate that the endothelial haematopoietic fate switch is actively repressed in a population of endothelial cells, and that derepression of these programs augments haematopoietic output. PMID:26204127

  6. Direct activation and anti-repression functions of GAL4-VP16 use distinct molecular mechanisms.

    PubMed Central

    Lyons, J G; Chambon, P

    1995-01-01

    In order to determine whether the molecular mechanisms used for direct activation by GAL4-VP16 are the same as those used for anti-repression, we have employed monoclonal antibodies specific for the VP16 activation domain. In the absence of added repressors, GAL4-VP16 was able to stimulate transcription from a template containing GAL4-binding sites, and the antibodies raised against the VP16 activation domain failed to inhibit this direct activation. GAL4-VP16 also was able to prevent histone H1-mediated repression by a mechanism that was strongly dependent on the presence of specific GAL4-binding elements in the promoter. However, in contrast to the assays conducted in the absence of repressors, the antibodies were strong inhibitors of GAL4-VP16-activated transcription in the presence of histone H1. Thus the binding of the antibodies distinguished between the direct activation and anti-repression functions of GAL4-VP16, indicating that these functions operate through distinct molecular mechanisms. The anti-repression-specific mechanism that is inhibitable by the antibodies acted at an early stage of preinitiation complex formation. Deletions of individual subdomains of the VP16 activation domain demonstrated that there was not a discrete subdomain responsible for the anti-repression function of GAL4-VP16. Thus, the inhibitory effect of the antibodies appeared to be due to the location of the epitope within the activator protein rather than to some inherent biochemical property of that region of the protein that is required specifically for anti-repression. The inhibitory effect of the antibodies also ruled out the possibility that steric exclusion of repressor proteins from the promoter was the sole means of anti-repression by the transcriptional activator. Images Figure 1 Figure 2 PMID:8554536

  7. GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats.

    PubMed Central

    Flick, J S; Johnston, M

    1991-01-01

    Growth of the yeast Saccharomyces cerevisiae on glucose leads to repression of transcription of many genes required for alternative carbohydrate metabolism. The GRR1 gene appears to be of central importance to the glucose repression mechanism, because mutations in GRR1 result in a pleiotropic loss of glucose repression (R. Bailey and A. Woodword, Mol. Gen. Genet. 193:507-512, 1984). We have isolated the GRR1 gene and determined that null mutants are viable and display a number of growth defects in addition to the loss of glucose repression. Surprisingly, grr1 mutations convert SUC2, normally a glucose-repressed gene, into a glucose-induced gene. GRR1 encodes a protein of 1,151 amino acids that is expressed constitutively at low levels in yeast cells. GRR1 protein contains 12 tandem repeats of a sequence similar to leucine-rich motifs found in other proteins that may mediate protein-protein interactions. Indeed, cell fractionation studies are consistent with this view, suggesting that GRR1 protein is tightly associated with a particulate protein fraction in yeast extracts. The combined genetic and molecular data are consistent with the idea that GRR1 protein is a primary response element in the glucose repression pathway and is required for the generation or interpretation of the signal that induces glucose repression. Images PMID:1922034

  8. Repression of Hybrid Dysgenesis in Drosophila Melanogaster by Individual Naturally Occurring P Elements

    PubMed Central

    Rasmusson, K. E.; Raymond, J. D.; Simmons, M. J.

    1993-01-01

    Individual P elements that were genetically isolated from wild-type strains were tested for their abilities to repress two aspects of hybrid dysgenesis: gonadal dysgenesis and mutability of a double-P element-insertion allele of the singed locus (sn(w)). These elements were also characterized by Southern blotting, polymerase chain reaction amplification and DNA sequencing. Three of the elements were 1.1-kb KP elements, one was a 1.2-kb element called D50, and one was a 0.5-kb element called SP. These three types of elements could encode polypeptides of 207, 204, and 14 amino acids, respectively. Gonadal dysgenesis was repressed by two of the KP elements (denoted KP(1) and KP(6)) and by SP, but not by the third KP element (KP(D)), nor by D50. Repression of gonadal dysgenesis was mediated by a maternal effect, or by a combination of zygotic and maternal effects generated by the P elements themselves. The mutability of sn(w) was repressed by the KP(1) and KP(6) elements, by D50 and by SP, but not by KP(D); however, the SP element repressed sn(w) mutability only when the transposase came from complete P elements and the D50 element repressed it only when the transposase came from the modified P element known as Δ2-3. In all cases, repression of sn(w) mutability appeared to be mediated by a zygotic effect of the isolated P element. Each of the isolated elements was also tested for its ability to suppress the phenotype of a P-insertion mutation of the vestigial locus (vg(21-3)). D50 was a moderate suppressor whereas SP and the three KP elements had little or no effect. These results indicate that each isolated P element had its own profile of repression and suppression abilities. It is suggested that these abilities may be mediated by P-encoded polypeptides or by antisense P RNAs initiated from external genomic promoters. PMID:8384145

  9. Androgen receptor repression of gonadotropin-releasing hormone gene transcription via enhancer 1.

    PubMed

    Brayman, Melissa J; Pepa, Patricia A; Mellon, Pamela L

    2012-11-01

    Gonadotropin-releasing hormone (GnRH) plays a major role in the hypothalamic-pituitary-gonadal (HPG) axis, and synthesis and secretion of GnRH are regulated by gonadal steroid hormones. Disruptions in androgen levels are involved in a number of reproductive defects, including hypogonadotropic hypogonadism and polycystic ovarian syndrome. Androgens down-regulate GnRH mRNA synthesis in vivo and in vitro via an androgen receptor (AR)-dependent mechanism. Methyltrienolone (R1881), a synthetic AR agonist, represses GnRH expression through multiple sites in the proximal promoter. In this study, we show AR also represses GnRH transcription via the major enhancer (GnRH-E1). A multimer of the -1800/-1766 region was repressed by R1881 treatment. Mutation of two bases, -1792 and -1791, resulted in decreased basal activity and a loss of AR-mediated repression. AR bound to the -1796/-1791 sequence in electrophoretic mobility shift assays, indicating a direct interaction with DNA or other transcription factors in this region. We conclude that AR repression of GnRH-E1 acts via multiple AR-responsive regions, including the site at -1792/-1791. PMID:22877652

  10. A highly conserved molecular switch binds MSY-3 to regulate myogenin repression in postnatal muscle

    PubMed Central

    Berghella, Libera; De Angelis, Luciana; De Buysscher, Tristan; Mortazavi, Ali; Biressi, Stefano; Forcales, Sonia V.; Sirabella, Dario; Cossu, Giulio; Wold, Barbara J.

    2008-01-01

    Myogenin is the dominant transcriptional regulator of embryonic and fetal muscle differentiation and during maturation is profoundly down-regulated. We show that a highly conserved 17-bp DNA cis-acting sequence element located upstream of the myogenin promoter (myogHCE) is essential for postnatal repression of myogenin in transgenic animals. We present multiple lines of evidence supporting the idea that repression is mediated by the Y-box protein MSY-3. Electroporation in vivo shows that myogHCE and MSY-3 are required for postnatal repression. We further show that, in the C2C12 cell culture system, ectopic MSY-3 can repress differentiation, while reduced MSY-3 promotes premature differentiation. MSY-3 binds myogHCE simultaneously with the homeodomain protein Pbx in postnatal innervated muscle. We therefore propose a model in which the myogHCE motif operates as a switch by specifying opposing functions; one that was shown previously is regulated by MyoD and Pbx and it specifies a chromatin opening, gene-activating function at the time myoblasts begin to differentiate; the other includes MYS-3 and Pbx, and it specifies a repression function that operates during and after postnatal muscle maturation in vivo and in myoblasts before they begin to differentiate. PMID:18676817

  11. Dual role of NRSF/REST in activation and repression of the glucocorticoid response.

    PubMed

    Abramovitz, Lilach; Shapira, Tamar; Ben-Dror, Iris; Dror, Vardit; Granot, Limor; Rousso, Tal; Landoy, Elad; Blau, Lior; Thiel, Gerald; Vardimon, Lily

    2008-01-01

    Restriction of glutamine synthetase to the nervous system is mainly achieved through the mutual function of the glucocorticoid receptor and the neural restrictive silencing factor, NRSF/REST. Glucocorticoids induce glutamine synthetase expression in neural tissues while NRSF/REST represses the hormonal response in non-neural cells. NRSF/REST is a modular protein that contains two independent repression domains, at the N and C termini of the molecule, and is dominantly expressed in nonneural cells. Neural tissues express however splice variants, REST4/5, which contain the repression domain at the N, but not at the C terminus of the molecule. Here we show that full-length NRSF/REST or its C-terminal domain can inhibit almost completely the induction of gene transcription by glucocorticoids. By contrast, the N-terminal domain not only fails to repress the hormonal response but rather stimulates it markedly. The inductive activity of the N-terminal domain is mediated by hBrm, which is recruited to the promoter only in the concomitant presence of GR. Importantly, a similar inductive activity is also exerted by the splice variant REST4. These findings raise the possibility that NRSF/REST exhibits a dual role in regulation of glutamine synthetase. It represses gene induction in nonneural cells and enhances the hormonal response, via its splice variant, in the nervous system. PMID:17984088

  12. The relationship of repression to the unconscious.

    PubMed

    Gillett, E

    1987-01-01

    I try to formulate the simplest topographic model that embodies current theoretical understanding. The repression mechanism is under the control of a single censorship located on the border of consciousness. I argue that neither the operation of the repression mechanism nor the decision process of the censorship, which controls the repression mechanism and other defence mechanisms, can be considered dynamically unconscious. I discuss the distinction drawn by Wallerstein, Sandler & Joffe between the experiential and the non-experiential, concluding that much of the non-conscious id, ego, and superego is non-experiential rather than dynamically unconscious. Within the experiential realm I present the reasons why the censorship is located on the edge of consciousness and the implications of this location for the distinction between the preconscious and the dynamic unconscious. PMID:3436713

  13. Repressive translational control in germ cells.

    PubMed

    Lai, Fangfang; King, Mary Lou

    2013-08-01

    The earliest stages of embryonic development in many animals proceed without zygotic transcription. Genetic control is executed by maternally inherited mRNAs that are under translational control. To set aside the future germ cell lineage, it is pivotal to both exert translational regulation of maternal germline mRNAs and to repress maternal signals in those same cells that drive somatic cell-fate determination. Here we review repressive translational regulation in the germline from the perspective of the conserved RNA binding proteins Pumilio and Nanos, and discuss common themes that have emerged. PMID:23408501

  14. SOD1 Integrates Signals from Oxygen and Glucose to Repress Respiration

    PubMed Central

    Reddi, Amit R.; Culotta, Valeria C.

    2012-01-01

    Summary Cu/Zn Superoxide Dismutase (SOD1) is an abundant enzyme that has been best studied as a regulator of antioxidant defence. Using the yeast S. cerevisiae, we report that SOD1 transmits signals from oxygen and glucose to repress respiration. The mechanism involves SOD1-mediated stabilization of two casein kinase 1-gamma (CK1γ) homologs, Yck1p and Yck2p, required for respiratory repression. SOD1 binds a C-terminal degron we identified in Yck1p/Yck2p, and promotes kinase stability by catalyzing superoxide conversion to peroxide. The effects of SOD1 on CK1γ stability are also observed with mammalian SOD1 and CK1γ and in a human cell line. Therefore in a single circuit, oxygen, glucose, and reactive oxygen can repress respiration through SOD1/ CK1γ signaling. Our data therefore may provide mechanistic insight into how rapidly proliferating cells and many cancers accomplish glucose-mediated repression in favour of aerobic glycolysis. PMID:23332757

  15. YB-1 regulates tiRNA-induced Stress Granule formation but not translational repression.

    PubMed

    Lyons, Shawn M; Achorn, Chris; Kedersha, Nancy L; Anderson, Paul J; Ivanov, Pavel

    2016-08-19

    Stress-induced angiogenin (ANG)-mediated tRNA cleavage promotes a cascade of cellular events that starts with production of tRNA-derived stress-induced RNAs (tiRNAs) and culminates with enhanced cell survival. This stress response program relies on a subset tiRNAs that inhibit translation initiation and induce the assembly of stress granules (SGs), cytoplasmic ribonucleoprotein complexes with cytoprotective and pro-survival properties. SG-promoting tiRNAs bear oligoguanine motifs at their 5'-ends, assemble G-quadruplex-like structures and interact with the translational silencer YB-1. We used CRISPR/Cas9-based genetic manipulations and biochemical approaches to examine the role of YB-1 in tiRNA-mediated translational repression and SG assembly. We found that YB-1 directly binds to tiRNAs via its cold shock domain. This interaction is required for packaging of tiRNA-repressed mRNAs into SGs but is dispensable for tiRNA-mediated translational repression. Our studies reveal the functional role of YB-1 in the ANG-mediated stress response program. PMID:27174937

  16. How targets select activation or repression in response to Wnt.

    PubMed

    Murgan, Sabrina; Bertrand, Vincent

    2015-01-01

    In metazoans, the Wnt signaling pathway plays a key role in the regulation of binary decisions during development. During this process different sets of target genes are activated in cells where the Wnt pathway is active (classic target genes) versus cells where the pathway is inactive (opposite target genes). While the mechanism of transcriptional activation is well understood for classic target genes, how opposite target genes are activated in the absence of Wnt remains poorly characterized. Here we discuss how the key transcriptional mediator of the Wnt pathway, the TCF family member POP-1, regulates opposite target genes during C. elegans development. We examine recent findings suggesting that the direction of the transcriptional output (activation or repression) can be determined by the way TCF is recruited and physically interacts with its target gene. PMID:27123368

  17. The role of COP1 in repression of photoperiodic flowering.

    PubMed

    Xu, Dongqing; Zhu, Danmeng; Deng, Xing Wang

    2016-01-01

    Plants use the circadian clock as a timekeeping mechanism to regulate photoperiodic flowering in response to the seasonal changes. CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), initially identified as a central repressor of seedling photomorphogenesis, was recently shown to be involved in the regulation of light input to the circadian clock, modulating the circadian rhythm and flowering. COP1 encodes a RING-finger E3 ubiquitin ligase and works in concert with SUPPRESSOR of phyA-105 (SPA) proteins to repress photoperiodic flowering by regulating proteasome-mediated degradation of CONSTANS (CO), a central regulator of photoperiodic flowering. In addition, COP1 and EARLY FLOWERING 3 (ELF3) indirectly modulate CO expression via the degradation of GIGANTEA (GI). Here, we summarize the current understanding of the molecular mechanisms underlying COP1's role in controlling of photoperiodic flowering. PMID:26949521

  18. Tristetraprolin Represses Estrogen Receptor α Transactivation in Breast Cancer Cells*

    PubMed Central

    Barrios-García, Tonatiuh; Tecalco-Cruz, Angeles; Gómez-Romero, Vania; Reyes-Carmona, Sandra; Meneses-Morales, Iván; León-Del-Río, Alfonso

    2014-01-01

    Estrogen receptor α (ERα) mediates the effects of 17β-estradiol (E2) in normal mammary gland, and it is a key participant in breast cancer tumor development. ERα transactivation activity is mediated by the synergistic interaction of two domains designated AF1 and AF2. The function of AF2 is to recruit coactivator and corepressor proteins that allow ERα to oscillate between the roles of transcriptional activator and repressor. In contrast, the mechanism responsible for AF-1 transcriptional activity is not completely understood. In this study, we identified tristetraproline (TTP) as a novel ERα-associated protein. TTP expression in MCF7 cells repressed ERα transactivation and reduced MCF7 cell proliferation and the ability of the cells to form tumors in a mouse model. We show that TTP transcriptional activity is mediated through its recruitment to the promoter region of ERα target genes and its interaction with histone deacetylases, in particular with HDAC1. TTP expression attenuates the coactivating activity of SRC-1, suggesting that exchange between TTP and other coactivators may play an important role in fine-tuning ERα transactivation. These results indicate that TTP acts as a bona fide ERα corepressor and suggest that this protein may be a contributing factor in the development of E2-dependent tumors in breast cancer. PMID:24737323

  19. In simple synthetic promoters YY1-induced DNA bending is important in transcription activation and repression.

    PubMed Central

    Kim, J; Shapiro, D J

    1996-01-01

    Depending on promoter context, YY1 can activate or repress transcription, or provide a site for transcription initiation. To investigate whether the ability of YY1 to induce DNA bending influenced its ability to activate and repress transcription, simple synthetic promoters were constructed in which the YY1 binding site was inserted between the TATA box and either the NF1 or AP1 recognition sequences. In transient transfections of COS cells, the NF1YY1TATA and NF1RYY1TATA promoters exhibited a dramatic 15-20-fold increase in correctly initiated transcription. These promoters exhibited even larger 60-80-fold increases in transcription in HeLa cells. Neither multiple copies of the YY1 binding site alone, nor placement of a YY1 site upstream of the NF1 site activated transcription. Deletion of 4 bp between the NF1 and YY1 sites, which changes the phase of the DNA bends, abolished the 16-fold activation of transcription by NF1YY1TATA. Insertion of the YY1 site between the AP1 site and the TATA box decreased transcription approximately 3-fold. Replacing the YY1 binding site with an intrinsic DNA bending sequence mimicked this transcription repression. Sequences of similar length which do not bend DNA fail to repress AP1-mediated transcription. Gel mobility shift assays were used to show that binding of YY1 to its recognition sequence did not repress binding of AP1 to its recognition sequences. Our data indicate that YY1-induced DNA bending may activate and repress transcription by changing the spatial relationships between transcription activators and components of the basal transcription apparatus. PMID:8932392

  20. Junk DNA and sectorial gene repression.

    PubMed

    Zuckerkandl, E

    1997-12-31

    Transcriptional repression in eukaryotes often involves tens or hundreds of kilobase pairs, two to three orders of magnitude more than the bacterial operator/repressor model does. Classical repression, represented by this model, was maintained over the whole span of evolution under different guises, and consists of repressor factors interacting primarily with promoters and, in later evolution, also with enhancers. The use of much larger amounts of DNA in the other mode of repression, here called the sectorial mode ('superrepression'), results in the conceptual transfer of so-called junk DNA to the domain of functional DNA. This contribution to the solution of the c-value paradox involves perhaps 15% of genomic 'junk,' and encompasses the bulk of the introns, thought to fill a stabilizing role in sectorially repressed chromatin structures. In the case of developmental genes, such structures appear to be heterochromatoid in character. However, solid clues regarding general structural features of superrepressed terminal differentiation genes remain elusive. The competition among superrepressible DNA sectors for sectorially binding factors offers, in principle, a molecular mechanism for developmental switches. Position effect variegation may be considered an abnormal manifestation of normal processes that underly development and involve heterochromatoid sectorial repression, which is apparently required for local elimination or modulation of morphological features (morpholysis). Sectorial repression of genes participating either in development or in terminal differentiation is considered instrumental in establishing stable cell types, and provides a basis for the distinction between determination and cell type specification. The gamut of possible stable cell types may have been broadened by the appearance in evolution of heavy isochores. Additional types of relatively frequent GC-rich cis-acting DNA motifs may offer reiterated binding sites to factors endowed with a

  1. Nimesulide, a cyclooxygenase-2 selective inhibitor, suppresses obesity-related non-alcoholic fatty liver disease and hepatic insulin resistance through the regulation of peroxisome proliferator-activated receptor γ

    PubMed Central

    Tsujimoto, Shunsuke; Kishina, Manabu; Koda, Masahiko; Yamamoto, Yasutaka; Tanaka, Kohei; Harada, Yusuke; Yoshida, Akio; Hisatome, Ichiro

    2016-01-01

    Cyclooxygenase (COX)-2 selective inhibitors suppress non-alcoholic fatty liver disease (NAFLD); however, the precise mechanism of action remains unknown. The aim of this study was to examine how the COX-2 selective inhibitor nimesulide suppresses NAFLD in a murine model of high-fat diet (HFD)-induced obesity. Mice were fed either a normal chow diet (NC), an HFD, or HFD plus nimesulide (HFD-nime) for 12 weeks. Body weight, hepatic COX-2 mRNA expression and triglyceride accumulation were significantly increased in the HFD group. Triglyceride accumulation was suppressed in the HFD-nime group. The mRNA expression of hepatic peroxisome proliferator-activated receptor γ (PPARγ) and the natural PPARγ agonist 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) were significantly increased in the HFD group and significantly suppressed in the HFD-nime group. Glucose metabolism was impaired in the HFD group compared with the NC group, and it was significantly improved in the HFD-nime group. In addition, the plasma insulin levels in the HFD group were increased compared with those in the NC group, and were decreased in the HFD-nime group. These results indicate that HFD-induced NAFLD is mediated by the increased hepatic expression of COX-2. We suggest that the production of 15d-PGJ2, which is mediated by COX-2, induces NAFLD and hepatic insulin resistance by activating PPARγ. Furthermore, the mRNA expression of tissue inhibitor of metalloproteinases-1 (TIMP-1), procollagen-1 and monocyte chemoattractant protein-1 (MCP-1), as well as the number of F4/80-positive hepatic (Kupffer) cells, were significantly increased in the HFD group compared with the NC group, and they were reduced by nimesulide. In conclusion, COX-2 may emerge as a molecular target for preventing the development of NAFLD and insulin resistance in diet-related obesity. PMID:27431935

  2. Origin and consequences of brain Toll-like receptor 4 pathway stimulation in an experimental model of depression

    PubMed Central

    2011-01-01

    Background There is a pressing need to identify novel pathophysiological pathways relevant to depression that can help to reveal targets for the development of new medications. Toll-like receptor 4 (TLR-4) has a regulatory role in the brain's response to stress. Psychological stress may compromise the intestinal barrier, and increased gastrointestinal permeability with translocation of lipopolysaccharide (LPS) from Gram-negative bacteria may play a role in the pathophysiology of major depression. Methods Adult male Sprague-Dawley rats were subjected to chronic mild stress (CMS) or CMS+intestinal antibiotic decontamination (CMS+ATB) protocols. Levels of components of the TLR-4 signaling pathway, of LPS and of different inflammatory, oxidative/nitrosative and anti-inflammatory mediators were measured by RT-PCR, western blot and/or ELISA in brain prefrontal cortex. Behavioral despair was studied using Porsolt's test. Results CMS increased levels of TLR-4 and its co-receptor MD-2 in brain as well as LPS and LPS-binding protein in plasma. In addition, CMS also increased interleukin (IL)-1β, COX-2, PGE2 and lipid peroxidation levels and reduced levels of the anti-inflammatory prostaglandin 15d-PGJ2 in brain tissue. Intestinal decontamination reduced brain levels of the pro-inflammatory parameters and increased 15d-PGJ2, however this did not affect depressive-like behavior induced by CMS. Conclusions Our results suggest that LPS from bacterial translocation is responsible, at least in part, for the TLR-4 activation found in brain after CMS, which leads to release of inflammatory mediators in the CNS. The use of Gram-negative antibiotics offers a potential therapeutic approach for the adjuvant treatment of depression. PMID:22053929

  3. Very low amounts of glucose cause repression of the stress-responsive gene HSP12 in Saccharomyces cerevisiae.

    PubMed

    de Groot, E; Bebelman, J P; Mager, W H; Planta, R J

    2000-02-01

    Changing the growth mode of Saccharomyces cerevisiae by adding fermentable amounts of glucose to cells growing on a non-fermentable carbon source leads to rapid repression of general stress-responsive genes like HSP12. Remarkably, glucose repression of HSP12 appeared to occur even at very low glucose concentrations, down to 0.005%. Although these low levels of glucose do not induce fermentative growth, they do act as a growth signal, since upon addition of glucose to a concentration of 0.02%, growth rate increased and ribosomal protein gene transcription was up-regulated. In an attempt to elucidate how this type of glucose signalling may operate, several signalling mutants were examined. Consistent with the low amounts of glucose that elicit HSP12 repression, neither the main glucose-repression pathway nor cAMP-dependent activation of protein kinase A appeared to play a role in this regulation. Using mutants involved in glucose metabolism, evidence was obtained suggesting that glucose 6-phosphate serves as a signalling molecule. To identify the target for glucose repression on the promoter of the HSP12 gene, a promoter deletion series was used. The major transcription factors governing (stress-induced) transcriptional activation of HSP12 are Msn2p and Msn4p, binding to the general stress-responsive promoter elements (STREs). Surprisingly, glucose repression of HSP12 appeared to be independent of Msn2/4p: HSP12 transcription in glycerol-grown cells was unaffected in a deltamsn2deltamsn4 strain. Nevertheless, evidence was obtained that STRE-mediated transcription is the target of repression by low amounts of glucose. These data suggest that an as yet unidentified factor is involved in STRE-mediated transcriptional regulation of HSP12. PMID:10708375

  4. PABP and the poly(A) tail augment microRNA repression by facilitated miRISC binding.

    PubMed

    Moretti, Francesca; Kaiser, Constanze; Zdanowicz-Specht, Agnieszka; Hentze, Matthias W

    2012-06-01

    Polyadenylated mRNAs are typically more strongly repressed by microRNAs (miRNAs) than their nonadenylated counterparts. Using a Drosophila melanogaster cell-free translation system, we found that this effect is mediated by the poly(A)-binding protein (PABP). miRNA repression was positively correlated with poly(A) tail length, but this stimulatory effect on repression was lost when translation was repressed by the tethered GW182 silencing domain rather than the miRNA-induced silencing complex (miRISC) itself. These findings are mechanistically explained by a notable function of PABP: it promotes association of miRISC with miRNA-regulated mRNAs. We also found that PABP association with mRNA rapidly diminished with miRISC recruitment and before detectable deadenylation. We integrated these data into a revised model for the function of PABP and the poly(A) tail in miRNA-mediated translational repression. PMID:22635249

  5. Repression and substitutive formation: the relationship between Freud's concepts reconsidered.

    PubMed

    Zepf, Siegfried

    2012-06-01

    This paper examines Freud's concept of repression and the relationship between repression and substitutive formation as it presents itself in Freud's writings. The author shows that Freud gives at least four different meanings to the term "repression": Freud uses it interchangeably with defense, as a consciously intended forgetting, as a specific unconscious mechanism of defense, and to describe the consequence of defense mechanisms leading to substitutive formations. The inconsistencies in this relationship are discussed and clarified, and Freud's economic and linguistic attempts at founding repression are subjected to critique; the need of a primal repression as a necessary condition for repression proper is pointed out. In developing Freud's linguistic foundation of repression further, the author presents defense as a semantic displacement. Ideas are excluded from the realm of the concepts that belong to them historically. These presentations become unconscious, that is, repressed, in that they can no longer be identified as "cases" of these conceptual internal contents. At the same time they are displaced into the extensions of concepts whose internal contents do not belong to them originally. It is by virtue of the internal contents of these concepts that the displaced elements as substitutive formations once again attain consciousness, albeit a false one. The author suggests dismissing repression as a specific defense mechanism of its own; to reversing Freud's thesis that repression, as a rule, creates a substitutive formation into its opposite; and recognizing that the mechanisms used to build substitutes, as a rule, create repression. PMID:22712593

  6. Conservation of uORF repressiveness and sequence features in mouse, human and zebrafish

    PubMed Central

    Chew, Guo-Liang; Pauli, Andrea; Schier, Alexander F.

    2016-01-01

    Upstream open reading frames (uORFs) are ubiquitous repressive genetic elements in vertebrate mRNAs. While much is known about the regulation of individual genes by their uORFs, the range of uORF-mediated translational repression in vertebrate genomes is largely unexplored. Moreover, it is unclear whether the repressive effects of uORFs are conserved across species. To address these questions, we analyse transcript sequences and ribosome profiling data from human, mouse and zebrafish. We find that uORFs are depleted near coding sequences (CDSes) and have initiation contexts that diminish their translation. Linear modelling reveals that sequence features at both uORFs and CDSes modulate the translation of CDSes. Moreover, the ratio of translation over 5′ leaders and CDSes is conserved between human and mouse, and correlates with the number of uORFs. These observations suggest that the prevalence of vertebrate uORFs may be explained by their conserved role in repressing CDS translation. PMID:27216465

  7. Feedback circuitry between miR-218 repression and RTK activation in Glioblastoma

    PubMed Central

    Mathew, Lijoy K; Huangyang, Peiwei; Mucaj, Vera; Lee, Samuel S.; Skuli, Nicolas; Eisinger-Mathason, T.S. Karin; Biju, Kevin; Li, Bo; Venneti, Sriram; Lal, Priti; Lathia, Justin D; Rich, Jeremy N; Keith, Brian; Simon, M Celeste

    2015-01-01

    Receptor tyrosine kinase (RTK) pathway signaling plays a central role in the growth and progression of Glioblastoma (GBM), a highly aggressive group of brain tumors. We recently reported that miR-218 repression, an essentially uniform feature of human GBM, directly promotes RTK hyperactivation by increasing the expression of key positive signaling effectors, including EGFR, PLCγ1, PIK3CA and ARAF (1). However, enhanced RTK signaling is known to activate compensatory inhibitory feedback mechanisms in both normal and cancer cells. We demonstrate here that miR-218 repression in GBM cells also increases the abundance of additional upstream and downstream signaling mediators, including PDGFRα, RSK2, and S6K1, which collectively function to alleviate inhibitory RTK feedback regulation. In turn, RTK signaling suppresses miR-218 expression via STAT3, which binds directly to the miR-218 locus, along with BCLAF1, to repress its expression. These data identify novel interacting feedback loops by which miR-218 repression promotes increased RTK signaling in high-grade gliomas. PMID:25943352

  8. PTEN represses RNA polymerase III-dependent transcription by targeting the TFIIIB complex.

    PubMed

    Woiwode, Annette; Johnson, Sandra A S; Zhong, Shuping; Zhang, Cheng; Roeder, Robert G; Teichmann, Martin; Johnson, Deborah L

    2008-06-01

    PTEN, a tumor suppressor whose function is frequently lost in human cancers, possesses a lipid phosphatase activity that represses phosphatidylinositol 3-kinase (PI3K) signaling, controlling cell growth, proliferation, and survival. The potential for PTEN to regulate the synthesis of RNA polymerase (Pol) III transcription products, including tRNAs and 5S rRNAs, was evaluated. The expression of PTEN in PTEN-deficient cells repressed RNA Pol III transcription, whereas decreased PTEN expression enhanced transcription. Transcription repression by PTEN was uncoupled from PTEN-mediated effects on the cell cycle and was independent of p53. PTEN acts through its lipid phosphatase activity, inhibiting the PI3K/Akt/mTOR/S6K pathway to decrease transcription. PTEN, through the inactivation of mTOR, targets the TFIIIB complex, disrupting the association between TATA-binding protein and Brf1. Kinetic analysis revealed that PTEN initially induces a decrease in the serine phosphorylation of Brf1, leading to a selective reduction in the occupancy of all TFIIIB subunits on tRNA(Leu) genes, whereas prolonged PTEN expression results in the enhanced serine phosphorylation of Bdp1. Together, these results demonstrate a new class of genes regulated by PTEN through its ability to repress the activation of PI3K/Akt/mTOR/S6K signaling. PMID:18391023

  9. Conservation of uORF repressiveness and sequence features in mouse, human and zebrafish.

    PubMed

    Chew, Guo-Liang; Pauli, Andrea; Schier, Alexander F

    2016-01-01

    Upstream open reading frames (uORFs) are ubiquitous repressive genetic elements in vertebrate mRNAs. While much is known about the regulation of individual genes by their uORFs, the range of uORF-mediated translational repression in vertebrate genomes is largely unexplored. Moreover, it is unclear whether the repressive effects of uORFs are conserved across species. To address these questions, we analyse transcript sequences and ribosome profiling data from human, mouse and zebrafish. We find that uORFs are depleted near coding sequences (CDSes) and have initiation contexts that diminish their translation. Linear modelling reveals that sequence features at both uORFs and CDSes modulate the translation of CDSes. Moreover, the ratio of translation over 5' leaders and CDSes is conserved between human and mouse, and correlates with the number of uORFs. These observations suggest that the prevalence of vertebrate uORFs may be explained by their conserved role in repressing CDS translation. PMID:27216465

  10. Ring1b bookmarks genes in pancreatic embryonic progenitors for repression in adult β cells.

    PubMed

    van Arensbergen, Joris; García-Hurtado, Javier; Maestro, Miguel Angel; Correa-Tapia, Miguel; Rutter, Guy A; Vidal, Miguel; Ferrer, Jorge

    2013-01-01

    Polycomb-mediated gene repression is essential for embryonic development, yet its precise role in lineage-specific programming is poorly understood. Here we inactivated Ring1b, encoding a polycomb-repressive complex 1 subunit, in pancreatic multipotent progenitors (Ring1b(progKO)). This caused transcriptional derepression of a subset of direct Ring1b target genes in differentiated pancreatic islet cells. Unexpectedly, Ring1b inactivation in differentiated islet β cells (Ring1b(βKO)) did not cause derepression, even after multiple rounds of cell division, suggesting a role for Ring1b in the establishment but not the maintenance of repression. Consistent with this notion, derepression in Ring1b(progKO) islets occurred preferentially in genes that were targeted de novo by Ring1b during pancreas development. The results support a model in which Ring1b bookmarks its target genes during embryonic development, and these genes are maintained in a repressed state through Ring1b-independent mechanisms in terminally differentiated cells. This work provides novel insights into how epigenetic mechanisms contribute to shaping the transcriptional identity of differentiated lineages. PMID:23271347

  11. Transforming Growth Factor-β1 Signaling Represses Testicular Steroidogenesis through Cross-Talk with Orphan Nuclear Receptor Nur77

    PubMed Central

    Park, Eunsook; Song, Chin-Hee; Park, Jae-Il; Ahn, Ryun-Sup; Choi, Hueng-Sik; Ko, CheMyong; Lee, Keesook

    2014-01-01

    Transforming growth factor- β1 (TGF-β1) has been reported to inhibit luteinizing hormone (LH) mediated-steroidogenesis in testicular Leydig cells. However, the mechanism by which TGF-β1 controls the steroidogenesis in Leydig cells is not well understood. Here, we investigated the possibility that TGF-β1 represses steroidogenesis through cross-talk with the orphan nuclear receptor Nur77. Nur77, which is induced by LH/cAMP signaling, is one of major transcription factors that regulate the expression of steroidogenic genes in Leydig cells. TGF-β1 signaling inhibited cAMP-induced testosterone production and the expression of steroidogenic genes such as P450c17, StAR and 3β-HSD in mouse Leydig cells. Further, TGF-β1/ALK5 signaling repressed cAMP-induced and Nur77-activated promoter activity of steroidogenic genes. In addition, TGF-β1/ALK5-activated Smad3 repressed Nur77 transactivation of steroidogenic gene promoters by interfering with Nur77 binding to DNA. In primary Leydig cells isolated from Tgfbr2flox/flox Cyp17iCre mice, TGF-β1-mediated repression of cAMP-induced steroidogenic gene expression was significantly less than that in primary Leydig cells from Tgfbr2flox/flox mice. Taken together, these results suggest that TGF-β1/ALK5/Smad3 signaling represses the expression of steroidogenic genes via the suppression of Nur77 transactivation in testicular Leydig cells. These findings may provide a molecular mechanism involved in the TGF-β1-mediated repression of testicular steroidogenesis. PMID:25140527

  12. Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically repress IL-6

    PubMed Central

    Gu, Yan; Li, Xia; Zhao, Dezhi; Liu, Yiqi; Wang, Chunmei; Zhang, Xiang; Su, Xiaoping; Liu, Juan; Ge, Wei; Levine, Ross L.; Li, Nan; Cao, Xuetao

    2015-01-01

    Epigenetic modifiers have fundamental roles in defining unique cellular identity through the establishment and maintenance of lineage-specific chromatin and methylation status1. Several DNA modifications such as 5-hydroxymethylcytosine (5hmC) are catalysed by the ten eleven translocation (Tet) methylcytosine dioxygenase family members2, and the roles of Tet proteins in regulating chromatin architecture and gene transcription independently of DNA methylation have been gradually uncovered3. However, the regulation of immunity and inflammation by Tet proteins independent of their role in modulating DNA methylation remains largely unknown. Here we show that Tet2 selectively mediates active repression of interleukin-6 (IL-6) transcription during inflammation resolution in innate myeloid cells, including dendritic cells and macrophages. Loss of Tet2 resulted in the upregulation of several inflammatory mediators, including IL-6, at late phase during the response to lipopolysaccharide challenge. Tet2-deficient mice were more susceptible to endotoxin shock and dextran-sulfate-sodium-induced colitis, displaying a more severe inflammatory phenotype and increased IL-6 production compared to wild-type mice. IκBζ, an IL-6-specific transcription factor, mediated specific targeting of Tet2 to the Il6 promoter, further indicating opposite regulatory roles of IκBζ at initial and resolution phases of inflammation. For the repression mechanism, independent of DNA methylation and hydroxymethylation, Tet2 recruited Hdac2 and repressed transcription of Il6 via histone deacetylation. We provide mechanistic evidence for the gene-specific transcription repression activity of Tet2 via histone deacetylation and for the prevention of constant transcription activation at the chromatin level for resolving inflammation. PMID:26287468

  13. Arsenic at very low concentrations alters glucocorticoid receptor (GR)-mediated gene activation but not GR-mediated gene repression: complex dose-response effects are closely correlated with levels of activated GR and require a functional GR DNA binding domain.

    PubMed

    Bodwell, Jack E; Kingsley, Lauren A; Hamilton, Joshua W

    2004-08-01

    Arsenic (As) contamination of drinking water is considered a principal environmental health threat throughout the world. Chronic intake is associated with an increased risk of cancer, diabetes, and cardiovascular disease, and recent studies suggest increased health risks at levels as low as 5-10 ppb. We report here that 0.05-1 microM (6-120 ppb) As showed stimulatory effects on glucocorticoid receptor (GR)-mediated gene activation in rat EDR3 hepatoma cells of both the endogenous tyrosine aminotransferase (TAT) gene and the reporter genes containing TAT glucocorticoid response elements. At slightly higher concentrations (1-3 microM), the effects of As became inhibitory. Thus, over this narrow concentration range, the effects of As changed from a 2- to 4-fold stimulation to a greater than 2-fold suppression in activity. Interestingly, the inhibitory effect of GR on both AP1- and NF-kappa B-mediated gene activation was not affected by As. The magnitude of GR stimulation and inhibition by As was highly dependent on the cellular level of hormone-activated GR. Mutational deletion studies indicated that the central DNA binding domain (DBD) of GR is the minimal region required for the As effect and does not require free sulfhydryls. Point mutations located within the DBD that have known structural consequences significantly altered the GR response to As. In particular, point mutations in the DBD that confer a DNA-bound GR confirmation abolished the low dose As stimulatory effect but enhanced the inhibitory response, further indicating that the DBD is important for mediating these As effects. PMID:15310238

  14. The Notch intracellular domain represses CRE-dependent transcription.

    PubMed

    Hallaq, Rania; Volpicelli, Floriana; Cuchillo-Ibanez, Inmaculada; Hooper, Claudie; Mizuno, Keiko; Uwanogho, Dafe; Causevic, Mirsada; Asuni, Ayodeji; To, Alvina; Soriano, Salvador; Giese, K Peter; Lovestone, Simon; Killick, Richard

    2015-03-01

    Members of the cyclic-AMP response-element binding protein (CREB) transcription factor family regulate the expression of genes needed for long-term memory formation. Loss of Notch impairs long-term, but not short-term, memory in flies and mammals. We investigated if the Notch-1 (N1) exerts an effect on CREB-dependent gene transcription. We observed that N1 inhibits CREB mediated activation of cyclic-AMP response element (CRE) containing promoters in a γ-secretase-dependent manner. We went on to find that the γ-cleaved N1 intracellular domain (N1ICD) sequesters nuclear CREB1α, inhibits cAMP/PKA-mediated neurite outgrowth and represses the expression of specific CREB regulated genes associated with learning and memory in primary cortical neurons. Similar transcriptional effects were observed with the N2ICD, N3ICD and N4ICDs. Together, these observations indicate that the effects of Notch on learning and memory are, at least in part, via an effect on CREB-regulated gene expression. PMID:25479589

  15. Pro-/Anti-inflammatory Dysregulation in Patients With First Episode of Psychosis: Toward an Integrative Inflammatory Hypothesis of Schizophrenia

    PubMed Central

    García-Bueno, Borja; Bioque, Miquel; Mac-Dowell, Karina S.; Barcones, M. Fe; Leza, Juan C.

    2014-01-01

    Background: Schizophrenia is a chronic syndrome of unknown etiology, predominantly defined by signs of psychosis. The onset of the disorder occurs typically in late adolescence or early adulthood. Efforts to study pathophysiological mechanisms in early stages of the disease are crucial in order to prompt intervention. Methods: Case-control study of first-episode psychotic (FEP) patients and matched controls. We recruited 117 patients during the first year after their FEP according to the DSM-IV criteria and recruited 106 gender-, race-, and age-matched controls between September 2010 and June 2011. Results: Biochemical studies carried out in peripheral mononuclear blood cells (PMBC) and plasma evidence a significant increase in intracellular components of a main proinflammatory pathway, along with a significant decrease in the anti-inflammatory ones. Multivariate logistic regression analyses identified the expression of inducible isoforms of nitric oxide synthase and cyclooxygenase in PMBC and homocysteine plasma levels as the most reliable potential risk factors and the inhibitor of the inflammatory transcription factor NFκB, IκBα, and the anti-inflammatory prostaglandin 15d-PGJ2 as potential protection factors. Discussion: Taken as a whole, the results of this study indicate robust phenotypical differences at the cellular machinery level in PMBC of patients with FEP. Although more scientific evidence is needed, the determination of multiple components of pro- and anti-inflammatory cellular pathways including the activity of nuclear receptors has interesting potential as biological markers and potential risk/protective factors for FEP. Due to its soluble nature, a notable finding in this study is that the anti-inflammatory mediator 15d-PGJ2 might be used as plasmatic biomarker for first episodes of psychosis. PMID:23486748

  16. Krüppel Homolog 1 Inhibits Insect Metamorphosis via Direct Transcriptional Repression of Broad-Complex, a Pupal Specifier Gene.

    PubMed

    Kayukawa, Takumi; Nagamine, Keisuke; Ito, Yuka; Nishita, Yoshinori; Ishikawa, Yukio; Shinoda, Tetsuro

    2016-01-22

    The Broad-Complex gene (BR-C) encodes transcription factors that dictate larval-pupal metamorphosis in insects. The expression of BR-C is induced by molting hormone (20-hydroxyecdysone (20E)), and this induction is repressed by juvenile hormone (JH), which exists during the premature larval stage. Krüppel homolog 1 gene (Kr-h1) has been known as a JH-early inducible gene responsible for repression of metamorphosis; however, the functional relationship between Kr-h1 and repression of BR-C has remained unclear. To elucidate this relationship, we analyzed cis- and trans elements involved in the repression of BR-C using a Bombyx mori cell line. In the cells, as observed in larvae, JH induced the expression of Kr-h1 and concurrently suppressed 20E-induced expression of BR-C. Forced expression of Kr-h1 repressed the 20E-dependent activation of the BR-C promoter in the absence of JH, and Kr-h1 RNAi inhibited the JH-mediated repression, suggesting that Kr-h1 controlled the repression of BR-C. A survey of the upstream sequence of BR-C gene revealed a Kr-h1 binding site (KBS) in the BR-C promoter. When KBS was deleted from the promoter, the repression of BR-C was abolished. Electrophoresis mobility shift demonstrated that two Kr-h1 molecules bound to KBS in the BR-C promoter. Based on these results, we conclude that Kr-h1 protein molecules directly bind to the KBS sequence in the BR-C promoter and thereby repress 20E-dependent activation of the pupal specifier, BR-C. This study has revealed a considerable portion of the picture of JH signaling pathways from the reception of JH to the repression of metamorphosis. PMID:26518872

  17. Repression of the integrated papillomavirus E6/E7 promoter is required for growth suppression of cervical cancer cells.

    PubMed

    Francis, D A; Schmid, S I; Howley, P M

    2000-03-01

    The human papillomavirus (HPV) E2 protein is an important regulator of viral E6 and E7 gene expression. E2 can repress the viral promoter for E6 and E7 expression as well as block progression of the cell cycle in cancer cells harboring the DNA of "high-risk" HPV types. Although the phenomenon of E2-mediated growth arrest of HeLa cells and other HPV-positive cancer cells has been well documented, the specific mechanism by which E2 affects cellular proliferation has not yet been elucidated. Here, we show that bovine papillomavirus (BPV) E2-induced growth arrest of HeLa cells requires the repression of the E6 and E7 promoter. This repression is specific for E2TA and not E2TR, a BPV E2 variant that lacks the N-terminal transactivation domain. We demonstrate that expression of HPV16 E6 and E7 from a heterologous promoter that is not regulated by E2 rescues HeLa cells from E2-mediated growth arrest. Our data indicate that the pathway of E2-mediated growth arrest of HeLa cells requires repression of E6 and E7 expression through an activity specified by the transactivation domain of E2TA. PMID:10684283

  18. Tristetraprolin Recruits Eukaryotic Initiation Factor 4E2 To Repress Translation of AU-Rich Element-Containing mRNAs

    PubMed Central

    Tao, Xianzun

    2015-01-01

    Tristetraprolin (TTP) regulates the expression of AU-rich element-containing mRNAs through promoting the degradation and repressing the translation of target mRNA. While the mechanism for promoting target mRNA degradation has been extensively studied, the mechanism underlying translational repression is not well established. Here, we show that TTP recruits eukaryotic initiation factor 4E2 (eIF4E2) to repress target mRNA translation. TTP interacted with eIF4E2 but not with eIF4E. Overexpression of eIF4E2 enhanced TTP-mediated translational repression, and downregulation of endogenous eIF4E2 or overexpression of a truncation mutant of eIF4E2 impaired TTP-mediated translational repression. Overexpression of an eIF4E2 mutant that lost the cap-binding activity also impaired TTP's activity, suggesting that the cap-binding activity of eIF4E2 is important in TTP-mediated translational repression. We further show that TTP promoted eIF4E2 binding to target mRNA. These results imply that TTP recruits eIF4E2 to compete with eIF4E to repress the translation of target mRNA. This notion is supported by the finding that downregulation of endogenous eIF4E2 increased the production of tumor necrosis factor alpha (TNF-α) protein without affecting the mRNA levels in THP-1 cells. Collectively, these results uncover a novel mechanism by which TTP represses target mRNA translation. PMID:26370510

  19. Repression of Igf1 expression by Ezh2 prevents basal cell differentiation in the developing lung.

    PubMed

    Galvis, Laura A; Holik, Aliaksei Z; Short, Kieran M; Pasquet, Julie; Lun, Aaron T L; Blewitt, Marnie E; Smyth, Ian M; Ritchie, Matthew E; Asselin-Labat, Marie-Liesse

    2015-04-15

    Epigenetic mechanisms involved in the establishment of lung epithelial cell lineage identities during development are largely unknown. Here, we explored the role of the histone methyltransferase Ezh2 during lung lineage determination. Loss of Ezh2 in the lung epithelium leads to defective lung formation and perinatal mortality. We show that Ezh2 is crucial for airway lineage specification and alveolarization. Using optical projection tomography imaging, we found that branching morphogenesis is affected in Ezh2 conditional knockout mice and the remaining bronchioles are abnormal, lacking terminally differentiated secretory club cells. Remarkably, RNA-seq analysis revealed the upregulation of basal genes in Ezh2-deficient epithelium. Three-dimensional imaging for keratin 5 further showed the unexpected presence of a layer of basal cells from the proximal airways to the distal bronchioles in E16.5 embryos. ChIP-seq analysis indicated the presence of Ezh2-mediated repressive marks on the genomic loci of some but not all basal genes, suggesting an indirect mechanism of action of Ezh2. We found that loss of Ezh2 de-represses insulin-like growth factor 1 (Igf1) expression and that modulation of IGF1 signaling ex vivo in wild-type lungs could induce basal cell differentiation. Altogether, our work reveals an unexpected role for Ezh2 in controlling basal cell fate determination in the embryonic lung endoderm, mediated in part by repression of Igf1 expression. PMID:25790853

  20. BZLF1 governs CpG-methylated chromatin of Epstein-Barr Virus reversing epigenetic repression.

    PubMed

    Woellmer, Anne; Arteaga-Salas, Jose M; Hammerschmidt, Wolfgang

    2012-09-01

    Epigenetic mechanisms are essential for the regulation of all genes in mammalian cells but transcriptional repression including DNA methylation are also major epigenetic mechanisms of defense inactivating potentially harmful pathogens. Epstein-Barr Virus (EBV), however, has evolved to take advantage of CpG methylated DNA to regulate its own biphasic life cycle. We show here that latent EBV DNA has an extreme composition of methylated CpG dinucleotides with a bimodal distribution of unmethylated or fully methylated DNA at active latent genes or completely repressed lytic promoters, respectively. We find this scenario confirmed in primary EBV-infected memory B cells in vivo. Extensive CpG methylation of EBV's DNA argues for a very restricted gene expression during latency. Above-average nucleosomal occupancy, repressive histone marks, and Polycomb-mediated epigenetic silencing further shield early lytic promoters from activation during latency. The very tight repression of viral lytic genes must be overcome when latent EBV enters its lytic phase and supports de novo virus synthesis in infected cells. The EBV-encoded and AP-1 related transcription factor BZLF1 overturns latency and initiates virus synthesis in latently infected cells. Paradoxically, BZLF1 preferentially binds to CpG-methylated motifs in key viral promoters for their activation. Upon BZLF1 binding, we find nucleosomes removed, Polycomb repression lost, and RNA polymerase II recruited to the activated early promoters promoting efficient lytic viral gene expression. Surprisingly, DNA methylation is maintained throughout this phase of viral reactivation and is no hindrance to active transcription of extensively CpG methylated viral genes as thought previously. Thus, we identify BZLF1 as a pioneer factor that reverses epigenetic silencing of viral DNA to allow escape from latency and report on a new paradigm of gene regulation. PMID:22969425

  1. Suppression of BRD4 inhibits human hepatocellular carcinoma by repressing MYC and enhancing BIM expression

    PubMed Central

    Zhang, Yi; Seng, Jing-Jing; Zhang, Hua-Peng; Ma, Xiu-Xian; Zhang, Gong; Li, Jie; Yan, Bing; Tang, Hong-Wei; Li, Shan-Shan; Wang, Li-Dong; Zhang, Shui-Jun

    2016-01-01

    Bromodomain 4 (BRD4) is an epigenetic regulator that, when inhibited, has anti-cancer effects. In this study, we investigated whether BRD4 could be a target for treatment of human hepatocellular carcinoma (HCC). We show that BRD4 is over-expressed in HCC tissues. Suppression of BRD4, either by siRNA or using JQ1, a pharmaceutical BRD4 inhibitor, reduced cell growth and induced apoptosis in HCC cell lines while also slowing HCC xenograft tumor growth in mice. JQ1 treatment induced G1 cell cycle arrest by repressing MYC expression, which led to the up-regulation of CDKN1B (P27). JQ1 also de-repressed expression of the pro-apoptotic BCL2L11 (BIM). Moreover, siRNA knockdown of BIM attenuated JQ1-triggered apoptosis in HCC cells, suggesting an essential role for BIM in mediating JQ1 anti-HCC activity. PMID:26575167

  2. Role of the proto-oncogene Pokemon in cellular transformation and ARF repression.

    PubMed

    Maeda, Takahiro; Hobbs, Robin M; Merghoub, Taha; Guernah, Ilhem; Zelent, Arthur; Cordon-Cardo, Carlos; Teruya-Feldstein, Julie; Pandolfi, Pier Paolo

    2005-01-20

    Aberrant transcriptional repression through chromatin remodelling and histone deacetylation has been postulated to represent a driving force underlying tumorigenesis because histone deacetylase inhibitors have been found to be effective in cancer treatment. However, the molecular mechanisms by which transcriptional derepression would be linked to tumour suppression are poorly understood. Here we identify the transcriptional repressor Pokemon (encoded by the Zbtb7 gene) as a critical factor in oncogenesis. Mouse embryonic fibroblasts lacking Zbtb7 are completely refractory to oncogene-mediated cellular transformation. Conversely, Pokemon overexpression leads to overt oncogenic transformation both in vitro and in vivo in transgenic mice. Pokemon can specifically repress the transcription of the tumour suppressor gene ARF through direct binding. We find that Pokemon is aberrantly overexpressed in human cancers and that its expression levels predict biological behaviour and clinical outcome. Pokemon's critical role in cellular transformation makes it an attractive target for therapeutic intervention. PMID:15662416

  3. Catabolite repression of the Bacillus subtilis hut operon requires a cis-acting site located downstream of the transcription initiation site.

    PubMed Central

    Wray, L V; Pettengill, F K; Fisher, S H

    1994-01-01

    Expression of the Bacillus subtilis hut operon is subject to regulation by catabolite repression. A set of hut-lacZ transcriptional fusions was constructed and used to identify two cis-acting sites involved in catabolite repression. The hutOCR1 operator site lies immediately downstream of the hut promoter and weakly regulates hut expression in response to catabolite repression. The downstream hutOCR2 operator site lies within the hutP gene, between positions +203 and +216, and is required for wild-type levels of catabolite repression. Both the hutOCR1 and hutOCR2 operators have sequence similarity to the sites which mediate catabolite repression of several other B. subtilis genes. Two mutations which relieve catabolite repression of hut expression were found to alter the nucleotide sequence of the hutOCR2 operator. Catabolite repression of hut expression was partially relieved in strains containing the ccpA mutation but not in strains containing either the pai or hpr mutation. Images PMID:8144455

  4. Repression in vitro, by human adenovirus E1A protein domains, of basal or Tat-activated transcription of the human immunodeficiency virus type 1 long terminal repeat.

    PubMed Central

    Song, C Z; Loewenstein, P M; Green, M

    1995-01-01

    Human adenovirus E1A proteins can repress the expression of several viral and cellular genes. By using a cell-free transcription system, we demonstrated that the gene product of the E1A 12S mRNA, the 243-residue protein E1A243R, inhibits basal transcription from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). The HIV-1 transactivator protein Tat greatly stimulates transcription from the viral promoter in vitro. However, E1A243R can repress Tat-activated transcription in vitro. Strong repression of both basal and Tat-activated transcriptions requires only E1A N-terminal amino acid residues 1 to 80. Deletion analysis showed that E1A N-terminal amino acids 4 to 25 are essential for repression, whereas amino acid residues 30 to 49 and 70 to 80 are dispensable. Transcriptional repression by E1A in the cell-free transcription system is promoter specific, since under identical conditions, transcription of the adenovirus major late promoter and the Rous sarcoma virus LTR promoter was unaffected. The repression of transcription by small E1A peptides in vitro provides an assay for investigation of molecular mechanisms governing E1A-mediated repression of both basal and Tat-activated transcriptions of the HIV-1 LTR promoter. PMID:7707515

  5. Repression of a matrix metalloprotease gene by E1A correlates with its ability to bind to cell type-specific transcription factor AP-2.

    PubMed Central

    Somasundaram, K; Jayaraman, G; Williams, T; Moran, E; Frisch, S; Thimmapaya, B

    1996-01-01

    Adenovirus E1A 243-amino acid protein can repress a variety of enhancer -linked viral and cellular promoters. This repression is presumed to be mediated by its interaction with and sequestration of p3OO, a transcriptional coactivator. Type IV 72-kDa collagenase is one of the matrix metalloproteases that has been implicated in differentiation, development, angiogenesis, and tumor metastasis. We show here that the cell type-specific transcription factor AP-2 is an important transcription factor for the activation of the type IV 72-kDa collagenase promoter and that adenovirus E1A 243-amino acid protein represses this promoter by targeting AP-2. Glutathione S-transferase-affinity chromatography studies show that the E1A protein interacts with the DNA binding/dimerization region of AP-2 and that the N-terminal amino acids of E1A protein are required for this interaction. Further, E1A deletion mutants which do not bind to p3OO can repress this collagenase promoter as efficiently as the wildtype E1A protein. Because the AP-2 element is present in a variety of viral and cellular enhancers which are repressed by E1A, these studies suggest that E1A protein can repress cellular and viral promoter/enhancers by forming a complex with cellular transcription factors and that this repression mechanism may be independent of its interaction with p3OO. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8610173

  6. From Sensorimotor Inhibition to Freudian Repression: Insights from Psychosis Applied to Neurosis

    PubMed Central

    Bazan, Ariane

    2012-01-01

    First, three case studies are presented of psychotic patients having in common an inability to hold something down or out. In line with other theories on psychosis, we propose that a key change is at the efference copy system. Going back to Freud’s mental apparatus, we propose that the messages of discharge of the motor neurons, mobilized to direct perception, also called “indications of reality,” are equivalent to the modern efference copies. With this key, the reading of the cases is coherent with the psychodynamic understanding of psychosis, being a downplay of secondary processes, and consequently, a dominance of primary processes. Moreover, putting together the sensorimotor idea of a failure of efference copy-mediated inhibition with the psychoanalytic idea of a failing repression in psychosis, the hypothesis emerges that the attenuation enabled by the efference copy dynamics is, in some instances, the physiological instantiation of repression. Second, we applied this idea to the mental organization in neurosis. Indeed, the efference copy-mediated attenuation is thought to be the mechanism through which sustained activation of an intention, without reaching it – i.e., inhibition of an action – gives rise to mental imagery. Therefore, as inhibition is needed for any targeted action or for normal language understanding, acting in the world, or processing language, structurally induces mental imagery, constituting a subjective unconscious mental reality. Repression is a special instance of inhibition for emotionally threatening stimuli. These stimuli require stronger inhibition, leaving (the attenuation of) the motor intentions totally unanswered, in order to radically prevent execution which would lead to development of excess affect. This inhibition, then, yields a specific type of motor imagery, called “phantoms,” which induce mental preoccupation, as well as symptoms which, especially through their form, refer to the repressed motor fragments

  7. From sensorimotor inhibition to freudian repression: insights from psychosis applied to neurosis.

    PubMed

    Bazan, Ariane

    2012-01-01

    First, three case studies are presented of psychotic patients having in common an inability to hold something down or out. In line with other theories on psychosis, we propose that a key change is at the efference copy system. Going back to Freud's mental apparatus, we propose that the messages of discharge of the motor neurons, mobilized to direct perception, also called "indications of reality," are equivalent to the modern efference copies. With this key, the reading of the cases is coherent with the psychodynamic understanding of psychosis, being a downplay of secondary processes, and consequently, a dominance of primary processes. Moreover, putting together the sensorimotor idea of a failure of efference copy-mediated inhibition with the psychoanalytic idea of a failing repression in psychosis, the hypothesis emerges that the attenuation enabled by the efference copy dynamics is, in some instances, the physiological instantiation of repression. Second, we applied this idea to the mental organization in neurosis. Indeed, the efference copy-mediated attenuation is thought to be the mechanism through which sustained activation of an intention, without reaching it - i.e., inhibition of an action - gives rise to mental imagery. Therefore, as inhibition is needed for any targeted action or for normal language understanding, acting in the world, or processing language, structurally induces mental imagery, constituting a subjective unconscious mental reality. Repression is a special instance of inhibition for emotionally threatening stimuli. These stimuli require stronger inhibition, leaving (the attenuation of) the motor intentions totally unanswered, in order to radically prevent execution which would lead to development of excess affect. This inhibition, then, yields a specific type of motor imagery, called "phantoms," which induce mental preoccupation, as well as symptoms which, especially through their form, refer to the repressed motor fragments. PMID:23162501

  8. Repression of somatic cell fate in the germline.

    PubMed

    Robert, Valérie J; Garvis, Steve; Palladino, Francesca

    2015-10-01

    Germ cells must transmit genetic information across generations, and produce gametes while also maintaining the potential to form all cell types after fertilization. Preventing the activation of somatic programs is, therefore, crucial to the maintenance of germ cell identity. Studies in Caenorhabditis elegans, Drosophila melanogaster, and mouse have revealed both similarities and differences in how somatic gene expression is repressed in germ cells, thereby preventing their conversion into somatic tissues. This review will focus on recent developments in our understanding of how global or gene-specific transcriptional repression, chromatin regulation, and translational repression operate in the germline to maintain germ cell identity and repress somatic differentiation programs. PMID:26043973

  9. Relationship between creativity, repression, and anxiety in first graders.

    PubMed

    Strauss, H; Hadar, M; Shavit, H; Itskowitz, R

    1981-08-01

    The present study dealt with the extent to which creativity may be identified in 71 first graders and raised the question of whether and how creativity is related to anxiety and repression at this young age. Furthermore, correlation of 0.62 was obtained between creativity and decrease in repression. The various subtests and the four dimensions of creativity were separately analyzed in relation to anxiety and repression, and the results were discussed. No relation was found between intelligence and the dynamic variables of anxiety and repression. PMID:7290875

  10. Variable requirements for DNA-binding proteins at polycomb-dependent repressive regions in human HOX clusters.

    PubMed

    Woo, Caroline J; Kharchenko, Peter V; Daheron, Laurence; Park, Peter J; Kingston, Robert E

    2013-08-01

    Polycomb group (PcG)-mediated repression is an evolutionarily conserved process critical for cell fate determination and maintenance of gene expression during embryonic development. However, the mechanisms underlying PcG recruitment in mammals remain unclear since few regulatory sites have been identified. We report two novel prospective PcG-dependent regulatory elements within the human HOXB and HOXC clusters and compare their repressive activities to a previously identified element in the HOXD cluster. These regions recruited the PcG proteins BMI1 and SUZ12 to a reporter construct in mesenchymal stem cells and conferred repression that was dependent upon PcG expression. Furthermore, we examined the potential of two DNA-binding proteins, JARID2 and YY1, to regulate PcG activity at these three elements. JARID2 has differential requirements, whereas YY1 appears to be required for repressive activity at all 3 sites. We conclude that distinct elements of the mammalian HOX clusters can recruit components of the PcG complexes and confer repression, similar to what has been seen in Drosophila. These elements, however, have diverse requirements for binding factors, which, combined with previous data on other loci, speaks to the complexity of PcG targeting in mammals. PMID:23775117

  11. Dexamethasone Induces Cardiomyocyte Terminal Differentiation via Epigenetic Repression of Cyclin D2 Gene.

    PubMed

    Gay, Maresha S; Dasgupta, Chiranjib; Li, Yong; Kanna, Angela; Zhang, Lubo

    2016-08-01

    Dexamethasone treatment of newborn rats inhibited cardiomyocyte proliferation and stimulated premature terminal differentiation of cardiomyocytes in the developing heart. Yet mechanisms remain undetermined. The present study tested the hypothesis that the direct effect of glucocorticoid receptor-mediated epigenetic repression of cyclin D2 gene in the cardiomyocyte plays a key role in the dexamethasone-mediated effects in the developing heart. Cardiomyocytes were isolated from 2-day-old rats. Cells were stained with a cardiomyocyte marker α-actinin and a proliferation marker Ki67. Cyclin D2 expression was evaluated by Western blot and quantitative real-time polymerase chain reaction. Promoter methylation of CcnD2 was determined by methylated DNA immunoprecipitation (MeDIP). Overexpression of Cyclin D2 was conducted by transfection of FlexiCcnD2 (+CcnD2) construct. Treatment of cardiomyocytes isolated from newborn rats with dexamethasone for 48 hours significantly inhibited cardiomyocyte proliferation with increased binucleation and decreased cyclin D2 protein abundance. These effects were blocked with Ru486 (mifepristone). In addition, the dexamethasone treatment significantly increased cyclin D2 gene promoter methylation in newborn rat cardiomyocytes. 5-Aza-2'-deoxycytidine inhibited dexamethasone-mediated promoter methylation, recovered dexamethasone-induced cyclin D2 gene repression, and blocked the dexamethasone-elicited effects on cardiomyocyte proliferation and binucleation. In addition, the overexpression of cyclin D2 restored the dexamethasone-mediated inhibition of proliferation and increase in binucleation in newborn rat cardiomyocytes. The results demonstrate that dexamethasone acting on glucocorticoid receptors has a direct effect and inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via epigenetic repression of cyclin D2 gene. PMID:27302109

  12. Laser Isotope Separation Employing Condensation Repression

    SciTech Connect

    Eerkens, Jeff W.; Miller, William H.

    2004-09-15

    Molecular laser isotope separation (MLIS) techniques using condensation repression (CR) harvesting are reviewed and compared with atomic vapor laser isotope separation (AVLIS), gaseous diffusion (DIF), ultracentrifuges (UCF), and electromagnetic separations (EMS). Two different CR-MLIS or CRISLA (Condensation Repression Isotope Separation by Laser Activation) approaches have been under investigation at the University of Missouri (MU), one involving supersonic super-cooled free jets and dimer formation, and the other subsonic cold-wall condensation. Both employ mixtures of an isotopomer (e.g. {sup i}QF{sub 6}) and a carrier gas, operated at low temperatures and pressures. Present theories of VT relaxation, dimerization, and condensation are found to be unsatisfactory to explain/predict experimental CRISLA results. They were replaced by fundamentally new models that allow ab-initio calculation of isotope enrichments and predictions of condensation parameters for laser-excited and non-excited vapors which are in good agreement with experiment. Because of supersonic speeds, throughputs for free-jet CRISLA are a thousand times higher than cold-wall CRISLA schemes, and thus preferred for large-quantity Uranium enrichments. For small-quantity separations of (radioactive) medical isotopes, the simpler coldwall CRISLA method may be adequate.

  13. MCRS2 represses the transactivation activities of Nrf1

    PubMed Central

    Wu, Jia-Long; Lin, Young-Sun; Yang, Chi-Chiang; Lin, Yu-Jen; Wu, Shan-Fu; Lin, Ying-Ting; Huang, Chien-Fu

    2009-01-01

    in the redistribution of Nrf1. This suggested the existence of Nrf1-MCRS2 complex in vivo. To further confirm the biological function, a reporter driven by CNC-bZIP protein binding sites was also shown to be repressed by MCRS2 in a transient transfection assay. An artificial reporter gene activated by LexA-Nrf1 was also specifically repressed by MCRS2. Conclusion From the results, we showed MCRS2, a new Nrf1-interacting protein, has a repression effect on Nrf1-mediated transcriptional activation. This was the first ever identified repressor protein related to Nrf1 transactivation. PMID:19187526

  14. The osmoregulatory pathway represses mating pathway activity in Saccharomyces cerevisiae: isolation of a FUS3 mutant that is insensitive to the repression mechanism.

    PubMed Central

    Hall, J P; Cherkasova, V; Elion, E; Gustin, M C; Winter, E

    1996-01-01

    Mitogen-activated protein (MAP) kinase cascades are conserved signal transduction pathways that are required for eukaryotic cells to respond to a variety of stimuli. Multiple MAP kinase pathways can function within a single cell type; therefore, mechanisms that insulate one MAP kinase pathway from adventitious activations by parallel pathways may exist. We have studied interactions between the mating pheromone response and the osmoregulatory (high-osmolarity glycerol response [HOG]) pathways in Saccharomyces cerevisiae which utilize the MAP kinases Fus3p and Hog1p, respectively. Inactivating mutations in HOG pathway kinases cause an increase in the phosphotyrosine content of Fus3p, greater expression of pheromone-responsive genes, and increased sensitivity to growth arrest by pheromone. Therefore, the HOG pathway represses mating pathway activity. In a HOG1+ strain, Fus3p phosphotyrosine increases modestly and transiently following an increase in the extracellular osmolarity; however, it increases to a greater extent and for a sustained duration in a hog1-delta strain. Thus, the HOG-mediated repression of mating pathway activity may insulate the mating pathway from activation by osmotic stress. A FUS3 allele whose gene product is resistant to the HOG-mediated repression of its phosphotyrosine content has been isolated. This mutant encodes an amino acid substitution in the highly conserved DPXDEP motif in subdomain XI. Other investigators have shown that the corresponding amino acid is also mutated in a gain-of-function allele of the MAP kinase encoded by the rolled locus in Drosophila melanogaster. These data suggest that the DPXDEP motif plays a role in the negative regulation of MAP kinases. PMID:8943326

  15. SAGA Complex Components and Acetate Repression in Aspergillus nidulans

    PubMed Central

    Georgakopoulos, Paraskevi; Lockington, Robin A.; Kelly, Joan M.

    2012-01-01

    Alongside the well-established carbon catabolite repression by glucose and other sugars, acetate causes repression in Aspergillus nidulans. Mutations in creA, encoding the transcriptional repressor involved in glucose repression, also affect acetate repression, but mutations in creB or creC, encoding components of a deubiquitination system, do not. To understand the effects of acetate, we used a mutational screen that was similar to screens that uncovered mutations in creA, creB, and creC, except that glucose was replaced by acetate to identify mutations that were affected for repression by acetate but not by glucose. We uncovered mutations in acdX, homologous to the yeast SAGA component gene SPT8, which in growth tests showed derepression for acetate repression but not for glucose repression. We also made mutations in sptC, homologous to the yeast SAGA component gene SPT3, which showed a similar phenotype. We found that acetate repression is complex, and analysis of facA mutations (lacking acetyl CoA synthetase) indicates that acetate metabolism is required for repression of some systems (proline metabolism) but not for others (acetamide metabolism). Although plate tests indicated that acdX- and sptC-null mutations led to derepressed alcohol dehydrogenase activity, reverse-transcription quantitative real-time polymerase chain reaction showed no derepression of alcA or aldA but rather elevated induced levels. Our results indicate that acetate repression is due to repression via CreA together with metabolic changes rather than due to an independent regulatory control mechanism. PMID:23173087

  16. SAGA complex components and acetate repression in Aspergillus nidulans.

    PubMed

    Georgakopoulos, Paraskevi; Lockington, Robin A; Kelly, Joan M

    2012-11-01

    Alongside the well-established carbon catabolite repression by glucose and other sugars, acetate causes repression in Aspergillus nidulans. Mutations in creA, encoding the transcriptional repressor involved in glucose repression, also affect acetate repression, but mutations in creB or creC, encoding components of a deubiquitination system, do not. To understand the effects of acetate, we used a mutational screen that was similar to screens that uncovered mutations in creA, creB, and creC, except that glucose was replaced by acetate to identify mutations that were affected for repression by acetate but not by glucose. We uncovered mutations in acdX, homologous to the yeast SAGA component gene SPT8, which in growth tests showed derepression for acetate repression but not for glucose repression. We also made mutations in sptC, homologous to the yeast SAGA component gene SPT3, which showed a similar phenotype. We found that acetate repression is complex, and analysis of facA mutations (lacking acetyl CoA synthetase) indicates that acetate metabolism is required for repression of some systems (proline metabolism) but not for others (acetamide metabolism). Although plate tests indicated that acdX- and sptC-null mutations led to derepressed alcohol dehydrogenase activity, reverse-transcription quantitative real-time polymerase chain reaction showed no derepression of alcA or aldA but rather elevated induced levels. Our results indicate that acetate repression is due to repression via CreA together with metabolic changes rather than due to an independent regulatory control mechanism. PMID:23173087

  17. Catabolite repression in Escherichia coli. A study of two hypotheses

    PubMed Central

    Moses, V.; Yudkin, M. D.

    1968-01-01

    1. Two hypotheses to account for general catabolite repression of the lactose enzymes in Escherichia coli were tested: the dilution model of Palmer & Moses (1967), and the specific catabolite repressor model of Loomis & Magasanik (1965, 1967). 2. The dilution model predicts that in mutants lacking the i–o regulation system the differential rate of β-galactosidase synthesis should increase when amino acid-synthesizing enzymes are repressed by the presence of amino acids in the medium. It also predicts that with such mutants the total absence of Pi from the medium should not result in the complete cessation of β-galactosidase synthesis that is observed with wild-type cells. 3. Neither prediction was confirmed experimentally, and it is concluded that this model cannot explain catabolite repression. 4. The specific repressor hypothesis depends on the properties of a strain of E. coli carrying the CR− mutation. It requires both that cells of this genotype should be totally resistant to general catabolite repression and that this resistance should be specific for the lactose enzymes. 5. In fact the synthesis of β-galactosidase by CR− cells, though showing resistance to catabolite repression by growth on glucose, was found to be repressed in several other circumstances. 6. Two other inducible enzymes, l-tryptophanase and d-serine deaminase, also showed resistance to repression by glucose in CR− cells. 7. It is concluded that this model, too, does not account for general catabolite repression. 8. Strains carrying deletions at either end of the lactose operon that extend into the structural genes of the operon continue to exhibit catabolite repression. 9. These experiments appear to eliminate the possibility that catabolite repression operates at the level of DNA transcription, and suggest that repression affects instead the translation of messenger RNA into protein. PMID:4881142

  18. Identification of actin as a 15-deoxy-Delta12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement.

    PubMed

    Aldini, Giancarlo; Carini, Marina; Vistoli, Giulio; Shibata, Takahiro; Kusano, Yuri; Gamberoni, Luca; Dalle-Donne, Isabella; Milzani, Aldo; Uchida, Koji

    2007-03-13

    A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization. PMID:17297918

  19. Active and Repressive Chromatin-Associated Proteome after MPA Treatment and the Role of Midkine in Epithelial Monolayer Permeability

    PubMed Central

    Khan, Niamat; Lenz, Christof; Binder, Lutz; Pantakani, Dasaradha Venkata Krishna; Asif, Abdul R.

    2016-01-01

    Mycophenolic acid (MPA) is prescribed to maintain allografts in organ-transplanted patients. However, gastrointestinal (GI) complications, particularly diarrhea, are frequently observed as a side effect following MPA therapy. We recently reported that MPA altered the tight junction (TJ)-mediated barrier function in a Caco-2 cell monolayer model system. This study investigates whether MPA induces epigenetic changes which lead to GI complications, especially diarrhea. Methods: We employed a Chromatin Immunoprecipitation-O-Proteomics (ChIP-O-Proteomics) approach to identify proteins associated with active (H3K4me3) as well as repressive (H3K27me3) chromatin histone modifications in MPA-treated cells, and further characterized the role of midkine, a H3K4me3-associated protein, in the context of epithelial monolayer permeability. Results: We identified a total of 333 and 306 proteins associated with active and repressive histone modification marks, respectively. Among them, 241 proteins were common both in active and repressive chromatin, 92 proteins were associated exclusively with the active histone modification mark, while 65 proteins remained specific to repressive chromatin. Our results show that 45 proteins which bind to the active and seven proteins which bind to the repressive chromatin region exhibited significantly altered abundance in MPA-treated cells as compared to DMSO control cells. A number of novel proteins whose function is not known in bowel barrier regulation were among the identified proteins, including midkine. Our functional integrity assays on the Caco-2 cell monolayer showed that the inhibition of midkine expression prior to MPA treatment could completely block the MPA-mediated increase in barrier permeability. Conclusions: The ChIP-O-Proteomics approach delivered a number of novel proteins with potential implications in MPA toxicity. Consequently, it can be proposed that midkine inhibition could be a potent therapeutic approach to prevent the

  20. Roles for the mitogen-activated protein kinase (MAPK) phosphatase, DUSP1, in feedback control of inflammatory gene expression and repression by dexamethasone.

    PubMed

    Shah, Suharsh; King, Elizabeth M; Chandrasekhar, Ambika; Newton, Robert

    2014-05-01

    Glucocorticoids act on the glucocorticoid receptor (NR3C1) to repress inflammatory gene expression. This is central to their anti-inflammatory effectiveness and rational improvements in therapeutic index depend on understanding the mechanism. Human pulmonary epithelial A549 cells were used to study the role of the mitogen-activated protein kinase (MAPK) phosphatase, dual-specificity phosphatase 1 (DUSP1), in the dexamethasone repression of 11 inflammatory genes induced, in a MAPK-dependent manner, by interleukin-1β (IL1B). Adenoviral over-expression of DUSP1 inactivated MAPK pathways and reduced expression of all 11 inflammatory genes. IL1B rapidly induced DUSP1 expression and RNA silencing revealed a transient role in feedback inhibition of MAPKs and inflammatory gene expression. With dexamethasone, which induced DUSP1 expression, plus IL1B (co-treatment), DUSP1 expression was further enhanced. At 1 h, this was responsible for the dexamethasone inhibition of IL1B-induced MAPK activation and CXCL1 and CXCL2 mRNA expression, with a similar trend for CSF2. Whereas, CCL20 mRNA was not repressed by dexamethasone at 1 h, repression of CCL2, CXCL3, IL6, and IL8 was unaffected, and PTGS2 repression was partially affected by DUSP1 knockdown. At later times, dexamethasone repression of MAPKs was unaffected by DUSP1 silencing. Likewise, 6 h post-IL1B, dexamethasone repression of all 11 mRNAs was essentially unaffected by DUSP1 knockdown. Qualitatively similar data were obtained for CSF2, CXCL1, IL6, and IL8 release. Thus, despite general roles in feedback inhibition, DUSP1 plays a transient, often partial, role in the dexamethasone-dependent repression of certain inflammatory genes. Therefore this also illustrates key roles for DUSP1-independent effectors in mediating glucocorticoid-dependent repression. PMID:24692548

  1. Repression of the Antioxidant NRF2 Pathway in Premature Aging.

    PubMed

    Kubben, Nard; Zhang, Weiqi; Wang, Lixia; Voss, Ty C; Yang, Jiping; Qu, Jing; Liu, Guang-Hui; Misteli, Tom

    2016-06-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare, invariably fatal premature aging disorder. The disease is caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A, leading, through unknown mechanisms, to diverse morphological, epigenetic, and genomic damage and to mesenchymal stem cell (MSC) attrition in vivo. Using a high-throughput siRNA screen, we identify the NRF2 antioxidant pathway as a driver mechanism in HGPS. Progerin sequesters NRF2 and thereby causes its subnuclear mislocalization, resulting in impaired NRF2 transcriptional activity and consequently increased chronic oxidative stress. Suppressed NRF2 activity or increased oxidative stress is sufficient to recapitulate HGPS aging defects, whereas reactivation of NRF2 activity in HGPS patient cells reverses progerin-associated nuclear aging defects and restores in vivo viability of MSCs in an animal model. These findings identify repression of the NRF2-mediated antioxidative response as a key contributor to the premature aging phenotype. PMID:27259148

  2. The kinase activity of PKR represses inflammasome activity.

    PubMed

    Yim, Howard C H; Wang, Die; Yu, Liang; White, Christine L; Faber, Pieter W; Williams, Bryan R G; Sadler, Anthony J

    2016-03-01

    The protein kinase R (PKR) functions in the antiviral response by controlling protein translation and inflammatory cell signaling pathways. We generated a transgenic, knock-in mouse in which the endogenous PKR is expressed with a point mutation that ablates its kinase activity. This novel animal allows us to probe the kinase-dependent and -independent functions of PKR. We used this animal together with a previously generated transgenic mouse that is ablated for PKR expression to determine the role of PKR in regulating the activity of the cryopyrin inflammasome. Our data demonstrate that, in contradiction to earlier reports, PKR represses cryopyrin inflammasome activity. We demonstrate that this control is mediated through the established function of PKR to inhibit protein translation of constituents of the inflammasome to prevent initial priming during innate immune signaling. These findings identify an important role for PKR to dampen inflammation during the innate immune response and caution against the previously proposed therapeutic strategy to inhibit PKR to treat inflammation. PMID:26794869

  3. Hypnotizability as a Function of Repression, Adaptive Regression, and Mood

    ERIC Educational Resources Information Center

    Silver, Maurice Joseph

    1974-01-01

    Forty male undergraduates were assessed in a personality assessment session and a hypnosis session. The personality traits studied were repressive style and adaptive regression, while the transitory variable was mood prior to hypnosis. Hypnotizability was a significant interactive function of repressive style and mood, but not of adaptive…

  4. Plant callus: mechanisms of induction and repression.

    PubMed

    Ikeuchi, Momoko; Sugimoto, Keiko; Iwase, Akira

    2013-09-01

    Plants develop unorganized cell masses like callus and tumors in response to various biotic and abiotic stimuli. Since the historical discovery that the combination of two growth-promoting hormones, auxin and cytokinin, induces callus from plant explants in vitro, this experimental system has been used extensively in both basic research and horticultural applications. The molecular basis of callus formation has long been obscure, but we are finally beginning to understand how unscheduled cell proliferation is suppressed during normal plant development and how genetic and environmental cues override these repressions to induce callus formation. In this review, we will first provide a brief overview of callus development in nature and in vitro and then describe our current knowledge of genetic and epigenetic mechanisms underlying callus formation. PMID:24076977

  5. Multiple Poliovirus Proteins Repress Cytoplasmic RNA Granules

    PubMed Central

    Dougherty, Jonathan D.; Tsai, Wei-Chih; Lloyd, Richard E.

    2015-01-01

    We have previously shown that poliovirus (PV) infection induces stress granule (SG) formation early in infection and then inhibits the formation of SG and disperses processing bodies (PBs) by the mid-phase of infection. Loss of SG was linked to cleavage of G3BP1 by viral 3C proteinase (3Cpro), however dispersal of PBs was not strongly linked to cleavage of specific factors by viral proteinases, suggesting other viral proteins may play roles in inhibition of SG or PB formation. Here we have screened all viral proteins for roles in inducing or inhibiting the formation of RNA granules by creating fusions with mCherry and expressing them individually in cells. Expression of viral proteins separately revealed that the capsid region P1, 2Apro, 3A, 3Cpro, the protease precursor 3CD and 3D polymerase all affect RNA granules to varying extents, whereas 2BC does not. 2Apro, which cleaves eIF4GI, induced SGs as expected, and entered novel foci containing the SG nucleating protein G3BP1. Of the two forms of G3BP, only G3BP1 is cleaved by a virus proteinase, 3Cpro, whereas G3BP2 is not cleaved by 3Cpro or 2Apro. Surprisingly, 3CD, which contains proteinase activity, differentially repressed PBs but not SGs. Further, both 2Apro and 3Cpro expression dispersed PBs, however molecular targets were different since PB dispersal due to 2Apro and heat shock protein (Hsp)90 inhibition but not 3Cpro, could be rescued by application of oxidative stress to cells. The data indicate that PV repression of SGs and PBs is multifactorial, though protease function is dominant. PMID:26610553

  6. Epigenetic repression of cardiac progenitor gene expression by Ezh2 is required for postnatal cardiac homeostasis

    PubMed Central

    Delgado-Olguín, Paul; Huang, Yu; Li, Xue; Christodoulou, Danos; Seidman, Christine E.; Seidman, J.G.; Tarakhovsky, Alexander; Bruneau, Benoit G.

    2011-01-01

    Adult-onset diseases can be associated with in utero events, but mechanisms for this remain unknown1,2. The polycomb histone methyltransferase, Ezh2, stabilizes transcription by depositing repressive marks during development that persist into adulthood3–9, but its function in postnatal organ homeostasis is unknown. We show that Ezh2 stabilizes cardiac gene expression and prevents cardiac pathology by repressing the homeodomain transcription factor Six1, which functions in cardiac progenitors but is stably silenced upon cardiac differentiation10. Ezh2 deletion in cardiac progenitors caused postnatal myocardial pathology and destabilized cardiac gene expression with activation of Six1-dependent skeletal muscle genes. Six1 induced cardiomyocyte hypertrophy and skeletal muscle gene expression. Furthermore, genetically reducing Six1 levels rescued the pathology of Ezh2-deficient hearts. Thus, Ezh2-mediated repression of Six1 in differentiating cardiac progenitors is essential for stable postnatal heart gene expression and homeostasis. Our results suggest that epigenetic dysregulation in embryonic progenitor cells predisposes to adult disease and dysregulated stress responses. PMID:22267199

  7. Genetic factors required to maintain repression of a paramutagenic maize pl1 allele.

    PubMed Central

    Hollick, J B; Chandler, V L

    2001-01-01

    A genetic screen identified two novel gene functions required to maintain mitotically and meiotically heritable gene silencing associated with paramutation of the maize purple plant 1 (pl1) locus. Paramutation at pl1 leads to heritable alterations of pl1 gene regulation; the Pl-Rhoades (Pl-Rh) allele, which typically confers strong pigmentation to juvenile and adult plant structures, changes to a lower expression state termed Pl'-mahogany (Pl'). Paramutation spontaneously occurs at low frequencies in Pl-Rh homozygotes but always occurs when Pl-Rh is heterozygous with Pl'. We identified four mutations that caused increased Pl' pigment levels. Allelism tests revealed that three mutations identified two new maize loci, required to maintain repression 1 (rmr1) and rmr2 and that the other mutation represents a new allele of the previously described mediator of paramutation 1 (mop1) locus. RNA levels from Pl' are elevated in rmr mutants and genetic tests demonstrate that Pl' can heritably change back to Pl-Rh in rmr mutant individuals at variable frequencies. Pigment levels controlled by two pl1 alleles that do not participate in paramutation are unaffected in rmr mutants. These results suggest that RMR functions are intimately involved in maintaining the repressed expression state of paramutant Pl' alleles. Despite strong effects on Pl' repression, rmr mutant plants have no gross developmental abnormalities even after several generations of inbreeding, implying that RMR1 and RMR2 functions are not generally required for developmental homeostasis. PMID:11139517

  8. Separate necdin domains bind ARNT2 and HIF1{alpha} and repress transcription

    SciTech Connect

    Friedman, Eitan R.; Fan Chenming

    2007-11-09

    PWS is caused by the loss of expression of a set of maternally imprinted genes including NECDIN (NDN). NDN is expressed in post-mitotic neurons and plays an essential role in PWS as mouse models lacking only the Ndn gene mimic aspects of this disease. Patients haploid for SIM1 develop a PW-like syndrome. Here, we report that NDN directly interacts with ARNT2, a bHLH-PAS protein and dimer partner for SIM1. We also found that NDN can interact with HIF1{alpha}. We showed that NDN can repress transcriptional activation mediated by ARNT2:SIM1 as well as ARNT2:HIF1{alpha}. The N-terminal 115 residues of NDN are sufficient for interaction with the bHLH domains of ARNT2 or HIF1{alpha} but not for transcriptional repression. Using GAL4-NDN fusion proteins, we determined that NDN possesses multiple repression domains. We thus propose that NDN regulates neuronal function and hypoxic response by regulating the activities of the ARNT2:SIM1 and ARNT2:HIF1{alpha} dimers, respectively.

  9. IL-7 signalling represses Bcl-6 and the TFH gene program.

    PubMed

    McDonald, Paul W; Read, Kaitlin A; Baker, Chandra E; Anderson, Ashlyn E; Powell, Michael D; Ballesteros-Tato, André; Oestreich, Kenneth J

    2016-01-01

    The transcriptional repressor Bcl-6 is linked to the development of both CD4(+) T follicular helper (TFH) and central memory T (TCM) cells. Here, we demonstrate that in response to decreased IL-2 signalling, T helper 1 (TH1) cells upregulate Bcl-6 and co-initiate TFH- and TCM-like gene programs, including expression of the cytokine receptors IL-6Rα and IL-7R. Exposure of this potentially bi-potent cell population to IL-6 favours the TFH gene program, whereas IL-7 signalling represses TFH-associated genes including Bcl6 and Cxcr5, but not the TCM-related genes Klf2 and Sell. Mechanistically, IL-7-dependent activation of STAT5 contributes to Bcl-6 repression. Importantly, antigen-specific IL-6Rα(+)IL-7R(+) CD4(+) T cells emerge from the effector population at late time points post influenza infection. These data support a novel role for IL-7 in the repression of the TFH gene program and evoke a divergent regulatory mechanism by which post-effector TH1 cells may contribute to long-term cell-mediated and humoral immunity. PMID:26743592

  10. Wingless blocks bristle formation and morphogenetic furrow progression in the eye through repression of Daughterless.

    PubMed

    Cadigan, Kenneth M; Jou, Austin D; Nusse, Roel

    2002-07-01

    In the developing eye, wingless activity represses proneural gene expression (and thus interommatidial bristle formation) and positions the morphogenetic furrow by blocking its initiation in the dorsal and ventral regions of the presumptive eye. We provide evidence that wingless mediates both effects, at least in part, through repression of the basic helix-loop-helix protein Daughterless. daughterless is required for high proneural gene expression and furrow progression. Ectopic expression of wingless blocks Daughterless expression in the proneural clusters. This repression, and that of furrow progression, can be mimicked by an activated form of armadillo and blocked by a dominant negative form of pangolin/TCF. Placing daughterless under the control of a heterologous promoter blocks the ability of ectopic wingless to inhibit bristle formation and furrow progression. hedgehog and decapentapleigic could not rescue the wingless furrow progression block, indicating that wingless acts downstream of these genes. In contrast, Atonal and Scute, which are thought to heterodimerize with Daughterless to promote furrow progression and bristle formation, respectively, can block ectopic wingless action. These results are summarized in a model where daughterless is a major, but probably not the only, target of wingless action in the eye. PMID:12091309

  11. Regulation of Peripheral Nerve Myelin Maintenance by Gene Repression through Polycomb Repressive Complex 2.

    PubMed

    Ma, Ki H; Hung, Holly A; Srinivasan, Rajini; Xie, Huafeng; Orkin, Stuart H; Svaren, John

    2015-06-01

    Myelination of peripheral nerves by Schwann cells requires coordinate regulation of gene repression as well as gene activation. Several chromatin remodeling pathways critical for peripheral nerve myelination have been identified, but the functions of histone methylation in the peripheral nerve have not been elucidated. To determine the role of histone H3 Lys27 methylation, we have generated mice with a Schwann cell-specific knock-out of Eed, which is an essential subunit of the polycomb repressive complex 2 (PRC2) that catalyzes methylation of histone H3 Lys27. Analysis of this mutant revealed no significant effects on early postnatal development of myelin. However, its loss eventually causes progressive hypermyelination of small-diameter axons and apparent fragmentation of Remak bundles. These data identify the PRC2 complex as an epigenomic modulator of mature myelin thickness, which is associated with changes in Akt phosphorylation. Interestingly, we found that Eed inactivation causes derepression of several genes, e.g., Sonic hedgehog (Shh) and Insulin-like growth factor-binding protein 2 (Igfbp2), that become activated after nerve injury, but without activation of a primary regulator of the injury program, c-Jun. Analysis of the activated genes in cultured Schwann cells showed that Igfbp2 regulates Akt activation. Our results identify an epigenomic pathway required for establishing thickness of mature myelin and repressing genes that respond to nerve injury. PMID:26041929

  12. Huckebein is part of a combinatorial repression code in the anterior blastoderm.

    PubMed

    Andrioli, Luiz Paulo; Digiampietri, Luciano Antonio; de Barros, Lilian Ponce; Machado-Lima, Ariane

    2012-01-01

    The hierarchy of the segmentation cascade responsible for establishing the Drosophila body plan is composed by gap, pair-rule and segment polarity genes. However, no pair-rule stripes are formed in the anterior regions of the embryo. This lack of stripe formation, as well as other evidence from the literature that is further investigated here, led us to the hypothesis that anterior gap genes might be involved in a combinatorial mechanism responsible for repressing the cis-regulatory modules (CRMs) of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) anterior-most stripes. In this study, we investigated huckebein (hkb), which has a gap expression domain at the anterior tip of the embryo. Using genetic methods we were able to detect deviations from the wild-type patterns of the anterior-most pair-rule stripes in different genetic backgrounds, which were consistent with Hkb-mediated repression. Moreover, we developed an image processing tool that, for the most part, confirmed our assumptions. Using an hkb misexpression system, we further detected specific repression on anterior stripes. Furthermore, bioinformatics analysis predicted an increased significance of binding site clusters in the CRMs of h 1, eve 1, run 1 and ftz 1when Hkb was incorporated in the analysis, indicating that Hkb plays a direct role in these CRMs. We further discuss that Hkb and Slp1, which is the other previously identified common repressor of anterior stripes, might participate in a combinatorial repression mechanism controlling stripe CRMs in the anterior parts of the embryo and define the borders of these anterior stripes. PMID:22027434

  13. Bile Acids Repress Hypoxia-Inducible Factor 1 Signaling and Modulate the Airway Immune Response

    PubMed Central

    Legendre, Claire; Reen, F. Jerry; Woods, David F.; Mooij, Marlies J.; Adams, Claire

    2014-01-01

    Gastroesophageal reflux (GER) frequently occurs in patients with respiratory disease and is particularly prevalent in patients with cystic fibrosis. GER is a condition in which the duodenogastric contents of the stomach leak into the esophagus, in many cases resulting in aspiration into the respiratory tract. As such, the presence of GER-derived bile acids (BAs) has been confirmed in the bronchoalveolar lavage fluid and sputum of affected patients. We have recently shown that bile causes cystic fibrosis-associated bacterial pathogens to adopt a chronic lifestyle and may constitute a major host trigger underlying respiratory infection. The current study shows that BAs elicit a specific response in humans in which they repress hypoxia-inducible factor 1α (HIF-1α) protein, an emerging master regulator in response to infection and inflammation. HIF-1α repression was shown to occur through the 26S proteasome machinery via the prolyl hydroxylase domain (PHD) pathway. Further analysis of the downstream inflammatory response showed that HIF-1α repression by BAs can significantly modulate the immune response of airway epithelial cells, correlating with a decrease in interleukin-8 (IL-8) production, while IL-6 production was strongly increased. Importantly, the effects of BAs on cytokine production can also be more dominant than the bacterium-mediated effects. However, the effect of BAs on cytokine levels cannot be fully explained by their ability to repress HIF-1α, which is not surprising, given the complexity of the immune regulatory network. The suppression of HIF-1 signaling by bile acids may have a significant influence on the progression and outcome of respiratory disease, and the molecular mechanism underpinning this response warrants further investigation. PMID:24914220

  14. Genetic analysis of transcriptional activation and repression in the Tn21 mer operon. [Bacteria

    SciTech Connect

    Ross, W.; Park, S.J.; Summers, A.O. )

    1989-07-01

    Transcription of the Tn21 mercury resistance operon (mer) is controlled by the toxic metal cation Hg(II). This control is mediated by the product of the merR gene, a 144-amino-acid protein which represses transcription of the structural genes (merTPCAD) in the absence of Hg(II) and activates transcription in the presence of Hg(II). We have used a mer-lac transcriptional fusion to obtain regulatory mutants in this metal-responsive system. Some mutants were defective in Hg(II)-induced activation while retaining repression function, others were defective in repression but not activation, and some had lost both functions. Mutations in three of the four cysteine residues of merR resulted in complete loss of Hg(II)-inducible activation but retention of the repressor function. Other lesions adjacent to or very near these cysteines exhibited severely reduced activation and also retained repressor function. There were two putative helix-turn-helix (HTH) domains in merR, and mutants in each had very different phenotypes. A partially dominant mutation in the more amino-terminal region of the two putative HTH regions resulted in loss of both activation and repression, consistent with a role for this region in DNA binding. Mutations in the more centrally located HTH region resulted only in loss of Hg(II)-induced activation. Lesions in the central and in the carboxy-terminal regions of merR exhibited both Hg(II)-independent and Hg(II)-dependent transcriptional activation. The sole cis-acting mutant obtained with this operon fusion strategy, a down-promoter mutation, lies in a highly conserved base in the -35 region of the merTPCAD promoter.

  15. Maintenance of Paternal Methylation and Repression of the Imprinted H19 Gene Requires MBD3

    PubMed Central

    Reese, Kimberly J; Lin, Shu; Verona, Raluca I; Schultz, Richard M; Bartolomei, Marisa S

    2007-01-01

    Paternal repression of the imprinted H19 gene is mediated by a differentially methylated domain (DMD) that is essential to imprinting of both H19 and the linked and oppositely imprinted Igf2 gene. The mechanisms by which paternal-specific methylation of the DMD survive the period of genome-wide demethylation in the early embryo and are subsequently used to govern imprinted expression are not known. Methyl-CpG binding (MBD) proteins are likely candidates to explain how these DMDs are recognized to silence the locus, because they preferentially bind methylated DNA and recruit repression complexes with histone deacetylase activity. MBD RNA and protein are found in preimplantation embryos, and chromatin immunoprecipitation shows that MBD3 is bound to the H19 DMD. To test a role for MBDs in imprinting, two independent RNAi-based strategies were used to deplete MBD3 in early mouse embryos, with the same results. In RNAi-treated blastocysts, paternal H19 expression was activated, supporting the hypothesis that MBD3, which is also a member of the Mi-2/NuRD complex, is required to repress the paternal H19 allele. RNAi-treated blastocysts also have reduced levels of the Mi-2/NuRD complex protein MTA-2, which suggests a role for the Mi-2/NuRD repressive complex in paternal-specific silencing at the H19 locus. Furthermore, DNA methylation was reduced at the H19 DMD when MBD3 protein was depleted. In contrast, expression and DNA methylation were not disrupted in preimplantation embryos for other imprinted genes. These results demonstrate new roles for MBD3 in maintaining imprinting control region DNA methylation and silencing the paternal H19 allele. Finally, MBD3-depleted preimplantation embryos have reduced cell numbers, suggesting a role for MBD3 in cell division. PMID:17708683

  16. The murine Sim-2 gene product inhibits transcription by active repression and functional interference.

    PubMed

    Moffett, P; Reece, M; Pelletier, J

    1997-09-01

    The Drosophila single-minded (Dsim) gene encodes a master regulatory protein involved in cell fate determination during midline development. This protein is a member of a rapidly expanding family of gene products possessing basic helix-loop-helix (bHLH) and hydrophobic PAS (designated a conserved region among PER, ARNT [aryl hydrocarbon receptor nuclear translocator] and SIM) protein association domains. Members of this family function as central transcriptional regulators in cellular differentiation and in the response to environmental stimuli such as xenobiotics and hypoxia. We have previously identified a murine member of this family, called mSim-2, showing sequence homology to the bHLH and PAS domains of Dsim. Immunoprecipitation experiments with recombinant proteins indicate that mSIM-2 associates with the arnt gene product. In the present work, by using fine-structure mapping we found that the HLH and PAS motifs of both proteins are required for optimal association. Forced expression of GAL4/mSIM-2 fusion constructs in mammalian cells demonstrated the presence of two separable repression domains within the carboxy terminus of mSIM-2. We found that mSIM-2 is capable of repressing ARNT-mediated transcriptional activation in a mammalian two-hybrid system. This effect (i) is dependent on the ability of mSIM-2 and ARNT to heterodimerize, (ii) is dependent on the presence of the mSIM-2 carboxy-terminal repression domain, and (iii) is not specific to the ARNT activation domain. These results suggest that mSIM-2 repression activity can dominantly override the activation potential of adjacent transcription factors. We also demonstrated that mSIM-2 can functionally interfere with hypoxia-inducible factor 1alpha (HIF-1alpha)/ARNT transcription complexes, providing a second mechanism by which mSIM-2 may inhibit transcription. PMID:9271372

  17. Celastrol Inhibits Tat-mediated Human Immunodeficiency Virus (HIV) Transcription and Replication

    PubMed Central

    Narayan, Vivek; Kodihalli, Ravindra C.; Chiaro, Chris; Cary, Daniele; Aggarwal, Bharat B.; Henderson, Andrew J.; Prabhu, K. Sandeep

    2011-01-01

    Current drugs used for anti-retroviral therapy against HIV have a narrow spectrum of activity, and more often have associated toxicities and severe side effects in addition to developing resistance. Thus, there is a need to develop new therapeutic strategies against HIV/AIDS to complement the already existing ones. Surprisingly, Tat, an early virus encoded protein required for the efficient transcription of the HIV genome, has not been developed as a target for small molecular therapeutics. We have previously described the ability of an endogenous Michael acceptor electrophile (MAE), 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), to inhibit Tat-dependent transcription, by targeting its cysteine (Cys) rich domain. In an effort to identify other MAEs possessing inhibitory activity against HIV-1 Tat, we tested a collection of plant-derived compounds with electrophilic properties, including curcumin, rosmarinic acid, and gambogic acid, for their ability to inhibit Tat. Celastrol (Cel), a triterpenoid MAE isolated from T. wilfordii, exhibited the highest inhibitory activity against Tat. Using biochemical techniques, we demonstrate that Cel by covalently modifying the cysteine thiols inhibits Tat transactivation function. Using circular dichroism (CD) spectroscopy, we show that alklylation of Tat brought about a change in the secondary structure of Tat, which inhibited the transcription elongation of the HIV proviral genome by effecting mechanisms other than Tat-TAR interaction. Our results demonstrate the underlying mechanism of anti-retroviral activity of the plant-derived MAEs, and suggest that Cel could serve as a lead compound to develop novel anti-viral therapeutics. PMID:21763500

  18. The CovR response regulator of group A streptococcus (GAS) acts directly to repress its own promoter.

    PubMed

    Gusa, Asiya A; Scott, June R

    2005-06-01

    The CovR/S (CsrR/S) two component system is a global regulator of virulence gene expression in the group A streptococcus (GAS, Streptococcus pyogenes). The response regulator, CovR, regulates about 15% of the genes of GAS, including its own operon. Using in vitro DNA binding assays with purified CovR protein, we found that CovR binds a DNA fragment including the covR promoter (Pcov). DNaseI footprint analyses showed that phosphorylation of CovR enhanced and extended the protected regions. The proposed CovR consensus binding sequence (ATTARA) was present at most, but not all protected regions. The effect of replacing the two thymine residues in the consensus binding sequence (CB) with guanine residues was evaluated both in vitro and in vivo. Most, but not all, CB mutations reduced binding of CovR in vitro. Using a transcriptional reporter introduced in single copy into the GAS chromosome, we found that mutations at each CB completely or partially relieved CovR-mediated repression in vivo. This suggests that CovR regulation of Pcov is direct. Further support for this conclusion comes from use of an in vitro GAS transcription system in which CovR was sufficient to mediate repression of Pcov. This repression was enhanced by phosphorylation of the protein. In addition, we found that the CovR binding region overlapping the promoter was essential for wild type repression of Pcov both in vitro and in vivo, suggesting that promoter occlusion is a primary mechanism of Pcov repression by CovR. PMID:15882414

  19. Recovered-memory therapy and robust repression: influence and pseudomemories.

    PubMed

    Ofshe, R J; Singer, M T

    1994-10-01

    A subset of the psychotherapists practicing trauma-focused therapy predicate their treatment on the existence of a newly claimed, powerful form of repression that differs from repression as used in the psychoanalytic tradition and from amnesia in any of its recognized forms. Recovered-memory specialists assist patients to supposedly retrieve vast quantities of information (e.g., utterly new dramatic life histories) that were allegedly unavailable to consciousness for years or decades. We refer to the hypothesized mental mechanism as "robust repression" and call attention to the absence of evidence documenting its validity and to the differences between it and other mental mechanisms and memory features. No recovered-memory practitioner has ever published a full specification of the attributes of this mechanism. That is, the properties it would have to have for the narratives developed during therapy to be historically accurate to any significant degree. This article reports a specification of the properties of the robust repression mechanism based on interviews with current and former patients, practitioners' writings, and reports to researchers and clinicians. The spread of reliance on the robust repression mechanism over the past 20 years through portions of the clinical community is traced. While involved in therapy, patients of recovered-memory practitioners come to believe that they have either instantly repressed large numbers of discrete events or simultaneously repressed all information about abuse they may have endured for as long as a decade.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7960294

  20. The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets.

    PubMed

    Huntzinger, Eric; Kuzuoglu-Öztürk, Duygu; Braun, Joerg E; Eulalio, Ana; Wohlbold, Lara; Izaurralde, Elisa

    2013-01-01

    Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact with poly(A)-binding protein (PABP) and the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins to interact with both PABP and deadenylases. To address these questions, we characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are triggered through the interactions of GW182 proteins with PABP and deadenylases. PMID:23172285

  1. Control of the Type 3 Secretion System in Vibrio harveyi by Quorum Sensing through Repression of ExsA ▿ ‡

    PubMed Central

    Waters, Christopher M.; Wu, Julie T.; Ramsey, Meghan E.; Harris, Rebecca C.; Bassler, Bonnie L.

    2010-01-01

    The type 3 secretion system (T3SS) genes of Vibrio harveyi are activated at low cell density and repressed at high cell density by quorum sensing (QS). Repression requires LuxR, the master transcriptional regulator of QS-controlled genes. Here, we determine the mechanism underlying the LuxR repression of the T3SS system. Using a fluorescence-based cell sorting approach, we isolated V. harveyi mutants that are unable to express T3SS genes at low cell density and identified two mutations in the V. harveyi exsBA operon. While LuxR directly represses the expression of exsBA, complementation and epistasis analyses reveal that it is the repression of exsA expression, but not exsB expression, that is responsible for the QS-mediated repression of T3SS genes at high cell density. The present work further defines the genes in the V. harveyi QS regulon and elucidates a mechanism demonstrating how multiple regulators can be linked in series to direct the expression of QS target genes specifically at low or high cell density. PMID:20543047

  2. Human THAP7 is a chromatin-associated, histone tail-binding protein that represses transcription via recruitment of HDAC3 and nuclear hormone receptor corepressor.

    PubMed

    Macfarlan, Todd; Kutney, Sara; Altman, Brian; Montross, Rebecca; Yu, Jiujiu; Chakravarti, Debabrata

    2005-02-25

    The identities of signal transducer proteins that integrate histone hypoacetylation and transcriptional repression are largely unknown. Here we demonstrate that THAP7, an uncharacterized member of the recently identified THAP (Thanatos-associated protein) family of proteins, is ubiquitously expressed, associates with chromatin, and represses transcription. THAP7 binds preferentially to hypoacetylated (un-, mono-, and diacetylated) histone H4 tails in vitro via its C-terminal 77 amino acids. Deletion of this domain, or treatment of cells with the histone deacetylase inhibitor TSA, which leads to histone hyperacetylation, partially disrupts THAP7/chromatin association in living cells. THAP7 coimmunoprecipitates with histone deacetylase 3 (HDAC3) and the nuclear hormone receptor corepressor (NCoR) and represses transcription as a Gal4 fusion protein. Chromatin immunoprecipitation assays demonstrate that these corepressors are recruited to promoters in a THAP7 dependent manner and promote histone H3 hypoacetylation. The conserved THAP domain is a key determinant for full HDAC3 association in vitro, and both the THAP domain and the histone interaction domain are important for the repressive properties of THAP7. Full repression mediated by THAP7 is also dependent on NCoR expression. We hypothesize that THAP7 is a dual function repressor protein that actively targets deacetylation of histone H3 necessary to establish transcriptional repression and functions as a signal transducer of the repressive mark of hypoacetylated histone H4. This is the first demonstration of the transcriptional regulatory properties of a human THAP domain protein, and a critical identification of a potential transducer of the repressive signal of hypoacetylated histone H4 in higher eukaryotes. PMID:15561719

  3. Repression of the locus of the enterocyte effacement-encoded regulator of gene transcription of Escherichia coli O157:H7 by Lactobacillus reuteri culture supernatants is LuxS and strain dependent.

    PubMed

    Jelcić, Ivan; Hüfner, Eric; Schmidt, Herbert; Hertel, Christian

    2008-05-01

    Culture supernatants of Lactobacillus reuteri ATCC 55730 repressed ler expression in Escherichia coli O157:H7 cells, but neither the strain's isogenic luxS mutant nor the L. reuteri 100-23C wild-type strain and its luxS mutant elicited a comparable effect. Furthermore, the epinephrine-mediated induction of ler expression was repressed by secreted substance(s) of L. reuteri ATCC 55730. PMID:18378666

  4. TNF-Alpha Represses Transcription of Human Bone Morphogenetic Protein-4 in Lung Epithelial Cells

    PubMed Central

    Zhu, Nian-Ling; Li, Changgong; Huang, Hao Hao; Sebald, Matthew; Londhe, Vedang A.; Heisterkamp, Nora; Warburton, David; Bellusci, Saverio; Minoo, Parviz

    2007-01-01

    Bone Morphogenetic Proteins are key signaling molecules in vertebrate development. Little is known about Bmp gene regulation in any organ. In Drosophila, the Bmp gene, dpp is regulated by Dorsal, the invertebrate homologue of Rel-NFkB. In this study we examined whether TNF-alpha, which activates NF-kB, can regulate Bmp4 gene expression. TNF-alpha reduced Bmp4 mRNA in lung adenocarcinoma A549 cells and repressed transcriptional activity of the human Bmp4 promoter in a dose-dependent manner. Similar repression was observed when the Bmp4 promoter was co-transfected with a p65 (RelA) expression vector in the absence of TNF-alpha treatment, suggesting that RelA mediates the effect of TNF-alpha. In support of this finding, the repressor effect of TNF-alpha on Bmp4 was abrogated by a co-transfected dominant negative mutant of IkB (S32A/S36A). The human Bmp4 promoter contains 3 putative consensus binding sites for NF-kB. Surprisingly, only one of the latter binding sites was capable of binding NFkB. Repressor effect of NFkB was not dependent on any of the three binding sites, but localized to a 122 bp fragment which bound both RelA and SP1. SP1stimulated transcription, whereas increasing doses of RelA opposed this effect. In vivo, TNF-alpha inhibited branching morphogenesis and LacZ gene expression in Bmp4-lacz transgenic lungs. These data support a model in which TNF-alpha-induced RelA interacts with SP1 to bring about transcriptional repression of Bmp4 gene. These findings provide a mechanistic paradigm for interactions between mediators of inflammation and morphogenesis with relevant implications for normal lung development and pathogenesis of disease. PMID:17350185

  5. Mutually repressing repressor functions and multi-layered cellular heterogeneity regulate the bistable Salmonella fliC census

    PubMed Central

    Stewart, Mary K.; Cookson, Brad T.

    2014-01-01

    Summary Bistable flagellar and virulence gene expression generates specialized Salmonella subpopulations with distinct functions. Repressing flagellar genes allows Salmonella to evade caspase-1 mediated host defenses and enhances systemic colonization. By definition, bistability arises when intermediate states of gene expression are rendered unstable by the underlying genetic circuitry. We demonstrate sustained bistable fliC expression in virulent Salmonella 14028 and document dynamic control of the distribution, or single-cell census, of flagellar gene expression by the mutually repressing repressors YdiV and FliZ. YdiV partitions cells into the fliC-OFF subpopulation, while FliZ partitions cells into the fliC-HIGH subpopulation at late timepoints during growth. Bistability of ΔfliZ populations and ydiV-independent FliZ control of flagellar gene expression provide evidence that the YdiV-FliZ mutually repressing repressor circuit is not required for bistability. Repression and activation by YdiV and FliZ (respectively) can shape the census of fliC expression independently, and bistability collapses into a predominantly intermediate population in the absence of both regulators. Metered expression of YdiV and FliZ reveals variable sensitivity to these regulators and defines conditions where expression of FliZ enhances fliC expression and where FliZ does not alter the fliC census. Thus, this evolved genetic circuitry coordinates multiple layers of regulatory heterogeneity into a binary response. PMID:25315056

  6. Transcriptional repression of MMP-1 by p21SNFT and reduced in vitro invasiveness of hepatocarcinoma cells.

    PubMed

    Bower, Kristen E; Fritz, Jamie M; McGuire, Kathleen L

    2004-11-18

    p21SNFT (21 kDa small nuclear factor isolated from T cells) is a human basic leucine zipper transcription factor that can repress AP-1-mediated transcription. We show here that overexpression of p21SNFT in HepG2 cells leads to repression of matrix metalloproteinase-1 by 70-80%. p21SNFT interacted with Jun at the matrix metalloproteinase-1 promoter -88 Ets/AP-1 enhancer element, where Jun is known to activate transcription via interaction with Fos and Ets proteins. When p21SNFT/Jun dimers bound the element in the presence of Ets, DNA was protected differently than when Fos was paired with Jun. The data suggest a difference in overall conformation between p21SNFT-containing and Fos-containing complexes that may be involved in the repression of matrix metalloproteinase-1 by p21SNFT. Overexpression of p21SNFT led to a reduction in invasiveness of HepG2 cells through type I collagen and reconstituted basement membrane, an effect similar to that obtained via direct immunodepletion of matrix metalloproteinase-1. The results indicate that the mechanism of repression of matrix metalloproteinase-1 by p21SNFT may be exploited in inhibiting pathological matrix remodeling during cancer progression in vivo. PMID:15467742

  7. Age-associated de-repression of retrotransposons in the Drosophila fat body, its potential cause and consequence.

    PubMed

    Chen, Haiyang; Zheng, Xiaobin; Xiao, Danqing; Zheng, Yixian

    2016-06-01

    Eukaryotic genomes contain transposable elements (TE) that can move into new locations upon activation. Since uncontrolled transposition of TEs, including the retrotransposons and DNA transposons, can lead to DNA breaks and genomic instability, multiple mechanisms, including heterochromatin-mediated repression, have evolved to repress TE activation. Studies in model organisms have shown that TEs become activated upon aging as a result of age-associated deregulation of heterochromatin. Considering that different organisms or cell types may undergo distinct heterochromatin changes upon aging, it is important to identify pathways that lead to TE activation in specific tissues and cell types. Through deep sequencing of isolated RNAs, we report an increased expression of many retrotransposons in the old Drosophila fat body, an organ equivalent to the mammalian liver and adipose tissue. This de-repression correlates with an increased number of DNA damage foci and decreased level of Drosophila lamin-B in the old fat body cells. Depletion of the Drosophila lamin-B in the young or larval fat body results in a reduction of heterochromatin and a corresponding increase in retrotransposon expression and DNA damage. Further manipulations of lamin-B and retrotransposon expression suggest a role of the nuclear lamina in maintaining the genome integrity of the Drosophila fat body by repressing retrotransposons. PMID:27072046

  8. CcpA-Dependent Carbon Catabolite Repression in Bacteria

    PubMed Central

    Warner, Jessica B.; Lolkema, Juke S.

    2003-01-01

    Carbon catabolite repression (CCR) by transcriptional regulators follows different mechanisms in gram-positive and gram-negative bacteria. In gram-positive bacteria, CcpA-dependent CCR is mediated by phosphorylation of the phosphoenolpyruvate:sugar phosphotransferase system intermediate HPr at a serine residue at the expense of ATP. The reaction is catalyzed by HPr kinase, which is activated by glycolytic intermediates. In this review, the distribution of CcpA-dependent CCR among bacteria is investigated by searching the public databases for homologues of HPr kinase and HPr-like proteins throughout the bacterial kingdom and by analyzing their properties. Homologues of HPr kinase are commonly observed in the phylum Firmicutes but are also found in the phyla Proteobacteria, Fusobacteria, Spirochaetes, and Chlorobi, suggesting that CcpA-dependent CCR is not restricted to gram-positive bacteria. In the α and β subdivisions of the Proteobacteria, the presence of HPr kinase appears to be common, while in the γ subdivision it is more of an exception. The genes coding for the HPr kinase homologues of the Proteobacteria are in a gene cluster together with an HPr-like protein, termed XPr, suggesting a functional relationship. Moreover, the XPr proteins contain the serine phosphorylation sequence motif. Remarkably, the analysis suggests a possible relation between CcpA-dependent gene regulation and the nitrogen regulation system (Ntr) found in the γ subdivision of the Proteobacteria. The relation is suggested by the clustering of CCR and Ntr components on the genome of members of the Proteobacteria and by the close phylogenetic relationship between XPr and NPr, the HPr-like protein in the Ntr system. In bacteria in the phylum Proteobacteria that contain HPr kinase and XPr, the latter may be at the center of a complex regulatory network involving both CCR and the Ntr system. PMID:14665673

  9. Multiple repressive mechanisms in the hippocampus during memory formation.

    PubMed

    Cho, Jun; Yu, Nam-Kyung; Choi, Jun-Hyeok; Sim, Su-Eon; Kang, SukJae Joshua; Kwak, Chuljung; Lee, Seung-Woo; Kim, Ji-il; Choi, Dong Il; Kim, V Narry; Kaang, Bong-Kiun

    2015-10-01

    Memory stabilization after learning requires translational and transcriptional regulations in the brain, yet the temporal molecular changes that occur after learning have not been explored at the genomic scale. We used ribosome profiling and RNA sequencing to quantify the translational status and transcript levels in the mouse hippocampus after contextual fear conditioning. We revealed three types of repressive regulations: translational suppression of ribosomal protein-coding genes in the hippocampus, learning-induced early translational repression of specific genes, and late persistent suppression of a subset of genes via inhibition of estrogen receptor 1 (ESR1/ERα) signaling. In behavioral analyses, overexpressing Nrsn1, one of the newly identified genes undergoing rapid translational repression, or activating ESR1 in the hippocampus impaired memory formation. Collectively, this study unveils the yet-unappreciated importance of gene repression mechanisms for memory formation. PMID:26430118

  10. MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures

    PubMed Central

    Muraoka, Naoto; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Isomi, Mari; Nakashima, Hanae; Akiyama, Mizuha; Wada, Rie; Inagawa, Kohei; Nishiyama, Takahiko; Kaneda, Ruri; Fukuda, Toru; Takeda, Shu; Tohyama, Shugo; Hashimoto, Hisayuki; Kawamura, Yoshifumi; Goshima, Naoki; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

    2014-01-01

    Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming. PMID:24920580

  11. Cytotype Regulation Facilitates Repression of Hybrid Dysgenesis by Naturally Occurring KP Elements in Drosophila melanogaster.

    PubMed

    Simmons, Michael J; Grimes, Craig D; Czora, Cody S

    2016-01-01

    P elements inserted in the Telomere Associated Sequences (TAS) at the left end of the X chromosome are determiners of cytotype regulation of the entire P family of transposons. This regulation is mediated by Piwi-interacting (pi) RNAs derived from the telomeric P elements (TPs). Because these piRNAs are transmitted maternally, cytotype regulation is manifested as a maternal effect of the TPs. When a TP is combined with a transgenic P element inserted at another locus, this maternal effect is strengthened. However, when certain TPs are combined with transgenes that contain the small P element known as KP, stronger regulation arises from a zygotic effect of the KP element. This zygotic effect is observed with transgenic KP elements that are structurally intact, as well as with KP elements that are fused to an ancillary promoter from the hsp70 gene. Zygotic regulation by a KP element occurs only when a TP was present in the maternal germ line, and it is more pronounced when the TP was also present in the grand-maternal germ line. However, this regulation does not require zygotic expression of the TP These observations can be explained if maternally transmitted piRNAs from TPs enable a polypeptide encoded by KP elements to repress P element transposition in zygotes that contain a KP element. In nature, repression by the KP polypeptide may therefore be facilitated by cytotype-mediating piRNAs. PMID:27172198

  12. HDAC8 and STAT3 repress BMF gene activity in colon cancer cells.

    PubMed

    Kang, Y; Nian, H; Rajendran, P; Kim, E; Dashwood, W M; Pinto, J T; Boardman, L A; Thibodeau, S N; Limburg, P J; Löhr, C V; Bisson, W H; Williams, D E; Ho, E; Dashwood, R H

    2014-01-01

    Histone deacetylase (HDAC) inhibitors are undergoing clinical trials as anticancer agents, but some exhibit resistance mechanisms linked to anti-apoptotic Bcl-2 functions, such as BH3-only protein silencing. HDAC inhibitors that reactivate BH3-only family members might offer an improved therapeutic approach. We show here that a novel seleno-α-keto acid triggers global histone acetylation in human colon cancer cells and activates apoptosis in a p21-independent manner. Profiling of multiple survival factors identified a critical role for the BH3-only member Bcl-2-modifying factor (Bmf). On the corresponding BMF gene promoter, loss of HDAC8 was associated with signal transducer and activator of transcription 3 (STAT3)/specificity protein 3 (Sp3) transcription factor exchange and recruitment of p300. Treatment with a p300 inhibitor or transient overexpression of exogenous HDAC8 interfered with BMF induction, whereas RNAi-mediated silencing of STAT3 activated the target gene. This is the first report to identify a direct target gene of HDAC8 repression, namely, BMF. Interestingly, the repressive role of HDAC8 could be uncoupled from HDAC1 to trigger Bmf-mediated apoptosis. These findings have implications for the development of HDAC8-selective inhibitors as therapeutic agents, beyond the reported involvement of HDAC8 in childhood malignancy. PMID:25321483

  13. Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes

    PubMed Central

    Cesarini, Elisa; Mozzetta, Chiara; Marullo, Fabrizia; Gregoretti, Francesco; Gargiulo, Annagiusi; Columbaro, Marta; Cortesi, Alice; Antonelli, Laura; Di Pelino, Simona; Squarzoni, Stefano; Palacios, Daniela; Zippo, Alessio; Bodega, Beatrice; Oliva, Gennaro

    2015-01-01

    Beyond its role in providing structure to the nuclear envelope, lamin A/C is involved in transcriptional regulation. However, its cross talk with epigenetic factors—and how this cross talk influences physiological processes—is still unexplored. Key epigenetic regulators of development and differentiation are the Polycomb group (PcG) of proteins, organized in the nucleus as microscopically visible foci. Here, we show that lamin A/C is evolutionarily required for correct PcG protein nuclear compartmentalization. Confocal microscopy supported by new algorithms for image analysis reveals that lamin A/C knock-down leads to PcG protein foci disassembly and PcG protein dispersion. This causes detachment from chromatin and defects in PcG protein–mediated higher-order structures, thereby leading to impaired PcG protein repressive functions. Using myogenic differentiation as a model, we found that reduced levels of lamin A/C at the onset of differentiation led to an anticipation of the myogenic program because of an alteration of PcG protein–mediated transcriptional repression. Collectively, our results indicate that lamin A/C can modulate transcription through the regulation of PcG protein epigenetic factors. PMID:26553927

  14. Cytotype Regulation Facilitates Repression of Hybrid Dysgenesis by Naturally Occurring KP Elements in Drosophila melanogaster

    PubMed Central

    Simmons, Michael J.; Grimes, Craig D.; Czora, Cody S.

    2016-01-01

    P elements inserted in the Telomere Associated Sequences (TAS) at the left end of the X chromosome are determiners of cytotype regulation of the entire P family of transposons. This regulation is mediated by Piwi-interacting (pi) RNAs derived from the telomeric P elements (TPs). Because these piRNAs are transmitted maternally, cytotype regulation is manifested as a maternal effect of the TPs. When a TP is combined with a transgenic P element inserted at another locus, this maternal effect is strengthened. However, when certain TPs are combined with transgenes that contain the small P element known as KP, stronger regulation arises from a zygotic effect of the KP element. This zygotic effect is observed with transgenic KP elements that are structurally intact, as well as with KP elements that are fused to an ancillary promoter from the hsp70 gene. Zygotic regulation by a KP element occurs only when a TP was present in the maternal germ line, and it is more pronounced when the TP was also present in the grand-maternal germ line. However, this regulation does not require zygotic expression of the TP. These observations can be explained if maternally transmitted piRNAs from TPs enable a polypeptide encoded by KP elements to repress P element transposition in zygotes that contain a KP element. In nature, repression by the KP polypeptide may therefore be facilitated by cytotype-mediating piRNAs. PMID:27172198

  15. HDAC8 and STAT3 repress BMF gene activity in colon cancer cells

    PubMed Central

    Kang, Y; Nian, H; Rajendran, P; Kim, E; Dashwood, W M; Pinto, J T; Boardman, L A; Thibodeau, S N; Limburg, P J; Löhr, C V; Bisson, W H; Williams, D E; Ho, E; Dashwood, R H

    2014-01-01

    Histone deacetylase (HDAC) inhibitors are undergoing clinical trials as anticancer agents, but some exhibit resistance mechanisms linked to anti-apoptotic Bcl-2 functions, such as BH3-only protein silencing. HDAC inhibitors that reactivate BH3-only family members might offer an improved therapeutic approach. We show here that a novel seleno-α-keto acid triggers global histone acetylation in human colon cancer cells and activates apoptosis in a p21-independent manner. Profiling of multiple survival factors identified a critical role for the BH3-only member Bcl-2-modifying factor (Bmf). On the corresponding BMF gene promoter, loss of HDAC8 was associated with signal transducer and activator of transcription 3 (STAT3)/specificity protein 3 (Sp3) transcription factor exchange and recruitment of p300. Treatment with a p300 inhibitor or transient overexpression of exogenous HDAC8 interfered with BMF induction, whereas RNAi-mediated silencing of STAT3 activated the target gene. This is the first report to identify a direct target gene of HDAC8 repression, namely, BMF. Interestingly, the repressive role of HDAC8 could be uncoupled from HDAC1 to trigger Bmf-mediated apoptosis. These findings have implications for the development of HDAC8-selective inhibitors as therapeutic agents, beyond the reported involvement of HDAC8 in childhood malignancy. PMID:25321483

  16. Catabolite Repression and Activation in Bacillus subtilis: Dependency on CcpA, HPr, and HprK

    PubMed Central

    Lorca, Graciela L.; Chung, Yong Joon; Barabote, Ravi D.; Weyler, Walter; Schilling, Christophe H.; Saier, Milton H.

    2005-01-01

    Previous studies have suggested that the transcription factor CcpA, as well as the coeffectors HPr and Crh, both phosphorylated by the HprK kinase/phosphorylase, are primary mediators of catabolite repression and catabolite activation in Bacillus subtilis. We here report whole transcriptome analyses that characterize glucose-dependent gene expression in wild-type cells and in isogenic mutants lacking CcpA, HprK, or the HprK phosphorylatable serine in HPr. Binding site identification revealed which genes are likely to be primarily or secondarily regulated by CcpA. Most genes subject to CcpA-dependent regulation are regulated fully by HprK and partially by serine-phosphorylated HPr [HPr(Ser-P)]. A positive linear correlation was noted between the dependencies of catabolite-repressible gene expression on CcpA and HprK, but no such relationship was observed for catabolite-activated genes, suggesting that large numbers of the latter genes are not regulated by the CcpA-HPr(Ser-P) complex. Many genes that mediate nitrogen or phosphorus metabolism as well as those that function in stress responses proved to be subject to CcpA-dependent glucose control. While nitrogen-metabolic genes may be subject to either glucose repression or activation, depending on the gene, almost all glucose-responsive phosphorus-metabolic genes exhibit activation while almost all glucose-responsive stress genes show repression. These responses are discussed from physiological standpoints. These studies expand our appreciation of CcpA-mediated catabolite control and provide insight into potential interregulon control mechanisms in gram-positive bacteria. PMID:16267306

  17. Transcriptional repression of SIRT1 by protein inhibitor of activated STAT 4 (PIAS4) in hepatic stellate cells contributes to liver fibrosis

    PubMed Central

    Sun, Lina; Fan, Zhiwen; Chen, Junliang; Tian, Wenfang; Li, Min; Xu, Huihui; Wu, Xiaoyan; Shao, Jing; Bian, Yaoyao; Fang, Mingming; Xu, Yong

    2016-01-01

    Interstitial fibrosis represents a key pathological process in non-alcoholic steatohepatitis (NASH). In the liver, fibrogenesis is primarily mediated by activated hepatic stellate cells (HSCs) transitioning from a quiescent state in response to a host of stimuli. The molecular mechanism underlying HSC activation is not completely understood. Here we report that there was a simultaneous up-regulation of PIAS4 expression and down-regulation of SIRT1 expression accompanying increased hepatic fibrogenesis in an MCD-diet induced mouse model of NASH. In cultured primary mouse HSCs, stimulation with high glucose activated PIAS4 while at the same time repressed SIRT1. Over-expression of PIAS4 directly repressed SIRT1 promoter activity. In contrast, depletion of PIAS4 restored SIRT1 expression in HSCs treated with high glucose. Estrogen, a known NASH-protective hormone, antagonized HSC activation by targeting PIAS4. Lentivirus-mediated delivery of short hairpin RNA (shRNA) targeting PIAS4 in mice ameliorated MCD diet induced liver fibrosis by normalizing SIRT1 expression in vivo. PIAS4 promoted HSC activation in a SIRT1-dependent manner in vitro. Mechanistically, PIAS4 mediated SIRT1 repression led to SMAD3 hyperacetylation and enhanced SMAD3 binding to fibrogenic gene promoters. Taken together, our data suggest SIRT1 trans-repression by PIAS4 plays an important role in HSC activation and liver fibrosis. PMID:27323886

  18. Bmi1 represses Ink4a/Arf and Hox genes to regulate stem cells in the rodent incisor

    PubMed Central

    Biehs, Brian; Hu, Jimmy Kuang-Hsien; Strauli, Nicolas B.; Sangiorgi, Eugenio; Jung, Heekyung; Heber, Ralf-Peter; Ho, Sunita; Goodwin, Alice F.; Dasen, Jeremy S.; Capecchi, Mario R.; Klein, Ophir D.

    2013-01-01

    The polycomb group gene Bmi1 is required for maintenance of adult stem cells in many organs1, 2. Inactivation of Bmi1 leads to impaired stem cell self-renewal due to deregulated gene expression. One critical target of BMI1 is Ink4a/Arf, which encodes the cell cycle inhibitors p16ink4a and p19Arf3. However, deletion of Ink4a/Arf only partially rescues Bmi1 null phenotypes4, indicating that other important targets of BMI1 exist. Here, using the continuously-growing mouse incisor as a model system, we report that Bmi1 is expressed by incisor stem cells and that deletion of Bmi1 resulted in fewer stem cells, perturbed gene expression, and defective enamel production. Transcriptional profiling revealed that Hox expression is normally repressed by BMI1 in the adult, and functional assays demonstrated that BMI1-mediated repression of Hox genes preserves the undifferentiated state of stem cells. As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated1, 2, 5–7, our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation. PMID:23728424

  19. Antenatal hypoxia induces epigenetic repression of glucocorticoid receptor and promotes ischemic-sensitive phenotype in the developing heart.

    PubMed

    Xiong, Fuxia; Lin, Thant; Song, Minwoo; Ma, Qingyi; Martinez, Shannalee R; Lv, Juanxiu; MataGreenwood, Eugenia; Xiao, Daliao; Xu, Zhice; Zhang, Lubo

    2016-02-01

    Large studies in humans and animals have demonstrated a clear association of an adverse intrauterine environment with an increased risk of cardiovascular disease later in life. Yet mechanisms remain largely elusive. The present study tested the hypothesis that gestational hypoxia leads to promoter hypermethylation and epigenetic repression of the glucocorticoid receptor (GR) gene in the developing heart, resulting in increased heart susceptibility to ischemia and reperfusion injury in offspring. Hypoxic treatment of pregnant rats from day 15 to 21 of gestation resulted in a significant decrease of GR exon 14, 15, 16, and 17 transcripts, leading to down-regulation of GR mRNA and protein in the fetal heart. Functional cAMP-response elements (CREs) at -4408 and -3896 and Sp1 binding sites at -3425 and -3034 were identified at GR untranslated exon 1 promoters. Hypoxia significantly increased CpG methylation at the CREs and Sp1 binding sites and decreased transcription factor binding to GR exon 1 promoter, accounting for the repression of the GR gene in the developing heart. Of importance, treatment of newborn pups with 5-aza-2'-deoxycytidine reversed hypoxia-induced promoter methylation, restored GR expression and prevented hypoxia-mediated increase in ischemia and reperfusion injury of the heart in offspring. The findings demonstrate a novel mechanism of epigenetic repression of the GR gene in fetal stress-mediated programming of ischemic-sensitive phenotype in the heart. PMID:26779948

  20. Ebf1 and c-Myb repress Rag transcription downstream of Stat5 during early B cell development

    PubMed Central

    Timblin, Greg A; Schlissel, Mark S

    2013-01-01

    The temporal control of recombination-activating gene (Rag) expression in developing lymphocytes prevents DNA breaks during periods of proliferation that could threaten genomic integrity. In developing B cells, the interleukin-7 receptor (IL-7R) and precursor B cell antigen receptor (pre-BCR) synergize to induce proliferation and the repression of Rag at the protein and mRNA levels for a brief period following successful immunoglobulin (Ig) heavy-chain gene rearrangement. While the mechanism of RAG2 protein downregulation is well-defined, little is known about the pathways and transcription factors that mediate transcriptional repression of Rag. Using Abelson Murine Leukemia Virus (AMuLV)-transformed B cells to model this stage of development, we identified Early B Cell Factor 1 (Ebf1) as a strong repressor of Rag transcription. shRNA-mediated knockdown of either Ebf1 or its downstream target c-Myb was sufficient to induce Rag transcription in these highly proliferative cells. Ebf1 and c-Myb antagonize Rag transcription by negatively regulating the binding of Foxo1 to the Rag locus. Ebf1 accomplishes this through both direct negative regulation of Foxo1 expression, and direct positive regulation of Gfi1b expression. Ebf1 expression is driven by the IL-7R downstream effector Stat5, providing a link between the negative regulation of Rag transcription by IL-7 and a novel repressive pathway involving Ebf1 and c-Myb. PMID:24068669

  1. COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells

    PubMed Central

    Marban, Céline; Redel, Laetitia; Suzanne, Stella; Van Lint, Carine; Lecestre, Dominique; Chasserot-Golaz, Sylvette; Leid, Mark; Aunis, Dominique; Schaeffer, Evelyne; Rohr, Olivier

    2005-01-01

    Human immunodeficiency virus type 1 (HIV-1) gene transcription is characterized by two temporally distinct phases. While the initial phase relies solely on cellular transcription factors, the subsequent phase is activated by the viral Tat transactivator. We have previously reported that the subsequent phase of viral gene transcription can be repressed by the chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2) in human microglial cells [O. Rohr, D. Lecestre, S. Chasserot-Golaz, C. Marban, D. Avram, D. Aunis, M. Leid and E. Schaeffer (2003), J. Virol., 77, 5415–5427]. Here, we demonstrate that CTIP proteins also repress the initial phase of HIV-1 gene transcription, mainly supported by the cellular transcription factors Sp1 and COUP-TF in microglial cells. We report that CTIP2 represses Sp1- and COUP-TF-mediated activation of HIV-1 gene transcription and viral replication as a result of physical interactions with COUP-TF and Sp1 in microglial nuclei. Using laser confocal microscopy CTIP2 was found to colocalize with Sp1, COUP-TF and the heterochromatin-associated protein Hp1α, which is mainly detected in transcriptionally repressed heterochromatic region. Moreover, we describe that CTIP2 can be recruited to the HIV-1 promoter via its association with Sp1 bound to the GC-box sequences of the long terminal repeat (LTR). Since our findings demonstrate that CTIP2 interacts with the HIV-1 proximal promoter, it is likely that CTIP2 promotes HIV-1 gene silencing by forcing transcriptionally repressed heterochromatic environment to the viral LTR region. PMID:15849318

  2. Induction, production, repression, and de-repression of exoglucanase synthesis in Aspergillus niger.

    PubMed

    Hanif, Atif; Yasmeen, Amber; Rajoka, M I

    2004-09-01

    The influence of carbon and nitrogen sources on the production of cellulases was investigated. The enzyme production was variable according to the carbon source. Levels of beta-cellobiohydrolase (CBH) were minimal in the presence of even low concentrations of glucose. Enzyme production was stimulated by other carbohydrates. The enzyme is subject to carbon source control by easily metabolizable sugars. Wheat bran and cellulose were the most effective promoters of beta-cellobiohydrolase and filter paperase (FPase) activities respectively, followed by rice bran. Exogenously supplied glucose inhibited the synthesis of the enzyme in cultures of A. niger growing on wheat bran. In defined medium with cellobiose, the cellobiohydrolase titres were 2- to 110-fold higher with cells growing on monomeric sugars and 1.5 times higher than cells growing on other disaccharides. It appeared that synthesis of beta-cellobiohydrolase varied under an induction mechanism, and a repression mechanism which changed the rate of synthesis of beta-cellobiohydrolase and FPase in induced over non-induced cultures. In this organism, substantial synthesis of beta-cellobiohydrolase can be induced by cellobiose, cellodextrin, cellulose or cellulose and hemi-cellulose containing substrates which showed low volumetric substrate uptake rate. The organism required limiting concentration of carbon, nitrogen or phosphorous for production of beta-cellobiohydrolase and FPase. During growth of A. niger on wheat bran, maximum volumetric productivities (Qp) of beta-cellobiohydrolase and FPase were 39.6 and 32.5 IU/lh and were significantly higher than the values reported for some other potent fungi and bacteria. The addition of actinomycin D (a repressor of transcription) and cycloheximide, (a repressor of translation) completely repressed CBH/FPase biosynthesis, suggested that the regulation of CBH synthesis in this organism occurs at both transcriptional and translational level. Thermodynamic studies

  3. Mechanism of promoter repression by Lac repressor-DNA loops.

    PubMed

    Becker, Nicole A; Peters, Justin P; Maher, L James; Lionberger, Troy A

    2013-01-01

    The Escherichia coli lactose (lac) operon encodes the first genetic switch to be discovered, and lac remains a paradigm for studying negative and positive control of gene expression. Negative control is believed to involve competition of RNA polymerase and Lac repressor for overlapping binding sites. Contributions to the local Lac repressor concentration come from free repressor and repressor delivered to the operator from remote auxiliary operators by DNA looping. Long-standing questions persist concerning the actual role of DNA looping in the mechanism of promoter repression. Here, we use experiments in living bacteria to resolve four of these questions. We show that the distance dependence of repression enhancement is comparable for upstream and downstream auxiliary operators, confirming the hypothesis that repressor concentration increase is the principal mechanism of repression loops. We find that as few as four turns of DNA can be constrained in a stable loop by Lac repressor. We show that RNA polymerase is not trapped at repressed promoters. Finally, we show that constraining a promoter in a tight DNA loop is sufficient for repression even when promoter and operator do not overlap. PMID:23143103

  4. CRISPR Technology for Genome Activation and Repression in Mammalian Cells.

    PubMed

    Du, Dan; Qi, Lei S

    2016-01-01

    Targeted modulation of transcription is necessary for understanding complex gene networks and has great potential for medical and industrial applications. CRISPR is emerging as a powerful system for targeted genome activation and repression, in addition to its use in genome editing. This protocol describes how to design, construct, and experimentally validate the function of sequence-specific single guide RNAs (sgRNAs) for sequence-specific repression (CRISPRi) or activation (CRISPRa) of transcription in mammalian cells. In this technology, the CRISPR-associated protein Cas9 is catalytically deactivated (dCas9) to provide a general platform for RNA-guided DNA targeting of any locus in the genome. Fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in mammalian cells. Delivery of multiple sgRNAs further enables activation or repression of multiple genes. By using scaffold RNAs (scRNAs), different effectors can be recruited to different genes for simultaneous activation of some and repression of others. The CRISPRi and CRISPRa methods provide powerful tools for sequence-specific control of gene expression on a genome-wide scale to aid understanding gene functions and for engineering genetic regulatory systems. PMID:26729910

  5. Rhizobacteria of Cotton and Their Repression of Seedling Disease Pathogens

    PubMed Central

    Hagedorn, C.; Gould, W. D.; Bardinelli, T. R.

    1989-01-01

    During the 1983 field season, the rhizobacteria (including organisms from rhizosphere soil and the root rhizoplane) of cotton plants at one location in Mississippi were inventoried at different plant growth stages. Isolates (1,000) were identified to the genus level and characterized for repression of Pythium ultimum and Rhizoctonia solani. Cotton seedlings were initially colonized by bacteria of many different genera, and populations quickly reached 108 CFU/g of root tissue. As the season progressed, the bacterial populations declined as root mass increased and the roots became more woodlike in consistency. Fluorescent pseudomonads were the most numerous gram-negative rhizobacterial isolates of those that were randomly collected and identified, and they provided the largest number of isolates with fungal repressive activity. Several other gram-negative bacterial genera were recovered throughout the growing season, and some gram-positive bacteria were also isolated routinely, but at lower numbers. There was no correlation between the proportion of rhizobacterial isolates that possessed fungal repressive activity and the plant growth stage from which the isolates were obtained. Approximately twice as many bacterial isolates demonstrated fungal repression in the agar assay compared with the inplanta assay, and isolates were found more frequently with fungal repressive activity against P. ultimum than against R. solai. PMID:16348043

  6. Glucocorticoid and TNF signaling converge at A20 (TNFAIP3) to repress airway smooth muscle cytokine expression.

    PubMed

    Sasse, Sarah K; Altonsy, Mohammed O; Kadiyala, Vineela; Cao, Gaoyuan; Panettieri, Reynold A; Gerber, Anthony N

    2016-08-01

    Airway smooth muscle is a major target tissue for glucocorticoid (GC)-based asthma therapies, however, molecular mechanisms through which the GC receptor (GR) exerts therapeutic effects in this key airway cell type have not been fully elucidated. We previously identified the nuclear factor-κB (NF-κB) inhibitor, A20 (TNFAIP3), as a mediator of cytokine repression by glucocorticoids (GCs) in airway epithelial cells and defined cooperative regulation of anti-inflammatory genes by GR and NF-κB as a key mechanistic underpinning of airway epithelial GR function. Here, we expand on these findings to determine whether a similar mechanism is operational in human airway smooth muscle (HASM). Using HASM cells derived from normal and fatal asthma samples as an in vitro model, we demonstrate that GCs spare or augment TNF-mediated induction of A20 (TNFAIP3), TNIP1, and NFKBIA, all implicated in negative feedback control of NF-κB-driven inflammatory processes. We applied chromatin immunoprecipitation and reporter analysis to show that GR and NF-κB directly regulate A20 expression in HASM through cooperative induction of an intronic enhancer. Using overexpression, we show for the first time that A20 and its interacting partner, TNIP1, repress TNF signaling in HASM cells. Moreover, we applied small interfering RNA-based gene knockdown to demonstrate that A20 is required for maximal cytokine repression by GCs in HASM. Taken together, our data suggest that inductive regulation of A20 by GR and NF-κB contributes to cytokine repression in HASM. PMID:27371733

  7. RFX family proteins differentially interact with HDACs to repress collagen alpha 2(I) gene (COL1A2) expression

    PubMed Central

    Xu, Y.; Sengupta, P.K.; Seto, E.; Smith, B.D.

    2006-01-01

    Our studies indicate that regulatory factor for X-box (RFX) family proteins repress collagen alpha2(I) gene (COL1A2) expression (1,2). In the present investigation, we examine the mechanism(s) underlying the repression of collagen gene by RFX proteins. Two members of the RFX family, RFX1 and RFX5, associate with distinct sets of co-repressors on the collagen transcription start site in vitro. RFX5 specifically interacts with histone deacetylase 2 (HDAC2) and the mammalian transcriptional repressor (mSin3B) whereas RFX1 preferably interacts with HDAC1 and mSin3A. HDAC2 cooperates with RFX5 to down-regulate collagen promoter activity while HDAC1 enhances inhibition of collagen promoter activity by RFX1. IFN-γ promotes the recruitment of RFX5/HDAC2/mSin3B to the collagen transcription start site but decreases the occupancy by RFX1/mSin3A as manifested by chromatin immunoprecipitation (ChIP) assay. RFX1 binds to methylated collagen sequence with much higher affinity than unmethylated sequence, recruiting more HDAC1 and mSin3A. The DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (aza-dC), that inhibits DNA methylation, reduces RFX1/HDAC1 binding to the collagen transcription start site in ChIP assays. Finally, both RFX1 and RFX5 are acetylated in vivo. TSA stimulates the acetylation of RFX proteins and activates the collagen promoter activity. Collectively, our data strongly indicate two separate pathways for RFX proteins to repress collagen gene expression: one for RFX5/HDAC2 in IFN-γ mediated repression, the other for RFX1/HDAC1 in methylation mediated collagen silencing. PMID:16464847

  8. A Novel POK Family Transcription Factor, ZBTB5, Represses Transcription of p21CIP1 Gene*

    PubMed Central

    Koh, Dong-In; Choi, Won-Il; Jeon, Bu-Nam; Lee, Choong-Eun; Yun, Chae-Ok; Hur, Man-Wook

    2009-01-01

    Transcriptional repression through chromatin remodeling and histone deacetylation has been postulated as a driving force for tumorigenesis. We isolated and characterized a novel POZ domain Krüppel-like zinc finger transcription repressor, ZBTB5 (zinc finger and BTB domain-containing 5). Serial analysis of gene expression (SAGE) analysis showed that ZBTB5 expression is higher in retinoblastoma and muscle cancer tissues. Immunocytochemistry showed that ZBTB5 was localized to the nucleus, particularly nuclear speckles. ZBTB5 directly repressed transcription of cell cycle arrest gene p21 by binding to the proximal GC-box 5/6 elements and the two distal p53-responsive elements (bp −2323 ∼ −2299; bp −1416 ∼ −1392). Chromatin immunoprecipitation assays showed that ZBTB5 and p53 competed with each other in occupying the p53 binding elements. ZBTB5 interacted with co-repressor-histone deacetylase complexes such as BCoR (BCL-6-interacting corepressor), NCoR (nuclear receptor corepressor), and SMRT (silencing mediator for retinoid and thyroid receptors) via its POZ domain. These interactions resulted in deacetylation of histones Ac-H3 and Ac-H4 at the proximal promoter, which is important in the transcriptional repression of p21. MTT (3-(4,5-di meth yl thi azol-2-yl)-2,5-diphenyltetrazolium bromide) assays and fluorescent-activated cell sorter analysis revealed that ZBTB5 stimulated both cell proliferation and cell cycle progression, significantly increasing the number of cells in S-phase. Overall, our data suggest that ZBTB5 is a potent transcription repressor of cell cycle arrest gene p21 and a potential proto-oncogene stimulating cell proliferation. PMID:19491398

  9. Genomic footprinting of the yeast zinc finger protein Rme1p and its roles in repression of the meiotic activator IME1.

    PubMed Central

    Shimizu, M; Li, W; Covitz, P A; Hara, M; Shindo, H; Mitchell, A P

    1998-01-01

    The zinc finger protein Rme1p is a negative regulator of the meiotic activator IME1 in Saccharomyces cerevisiae . Prior studies have shown that Rme1p binds in vitro to a site near nt -2030 in the IME1 upstream region, but a genomic mutation in that site has little effect on repression of IME1 . To identify Rme1p binding sites in vivo , we have examined the binding of Rme1p to genomic sites through in vivo footprinting. We show that Rme1p binds to two sites in the IME1 upstream region, near nt -1950 and -2030. Mutations in both binding sites abolish repression of chromosomal IME1 by Rme1p, whereas a mutation in either single site causes partial derepression. Therefore, both Rme1p binding sites are essential for repression of IME1 . Prior studies have shown that repression by Rme1p depends upon RGR1 and SIN4 , which specify RNA polymerase II mediator subunits that are required for normal nucleosome density. We find that RGR1 and SIN4 are not simply required for Rme1p to bind to DNA in vivo . These results suggest that Rme1p functions directly as a repressor of IME1 and that Rgr1p and Sin4p are required for DNA-bound Rme1p to exert repression. PMID:9580682

  10. Orphan nuclear receptor small heterodimer partner inhibits transforming growth factor-beta signaling by repressing SMAD3 transactivation.

    PubMed

    Suh, Ji Ho; Huang, Jiansheng; Park, Yun-Yong; Seong, Hyun-A; Kim, Dongwook; Shong, Minho; Ha, Hyunjung; Lee, In-Kyu; Lee, Keesook; Wang, Li; Choi, Hueng-Sik

    2006-12-22

    Orphan nuclear receptor small heterodimer partner (SHP) is an atypical member of the nuclear receptor superfamily; SHP regulates the nuclear receptor-mediated transcription of target genes but lacks a conventional DNA binding domain. In this study, we demonstrate that SHP represses transforming growth factor-beta (TGF-beta)-induced gene expression through a direct interaction with Smad, a transducer of TGF-beta signaling. Transient transfection studies demonstrate that SHP represses Smad3-induced transcription. In vivo and in vitro protein interaction assays revealed that SHP directly interacts with Smad2 and Smad3 but not with Smad4. Mapping of domains mediating the interaction between SHP and Smad3 showed that the entire N-terminal domain (1-159 amino acids) of SHP and the linker domain of Smad3 are involved in this interaction. In vitro glutathione S-transferase pulldown competition experiments revealed the SHP-mediated repression of Smad3 transactivation through competition with its co-activator p300. SHP also inhibits the activation of endogenous TGF-beta-responsive gene promoters, the p21, Smad7, and plasminogen activator inhibitor-1 (PAI-1) promoters. Moreover, adenovirus-mediated overexpression of SHP decreases PAI-1 mRNA levels, and down-regulation of SHP by a small interfering RNA increases both the transactivation of Smad3 and the PAI-1 mRNA levels. Finally, the PAI-1 gene is expressed in SHP(-/-) mouse hepatocytes at a higher level than in normal hepatocytes. Taken together, these data indicate that SHP is a novel co-regulator of Smad3, and this study provides new insights into regulation of TGF-beta signaling. PMID:17074765

  11. Reduced specificity of negative autobiographical memories in repressive coping.

    PubMed

    Geraerts, Elke; Dritschel, Barbara; Kreplin, Ute; Miyagawa, Liv; Waddington, Joanne

    2012-12-01

    The current study examined memory specificity of autobiographical memories in individuals with and without a repressive coping style. It seems conceivable that reduced memory specificity may be a way to reduce accessibility of negative experiences, one of the hallmark features of a repressive coping style. It was therefore hypothesized that repressors would show reduced specificity when retrieving negative memories. In order to study memory specificity, participants (N = 103) performed the autobiographical memory test. Results showed that individuals with a repressive coping style were significantly less specific in retrieving negative experiences, relative to control groups of low anxious, high anxious, and defensive high anxious individuals. This result was restricted to negative memory retrieval, as participants did not differ in memory specificity for positive experiences. These results show that repressors retrieve negative autobiographical memories in an overgeneral way, possibly in order to avoid negative affect. PMID:23200428

  12. Repression-sensitization, stress, and perception of pain in others.

    PubMed

    Von Baeyer, C

    1982-08-01

    To assess the influence of individuals' defensive style on perception of pain in others, 60 undergraduate women rated the amount of pain expressed in slides of people displaying high or low pain. Subjects were categorized as high or low on Byrne's Repression-Sensitization Scale, and their level of stress was varied by presentation of an anxiety-provoking film (stress condition) or a neutral film (control condition) prior to the rating task. A significant interaction between Repression-Sensitization and slide category (high versus low pain) indicated that sensitizers assigned higher ratings of pain than repressers to slides that were relatively low in rated expressiveness of pain. Individual differences in readiness to recognize potentially threatening stimuli seem most evident when the stimuli are relatively ambiguous. The manipulation of stress produced no significant effects on ratings of pain. PMID:7133920

  13. Induction of proliferation by 15-deoxy-delta12,14-prostaglandin J2 and the precursors in monocytic leukemia U937.

    PubMed

    Azuma, Yasutaka; Watanabe, Kyoko; Date, Masataka; Daito, Michiharu; Ohura, Kiyoshi

    2004-08-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in several human tumors including gastric, lung, colon, prostate and breast. However, the role of PPARgamma signals in leukemia is still unclear. The aim of this study is to evaluate the ability of 15-deoxy-Delta12,14-prostaglandin J2 (15dPGJ2), that is a ligand for PPARgamma, on proliferation of human leukemia cell line U937. 15dPGJ2 at 5 micromol/l stimulated the proliferation. In contrast, 15dPGJ2 at concentrations of >10 micromol/l inhibited the proliferation through the induction of apoptosis. PGD2, PGJ2 and Delta12-PGJ2 (DeltaPGJ2), those are precursors of 15dPGJ2, had similarly proliferative effects, whereas they showed antiproliferative effects at high concentrations. FACScan analysis revealed that PGD2 at 5 micromol/l, PGJ2 at 1 micromol/l, DeltaPGJ2 at 1 micromol/l and 15dPGJ2 at 5 micromol/l, all accelerated cell cycle progression. Immunoblotting analysis revealed that PGD2 at 5 micromol/l and 15dPGJ2 at 5 micromol/l inhibited the expression of phospho-p38, phospho-MKK3/MKK6 and phospho-ATF-2, and the expression of Cdk inhibitors including p18, p27. In contrast, PGJ2 at 1 micromol/l and DeltaPGJ2 at 1 micromol/l did not affect the expression of them. These results suggest that 15dPGJ2 and PGD2 may, through inactivation of the p38 MAPK pathway, inhibit the expression of Cdk inhibitors, leading to acceleration of proliferation. PMID:15240994

  14. PTH and Vitamin D Repress DMP1 in Cementoblasts.

    PubMed

    Wang, L; Tran, A B; Nociti, F H; Thumbigere-Math, V; Foster, B L; Krieger, C C; Kantovitz, K R; Novince, C M; Koh, A J; McCauley, L K; Somerman, M J

    2015-10-01

    A complex feedback mechanism between parathyroid hormone (PTH), 1,25(OH)2D3 (1,25D), and fibroblast growth factor 23 (FGF-23) maintains mineral homeostasis, in part by regulating calcium and phosphate absorption/reabsorption. Previously, we showed that 1,25D regulates mineral homeostasis by repressing dentin matrix protein 1 (DMP1) via the vitamin D receptor pathway. Similar to 1,25D, PTH may modulate DMP1, but the underlying mechanism remains unknown. Immortalized murine cementoblasts (OCCM.30), similar to osteoblasts and known to express DMP1, were treated with PTH (1-34). Real-time quantitative polymerase chain reaction (PCR) and Western blot revealed that PTH decreased DMP1 gene transcription (85%) and protein expression (30%), respectively. PTH mediated the downregulation of DMP1 via the cAMP/protein kinase A (PKA) pathway. Immunohistochemistry confirmed the decreased localization of DMP1 in vivo in cellular cementum and alveolar bone of mice treated with a single dose (50 µg/kg) of PTH (1-34). RNA-seq was employed to further identify patterns of gene expression shared by PTH and 1,25D in regulating DMP1, as well as other factors involved in mineral homeostasis. PTH and 1,25D mutually upregulated 36 genes and mutually downregulated 27 genes by ≥2-fold expression (P ≤ 0.05). Many identified genes were linked with the regulation of bone/tooth homeostasis, cell growth and differentiation, calcium signaling, and DMP1 transcription. Validation of RNA-seq results via PCR array confirmed a similar gene expression pattern in response to PTH and 1,25D treatment. Collectively, these results suggest that PTH and 1,25D share complementary effects in maintaining mineral homeostasis by mutual regulation of genes/proteins associated with calcium and phosphate metabolism while also exerting distinct roles on factors modulating mineral metabolism. Furthermore, PTH may modulate phosphate homeostasis by downregulating DMP1 expression via the cAMP/PKA pathway. Targeting

  15. Repression of apical homeobox genes is required for embryonic root development in Arabidopsis.

    PubMed

    Grigg, Stephen P; Galinha, Carla; Kornet, Noortje; Canales, Claudia; Scheres, Ben; Tsiantis, Miltos

    2009-09-15

    Development of seed plant embryos is polarized along the apical-basal axis. This polarization occurs in the absence of cell migration and culminates in the establishment of two distinct pluripotent cell populations: the shoot apical meristem (SAM) and root meristem (RM), which postembryonically give rise to the entire shoot and root systems of the plant. The acquisition of genetic pathways that delimit root from shoot during embryogenesis must have played a pivotal role during land plant evolution because roots evolved after shoots in ancestral vascular plants and may be shoot-derived organs. However, such pathways are very poorly understood. Here we show that RM establishment in the model plant Arabidopsis thaliana requires apical confinement of the Class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) proteins PHABULOSA (PHB) and PHAVOLUTA (PHV), which direct both SAM development and shoot lateral organ polarity. Failure to restrict PHB and PHV expression apically via a microRNA-dependent pathway prevents correct elaboration of the embryonic root development program and results in embryo lethality. As such, repression of a fundamental shoot development pathway is essential for correct root development. Additionally, our data suggest that a single patterning process, based on HD-ZIP III repression, mediates both apical-basal and radial polarity in the embryo and lateral organ polarity in the shoot. PMID:19646874

  16. Activation and repression by oncogenic MYC shape tumour-specific gene expression profiles.

    PubMed

    Walz, Susanne; Lorenzin, Francesca; Morton, Jennifer; Wiese, Katrin E; von Eyss, Björn; Herold, Steffi; Rycak, Lukas; Dumay-Odelot, Hélène; Karim, Saadia; Bartkuhn, Marek; Roels, Frederik; Wüstefeld, Torsten; Fischer, Matthias; Teichmann, Martin; Zender, Lars; Wei, Chia-Lin; Sansom, Owen; Wolf, Elmar; Eilers, Martin

    2014-07-24

    In mammalian cells, the MYC oncoprotein binds to thousands of promoters. During mitogenic stimulation of primary lymphocytes, MYC promotes an increase in the expression of virtually all genes. In contrast, MYC-driven tumour cells differ from normal cells in the expression of specific sets of up- and downregulated genes that have considerable prognostic value. To understand this discrepancy, we studied the consequences of inducible expression and depletion of MYC in human cells and murine tumour models. Changes in MYC levels activate and repress specific sets of direct target genes that are characteristic of MYC-transformed tumour cells. Three factors account for this specificity. First, the magnitude of response parallels the change in occupancy by MYC at each promoter. Functionally distinct classes of target genes differ in the E-box sequence bound by MYC, suggesting that different cellular responses to physiological and oncogenic MYC levels are controlled by promoter affinity. Second, MYC both positively and negatively affects transcription initiation independent of its effect on transcriptional elongation. Third, complex formation with MIZ1 (also known as ZBTB17) mediates repression of multiple target genes by MYC and the ratio of MYC and MIZ1 bound to each promoter correlates with the direction of response. PMID:25043018

  17. An epigenetic switch ensures transposon repression upon dynamic loss of DNA methylation in embryonic stem cells

    PubMed Central

    Walter, Marius; Teissandier, Aurélie; Pérez-Palacios, Raquel; Bourc'his, Déborah

    2016-01-01

    DNA methylation is extensively remodeled during mammalian gametogenesis and embryogenesis. Most transposons become hypomethylated, raising the question of their regulation in the absence of DNA methylation. To reproduce a rapid and extensive demethylation, we subjected mouse ES cells to chemically defined hypomethylating culture conditions. Surprisingly, we observed two phases of transposon regulation. After an initial burst of de-repression, various transposon families were efficiently re-silenced. This was accompanied by a reconfiguration of the repressive chromatin landscape: while H3K9me3 was stable, H3K9me2 globally disappeared and H3K27me3 accumulated at transposons. Interestingly, we observed that H3K9me3 and H3K27me3 occupy different transposon families or different territories within the same family, defining three functional categories of adaptive chromatin responses to DNA methylation loss. Our work highlights that H3K9me3 and, most importantly, polycomb-mediated H3K27me3 chromatin pathways can secure the control of a large spectrum of transposons in periods of intense DNA methylation change, ensuring longstanding genome stability. DOI: http://dx.doi.org/10.7554/eLife.11418.001 PMID:26814573

  18. SUMOylation Regulates the Transcriptional Repression Activity of FOG-2 and Its Association with GATA-4

    PubMed Central

    Perdomo, José; Jiang, Xing-Mai; Carter, Daniel R.; Khachigian, Levon M.; Chong, Beng H.

    2012-01-01

    Friend of GATA 2 (FOG-2), a co-factor of several GATA transcription factors (GATA-4, -5 and 6), is a critical regulator of coronary vessel formation and heart morphogenesis. Here we demonstrate that FOG-2 is SUMOylated and that this modification modulates its transcriptional activity. FOG-2 SUMOylation occurs at four lysine residues (K312, 471, 915, 955). Three of these residues are part of the characteristic SUMO consensus site (ψKXE), while K955 is found in the less frequent TKXE motif. Absence of SUMOylation did not affect FOG-2′s nuclear localization. However, mutation of the FOG-2 SUMOylation sites, or de-SUMOylation, with SENP-1 or SENP-8 resulted in stronger transcriptional repression activity in both heterologous cells and cardiomyocytes. Conversely, increased FOG-2 SUMOylation by overexpression of SUMO-1 or expression of a SUMO-1-FOG-2 fusion protein rendered FOG-2 incapable of repressing GATA-4-mediated activation of the B-type natriuretic peptide (BNP) promoter. Moreover, we demonstrate both increased interaction between a FOG-2 SUMO mutant and GATA-4 and enhanced SUMOylation of wild-type FOG-2 by co-expression of GATA-4. These data suggest a new dynamics in which GATA-4 may alter the activity of FOG-2 by influencing its SUMOylation status. PMID:23226341

  19. Parallel pathways of repression and antirepression governing the transition to stationary phase in Bacillus subtilis.

    PubMed

    Banse, Allison V; Chastanet, Arnaud; Rahn-Lee, Lilah; Hobbs, Errett C; Losick, Richard

    2008-10-01

    The AbrB protein of the spore-forming bacterium Bacillus subtilis is a repressor of numerous genes that are switched on during the transition from the exponential to the stationary phase of growth. The gene for AbrB is under the negative control of the master regulator for entry into sporulation, Spo0A-P. It has generally been assumed that derepression of genes under the negative control of AbrB is achieved by Spo0A-P-mediated repression of abrB followed by rapid degradation of the AbrB protein. Here, we report that AbrB levels do decrease during the transition to stationary phase, but that this decrease is not the entire basis by which AbrB-controlled genes are derepressed. Instead, AbrB is inactivated by the product of a uncharacterized gene, abbA (formerly ykzF), whose transcription is switched on by Spo0A-P. The abbA gene encodes an antirepressor that binds to AbrB and prevents it from binding to DNA. Combining our results with previous findings, we conclude that Spo0A-P sets in motion two parallel pathways of repression and antirepression to trigger the expression of diverse categories of genes during the transition to stationary phase. PMID:18840696

  20. Chronic idiopathic urticaria, psychological co-morbidity and posttraumatic stress: the impact of alexithymia and repression.

    PubMed

    Hunkin, Victoria; Chung, Man Cheung

    2012-12-01

    The objective of this study was to investigate the interrelationship between chronic idiopathic urticaria (CIU), psychological co-morbidity, posttraumatic stress, repression and alexithymia. 89 participants with CIU and 105 without CIU responded to an online questionnaire. Both groups completed the general health questionnaire-12, the perceived stress scale, the posttraumatic stress diagnostic scale and the Toronto alexithymia scale-20 and were categorised into four defence mechanism groups (repressive, defensive, high-anxious, low-anxious). CIU participants also completed the Skindex-17 and a self-report severity measure. CIU participants reported higher levels of alexithymia than the control group and their defence mechanism was most likely to be categorised as defensive, with conscious self-image management reported alongside high manifest anxiety. Partial least squares analysis revealed significant paths between posttraumatic stress and CIU severity and psychological co-morbidity. Posttraumatic stress was associated with alexithymia and type of defence mechanism. Only being in the high-anxious group partially mediated the relationship between posttraumatic stress and CIU severity. In conclusion, there is evidence for a relationship between CIU and trauma. The severity of posttraumatic symptoms varies depending upon alexithymic traits and defence mechanisms used. Disease severity and psychological co-morbidity are differentially influenced by the relationships between trauma, alexithymic traits and defence mechanisms. PMID:22362490

  1. Histone demethylase Lsd1 represses hematopoietic stem and progenitor cell signatures during blood cell maturation

    PubMed Central

    Kerenyi, Marc A; Shao, Zhen; Hsu, Yu-Jung; Guo, Guoji; Luc, Sidinh; O'Brien, Kassandra; Fujiwara, Yuko; Peng, Cong; Nguyen, Minh; Orkin, Stuart H

    2013-01-01

    Here, we describe that lysine-specific demethylase 1 (Lsd1/KDM1a), which demethylates histone H3 on Lys4 or Lys9 (H3K4/K9), is an indispensible epigenetic governor of hematopoietic differentiation. Integrative genomic analysis, combining global occupancy of Lsd1, genome-wide analysis of its substrates H3K4 monomethylation and dimethylation, and gene expression profiling, reveals that Lsd1 represses hematopoietic stem and progenitor cell (HSPC) gene expression programs during hematopoietic differentiation. We found that Lsd1 acts at transcription start sites, as well as enhancer regions. Loss of Lsd1 was associated with increased H3K4me1 and H3K4me2 methylation on HSPC genes and gene derepression. Failure to fully silence HSPC genes compromised differentiation of hematopoietic stem cells as well as mature blood cell lineages. Collectively, our data indicate that Lsd1-mediated concurrent repression of enhancer and promoter activity of stem and progenitor cell genes is a pivotal epigenetic mechanism required for proper hematopoietic maturation. DOI: http://dx.doi.org/10.7554/eLife.00633.001 PMID:23795291

  2. Runx1 repression by histone deacetylation is critical for Setbp1-induced mouse myeloid leukemia development

    PubMed Central

    Vishwakarma, Bandana A.; Nguyen, Nhu; Makishima, Hideki; Hosono, Naoko; Gudmundsson, Kristbjorn O.; Negi, Vijay; Oakley, Kevin; Han, Yufen; Przychodzen, Bartlomiej; Maciejewski, Jaroslaw P.; Du, Yang

    2015-01-01

    Abnormal activation of SETBP1 through overexpression or missense mutations is highly recurrent in various myeloid malignancies; however, it is unclear whether such activation alone is able to induce leukemia development. Here we show that Setbp1 overexpression in mouse bone marrow progenitors through retroviral transduction is capable of initiating leukemia development in irradiated recipient mice. Before leukemic transformation, Setbp1 overexpression significantly enhances the self-renewal of hematopoietic stem cells (HSCs) and expands granulocyte macrophage progenitors (GMPs). Interestingly, Setbp1 overexpression also causes transcriptional repression of critical hematopoiesis regulator gene Runx1 and this effect is crucial for Setbp1-induced transformation. Runx1 repression is induced by Setbp1-mediated recruitment of a nucleosome remodeling deacetylase (NuRD) complex to Runx1 promoters and can be reversed by treatment with histone deacetylase (HDAC) inhibitors Entinostat and Vorinostat. Moreover, treatment with these inhibitors caused efficient differentiation of Setbp1 activation-induced leukemia cells in vitro, and significantly extended the survival of mice transplanted with such leukemias, suggesting that HDAC inhibition could be an effective strategy for treating myeloid malignancies with SETBP1 activation. PMID:26205084

  3. Yeast nitrogen catabolite repression is sustained by signals distinct from glutamine and glutamate reservoirs.

    PubMed

    Fayyad-Kazan, Mohammad; Feller, A; Bodo, E; Boeckstaens, M; Marini, A M; Dubois, E; Georis, I

    2016-01-01

    Nitrogen catabolite repression (NCR) is a wide transcriptional regulation program enabling baker's yeast to downregulate genes involved in the utilization of poor nitrogen sources when preferred ones are available. Nowadays, glutamine and glutamate, the major nitrogen donors for biosyntheses, are assumed to be key metabolic signals regulating NCR. NCR is controlled by the conserved TORC1 complex, which integrates nitrogen signals among others to regulate cell growth. However, accumulating evidence indicate that the TORC1-mediated control of NCR is only partial, arguing for the existence of supplementary regulatory processes to be discovered. In this work, we developed a genetic screen to search for new players involved in NCR signaling. Our data reveal that the NADP-glutamate dehydrogenase activity of Gdh1 negatively regulates NCR-sensitive gene transcription. By determining the total, cytoplasmic and vacuolar pools of amino acids, we show that there is no positive correlation between glutamine/glutamate reservoirs and the extent of NCR. While our data indicate that glutamine could serve as initial trigger of NCR, they show that it is not a sufficient signal to sustain repression and point to the existence of yet unknown signals. Providing additional evidence uncoupling TORC1 activity and NCR, our work revisits the dogmas underlying NCR regulation. PMID:26419331

  4. Translational repression by the human iron-regulatory factor (IRF) in Saccharomyces cerevisiae.

    PubMed Central

    Oliveira, C C; Goossen, B; Zanchin, N I; McCarthy, J E; Hentze, M W; Stripecke, R

    1993-01-01

    The regulation of the synthesis of ferritin and erythroid 5-aminolevulinate synthase in mammalian cells is mediated by the interaction of the iron regulatory factor (IRF) with a specific recognition site, the iron responsive element (IRE), in the 5' untranslated regions (UTRs) of the respective mRNAs. A new modular expression system was designed to allow reconstruction of this regulatory system in Saccharomyces cerevisiae. This comprised two components: a constitutively expressed reporter gene (luc; encoding luciferase) preceded by a 5' UTR including an IRE sequence, and an inducibly expressed cDNA encoding human IRF. Induction of the latter led to the in vivo synthesis of IRF, which in turn showed IRE-binding activity and also repressed translation of the luc mRNA bearing an IRE-containing 5' UTR. The upper stem-loop region of an IRE, with no further IRE-specific flanking sequences, sufficed for recognition and repression by IRF. Translational regulation of IRE-bearing mRNAs could also be demonstrated in cell-free yeast extracts. This work defines a minimal system for IRF/IRE translational regulation in yeast that requires no additional mammalian-specific components, thus providing direct proof that IRF functions as a translational repressor in vivo. It should be a useful tool as the basis for more detailed studies of eukaryotic translational regulation. Images PMID:8265343

  5. The transcription factor TBX2 regulates melanogenesis in melanocytes by repressing Oca2.

    PubMed

    Chen, Yu; Pan, Li; Su, Zhongyuan; Wang, Jing; Li, Huirong; Ma, Xiaoyin; Liu, Yin; Lu, Fan; Qu, Jia; Hou, Ling

    2016-04-01

    The T-box transcription factor TBX2 is known for its role as a critical regulator of melanoma cell proliferation, but its role in regulating melanogenesis has not been widely studied. Here we use a series of experiments to show in primary and immortalized mouse melanocytes that TBX2 acts as regulator of melanogenesis by repressing the expression of the gene encoding the melanosomal protein OCA2. We find that α-MSH or forskolin, both of which stimulate melanogenesis, also reduce TBX2 expression, and that specific knockdown of TBX2 increases melanogenesis. This effect primarily involves an increase in Oca2 expression as the combined knockdown of both Tbx2 and Oca2 interferes with the Tbx2 knockdown-mediated increase in melanogenesis. Standard chromatin immunoprecipitation and reporter assays suggest that TBX2 represses Oca2 at least in part directly. Hence, the results suggest that TBX2 may act as a nexus linking cell proliferation and melanogenesis. PMID:26971330

  6. Piwi Modulates Chromatin Accessibility by Regulating Multiple Factors Including Histone H1 to Repress Transposons.

    PubMed

    Iwasaki, Yuka W; Murano, Kensaku; Ishizu, Hirotsugu; Shibuya, Aoi; Iyoda, Yumiko; Siomi, Mikiko C; Siomi, Haruhiko; Saito, Kuniaki

    2016-08-01

    PIWI-interacting RNAs (piRNAs) mediate transcriptional and post-transcriptional silencing of transposable element (TE) in animal gonads. In Drosophila ovaries, Piwi-piRNA complexes (Piwi-piRISCs) repress TE transcription by modifying the chromatin state, such as by H3K9 trimethylation. Here, we demonstrate that Piwi physically interacts with linker histone H1. Depletion of Piwi decreases H1 density at a subset of TEs, leading to their derepression. Silencing at these loci separately requires H1 and H3K9me3 and heterochromatin protein 1a (HP1a). Loss of H1 increases target loci chromatin accessibility without affecting H3K9me3 density at these loci, while loss of HP1a does not impact H1 density. Thus, Piwi-piRISCs require both H1 and HP1a to repress TEs, and the silencing is correlated with the chromatin state rather than H3K9me3 marks. These findings suggest that Piwi-piRISCs regulate the interaction of chromatin components with target loci to maintain silencing of TEs through the modulation of chromatin accessibility. PMID:27425411

  7. Pleiotropic Properties of a Yeast Mutant Insensitive to Catabolite Repression

    PubMed Central

    Stark, Helene Cherrick; Fugit, Donna; Mowshowitz, Deborah Bernhardt

    1980-01-01

    The flk1 mutation, which was originally isolated in the yeast Saccharomyces carlsbergenesis, causes insensitivity to catabolite repression. This mutation has been further characterized and mapped. The gene flk1 is located on chromosome III between thr4 and MAL2, 14 centimorgans from MAL2. flk1 is shown to be allelic to the pleiotropic mutants tup1, cyc9, and umr7; and flk1 is shown to exhibit an array of pleiotropic properties common to tup1, cyc9 and umr7; These results suggest that the flk1 mutation is not a specific lesion affecting catabolite repression. PMID:17249023

  8. Evolutionarily conserved multisubunit RBL2/p130 and E2F4 protein complex represses human cell cycle-dependent genes in quiescence.

    PubMed

    Litovchick, Larisa; Sadasivam, Subhashini; Florens, Laurence; Zhu, Xiaopeng; Swanson, Selene K; Velmurugan, Soundarapandian; Chen, Runsheng; Washburn, Michael P; Liu, X Shirley; DeCaprio, James A

    2007-05-25

    The mammalian Retinoblastoma (RB) family including pRB, p107, and p130 represses E2F target genes through mechanisms that are not fully understood. In D. melanogaster, RB-dependent repression is mediated in part by the multisubunit protein complex Drosophila RBF, E2F, and Myb (dREAM) that contains homologs of the C. elegans synthetic multivulva class B (synMuvB) gene products. Using an integrated approach combining proteomics, genomics, and bioinformatic analyses, we identified a p130 complex termed DP, RB-like, E2F, and MuvB (DREAM) that contains mammalian homologs of synMuvB proteins LIN-9, LIN-37, LIN-52, LIN-54, and LIN-53/RBBP4. DREAM bound to more than 800 human promoters in G0 and was required for repression of E2F target genes. In S phase, MuvB proteins dissociated from p130 and formed a distinct submodule that bound MYB. This work reveals an evolutionarily conserved multisubunit protein complex that contains p130 and E2F4, but not pRB, and mediates the repression of cell cycle-dependent genes in quiescence. PMID:17531812

  9. Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway.

    PubMed

    Foda, Bardees M; Singh, Upinder

    2015-08-21

    RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5'-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. PMID:26149683

  10. STAT3 Represses Nitric Oxide Synthesis in Human Macrophages upon Mycobacterium tuberculosis Infection

    PubMed Central

    Queval, Christophe J.; Song, Ok-Ryul; Deboosère, Nathalie; Delorme, Vincent; Debrie, Anne-Sophie; Iantomasi, Raffaella; Veyron-Churlet, Romain; Jouny, Samuel; Redhage, Keely; Deloison, Gaspard; Baulard, Alain; Chamaillard, Mathias; Locht, Camille; Brodin, Priscille

    2016-01-01

    Mycobacterium tuberculosis is a successful intracellular pathogen. Numerous host innate immune responses signaling pathways are induced upon mycobacterium invasion, however their impact on M. tuberculosis replication is not fully understood. Here we reinvestigate the role of STAT3 specifically inside human macrophages shortly after M. tuberculosis uptake. We first show that STAT3 activation is mediated by IL-10 and occurs in M. tuberculosis infected cells as well as in bystander non-colonized cells. STAT3 activation results in the inhibition of IL-6, TNF-α, IFN-γ and MIP-1β. We further demonstrate that STAT3 represses iNOS expression and NO synthesis. Accordingly, the inhibition of STAT3 is detrimental for M. tuberculosis intracellular replication. Our study thus points out STAT3 as a key host factor for M. tuberculosis intracellular establishment in the early stages of macrophage infection. PMID:27384401

  11. Long range repression conferring boundaries of Ultrabithorax expression in the Drosophila embryo.

    PubMed Central

    Müller, J; Bienz, M

    1991-01-01

    In an attempt to reconstruct the embryonic expression pattern of the homeotic gene Ultrabithorax (Ubx) by stable integration of fusion constructs, we identified three key control regions called PBX, ABX and BXD. Each of these confers an expression pattern mimicking certain aspects of Ubx expression. The PBX and ABX patterns are limited to the Ubx domain with anterior boundaries at parasegments 6 and 5. In contrast, the BXD pattern extends from head to tail. PBX or ABX expression boundaries are imposed on the BXD pattern, if PBX or ABX is linked to BXD. These boundaries, although not the PBX and ABX expression limits themselves, are dependent on Polycomb function. We conclude that PBX and ABX are recognized by repressors which act across large distances to suppress BXD activity. Stable and heritable Ubx expression boundaries are thus mediated by this process of long range repression. Images PMID:1680676

  12. ZBTB7A acts as a tumor suppressor through the transcriptional repression of glycolysis

    PubMed Central

    Liu, Xue-Song; Haines, Jenna E.; Mehanna, Elie K.; Genet, Matthew D.; Ben-Sahra, Issam; Asara, John M.; Manning, Brendan D.

    2014-01-01

    Elevated glycolysis is a common metabolic trait of cancer, but what drives such metabolic reprogramming remains incompletely clear. We report here a novel transcriptional repressor-mediated negative regulation of glycolysis. ZBTB7A, a member of the POK (POZ/BTB and Krüppel) transcription repressor family, directly binds to the promoter and represses the transcription of critical glycolytic genes, including GLUT3, PFKP, and PKM. Analysis of The Cancer Genome Atlas (TCGA) data sets reveals that the ZBTB7A locus is frequently deleted in many human tumors. Significantly, reduced ZBTB7A expression correlates with up-regulation of the glycolytic genes and poor survival in colon cancer patients. Remarkably, while ZBTB7A-deficient tumors progress exceedingly fast, they exhibit an unusually heightened sensitivity to glycolysis inhibition. Our study uncovers a novel tumor suppressor role of ZBTB7A in directly suppressing glycolysis. PMID:25184678

  13. E2F-Rb Complexes Assemble and Inhibit cdc25A Transcription in Cervical Carcinoma Cells following Repression of Human Papillomavirus Oncogene Expression

    PubMed Central

    Wu, Lingling; Goodwin, Edward C.; Naeger, Lisa Kay; Vigo, Elena; Galaktionov, Konstantin; Helin, Kristian; DiMaio, Daniel

    2000-01-01

    Expression of the bovine papillomavirus E2 protein in cervical carcinoma cells represses expression of integrated human papillomavirus (HPV) E6/E7 oncogenes, followed by repression of the cdc25A gene and other cellular genes required for cell cycle progression, resulting in dramatic growth arrest. To explore the mechanism of repression of cell cycle genes in cervical carcinoma cells following E6/E7 repression, we analyzed regulation of the cdc25A promoter, which contains two consensus E2F binding sites and a consensus E2 binding site. The wild-type E2 protein inhibited expression of a luciferase gene linked to the cdc25A promoter in HT-3 cervical carcinoma cells. Mutation of the distal E2F binding site in the cdc25A promoter abolished E2-induced repression, whereas mutation of the proximal E2F site or the E2 site had no effect. None of these mutations affected the activity of the promoter in the absence of E2 expression. Expression of the E2 protein also led to posttranscriptional increase in the level of E2F4, p105Rb, and p130 and induced the formation of nuclear E2F4-p130 and E2F4-p105Rb complexes. This resulted in marked rearrangement of the protein complexes that formed at the distal E2F site in the cdc25A promoter, including the replacement of free E2F complexes with E2F4-p105Rb complexes. These experiments indicated that repression of E2F-responsive promoters following HPV E6/E7 repression was mediated by activation of the Rb tumor suppressor pathway and the assembly of repressing E2F4-Rb DNA binding complexes. Importantly, these experiments revealed that HPV-induced alterations in E2F transcription complexes that occur during cervical carcinogenesis are reversed by repression of HPV E6/E7 expression. PMID:10982822

  14. Intellectual Performance as a Function of Repression and Menstrual Cycle.

    ERIC Educational Resources Information Center

    Englander-Golden, Paula; And Others

    Performance on complex (Space Relations and Verbal Reasoning) and simple (Digit Symbol) tests was investigated as a function of Byrne's Repression-Sensitization (RS) dimension, phase of menstrual cycle and premenstrual-menstrual (PM) symptomatology in a group of females not taking oral contraceptives. Two control groups, consisting of males and…

  15. Addressing the repressed needs of the Arabic client.

    PubMed

    Dwairy, M

    1997-01-01

    In comparison to families in Western society, the traditional Arabic family plays a relatively greater role in providing support for adult progeny. This serves to condition adult offspring to continue to comply with the will and values of the family. Therefore, in exchange for familial support, Arabic individuals learn to repress authentic needs and emotions, and within that process they relinquish the need for self-actualization. Arabic society discourages individualism and opposes self-actualization by means of simultaneous punishment and moralization. Thus, there is a relatively greater development of the social value system (or superego) and comparatively less development of the self (or ego). In comparison to Western society, Arabic individuals continue to experience greater oppression during adulthood. Given these cultural differences, the processes of reliving and activating repressed needs and emotions, which ultimately serves to promote self-actualization, will transform intrapsychic conflicts into interpersonal and social ones. Thus, personal actions typically encouraged during Western psychotherapy are likely to produce significant social oppression. Indeed, promoting awareness of repressed needs and emotions often leads the Arabic client to become more helpless, because such wishes will rarely be socially sanctioned or satisfactorily fulfilled. Therefore, when addressing repressed needs and emotions in psychotherapy, ego strength, cultural identity, and degree of strictness of the client's family of origin must be considered. PMID:9231529

  16. Maltose and Maltodextrin Utilization by Listeria monocytogenes Depend on an Inducible ABC Transporter which Is Repressed by Glucose

    PubMed Central

    Gopal, Shubha; Berg, Daniela; Hagen, Nicole; Schriefer, Eva-Maria; Stoll, Regina; Goebel, Werner; Kreft, Jürgen

    2010-01-01

    Background In the environment as well as in the vertebrate intestine, Listeriae have access to complex carbohydrates like maltodextrins. Bacterial exploitation of such compounds requires specific uptake and utilization systems. Methodology/Principal Findings We could show that Listeria monocytogenes and other Listeria species contain genes/gene products with high homology to the maltodextrin ABC transporter and utilization system of B. subtilis. Mutant construction and growth tests revealed that the L. monocytogenes gene cluster was required for the efficient utilization of maltodextrins as well as maltose. The gene for the ATP binding protein of the transporter was located distant from the cluster. Transcription analyses demonstrated that the system was induced by maltose/maltodextrins and repressed by glucose. Its induction was dependent on a LacI type transcriptional regulator. Repression by glucose was independent of the catabolite control protein CcpA, but was relieved in a mutant defective for Hpr kinase/phosphorylase. Conclusions/Significance The data obtained show that in L. monocytogenes the uptake of maltodextrin and, in contrast to B. subtilis, also maltose is exclusively mediated by an ABC transporter. Furthermore, the results suggest that glucose repression of the uptake system possibly is by inducer exclusion, a mechanism not described so far in this organism. PMID:20436965

  17. NSun2 delays replicative senescence by repressing p27 (KIP1) translation and elevating CDK1 translation

    PubMed Central

    Xing, Junyue; Liu, Zhenyun; Jiang, Bin; Dou, Yali; Gorospe, Myriam; Wang, Wengong

    2015-01-01

    A rise in the levels of the cyclin-dependent kinase (CDK) inhibitor p27KIP1 is important for the growth arrest of senescent cells, but the mechanisms responsible for this increase are poorly understood. Here, we show that the tRNA methyltransferase NSun2 represses the expression of p27 in replicative senescence. NSun2 methylated the 5′-untranslated region (UTR) of p27 mRNA at cytosine C64 in vitro and in cells, thereby repressing the translation of p27. During replicative senescence, increased p27 protein levels were accompanied by decreased NSun2 protein levels. Knockdown of NSun2 in human diploid fibroblasts (HDFs) elevated p27 levels and reduced the expression of CDK1 (encoded by CDK1 mRNA, a previously reported target of NSun2), which in turn further repressed cell proliferation and accelerated replicative senescence, while overexpression of NSun2 exerted the opposite effect. Ectopic overexpression of the p27 5′UTR fragment rescued the effect of NSun2 overexpression in lowering p27, increasing CDK1, promoting cell proliferation, and delaying replicative senescence. Our findings indicate that NSun2-mediated mRNA methylation regulates p27 and CDK1 levels during replicative senescence. PMID:26687548

  18. Let-7 Represses Carcinogenesis and a Stem Cell Phenotype in the Intestine via Regulation of Hmga2

    PubMed Central

    Madison, Blair B.; Jeganathan, Arjun N.; Mizuno, Rei; Winslow, Monte M.; Castells, Antoni; Cuatrecasas, Miriam; Rustgi, Anil K.

    2015-01-01

    Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2. PMID:26244988

  19. GATA3 induces human T-cell commitment by restraining Notch activity and repressing NK-cell fate.

    PubMed

    Van de Walle, Inge; Dolens, Anne-Catherine; Durinck, Kaat; De Mulder, Katrien; Van Loocke, Wouter; Damle, Sagar; Waegemans, Els; De Medts, Jelle; Velghe, Imke; De Smedt, Magda; Vandekerckhove, Bart; Kerre, Tessa; Plum, Jean; Leclercq, Georges; Rothenberg, Ellen V; Van Vlierberghe, Pieter; Speleman, Frank; Taghon, Tom

    2016-01-01

    The gradual reprogramming of haematopoietic precursors into the T-cell fate is characterized by at least two sequential developmental stages. Following Notch1-dependent T-cell lineage specification during which the first T-cell lineage genes are expressed and myeloid and dendritic cell potential is lost, T-cell specific transcription factors subsequently induce T-cell commitment by repressing residual natural killer (NK)-cell potential. How these processes are regulated in human is poorly understood, especially since efficient T-cell lineage commitment requires a reduction in Notch signalling activity following T-cell specification. Here, we show that GATA3, in contrast to TCF1, controls human T-cell lineage commitment through direct regulation of three distinct processes: repression of NK-cell fate, upregulation of T-cell lineage genes to promote further differentiation and restraint of Notch activity. Repression of the Notch1 target gene DTX1 hereby is essential to prevent NK-cell differentiation. Thus, GATA3-mediated positive and negative feedback mechanisms control human T-cell lineage commitment. PMID:27048872

  20. Transcriptional repression by RING finger protein TIF1 beta that interacts with the KRAB repressor domain of KOX1.

    PubMed Central

    Moosmann, P; Georgiev, O; Le Douarin, B; Bourquin, J P; Schaffner, W

    1996-01-01

    Many of the vertebrate zinc finger factors of the Kruppel type (C2H2 zinc fingers) contain in their N-terminus a conserved sequence referred to as the KRAB (Kruppel-associated box) domain that, when tethered to DNA, efficiently represses transcription. Using the yeast two-hybrid system, we have isolated an 835 amino acid RING finger (C3HC4 zinc finger) protein, TIF1 beta (also named KAP-1), that specifically interacts with the KRAB domain of the human zinc finger factor KOX1/ZNF10. TIF1 beta, TIF1 alpha, PML and efp belong to a characteristic subgroup of RING finger proteins that contain one or two other Cys/His-rich clusters (B boxes) and a putative coiled-coil in addition to the classical C3HC4 RING finger motif (RBCC configuration). Like TIF1 alpha, TIF1 beta also contains an additional Cys/His cluster (PHD finger) and a bromo-related domain. When tethered to DNA, TIF1 beta can repress transcription in transiently transfected mammalian cells both from promoter-proximal and remote (enhancer) positions, similarly to the KRAB domain itself. We propose that TIF1 beta is a mediator of the transcriptional repression exerted by the KRAB domain. PMID:9016654

  1. Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2

    PubMed Central

    Justin, Neil; Zhang, Ying; Tarricone, Cataldo; Martin, Stephen R.; Chen, Shuyang; Underwood, Elizabeth; De Marco, Valeria; Haire, Lesley F.; Walker, Philip A.; Reinberg, Danny; Wilson, Jon R.; Gamblin, Steven J.

    2016-01-01

    Polycomb repressive complex 2 (PRC2) silences gene expression through trimethylation of K27 of histone H3 (H3K27me3) via its catalytic SET domain. A missense mutation in the substrate of PRC2, histone H3K27M, is associated with certain pediatric brain cancers and is linked to a global decrease of H3K27me3 in the affected cells thought to be mediated by inhibition of PRC2 activity. We present here the crystal structure of human PRC2 in complex with the inhibitory H3K27M peptide bound to the active site of the SET domain, with the methionine residue located in the pocket that normally accommodates the target lysine residue. The structure and binding studies suggest a mechanism for the oncogenic inhibition of H3K27M. The structure also reveals how binding of repressive marks, like H3K27me3, to the EED subunit of the complex leads to enhancement of the catalytic efficiency of the SET domain and thus the propagation of this repressive histone modification. PMID:27121947

  2. Glucose lowers CRP* levels resulting in repression of the lac operon in cells lacking cAMP.

    PubMed

    Tagami, H; Inada, T; Kunimura, T; Aiba, H

    1995-07-01

    CRP-cAMP-dependent operons of Escherichia coli can be expressed in cells lacking functional adenylate cyclase when they carry a second-site mutation in the crp gene (crp*). It is known that the expression of these operons is repressed by glucose, but the molecular mechanism underlying this cAMP-independent catabolite repression has been a long-standing mystery. Here we address the question of how glucose inhibits the expression of beta-galactosidase in the absence of cAMP. We have isolated several mutations in the crp gene that confer a CRP* phenotype. The expression of beta-galactosidase is reduced by glucose in cells carrying these mutations. Using Western blotting and/or SDS-PAGE analysis, we demonstrate that glucose lowers the cellular concentration of CRP* through a reduction in crp* mRNA levels. The level of CRP* protein correlates with beta-galactosidase activity. When the crp promoter is replaced with the bla promoter, the inhibitory effect of glucose on crp* expression is virtually abolished. These data strongly suggest that the lowered level of CRP* caused by glucose mediates catabolite repression in cya- crp* cells and that the autoregulatory circuit of the crp gene is involved in the down-regulation of CRP* expression by glucose. PMID:7494474

  3. Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2.

    PubMed

    Justin, Neil; Zhang, Ying; Tarricone, Cataldo; Martin, Stephen R; Chen, Shuyang; Underwood, Elizabeth; De Marco, Valeria; Haire, Lesley F; Walker, Philip A; Reinberg, Danny; Wilson, Jon R; Gamblin, Steven J

    2016-01-01

    Polycomb repressive complex 2 (PRC2) silences gene expression through trimethylation of K27 of histone H3 (H3K27me3) via its catalytic SET domain. A missense mutation in the substrate of PRC2, histone H3K27M, is associated with certain pediatric brain cancers and is linked to a global decrease of H3K27me3 in the affected cells thought to be mediated by inhibition of PRC2 activity. We present here the crystal structure of human PRC2 in complex with the inhibitory H3K27M peptide bound to the active site of the SET domain, with the methionine residue located in the pocket that normally accommodates the target lysine residue. The structure and binding studies suggest a mechanism for the oncogenic inhibition of H3K27M. The structure also reveals how binding of repressive marks, like H3K27me3, to the EED subunit of the complex leads to enhancement of the catalytic efficiency of the SET domain and thus the propagation of this repressive histone modification. PMID:27121947

  4. Arabidopsis DE-ETIOLATED1 Represses Photomorphogenesis by Positively Regulating Phytochrome-Interacting Factors in the Dark[C][W

    PubMed Central

    Dong, Jie; Tang, Dafang; Gao, Zhaoxu; Yu, Renbo; Li, Kunlun; He, Hang; Terzaghi, William; Deng, Xing Wang

    2014-01-01

    Arabidopsis thaliana seedlings undergo photomorphogenic development even in darkness when the function of DE-ETIOLATED1 (DET1), a repressor of photomorphogenesis, is disrupted. However, the mechanism by which DET1 represses photomorphogenesis remains unclear. Our results indicate that DET1 directly interacts with a group of transcription factors known as the phytochrome-interacting factors (PIFs). Furthermore, our results suggest that DET1 positively regulates PIF protein levels primarily by stabilizing PIF proteins in the dark. Genetic analysis showed that each pif single mutant could enhance the det1-1 phenotype, and ectopic expression of each PIF in det1-1 partially suppressed the det1-1 phenotype, based on hypocotyl elongation and cotyledon opening angles observed in darkness. Genomic analysis also revealed that DET1 may modulate the expression of light-regulated genes to mediate photomorphogenesis partially through PIFs. The observed interaction and regulation between DET1 and PIFs not only reveal how DET1 represses photomorphogenesis, but also suggest a possible mechanism by which two groups of photomorphogenic repressors, CONSTITUTIVE PHOTOMORPHOGENESIS/DET/FUSCA and PIFs, work in concert to repress photomorphogenesis in darkness. PMID:25248553

  5. GATA3 induces human T-cell commitment by restraining Notch activity and repressing NK-cell fate

    PubMed Central

    Van de Walle, Inge; Dolens, Anne-Catherine; Durinck, Kaat; De Mulder, Katrien; Van Loocke, Wouter; Damle, Sagar; Waegemans, Els; De Medts, Jelle; Velghe, Imke; De Smedt, Magda; Vandekerckhove, Bart; Kerre, Tessa; Plum, Jean; Leclercq, Georges; Rothenberg, Ellen V.; Van Vlierberghe, Pieter; Speleman, Frank; Taghon, Tom

    2016-01-01

    The gradual reprogramming of haematopoietic precursors into the T-cell fate is characterized by at least two sequential developmental stages. Following Notch1-dependent T-cell lineage specification during which the first T-cell lineage genes are expressed and myeloid and dendritic cell potential is lost, T-cell specific transcription factors subsequently induce T-cell commitment by repressing residual natural killer (NK)-cell potential. How these processes are regulated in human is poorly understood, especially since efficient T-cell lineage commitment requires a reduction in Notch signalling activity following T-cell specification. Here, we show that GATA3, in contrast to TCF1, controls human T-cell lineage commitment through direct regulation of three distinct processes: repression of NK-cell fate, upregulation of T-cell lineage genes to promote further differentiation and restraint of Notch activity. Repression of the Notch1 target gene DTX1 hereby is essential to prevent NK-cell differentiation. Thus, GATA3-mediated positive and negative feedback mechanisms control human T-cell lineage commitment. PMID:27048872

  6. Staphylococcus aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism

    PubMed Central

    Boisset, Sandrine; Geissmann, Thomas; Huntzinger, Eric; Fechter, Pierre; Bendridi, Nadia; Possedko, Maria; Chevalier, Clément; Helfer, Anne Catherine; Benito, Yvonne; Jacquier, Alain; Gaspin, Christine; Vandenesch, François; Romby, Pascale

    2007-01-01

    RNAIII is the intracellular effector of the quorum-sensing system in Staphylococcus aureus. It is one of the largest regulatory RNAs (514 nucleotides long) that are known to control the expression of a large number of virulence genes. Here, we show that the 3′ domain of RNAIII coordinately represses at the post-transcriptional level, the expression of mRNAs that encode a class of virulence factors that act early in the infection process. We demonstrate that the 3′ domain acts primarily as an antisense RNA and rapidly anneals to these mRNAs, forming long RNA duplexes. The interaction between RNAIII and the mRNAs results in repression of translation initiation and triggers endoribonuclease III hydrolysis. These processes are followed by rapid depletion of the mRNA pool. In addition, we show that RNAIII and its 3′ domain mediate translational repression of rot mRNA through a limited number of base pairings involving two loop–loop interactions. Since Rot is a transcriptional regulatory protein, we proposed that RNAIII indirectly acts on many downstream genes, resulting in the activation of the synthesis of several exoproteins. These data emphasize the multitude of regulatory steps affected by RNAIII and its 3′ domain in establishing a network of S. aureus virulence factors. PMID:17545468

  7. Gfi1b negatively regulates Rag expression directly and via the repression of FoxO1

    PubMed Central

    Schulz, Danae; Vassen, Lothar; Chow, Kwan T.; McWhirter, Sarah M.; Amin, Rupesh H.; Möröy, Tarik

    2012-01-01

    Precise regulation of Rag (recombination-activating gene) expression is crucial to prevent genomic instability caused by the generation of Rag-mediated DNA breaks. Although mechanisms of Rag activation have been well characterized, the mechanism by which Rag expression is down-regulated in early B cell development has not been fully elucidated. Using a complementary DNA library screen, we identified the transcriptional repressor Gfi1b as negative regulator of the Rag locus. Expression of Gfi1b causes repression of Rag1 and Rag2 in cell lines and primary mouse cells. Conversely, Gfi1b-deficient cell lines exhibit increased Rag expression, double-strand breaks and recombination, and cell cycle defects. In primary cells, transcription of Gfi1b inversely correlates with Rag transcription, and simultaneous inactivation of Gfi1 and Gfi1b leads to an increase in Rag transcription early in B cell development. In addition, deletion of Gfi1 and Gfi1b in vivo results in a severe block in B cell development. Gfi1b orchestrates Rag repression via a dual mechanism. Direct binding of Gfi1b to a site 5′ of the B cell–specific Erag enhancer results in epigenetic changes in the Rag locus, whereas indirect inhibition is achieved through repression of the trans-activator Foxo1. Together, our experiments show that Gfi family members are essential for normal B cell development and play an important role in modulating expression of the V(D)J recombinase. PMID:22201127

  8. Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression.

    PubMed Central

    Miyada, C G; Stoltzfus, L; Wilcox, G

    1984-01-01

    The araC gene encodes a positive regulatory protein required for L-arabinose utilization in Escherichia coli. Transcription from the araC promoter has been shown to be under positive control by cAMP receptor protein and under negative control by its protein product (autoregulation). This work describes the identification of the region of the araC promoter that interacts with the cAMP receptor protein to mediate catabolite repression. A 3-base-pair deletion centered 60 base pairs from the transcriptional initiation site results in a mutant araC promoter that, in the absence of araC protein, reduces transcriptional activity when compared with the wild-type promoter and is unresponsive to various concentrations of intracellular cAMP in vivo. The same deletion results in a lowered affinity of the araC promoter for cAMP receptor protein in vitro. However, this lowered affinity for the mutant araC promoter does not result in substantial reduction of intracellular araC protein because autoregulation of the araC gene dominates catabolite repression. The 3-base-pair deletion in the cAMP receptor protein binding site of the araC promoter does not affect catabolite repression of the adjacent araBAD operon. The implications of these results on current models for expression of the araBAD operon and the araC gene are discussed. Images PMID:6377308

  9. Msx1 Homeodomain Protein Represses the αGSU and GnRH Receptor Genes During Gonadotrope Development

    PubMed Central

    Xie, Huimin; Cherrington, Brian D.; Meadows, Jason D.; Witham, Emily A.

    2013-01-01

    Multiple homeodomain transcription factors are crucial for pituitary organogenesis and cellular differentiation. A homeodomain repressor, Msx1, is expressed from the ventral aspect of the developing anterior pituitary and implicated in gonadotrope differentiation. Here, we find that Msx1 represses transcription of lineage-specific pituitary genes such as the common α-glycoprotein subunit (αGSU) and GnRH receptor (GnRHR) promoters in the mouse gonadotrope-derived cell lines, αT3-1 and LβT2. Repression of the mouse GnRHR promoter by Msx1 is mediated through a consensus-binding motif in the downstream activin regulatory element (DARE). Truncation and mutation analyses of the human αGSU promoter map Msx1 repression to a site at −114, located at the junctional regulatory element (JRE). Dlx activators are closely related to the Msx repressors, acting through the same elements, and Dlx3 and Dlx2 act as transcriptional activators for GnRHR and αGSU, respectively. Small interfering RNA knockdown of Msx1 in αT3-1 cells increases endogenous αGSU and GnRHR mRNA expression. Msx1 gene expression reaches its maximal expression at the rostral edge at e13.5. The subsequent decline in Msx1 expression specifically coincides with the onset of expression of both αGSU and GnRHR. The expression levels of both αGSU and GnRHR in Msx1-null mice at e18.5 are higher compared with wild type, further confirming a role for Msx1 in the repression of αGSU and GnRHR. In summary, Msx1 functions as a negative regulator early in pituitary development by repressing the gonadotrope-specific αGSU and GnRHR genes, but a temporal decline in Msx1 expression alleviates this repression allowing induction of GnRHR and αGSU, thus serving to time the onset of gonadotrope-specific gene program. PMID:23371388

  10. Salt stress represses production of extracellular proteases in Bacillus pumilus.

    PubMed

    Liu, R F; Huang, C L; Feng, H

    2015-01-01

    Bacillus pumilus is able to secrete subtilisin-like prote-ases, one of which has been purified and characterized biochemically, demonstrating great potential for use in industrial applications. In the current study, the biosynthesis and transcription of extracellular pro-teases in B. pumilus (BA06) under salt stress were investigated using various methods, including a proteolytic assay, zymogram analysis, and real-time PCR. Our results showed that total extracellular proteolytic activity, both in fermentation broth and on milk-containing agar plates, was considerably repressed by salt in a dosage-dependent manner. As Bacillus species usually secret multiple extracellular proteases, a vari-ety of individual extracellular protease encoding genes were selected for real-time PCR analysis. It was shown that proteases encoded by the aprE and aprX genes were the major proteases in the fermentation broth in terms of their transcripts in B. pumilus. Further, transcription of aprE, aprX, and epr genes was indeed repressed by salt stress. In con-trast, transcription of other genes (e.g., vpr and wprA) was not repressed or significantly affected by the salt. Conclusively, salt stress represses total extracellular proteolytic activity in B. pumilus, which can largely be ascribed to suppression of the major protease-encoding genes (aprE, aprX) at the transcriptional level. In contrast, transcription of other pro-tease-encoding genes (e.g., vpr, wprA) was not repressed by salt stress. PMID:25966269

  11. ZFP36L1 promotes monocyte/macrophage differentiation by repressing CDK6

    PubMed Central

    Chen, Ming-Tai; Dong, Lei; Zhang, Xin-Hua; Yin, Xiao-Lin; Ning, Hong-Mei; Shen, Chao; Su, Rui; Li, Feng; Song, Li; Ma, Yan-Ni; Wang, Fang; Zhao, Hua-Lu; Yu, Jia; Zhang, Jun-Wu

    2015-01-01

    RNA binding proteins (RBPs)-mediated post-transcriptional control has been implicated in influencing various aspects of RNA metabolism and playing important roles in mammalian development and pathological diseases. However, the functions of specific RBPs and the molecular mechanisms through which they act in monocyte/macrophage differentiation remain to be determined. In this study, through bioinformatics analysis and experimental validation, we identify that ZFP36L1, a member of ZFP36 zinc finger protein family, exhibits significant decrease in acute myeloid leukemia (AML) patients compared with normal controls and remarkable time-course increase during monocyte/macrophage differentiation of PMA-induced THP-1 and HL-60 cells as well as induction culture of CD34+ hematopoietic stem/progenitor cells (HSPCs). Lentivirus-mediated gain and loss of function assays demonstrate that ZFP36L1 acts as a positive regulator to participate in monocyte/macrophage differentiation. Mechanistic investigation further reveals that ZFP36L1 binds to the CDK6 mRNA 3′untranslated region bearing adenine-uridine rich elements and negatively regulates the expression of CDK6 which is subsequently demonstrated to impede the in vitro monocyte/macrophage differentiation of CD34+ HSPCs. Collectively, our work unravels a ZFP36L1-mediated regulatory circuit through repressing CDK6 expression during monocyte/macrophage differentiation, which may also provide a therapeutic target for AML therapy. PMID:26542173

  12. Repression of multiple CYP2D genes in mouse primary hepatocytes with a single siRNA construct.

    PubMed

    Elraghy, Omaima; Baldwin, William S

    2015-01-01

    The Cyp2d subfamily is the second most abun-dant subfamily of hepatic drug-metabolizing CYPs. In mice, there are nine Cyp2d members that are believed to have redundant catalytic activity. We are testing and optimizing the ability of one short interfering RNA (siRNA) construct to knockdown the expression of multiple mouse Cyp2ds in primary hepatocytes. Expression of Cyp2d10, Cyp2d11, Cyp2d22, and Cyp2d26 was observed in the primary male mouse hepatocytes. Cyp2d9, which is male-specific and growth hormone-dependent, was not expressed in male primary hepatocytes, potentially because of its dependence on pulsatile growth hormone release from the anterior pituitary. Several different siRNAs at different concentrations and with different reagents were used to knockdown Cyp2d expression. siRNA constructs designed to repress only one construct often mildly repressed several Cyp2d isoforms. A construct designed to knockdown every Cyp2d isoform provided the best results, especially when incubated with transfection reagents designed specifically for primary cell culture. Interestingly, a construct designed to knockdown all Cyp2d isoforms, except Cyp2d10, caused a 2.5× increase in Cyp2d10 expression, presumably because of a compensatory response. However, while RNA expression is repressed 24 h after siRNA treatment, associated changes in Cyp2d-mediated metabolism are tenuous. Overall, this study provides data on the expression of murine Cyp2ds in primary cell lines, valuable information on designing siRNAs for silencing multiple murine CYPs, and potential pros and cons of using siRNA as a tool for repressing Cyp2d and estimating Cyp2d's role in murine xenobiotic metabolism. PMID:25124873

  13. ΔNp63α represses anti-proliferative genes via H2A.Z deposition

    PubMed Central

    Gallant-Behm, Corrie L.; Ramsey, Matthew R.; Bensard, Claire L.; Nojek, Ignacio; Tran, Jack; Liu, Minghua; Ellisen, Leif W.; Espinosa, Joaquín M.

    2012-01-01

    ΔNp63α is a member of the p53 family of transcription factors that functions as an oncogene in squamous cell carcinomas (SCCs). Because ΔNp63α and p53 bind virtually identical DNA sequence motifs, it has been proposed that ΔNp63α functions as a dominant-negative inhibitor of p53 to promote proliferation and block apoptosis. However, most SCCs concurrently overexpress ΔNp63α and inactivate p53, suggesting the autonomous action of these oncogenic events. Here we report the discovery of a novel mechanism of transcriptional repression by ΔNp63α that reconciles these observations. We found that although both proteins bind the same genomic sites, they regulate largely nonoverlapping gene sets. Upon activation, p53 binds all enhancers regardless of ΔNp63α status but fails to transactivate genes repressed by ΔNp63α. We found that ΔNp63α associates with the SRCAP chromatin regulatory complex involved in H2A/H2A.Z exchange and mediates H2A.Z deposition at its target loci. Interestingly, knockdown of SRCAP subunits or H2A.Z leads to specific induction of ΔNp63α-repressed genes. We identified SAMD9L as a key anti-proliferative gene repressed by ΔNp63α and H2A.Z whose depletion suffices to reverse the arrest phenotype caused by ΔNp63α knockdown. Collectively, these results illuminate a molecular pathway contributing to the autonomous oncogenic effects of ΔNp63α. PMID:23019126

  14. Unintended Consequences of Repression: Alliance Formation in South Korea's Democracy Movement (1970-1979)

    ERIC Educational Resources Information Center

    Chang, Paul Y.

    2008-01-01

    Research regarding the impact of repression on social movements has yielded conflicting findings; some argue that repression decreases the total quantity of protest events while others argue that it motivates protest. To move beyond this impasse, various scholars have suggested exploring how repression influences the quality of social movements.…

  15. Possible involvement of p38 in mechanisms underlying acceleration of proliferation by 15-deoxy-Delta(12,14)-prostaglandin J2 and the precursors in leukemia cell line THP-1.

    PubMed

    Azuma, Yasutaka; Watanabe, Kyoko; Date, Masataka; Daito, Michiharu; Ohura, Kiyoshi

    2004-03-01

    15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ2), which is a ligand for peroxisome proliferator-activated receptor gamma (PPARgamma), induced apoptosis of several human tumors including gastric, lung, colon, prostate, and breast. However, the role of PPARgamma signals in other types of cancer cells (e.g., leukemia) except solid cancer cells is still unclear. The aim of this study is to evaluate the ability of 15dPGJ2 to modify the proliferation of the human leukemia cell line THP-1. 15dPGJ2 at 5 microM stimulated the proliferation in THP-1 at 24 to 72 h after incubation. In contrast, 15dPGJ2 at concentrations above 10 microM inhibited the proliferation through the induction of apoptosis. PGD2, PGJ2, and Delta12-PGJ2 (DeltaPGJ2), precursors of 15dPGJ2, had similar proliferative effects at lower concentrations, whereas they induced apoptosis at high concentrations. 15dPGJ2 and three precursors failed to induce the differentiation in THP-1 as assessed by using the differentiation marker CD11b. FACScan analysis revealed that PGD2 at 5 microM, PGJ2 at 1 microM, DeltaPGJ2 at 1 microM and 15dPGJ2 at 5 microM all accelerated cell cycle progression in THP-1. Immunoblotting analysis revealed that PGD2 at 5 microM and 15dPGJ2 at 5 microM inhibited the expression of phospho-p38, phospho-MKK3/MKK6, and phospho-ATF-2, and the expression of Cdk inhibitors including p18, p21, and p27 in THP-1. In contrast, PGJ2 at 1 microM and DeltaPGJ2 at 1 microM did not affect their expressions. These results suggest that 15dPGJ2 and PGD2 may, through inactivation of the p38 mitogen-activated protein kinase pathway, inhibit the expression of Cdk inhibitors, leading to acceleration of the THP-1 proliferation. PMID:15037811

  16. The Involvement of Mig1 from Xanthophyllomyces dendrorhous in Catabolic Repression: An Active Mechanism Contributing to the Regulation of Carotenoid Production.

    PubMed

    Alcaíno, Jennifer; Bravo, Natalia; Córdova, Pamela; Marcoleta, Andrés E; Contreras, Gabriela; Barahona, Salvador; Sepúlveda, Dionisia; Fernández-Lobato, María; Baeza, Marcelo; Cifuentes, Víctor

    2016-01-01

    The red yeast X. dendrorhous is one of the few natural sources of astaxanthin, a carotenoid used in aquaculture for salmonid fish pigmentation and in the cosmetic and pharmaceutical industries for its antioxidant properties. Genetic control of carotenogenesis is well characterized in this yeast; however, little is known about the regulation of the carotenogenesis process. Several lines of evidence have suggested that carotenogenesis is regulated by catabolic repression, and the aim of this work was to identify and functionally characterize the X. dendrorhous MIG1 gene encoding the catabolic repressor Mig1, which mediates transcriptional glucose-dependent repression in other yeasts and fungi. The identified gene encodes a protein of 863 amino acids that demonstrates the characteristic conserved features of Mig1 proteins, and binds in vitro to DNA fragments containing Mig1 boxes. Gene functionality was demonstrated by heterologous complementation in a S. cerevisiae mig1- strain; several aspects of catabolic repression were restored by the X. dendrorhous MIG1 gene. Additionally, a X. dendrorhous mig1- mutant was constructed and demonstrated a higher carotenoid content than the wild-type strain. Most important, the mig1- mutation alleviated the glucose-mediated repression of carotenogenesis in X. dendrorhous: the addition of glucose to mig1- and wild-type cultures promoted the growth of both strains, but carotenoid synthesis was observed only in the mutant strain. Transcriptomic and RT-qPCR analyses revealed that several genes were differentially expressed between X. dendrorhous mig1- and the wild-type strain when cultured with glucose as the sole carbon source. The results obtained in this study demonstrate that catabolic repression in X. dendrorhous is an active process in which the identified MIG1 gene product plays a central role in the regulation of several biological processes, including carotenogenesis. PMID:27622474

  17. Repressing Notch Signaling and Expressing TNFα Are Sufficient to Mimic Retinal Regeneration by Inducing Müller Glial Proliferation to Generate Committed Progenitor Cells

    PubMed Central

    Conner, Clay; Ackerman, Kristin M.; Lahne, Manuela; Hobgood, Joshua S.

    2014-01-01

    Retinal damage in teleosts, unlike mammals, induces robust Müller glia-mediated regeneration of lost neurons. We examined whether Notch signaling regulates Müller glia proliferation in the adult zebrafish retina and demonstrated that Notch signaling maintains Müller glia in a quiescent state in the undamaged retina. Repressing Notch signaling, through injection of the γ-secretase inhibitor RO4929097, stimulates a subset of Müller glia to reenter the cell cycle without retinal damage. This RO4929097-induced Müller glia proliferation is mediated by repressing Notch signaling because inducible expression of the Notch Intracellular Domain (NICD) can reverse the effect. This RO4929097-induced proliferation requires Ascl1a expression and Jak1-mediated Stat3 phosphorylation/activation, analogous to the light-damaged retina. Moreover, coinjecting RO4929097 and TNFα, a previously identified damage signal, induced the majority of Müller glia to reenter the cell cycle and produced proliferating neuronal progenitor cells that committed to a neuronal lineage in the undamaged retina. This demonstrates that repressing Notch signaling and activating TNFα signaling are sufficient to induce Müller glia proliferation that generates neuronal progenitor cells that differentiate into retinal neurons, mimicking the responses observed in the regenerating retina. PMID:25339752

  18. Modulation of mammary cancer cell migration by 15-deoxy-Δ12,14-prostaglandin J2: implications for anti-metastatic therapy

    PubMed Central

    Diers, Anne R.; Dranka, Brian P.; Ricart, Karina C.; Oh, Joo Yeun; Johnson, Michelle S.; Zhou, Fen; Pallero, Manuel A.; Bodenstine, Thomas M.; Murphy-Ullrich, Joanne E.; Welch, Danny R.; Aimee, Landar

    2010-01-01

    SYNOPSIS Recently, a number of steps in the progression of metastatic disease have been shown to be regulated by redox signaling. Electrophilic lipids affect redox signaling through the post-translational modification of critical cysteine residues in proteins. However, the therapeutic potential as well as the precise mechanisms of action of electrophilic lipids in cancer cells is poorly understood. In this study, we investigate the effect of the electrophilic prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) on metastatic properties of breast cancer cells. 15d-PGJ2 was shown to decrease migration, stimulate focal adhesion disassembly and cause extensive F-actin reorganization at low concentrations (0.03-0.3 μM). Importantly, these effects seem to be independent of PPARγ and modification of actin or Keap1, which are known protein targets of 15d-PGJ2 at higher concentrations. Interestingly, the p38 inhibitor SB203580 was able to prevent both 15d-PGJ2-induced F-actin reorganization and focal adhesion disassembly. Taken together, our results suggest that electrophiles such as 15d-PGJ2 are potential anti-metastatic agents which exhibit specificity for migration and adhesion pathways at low concentrations where there are no observed effects on Keap1 or cytotoxicity. PMID:20536428

  19. Glucocorticoid Repression of Inflammatory Gene Expression Shows Differential Responsiveness by Transactivation- and Transrepression-Dependent Mechanisms

    PubMed Central

    King, Elizabeth M.; Chivers, Joanna E.; Rider, Christopher F.; Minnich, Anne; Giembycz, Mark A.; Newton, Robert

    2013-01-01

    Binding of glucocorticoid to the glucocorticoid receptor (GR/NR3C1) may repress inflammatory gene transcription via direct, protein synthesis-independent processes (transrepression), or by activating transcription (transactivation) of multiple anti-inflammatory/repressive factors. Using human pulmonary A549 cells, we showed that 34 out of 39 IL-1β-inducible mRNAs were repressed to varying degrees by the synthetic glucocorticoid, dexamethasone. Whilst these repressive effects were GR-dependent, they did not correlate with either the magnitude of IL-1β-inducibility or the NF-κB-dependence of the inflammatory genes. This suggests that induction by IL-1β and repression by dexamethasone are independent events. Roles for transactivation were investigated using the protein synthesis inhibitor, cycloheximide. However, cycloheximide reduced the IL-1β-dependent expression of 13 mRNAs, which, along with the 5 not showing repression by dexamethasone, were not analysed further. Of the remaining 21 inflammatory mRNAs, cycloheximide significantly attenuated the dexamethasone-dependent repression of 11 mRNAs that also showed a marked time-dependence to their repression. Such effects are consistent with repression occurring via the de novo synthesis of a new product, or products, which subsequently cause repression (i.e., repression via a transactivation mechanism). Conversely, 10 mRNAs showed completely cycloheximide-independent, and time-independent, repression by dexamethasone. This is consistent with direct GR transrepression. Importantly, the inflammatory mRNAs showing attenuated repression by dexamethasone in the presence of cycloheximide also showed a significantly greater extent of repression and a higher potency to dexamethasone compared to those mRNAs showing cycloheximide-independent repression. This suggests that the repression of inflammatory mRNAs by GR transactivation-dependent mechanisms accounts for the greatest levels of repression and the most potent

  20. Repression by ARP-1 sensitizes apolipoprotein AI gene responsiveness to RXR alpha and retinoic acid.

    PubMed Central

    Widom, R L; Rhee, M; Karathanasis, S K

    1992-01-01

    The gene coding for apolipoprotein AI (apoAI), a lipid binding protein involved in the transport of cholesterol and other lipids in the plasma, is expressed in mammals predominantly in the liver and the intestine. Liver-specific expression is controlled by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located between nucleotides -222 and -110 upstream of the apoAI gene transcription start site (+1). Previous studies in our laboratory have shown that ARP-1, a member of the nuclear receptor superfamily whose ligand is unknown (orphan receptor), binds to site A and represses transcription of the apoAI gene in liver cells. In a more recent series of experiments, we found that site A is a retinoic acid (RA) response element that responds preferentially to the recently identified RA-responsive receptor RXR alpha over the previously characterized RA receptors RAR alpha and RAR beta. In this study we investigated the combined effects of ARP-1 and RXR alpha on apoAI gene expression in liver cells. Transient transfection assays showed that site A is necessary and sufficient for RXR alpha-mediated transactivation of the apoAI gene basal promoter in human hepatoma HepG2 cells in the presence of RA and that this transactivation is abolished by increasing amounts of cotransfected ARP-1. Electrophoretic mobility shift assays and subsequent Scatchard analysis of the data revealed that ARP-1 and RXR alpha bind to site A with similar affinities. These assays also revealed that ARP-1 and RXR alpha bind to site A as heterodimers with an affinity approximately 10 times greater than that of either ARP-1 or RXR alpha alone. Further transfection assays in HepG2 cells, using as a reporter a construct containing the apoAI gene basal promoter and its upstream regulatory elements (including site A) in their natural context, revealed that RXR alpha

  1. Repression of harmful meiotic recombination in centromeric regions.

    PubMed

    Nambiar, Mridula; Smith, Gerald R

    2016-06-01

    During the first division of meiosis, segregation of homologous chromosomes reduces the chromosome number by half. In most species, sister chromatid cohesion and reciprocal recombination (crossing-over) between homologous chromosomes are essential to provide tension to signal proper chromosome segregation during the first meiotic division. Crossovers are not distributed uniformly throughout the genome and are repressed at and near the centromeres. Rare crossovers that occur too near or in the centromere interfere with proper segregation and can give rise to aneuploid progeny, which can be severely defective or inviable. We review here how crossing-over occurs and how it is prevented in and around the centromeres. Molecular mechanisms of centromeric repression are only now being elucidated. However, rapid advances in understanding crossing-over, chromosome structure, and centromere functions promise to explain how potentially deleterious crossovers are avoided in certain chromosomal regions while allowing beneficial crossovers in others. PMID:26849908

  2. Repression predicts outcome following multidisciplinary treatment of chronic pain.

    PubMed

    Burns, J W

    2000-01-01

    This study examined whether repression predicts outcome following multidisciplinary treatment for chronic pain and whether links between anxiety and outcome are obscured by repressors. Ninety-three chronic pain patients completed a 4-week pain program. Lifting capacity, walking endurance, depression, pain severity, and activity were measured at pre- and posttreatment. Low-anxious, repressor, high-anxious, and defensive/high-anxious groups were formed from median splits of Anxiety Content (ACS) and Lie scales of the Minnesota Multiphasic Personality Inventory-2 (MMPI-2; Butcher, Dahlstrom, Graham, Tellegen, & Kaemmer, 1989). Significant ACS x Lie interactions were found for lifting capacity, depression, and pain severity changes. Planned comparisons showed that both repressors and high-anxious patients performed poorly on lifting capacity; repressors alone recovered poorly on depression and pain severity. Results imply that repression may interfere with the process and outcome of pain programs. PMID:10711590

  3. Recovering a repressed memory, and representational shift in an adolescent.

    PubMed

    Szajnberg, N M

    1993-01-01

    This case report focuses on the disappearance of a fixed pictorial iconic representation and transformation to symbolic (word) representation following the recovery of a repressed memory in a twelve-and-a-half-year-old after the first eleven months of psychoanalysis. There is a substantial body of psychoanalytic literature on the importance of language, naming or articulation of experiences associated with transformation of psychic structure (Fenichel, 1941; Glover, 1955; Kris, 1956a, 1956b; Werner and Kaplan, 1963; Schafer, 1976, 1978a, 1978b, 1980; Shapiro, 1979; Spence, 1982; Stern, 1989) and a relatively autonomous literature on the communicative representational power of children's play or visual portrayals (Piaget, 1962; A. Freud, 1947; Klein, 1964; Axeline, 1947; Esman, 1983; Neubauer et al., 1987; Krall, 1989). To our knowledge, there is no report of the structural effect of the recovery of a repressed traumatic experience, in this case, with subsequent parental confirmation of the traumatic event. PMID:8354843

  4. Carbon catabolite repression of maltase synthesis in Saccharomyces carlsbergensis.

    PubMed Central

    Federoff, H J; Eccleshall, T R; Marmur, J

    1983-01-01

    Carbon catabolite repression of maltase gene expression is brought about by the addition of glucose, resulting in a drastic inhibition of the induction of maltase. When added to induced cells, glucose leads to the inhibition of maltase synthesis within 30 min, which can be accounted for by the disappearance of hybridizable maltase RNA sequences. The loss of maltase-specific RNA due to catabolite repression can be traced to the combined effects of a 15-fold decrease in the rate of transcription of the maltase structural gene 15 to 20 min after the addition of glucose and a change in the half-life of maltase mRNA. However, the stability of maltase, once induced, is not affected by the addition of glucose. Images PMID:6352680

  5. Notochord repression of endodermal Sonic hedgehog permits pancreas development

    PubMed Central

    Hebrok, Matthias; Kim, Seung K.; Melton, Douglas A.

    1998-01-01

    Notochord signals to the endoderm are required for development of the chick dorsal pancreas. Sonic hedgehog (SHH) is normally absent from pancreatic endoderm, and we provide evidence that notochord, in contrast to its effects on adjacent neuroectoderm where SHH expression is induced, represses SHH expression in adjacent nascent pancreatic endoderm. We identify activin-βB and FGF2 as notochord factors that can repress endodermal SHH and thereby permit expression of pancreas genes including Pdx1 and insulin. Endoderm treatment with antibodies that block hedgehog activity also results in pancreatic gene expression. Prevention of SHH expression in prepancreatic dorsal endoderm by intercellular signals, like activin and FGF, may be critical for permitting early steps of chick pancreatic development. PMID:9620856

  6. Prox1 Directly Interacts with LSD1 and Recruits the LSD1/NuRD Complex to Epigenetically Co-Repress CYP7A1 Transcription

    PubMed Central

    Liu, Yanfeng; Xie, Youhua; Liu, Jing

    2013-01-01

    Cholesterol 7α-hydroxylase (CYP7A1) catalyzes the first and rate-limiting step in the classical pathway of bile acids synthesis in liver and is crucial for maintaining lipid homeostasis. Hepatocyte nuclear factor 4α (HNF4α) and α1-fetoprotein transcription factor (FTF) are two major transcription factors driving CYP7A1 promoter activity in hepatocytes. Previous researches have shown that Prospero-related homeobox (Prox1) directly interacts with both HNF4α and FTF and potently co-represses CYP7A1 transcription and bile acid synthesis through unidentified mechanisms. In this work, mechanisms involved in Prox1-mediated co-repression were explored by identifying Prox1-associated proteins using immunoprecipitation followed by mass spectrometry (IP-MS) methodology. Multiple components of the epigenetically repressive lysine-specific demethylase 1 (LSD1)/nucleosome remodeling and histone deacetylase (NuRD) complex, most notably LSD1 and histone deacetylase 2 (HDAC2), were found to be associated with Prox1 and GST pulldown assay demonstrated that Prox1 directly interacts with LSD1. Sequential chromatin immunoprecipitation (ChIP) assays showed that Prox1 co-localizes with HNF4α, LSD1 and HDAC2 on CYP7A1 promoter in HepG2 cells. Furthermore, by using ChIP assay on HepG2 cells with endogenous Prox1 knocked down by RNA interference, Prox1 was shown to recruit LSD1 and HDAC2 onto CYP7A1 promoter and cause increased H3K4 demethylation. Finally, bile acids treatment of HepG2 cells, which significantly repressed CYP7A1 transcription, resulted in increased Prox1 and LSD1/NuRD complex occupancy on CYP7A1 promoter with a concurrent increase in H3K4 demethylation and H3/H4 deacetylation. These results showed that Prox1 interacts with LSD1 to recruit the repressive LSD1/NuRD complex to CYP7A1 promoter and co-represses transcription through epigenetic mechanisms. In addition, such Prox1-mediated epigenetic repression is involved in the physiologically essential negative feedback

  7. CsrA Participates in a PNPase Autoregulatory Mechanism by Selectively Repressing Translation of pnp Transcripts That Have Been Previously Processed by RNase III and PNPase

    PubMed Central

    Park, Hongmarn; Yakhnin, Helen; Connolly, Michael; Romeo, Tony

    2015-01-01

    ABSTRACT Csr is a conserved global regulatory system that represses or activates gene expression posttranscriptionally. CsrA of Escherichia coli is a homodimeric RNA binding protein that regulates transcription elongation, translation initiation, and mRNA stability by binding to the 5′ untranslated leader or initial coding sequence of target transcripts. pnp mRNA, encoding the 3′ to 5′ exoribonuclease polynucleotide phosphorylase (PNPase), was previously identified as a CsrA target by transcriptome sequencing (RNA-seq). Previous studies also showed that RNase III and PNPase participate in a pnp autoregulatory mechanism in which RNase III cleavage of the untranslated leader, followed by PNPase degradation of the resulting 5′ fragment, leads to pnp repression by an undefined translational repression mechanism. Here we demonstrate that CsrA binds to two sites in pnp leader RNA but only after the transcript is fully processed by RNase III and PNPase. In the absence of processing, both of the binding sites are sequestered in an RNA secondary structure, which prevents CsrA binding. The CsrA dimer bridges the upstream high-affinity site to the downstream site that overlaps the pnp Shine-Dalgarno sequence such that bound CsrA causes strong repression of pnp translation. CsrA-mediated translational repression also leads to a small increase in the pnp mRNA decay rate. Although CsrA has been shown to regulate translation and mRNA stability of numerous genes in a variety of organisms, this is the first example in which prior mRNA processing is required for CsrA-mediated regulation. IMPORTANCE CsrA protein represses translation of numerous mRNA targets, typically by binding to multiple sites in the untranslated leader region preceding the coding sequence. We found that CsrA represses translation of pnp by binding to two sites in the pnp leader transcript but only after it is processed by RNase III and PNPase. Processing by these two ribonucleases alters the m

  8. Role of an inhibitory pyrimidine element and polypyrimidine tract binding protein in repression of a regulated alpha-tropomyosin exon.

    PubMed

    Gooding, C; Roberts, G C; Smith, C W

    1998-01-01

    Splicing of exons 2 and 3 of a-tropomyosin (TM) involves mutually exclusive selection of either exon 3, which occurs in most cells, or of exon 2 in smooth muscle (SM) cells. The SM-specific selection of exon 2 results from the inhibition of exon 3. At least two essential cis-acting elements are required for exon 3 inhibition, the upstream and downstream regulatory elements (URE and DRE). These elements are essential for repression of TM exon 3 in SM cells, and also mediate a low level of repression of exon 3 in an in vitro 5' splice site competition assay in HeLa extracts. Here, we show that the DRE consists of at least two discrete components, a short region containing a number of UGC motifs, and an essential pyrimidine-rich tract (DY). We show that the specific sequence of the DY element is important and that DY is able to bind to factors in HeLa nuclear extracts that mediate a low background level of exon 3 skipping. Deletion of a sequence within DY identified as an optimal binding site for PTB impairs (1) regulation of splicing in vivo, (2) skipping of exon 3 in an in vitro 5' splice site competition, (3) the ability of DY competitors to affect the 5' splice site competition in vitro, and (4) binding of PTB to DY. Addition of recombinant PTB to in vitro splicing reactions is able to partially reverse the effects of the DY competitor RNA. The data are consistent with a model for regulation of TM splicing that involves the participation of both tissue-specific and general inhibitory factors and in which PTB plays a role in repressing both splice sites of exon 3. PMID:9436911

  9. Thanatos-associated protein 7 associates with template activating factor-Ibeta and inhibits histone acetylation to repress transcription.

    PubMed

    Macfarlan, Todd; Parker, J Brandon; Nagata, Kyosuke; Chakravarti, Debabrata

    2006-02-01

    The posttranslational modifications of histones on chromatin or a lack thereof is critical in transcriptional regulation. Emerging studies indicate a role for histone-binding proteins in transcriptional activation and repression. We have previously identified template-activating factor-Ibeta (TAF-Ibeta, also called PHAPII, SET, and I(2)(pp2A)) as a component of a cellular complex called inhibitor of acetyltransferases (INHAT) that masks histone acetylation in vitro and blocks histone acetyltransferase (HAT)-dependent transcription in living cells. TAF-Ibeta has also been shown to associate with transcription factors, including nuclear receptors, to regulate their activities. To identify novel interactors of TAF-Ibeta, we employed a yeast two-hybrid screen and identified a previously uncharacterized human protein called thanatos-associated protein-7 (THAP7), a member of a large family of THAP domain-containing putative DNA-binding proteins. In this study we demonstrate that THAP7 associates with TAF-Ibeta in vitro and map their association domains to a C-terminal predicted coiled-coil motif on THAP7 and the central region of TAF-Ibeta. Similarly, stably transfected THAP7 associates with endogenous TAF-Ibeta in intact cells. Like TAF-Ibeta, THAP7 associates with histone H3 and histone H4 and inhibits histone acetylation. The histone-interacting domain of THAP7 is sufficient for this activity in vitro. Promoter-targeted THAP7 can also recruit TAF-Ibeta and silencing mediator of retinoid and thyroid receptors/nuclear hormone receptor corepressor (NCoR) proteins to promoters, and knockdown of TAF-Ibeta by small interfering RNA relieves THAP7-mediated repression, indicating that, like nuclear hormone receptors, THAP7 may represent a novel class of transcription factor that uses TAF-Ibeta as a corepressor to maintain histones in a hypoacetylated, repressed state. PMID:16195249

  10. TBX2 represses PTEN in rhabdomyosarcoma and skeletal muscle

    PubMed Central

    Zhu, Bo; Zhang, Meiling; Williams, Elizabeth M.; Keller, Charles; Mansoor, Atiya; Davie, Judith K.

    2015-01-01

    Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma in children that shares many features of developing skeletal muscle. TBX2, a T-box family member, is highly up regulated in tumor cells of both major RMS subtypes where it functions as an oncogene. TBX2 is a repressor that is often over expressed in cancer cells and functions in bypassing cell growth control, including the repression of the cell cycle regulators p14 and p21. We have found that TBX2 directly represses the tumor suppressor PTEN in both RMS and normal muscle. Exogenous expression of TBX2 in normal muscle cells down regulates PTEN, and depletion or interference with TBX2 in RMS cells up regulates PTEN. Human RMS tumors show high levels of TBX2 and correspondingly low levels of PTEN. The expression of PTEN in clinical RMS samples is relatively uncharacterized and we establish that suppression of PTEN is a frequent event in both subtypes of RMS. TBX2 represses PTEN by directly binding to the promoter and recruiting the histone deacetylase, HDAC1. RMS cells have high levels of activated AKT due to the deregulation of PI3K signaling, and depletion or interference with TBX2, which up regulates PTEN, results in a reduction of phospho-AKT. We have also found that the highly related T-box family member TBX3 does not repress PTEN in the muscle lineage. This work suggests that TBX2 is a central component of the PTEN/PI3K/AKT signaling pathway deregulation in RMS cells and that targeting TBX2 in RMS tumors may offer a novel therapeutic approach for RMS. PMID:26686089

  11. Rest represses maturation within migrating facial branchiomotor neurons

    PubMed Central

    Love, Crystal E.; Prince, Victoria E.

    2015-01-01

    The vertebrate brain arises from the complex organization of millions of neurons. Neurogenesis encompasses not only cell fate specification from neural stem cells, but also the terminal molecular and morphological maturation of neurons at correct positions within the brain. RE1-silencing transcription factor (Rest) is expressed in non-neural tissues and neuronal progenitors where it inhibits the terminal maturation of neurons by repressing hundreds of neuron-specific genes. Here we show that Rest repression of maturation is intimately linked with the migratory capability of ze